2021 한국생물공학회 춘계학술발표대회 및 국제심포지엄

2021 한국생물공학회 춘계학술발표대회

및 국제심포지엄

2021 KSBB Spring Meeting and

International Symposium

2021. 4. 14(수) ~ 16(금)

April 14(Wed) ~ 16(Fri), 2021

Emerging Trends in Biotechnology after the Pandemic2021 한국생물공학회 춘계학술발표대회 및 국제심포지엄2021 KSBB Spring Meeting and International Symposium

Plenary Lecture

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S111 Ideonella sakaiensis, PETase and MHETase: From Identification of Microbial PET Degradation to the Current Situation

Kohei ODA Kyoto Institute of Technology, Matsugasaki, Kyoto, Japan Corresponding Author Email : [email protected] Poly(ethylene terephthalate) (PET) is used extensively worldwide, and its accumulation in the environment threatens ecosystems. We succeeded in isolation from a microbial consortium of Ideonella sakaiensis 201-F6, microorganism that is able to use PET as its energy and carbon sources, and characterization of the key enzymes, PETase and MHETase (Yoshida et al., Science 2016). This discovery accelerated research on not only these enzymes, but also on cutinases known to degrade PET. As a result, cutinase LCC is close to its use for industrial-scale treating of waste PET. In this symposium, the current status of this research area will be introduced. Keywords : Poly(ethylene terephthalate) (PET), Microbial consortium, Ideonella sakaiensis, PET hydrolase (PETase), Mono(2-hydroxyethyl) terephthalic acid hydrolase (MHETase), PET hydrolytic enzyme (PHE) References 1. Shosuke Yoshida, Kazumi Hiraga, Toshihiko Takehana, Ikuo Taniguchi, Hironao Yamaji, Yasuhito Maeda,

Kiyotsuna Toyohara, Kenji Miyamoto, Yoshiharu Kimura, Kohei Oda. A bacterium that degrades and assimilates poly(ethylene terephthalate), Science, 351, 1196-1199 (2016).

2. Shosuke Yoshida, Kazumi Hiraga, Toshihiko Takehana, Ikuo Taniguchi, Hironao Yamaji, Yasuhito Maeda, Kiyotsuna Toyohara, Kenji Miyamoto, Yoshiharu Kimura, Kohei Oda. Response to comment on “A bacte-rium that degrades and assimilates poly(ethylene terephthalate)”, Science, 353, 759-e (2016).

3. Ikuo Taniguchi, Shosuke Yoshida, Kazumi Hiraga, Kenji Miyamoto, Yoshiharu Kimura, Kohei Oda. Bio-degradation of PET: Current status and application aspects, ACS Catalysis, 9, 4089-4105 (2019).

4. Kazumi Hiraga, Ikuo Taniguchi, Shosuke Yoshida, Yoshiharu Kimura, Kohei Oda. Biodegradation of waste PET A sustainable solution for dealing with plastic pollution, EMBO Reports, e49365 (2019).

5. Shosuke Yoshida, Kazumi Hiraga, Ikuo Taniguchi, Kohei Oda. Methods in Enzymology, Vol. 648, Enzymatic Plastic Degradation, Chapter 9. Ideonella sakaiensis, PETase, and MHETase: From identi-fication of microbial PET degradation to enzyme characterization, edited by Gert Weber, Uwe T. Bornscheur and Ren Wei, Academic Press (2021).

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S112 Engineering Enzyme Therapeutics for Cancer Immunotherapy and Inborn Errors of Metabolism

George GEORGIOU Depts of Chemical Engineering, Molecular Biosciences, Biomedical Engineering, and Oncology, University of Texas, Austin, USA Corresponding Author Email : [email protected] Cancer is predicated on the suppression of adaptive immune responses. A key mechanism for immune escape in cancer arises from genetic changes and/or metabolic re-programming that result in the aberrant accumulation of metabolites that potently suppress lymphocyte function. Our lab has pioneered the concept of immune check-point enzyme therapy whereby systemic administration of pharmacologically optimized, human engineered enzymes is employed to activate the immune system leading to cancer eradication. We have invented and led the development of 5 such engineered enzymes that are now either in clinical or late-stage preclinical evaluation. In this presentation I will describe the mechanism of action for two of these checkpoint enzymes and the respective protein engineering campaigns that led to the design of the clinical candidate molecules for human studies. I will also briefly mention our related work on engineered human enzymes for the treatment of inborn error of metabolism diseases. References 1. Cramer, S.L., et al., “Systemic Depletion of L-Cyst(e)ine with Cyst(e)inase Increases Reactive Oxygen

Species and Suppresses Tumor Growth,” Nature Medicine 23120-127 (2017). 2. Triplett, T.A. et al., “Reversal of Indoleamie 2,3-Dioxygenase-Mediated Cancer Immune Suppression by

Systemic Kynurenine Depletion with a Therapeutic Enzyme,” Nature Biotechnology 36:758-764 (2018). 3. Wang, W., et al., “CD8+ T Cells Regulate Tumour Ferroptosis During Cancer Immunotherapy” Nature

569:270-274 (2019). 4. Lu, W-C. et al., “Enzyme-Mediated Depletion of Serum L-Met Abrogates Prostate Cancer Growth via

Multiple Mechanisms without Evidence of Systemic Toxicity, “Proc. Natl. Acad. Sci. USA 117:532-540 (2020).

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S113 Emergence of Genome Engineering in the 2020s

Bernhard PALSSON University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA Corresponding Author Email : [email protected] Background: We have reached the point in time where we can realistically contemplate the design of entire genomes. This will open a new and fundamental chapter in the history of engineering applied to life science. One can thus hypothesize that a new fundamental engineering discipline will emerge during the 2020s, and most likely the first Departments of Genome Engineering will be in operation at leading universities around the world by 2030. Foundations of engineering disciplines: Engineering disciplines are built on similar foundations, including, definition, measurement, modeling (mechanistic and phenomenological), construction, and control. We now have genome-wide capabilities representing these foundations for Genome Engineering. Need for modeling and prediction: The need to integrate knowledge types into big data analytics, generally referred to as explanatory-artificial-intelligence, is growing. Existing genome-scale modeling tools are well positioned to play an important role, but they must be integrated with big data analytics. This talk will describe progress with three approaches to such knowledge enrichment: 1) the use of Independent Component Analysis (ICA) to define independently modulated sets of genes in bacterial transcriptomes, 2) the use of pangenome analysis for the thousands of bacterial genome sequences being generated, and 3) the use of machine learning methods for the analysis of binary phenotypes, such as antimicrobial resistance.

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S114 Type III CRISPR-Cas - from Structure-function Analyses to Diagnostics

Jurre A. STEENS, Yifan ZHU, John VAN DER OOST, Raymond H.J. STAALS Laboratory of Microbiology, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands Corresponding Author Email : [email protected] Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3’ end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5’ end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range. Keywords : CRISPR-Cas, type III, Cmr complex, Cas10, seed, cyclic oligoadenylates, diagnostics, SARS-CoV-2 References 1. Steens, Zhu et al. (2021) SCOPE: Flexible targeting and stringent CARF activation enables type III

CRISPR-Cas diagnostics. BioRxiv, https://doi.org/10.1101/2021.02.01.429135.

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S115 Unlocking the Power of Next-Generation Sequencing

Alex ARAVANIS Senior Vice President and Chief Technology Officer, Illumina, USA Corresponding Author Email : [email protected] The massively parallel sequencing technology known as next-generation sequencing (NGS) has revolutionized the biological sciences. With its ultra-high throughput, scalability, and speed, NGS enables researchers to perform a wide variety of applications and study biological systems at a level never before possible. Illumina’s Chief Technology Officer, Alex Aravanis PhD, MD, presents on the power and potential of next-generation sequencing technology and the opportunities to accelerate innovation in the genomics industry in the clinic and beyond. Keywords : Illumina, Next Generation Sequencing, Technology

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S116 Multiomics Approach for Targeting Novel Microbiome Therapeutics against Chronic Diseases

GwangPyo KO Graduate School of Public Health, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Recently, there were dramatically increased interests on human microbiome research worldwide. While human ecosystems is maintained through mutualistic relationship between human and microbiota, the effect of gut microbiota and associated molecular mechanisms on metabolic and immunological diseases has been not much studied. We have determined the effects of microbiome and determined the genes and pathways of human gut microbiome using both human populations and animal models. The specific aims of this presentation are 1) to determine and characterize the composition of human microbiome as related to target chronic diseases, 2) to investigate the biomarkers for predicting chronic based on microbiome and associated molecular components. Our data suggest that combination of specific microbiome and associated molecular mechanisms in the gut predicts the incidences of chronic metabolic diseases. Our research will help us to understand the association between human microbiome and metabolic diseases and to provide microbiome-based diagnostics and therapeutics. References 1. HS Yoon, CH Cho, MS Yun, HJ You, DH Han, KH Cha, SJ J, SH Moon, K Lee, TW Nam, Ko G. 2021.

Akkermansia muciniphila secretes a glucagon-like peptide-1-inducible protein that improve glucose homeostasis and ameliorate metabolic disease. Nature Microbiol. In press.

2. Lee G, You HJ, Bajai JS, Joo SK, Yu J, Park S, Kang H, Park JH, Kim JH, Lee DH, Lee S, Kim W, and Ko G. 2020. Distinct signatures of gut microbiome and metabolites associated with significant fibrosis in non-obese NAFLD. Nature Comm. 10.1038.

3. Seo B, Jeon K, Moon S, Lee K, Kim WK, Jeong H, Cha KH, Lim MY, Kang W, Kweon MN, Sung J, Kim W, Park JH and Ko G. 2020. Roseburia spp. ameliorate alcoholic fat liver via restoration of gut barrier functions. Cell Host & Microbe. 8:27(1):25-40.

4. Si J, You HJ, Sung J, and Ko G. 2017. Prevotella as a hub for vaginal microbiota under the influence of host genetics and their association with obesity. Cell Host & Microbe. 21(1):97-105.

5. Lim MY, You HJ, Yoon HS, Kwon B, Lee KY, Lee S, Song YM, Sung J, Ko G. 2017. The effect of heritability and host genetics on the gut microbiota and metabolic syndrome. Gut, Apr 6, 2015-311326.

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S117 Natural Products Biosynthesis and Their Modification in the Context of Trends in Biocatalysis and Protein Engineering

Byung-Gee KIM Seoul National University, School of Chemical and Biological Engineering / Institute of Molecular Biology and Genetics / Bio-MAX Institute, Seoul, Korea Corresponding Author Email : [email protected] In the past 30 years, although many new discoveries and concepts have been developed in the field of biotechnology, development of biocatalysis and protein engineering was recognized as a process development of bio-reaction systems and protein catalysts used in the synthesis of fine chemicals in organic chemistry and bioindustry. As the turn-around time of custom-made biocatalysts development becomes very rapidly shortened due to variety of technology innovations, and diverse applications of biocatalysts in chemical and bio-industry are newly opened, biocatalysis already became a good alternative means to substitute chemical catalysis in the field of organic chemistry, and its coverage and its influence in chemical industry will be rapidly increased and broadly expanded in the near future. In this lecture, first, a short review of development of such protein engineering and in vitro evolution will be presented to understand advances in protein in vitro evolution and mutagenesis during the last 30 years. In the trends of these protein evolutionary studies, our lab has focused on natural products biosynthesis and their modifications using the enzymes involved in natural product biosynthesis reactions, such as oxidoreductases (tyrosinase, reductase, P450 and FAD-dependent monooxygenase), glycosyltransferase and halogenase, and to overcome any problems and hurdles we have met in the studies, always protein engineering and/or evolution were required to improve their desired properties, such as activity, enantioselectivity, substrate specificity, and stability. Here, some examples of related our research activities on synthesis of natural products will be presented: i) isoflavonoids bioconversion(ortho-hydroxylation, oxygenation, isomerization and reductions), ii) human milk oligosaccharide production, and iii) indigoids biosynthesis. Stories of such examples will be in perspectives to evaluate future advances in enzymatic approaches for natural product biosynthesis. Keywords : Biocatalysis, Protein engineering, in vitro evolution, natural product synthesis, oxidoreductase, glycosyltransferase, halogenase References 1. Jeongchan Lee, Joonwon Kim, Ji Eun Song, Won-Suk Song, Eun-Jung Kim, Yun-Gon Kim, Hee-Jin Jeong,

Hye Rim Kim, Kwon-Young Choi, and Byung-Gee Kim, “Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli,” (2021) Nature Chemical Biology, 17:104-112.

2. Yun Hee Choi, Bum Seok Park, Joo‐Hyun Seo, Byung‐Gee Kim, “Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β-galactosidase modification,” (2019) Biotechnology and Bioengi-neering, 116:3324-3332.

3. Pyung-Gang Lee, Joonwon Kim, Eun-Jung Kim, Sang-Hyuk Lee, Kwon-Young Choi, Romas J. Kazlauskas, and Byung-Gee Kim, “Biosynthesis of (-)-5-hydroxy-equol and 5-hydroxy-dehydroequol from soy isoflavone, genistein using microbial whole cell bioconversion”, (2017) ACS Chemical Biology, 12, 2883-2890.

4. Sang-Hyuk Lee, Kiheon Baek, Ju-Eun Lee, Byung-Gee Kim, “Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation”, (2016) Biotechnology and Bioengineering, 113(4):735-743.

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[S3] 기업특별세션:

Microbiome and

Pre/pro/postbiotics

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S311 Microbiome and Precision Medicine

Hansoo PARK Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Korea Corresponding Author Email : [email protected] The gut microbiome of patients with disease is believed to influence the development of disease, as well as the efficacy of the drugs. Human Microbiome research was conducted to understand how microbial communities impact on human health. However, the genomic characteristics and detailed functions of effective bacterial strains have not been fully clarified. In this study, we utilized an integrated approach, involving metagenome, bacterial whole genome/transcriptome, mouse intestinal transcriptome, and mouse serum metabolome analysis, to decipher whether bacterial strain-specific differences influence the disease susceptibilities and efficacy of therapeutics in various diseases. Keywords : Precision Medicine, Microbiome, Immuno-oncology, Obesity

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S312 Microbiome and Probiotics research in Cell Biotech

Sanghyun LIM Cell Biotech R&D Center, Gimpo, Korea Corresponding Author Email : [email protected] Cell Biotech, microbiome and probiotics leading company, was established in 1995. One of specialties in Cell Biotech is one-stop solution system, which means that we are doing all steps from R&D to sales and education to keep high-quality of products. In addition, Dual-coating is very-well known technology worldwide. Probiotics confers a health benefit on host. Many researches have proved the health benefits of probiotics on the various diseases such as inflammatory disorders and metabolic diseases. Microbiome research is incredibly increasing with increasing beneficial effects of probiotics on unhealthy subjects. Such research reports that ingestion of probiotics causes changes in intestinal microbiome, suggesting the possibility of the development of pharmabiotics for patients. Here I would like to introduce what kinds of research are doing in Cell Biotech. Keywords : Microbiome, Probiotics, Cell Biotech

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S313 Improve the Quality of Your Life with Human Milk Oligosaccharides

Chulsoo SHIN Advanced Protein Technologies Corp., Suwon, Korea Corresponding Author Email : [email protected] Advanced Protein Technologies Corp. (APTech), is a bio-venture company specialized in developing functional materials and manufacturing recombinant proteins for biopharmaceutical use with its expertise in microbial metabolic engineering technology. In 2016, APTech received the production technology of 2'-Fucosyllactose (2'-FL), a human milk oligosaccharide available in the highest concentration in human milk, was transferred, from Professor Seo Jin-ho's team at Seoul National University worked towards commercializing it. Human breast milk contains full of bioactive components to support the healthy growth and development of newborn babies in their early stage of life. These bioactive components are consisted of essential microbes, human milk oligosaccharides (HMOs), immunoglobulins, lactoferrin, cytokines, and fatty acids. HMOs are the third most abundant solid components after lactose and lipids. HMOs are known over 200 derivatives, combined with lactose linked to fucose, sialic acids and/or N-acetyl glucosamine. The most abundant HMO is 2’-FL, which constitutes nearly 30% the total HMOs. Several biological functions have reported the health benefits of HMOs, which modulation of the microbiota as probiotics, anti-pathogenic effect, anti-viral effect, modulation of the intestinal epithelial cell response, and development of the immune system. Recently, APTech received the approval for 2’-FL to be used as a food ingredient in Vietnam, Thailand, and Malaysia, and US FDA GRAS. Concurrently, 2’-FL is also under review by EFSA for Novel Food approval in Europe and an application to submit a dossier to China is in preparation. Likewise, food ingredient registrations for Latin America, Eastern Europe, Japan, and India are also in progress. With 2’-Fucosyllactose as a starting point, APTech plans to complete a portfolio of the 7 major Human Milk Oligosaccharides including 3-fuco-syllactose (3-FL), Lacto-N-Tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-FucoPentaose (LNFP), 3'-Sialyllatose (3'-SL), and 6'-Sialyllactose (6'-SL). APTech believed that human milk oligosaccharide will be promising health ingredient and improve the quality of life. Keywords : 2’-fucosyllactose, Human milk oligosaccharides, food ingredient, Health benefit, APTech

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S314 The Prebiotics, a Mushroom (Schizophyllum commune) Derived β-glucan Effect Beneficially the Gut Microbiota on Low-fiber Diet or High-fat Diet Mice Model

Sunghong KIM1,2, Moonjae CHO1,2 1Quegen Biotech, Seoul, Korea, 2Jeju National University, Jeju, Korea Corresponding Author Email : [email protected] β-glucan has been studied for various efficacy and can be consumed through grains or mushrooms. But the intake of β-glucan has not been enough to show its effect. Recently, the development of biotechnology has led to the continuous efforts to produce pure β-glucan through the cultivation of yeast, bacteria or mushroom mycelium and introduce it as a health functional food. In particular, β-glucan has been known to have anti-cancer and anti-inflammatory effects, and recently, interest in immune function has been increasing due to COVID19 pandemic. In addition, the function as prebiotics is known along with probiotics, and it is attracting much attention in the development of Synbiotics. Since 2018, Quegen Biotech has been conducting research on β-glucan intestinal function with Jeju National University. After completing clinical trials at Jeonbuk National University Hospital last year, it is now analyzing the usefulness of the human body. In this study, we present the results of changes in gut microbiota according to the intake of β-glucan in mice fed HFD (high-fat diet) or LFD(low-fiber diet). Keywords : Prebiotics, β-glucan, gut microbiota, Synbiotics, high-fat diet mice References 1. Karthika Muthuramalingam, Moonjae Cho, Dietary intervention using (1,3)/(1,6)-β-glucan, a fungus-

derived soluble prebiotic ameliorates high-fat diet-induced metabolic distress and alters beneficially the gut microbiota in mic model (2019), European Journal of Nutrition, 59(6), 2617-2629.

2. Karthika Muthuramalingam, Moonjae Cho, Effect of mushroom derived β-glucan on low-fiber diet induced gut dysbiosis (2019), J Appl Biol Chem, 62(2), 211-217.

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International Program

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[S4-1]

Recent Advances in

Genome Editing Technology

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S411 Development of the Base Editing Technology Target-AID and Its Applications

Keiji NISHIDA Graduate School of Science, Technology and Innovation Kobe University, Japan Corresponding Author Email : [email protected] Genome editing is the technology that enables highly efficient and precise modification of genomic DNA information. Conventional genome editing involves nuclease-mediated DNA double strand breaks at the targeted site of the genome, which then lead mutagenesis through repair mechanisms of the cell. However, DNA double strand breaks are toxic to the cell and resultant mutation was random. In place of nuclease activity of conventional genome editing, newly developed base editing technology employs DNA base-modifying enzymes to perform direct introduction of point mutations at the target site. Deaminase-mediated base editing (BE and Target-AID) has been developed by tethering DNA cytidine deaminases to nuclease-deficient CRISPR-Cas9 system, enabling pinpoint C to T mutagenesis within the range of 3-5 bases. It shows much less toxicity compared to full nuclease Cas9 and does not require template DNA for precise genome editing. It is now applicable to wide range of organisms and keep improving. Recent developments and applications of the base editing technology will be discussed. Keywords : Genome editing, Base editing, Synthetic Biology References 1. Banno S, Nishida K*, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in

Escherichia coli. Nature Microbiolog. Feb 5. doi: 10.1038/s41564-017-0102-6. (2018). 2. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu

H, Miura K, Ezura H*, Nishida K*, Ariizumi T*, Kondo A. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nature Biotechnology. Mar 27. doi: 10.1038/nbt.3833. (2017).

3. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, Aug 4. pii: aaf8729. (2016).

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S412 Virus-induced Plant Genome Editing

Sang-Gyu KIM Department of Biological Sciences, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Plant viruses have been engineered to express heterologous proteins and RNAs in plants for several decades. This viral system can now be applied to editing plant genomes. Virus vectors can deliver Cas proteins and guide RNAs, two key components of the CRISPR gene-editing system, into a plant cell without a complicated experimental procedure. In some cases, plant viruses move to meristematic cells and express gene-editing components in the cell, which results in the production of mutant seeds. Here, we focus on three main issues of the virus-induced genome editing (VIGE) technology in plants: (1) how to express the relatively large size of Cas proteins, (2) how to express guide RNA, and (3) how to increase the efficiency with which viruses are delivered into meristematic cells. We highlight recent advances in how plant virus vectors can be used efficiently in plant-genome editing. Keywords : CRISPR, Plant Genome Editing, Virus References 1. Youngbin Oh, Hyeonjin Kim, Sang-Gyu Kim, Virus-induced plant genome editing, Current Opinion in

Plant Biology (2021).

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S413 Current Status and Challenges of Genome Editing Tools

Sangsu BAE Department of Chemistry, Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] Genome editing tools such as ZFNs, TALENs, and CRISPR-Cas9/Cpf1 derived RNA-guided endonucleases have been broadly used for biomedical research, biotechnology, and plant transformation. CRISPR nucleases are widely exploited due to the ease of use and inexpensive cost; researchers can induce gene editing at different sites by simply altering guide RNAs. Ultimately, the Nobel Prize in Chemistry 2020 were awarded for discovering one of gene technology's sharpest tools. However, the genome editing tools are constantly being developed. DNA base editing tools, including cytosine base editors (CBEs) and adenine base editors (ABEs), enable the direct conversion of DNA bases without producing double-stranded breaks of DNA were developed. Furthermore, a prime editor (PE) that enables generating small insertion and deletion in addition to substitution of several nucleotides at target sites, was recently developed. While the gene editing mechanism is different for each tool, all tools have been developed based on the CRISPR effectors. Here I present current trends in gene editing tools along with on-going studies of my group such as development of web-based programs in CRISPR RGEN Tools (www.rgenome.net), protein engineering for enhancing specificity of ABEs, and versatile application of CRISPR tools for plant transformation and gene editing therapy. Keywords : CRISPR, Base editing, Prime editing, Gene editing therapy, Web-based programs References 1. Jeong YK, Lee SH, Hwang G-H, Hong S-A, Park S-e, Kim J-S, Woo J-S* and Bae S*, “Precise adenine

base editors that exhibit minimized cytosine catalysis” (In revision). 2. Kim Y, Yu J, Hong S-A, Eom J, Jang K, Lee S-N, Woo J-S, Jeong J*, Bae S* and Choi D*, “Ex vivo

therapeutic base and prime editing using chemically derived hepatic progenitors in a mouse model of tyrosinemia type 1” (In revision).

3. Kim C, Jeong YK, Yu J, Shin HJ, Ku KB, Cha HJ, Han JH, Hong S-A, Kim B-T, Kim S-J*, Woo J-S* and Bae S*, “Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens,” Advanced Biology (In press). (Featured on the front cover)

4. Jeong YK, Song B and Bae S*, “Current status and challenges of DNA base editing tools” Molecular Therapy 28, 1938-1952 (2020).

5. Yu J, Cho E, Choi Y-G, Jeong YK, Na Y, Kim J-S, Cho S-R*, Woo J-S* and Bae S*, “Purification of an intact human protein overexpressed from its endogenous locus via direct genome engineering” ACS Synthetic Biology 9, 1591-1598 (2020). (Cover article)

6. Kim HS, Jeong YK, Hur JK, Kim J-S* and Bae S*, “Adenine base editors catalyze cytosine conversions in human cells” Nature Biotechnology 37, 1145-1148 (2019).

7. Jeon Y, Choi YH, Jang Y, Yu J, Goo J, Lee G, Jeong YK, Lee SH, Kim I-S, Kim J-S, Jeong C*, Lee S* and Bae S*, “Direct observation of DNA target searching and cleavage by CRISPR-Cas12a” Nature Communi-cations 9, 2777 (2018).

8. Park J, Childs L, Kim D, Hwang G-H, Kim S, Kim S-T, Kim J-S* and Bae S*, “Digenome-seq web tool for profiling CRISPR specificity” Nature Methods 14, 548-549 (2017).

9. Lim Y, Bak SY, Sung K, Jeong E, Lee SH, Kim J-S, Bae S* and Kim SK*, “Structural roles of guide RNA in the nuclease activity of Cas9 endonuclease” Nature Communications 7, 13350 (2016).

10. Bae S, Kweon J, Kim HS and Kim J-S, “Microhomology-based choice of Cas9 nuclease target sites” Nature Methods 11, 705-706 (2014).

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S414 Supercharging Engineered T cell Therapies for Cancer with Genome Editing

Jae young LEE ToolGen Inc., Seoul, Korea Corresponding Author Email : [email protected] Immune cell-based therapy, particularly genetically modified T cells such as CAR-engineered T cells has shown promise in treating certain blood cancers including acute lymphoblastic leukemia and non-Hodgkin lymphomas. However, clinical utility of CAR-engineered T cells in the context of chronic lymphoblastic leukemia and solid cancer has been questionable. Efficacious and persistent CAR-T cells should be resistant to tumor-associated deleterious immunosuppressive molecules such as TGFβ and/or Prostaglandin E2 (PGE2) and effectively establish long-term memory T cells without exhaustion. Tumor-associated inhibitory signals can dampen the activity of T cells, impeding the anticancer functions of T cells. Many studies have focused on PD-1 or CTLA-4 blockade to invigorate T-cell functions through CD28 signaling, however obtaining robust clinical outcomes remains challenging. To overcome inhibitory tumor-associated microenvironment, we propose downregulation of diacylglycerol kinase (DGK), as it metabolizes diacylglycerol (DAG) to phosphatidic acid (PA) to regulate T cell function. For this, we utilized CRISPR/Cas9 to knockout of two different isoforms of DGKs (DGKα and DGKζ) that are expressed in human T cells. Knockout of DGKs augmented the effector functions of CAR-T cells in vitro via increased TCR signaling. DGKs knockout from CAR-T cells rendered them resistant to soluble immunosuppressive factors such as TGFβ and PGE2 and sustained effector functions under conditions of repeated tumor stimulation. Moreover, DGK knockout caused significant regression of several solid tumor models through enhanced effector functions of either CAR-engineered or TCR-engineered T cells in a xenograft mouse model. Furthermore, DGK knockout CAR-T cells also showed long-term memory phenotype in xenograft mouse model even with repeated tumor challenge. Collectively, our study shows that editing of DGKs effectively enhances the effector functions of CAR-T cells, suggesting that CRISPR/Cas9-mediated knockout of DGK could be applicable as part of a multifaceted clinical strategy to treat various cancers. Keywords : CAR-T, TCR-T, CRISPR/Cas9, Cell Therapy, Cancer

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[S4-2]

Protein and Enzyme Engineering for

Therapeutic Applications

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S421 Design of Bioconjugates that Function at Biological Interface

Noriho KAMIYA1,2 1Division of Biotechnology, Center for Future Chemistry, Kyushu University, Japan, 2Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan Corresponding Author Email : [email protected] By combining the functions of the major components of living organisms, such as proteins, nucleic acids, lipids, and carbohydrates, it will be possible to design value-added novel bioconjugates. The introduction of synthetic compounds to naturally occurring components can further expand the utility of the intrinsic biological functions and even lead to generate new functions. There is growing interest in the use of enzymes that catalyze site-specific crosslinking reactions for this purpose in order to minimize the impact of chemical modification on the structure and function of biomolecules upon bioconjugation. In this context, we have demonstrated the potential of oxidoreductases such as horseradish peroxidase (HRP)1 and laccase2 for crosslinking reaction that is specific to a tyrosine residue genetically introduced into the protein of interest. Microbial transglutaminase (MTG) has also been widely used to crosslink a variety of functional molecules such as biotin3, nucleic acids4, synthetic polymers5, lipids6 and drugs7 with proteins. In this presentation, our on-going efforts to create novel biocon-jugates that function at the biological interface will be introduced. Keywords : Enzymatic crosslinking, Horseradish peroxidase Microbial transglutaminase, Laccase, Lipidization References 1. K. Minamihata, M. Goto, N. Kamiya, Site-specific conjugation of an antibody binding protein catalyzed by

horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG. (2015) Biotechnol. J., 10, 222-226.

2. D. Permana, K. Minamihata, R. Sato, R. Wakabayashi, M. Goto, N. Kamiya, Linear polymerization of protein by sterically-controlled enzymatic cross-linking with a tyrosine-containing peptide loop. (2020) ACS Omega., 5, 5160-5169.

3. Y. Mori, H. Nakazawa, G.A.L. Goncalves, T. Tanaka, M. Umetsu, N. Kamiya, One-dimensional assembly of functional proteins: toward the design of an artificial cellulosome. (2016) Mol. Syst. Des. Eng., 1, 66-73.

4. M. Takahara, R. Wakabayashi, K. Minamihata, M. Goto, N. Kamiya, Primary amine-clustered DNA aptamer for DNA-protein conjugation catalyzed by microbial transglutaminase. (2017) Bioconjugate Chem., 28, 2954-2961.

5. R. Wakabayashi, K. Yahiro, K. Hayashi, M. Goto, N. Kamiya, Protein-grafted polymers prepared through a site-specific conjugation by microbial transglutaminase for an immunosorbent assay. (2017) Biomacro-molecules, 18, 422-430.

6. [1] M. Takahara, R. Wakabayashi, N. Fujimoto, K. Minamihata, M. Goto, N. Kamiya, Enzymatic cell-surface decoration with proteins using amphiphilic lipid-fused peptide substrates. (2019) Chem. Eur. J., 25, 7315–7321.; [2] M. Takahara, S. Mochizuki, R. Wakabayashi, K. Minamihata, M. Goto, K. Sakurai, N. Kamiya, Extending the half-life of a protein in vivo by enzymatic labeling with amphiphilic lipopeptides. (2021) Bioconjugate Chem. DOI: 10.1021/acs.bioconjchem.1c00027.

7. P. Strop, Versatility of Microbial Transglutaminase (2014) Bioconjugate Chem., 25, 855-862.

21


S422 Repurposing Biology through Synthetic Enzymology

Wen Shan YEW Department of Biochemistry, Yong Loo Lin School of Medicine, and Synthetic Biology for Clinical and Techno-logical Innovation, National University of Singapore, Singapore Corresponding Author Email : [email protected] Synthetic Enzymology has been increasingly used to engineer enzymes within biological systems for the repurposing of biology for purposeful function. On that note, the talk will touch on the Synthetic Cannabinoid Biology Programme (SCBP) that is part of the National Research Foundation’s (NRF) Synthetic Biology R&D Programme. The SCBP aims to discover new biosynthetic pathways that produce cannabinoid compounds in a sustainable manner. The SCBP does not study the psychoactive compounds of the cannabis plant, such as tetrahydrocannabinol (THC), which is illegal in Singapore. Instead, it identifies cannabinoid genes for the sustainable production of cannabinoids, such as cannabidiol (CBD). The SCBP does not use the cannabis plant as the aim is to produce non-psychoactive cannabinoids through synthetic biology, so that benefits of cannabinoid pharmaceuticals can be reaped without the use of the plant. In this talk, we will highlight some recent efforts in the sustainable production of cannabinoids using synthetic biology. In addition, the use of mechanistic and engineering principles in enzymology will also be highlighted to demonstrate the utility of synthetic enzymology in a range of applications from therapeutics to sustainable manufacturing to bioreme-diation. Keywords : Synthetic Cannabinoid Biology, Synthetic Enzymology, Synthetic Biology

22


S423 Discovery and Engineering of Human Antibodies Against G Protein-Coupled Receptors

Sang Taek JUNG Department of Biomedical Sciences, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Antibody molecules have been widely used for therapeutics, diagnostics, and biomedical research reagents due to their outstanding target specificity and evolvability. For efficient isolation of monoclonal antibodies against highly valuable targets (e.g., soluble oncogenic targets, receptors, toxins, etc.) in a high throughput manner, we have constructed a series of human antibody libraries, which have a vast diversity of over 1× 1011, and have successfully utilized them for the isolation of monoclonal antibodies against highly challenging target antigens including G protein-coupled receptors (GPCRs). GPCR plays pivotal roles in proliferation and metastasis of many types of cancer, and its expression is highly correlated with prognosis and survival rate of cancer patients. In this study, we have successfully prepared endothelin receptor type A antigen, and screening of our in-house human antibody library could generate anti-ETA antibodies antagonizing the ETA-mediated cellular signaling. In vitro and in vivo anti-tumor potency of the resulting antibodies have been thoroughly evaluated and the results will be presented. Keywords : Antibody Engineering, G Protein-Coupled Receptors, Fc Engineering

23


S424 Multivalent Albumin-FcRn Interactions for the Significantly Prolonged Serum Half-life of a Therapeutic Protein

Inchan KWON School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] Human serum albumin (HSA) has been investigated as a serum half-time extender of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein leading to a limited extension of serum half-life. We hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic enzyme used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bioorthogonal chemistry, strain promoted azide-alkyne cycloaddition. To increase the number of HSA molecules conjugated to one Uox, we employed the faster bioorthogonal chemistry. We prepared Uox variants conjugated to one, two, or four HSA molecules. The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. Keywords : FcRn-mediated recycling, half-life extension, serum albumin, site-specific albumin conjugation, urate oxidase, bioorthogonal chemistry

24


S425 Engineering Antibodies for Superior Pharmacokinetic Properties

Tae Hyun KANG School of Applied Chemistry, Kookmin University, Seoul, Korea Corresponding Author Email : [email protected] Antibodies are a cornerstone molecule in humoral immunity. Fc domain of antibodies takes part in two major functions, which are i) effector functions of immune effector cells and ii) serum half-life of IgG antibodies via Fc receptors (FcRs) binding. Thus, engineering Fc towards selective FcRs affinity will lead to antibodies with guided effector functions or with superior pharmacokinetic properties. In this seminar I am going to talk about engineering antibody Fc for increasing serum half-life, capitalizing on sophisticated directed evolution strategies. Therapeutic potencies of the biochemically evolved Fc’s are validated using human neonatal Fc receptor (hFcRn)-transgenic mice in vivo. Keywords : Antibody Engineering, Fc receptors (FcRs), Pharmacokinetics

25


[S4-3]

Trends in Microbiome Engineering

26


S431 Engineering Microbes to Rewire Host-microbiome Interactions

Matthew CHANG NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), and Synthetic Biology Translational Research Program and Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Corresponding Author Email : [email protected] The wealth of knowledge on the human microbiota composition and its roles in health and disease has recently spurred the development of novel therapeutic strategies. Moreover, with an array of genetic tools that are readily available, programmable genetic circuits can be designed, genomes can be edited and rewritten, and cells can be reprogrammed to foster novel microbiota-based interventions. In this talk, our recent work on engineering gut-resident microbes as versatile platforms equipped with clinically relevant functionalities will be presented. A particular emphasis will be placed on our efforts to transform gut microbes into live biotherapeutics with prophylactic and therapeutic efficacy against pathogenic infections and chronic metabolic diseases. This work provides a strong foundation for engineering microbes to modulate host-microbiome interactions and supports the use of live biotherapeutics as a viable strategy for clinical intervention.

27


S432 Genome-wide Multi-omics Analysis of Mucin-degrading Bacteria Reveals Their Nutrient-dependent Distinct Metabolic Features in the Human Gut

Kyoung Su KIM, Dong-Woo LEE Department of Biotechnology, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Diet-driven gut microbiota is highly associated with human health and diseases. Mucin-degrading (MD) bacteria play a crucial role in shaping commensal microbiota's structure and function in the human gut, but their metabolic features remain elusive. Herein we performed a genome-wide analysis to investigate the nutrient-dependent physiology and metabolism of MD bacteria. The genome sequences of Ruminococcus gnavus ATCC 29149 (RG, 3.55 Mb) and Akkermancia muciniphila ATCC BAA-835 (AM, 2.66 Mb) reveal that they contained 3,345 and 2246 protein-encoding genes, respectively, according to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). However, the PGAP annotations exhibited >60% mismatches with those from different pipeline configurations. Accordingly, we revised the genome annotations of both MD bacteria using the UniProtKB/Swiss-Prot database, followed by additional manual curation based on sequence and structural domain similarity searches. Consequently, we revised 25 genes for RG and 47 genes for AM involved in mucin-derived fermentation pathways and their distinct MD enzymes. To validate the reconstructed pathways, we investigated MD bacteria's nutrient-dependent physiological features and their fermentative profiles using comparative transcriptomic analysis. As a result, we identified their distinct propionate fermentation pathways together with mucin-derived microbial metabolites such as indole-3-lactate and leucic acid by LC-MS/MS spectrometry. Our multi-omics analyses integrated with physiological characterization under anaerobic conditions reveal MD bacteria's differential metabolic features, providing insight into their metabolic consequences of nutrient-dependent phenotypes on microbiota networks with commensal bacteria in the human gut. Keywords : Mucin-degrading bacteria, genome annotation, anaerobic fermentation, Ruminococcus gnavus, Akkermansia muciniphila

28


S433 Strict Anaerobic Microbiota to Clinical Application

Byoung-Chan KIM Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea / 114 Bioventure Center, HealthBiome, Inc., Daejeon, Korea Corresponding Author Email : [email protected] The study of the human microbiome has come to the forefront of biological research. The human symbiotic microbiota and its metabolites influence not only gastrointestinal and nutritional diseases such as Clostridium difficile infection (CDI), irritable bowel syndrome, inflammatory bowel disease, obesity and diabetes but also a host of other human diseases including cancer, arteriosclerosis, Alzheimer’s (AD), and Parkinson’s (PD) disease. However, most research into the human microbiome thus far has focused on microbial diversity and metagenomic analysis through next-generation sequencing (pyrosequencing) techniques. In the next phase, research should focus on the isolation of foundation species and their clinical applications for preventing and curing these microbiota-related human diseases. Recent research has shown that obligate anaerobes occupy more than 99% of total gut microbes and they have a more profound impact on human diseases than facultative ones. Several global drug companies have created cocktails of human gut strict anaerobes for use in faecal microbiota transplantation (FMT); some of these cocktails are currently undergoing clinical trials as treatments for human diseases, and are almost ready for commercial release. Here, I will show one example of the importance of strict anaerobe for regulating neuro-related human disease such as AD, PD, autism spectrum disorder (ASD) and depression. Agathobaculum butyriciproducens (Abut) is a strict anaerobic and butyric acid-producing bacteria. Abut administration to AD, PD, ASD and depression mouse models, improved the phenotypic mouse behaviours and relieve the relative disease symptoms. In conclusion, Abut can be a candidate of Live Bio therapeutic Product (LBP) for preventing or even curing human neuro-related diseases. Keywords : Microbiome, Strict Anaerobes, Agathobaculum, LBP References 1. Go et al., Human gut microbiota Agathobaculum butyriciproducens improves cognitive impairment in LPS-

induced and APP/PS1 mouse models of Alzheimer's disease (2021), Nutrition research, 86, 96-108. 2. Eckburg et al., Diversity of the Human Intestinal Microbial Flora (2005), Science, 308, 1635-1638.

29


S434 Engineering Genetic Circuits for Sensing Gut Inflammation

Dae-Hee LEE Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Corresponding Author Email : [email protected] Trillions of microbes are living and interacting with host cells in the human gut. Since they are essential to human health, changes in the composition of this gut microbiome cause disease-associated symptoms, which motivates the engineering of microbes to sense, treat, or prevent disease. Microbes reprogrammed using advanced genetic circuits are envisaged as emerging living diagnostics for a wide range of diseases and play key roles in regulating gut microbiota to treat disease in a non-invasive manner. Here, we developed a smart Escherichia coli that senses and responds to nitrate, a biomarker of gut inflammation. To this end, we first employed the NarX-NarL two-component regulatory system in E. coli to construct a nitrate-responsive genetic circuit. Next, we optimized this genetic circuit for the best performance using measures of sensitivity and specificity. We then introduced the nitrate genetic circuit into a well-known probiotic E. coli Nissle 1917. We demonstrated that the designed genetic circuit could sense elevated nitrate levels during gut inflammation in mice with native gut microbiota. Moreover, using Boolean AND gate, we generated a genetic circuit for simultaneous sensing of the thiosulfate and nitrate biomarkers, thus increasing the tool’s specificity for diagnosing gut inflammation. The nitrate-responsive genetic circuit will enable new approaches for non-invasive diagnostics of inflammation-associated diseases. Keywords : Genetic circuit, Gut inflammation, Designer probiotic, Nitrate sensing, Thiosulfate sensing, Boolean AND gate References 1. Seung-Gyun Woo et al., A designed whole-cell biosensor for live diagnosis of gut inflammation through

nitrate sensing (2020), Biosensors and Bioelectronics, 168, 112523.

30


[S4-4]

Recent Progress of

Bio-based Plastics

31


S441 Closing Cycles in the Plastics Bioeconomy Using Extremophilic Bacteria

Nick WIERCKX Jülich Research Centre, Institute for Bio- and Geosciences IBG-1:Biotechnology, Jülich, Germany Corresponding Author Email : [email protected] The environmental impact of fossil-based plastics has been broadly discussed. The carbon footprint of traditional plastics contributes to climate change, and especially for more complex polymers or mixed waste stream, technical recycling options are still extremely limited. On the one hand, we produce bio-based plastic monomers using the extremely solvent-tolerant Pseudomonas taiwanensis. This strain was engineered to produce drop-in materials such as phenol, 4-hydroxybenzoate, and styrene from glucose or glycerol (1). These developments can enable the production of novel and more sustainable polymers, but they don’t necessarily solve the problem of end-of-life plastic pollution. Thus, on the other hand, we work to establish plastic waste as a novel bulk carbon source for industrial biotechnology (2). We have engineered Pseudomonas to metabolize ethylene glycol (3), 1,4-butanediol (4), and adipic acid as aliphatic monomers of many polyesters and polyurethanes. The resulting strains were genomically and biochemically characterized. The gained insights enable engineering of plastic monomer metabolism in different Pseudomonas strains that produce various value-added products. By tackling both the bio-based production of plastics and the biodegradation of plastics we can create new value cycles that incentivize waste collection and therefore reduce the burden on the environment. Keywords : Plastic hydrolysates, plastic monomers, upcycling, Pseudomonas References 1. T. Schwanemann, M. Otto, N. Wierckx, B. Wynands. 2020. Pseudomonas as versatile aromatics cell

factory. Biotechnol. J. 15:1900569. 2. N. Wierckx, M. Auxiliadora Prieto, P. Pomposiello, V. de Lorenzo, K. O’Connor, L.M. Blank. 2015. Plastic

waste as a novel substrate for Industrial Biotechnology. Microb. Biotechnol. 8:900-903. 3. W.J. Li, L.N. Jayakody, M.A. Franden, M. Wehrmann, T. Daun, B. Hauer, L.M. Blank, G.T. Beckham, J.

Klebensberger, N. Wierckx. 2019. Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism by Pseudomonas putida KT2440. Environ. Microbiol. 21:3669-3682.

4. W.J. Li, T. Narancic, S.T. Kenny, P.-J. Niehoff, K. E. O'Connor, L.M. Blank, N. Wierckx. 2020. Unraveling 1,4-butanediol metabolism in Pseudomonas putida KT2440. Front. Microbiol. 11:382.

32


S442 Utilizing Formate for Biosynthesis of Polyhydorxyalkanoate (PHA)

Jihee YOON, Min-Kyu OH Department of Chemical and Biological Engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Formate is a promising and sustainable feedstock synthesized from carbon monoxide (CO) or carbon dioxide. Methylorubrum extorquens AM1 is a type II methylotroph that can use formate as a sole carbon source. In this study, we engineered this strain to convert formate into poly-3-hydroxybutyrate (PHB), a promising degradable biopolymer. First, we developed two-stage bioreactor. Acetobacterium woodii was used to transform CO to formate with a whole cell catalysis first-stage reactor. When the strain’s energy metabolism was blocked to suppress acetate production, formate was produced from CO with yield of 100%. The resulting formate was separated and fed to a second-stage reactor. In this bioreactor, an engineered Methylbacterium extorquens AM1 was used for biopolymer synthesis. With optimized pH, formate uptake, and biopolymer synthetic pathway, a high rate production of PHB was conducted. Although PHB production from CO was realized by two stage bioprocess, mechanical property of PHB is very poor to substitute plastics. Therefore, production of PHA copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), is desired. For this purpose, the endo-genous PhaA and PhaC were replaced by bktB and phaC2, with broader substrate specificities. Also, PhaJ was additionally expressed. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, 3HV portions was increased up to 70.6% when cheap SCFA’s were supplied. This study showed that a PHBV can be produced from CO through metabolic engineering and controlled bioprocesses. Keywords : Biopolymer, Formate, Polyhydorxyalkanoate (PHA), Metabolic engineering, Carbon monoxide References 1. H.W. Hwang, J. Yoon, K. Min, M.S. Kim, S.J. Kim, D.H. Cho, H. Susila, J.G. Na, M.K. Oh, Y.H. Kim,

Chem. Eng. J. 389:124394 (2020). 2. J. Yoon, W. Chang, S.H. Oh, S.H. Choi, Y.H. Yang, M.K. Oh, Int. J. Biol. Macromol. 177:284-293 (2021).

33


S443 Development of Biodegradable Plastics Using Bio Based Chemicals

Hoon RYU Industrial Biotechnology PU, Samyang Corp., Seoul, Korea Corresponding Author Email : [email protected] Global warming and waste plastics issues are recognized as important environmental issues. One of important materials to cope with these issues is eco-friendly plastics. Eco-friendly plastics can be divided into non-degradable bioplastics and biodegradable plastics. Non-degradable bioplastics which use biomass derived chemicals play an important role in global carbon neutrality. Bio-degradable plastics are mainly produced using monomers derived from biomass or fermentation product. Biodegradable plastics using bio based chemicals are very important in terms of global warming delay by reducing carbon dioxide in the atmosphere and decrease in waste plastic issues. In this presentation, the development and applications of biodegradable plastics using bio based chemical materials such as isosorbide along with the market prospect of eco-friendly plastics will be introduced. Keywords : Bio based chemical, biodegradable plastics, Global warming, Waste plastic, Isosorbide

34


S444 Chemo- and Bio-cascade Catalysis to Produce Plastic Monomers from Polyethylene Terephthalate

Jeong Chan JOO1, Si Jae PARK2, Kyoung Heon KIM3, Dongyeop OH4, Bong Keun SONG4, Hyun Gil CHA4, Bong Hyun SUNG5, Young Joo YEON6, Hee Taek KIM7 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea, 2Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea, 3Department of Biotechnology, Graduate School, Korea University, Seoul, Korea, 4Research Center for Bio-based Chemicals, Korea Research Institute of Chemical Technology, Ulsan, Korea, 5Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, 6Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Korea, 7Department of Food Science and Technology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Biological depolymerization of polyethylene terephthalate into monomers has gained great attention to reutilize PET waste as renewable materials. Recently, PETases, MHEtases, and cutinases have been discovered and engineered to efficiently degrade PET into monomers for regeneration PET. However, these PET-hydrolytic enzymes still have some limitations in PET depolymerization such as low activity and solubility. Chemo- and bio-cascade depolymerization of PET can be used to overcome limitations of biological depolymerization. In this talk, combination of chemical catalyst and biocatalyst will be presented for the upcycling of PET into value-added chemicals such as plastic monomers or coating agent. Keywords : Chemo- and bio catalysis, cascade reaction, biological upcycling, PET (polyethylene terephthalate) References 1. Kang M.J., Kim H.T., Lee M., Kim K.A., Khang T.U., Song H.M., Park S.J., Joo J.C., and Cha H.G. Green

Chemistry 22 (11), 3461-3469 (2020). 2. Kim H.T., Kim J.K., Cha H.G., Kang M.J., Lee H.S., Khang T.U., Yun E.J., Lee D.-H., B.K. Song, Park

S.J., Joo J.C., and Kim K.H. ACS Sustainable Chemistry & Engineering, 7 (24), 19396-1940 (2019).

35


[S4-5]

BEST-KSBB Joint Symposium

36


S451 The Challenges and Opportunities of Synthetic Biology

I-Son Grace NG Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan Corresponding Author Email : [email protected] In the past seven years, I initiated my “Functional Genes and Proteomics Laboratory” and “iGEM_NCKU_ Tainan for undergraduate students” in National Cheng Kung University. Our mission is to solve the problems by using technologies and concepts of synthetic biology for the future. In this talk, I would introduce our research interests which including: (1) Chemical production of 5-aminolevulinic acid by “AI Cell”, which we engineered a new strain of Escherichia coli, called PIECE. In this strain, the super salvage pathway was fine-tuned to enhance the regeneration of pyridoxal 5’-phosphate (PLP), on which the activity of ALA synthase (ALAS) is highly dependent. The metabolic flux analysis indicated that PIECE would redirect more carbon into the tricarboxylic acid (TCA) cycle and generate more glycine and glutamate, thereby increasing the ALA and biomass. Finally, the maximum ALA yield of 8.21 g/L and a productivity of 0.228 g/L/h were obtained. The results showed that PIECE was not only feasible for high-level ALA production but also had significant potential to reprogram the carbon flux in the TCA cycle to produce value-added chemicals. (2) We developed the “whole-cell biosensor for diagnostic and prevention” for diabetes. A synthetic cell which coupling plasmid-driven T7RNA polymerase (PDT7) and NIMPLY-mediated CRISPRi would increase the sensitivity for diagnostic and finally was applied to an integrative and portable system for daily urine glucose inspection. (3) We used CRISPR/Cas9 system to enhance lipid accumulation in microalgae, Chlorella vulgaris. the Agrobacterium-mediated plasmid is suitable for gene insertion in Chlorella species. The left-border and right-border assisted Cas9 with sgRNA designed on omega-3 fatty acid desaturase (fad3) gene to obtain a higher accumulation of lipid content by 46% (w/w) in C. vulgaris FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in Chlorella. Finally, I would share 4 projects done by iGEM_NCKU_Tainan from 2016 to 2019, especially for the project of “Oh My Gut” which we got the Undergraduate Grand Prize Winner in 2019. No matter what kinds of challenges we will face, synthetic biology is one of the attractive opportunities to the next generation. Keywords : Synthetic biology, 5-aminolevulinic acid, biosensor, CRISPR/Cas9, microalgae, iGEM References 1. Chengfeng Xue, Tzu-Hsuan Yu, I-Son Ng, Engineering pyridoxal kinase PdxY-integrated Escherichia coli

strain and optimization for high-level 5-aminolevulinic acid production. Journal of the Taiwan Institute of Chemical Engineers, (2021) in press.

2. Shih-I Tan, I-Son Ng, CRISPRi-mediated NIMPLY logic gate for fine-tuning the whole-cell sensing toward simple urine glucose detection. ACS Synthetic Biology, (2021) 10(2), 412-421.

3. Way-Rong Lin, I-Son Ng, Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation. Enzyme and Microbial Technology, (2020) 133: 109458.

4. Way-Rong Lin, Shih-I Tan, Chuan-Chieh Hsiang, Po-Kuei Sung, I-Son Ng, Challenges and opportunity of recent genome editing and multi-omics in cyanobacteria and microalgae for bio-refinery. Bioresource Technology, (2019) 291, 121932.

5. https://2019.igem.org/Team:NCKU_Tainan

37


S452 On-site pre-detection of pathogens using colorimetric DNA biosensors

Byeong Hee HWANG Division of Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] On-site pre-detection of pathogens including COVID-19 could significantly decrease of a disease outbreak or national loss in most of the countries. However, conventional detection techniques were limited in use for on-site detection due to the necessity of specialized skill or equipment. Therefore, it is necessary to develop a new technique that can pre-detect pathogens in the field without special skills or equipment. Here, we presented a novel genetic biosensor based on enzyme-free dual-amplification of universal hybridization chain reaction (uHCR) and hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme and a DNAzyme strategy to control a plasmonic biosensor for rapid and simple visual detection of Salmonella Choleraesuis. As a result, the proposed biosensors could visually detect single nucleotide polymorphisms or pathogens without unstable enzymes, a specialized technique, or equipment. Therefore, these advantages could allow that this biosensor would be used for on-site pre-detection to lower the risk of transmission of infectious diseases including COVID-19. Keywords : On-site detection, Colorimetric, DNA Biosensor, Pathogens

38


S453 Programming Cells for Detecting and Degrading Phenolic Compounds

Shen-Long TSAI Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan With industrialization and urbanization, our daily life has become much more convenient than ever. However, problems associated with such modernization are gradually appearing. Especially, environmental pollution is one of the most major problems people are facing now. 1 Therefore, our greatest challenge is to find means to deal with the issue. In my lab, we have employed genetic engineering techniques to address these issues. In this talk, I am going to demonstrate how we can reprogram a bacterial strain to sense, seek and destroy phenolic compounds. Via harnessing the transcription factors, 2 we systematically created several biological crafts to detect phenols. To control the cell movement, the chemotaxis system was analyzed and remodeled. 3 Finally, a phenol utilization pathway was appended for the degradation of phenol. Keywords : Phenol, biodegradation, detection, chemotaxis References 1. Maurya, D. K., Kumar, A., Chaurasiya, U., Hussain, T., Singh, S. K. Modern era of microbial biotechno-

logy: opportunities and future prospects. (2021) Microbiomes and Plant Health, 317-343. 2. Mitchler, M. M., Garcia, J. M., Montero, N. E., Williams, G. J. Transcription factor-based biosensors: a

molecular-guided approach for natural product engineering. (2021) Current Opinion in Biotechnology, 69, 172-181.

3. Arumugam, G., Tyagi, J. Keller-Segel chemotaxis models: a review. (2021) Acta Applicandae Mathe-maticae, 171(1), 1-82.

4. Zhou, Weiwei, et al. Metabolomics analysis reveals potential mechanisms of phenolic accumulation in lettuce (Lactuca sativa L.) induced by low nitrogen supply. (2021) Plant Physiology and Biochemistry 158, 446-453.

39


S454 Mechanical Force-Responsive Microcapsules for Self-Regulated Therapeutics Release in Dynamically Loaded Environments

Yun Kee JO Department of Biomedical Convergence Science and Technology, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement and other complications. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of a non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMC-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approaches will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices. Keywords : stretch-responsive, mechano-activation, adhesive microcapsules, fabric wound dressing, antibiotics delivery References 1. Y. K. Jo, D. Lee, Small 1903736 (2020). 2. A. P. Perodo, Y. K. Jo, G. Duan, G. R. Dodge, D. Lee, R. L. Mauck, Biomaterials 265, 120255 (2021).

40


[S4-6]

Emerging NGS Tools for

Industrial Biotechnology

41


S461 FACS-seq as a Powerful Tool for Profiling the Dose-response Curves of Biosensors in a Massively Parallel Manner

Chong ZHANG Department of Chemical Engineering, Tsinghua University, Beijing, China Corresponding Author Email : [email protected] Living organisms have a variety of mechanisms to sense and respond to the environmental signals by dyna-mically regulating their genetic expression networks. Harnessing this ability, genetically encoded biosensors, mimicking natural regulation networks by assembling basic biological parts like promoter, ribosome binding site, operator, reporter etc. into genetic circuit, are developed to recognize the analytes and transduce their signals to a measurable output. Dose-response curve, mapping the genetic circuits to their function, shapes how individual biosensors respond to the specific signals, which is crucial not only for the specific usage scenario of genetic encoded biosensor, but also for illustrating their regulatory functions in living cells. In this report, we would like show our recent attempts on the application of a dose-response profiling method, namely fluorescence-activated cell sorting coupled with next-generation sequencing (FACS-seq), in generating accurate dose-response curves for thousands of biosensors in a massively parallel manner, which provides a powerful platform for dissecting the mechanistic basis of the regulatory elements in living cells, and for the fine tuning of biosensors in a customized and low-cost manner. As the first example, we focused on tnaC, which encodes the tryptophan operon leader peptide in bacteria and is a model of macromolecular-machinery-dynamics-dependent regulatory elements. Working as a molecular sensor, tnaC responds to intracellular tryptophan (Trp) and regulates the biosynthesis of indol. We used FACS-seq to generate accurate response curves for all possible codon substitutions in tnaC. The FACS-seq results allowed us to generate comprehensive genotype-phenotype maps for 1,450 tnaC variants in living cells. The results clarified the nature of several transient, previously enigmatic states involving RNAP and the ribosome, and these states play important roles in the tnaC sensor response. Using in silico modeling and additional experiment, we further demonstrated the molecular basis of the quantitative relation between basal and inductive response, as well as the range of detection of the sensor. In the second example, we developed a novel biological parts assembly workflow to encode genetic circuits with short DNA barcodes, which make sure one-to-one correspondence of the barcodes and biological parts combinations, enabling high-throughput generation of dose-response curves of higher-order combinatorial biosensor space. As a proof of concept, we applied our novel workflow for the fine-tuning of the dose-response curve of malonyl-CoA biosensor based on FapR-fapO system in Saccharomyces cerevisiae. With our 5-step encoding workflow, a trackable combinatorial library contained 5155/5184 combinations with 6 levels of TF dosage, 4 different operator positions, and 216 possible UAS designs, was constructed. FACS-seq successfully characterized the response curve of 2,632 biosensors out of 5184 combinations, providing large-scale genotype-phenotype association data of the designed biosensors. Machine-learning algorithms were then developed to predict uncharacterized dose-response curves and identify key features in the whole combinatorial library, generating a panoramic scanning map of the combinatorial space. With the assistance of our novel workflow, 3755 dose-response curves were obtained at a cost of $1.37 per curve, and a malonyl-CoA biosensor with the largest dynamic response range reported so far was successfully acquired. Keywords : FACS-seq, biosensor, dose-response curve, tryptophan, malonyl-CoA References 1. Wang, T.*, Zheng, X., Ji, H., Wang, T. L., Xing, X. H., & Zhang, C.*. Dynamics of transcription-

translation coordination tune bacterial indole signaling. Nature Chemical Biology 16, 440-449 (2020).

42


S462 The Fur Pan-regulon Uncovers the Complexity and Diversity of Transcriptional Regulation in Escherichia coli

Donghyuk KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Global transcription factors (TFs) have been studied extensively in the reference strain Escherichia coli K-12 MG1655 for the past few decades. However, conservation and possible diversity of regulons has not been experimentally investigated. With experimentally-derived pan-regulon definition for the highly conserved global Ferric uptake regulator (Fur), involved in iron homeostasis, we determined Fur regulons in nine E. coli strains of different phylogroups with ChIP-exo and RNA-seq. Thirty-six of the 469 target genes (8%) found belong to the core regulon, comprising genes involved in ion transport/metabolism, energy production/conversion, and amino acid transport/metabolism. Other 158 target genes (34%) comprise the accessory regulon, most of which were related to cell wall structure/biogenesis, and virulence factor pathways. The remaining target genes (58%) were unique to each strain, with largely unknown gene functions. The pan-regulon is applied to: 1) link strain-specific phenotypes (growth imparied, siderophore production, antibiotic resistance) to underlying molecular mechanisms; and 2) reveal that Fur has a disparate of target genes between the nine strains. Taken together, the Fur transcriptional regulation varies in the same E. coli species, though the binding pattern is broadly conserved. We thus, for the first time, provide experimental evidence of the Fur pan-regulon that shows surprisingly high diversity in regulon genes amongst strains of the same species. Keywords : Pan-genome, Pan-regulon, transcription factor binding sites, transcription regulation network

43


S463 Synthetic Biosensor Accelerates Evolution by Rewiring Carbon Metabolism toward Specific Metabolite

Sang Woo SEO School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolved cells under the selective condition toward the acquisition of genotypes that optimally reallocated cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we combined genome-seq, RNA-seq, and ChIP-exo analyses, and deter-mined that mutations in the conserved regions of proteins involved in global transcriptional regulation altered the expression of several genes associated with central carbon metabolism. These changes rewired central carbon flux towards the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a new perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization. Keywords : NGS, Biosensor, ALE

44


S464 Sensitive One-pot Isothermal Detection of Pathogen-derived RNAs

Jeong Wook LEE Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] The recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and simple onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times. Keywords : SASR-CoV-2, molecular diagnosis, isothermal, one-pot

45


[S4-7]

Frontiers in Biomedical Studies

46


S471 Biochemical Engineering in Animal Cell Technology

Takeshi OMASA Department of Biotechnology Graduate School of Engineering, Osaka University, Osaka, Japan Corresponding Author Email : [email protected] Therapeutic antibodies are now the workhorse for the pharmaceutical industry in 21 century. Mammalian cell lines are important host cells for the industrial production of therapeutic antibodies because of their capacity for correct folding, assembly, and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic antibodies. In this presentation, I introduced our recent approach for CHO genome instability, a bottle-neck process for antibody production, and to prevent antibody aggregation in CHO cell cultivation. Moreover, I will introduce the integrated research and developments in manufacturing technologies for therapeutic protein and cell and gene therapy at the MAB organization. Keywords : biochemical engineering, mammalian cell, biopharmaceuticals, Chinese hamster ovary cell References 1. K. Kaneyoshi, et al., J. Biosci. Bioeng. 127: 107-113 (2019). 2. M. Onitsuka*, et al., J. Biosci. Bioeng., 127:752-757 (2019). 3. N. Yamano-Adachi*, et al., J. Biosci. Bioeng., 129:121-128 (2020). 4. T. Omasa, J. Biosci. Bioeng., 94:600-605 (2002). (Review).

47


S472 Epidural Electrotherapy for Brain Disorders

Sunggu YANG Department of Nano-bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Penetrating electronics have been used for treating epilepsy, yet their therapeutic effects are debated largely due to the lack of a large scale, real-time, safe recording/stimulation. Here, our proposed technology integrates ultrathin epidural electronics into an electrocorticography (ECoG) array, therein simultaneously sampling brain signals in a large area for diagnostic purposes and delivering electrical pulses for treatment. The system was empirically tested to record the ictal-like activities of the thalamocortical network in vitro and in vivo using our epidural electronics. Also, it is newly demonstrated that the electronics selectively diminish epileptiform activities, but not normal signal transduction, in live animals. We propose that this technology heralds a new generation of diagnostic and therapeutic brain-machine interfaces. Such an electronic system can be applicable for several brain diseases such as tinnitus, Parkinson’s disease, Huntington’s disease, depression, and schizo-phrenia. Keywords : Electrotherapy, Brain disorders, Graphene, E-skin References 1. SW. Park, et al., Small 14 (30) (2018).

48


S473 Development of in vitro Human Skeletal Muscle Models Using Microfabricated Devices

Kazunori SHIMIZU Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan Corresponding Author Email : [email protected] In vivo animal experiments have been employed to understand disease mechanisms and check efficacy, toxicity and irritancy of drugs, chemicals, and cosmetics. However, there is a growing need for alternatives to the animal experiments because of the difference of physiological responses between animals and humans, and ethical issues for the use of animals. One of the major alternatives is an in vitro cell culture. Although two-dimensional (2D) cell culture has been employed conventionally, the cultured cells in flat layers on plastic surfaces does not accurately model the in vivo state. In addition, it is difficult to evaluate tissue/organ-like functions including barrier functions, adsorption functions and contractile properties by the conventional 2D culture technique. Thus, better in vitro cell culture techniques to mimic and evaluate tissue/organ-level functions are needed. Skeletal muscle is the most abundant tissue in our body and does not only function as an actuator but involves in multiple physiological functions such as glucose homeostasis and body temperature maintenance, which are all influenced by muscle diseases. Therefore, the functional deterioration of skeletal muscles directly causes a reduced exercise capacity as well as metabolic abnormality, in turn worsening the quality of life significantly. Especially, in super aging society, diseases causing muscle weakness including sarcopenia and disuse atrophy are received much attention. We have developed in vitro human skeletal muscle models (1-7). In this presentation, I will mainly talk about 1) microdevices for contractile force measurement of tissue engineered skeletal muscle and their application for phenotypic drug screening against skeletal muscle atrophy and 2) microdevices for contractile force measure-ment of tissue engineered skeletal muscle innervated by iPSC derived motor neurons (7). Keywords : In vitro models, Skeletal muscle cells, Motor neurons, Human iPSCs, Contractile force, Tissues/ Organs-on-a-Chip References 1. Shimizu, K., Genma, R., Goto, Y., Nagasaka, S., Honda, H.: Bioengineering, 4(2):56 (2017). 2. Yamaoka, N., Shimizu, K., Imaizumi, Y., Ito, T., Okada, Y., Honda, H.: BioChip Journal, 13(2): 127-132

(2019). 3. Shimizu, K., Ohsumi, S., Kishida, T., Mazda, O., Honda, H.: Journal of Bioscience and Bioengineering,

129(5): 632-637 (2020). 4. Nagashima, T., Hadiwidjaja, S., Ohsumi, S., Murata, A., Hisada, T., Kato, R., Okada, Y., Honda, H.,

Shimizu, K.: Advanced Biosystems, 4: 2000121 (2020). 5. Ding, R., Horie, M., Nagasaka, S., Ohsumi, S., Shimizu, K., Honda, H., Nagamori, E., Fujita, H.,

Kawamoto, T.: Journal of Bioscience and Bioengineering, 130(1): 98-105 (2020). 6. Yoshioka, K., Ito, A., Arifuzzaman, Md., Yoshigai, T., Fan, F., Sato, K., Shimizu, K., Kawabe, Y.,

Kamihira, M.: Journal of Bioscience and Bioengineering, in press. 7. Yamamoto, K., Yamaoka, N., Imaizumi, Y., Nagashima, T., Furutani, T., Ito, T., Okada, Y., Honda, H.,

Shimizu, K.: bioRxiv, doi.org/10.1101/2021.01.07.424253.

49


S474 Dissolving Microneedle for Chronic Inflammatory Skin Diseases

Hyungil JUNG Department of Biotechnology, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Triamcinolone acetonide (TA) is one of the most widely prescribed drugs for relieving atopic dermatitis (AD). Since its conventional injection method referred to as TA intralesional injection causes severe pain to patients, applying dissolving microneedles (DMNs) on skin to deliver TA can be an alternative method for treating AD. In this presentation, I introduce our recent approach to overcome TA’s poor dissolving nature which makes it difficult to design and fabricate DMNs containing therapeutic dosage of TA. Moreover, I will introduce the design and fabrication of candlelit shaped TA-loaded DMNs (Candlelit-DMN) which enabled TA evenly distributed across target layer in human skin tissue, and effectively alleviated inflammation. Keywords : Triamcinolone acetonide, Atopic dermatitis, Dissolving microneedles, Candlelit-DMN References 1. M. Jang, et al., Adv. Healthcare Mater., 202001691 (2021). 2. M. King, et al., J. Clin. Aesthet. Dermatol., 13.1: E53 (2020). 3. M. J. Ho, et al., Pharmaceutics., 11.8:419 (2019). 4. Y.C. Kim, et al., Adv. Drug Delivery., 64:1547 (2012). (Review) 5. S.F. Lahiji, et al., Sci. Rep., 9:1 (2019).

50


[S4-8]

Systems Biology for Industrial Applications

51


S481 The Molecular Treasure Hunt – An ‘Omics-based Approach to Find New Bioactive Compounds

Tilmann WEBER The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark Corresponding Author Email : [email protected] Genome analyses of many microorganisms but also higher organisms indicate that the genetic potential to synthesize specialized metabolites is far beyond the number of molecules observed in traditional screenings. With the availability of cheap and easy-to-obtain whole genome sequences, in silico genome mining has become an indispensable tool to complement the classical chemistry-centered approach to identify and characterize novel secondary / specialized metabolites. Since the initial release in 2011, the open source genome mining pipeline antiSMASH(1) (https://antismash.secondarymetabolites.org), which we develop in collaboration with the group of M. Medema (U. Wageningen, Netherlands) and many international contributors, has become one of the most widely used tools. We recently released version 6 of antiSMASH, including an improved user interface, new detection modules, a new cluster comparison tool, and many internal optimizations. Specialist and non-specialist users can easily analyze genomic sequences for the presence of secondary metabolite biosynthetic gene clusters with antiSMASH. To provide extensive analysis options of the data generated with antiSMASH, we have extended the framework with several databases (2-4). The antiSMASH database, (https://antismash-db.secondarymetabolites.org/)(2), contains 147,517 high quality BGC regions from 388 archaeal, 25,236 bacterial and 177 fungal “high-quality” genomes. These genome mining technologies build the foundation of further in silico studies towards a more compre-hensive “Genome Analytics” platform, which we use to streamline our natural product discovery and charac-terization efforts. Albeit streptomycetes are studied for many decades as proficient producers of bioactive compounds, there are still severe limitations concerning efficiency of mutagenesis protocols that often hamper systems metabolic engineering and Synthetic Biology approaches. We have therefore developed an extensive CRISPR/Cas9-based toolkit (5-7) for streptomycetes that now also includes tools that utilize multiplexing and DSB-free base editing technology to highly effectively engineer actinomycetes. Keywords : Genome mining, antibiotics, actinomycetes, Streptomyces, CRISPR References 1. Blin, K., Shaw, S., Steinke, K., Villebro, R., Ziemert, N., Lee, S.Y., Medema, M.H. and Weber, T. (2019)

antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res., 47, W81-W87.

2. Blin, K., Shaw, S., Kautsar, S.A., Medema, M.H. and Weber, T. (2021) The antiSMASH database version 3: increased taxonomic coverage and new query features for modular enzymes. Nucleic Acids Res., 49, D639-D643.

3. Kautsar, S.A., Blin, K., Shaw, S., Weber, T. and Medema, M.H. (2021) BiG-FAM: the biosynthetic gene cluster families database. Nucleic Acids Res., 49, D490-D497.

4. Kautsar, S.A., Blin, K., Shaw, S., Navarro-Munoz, J.C., Terlouw, B.R., van der Hooft, J.J.J., van Santen, J.A., Tracanna, V., Suarez Duran, H.G., Pascal Andreu, V. et al. (2020) MIBiG 2.0: a repository for biosynthetic gene clusters of known function. Nucleic Acids Res., 48, D454-D458.

5. Tong, Y., Whitford, C.M., Blin, K., Jorgensen, T.S., Weber, T. and Lee, S.Y. (2020) CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes. Nat. Protoc., 15, 2470-2502.

6. Tong, Y., Whitford, C.M., Robertsen, H.L., Blin, K., Jorgensen, T.S., Klitgaard, A.K., Gren, T., Jiang, X., Weber, T. and Lee, S.Y. (2019) Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST. Proc. Natl. Acad. Sci. U. S. A., 116, 20366-20375. 7. Tong, Y., Charusanti, P., Zhang, L., Weber, T. and Lee, S.Y. (2015) CR

7. Tong, Y., Charusanti, P., Zhang, L., Weber, T. and Lee, S.Y. (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth. Biol., 4, 1020-1029.

52


S482 Systems Biological Model-guided Design and Engineering of Personalized Probiotics with Host and Microbiome Interactions

Dong-Yup LEE School of Chemical Engineering, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] Probiotics are live beneficial microorganisms that can be consumed in the form of dairy and food products as well as dietary supplements to promote a healthy balance of gut bacteria in humans. Now, one of the main scientific and industrial challenges is to identify and select promising strains and formulate multi-strain probiotic blend with consistent efficacy which is highly dependent on individual dietary regimes, gut environments, and health conditions [1]. Interestingly, the limitations of current in vivo and in vitro methods for testing probiotic strains can be overcome by in silico model guided systems biology approach since genome scale metabolic models (GEMs) can be used to describe and predict their cellular behaviours, metabolic states and community interactions under various gut environments. In this talk, I will summarize currently available GEMs of microbial strains with probiotic potentials, and present a knowledge-based platform to evaluate their capabilities on the basis of probiotic criteria in terms of metabolic characteristics, stability, safety, colonization, postbiotics, interaction with the gut microbiome and variation across diets. Its applicability is demonstrated by a case study where genome-wide metabolic landscapes in several lactic acid bacteria (LAB) are systematically evaluated to elucidate diet-induced and strain specific probiotic features. In addition, I will show how their metabolic interactions with the host and gut microbiome can be investigated to propose new strategies for personalized probiotics design, thereby ameliorating lifestyle diseases. Keywords : Systems biology, smart probiotics, lactic acid bacteria (LAB), microbiome, genome-scale metabolic model References 1. Choi, Y. M., Y. Q. Lee, H.-S. Song and D.-Y. Lee. 2020. Genome scale metabolic models and analysis for

evaluating probiotic potentials. Biochem. Soc. Trans., 48(4): 1309-1321.

53


S483 Systems Biology Applications in Precision Medicine: Multi-omics Approaches

Sunjae LEE School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea Corresponding Author Email : [email protected] “data-driven” life sciences. Recent advance in high throughput technology has enabled us in-depth molecular profiling of individuals with reasonable economic costs and provided the integrated understanding of clinical trials and disease progressions. Systems biology tools have accelerated the integration of multi-omics data and helped the identifications of druggable targets and biomarkers of chronic diseases. For example, molecular signatures of hundred individuals has been traced over a year (Swedish wellness study) and found significant signatures of pre-disease states by systems biology approaches (Abdella Tebani et al., 2020, Nature Communications). Clinical interventions of fatty liver diseases by isocaloric low carbohydrate diets was explored with multi-omics data and elucidated underlying molecular mechanisms that boosted the fat burning during the interventions (Adil Mardinoglu et al., 2018, Cell Metabolism). Furthermore, systems biology identified the biomarkers of insulin resistance, plasma mannose, which was found more powerful than blood glucose, and druggable targets of fatty liver diseases, pyruvate kinase L/R (Sunjae Lee et al., 2017, Molecular Systems Biology; Sunjae Lee et al., 2016, Cell Metabolism). In this talk, the speaker will address the current systems biology applications in precision medicine, especially multi-omics analysis of chronic diseases. Especially recent efforts of understanding human gut microbiome by systems biology approaches, such as pan-disease metagenomic association studies, will be presented. Keywords : systems biology, precision medicine, human metabolism, human microbiome References 1. Abdellah Tebani et al., “Integration of molecular profiles in a longitudinal wellness profiling cohort”

(2020), Nature Communications. 2. Adil Mardinoglu et al., “An integrated understanding of the rapid metabolic benefits of a carbohydrate-

restricted diet on hepatic steatosis in humans” (2018), Cell Metabolism. 3. Sunjae Lee et al., “Network analyses identify liver‐specific targets for treating liver diseases” (2017),

Molecular Systems Biology. 4. Sunjae Lee et al., “Integrated network analysis reveals an association between plasma mannose levels and

insulin resistance” (2016), Cell Metabolism.

54


S484 Retrobiosynthesis for Metabolic Engineering

Hyun Uk KIM Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea Corresponding Author Email : [email protected] Retrobiosynthesis helps systematically design novel biosynthetic pathways for the production of chemicals and materials through metabolic engineering. Retrobiosynthesis is typically implemented by using a set of biochemical reaction rules that describe chemical transformation patterns between substrates and products at an atomic level. Despite its great potential, experimental validation of the retrobiosynthetic predictions has been conducted only for a few chemicals. Here, application of retrobiosynthesis for the production of multiple short-chain primary amines (SCPAs) using Escherichia coli will be presented [1]. SCPAs have a wide range of industrial applications, for example, as a precursor of pharmaceuticals, agrochemicals, and solvents. Also, a deep learning model named DeepRFC will be introduced that examines the feasibility of a large number of retrobiosynthesis-derived enzymatic reactions [2]. DeepRFC will help reduce the number of the initially predicted reactions for experimental validation. These studies will allow more active application of retrobio-synthesis in metabolic engineering. Keywords : Metabolic engineering, Systems biology, Retrobiosynthesis, Short-chain primary amines, DeepRFC References 1. Kim et al. Microbial production of multiple short-chain primary amines via retrobiosynthesis. Nature

Communications 12, 173 (2021). 2. Kim et al. A deep learning approach to evaluate the feasibility of enzymatic reactions generated by

retrobiosynthesis. Biotechnology Journal. In press.

55


[S4-9]

New Application of Biotechnology

in Food Industry

56


S491 Engineering Yeast Strains for Producing Food Ingredients and Value-added Chemicals

Eun Joong OH Department of Food Science, Purdue University, West Lafayette, USA Corresponding Author Email : [email protected] Microbes have recently been engineered to be cellular factories with metabolic pathways designed to convert carbon sources, such as sugars, into value-added chemicals and food materials. However, not all fermentation processes are currently applicable for industrial-scale production. This is due to several challenges, such as high feedstock costs and low productivity. Metabolic engineering is an applied science that employs recombinant DNA technology to improve cellular activity. Metabolic engineering has been advanced as an effective tool with which to improve industrial fermentation processes. My talk will focus on fermentation science and the biotechnological production of fuels and value-added chemicals using engineered yeast strains. I propose metabolic engineering and fermentation research using rational and combinatorial tools to develop and optimize engineered strains capable of rapid and efficient fermentation of abundant sugars in non-food plant biomass, including cellobiose and xylose. I will also highlight multiplex CRISPR/Cas9 gene editing coupled with high-throughput screening and deep sequencing in the field of genome engineering, leading to a better understanding of genotype-phenotype correlations. Keywords : Yeast, metabolic engineering, CRISPR/Cas9 References 1. Oh, E. J., Liu, R., Liang, L., Freed, E. F., Eckert, C. A., and Gill, R. T. 2020. Multiplex evolution of

antibody fragments utilizing a yeast surface display platform. ACS Synthetic Biology 9(8), 2197-2202. 2. Oh, E. J., Skerker, J. M., Kim, S. R., Wei, N., Turner, T. L., Maurer, M., Arkin, A. P., and Jin, Y. S. 2016.

Gene amplification on demand accelerates cellobiose utilization in engineered Saccharomyces cerevisiae. Applied and Environmental Microbiology 82, 3631-3639.

3. Oh, E. J., Ha, S. J., Kim, S. R., Lee, W. H., Galazka, J. M., Cate, J. H. D., and Jin, Y. S. 2013. Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccha-romyces cerevisiae. Metabolic Engineering 15, 226-234.

57


S492 Biosynthesis of Functional Food Materials by Amylosucrase

Dong-Ho SEO Department of Food Science and Technology, College of Agriculture and Life Sciences, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Amylosucrase (ASase; EC 2.4.1.4) is an enzyme that has great potential in the biotechnology and food industries, due to its multifunctional enzyme activities. It can synthesize α-1,4-glucans, like amylose, from sucrose as a sole substrate, but importantly, it can also utilize various other molecules as acceptors. It efficiently synthesizes modified starch with increased ratios of slow digestive starch (SDS) and resistant starch (RS), and glucosylated functional compounds with increased water solubility and stability. First, novel modified chestnut starch was synthesized by ASase from Deinococcus geothermalis (DGAS). DGAS-modification of chestnut starch increased non-digestible resistant starch and it ameliorated diet-induced obesity via GPR43-mediated suppression of insulin signaling thereby presumably reducing fat accumulation in white adipose tissue. Second. α-flavone glycosides have beneficial properties for applications in the pharmaceutical, cosmetic, and food industries. However, their chemical syntheses are often limited by a low efficiency or scarcity of substrates. α-flavone glucosides were enzymatically synthesized by DGAS using sucrose and various flavones as a donor for glucosyl units and acceptors, respectively. Finally, the DGAS expressed in Corynebacterium glutamicum (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in Escherichia coli (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity was greater in cDGAS than in eDGAS. Furthermore, The transglycosylation activity of cDgAS was optimized for luteolin glycoside synthesis using purified enzymes (PEs), whole cells (WCs) suspended in buffer (WCB), and WCs in ethanol (WCE). The reaction parameters for PE, WCE, WCB, and IC transglycosylation were optimized using response surface methodology, and ICs required lower concentrations of substrates while synthesizing higher concentrations of reaction products in addition to producing the novel compound luteolin maltotrioside after several cycles of reuse. Keywords : Amylosucrase, Transglycosylation, Deinococcus geothermalis, Corynebacterium glutamicum References 1. D. H. Seo, S. H. Yoo, S. J. Choi, Y. R. Kim and C. S. Park, Food Sci. Biotechnol. 29(1):1-16 (2020). 2. E. S. Lee, B. H. Lee, D. U. Shin, M. Y. Lim, W. H. Chung, C. S. Food Hydrocolloids. 75:22-23 (2018)Park,

M. Y. Baik, Y. D. Nam, D. H. Seo. 3. S. W. Jang, C. H. Cho, Y. S. Jung, C. Rha, T. G. Nam, D. O. Kim, Y. G. Lee, N. I. Baek, C. S. Park, B. H.

Lee, S. Y. Lee, H. S. Shin, D. H. Seo, Plos One. 13(11): e0207466 (2018). 4. Y. W. Chin, S. W. Jang, H. S. Shin, T. W. Kim, S. K. Kim, C. S. Park, D. H. Seo, Enzyme Microb. Technol.

135:109505 (2020).

58


S493 Future Muscle Food : Status and Prospects of Cultured Meat

Cheorun JO Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Cellular agriculture is an emerging research field of agribiotechnology. Stem cell and tissue engineering makes it possible to produce agricultural products and by-products without a sacrifice of animals. One of the representative technologies in cellular agriculture, cultured muscle tissue-based protein products, known as cultured meat, are produced through in vitro myogenesis involving muscle stem cell culture and differentiation, and mature muscle cell processing for flavor and texture. The idea of cultured meat has been developed to resolve some problems related to industrial livestock farming by lessening undesirable results from traditional animal farming. In this presentation, the background of development, status and prospects of cultured meat will be discussed at international and national levels. Keywords : Cultured meat, muscle food, cellular agriculture, myogenesis References 1. Mark J. Post, Shulamit Levenberg, David L. Kaplan, Nicholas Genovese, Jianan Fu, Christopher J. Bryant,

Nicole Negowetti, Karin Verzijden, and Panagiota Moutsatsou, Scientific, sustainability and regulatory challenges of cultured meat (2020) Nature Food, 1, 403-415.

2. Kwang-Hwan Choi, Ji Won Yoon, Minsu Kim, Hyun Jung Lee, Jinsol Jeong, Minkyung Ryu, Cheorun Jo, Chang-Kyu Lee, Muscle stem cell isolation and in vitro culture for meat production: A methodological review (2021) Comprehensive Review in Food Science and Food Safety, 20, 429-457.

59


S494 Potential of Enhancing Cellulose Microfibrillation or Nanocrystallization to Use as a Functional Biomaterial

Young Hoon JUNG School of Food Science & Biotechnology, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] Cellulose is one of the most abundant biopolymers in the world. Nano-sized cellulose, which can be obtained through extraction of either fibrils by physical treatment or crystals by chemical treatment from cellulose, has much wide surface area in comparison with cellulose. Thereby, the nanocellulose can give special properties to the final materials when using as an additive. In this talk, we prepared microfibrillated cellulose and nanocrystal cellulose from a red algal biomass, Gelidium amansii. Through consecutive steps such as removal of other compounds except cellulose and physical or chemical pretreatment, each form of nanocellulose was obtained. Then, their surface characteristics including size dimensions, morphologies, degree of fibrillation, etc. were widely investigated. Also, the effect of those nanocellulose as an anti-inflammatory material on human keratinocytes and mice skin was evaluated. In conclusion, both microfibrillated cellulose and nanocrystal cellulose derived from Gelidium amansii migh be promising cosmeceutical agents. Keywords : Nanocellulose, Microfibrillated cellulose, Nanocrystal cellulose, Anti-inflammation, Cosmeceutical, Gelidium amansii

60


S495 Advanced Electrochemical Sensor Modified with Affinity Peptides for Sensitive Norovirus Detection

Jong Pil PARK Department of Food Science and Technology, Chung -Ang University, Anseong, Korea Corresponding Author Email : [email protected] Electrochemical-based biosensors have been widely applied in many fields because they have high sensitivity, are easy to control, and allow for label-free detection and miniaturization in a point-of-care testing. However, target-specific probes such as DNA, antibody, and protein on the working electrode surface is needed to enable specific detection of targets in electrochemical analysis. Antibodies serve as important capture reagents in several fields such as the clinical or pharmaceutical analysis, diagnosis, and treatment of various diseases. However, they have limitations such as being relatively large in size and having cross-reactivity to be occurred in immunoassay tests. In contrast, affinity peptides have a small size and simple structure and are relatively stable in a variety of environmental conditions. The aim of this talk is to show unique affinity peptides which would recognize norovirus with high specificity and selectivity and to explore the binding interactions using several analytical methods. To improve the abilities of affinity peptides, we used a rational design approach with in silico modeling and tried to synthesize advanced affinity peptides for an electrochemical sensor. Keywords : affinity peptide, binding affinity, modification, engineering, norovirus detectioin

61


Special Program

62


[S5-1]

바이오 소재분야 부상기술 연구동향

분석사업 성과발표회

63


S511 바이오 의약 소재

최종훈

중앙대학교 융합공학부 바이오메디컬공학전공

Corresponding Author Email : [email protected] 본 발표에서는 바이오 의약 분야 3 대 부상기술에 대해 논하고자 함. 나노바이오 기반 진단기술에서는

최근 부상하고 있는 생체 내 자연적으로 분비되는 세포외소포체 (엑소좀)를 이용한 질병의 조기진단

기술개발에 대해 현재의 상황과 미래를 예측하고자 함. 두 번째로 차세대 백신 기술로서 대두되고 있

는 mRNA 백신 기술의 현재 동향에 대해 논하고자 함. 마지막으로 성공적으로 유해 박테리아를 제거

하고 바이오필름의 형성을 예방하기 위해서 균주의 독성과 항생물질에 대해 내성이 생기는 문제점을

해결하고자 하는 항균 나노 신소재들에 대해 그 현재와 미래를 다뤄보고자 함.

Keywords : 세포외소포체 진단, mRNA 백신, 항균 나노 신소재

64


S512 바이오 생리활성 분야 국내외 유망기술 및 산업 동향 분석 서승오

가톨릭대학교 식품영양학과

Corresponding Author Email : [email protected] 글로벌 팬더믹 시대에 인류의 건강 증진과 지속가능한 삶을 구현하기 위한 사회적 요구와 산업적 수

요가 증가함에 따라 감염병에 효과적으로 대처하는 다양한 유망 바이오 생리활성 소재 및 기반 기술

이 급격하게 성장하고 있다. 이러한 포스트 코로나 시대의 바이오산업 패러다임 변화에 대응하기 위

해 인류의 건강과 밀접한 관련이 있는 바이오 생리활성 소재 분야의 국내외 유망 기술 및 산업 동향

에 대해 분석하였다. 특히, 면역력 증강 등과 같은 건강기능성을 바탕으로 감염병에 대응할 수 있는

다양한 바이오 생리활성 소재를 생물공학적으로 제조하는 기술이 아래와 같이 3 대 부상기술로 선정

되었다.

1. 합성비타민 대체 바이오비타민 소재 제조기술

2. 미세조류 유래 차세대 생리활성 소재 제조기술

3. 글로벌 팬더믹 대응 미래 바이오 웰빙/메디칼 소재 제조기술

각 분야의 전문가들을 대상으로 한 설문조사와 더불어 국내외 최근 연구동향 및 산업동향 분석을 통

해 해당 부상 기술의 개념, 기술의 중요성, 산업적 필요성, 국내외 기술수준 및 격차, 정부지원 방안,

해외협력 방안 등을 도출하였다.

Keywords : 바이오소재 부상기술, 바이오 생리활성 소재, 바이오비타민, 미세조류, 웰빙/메디칼 소재

References 1. 바이오소재 분야 부상기술 연구동향 분석 연구 최종보고서, 한국생물공학회, 2021.03.15

65


S513 바이오 화학

김희택

충남대학교 농업생명과학대학 식품공학과

Corresponding Author Email : [email protected] 최근, 전 세계적으로 플라스틱 폐기물과 지구 온난화가 주요한 사회문제로 대두되고 있습니다. 이와

같은 문제를 해결하기 위해 생분해성 플라스틱 개발, 플라스틱 업사이클링 개발등 다양한 접근법들이

제시되고 있지만, 제한적인 범위의 플라스틱 생산 및 이산화탄소 배출 문제 등 여러 현안들이

남아있습니다. 이와 같은 문제를 근원적으로 해결하기 위한 부상기술에 대한 연구가 최근 활발히

이루어 지고 있습니다. 본 발표에서는 플라스틱 문제에 대한 현 상황을 공유하고 부상기술로서 Carbon

close loop 에 입각하여 도출된 바이오매스 기반 차세대 플라스틱 소재, 플라스틱 기능향상 소재 및

플라스틱의 생물학적 업사이클링 기술에 대하여 소개하도록 하겠습니다.

Keywords : 바이오매스, 플라스틱 소재, 기능향상 소재, 생물학적 업사이클링 기술

66


S514 바이오소재 분야 부상기술 연구동향 분석 – 바이오기반기술

연영주 1, 이대희 2, 안정오 3 1강릉원주대학교 생명화학공학과, 2한국생명공학연구원 합성생물학전문연구단, 3한국생명공학연구원 바이오상용화지원센터

Corresponding Author Email : [email protected] 본 발표에서는 ‘바이오기반기술’ 분야의 3 대 부상기술에 대해 소개한다. 첫 번째 기술은 ‘AI 기반

스마트 바이오공정 개발 기술’로, 바이오소재 생산 관련 다양한 바이오공정으로부터 빅데이터 수집 및

플랫폼 모델 구축 빅데이터를 수집하고, 이를 활용하여 바이오공정별 과업을 스스로 판단해 실행할 수

있는 스마트 머신 개발과, 사물인터넷을 통한 스마트 머신 간의 상호 소통 체계 구축으로 최적의

바이오의약품 및 바이오소재 제품 생산을 위한 스마트 바이오팩토리를 구현하는 기술이다. 두 번째

기술은 ‘바이오파운드리 기반 산업용 고효율 미생물 초고속 개발 기술’로, 합성생물학 기술에 컴퓨터

기반 DNA 설계, 자동화, 인공지능을 융합하여 바이오산업의 오랜 난제인 속도, 규모, 효율을 혁신할 수

있는 바이오파운드리 시스템을 구축하고, 이를 산업용 고효율 미생물을 초고속으로 개발하고 상용화에

도입할 수 있는 기술 개발을 소개한다. 세 번째는 ‘차세대 분자설계 기술 기반 스마트 바이오촉매 개발

기술’로, 기존 분자모델링 및 단백질공학적 효소개량 기술에 빅데이터 및 인공지능 기술을 도입하여

기존 기술 대비 설계 정확도 개선, 개량 시간 단축, 투자비용 절감 등의 효과를 제공할 수 있는

분자설계 기술을 개발하고, 이를 통해 산업적 응용에서 요구되는 주요 성능지표들이 동시에 향상된

스마트 바이오 촉매를 개발하는 기술에 대해 소개한다. 상기 3 대 부상기술의 연구동향 및 중요성에

대해 소개할 것이며, 기술적 측면 뿐만 아니라 경제/사회적 및 정부/정책적 영향에 대해서도 논의될

것이다.

Keywords : 부상기술, 바이오기반기술, 인공지능, 빅데이터, 스마트 바이오공정, 바이오파운드리, 차세대

분자설계, 스마트 바이오촉매

67


[S5-2]

신진연구자를 위한 포럼

68


S521 Peptide-Nanoparticle Conjugates for Immune Checkpoint Blockade

Woo-jin JEONG Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] The majority of anti PD-1/PD-L1 agents currently approved or under development are based on full-size monoclonal antibodies. Despite their demonstrated efficacy, the widespread use of the antibody drugs has been hindered due to their high cost and complexity in manufacturing and low thermodynamic stability. Herein, we introduce a peptide-dendrimer conjugate (PDC) approach that stabilizes the β-hairpin structure of peptides via intermolecular forces and the excluded volume effect as well as exploits the multivalent binding effect. Because of the synergistic advantages, the PDCs based on a β-hairpin peptide isolated from an engineered PD-1 protein showed significantly higher affinity (avidity) to PD-L1, as compared to free peptides (by up to 5 orders of magnitude). The enhanced binding kinetics with high selectivity was translated into an improved immune checkpoint inhibitory effect in vitro, at a level comparable to (if not better than) that of a full-size monoclonal antibody. The results demonstrate the potential of the PDC system as a novel class of inhibitors targeting β-strand-rich protein surfaces, such as PD-1 and PD-L1, displaying its potential as a new cancer immunotherapy platform. Keywords : Peptide-nanoparticle conjugates, Cancer immunotherapy, Immune Checkpoint Blockade, Multivalent binding effect, β-hairpin stabilization

69


S522 Tailoring Biomaterials for Cancer Immunotherapy

Kye Il JOO Department of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Despite the great promise of immune checkpoint blockade (ICB) therapy for cancer treatment, the currently available options for ICB treatment pose major clinical challenges, including the risk of severe systemic autoimmune responses. Here, we developed a novel localized delivery platform, immuno-bioglue (imuGlue), which is inspired by the intrinsic underwater adhesion properties of marine mussels and can allow the optimal retention of anti-PD-L1 drugs at tumor sites and the on-demand release of drugs in response to the tumor microenvi-ronment. Using a triple-negative breast cancer and melanoma models, we found that imuGlue could signify-cantly enhance anti-tumor efficacy by eliciting a robust T cell-mediated immune response while reducing systemic toxicity by preventing the rapid diffusion of anti-PD-L1 drugs into the systemic circulation and other tissues. It was also demonstrated that imuGlue could be successfully utilized for combination therapy with other immunomodulatory drugs to enhance the anti-tumor efficacy of ICB-based immunotherapy, demonstrating its versatility as a new treatment option for cancer immunotherapy. Keywords : Biomaterial Design, Cancer Immunotherapy, Nano Immuno-Engineering

70


S523 Nanotechnology Applied in Prevention, Diagnosis and Treatment of Infectious Diseases and Cancer

Hyun-Ouk KIM1,2 1Department of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Korea 2Biohealth-machinery Convergence Engineering, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] The worst pandemic in history was the Black Death, which killed a third of the European population in the Middle Ages. Following the Spanish flu in 1918, the Asian flu in 1957, the Hong Kong flu in 1968, and influenza A (H1N1), also called the swine flu in 2009, the World Health Organization (WHO) declared a pandemic over COVID-19 after 11 years. Despite the development of diagnosis, treatment, and vaccines for infectious diseases around the world, overseas movement of people and animals is becoming easier, and the occurrence of emerging, newly emerging, and exotic malignant contagious infectious diseases is increasing due to urban development, environmental destruction, and so on. Artificial nanostructures have not only physical properties with high surface area, but also various physic-chemical properties (conductivity, catalytic activity, enzyme activity, optical properties, etc.), and can support vaccines and therapeutic agents. Since the formation of various nanostructures can be used for prevention, diagnosis and treatment, the convergence of nano and biotechnology is very important. Therefore, there is a need to solve the problem of prevention, early diagnosis, and treatment of emerging, newly emerging, exotic malignant contagious infectious diseases with nano and biotechnology convergence technology. Keywords : Cancer, Diagnosis, Infectious Diseases, Nanoparticle, Prevention, Treatment

71


S524 Cationic Polymer Development for mRNA Delivery and their Application for CRISPR-Cas Technology

HyunJin KIM Department of Biological Engineering, College of Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Efficient nonviral vector gradually become an essential requisite for successful gene editing therapy via CRISPR-Cas technology, which Cas9 protein edits guide RNA (gRNA)-complementary target chromosome DNA. The nonviral vector in the local administration can accomplish gene editing in the area of interest without inducing undesirable gene editing in the off-region. We have developed amphiphilic polyaspartamide deriva-tives (PAsp(DET/CHE)) to induce transient and high Cas9 protein expression from messenger RNA (mRNA). Diethylenetriamine (DET) moiety provides high endosomal escape ability and associates with negatively charged mRNA. 2-Cyclohexylethyl (CHE) moiety optimizes polymer hydrophobicity between mRNA nanoparticle stability in the extracellular condition and mRNA release in the cytoplasm. CHE moiety was selected from the highest luciferase mRNA expression in cultured cells among ~20 aliphatic groups between 5 and 10 carbon atoms. PAsp(DET/CHE) was synthesized via two-step process. The precursor polymer poly(β-benzyl-L-aspartate) (PBLA) were synthesized with ring opening polymerization between n-butylamine and β-benzyl-L-aspartate N-carboxy-anhydride (BLA-NCA) to have degree of polymerization (DP) of 26. PAsp(DET/CHE) was synthesized by co-aminolysis of DET and CHE into PBLA to contain 15 DET and 11 CHE moieties. Interestingly, the mRNA translation efficacy was not affected by DP of PAsp(DET/CHE) from 26 to 121 but by the amounts of CHE moieties in the single polymer. The PAsp(DET/CHE) with less than 8 CHE introduction amounts did not increase mRNA translation efficacy at all but dramatically enhanced the translation efficacy with more than 11 CHE introduction amounts. We analyzed in vitro cellular uptakes, endosomal escape ability, and release kinetics of mRNA in cytoplasm to clarify the mechanism of high mRNA translation of PAsp(DET/CHE). Gene editing was evaluated in Ai9 mouse, which red fluorescence occurs in the results of Cas9/gRNA based STOP cassette cleavages. The nano-particles were prepared with mixing between PAsp(DET/CHE) and Cas9 mRNA (or gRNA). The nanoparticles (1.5 ug Cas9 mRNA and 1.5 ug sgRNA) were locally injected into ventricular area of the Ai9 mouse. At 2 day post-administration, the brain was dissected and observed by using confocal laser scanning microscope (CLSM). Gene editing occurred in the ependymal layer and choroid plexus of almost all brain area, which indicated high delivery efficiency of PAsp(DET/CHE) into the brain. PAsp(DET/CHE) has high gene editing efficacy in the local delivery into brain, nose, and muscle. These indicate high potential of the hydrophobicity optimization of polyaspartamide derivatives and demonstrates the strong potential of PAsp(DET/CHE) for in vitro and in vivo Cas9 mRNA/gRNA delivery. Keywords : mRNA delivery, cationic polymer, gene editing, amphiphile

72


S525 Ribocomputing: Leveraging RNA for Computation in the Cell

Jongmin KIM Department of Life Sciences, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] Synthetic biology aims to develop engineering-driven approaches to the programming of cellular functions that could yield transformative technologies. Synthetic gene circuits that combine DNA, protein, and RNA com-ponents have demonstrated a range of functions such as bistability, oscillation, feedback, and logic capabilities. Despite many advances, technical challenges remain for scaling up the complexity of these networks due to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and substantial resource requirements for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Such ‘ribocomputing’ systems are composed of de novo designed parts and operate via predictable and designable base-pairing rules, allowing for effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. We demonstrate that these ribocomputing devices in Escherichia coli can evaluate two-input logic expressions with dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input logic expression [1]. We further demonstrate that ribocomputing design strategy can be used to develop a large library of high-performance translational repressors and 4-input NAND logic gates [2]. Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings. Keywords : RNA synthetic biology, logic circuits, molecular computation References 1. Alexander A. Green, Jongmin Kim, et al., Complex cellular logic computation using ribocomputing devices

(2017), Nature, 548(7665), 117-121. 2. Jongmin Kim, et al., De novo-designed translation-repressing riboregulators for multi-input cellular logic

(2019), Nature Chemical Biology, 15(12), 1173-1182.

73


S526 Synthetic Evolutionary Bioengineering: Cooperation of Nature’s Power and Human Design

Sungho JANG1,2 1Division of Bioengineering, Incheon National University, Incheon, Korea, 2Department of Bioengineering and Nano-Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Biological systems have the potential to provide solutions for urgent challenges that we are facing from energy, environment, and human health. Engineering biological systems, however, frequently involves screening of extremely large solution space in the hope of finding useful phenotypes such as proteins with new functions or metabolic pathways for chemical production. In this talk, I am going to introduce examples showing how human design can cooperate with nature’s power to provide valuable solutions, focusing on the metabolic engineering of microorganisms. In particular, the development and applications of synthetic regulatory RNAs will be discussed which were utilized as molecular tools to guide the evolution of metabolite-producing recombinant bacteria. Additionally, the rationally-designed molecular program for rapid diagnosis of pathogens called SENSR will be discussed. Keywords : Synthetic Biology, Artificial Evolution, Metabolic Engineering, Molecular Diagnosis References 1. S. Jang, S. Jang, Y. Xiu, T. J. Kang, S.-H. Lee, M.A.G. Koffas, G. Y. Jung, ACS Synth. Biol. 6(11), 2077-

2085 (2017). 2. S. Jang, S. Jang, D.-K. Im, T. J. Kang, M.-K. Oh, G. Y. Jung, ASC Synth. Biol. 8(6), 1729-1283 (2019). 3. S. Jang, S. Jang, J. Yang, S. W. Seo, G. Y. Jung, Curr. Opin. Biotechnol. 53, 1-11 (2018). 4. H. G. Lim, S. Jang, S. Jang, S. W. Seo, G. Y. Jung, Curr. Opin. Biotechnol. 54, 18-25 (2018). 5. C. H. Woo, S. Jang, G. Shin, G. Y. Jung, J. W. Lee, Nat. Biomed. Eng. 4(12), 1168-1179 (2020).

74


S527 Expanding the Limits of the Second Genetic Code with Ribozymes

Joongoo LEE Department of Chemical Engineering, POSTECH, Pohang, Korea Corresponding Author Email : [email protected] From a chemical engineer’s perspective, biology is technology. As an example, biology produces materials that no other human-made technology and chemistry can, and operates numerous complex chemical/enzymatic reactions across a wide variety of lengths of scales from the single-molecule to the macro-scale level. Thus, scientists have engineered living organisms to harness the advanced technologies that are embedded in biology and manufacture next-generation commodities on demand. However, engineering living organisms to produce high-value molecules remains costly and slow, and new tools are needed to understand and expand the powerful capabilities of biotechnology. In this presentation, I will discuss my strategy to address this issue and broaden the scope of biological material-catalyzed transformations. In the first portion of my talk, I will describe our recent efforts to expand the genetic code using ribozymes for the synthesis of bio-based products that extend beyond natural limits. In the second part, I will explain my investigation into engineering the translational apparatus that has been evolutionarily optimized to accept natural amino acid building blocks. Overall, my work aims to deepen a fundamental understanding of the origin of life and enable new classes of functional materials such as precision polymers, therapeutics, and biosensors. Keywords : ribosome, ribozyme, unnatural amino acids, non-canonical chemical substrates, cell-free protein expression, protein translation, DNA, RNA, polymers, non-amide backbone

75


S528 Repetitive Sequence Analysis of Fibrous Proteins Using Bioinformatics and the Roles of KISTEP for Researchers

Dooyup JUNG Center for Growth Engine R&D Coordination, Korea Institute of S&T Evaluation and Planning, Eumseong, Korea Corresponding Author Email : [email protected] This talk will be organized as two parts. The first part is the amino acid sequence analysis of fibrous proteins harboring repeating units using bioinformatics. Because there are close relationships among sequences, molecular structures, and mechanical properties, appropriate computational methods are necessary to efficiently analyze, find, store, and utilize fibrous protein sequences. However, pre-established bioinformatical tools do not suitably contribute to fibrous proteins due to their sequence repetitiveness. Therefore, in this talk, the development of novel bioinformatical platform for fibrous protein sequences will be introduced from spider silk sequence analysis with spider evolution and fiber property to fibrous protein fingerprint database construction. The second part is about the roles of KISTEP to support R&D policy. Especially, the process and method of government R&D budget allocation and coordination in Korea will be briefly explained, mainly dealing with materials field as an example. In addition, the information produced or edited by KISTEP, such as statistics and publications, that can be helpful for researchers to plan their own projects will be explained. Keywords : fibrous proteins, repetitive sequence, bioinformatics, spider silk, evolution, mechanical property, KISTEP References 1. D. Jung, Y. J. Yang, and H. J. Cha, Biotechnol. J. 14, 1900138 (2019).

76


[S5-3]

일반특강

77


S531 Development of L-Ornithine Hydrochloride and Its Application Process to KFDA as A Food Additive

Keumsoo HAN, Minhong KIM MH2 Biochemical Co. Ltd., Korea Corresponding Author Email : [email protected] Ornithine is a compound that exists in most living organisms and is a non-protein amino acid. Ornithine is a proline precursor that is a component of collagen in the skin tissue and is involved in cell growth and differentiation, which is also involved in the release of ammonia produced during protein metabolism out of the body. Currently, ornithine is used for the treatment of encephalopathy due to poor liver function and is used as a special purpose food for baby formula and as a flavor enhancer to offset the bitterness of foods. The manufacture of Ornithine has begun by Kyowa Hakko in Japan since 1957, and is manufactured by Ajinomoto and Evonik in Germany using fermentation processes. In 2005, the company manufactured ornithine by enzymatic processes and exported it mainly abroad ever since. Although it is not yet permitted as a food additive in Korea, it has been listed in Codex since 2013 and is mainly applied as a flavor enhancer in Japan, the United States, and Thailand. In order to be enlisted as a food additive in Korea, food additives draft must be applied to KFDA. The draft must include the status of enlistment in other foreign countries, development details, manufacturing data, the food additive specification, technical needs of the additive, human safety, and dosage in foods used for. In this presentation, I would like to compare the application processes of food additives with foreign countries and to suggest some consideration of preparing experimental data. Keywords : L-Ornithine Hydrochloride, Food Additive, Health Benefit, Safety, KFDA References 1. Evaluation of Certain Food Additives (Seventy-sixth Report of the Joint FAO/WHO Expert Committee on

Food Additives) WHO Technical report Series 974, 2012. 2. Shigeru Ishida, M. Sarada, H. Seki, L. McGirr, A. Lau, K. Morishida, Regulatory Toxicology and

Pharmacology 67, 36 (2013).

78


S532 Development and Commercialization of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay (MESIA) for Point-of-Care Testing

Hyundoo HWANG BBB Inc., Seoul, Korea Corresponding Author Email : [email protected] Immunoassay has been one of key technologies for determination of biomarkers in complex biological fluids such as blood, urine, and saliva. Enzyme-linked immunosorbent assay (ELISA), which is the most widely used immunoassay technique, is the gold standard for quantitative analysis of protein biomarkers. However, it takes several hours of analysis, and requires huge and expensive instruments as well as intervention of experts. Rapid diagnostic test (RDT), which is based on lateral flow immunoassay (LFIA), allows low-cost, rapid detection of biomarkers. However, it is neither accurate nor precise, thus usually applied for qualitative screening of non-critical biomarkers. Recently, bench-top immunoassay systems based on microfluidics and fluorescence have been introduced to the market. They are more precise than RDT, but require expensive optical components sensitive to dusts and vibration, as well as additional steps with external instruments for sample preparation. Therefore, the commercial bench-top systems are not appropriate for the point-of-care testing and self-monitoring of disease markers. Here we introduce a novel immunoassay technique, called MESIA (magnetic force-assisted electrochemical sandwich immunoassay). MESIA provides both high accuracy and usability, enabling self-tests at the point of care with a drop of finger-prick blood. Once a blood sample is loaded into a tiny cartridge, all the processes from sample preparation to analysis are automatically conducted in a hand-held reader system. The analytical and clinical performances of MESIA for quantification of blood biomarkers were evaluated according to the CLSI guidelines. They showed high reproducibility, sensitivity, and specificity as well as excellent agreement with commercial hospital immunoassay systems. Recently, we developed and commercialized the COVID-19 antigen kit and obtained FDA EUA for the first time among the products made in Korea. MESIA is now recognized as one of new categories for immunoassay technologies in addition to ELISA and LFIA. In this talk, we present the history of development and commercialization of MESIA, and discuss the future outlooks of the new technology. Keywords : Immunoassay, In vitro diagnostics, Lab-on-a-chip, Point-of-care, Biomarkers

79


S533 O2-tolerant Ni–Fe Carbon Monoxide Dehydrogenase by Subsrate Tunnel Engineering

Suk Min KIM, Jinhee LEE, Seung Hyuk KANG, Yong Hwan KIM Department of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] The extremely O2-sensitive Ni–Fe carbon monoxide dehydrogenase (CODH) is one of the most attractive enzymes for toxic CO removals from nature and air pollutants [1], since these enzymes can catalyse the oxidation of CO to CO2 at high rate (>10,000 s-1). To improve O2-tolerance of these CODHs is a big challenge for biocatalyst use in industrial combustion processes, such as steel mills. Here we showed that the characteristic change of substrate tunnel close to the active site (C-cluster) of ChCODH-II (Carboxydothermus hydrogeno-formans), enables to increase O2-tolerance of Ni–Fe CODH. Our experiments revealed that increase of O2-tolerance compared to that of wild type is achieved for the redesigned ChCODH-II mutant near atmospheric condition. Moreover, this mutant efficiently catalyzed the oxidation of CO contained in the real waste gases of LDG (Linz–Donawiz converter Gas) discharged in the steel mill. Our newly designed enzyme will be definitely a working horse in the removal of toxic gas from environmental pollutants as well as the conversion of waste CO gas into value-added chemicals. Keywords : Carbon monoxide, Ni-Fe CO dehydrogenase, Oxygen tolerance, Gas tunnel, Waste gas References 1. S. W. Ragsdale, Crit. Rev. Biochem. Mol. Biol. 39, 165 (2004).

80


S534 Antibacterial Activity of Metal Nanowire and Metal Oxide Thin Films

Ji-Hyeon KIM, Junfei MA, Ga-Hyun LEE, Seunghun LEE, Chang Su KIM Advanced Nano-Surface Department, Korea Institute of Materials Science, Changwon, Korea Corresponding Author Email : [email protected] Zinc oxide (ZnO) and silver nanowire (AgNW), identified as promising antibacterial agents, were investigated on the antibacterial performance and associated properties. Firstly, to evaluate the functional sustainment of ZnO antibacterial film in an ozone disinfection system, ZnO sol-gel films were subjected to ultraviolet–ozone treatment for different periods. Despite the morphological and chemical changes, the satisfactory antibacterial-activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was sustained even after 2 h treatment. Secondly, the antibacterial activity of a AgNW spin-coated film were enhanced by 1200 kGy electron beam irradiation. Silver-oxide islands generated by the irradiation improved the antibacterial activity from 93.5 to 97.3 % for S. aureus and 96.1 to 99.9 % for E. coli. Thirdly, the antibacterial activity of AgNW spin-coated films was improved by post-heat treatment. AgNW films were heated at various temperatures for 30 min. With increasing the heating temperature, the AgNWs broke into more segments, and became more oxidized, and hence the antibacterial activity against E. coli increased from 92.6 to 95.7 %. Keywords : antibacterial film, zinc oxide, silver nanowire

81


[S5-4]

From Lab. Research to

Plant Design: Bioprocess

Modeling and Analysis

82


S541 Integrated Biorefinery Process from Process Optimization to Process Design for Production of Value-added Products

Chulhwan PARK Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] The biorefinery is well known platform that converts various biomass to value-added products (bioenergy, biochemical and biomaterial etc.). Various applications such as selection of biomass, optimization of pretreat-ment, improvement of process efficiency and design of hybrid process can be applied for the sustainable utilization of biomass. In this presentation, the following four categories will be presented briefly; (1) bioenergy and biochemical production from lignocellulosic biomass, (2) enzymatic coproduction of value-added products from vegetable oil, (3) formate ester production based on C1 gas, and (4) process design and evaluation for value-added chemicals production. Keywords : biorefinery, biomass, optimization, value-added chemical, process design References 1. H. Kim et al., Improved production of bacterial cellulose through investigation of effects of inhibitory

compounds from lignocellulosic hydrolysates, Global Change Biology Bioenergy, 13, 3, 436-444 (2021). 2. M. Shin et al., Novel and efficient synthesis of phenethyl formate via enzymatic esterification of formic

acid, Biomolecules, 10(1), 70 (2020). 3. C. Park et al., Eco-design and evaluation for production of 7-aminocephalosporanic acid from carbohydrate

wastes discharged after microalgae-based biodiesel production, Journal of Cleaner Production, 133, 511-517 (2016).

4. J. Joo et al., Improved fermentation of lignocellulosic hydrolysates to 2,3-butanediol through investigation of effects of inhibitory compounds by Enterobacter aerogenes, Chemical Engineering Journal, 306, 916-924 (2016).

83


S542 Development of Biomass Production Process using Ettlia sp. for Operational Expenditure (OPEX) Reduction

Minsik KIM Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Corresponding Author Email : [email protected] In recent decades, carbon dioxide emission has been striking the globe and carbon-neutral energy sources have become necessary. Biomass has been spotlighted to solve the greenhouse effect and microalgal biomass especially is gaining attention with advantages regarding food-ethics and deforestation. In this study, a cultivation process for a microalga species, namely Ettlia sp. YC001, was statistically optimized. The optimi-zation under heterotrophy increased biomass and lipid productivities by 4.8-fold and 4.1-fold, respectively. Subsequently, two feedstocks, i.e., Helianthus tuberosus and algal residue were utilized as alternative nutrient sources. Furthermore, a sequential hydrolysis process was developed to utilize both feedstocks efficiently. The sequential hydrolysis process generated a low-cost medium which resulted in almost identical biomass productivity and increased lipid productivity relative to the statistically optimized medium. Additionally, an analogous techno-economic analysis for both processes using the optimized medium and low-cost medium was conducted to examine the effectiveness of the developed processes. The result showed that hydrolysate medium decreased two-third and three-fourth of required operational expenditures for the optimized media to produce Ettlia sp. biomass and lipid, respectively. However, the calculation showed that an excessive amount of capital expenditure was required to utilize microalgal biomass as biofuel, which in turn requires further research on decreasing capital expenditure. Keywords : Acid hydrolysis, Algal residue, Biomass, Ettlia sp., Plackett-Burman Design, Response surface method, Techno-economic analysis References 1. M. Kim, B.Lee, H. S. Kim, K. Nam, M. Moon, H.-M. Oh*, and Y. K. Chang*, Sci. Rep. 9, 6830 (2019). 2. M. Kim, J. M. Cho, H. S. Kim, H. Lee, H.-M. Oh*, and Y. K. Chang, Energy Convers. Manag. 211, 112769

(2020).

84


S543 Toward Biomass-derived Biodegradable Plastics: Process Development and Analyses

Wangyun WON Department of Chemical Engineering, Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Lactic acid (LA) is an important bio-based precursor of polylactic acid (PLA), which is biodegradable and biocompatible plastic that can replace conventional non-biodegradable plastics. In this study, we propose two strategies for the production of LA, one from lignocellulosic biomass (Strategy A) and the other from sugar (Strategy B), and conduct integrative analyses such as techno-economic analysis, pioneer plant analysis, uncertainty analysis, and life-cycle assessment to investigate the comprehensive feasibility and sustainability for the proposed strategies. To reduce utility consumption, we perform heat integration. The minimum selling prices of LA, which are defined as a selling price at breakeven point, for the strategies A and B are estimated to be $1,498/ton and $1,491/ton, respectively, which are lower than current market price of LA, i.e., $1,526/ton, indicating that LA produced from the proposed strategies is cost-competitive. These MSPs can increase to $2,340/ton and $1,641/ton for strategies A and B, respectively, under the assumption of pioneer plant. The uncertainty analysis using Monte-Carlo simulation shows risk and uncertainty in our new strategies by means of cumulative probability and frequency graphs. Through the life-cycle assessment, we quantify the environmental impact of the proposed process. Keywords : biodegradable, economics, sustainability

85


S544 Process Design and Assessment of Electrochemical Carbon and Nitrogen Reduction Processes

Juyeon KIM, Jonggeol NA Department of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Electrochemical processes–converting carbon dioxide and nitrogen to value-added precursors such as ethylene and ammonia–are promising techniques to escape from the petrochemical-dependent industrial structure. However, assessments of the economics of overall processes simultaneously are difficult due to complicated reaction networks and product combinations affecting optimal process design. Here, we introduce a general techno-economic analysis of electrochemical carbon dioxide and nitrogen reduction processes through a superstructure-based automated process synthesis platform and thereby considering the overall process design combinations simultaneously, which leads to the rigorous assessment. The economical global sensitivity according to current density, Faraday efficiency, and overpotential is evaluated. The analysis highlights the promise optimal design for 1) coupled process of carbon dioxide reduction with organic oxidation reaction, and 2) nitrate production through electrochemical nitrogen oxidation and ammonia production through nitrate reduction in a single reaction system. Keywords : electrochemical, nitrogen reduction, carbon dioxide reduction, carbon capture and utilization, process systems engineering, techno-economic analysis References 1. Na, J., Seo, B., Kim, J. et al., Nat Commun. 10, 5193 (2019).

86


[S5-5]

Recent Advances of

Electrochemical Biotechnology

for Energy and Environment

87


S551 Nanobiocatalysis for CO2 Conversion and Utilization

Jungbae KIM Department of Chemical and Biological Engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Even though enzymes can be employed and newly proposed for various applications such as electrochemical biosensors, membrane antifouling and CO2 conversion, their successes in practical applications are often hampered due to their poor stability. Nanobiocatalysis, immobilizing enzymes using various nanomaterials, has demonstrated its successes in stabilizing the enzyme activity for various enzyme applications such as biosensors, biofuel cells, enzyme-linked immunosorbent assay, membrane antifouling, and CO2 conversion. This presenta-tion will mainly cover nanobiocatalytic stabilization of carbonic anhydrases for their potential successes in CO2 conversion. Especially, the nanobiocatalytic stabilization of carbonic anhydrase, catalyzing the hydration of CO2 to bicarbonate at a 105-6 turnover number, has made an unprecedented success by maintaining 83% of initial enzyme activity after incubation in aqueous solution under shaking at 200 rpm for two years. Stabilized carbonic anhydrase was successfully employed for the effective conversion of CO2 to bicarbonate, which was further used for expedited microalgae growth and improved calcium carbonate production. If time permits, several other examples of nanobiocatalytic applications together with several other research areas will be introduced. Keywords : Nanobiocatalysis, carbonic anhydrase, CO2 conversion, expedited microalgae growth, calcium carbonate formation References 1. Han Sol Kim, Han Sol Kim, Sung-Gil Hong, Kie Moon Woo, Vanesa Teijeiro Seijas, Seongbeen Kim,

Jinwoo Lee and Jungbae Kim, ACS Catal. 8, 6526-6536 (2018). 2. Han Sol Kim, Sung-Gil Hong, Jusang Yang, Youngjun Ju, Joongbok Ok, Seok-Joon Kwon, Kyung-Min

Yeon, Jonathan S. Dordick, Jungbae Kim, J. CO2 Util., 38, 291-298 (2020). 3. Seung-Hyun Jun, Jusang Yang, Hancheol Jeon, Han Sol Kim, Seung Pil Pack, EonSeon Jin, and Jungbae

Kim, Environ. Sci. Technol., 54, 1223-1231 (2020).

88


S552 Electrochemical Biosensors Using Redox Cycling

Haesik YANG Department of Chemistry, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] High signal amplification is essential for ultrasensitive detection of biomolecules. In recent years, we have developed new redox cycling schemes, combined with enzymatic or nanozymatic amplification, that allow for high signal amplification in a simple format along with low background levels. To minimize unwanted side reactions, all involved species were properly selected. In this presentation, the basic concept of the developed redox-cycling schemes will be explained, and their application to the detection of microorganisms including Escherichia coli (E. coli) and Aspergillus niger (A. niger) will be introduced. The E. coli detection method harnesses the intracellular β-galactosidase (Gal) activity of E. coli along with the signal amplification based on redox cycling. The Gal expression level is increased by treatment with a Gal expression-inducer; the enzymatic reaction of Gal is facilitated by the permeabilization treatment involving the use of chloroform and sodium dodecyl sulfate; the electrochemical signal is amplified by the electrochemical–chemical–chemical and chemical–chemical redox cycling involving the Gal product. A sensitive detection method specific to A. niger is based on a single-mediator system combined with electrochemical-chemical redox cycling. Intracellular NAD(P)H-oxidizing enzymes in A. niger converts electro-inactive 4-nitro-1-naphthol into electro-active 4-amino-1-naphthol. Since the membrane and wall of A. niger is well permeable to both 4-nitro-1-naphthol and 4-amino-1-naphthol in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of A. niger. Keywords : Biosensors, Escherichia coli, Aspergillus niger References 1. H. Yang, Curr. Opin. Chem. Biol. 16, 422 (2012). 2. S. Noh, Y. Choe, V. Tamilavan, M. H. Hyun, H. Y. Kang, H. Yang, Sens. Actuators B: Chemical 209, 951

(2015). 3. J. Kwon, E.-M. Cho, P. Nandhakumar, S. I. Yang, H. Yang, Anal. Chem. 90, 13941 (2018). 4. P. Nandhakumar, G. Kim, S. Park, S. Kim, S. Kim, J. K. Park, N.-S. Lee, Y. H. Yoon, H. Yang, Angew.

Chem. Int. Ed. 59, 22419 (2020).

89


S553 Synthetic Analogs of Mo/W Enzymes and Reactivity with CO2 and Protons

Junhyeok SEO Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, Korea Corresponding Author Email : [email protected] Synthetic modeling of metalloenzyme active sites can provide understandings of enzyme reactivity, as well as research insights into the development of catalysts for analogous reactions. We are interested in transition metal complexes having CO2-conversion reactivity, and as a research approach, we study synthetic analogs of formate dehydrogenases (FDHs) active sites. In some prokaryotes, the FDHs, containing molybdenum (Mo) or tungsten (W) cofactors, facilitate the reversible conversion of formate/CO2 (HCO2

– CO2 + H+ + 2e–). Remarkably, the enzyme operates near the thermodynamic potential of the reduction of CO2 to formate (–0.43 V vs. NHE at pH = 7). The FDH active site has a high valent Mo/W(IV,VI) center coordinated by equatorial bis-dithiolene and axial chalcogenide ligands. The dithiolene structure plays a role as an electron sink to accept/donate electrons from/to the metal ions while stabilizing the high valent reaction core, and the chalcogenide ligand determines the reactivity with substrates. A synthetic model, [WIV(OH)(S2C2Ph2)2]– complex, where a hydroxy (OH) group coordinates to the W ion in the axial position, reacted with CO2 in the fashion of O-atom exchange between W-OH/CO2 through a [W-CO3H] intermediate. The nucleophilic hydroxide group seems to initiate the reaction, however, the next step to the formate generation was not achieved by the current model. On the other hand, we investigated the CO2 reactivity of a thermally stable [WIV(O)(S2C2Me2)2]2–, which showed stability at ~100 °C under N2 atmosphere, but interestingly, the W(IV) ion was oxidized to W(V) while reacting with CO2, and the complex was structurally reorganized to a triply bridged dinuclear W(V) complex, [W2O2(μ-S)(μ-S2C2Me2)(S2C2Me2)2]2–. During the oxidative conversion, the stoichiometric amount of CO2 was reduced to formate in the presence of a proton source. In this talk, I will discuss the experimental data and current understandings of the (electro)chemical reactivities of the Mo/W-bis(dithiolene) complexes. Keywords : formate dehydrogenase, synthetic analogs, Mo/W dithiolene complexes References 1. Seo. J.*, Shearer, J.; Williard, P. G.; Kim, E.*, Dalton Trans., 48, 17441 (2019).

90


S554 Prevention of Electrochemical Degradation in Bioelectrochemical System by Electrical Control

Taeyoung KIM Department of Environmental Engineering, Chosun University, Gwangju, Korea Corresponding Author Email : [email protected] The bioelectrochemical system is recognized for its potential as a useful future environmental-energy techno-logy in terms of sustainable wastewater treatment and green energy production1,2). Though the bioelectroche-mical technology has rapidly developed over the past 20 years, until now, the scale-up of the bioelectrochemical system to the level of industrial application in the real world has not been achieved. The problems in bioelectrochemical degradation such as voltage reversal phenomenon and power overshoot should be overcome in order to achieve a proven level of wastewater treatment efficiency and power generation3). In this study, three multi-electrode-embedded microbial fuel cells (MFCs) were sequentially connected and operated in series and parallel modes, fed by effleunt of an anaerobic digester continuously operated using swine wastewater. The MFCs connected in parallel showed the higher power density compared to the series mode. The bioelectroche-mical degradations were prevented by MFCs connected in parallel and connected with power management system despite inevitable electrical malfunction conditions by imbalance organic loadings. These findings can lead to be closer real application of bioelectrochemical systems for sustainable wastewater treatment and bioenergy generation. Keywords : bioelectrochemical system, microbial fuel cell, wastewater treatment, voltage reversal, power management system References 1. Bruce E. Logan, Bert Hamelers, René Rozendal, Uwe Schröder, Jürg Keller, Stefano Freguia, Peter

Aelterman, Willy Verstraete, and Korneel Rabaey, Microbial fuel cells: methodology and technology (2006), Environmental Science & Technology, 40, 5181-5192.

2. René A. Rozendal, Hubertus V.M. Hamelers, Korneel Rabaey, Jurg Keller, Cees J.N. Buisman, Towards practical implementation of bioelectrochemical wastewater treatmetn(2008), 26, 450-459.

3. Bongkyu Kim, S. Venkata Mohan, Deby Fapyane, In Seop Chang, Controlling voltage reversal in microbial fuel cells(2020), Trends in Biotechnology, 38(6), 667-678.

91


[S5-7]

Frontiers in C1 Gas Refinery

92


S571 Methanotrophic Platform Cell Factory for Methane Bioconversion

Eun Yeol LEE Department of Chemical Engineering, Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Methane is a low-priced next-generation carbon feedstock for industrial biotechnology. Methanotrophs can convert methane to value-added products. Methane bioconversion using methanotrophic platform cell factory can valorize methane and mitigate methane as greenhouse gas. With recent advances in molecular biology-based tool development and system biology-based understanding of methanotrophs’ physiology, methanotrophs can be engineered to produce various target products from methane. In this presentation, technical challenges and issues of methanotrophs as a platform cell factory for methane bioconversion will be discussed. Keywords : Methanotrophs, methane, bioconversion References 1. Anh Duc Nguyen, Eun Yeol Lee, Engineered methanotrophy: a sustainable solution for methane-based

industrial biomanufacturing (2021). Trend. Biotechnol. doi: 10.1016/j.tibtech.2020.07.007 2. Diep Thi Ngoc Nguyen, Ok Kyung Lee, Nguyen Thi Thu and Eun Yeol Lee, Type II methanotrophs: a

promising microbial cell-factory platform for bioconversion of methane to chemicals (2021). Biotechnol. Adv., 47, 107700.

93


S572 Synthetic Microbial Consortium for the Production of High Value Chemicals from Carbon Monoxide

Gyoo Yeol JUNG1,2 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea, 2Department of Chemical Engineering, POSTECH, Pohang, Korea Corresponding Author Email : [email protected] Carbon monoxide is produced from iron smelting as a byproduct and a large quantity of CO byproduct is also formed during the oxidative processes for the production of chemicals. For this reason, it is important to develop a process for the conversion of CO to high value chemicals such as bioactive compounds, animal feeds, and platform chemicals. Biological process is known as the most efficient method to transform from CO to high value products because some microorganisms can grow by using CO as sole carbon source. Naturally available CO consuming microorganisms are, however, hard to be engineered due to lack of genetic tools and their biological information. In this presentation, synthetic microbial consortium using novel synthetic biology tools for the development of efficient biological process converting CO to high value chemicals. Keywords : Microbial consortia, Carbon monoxide, Fermentation stability, 3-Hydroxypropionic acid, Itaconic acid References 1. Sanghak Cha et al, Design of mutualistic microbial consortia for stable conversion of carbon monoxide to

value-added chemicals (2021), Metabolic Engineering, 64, 146-153.

94


S573 Biotransformation of Formaldehyde into Glycolic Acid, Ethylene Glycol, and Ethanol Amine via Glycolaldehyde by using Glyoxylate Carboligase as a Key Enzyme

Jeong-Sun KIM1, Jin-Byung PARK2 1Department of Chemistry, Chonnam National University, Gwangju, Korea, 2Dapartment of Food science and Engineering, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Formaldehyde (HCHO) is an emerging C1 source, because it could be prepared from CH4, CH3OH, CO, and CO2 by biological and/or chemical means [1]. A very simple biocatalytic system for the synthesis of the industrially relevant C2 chemicals (e.g., glycolic acid, ethylene glycol, and ethanol amine [2]) from formal-dehyde was established. The biocatalytic system consisted of a newly discovered thermostable glyoxylate carboligase from Escherichia coli K-12 (EcGCL) and an aldehyde dehydrogenase (e.g., α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense) or an alcohol dehydrogenase (i.e., 1,3-propanediol dehydrogenase (DhaT) of Klebsiella pneumoniae) or ω-transaminase (ω-TA) from Silicibacter pomeroyi [2]. The combination of EcGCL and KGSADH allowed to produce glycolic acid from formaldehyde via glycolaldehyde to a conversion of 62%, while the serial reaction of EcGCL and DhaT led to formation of ethylene glycol to a conversion of 64%. The combination of EcGCL and ω-TA enabled the production of ethanol amine to a conversion of 64%. This study will contribute to enzymatic synthesis of glycolic acid, ethylene glycol, and ethanol amine from C1 compounds in an environment-friendly way. Keywords : Formaldehyde, Glyoxylate carboligase, Glycolaldehyde References 1. Sarah Desmons, Régis Fauré, Sébastien Bontemps, Formaldehyde as a promising C1 source: the instru-

mental role of biocatalysis for stereocontrolled reactions (2019) ACS Catalysis, 9, 9575-9588. 2. Laura Salusjärvi, Sami Havukainen, Outi Koivistoinen, Mervi Toivari, Biotechnological production of

glycolic acid and ethylene glycol: current state and perspectives (2019) Applied Microbiology and Bio-technology, 103, 2525-2535.

95


S574 Systems Biology of Acetogenic Bacteria for Sustainable Bioproduction from C1 Feedstocks

Byung-Kwan CHO Department of Biological Sciences, KAIST, Dajeon, Korea Corresponding Author Email : [email protected] The C1 gases generated in various industrial processes consist of carbon dioxide (CO2), carbon monoxide (CO), and methane (CH4). Because these gases cause global climate change, such as the greenhouse effect and environmental pollution, acetogenic bacteria (acetogens) have emerged as a biocatalyst to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl (WL) pathway. However, despite the advantage of applying acetogens as biocatalysts, lack of systemic understanding such as the regulation mechanism of the WL pathway or the transfer process of an external electron to fix C1 compounds has limited the construction of a cellular factory optimized. Instead, most previous studies focused on understanding the biochemistry of WL pathway and physiological change of acetogen. To overcome the limitation of lack of knowledge about the genetic landscape, we have considered understanding the C1 fixation mechanism in acetogen at the molecular-levels based on the system biology. Through this approach, we understand the WL pathway's regulation mechanism, discovered a novel C1 fixation pathway, or demonstrated how the redox energy generated from the external electrode could be utilized to fix the C1 gas. These research results will suggest the direction of strain improvement necessary for constructing an efficient C1 gas fixing system using acetogen. Keywords : Acetogenic bacteria, Wood-Ljundahl pathway, System biology References 1. Y. Song et al., Functional cooperation of the glycine synthase-reductase and Wood–Ljungdahl pathways for

autotrophic growth of Clostridium drakei (2020), PNAS, 117(13). 2. S. Jin et al., Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth

(2021), PNAS, 118(9).

96


부문위원회 및 학생세션

97


[S6-1]

부문위원회 및 학생세션 I:

Fresh Ph.D.

98


S6101 A Novel CO2-free Approach for the pH-control and Recycling of Bicarbonate-carbonate-buffered Culture Media in Microalgae Production Systems

Niklas ZELL1, Sunhwa JUNG1, Fabian BOßLE1, Marc HERDEN1, Peter KURZWEIL1, Franz BISCHOF1, Aelxander JAHN2, Chrsitoph LINDENBERGER1 1OTH Amberg-Weidern, Department of Mechanical Engineering and Environmental Engineering, Amberg, Germany, 2German Engineering Research and Development Center LSTME Busan, Busan, Korea Corresponding Author Email : [email protected] Over the past decades, microalgae have developed into a promising source of biofuels and -materials as well as a possible carbon sink for carbon capture technologies and yet their cultivation still poses economic challenges, especially regarding their carbon supply. Owing its simplicity, the cultivation in highly saline bicarbonate-carbonate-buffered media is an appealing way to address this problem, but only as long as the media’s pH can be kept low enough to avoid the shift of available bicarbonate into worthless carbonate. Most methods proposed for such a pH-control rely on the use of CO2- or flue-gas, which is limited by the relatively low mass transfer. Therefore, a different approach is presented, that is based upon perm-selective cation-exchange-membranes and completely omits the use of gaseous CO2-supply for pH-control. During the process, the cultivation media flows over the membrane that separates the broth from an acid reservoir (here hydrochloric acid). The H+ from the acid pass through the membrane, lowering the broth’s pH without the introduction of additional anions. The transfer of Na+ and K+ from the media to the acid balances the electrical flow. Using a lab-scale membrane-module, this method was applied to the used cultivation media of Arthrospira platensis cultures, which has risen to pH 12, resulting in a low vitality of the culture. After lowering the pH back to 8 again, thereby shifting all the carbonates back to available bicarbonates, the medium was used for a second batch culture of A. platensis. The growth in the regenerated media coincided well with that of the cultures fed with fresh medium, thus clearly demonstrating the effectiveness of this method in terms of pH-control, overall medium-recycling and improved substrate-use efficiency. Keywords : Microalgae, cation exchange membrane, pH control, CO2 free, media regeneration

99


S6102 Numerical Investigation of Fluid Flow, Light Distribution and Cellular Growth Kinetics in Bubble Column Photobioreactors

Giovanni LUZI1, Christopher MCHARDY2, Bastian EYSEL2, Christoph LINDENBERGER3, Cornelia RAUH1,2,4, Antonio DELGADO1,4 1LSTME Busan Branch, Busan, Korea, 2Department of Food Biotechnology and Food Process Engineering, Technical University of Berlin, Berlin, Germany, 3Department of Mechanical Engineering and Environmental Engineering, OTH Amberg, Kaiser-Wilhelm-Ring, Amberg, Germany, 4Institute of Fluid Mechanics, University of Erlangen-Nuremberg, Cauerstraße, Erlangen, Germany Corresponding Author Email : [email protected] One of the most promising potential choices for sustainable production of food, feed, or fuels is represented by microalgae. These microorganisms are cultivated in photobioreactors (PBR) where the maximum culture density is limited to few grams per liters. To increase the PBR productivity, researchers suggest promoting flashing light conditions, either by intensive mixing or by applying illumination strategies like light pulsation. Independently of the chosen approach, the light distribution represents a critical parameter for the growth of phototrophic microorganisms, and it is mostly affected by the microorganism concentration and by the distribution of gas bubbles. Utilizing numerical simulations, we first investigated the influence of gas bubbles on the light field at different gas superficial velocities, concluding that the presence of gas bubbles affects both the spatial distribution and the magnitude of the light intensity field only at biomass concentrations less than 1 kg/m3, but generally to a very small extent. Afterward, we compared pneumatic mixing and flashing LED sources regarding their feasibility to promote the flashing light effect. On the one hand, we found that at the investigated operating conditions pneumatic mixing alone has no impact on the growth rate and a further increase of the mixing intensity might be limited by the shear sensitivity of the cells. On the other hand, numerical simulations clearly show that illumination with flashing LED promotes an increase of the growth rate up to a factor of 2.5 if proper flashing frequencies are selected. Our numerical simulations integrate the computation of the three-dimensional fluid flow and light field, as well as the computation of the growth kinetics of algal cells. In our studies, we selected a 5 cm diameter bubble column PBR filled with a suspension of Chlamydomonas reinhardtii, which is a reactor size relevant for industrial conditions. Keywords : Bubble Column, Numerical Simulations, Flashing LED, Growth Kinetics

100


S6103 Development of an in vitro Gut Simulation Device for the Investigation of Host-microbe Interaction by Integrative Multiomics Analysis

Won-Suk SONG, Sung-Hyun JO, Jae-Seung LEE, Hyo-Jin JEON, Ji-Eun KWON, Ji-Hyeon PARK, Ye-Rim KIM, Yun-Gon KIM Department of Chemical Engineering, Soongsil University, Seoul, Korea Corresponding Author Email : [email protected] The commensal gut bacterium Akkermansia muciniphila is in the spotlight as a promising probiotic candidate that improves host health and prevents diseases. However, the biological interaction of A. muciniphila with human gut epithelial cells has rarely been explored for use in biotherapeutics. Here, this study elaborates on the development of the in vitro device that simulates the gut epithelium to elucidate the biological effects of living anaerobic bacteria via multiomics analysis: the Mimetic Intestinal host-Microbe Interaction Coculture System (MIMICS). Both human gut epithelial cells (Caco-2) and the anaerobic bacterium, Akkermansia muciniphila, can remain viable for 12 hours after coculture in the MIMICS. The changes of transcriptomics and proteomics (i.e. cell-cell junctions, immune responses, and mucin secretion) in gut epithelial cells after the treatment with A. muciniphila closely correspond with those reported in previous animal studies. In addition, multiomics results revealed that A. muciniphila activates glucose and lipid metabolism in gut epithelial cells, leading to the increase in ATP production. This study suggests that the MIMICS may be an effective general tool for evaluating the effects of anaerobic bacteria on gut epithelial cells. Keywords : Akkermansia muciniphila, Host-microbe interaction, multiomics analysis, gut simulation device, LC-MS/MS References 1. W. Song et al, Biotechnol. Bioeng. Online published (2021).

101


S6104 Gold Nanoparticle-assisted SELEX as a Visual Monitoring Platform for Rapid Discovery of Small Molecule-binding DNA Aptamers

Eun-Song LEE1,2, Jeong Min LEE1, Hea-Jin KIM1, Gyeongmin KIM1, Young-Pil KIM1,2 1Department of Life Science, Hanyang University, Seoul, Korea, 2Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual and straightforward monitoring platform for the rapid discovery of small molecule-binding single-stranded DNA (ssDNA) aptamers. Through the colorimetric changes within and between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor post-SELEX monitoring process. We also identified ssDNA aptamers with high selectivity and binding affinity by targeting two hormones (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is directly useful as a self-monitoring platform to discover ssDNA aptamers for diverse targets in a rapid and simple way. Keywords : Gold Nanoparticle, Aptamer, SELEX, Brassinolide, Bisphenol A

102


S6105 Local Therapeutic Nanoplatform Based on Stimuli-responsive Mussel Adhesive Protein For Highly Efficient Cancer Therapy

Yeonsu JEONG1, Yun Kee JO2, Hyung Joon CHA1 1Biomaterials Research Center, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2Department of Biomedical Convergence Science and Technology, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] Nanoparticles (NPs)-based drug delivery system has been developed with advantages of passive targeting ability and controlled release property, however, systemic approaches have several limitations such as insufficient accumulation and systemic toxicity. In addition, harsh microenvironments including flow of body fluids have been major challenges in effective bioavailability of therapeutics. Thus, local therapeutic system requires innovative NPs with great adhesion, stimuli-sensitivity, and high biocompatibility to improve therapeutic absorption. Here, we propose mussel adhesive protein (MAP)-based pH- and photo-responsive NPs to provide prolonged retention on tumor tissue and facilitate the controlled release of therapeutics for local cancer therapy. The MAP NPs showed superior surface adhesion and selective drug release properties responding to pH and light, enabling localized chemo-, gas-, and photothermal-therapy. The locally-treated MAP NPs exhibited significant anticancer effects in vivo devoted by extended retention on tumor sites. We anticipate that the MAP-based nanoplatform can offer the versatile approaches for effective cancer therapy with few risk of adverse effects. Keywords : Mussel adhesive protein, Adhesive nanoparticle, Stimuli-responsive nanoplatform, Local drug delivery References 1. Y. Jeong, Y. K. Jo, B. J. Kim, B. Yang, K. I. Joo, and H. J. Cha, ACS Nano 12, 8909 (2018). 2. Y. Wang, Y. Deng, H. Luo, A. Zhu, H. Ke, H. Yang, and H. Chen, ACS Nano 11, 12134 (2017). 3. K. Park, ACS Nano 7, 7442 (2013).

103


S6106 A Novel Bicelle Preparation Method Using Microfluidic Hydrodynamic Focusing

Sunghak CHOI1, Bongsu KANG2, Keesung KIM3, Ho-Sup JUNG1 1Dept. of Food Science and Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul, Korea, 2Dept. of Mechanical Engineering, Kyungpook National University, Daegu, Korea, 3College of Engineering, Research Institute of Advanced Materials, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] A bilayered micelle or so-called bicelle offers a variety of potential applications in the biomedical field due to its high stability and similarity with biological membranes [1]. Thin-film hydration method which is the conventional method to prepare bicelles demands complicated manufacturing processes so that enhancing productivity is a significant issue. Moreover, it accompanies harsh conditions such as repeating freezing and thawing cycles, in which the physicochemical characteristics of functional materials could be denatured [2]. Here, we propose a novel method for bicelle synthesis using a microfluidic chip without any external energy exertion. Bicelles were successfully prepared using this continuous method, with enhanced physicochemical properties. The critical condition for bicelle formation in this microfluidic system is deduced through experimental and analytical studies in terms of concentration and mixing time. Furthermore, the size of bicelle can be controlled, which crucially influences cellular uptake, transportation, and accumulation behavior. Keywords : Bicelle, Microfluidic Chip, Hydrodynamic Focusing References 1. M. Beaugrand, A. A. Arnold, J. Hénin, D. E. Warschawski, P. T. F. Williamson, and I. Marcotte, Langmuir.

30, 6162-6170 (2014). 2. S. Taguchi, K. Suga, K. Hayashi, Y. Okamoto,H.-S. Jung, H. Nakamura, H. Umakoshi, Colloids and

Interfaces. 2, 73 (2013).

104


S6107 Glycan Chip Based on i-motif DNA Linker with pH-responsive Structural Change for On-surface Biosynthesis of Cancer-associated Glycans

Hye Ryoung HEO1, Jeong Hyun SEO2, Chang Sup KIM3, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2School of Chemical Engineering, Yeungnam University, Gyeongsan, Korea, 3School of Chemistry and Biochemistry, Yeungnam University, Gyeongsan, Korea Corresponding Author Email : [email protected], [email protected] On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for applying glycan-involved applications simultaneously. However, current methods cannot provide quantitative information on synthesized glycans, resulting in undefined structures on a surface, which leads to unequable and unreliable results. In this work, novel glycan chip was developed by introducing pH-responsive i-motif DNA linker capable of reversibly controlling the immobilization and separation of glycans on chip surface in pH-dependent manner. On-chip enzymatic glycosylations were optimized for synthesizing cancer-associated glycans such as Globo H hexasaccharide by analyzing quantitatively products isolated from the surface. This enabled to construct glycan chip composed of structurally defined cancer-associated glycans. Interaction analysis of anti-Globo H antibody and Globo H-related glycans biosynthesized on the chip showed that VK9 strongly interacts with biosynthesized Globo H hexasaccharide. Furthermore, we investigated glycan-binding specificity of MCF-7 breast cancer cells using glycan chip consisting of biosynthesized Globo H hexasaccharide and its related glycans to identify a high-affinity glycan ligand. The cancer cell strongly recognized Globo H hexasaccharide, suggesting that the specific receptor for Globo H hexasaccharide can be present on the surface of breast cancer cell. These results demonstrated the feasibility of the DNA linker-based glycan chip for on-surface complex glycan biosynthesis and glycan-involved applications. Keywords : glycan chip, on-chip synthesis, enzymatic glycosylation

105


[S6-2]

부문위원회 및 학생세션 II:

Fresh Ph.D.

106


S6201 Amino Acids Detection Using a Complementary Cell-free Protein Synthesis System

Hye Jin LIM, Kyung-Ho LEE, Dong-Myung KIM Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Amino acids are building blocks of protein synthesis, and also play important roles in maintaining the physiological homeostasis. For example, changes in amino acid concentrations are closely related with cell signaling, gene expression, and protein phosphorylation cascade. In addition, many amino acids serve as the precursors of key metabolites, including hormones and polyamines. Therefore, quantification of amino acids can provide important information for disease diagnosis. The detection and quantification of amino acids have conventionally employed chromatography technologies, including high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography (GC). However, these methods require complicated equipment setup, highly trained operator, and long process time. In this study, we developed a rapid and cost-effective assay method that harnesses the molecular process of protein synthesis for quantitative detection of amino acids. In this scheme, dubbed complementary cell-free protein synthesis (CCFPS) assay, a reaction mixture for cell-free protein synthesis is prepared without amino acids that need to be analyzed. When programmed with a reporter gene, and mixed with an assay sample containing the missing amino acids, the reaction mixture for this CCFPS assay produces detection signal. With different reporter enzymes, the intensity of the signal from the CCFPS assay showed linear correlations with the concentrations of amino acids in assay samples. Furthermore, we could harness the endogenous enzymes of cell extract for the measurement of non-proteinogenic amino acids by the CCFPS assay. Keywords : Cell-free protein synthesis, Amino acids, Amino acid analysis, Diagnosis References 1. M. C. Waldhier, M. A. Gruber, K. Dettmer, and P. J. Oefner, Anal. Bioanal. Chem. 394, 695 (2009). 2. Y. J. Jang, K. H. Lee, T. H. Yoo, D. M. Kim (2017) Anal. Chem., 89, 9638-9642. 3. Y. J. Jang, K. H. Lee, T. H. Yoo, D. M. Kim (2019) Anal. Chem., 91, 2531-2535. 4. Y. J. Jang, K. H. Lee, T. H. Yoo, D. M. Kim 2020) Anal. Chem., 92, 11505-11510.

107


S6202 Resolving the Mutually Exclusive Immune Activities of Chitosan

Suyoung LEE1, Seohyun BYUN2, Sin-Hyeog IM2, Dong Soo HWANG1 1Division of Environmental Science and Engineering, POSTECH, Pohang, Korea, 2Division of Integrative Bioscience and Biotechnology, Department of Life Science, POSTECH, Pohang, Korea Corresponding Author Email : [email protected] Chitosan has multifaceted functions displayed by both pro- and anti-inflammatory properties which hinder its effective development as an immunomodulatory agent. Herein, the contributions of molecular weight (MW) of chitosan with regard to its immune regulatory properties towards inflammation are investigated. The anti-inflammatory properties of low and relatively high MW chitosan are compared by utilizing immunological assays and nanomechanics based experiments on the surface forces apparatus (SFA). Interestingly, compared with relatively high MW chitosan, low MW chitosan significantly increases the differentiation of anti-inflammatory regulatory T cells (Tregs) through the Dectin-1-dependent pattern recognition receptor (PRR) on antigen-presenting cells in mice. SFA analyses also show a similar trend of interaction forces between chitosan and diverse PRRs depending on its MW. The results obtained in the immunological and nanomechanical experiments are consistent and imply that the binding features of PRRs vary depending on the MW of chitosan, which may alter immune activity. In accordance, in vivo administration of only low and not high MW chitosan, repress inflammatory responses and suppress the progression of experimental colitis. This study elucidates a previously unexplored size-dependent immunoregulatory property of chitosan, and suggests the applicability of low MW chitosan as a pharmaceutical ingredient to treat diverse inflammatory disorders. Keywords : chitosan, immune activities, pattern recognition receptors, anti-inflammation References 1. C. L. Bueter, C. K. Lee, J. P. Wang, G. R. Ostroff, C. A. Specht, S. M. Levitz, Journal of immunology, 192,

5943 (2014). 2. K. Azuma, T. Osaki, S. Kurozumi, M. Kiyose, T. Tsuka, Y. Murahata, T. Imagawa, N. Itoh, S. Minami, K.

Sato, Y. Okamoto, Carbohydrate polymers, 115, 448 (2015). 3. T. A. Reese, H. E. Liang, A. M. Tager, A. D. Luster, N. Van Rooijen, D. Voehringer, R. M. Locksley,

Nature, 447, 92 (2007). 4. Carla A. Da Silva, Dominik Hartl, Wei Liu, Chun G. Lee, Jack A. Elias, J Immunol. 181, 4279 (2008).

108


S6203 Genome-scale Analysis of Acetobacterium woodii Identifies Translational Regulation of Acetogenesis

Jongoh SHIN, Yoseb SONG, Seulgi KANG, Sangrak JIN, Sun Chang KIM, Suhyung CHO, Byung-Kwan CHO Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Acetogens synthesise acetyl-CoA via the CO2-fixing Wood–Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon metabolism in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. In particular, Wood-Ljungdahl pathway genes are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas the carbonyl-branch presents an increased translation level in comparison to the methyl-branch under heterotrophic growth conditions. The translation efficiency of genes in these pathways is differentially regulated by 5′-untranslated regions and ribosome-binding sequences under the two growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas fermenting acetogenic bacteria for better productivity. ** This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project 2011-0031957 and C1 Gas Refinery Program 2018M3D3A1A01055733 of the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning. Keywords : Acetogenesis, Acetogen, Translational regulation, Wood-Ljungdahl pathway

109


S6204 Improving CO2 Tolerance by Expression of a Single H+ Pump Enables Microalgae-based Industrial Flue Gas Valorization

Hong Il CHOI1, Sung-Won HWANG2, Sang Jun SIM1 1Department of Chemical and Biological Engineering, Korea University, Seoul, Korea, 2Department of Chemical Engineering, University of Michigan, MI, USA Corresponding Author Email : [email protected] Microalgae have garnered much attention as a promising biological platform for CO2 reduction for their ability to photosynthetically produce various carbon-neutral products using CO2 as a sole carbon source. However, the real-world application has been severely limited since they are ironically susceptible to elevated CO2. In order to address this issue, we scrutinized the transcriptome of a CO2 intolerant model microalga, Chlamydomonas reinhardtii, in response to extremely high CO2 environments to pinpoint the genetic cause and found that aberrantly low expression of plasma membrane H+-ATPases (PMAs), would be a primary reason for the susceptibility. Subsequently, we constitutively overexpressed a universally expressible PMA protein in C. reinhardtii. The single gene overexpression yielded a microalgal strain that exhibit 3.2-fold increased autotrophic production against high CO2 milieus. The reproducibility of the improvement was confirmed by proof-of-concept outdoor cultivation with high CO2-containing flue gas emitted from a power plant. The results suggest that flue gas streams can be valorized by the microalgae when conjugated with this genetic engineering strategy. Keywords : Microalgae, CO2 utilization, CO2 tolerance References 1. A. Solovchenko and I. Khozin-Goldberg, Biotechnol. Lett. 35, 1745 (2013). 2. L. Wei et al., Metab. Eng. 54, 95 (2019).

110


S6205 The Entner–doudoroff Pathway (EDP) Intermediate-inducible Expression System for 2,3-butanediol Production in Escherichia coli

SATHESH-PRABU CHANDRAN, Sung Kuk LEE School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Recent biotechnological progress, particularly in the areas of genetic/genomic engineering, synthetic biology, systems biology, and metabolic engineering, offers novel tools to customize cell factories for bio-based production. The production of bio-based bulk chemicals from renewable bioresources has gained great attention in the scientific community as the process could avoid and/or minimize the negative impacts of the conventional chemical-based productions on the environment and depleting resources of natural petrochemicals. An inducible promoter is a crucial component for microbial-based chemical production. The induction of promoter expression by the addition of chemical inducers such IPTG is considered the most efficient method. However, it has the following limitations, such as the requirement of cell growth monitoring for the addition of chemical inducers at the optimal cell density, not feasible with industrial scale-up, and undesirable because of its toxicity and cost. Moreover, constitutive expression or strong expression of synthetic pathway genes can cause growth inhibition, less glucose consumption, and reduce product yield. Thus, inexpensive carbon source- or substrate-inducible systems are considered as one of the efficient systems for biosynthesis of chemicals, especially at industrial scale. 2,3-Butanediol (BDO), an important platform chemical, has many industrial applications and is used as a solvent, a high-grade aviation fuel because of its high-octane number, and a precursor of many synthetic polymers and resins. In addition, BDO is used in the manufacture of perfumes, fumigants, printing inks, moistening and softening agents, antifreeze, lubricants, fuel additives, explosives, plasticizes, and pharmaceu-tical carriers. In the present study, a glucose-inducible gene expression system has been developed using HexR-Pzwf1 of Pseudomonas putida to induce the metabolic pathways. Since the system is controlled by an Entner–Doudoroff pathway (EDP) intermediate, the EDP of Escherichia coli was activated by deleting pfkA and gntR genes. Growth experiment with GFP as a reporter indicated that the induction of this system was tightly controlled over a wide range of glucose in E. coli without adding any inducer. 2,3-butanediol (BDO) synthetic pathway genes were expressed by this system in the pfkA-gntR-deleted strain. The resultant engineered strain harbouring this system efficiently produced BDO with a 71% increased titer than the control strain. The strain was also able to produce BDO from a mixture of glucose and xylose which is comparable to glucose alone. Further, the strain produced 11 g/L of BDO at a yield of 0.48 g/g from the hydrolysate of empty palm fruit bunches. This system can also be applied in many other bio-production processes from lignocellulosic biomass. Keywords : 2,3-Butanediol (BDO), Entner-Doudoroff pathway, Pseudomonas putida, HexR

111


S6206 Genome Mining and Reassessment of Streptomyces venezuelae Strains Harboring Novel Secondary Metabolites Biosynthetic Gene Clusters

Namil LEE1,2, Mira CHOI3, Woori KIM1,2, Suhyung CHO1,2, Bernhard PALSSON4,5,6, Kyoung-Soon JANG3,7, Byung-Kwan CHO1,2 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Innovative Biomaterials Research Center, KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Bio-Chemical Analysis Team, Korea Basic Science Institute, Cheongju, Korea, 4Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 5Department of Pediatrics, University of California San Diego, La Jolla, CA, USA, 6Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark, 7Division of Bio-Analytical Science, University of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Streptomyces strains have been traditionally classified based on morphological and physiological observations. Therefore, considerable inaccuracies have accumulated in Streptomyces taxonomy, causing the misclassification of a large number of Streptomyces strains. Here, we present the highly resolved classification of 10 Streptomyces venezuelae strains, using 16S rRNA sequencing, MALDI-TOF MS protein profiling, and whole-genome mining. The results revealed that only three (ATCC 10595, 21113, and 10712) of the 10 strains could be classified as S. venezuelae species. Accurate classification, following secondary metabolite biosynthetic gene cluster mining and targeted LC-MS/MS-based metabolite screening, enabled to identify of the production of 126 secondary metabolites from the 10 S. venezuelae strains. In particular, the comparison of biosynthetic gene clusters for pyrrolamide-type antibiotics of four S. venezuelae strains revealed that the novel gene, athv28, is critical in the synthesis of the anthelvencin precursor, 5-amino-3,4-dihydro-2H-pyrrol-2-carboxylate. Our findings illustrate the importance of the accurate and reliable classification of Streptomyces and better utilization of misclassified Streptomyces strains for the discovery of novel smBGCs. ** This work was supported by the Bio & Medical Technology Development Program (grant no. 2018M 3A9F3079664 to B.-K.C.) through the National Research Foundation (NRF) funded by the Ministry of Science and ICT (MSIT). This work was also supported by a grant from the Novo Nordisk Foundation (grant no. NNF10CC1016517) and KBSI grant (C030440). Keywords : Streptomyces, classification, genomics, secondary metabolite

112


S6207 Pig Sera-derived Anti-SARS-CoV-2 Antibodies with Controlled Specificity for Competitive Immunoassay

Ji-Hong BONG, Jaeyong JUNG, Jeong Soo SUNG, Chang Kyu LEE, Jae-Chul PYUN Department of Materials Science and Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies against nucleoprotein (NP) were purified from pig sera through two steps: (1) isolation of anti-SARS-CoV-2 NP antibodies using magnetic beads with immobilized SARS-CoV-2 NP and (2) filtration of anti-SARS-CoV-2 spike protein (SP). The binding activities of the isolated antibodies were confirmed using immunoassay and immunostaining. The binding constant (Kd) of the purified anti-SARS-CoV-2 NP antibodies was estimated using a surface plasmon resonance biosensor (SPR) and calculated to be 185 pM. From the dose-response curve, the limit of detection was estimated to be 1.02 pM. Finally, a competitive immunoassay was developed for detecting SARS-CoV-2 NP as well as SARS-CoV-2 in the culture fluid using magnetic beads with immobilized anti-SARS-CoV-2 NP antibodies. The results demonstrated (a) the detection of SARS-CoV-2 in viral fluid and (b) the discrimination of SARS-CoV-2 from SARS-CoV, MERS-CoV, and CoV strain 229E in viral fluids. Additionally, anti-SARS-CoV-2 antibodies against SP were obtained when the isolation process was performed using the magnetic beads with immobilized human SARS-CoV-2 SP. Keywords : SARS-CoV-2, COVID-19, Antibody, Nucleoprotein, Spike protein, Surface plasmon resonance, Immunoassay

113


[S6-3]

부문위원회 및 학생세션 III

114


S6301 Bioproduction of Propionic Acid from Engineered Pseudomonas putida Strain

Rameshwar TIWARI, Sung Kuk LEE School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Propionic acid is an industrially relevant organic acid used in various applications like food, agricultural, and pharmaceutical industries. However, petrochemical based unsustainable propionic acid production leads to the development of microbial bioprocess from renewable substrates like lignocellulosic biomass. Levulinic acid is a γ-keto acid (C5) platform chemical produced by acid-catalyzed dehydration and the hydrolysis of sugars obtained from lignocellulosic biomass without using expensive hydrolytic enzymes. The metabolic pathway of levulinic acid utilization was discovered in Pseudomonas putida strain and further used for levulinic acid-based biorefinery to produce biologically diverse chemicals. Moreover, P. putida is recognized as a safe microbial host and is emerging as a next-generation industrial workhorse due to its metabolic versatility and remarkable tolerance to oxidative stress, organic solvents, and aromatic compounds. In present work, levulinic acid assimilation pathway of P. putida EM42 strain was directed to produce propionic acid by blocking side pathways and its interconversion into propionyl-CoA. Further, levulinic acid-inducible expression system was used to express metabolic genes for propionic acid production. Metabolic engineered P. putida strain was used to optimize the bioprocess for higher production yield of propionic acid. This study revealed the exploitation of levulinic acid catabolic pathway from P. putida as an alternative route for the sustainable and industrial production of propionic acid. Keywords : Propionic acid, Pseudomonas putida, levulinic acid, metabolic engineering

115


S6302 Metabolic Engineering of Corynebacterium glutamicum for the Enhanced Production of a Natural Blue Pigment Indigoidine as Sustainable Fabric Dyes

Mohammad Rifqi GHIFFARY1,2, Cindy Pricilia Surya PRABOWO2,3, Komal SHARMA1,2, Sang Yup LEE2,3,4, Hyun Uk KIM1,2,4 1Systems Biology and Medicine Laboratory, Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon, Korea, 3Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engi-neering, KAIST Institute for BioCentury, KAIST, Daejeon, Korea, 4KAIST Institute for Artificial Intelligence, BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] The industrial production of dyes for textiles mainly involves numerous toxic chemicals which causes severe water pollution. For this, indigoidine has attracted attention as an alternative natural blue dye, but it is necessary to achieve a high-level production to compete with existing synthetic blue dyes. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing indigoidine to a high concentration with high productivity. First, blue-pigment indigoidine synthetase (bpsA) gene from Streptomyces lavendulae was expressed in C. glutamicum, which carries strong fluxes toward L-glutamate, a precursor of indigoidine. Production performance of this base strain, already producing 7.3 ± 0.3 g/L indigoidine from flask, was further improved by streamlining intracellular supply of the precursors L-glutamate and L-glutamine, strengthening phosphotransferase system-independent glucose uptake system, channeling carbon fluxes from glycolysis to tricarboxylic acid (TCA) cycle, and minimizing byproducts formation. The final strain named BIRU11 produced 49.30 g/L indigoidine with the productivity of 0.96 g/L/h from fed-batch fermentation, the highest titer and productivity to date. Finally, indigoidine from the fed-batch fermentation of the BIRU11 strain was used to dye white cotton fabrics to examine its color and performance. This study demonstrates the potential of producing fabric dyes in a sustainable manner by using a metabolically engineered bacterium. Keywords : Corynebacterium glutamicum, fabric dye, indigoidine, metabolic engineering, natural blue pigment, nonribosomal peptide

116


S6303 Bone Substitute Biomineral Complex Co-derived from Marine Biocalcification and Biosilicification

Jinyoung YUN1, Yeonsu JEONG1, Onyou NAM2, Ki Baek YEO3, Yun Kee JO4, Hye Ryoung HEO1, Chang Sup KIM5, Kye Il JOO1, Seung Pil PAC3, EonSeon JIN2, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Life Science, Hanyang University, Seoul, Korea, 3Department of Biotechnology and Bioinfor-matics, Korea University, Sejong, Korea, 4Department of Biomedical Convergence Science and Technology, Kyungpook National University, Daegu, Korea, 5School of Chemistry and Biochemistry, Yeungnam University, Gyeongsan, Korea Corresponding Author Email : [email protected] With aging population, the need for the use of bone graft is increasing to promote healing process of damaged bone [1]. Bone substitute materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds [2]. To improve bone reconstruction, the combination of several materials have been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials [3]. In this work, a marine-inspired biomineral complex was suggested as a novel potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive protein show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its feasibility as a bone graft material was determined by evaluating the in vitro osteogenic behaviors of preosteoblast cells and in vivo bone regeneration in rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used in the field of bone regenerative medicine. Keywords : coccoliths, biosilica, biomineral, bone substitutes, bone regeneration References 1. C. J. Damien and J. R. Parsons, J. Appl. Biomater., 2, 187 (1991); W. H. Hiatt et al., J. Periodontology, 49,

495 (1978). 2. A. Brydone, D. Meek, S. Maclaine, Proc. Inst. Mech. Eng., Part H, 224, 1329 (2010). 3. Y. Khan et al, J. Bone Jt. Surg, 90, 36 (2008).

117


S6304 Amine-catechol Pair Effect on Underwater Adhesion of Marine Mussel

Mincheol SHIN1, Ji Yeon SHIN2, Kyeounghak KIM1, Byeongseon YANG1, Yeonju PARK3, Sila JIN4, Young Mee JUNG3,4, Jeong Woo HAN1, Nak-Kyoon KIM2, Hyung Joon CHA1 1Department of chemical engineering, POSTECH, Pohang, Korea, 2Advanced analysis center, KIST, Seoul, Korea, 3Kangwon radiation convergence research support center, Kangwon National University, Chuncheon, Korea, 4Department of chemistry, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Non-covalent interactions have attained many attentions in various areas including molecular recognition, biological self-assembly, and molecular adhesion. Among them, cation-π interaction has been suggested as crucial intermolecular interaction in underwater adhesion recently. In specific, several studied have shown that cation-π interaction is the main contributor to the liquid-liquid phase separation (LLPS) in underwater for mussel-inspired materials. Also, molecular cohesion forces found out to be tuned by changing π candidate in underwater. In this study, we focused on how the position of cation sources could affect intermolecular cation-π interactions in underwater using surface forces apparatus (SFA), Raman spectroscopy, and NMR. Model peptides were synthesized for nanomechanical studies. Underwater cation-pi interactions were measured by (1) different aromatic π sources, (2) salt concentrations, and (3) reversibility of non-covalent interactions. This study could suggest that cation sources might deteriorate intermolecular cation-π interactions and could give insight on designing biomaterials using in underwater. Keywords : Underwater adhesion, Mussel adhesion, Dopa, Amine-catechol synergy, Cation-pi interaction, Noncovalent interaction

118


S6305 Redox Regulation of Dopa in Interfacial Underwater Adhesion of Marine Mussel

Tae Hee YOON, Mincheol SHIN, Byeongseon YANG, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] Underwater adhesion has got attention due to its versatility in biomedical applications. Since many biomedical applications face with a wet environment, underwater adhesion should be applied for adequate contact with watery surfaces such as tissues. Researchers has focused on marine organisms such as mussel which facilitates wet adhesion in daily life. Many studies revealed that 3,4-dihydroxyphenylalanine (Dopa) is a key molecule in underwater adhesion. Dopa can participate in both surface adhesion and cohesion according to its oxidative states. However, chemical state of Dopa and their role in adhesion is hard to control because it is easily oxidized in physiological environment and has complicated oxidative pathway. Therefore, to make successful underwater glue using Dopa, understanding how marine mussel regulates the redox of Dopa in the underwater adhesive system is essential. In this study, we investigated effect of redox protein on interfacial adhesive protein in mussel adhesion. Keywords : Underwater adhesion, 3,4-dihydroxyphenylalanine, Redox regulation

119


S6306 Electrospun Surgical Dressing with Natural Proteins for Dual Function as Topical Hemostatic Agent and Anti-adhesion Barrier

Jaeyun LEE1, Eun Jin KIM2, Ki Joo KIM2, Jong Won RHIE2, Kye Il JOO1,3, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Plastic and Reconstructive Surgery, The Catholic University of Korea, Seoul, Korea, 3Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Visceral surgery is accompanied by a risk of severe hemorrhage that cannot be controlled by patients’ natural coagulation ability. Furthermore, the coagulopathies from chronic bleeding or anticoagulant drugs may fall into acidosis and hypothermia which leading them to fatal trauma triad at emergent state. In addition, wounds with excessive bleeding may also cause undesired adhesion with surrounding organs, inflammation, infection, and neuralgia, worsening the surgical prognosis. Therefore, it is necessary for surgical dressing to halt bleeding on the side contact with wound and prevent adhesion on the outer side. However, most commercial products do not have both features as topical hemostatic agents and anti-adhesion barriers. There are recent approaches introducing hydrophobic paraffin, silica, synthetic polymer or carbon nanofiber to existing hemostatic materials for minimizing adhesion and infection, but these materials are limited to skin wounds due to their low biocompatibility. In this study, novel dressing for internal use with both blood coagulation and anti-adhesion function was developed by electrospinning silk fibroin and mussel adhesive protein (MAP). Fibroin has feasible mechanical property as wound dressing, and its hydrophobicity can be controlled through additional solvent treatment after electrospinning. DOPA residues present in bioengineered MAP have underwater adhesion ability to aggregate blood plasma components and activate platelets. The functionality, biocompatibility, and biodegradability of the both proteins in the nanofiber were verified through in vitro and in vivo experiments. In anticoagulated animal tests, the nanofibrous membrane promoted blood clotting and protected wound areas, confirming the feasibility as a novel surgical dressing. Keywords : silk fibroin, mussel adhesive protein, topical hemostatic agent, anti-adhesion barrier, electrospun nanofiber

120


S6307 A Simple and Efficient PCR-based Genome Walking Technique to Identify Transgene Integration Sites in CHO Cells

Hye-Jin HAN, Jong Youn BAIK Department of Biological Sciences and Bioengineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Chinese hamster ovary (CHO) cell lines have usually been developed by random integration, yielding heterogeneous clones with various expression level and stability characteristics due to the transgene insertion sites in genome. So, it is worth analyzing them for better characterization of cell lines [1]. While next-generation sequencing is commonly used to locate the insertion site, it requires complicated process and analysis of sequence reads. Moreover, it is a time-consuming (4-6 weeks) and costly ($2,000-3,000) method. Hence, we adapted splinkerette-PCR to identify the transgene insertion site with less resources (both time and cost). As a proof-of-concept experiment, a plasmid vector was cleaved using restriction enzymes, ligated with splinkerette adaptors that inhibit adaptor-adaptor amplificon [2], PCR-amplified, and run on a DNA gel to visualize the PCR product with an expected size. After the selection of restriction enzymes that have the highest cutting frequencies using a bioinformatics tool, this technique was then applied to the CHO genome to analyze the integration sites of a recombinant protein gene. In conclusion, this method would help to provide the genomic information with more affordable options in the process of cell line development. Keywords : Chinese hamster ovary (CHO) cells, Integration site, Unknown DNA sequence, Splinkerette-PCR References 1. L. M. Grav, D. Sergeeva, J. S. Lee, I. M. D. Mas, N. E. Lewis, M. R. Andersen, L. K. Nielsen, G. M. Lee

and H. F. Kildegaard, ACS Synth. Biol. 7, 2148-2159 (2018). 2. A. G. Uren, H. Mikkers, J. Kool, L. V. D. Weyden, A. H. Lund, G. H. Wilson, R. Rance, J. Jonkers, M. V.

Lohuizen, A. Berns and D. J. Adams, Nat. Protoc. 4(5), 789-798 (2009).

121


S6308 Bilirubin-encapsulated Albumin Nanoparticles for Cancer Treatment; Synthesis, Characterization, and in-vitro Cellular Assay

Seyedmohammad MOOSAVIZADEH1, Gyu Hyeok LEE2, Dae Hee LEE2, Sung In LIM1 1Department of Chemical Engineering, Pukyong National University, Busan, Korea, 2Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung, Korea Corresponding Author Email : [email protected] Bilirubin (BR) is the end-product of heme catabolism which occurs abundantly in the blood plasma. Bilirubin is an endogenous antioxidant capable of scavenging different reactive oxygen species (ROS), thereby playing a role in protecting cells and tissues from oxidative damage. Indeed, numerous experimental studies proved the potential of bilirubin for anti-inflammatory and anti-cancer activities. However, its low water-solubility and high sensitivity to oxygen restrict further clinical developments, requiring an appropriate drug carrier capable of stably delivering a defined dosage to the site of action. Albumin is the most abundant protein in blood with a long circulatory half-life, with the capability to bind nutrients, metabolites, and metals, lending itself to a promising drug carrier. Moreover, albumin is specifically localized to tumors or inflamed tissues in either an active or a passive manner. Recently, serum albumin nanoparticles, i.e., BSA-NPs, have been used for delivering various types of therapeutic agents, encapsulated inside or modified on the surface. In this study, we explored how to synthesize and characterize albumin nanoparticles containing hydrophobic bilirubin by the Desolvation method and measure its toxicity on normal and cancerous cells. Using this method, albumin nanoparticles encapsulated bilirubin with a stable size around 120 nm, and a spherical morphology were obtained. The entrapment efficiency (EE) was around 60%, and the drug loading (DL) was around 10%. The in vitro cell assay of nanoparticles on normal and cancerous cells showed low toxicity on normal cells, under 10%, in contrast to more than 60% toxicity on cancerous cells. Keywords : Albumin nanoparticle, drug delivery, cancer therapy References 1. C. Weber, et al. Int. J. Pharm. 194, 91-102 (2000). 2. A. Jahanban-Esfahlan, et al. Int. J. Biol. Macromol. 91, 703-709 (2016). 3. Lee Y, et al. Angew Chemie-Int. Ed. 55(36):10676-10680 (2016).

122


S6309 Assessing the Role of Cellular dsRNAs in the Pathogenesis of Sjogren's Syndrome and as the Therapeutic Target in Autoimmunity

Jimin YOON1, Min-Seok LEE1, Ahsan Ausaf ALI1, Ye Rim OH2, Yong Seok CHOI2, Namseok LEE1, Seunghee CHA3, Joon Young HYON4, Yun Jong LEE5,6, Sung Gap IM1,7, Yoosik KIM1,8 1Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 2Medical Science Research Institute, Seoul National University Bundang Hospital, Seongnam, Korea, 3Department of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL, USA, 4Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Korea, 5Division of Rheumatology, Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea, 6Department of Internal Medicine, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea, 7KI for NanoCentury (KINC), KAIST, Daejeon, Korea, 8KI for Health Science and Technology (KIHIST), KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Sjӧgren’s syndrome (SS) is a systemic autoimmune disease that mainly targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, SS is under-diagnosed due to complex immunopathogenesis and the lack of biomarkers. Here, we investigated the role of cellular double-stranded RNAs, especially originated from the mitochondria (mt-dsRNAs) in pathogenesis of SS. We found that mt-dsRNAs were elevated in saliva/tears of SS patients and were related to exocrine dysfunction and glandular lymphocytic infiltration. Using our unique 3D culture system, we showed that dsRNA stress led to increased mt-dsRNAs and their cytosolic export, accompanied by characteristic molecular phenotypes of SS. Moreover, mt-dsRNA induction occurred downstream of the JAK1 pathway. Interestingly, suppression of mtRNA induction by antioxidant, JAK1 inhibitor, or mtRNA polymerase inhibitor reversed the molecular phenotypes of SS. Altogether, our study underscores the importance of mtRNA upregulation, and presents mt-dsRNAs as the potential therapeutic target to prevent promotion of SS pathogenesis. Keywords : Autoimmune disease, Mitochondrial RNA, double-stranded RNA, Sjogren's syndrome, 3D cell culture References 1. Qin, B. et al. Ann Rheum Dis. 74. 1983-1989 (2015). 2. Groeger, S. and Meyle J. Front Immunol. 10, 208 (2019). 3. Lee, B.H. et al. PLos One. 8, 253113 (2013). 4. Steinfeld, S. et al. Lab Invest. 81, 143-148 (2001). 5. Kim, S. et al. bioRxive. 2020.2006.2017.156323 (2020).

123


[S6-4]

부문위원회 및 학생세션 IV

124


S6401 Development of Novel Fruit Juice Concentration Process Using Gas Hydrate Technology

Soebiakto LOEKMAN1, Timo CLAßEN2, Alexander RUDOLPH3, Giovanni LUZI1, Bernhard GATTERNIG2, Christopher MCHARDY3, Chae-Gook LEE4, Da-Yun PARK4, Man-Gi CHO1,4, Cornelia RAUH1,3, Antonio DELGADO1,2 1German Engineering Research and Development Center, LSTME Busan, Busan, Korea, 2Institute of Fluid Mechanics, FAU Erlangen-Nürnberg, Erlangen, Germany, 3Department of Food Biotechnology and Food Process Engineering, Technical University Berlin, Berlin, Germany, 4Department of Biotechnology, Division of Energy and Bioengineering, Dongseo University, Busan, Korea Corresponding Author Email : [email protected] Non-thermal food processing has gained interest as it enables the conservation of food properties & sensorial characteristics, such as flavor, color, & mouthfeel. Thermosensitive substances, e.g., vitamin, polyphenol & microbial load are also preserved. Also, the process energy requirement can be drastically reduced. CO2 is an inert gas & can form gas hydrates with water. CO2 is an ideal compound for realizing juices concentration. In the conventional process, juices need to be heated up to 80°C to achieve a degree of concentration between 65–85%, with an energy demand of 180–2,160 kJ/kg H2O. There is a possibility to lose heat-sensitive & volatile components. The freeze concentration process, which acts gentler towards the product, requires an energy demand of 936–1,800 kJ/kg H2O to achieve a concentration of up to 55%. CO2 gas hydrate technology offers a more energy-efficient process with an energy demand of 252–360 kJ/kg H2O. The products can theoretically reach a degree of concentration up to 99%. In this work, the phase equilibrium curve for three fruit juices was established. The results of juice concen-tration, carried out in two different reactor types, will also be reported. Keywords : Food Technology, CO2 Gas Hydrate, Fruit Juices, Non-thermal Process, Concentration Process, Bubble Column, Stirred-Tank Reactor References 1. B. Guignon, C. Aparicio, P.D. Sanz, and L. Otero, Food Res. Int. 46, 83-91 (2012). 2. B. Jiao, A. Cassano, and E.J. Drioli, Food Eng. 63, 303-324 (2004). 3. B. Taylor, Fruit and juice processing Chemistry and Technology of Soft Drinks and Fruit Juices. (2007). 4. M. Cheryan, Handb. food Eng. 2nd edn. CRC Press. Boca Rat. 590-595 (2007). 5. S. Li, Y. Shen, D. Liu, L. Fan, and Z. Tan, Chem. Eng. Res. Des. 93, 773-778 (2015).

125


S6402 A Gas Uptake and Holdup Based Method for Biomass Estimation of the Cyanobacteria Chlamydomonas asymmetrica in Flat Panel Biofilm Photobioreactor

Gamze ER1,2, Alexander JAHN1, Shah Abid ALI3, Man-Gi CHO3 1German Engineering Research and Development Center LSTME Busan, Korea, 2Department of Food Biotechnology and Food Process Engineering, Technical University Berlin, Germany, 3Department of Biotechnology, Division of Energy and Bioengineering, Dongseo University, Busan, Korea Corresponding Author Email : [email protected] Biofilm-based Photobioreactors (PBRs) use microalgal biomass immobilized on a substrate with reduced water content and consumption when compared to conventional suspended cultivation. CO2 and carbonate are used by microalgae as inorganic carbon source and their consumption is an indicator for metabolic activity and biomass accumulation. Here CO2 uptake, holdup and release in and from the culture were measured and correlated with gravimetrically determined biofilm mass. For this, a wicking based flat panel biofilm PBR including CO2/O2 gas control was developed, and the CO2 uptake and holdup determined by CO2 NIR off-gas analysis. The cyanobacteria Chlamydomonas asymmetrica were cultivated on hydrophilic modified expanded UHMWPE plates over circulating AF6 medium at 30°C and fluorescent light illumination about 710.01 µW/cm2. CO2 holdup in the system was subtracted from values determined during biofilm cultivation at stationary and growth conditions and correlated with measured biomass. From the gas holdup a minimum feasible cultivation size, dependent on the NIR-CO2 sensor sensitivity, for the use of the developed method was estimated. Results suggest the potential use of gas holdup measurement for biofilm growth assessment in medium and large-scale closed cultivations. Keywords : Cyanobacteria, Biofilm Photobioreactor, Biomass, Off-gas analysis, CO2 uptake

126


S6403 Yeast-derived Vacuoles as a Novel Carrier with Enhanced the Blood-brain Barrier Penetrating for Targeted Neurodegenerative Therapy

Thi Ngoc-Han NGUYEN1, Ngoc-Tu NGUYEN2, Jiho MIN1,2 1Department of Bioprocess Engineering, Jeonbuk National University, Jeonju, Korea, 2Division of Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Blood-brain barrier is a brain protection structure that restrict the drug delivery from blood to the central nervous system. Thus, we have developed a novel drug carrier that can overcome this issue using yeast vacuole. The purpose of this study is to assess the drug transporting ability of yeast vacuoles through an in vitro blood-brain barrier (BBB) model. Here, we used Daunorubicin (DNR) as a microtubule targeting agent with its disaggregating ability of pre-form fibril and prevention of Tau fibrillization. The in vitro model of the BBB is developed by culturing the human cerebral microvascular endothelial cell line (hCMEC /D3) on transwell inserts in EBM-2 Endothelial basal medium until cells formed a monolayer. After that, the nano-sized yeast vacuole was loaded with drug DNR, and then the signals inside and outside the BBB model were detected using The Fluoroxmeter GloMax® Explorer. The DNR was essentially penetrated through the cell monolayer regulates by endocytosis via receptor-mediated endocytosis on the surface of the cell. Besides, the confocal imaging data show a significant increase in intracellular DNR fluorescence when the cell was treated with vacuole-encapsulated-drug. Our results indicated that the penetration of drugs using yeast vacuole is a promising therapeutic strategy for the treatment of neurodegenerative in general and for the cure Alzheimer’s disease in particular. Keywords : Alzheimer’s Disease, Yeast Vacuole, Blood-Brain Barrier Penetration, Drug Carrier, Neuro-degenerative Disease

127


S6404 Sutureless Transplantation of Amniotic Membrane Using a Visible Light-curable Protein Bioadhesive for Ocular Surface Reconstruction

Seong-Woo MAENG1, Tae Yoon PARK1, Ji Sang MIN2, Longyu JIN2, Woo Chan PARK2, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Ophthalmology, Dong-A University College of Medicine, Busan, Korea Corresponding Author Email : [email protected] The conjunctiva is a thin mucous membrane of the eye. Pterygium, a commonly appearing disease on the ocular surface, requires surgery to excise the conjunctiva to prevent visual deterioration. Recently, transplantation of the amniotic membrane (AM), which is the innermost membrane of the placenta, has been highlighted as an efficient method to cure conjunctiva defects due to its lack of side effects and scarring at the donor site compared to mitomycin C treatment and conjunctival autografting. However, to minimize additional damage to the ocular surface by the suturing process, AM transplantation (AMT) needs to be simplified by using a less invasive, time-saving method. In this work, we apply a visible light-curable protein bioadhesive (named FixLight) for efficient sutureless AMT. FixLight, which is based on bioengineered mussel adhesive protein, is easily applied between damaged ocular surfaces and transplanted AM, and rapidly cured by harmless blue light. Through in vivo evaluation using a rabbit model, we are demonstrating that FixLight enabled the facile, fast, and strong attachment of AM on scleral tissue and promoted ocular surface reconstruction with good biocompatibility. Keywords : ocular surface reconstructions, conjunctivas, amniotic membrane transplantations, mussel adhesive proteins, visible light-curable bioadhesives

128


S6405 Domains Fusion in Glycerol Dehydratase Stabilize the Enzyme Structure and Coenzyme B12 Interface, Which May Prolong Its Enzymatic Activity: a Mechanistic Approach

Abdul NASIR1, Tae Hyeon YOO1,2 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] The production of biodiesel has become an important strategy to address the fading petroleum reservoirs and environmental issues. Recently biological processes to produce high-value products from glycerol have drawn much attention. Glycerol dehydratase (GDHt) is involved in the dehydration of glycerol to form 3-hydroxypropanal (3-HPA), and the intermediate can be converted into 1,3-propandiol or 3-hydroxypropionic acid. The dissociation of β subunit in purification has been circumvented through the linking of α and β subunits which interestingly increases the catalytic activity of the linked enzyme. Here we took advantage of the previous knowledge about the domain fusion-based activity enhancement of the GDHt and proposed a computational model to explore the structural and conformational changes in the non-fused (WT) and fused form of GDHt (fGDHt) complex. Protein modeling, molecular mechanics, and thermodynamics computational approaches were utilized to comparatively monitor the intra- and inter-subunits interface changes. Finally, the cloning, expression, and activity assay were performed on the WT and fGDHt to evaluate the enzyme activity and its biochemical properties. Overall, our findings suggest that linker-based fusion of the α and β subunits affect the stability of GDHt and the bound coenzymes, which may delay the inactivation of the enzyme. Keywords : Glycerol dehydratase, MD simulation, Coenzyme B12, Enzyme engineering

129


S6406 Melanin Decolorization with Effective Lignin Peroxidase Under the Human Skin Environment

Hyeryeong GYE, Seung Hyun HAN, TRANG VU THIEN NGUYEN, Heeyeon BAEK, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Recently, there is a growing trend of longing for skin whitening. Therefore, many studies related to the techniques for skin whitening are being conducted, and representative of them are blocking UV or inhibiting the tyrosinase and decomposing or decolorizing melanin that has already been produced and accumulated at the skin. However, because of the side effects caused by chemical reagents inhibiting tyrosinase, currently, studies on melanin decolorization by enzymes are taking attention as alternative owing to less toxic and selective degrading/decoloring melanin. Lignin peroxidase used in this study is known to have enough higher redox potential than other peroxidases to decolorize melanin. Being applied to the human skin as whitening cosmetics, the stability and activity of lignin peroxidase at pH 5.5, which is close to the normal pH of the human skin need to be high. In addition, the activity and stability of lignin peroxidase under human body temperature around 35~37 degrees Celsius need to be maintained for the actual use of whitening cosmetics. So, not only using the enzyme LiPH8 that prepared in our previous report, in this study, but also the other different lignin peroxidase isozymes were expressed and purified as recombinant enzyme and selected with the longest stability and highest activity under human skin environment. Keywords : Melanin, Decolorization, Lignin peroxidase, Skin whitening

130


S6407 Improving Oxidative Stability of Baeyer-villiger Monooxygenase from Thermobifida fusca by Engineering Enzyme Access Tunnel

So-Yeon PARK, Myeong-Ju KIM, Eun-Ji SEO, Jeong-Hoo LEE, Jin-Byung PARK Department of Food science and Engineering, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Baeyer-Villiger monooxygenases (BVMOs) are flavoproteins that catalyze the oxidation of ketones into esters using NADPH and oxygen [1]. BVMOs can be applied for the production of high-value fine chemicals such as bioplastic monomers and chiral pharmaceutical intermediates [2-4]. Unfortunately, the BVMOs generally have low stability because H2O2 can be formed from decay of the peroxyflavin intermediate [1]. In this study, we established a rational approach based on in silico analysis of the enzyme structure. A phenylacetone mono-oxygenase variant from Thermobifida fusca (PAMO_C65D), which has NADPH oxidation activity, was used as a model system. By using Caver and Protein Energy Landscape Exploration (PELE) program, we predicted the H2O2 migration path in the PAMO and identified the hot-spot residues in tunnels to improve oxidative stability. The triple mutants (i.e., PAMO_C65D/M446I/Y495I) showed ca. 6-fold greater H2O2 resistance as compared to the PAMO_C65D. This study will contribute to engineering of H2O2 resistant enzymes. Keywords : Baeyer-Villiger monooxygenases, oxidative stability, H2O2 migration path References 1. Fürst, Maximilian JLJ, et al., ACS Catalysis, 9, 12 (2019). 2. Zhang, Wuyuan, et al., Nature communications, 11,1 (2020). 3. Cha, H.-J., S.-Y. Hwang, et al., Angewandte Chemie. International Edition, 59 (2020). 4. Kim, Tae-Hun, et al., ACS Catalysis, 10, 9 (2020).

131


S6408 Versatile Formate Production Through Enzyme-catalyzed Carbon Monoxide Hydration Reaction

Jinhee LEE, Ho Won HWANG, Suk Min KIM, Sung-heuck KANG, Byoung Wook JEON, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Carbon monoxide is hugely emitted from industrial processes as a part of waste gas in modern age. This research suggests the CO (carbon monoxide) hydration reaction which works at room temperature without additional energy supply and even produce versatile formate. This reaction is catalyzed by the combined action of CODH (CO dehydrogenase), FDH (formate dehydrogenase) and electron mediator. The CODH oxidizes CO to CO2 (carbon dioxide) through water split and yields electrons which subsequently used to reduce electron mediators. Then, the reduced mediators transfer electrons to FDH catalyzing the CO2 to formate. Isotope analysis of 13CO and D2O supports the production of formate from CO. As a result, no additional energy is necessary because CO was the sole source of electron as well as CO2. Furthermore, this reaction system was robust to produce over 1 M of formate and stable reusability was shown with real LDG (Linz-Donawiz converter gas) from Hyundai Steel without any pretreatment. Keywords : carbon monoxide, formate, carbon monoxide dehydrogenase, formate dehydrogenase

132


S6409 Engineering of Glyoxylate Carboligase from Escherichia coli K-12 for the Production of Glycolaldehyde from Formaldehyde

Ye-Na KIM1, Hye-Jin JO1, Hanna YU1, Huijin CHEON1, Jun-Hong KIM2, Jeong-Sun KIM2, Jin-Byung PARK1 1Department of Food science and Engineering, Ewha Womans University, Seoul, Korea, 2Department of Chemistry, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Formaldehyde (HCHO) is an emerging C1 source, because it could be prepared from CH4, CH3OH, CO, and CO2 by biological and/or chemical means [1]. Here, we have examined the glyoxylate carboligase from Escherichia coli K-12 (EcGCL) for the condensation of two molecules of formaldehyde into one molecule of glycolaldehyde. The EcGCL showed a catalytic efficiency (kcat/KM) of 16 M−1·s−1. The catalytic efficiency was enhanced by engineering of the active site and substrate entrance site based on crystal structures and substrate docking simulation. One of the variants (i.g., EcGCL_R484M/N283Q/L478M) exhibited the catalytic efficiency of 33 M−1·s−1. This value is ca. 5-fold greater than that of the glycolaldehyde synthase, which was previously reported to catalyze the reaction [2]. This study will contribute to synthesis of the industrially relevant C2 chemicals (e.g., glycolaldehyde, glycolic acid, ethylene glycol, and ethanol amine [3]) from formaldehyde in an environment-friendly way. Keywords : Formaldehyde, carboligating activity, EcGCL, Glycolaldehyde References 1. S. Desmons, R. Fauré, S. Bontemps.ACS Catalysis,9(10), 9575-9588(2019). 2. X. Lu, Y. Liu, Y. Yang, S. Wang, Q. Wang, X. Wang at al.,Nature communications.10(1), 1-10(2019). 3. L. Salusjärvi, S. Havukainen, O. Koivistoinen and M.Toivari, Applied Microbiology and Biotechnology.

103(6), 2525-2535(2019).

133


S6410 Molar-scale Formation of Formate from CO2 in a Reaction Catalyzed by a Noncanonical Formate Dehydrogenase

Byoung Wook JEON1, Yoonyoung HEO2, Junsun PARK3, Jang Hyuk AHN1,4, Jun Hee LEE1,4, Jungki RYU1,4, Soung-Hun ROH3, Hyung Ho LEE2, Yong Hwan KIM1 1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea, 2Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Korea, 3Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea, 4Department of Energy Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Electrochemical reduction of CO2 to value-added chemicals is a challenging but urgent task to mitigate global climate crisis. Despite the high turnover frequency and a near-unity selectivity, poor oxygen tolerance and unfavorable reversibility of biocatalysts hampered the practical application in CO2 conversion. Here, we present the oxygen-tolerant formate dehydrogenase from Methylobacterium extorquens AM1 (MeFDH1) with superior activity towards CO2 reduction to produce molarity scale of formate. In electrochemical properties, the initial formate productivity was increased from 2.43 (± 0.15) to 20.17 (± 2.03) mM/h with approximately 100 % of the Faradaic efficiency (FE) in the applied potential from -0.014 to -0.364 V. To demonstrate this significant activity for CO2 reduction, analysis for enzyme kinetics was investigated. The kinetic equilibrium constant (Keq) between CO2 reduction and formate oxidation of MeFDH1 was about 63 while most of the reported FDHs have Keq values on the scale of 10-4 to 10-2. For the long-term stability of CO2 reduction, the immobilization improved the long-term formate productivity with a near-unity FE: the maximum formate concentration of 1.74 M after 216 h and final FE of 100.16 %. Cryo-EM structure of MeFDH1 and Density-functional theory calculation was supported to explain the O2 tolerance and catalytic properties of MeFDH1, comparing with other FDHs. Keywords : CO2 reduction, formate, formate dehydrogenase, electrochemical, O2 tolerance, W-bis-MGD

134


[S6-5]

부문위원회 및 학생세션 V

135


S6501 The Efficient Carbon Monoxide Removal Using Engineered Carbon Monoxide Dehydrogenase

Sung-heuck KANG1, Suk Min KIM2, Jinhee LEE2, Ji-Sook HAHN1, Yong Hwan KIM2 1The School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 2School of Energy and Chemical Engineering, UNITS, Ulsan, Korea Corresponding Author Email : [email protected] The carbon monoxide, which is one of major pollutant, is a toxic gas that cause health hazard. For biological removal of carbon monoxide, carbon monoxide dehydrogenase (CODH), which is the only enzyme that oxidize carbon monoxide to nontoxic carbon dioxide, could be a powerful candidate. For this reaction, CODH requires mediator which could act as electron acceptor for oxidation of carbon monoxide. Among various CODH, anaerobic CODHs, which show high reaction rate, could be a powerful candidate for efficient removal of carbon monoxide. However, Michaelis constants for mediator of anaerobic CODHs are generally high compare to the constants for carbon monoxide. Because of this, high concentration of mediator is required to show sufficient reaction rate, and expensive price of mediators became the barrier that block the industrial application of anaerobic CODH for oxidation of carbon monoxide. So improving Micaelis constant for mediator CODH is crucial for economically plausible oxidation of carbon monoxide. In this study, we have discovered putative binding site of mediator in CODH and mutants which showed improved kinetic constants are found experimentally. Keywords : CO, CODH, Micaelis constant, docking simulation

136


S6502 Lab Evolution of Non-heme Alkane Monooxygenase with in vivo Screening System

Hee-Wang YOO1,2, Jin Young KIM1,2, Beom Gi PARK2,3, Hyungdon YUN4, Byung-Gee KIM1,2,3,5,6 1Interdisciplinary Program of Bioengineering, Seoul National University, Seoul, Korea, 2Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea, 3School of Chemical and Biological Engi-neering, Seoul National University, Seoul, Korea, 4Department of Systems Biotechnology, Konkuk University, Seoul, Korea, 5Bio-MAX/N-Bio, Seoul National University, Seoul, Korea, 6Institute for Sustainable Development (ISD), Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Enzyme engineering is conducted by many groups to find better enzymes. Screening methods for selecting better enzymes include in vitro methods of purifying enzymes and in vivo screening for selection within cells. In vivo screening has the advantage of being able to simultaneously check the optimal expression and activity of enzymes in cells. In this study, the evolution of non-heme alkane monooxygenase was progressed through in vivo screening. Library of monooxygenase was constructed with error-prone PCR. Here, we construct in vivo screening system with PAR promoter and FadR system to select for high-performing variants.1 Also, since FadR from E. coli cannot binding with lauroyl-CoA we introduced FadR from Thermus thermophilus and putative binding sequence of FadR from Thermus thermophilus in PAR to screening toward dodecanoic acid. We apply this system to express TetA with PAR promoter when dodecanoic acid was produced by mono-oxygenase from dodecane to able growth in the LB media with tetracycline. We demonstrate about 2-fold enhanced production of 1-dodecanol and dodecanoic acid from dodecane. Keywords : In vivo screening, Error-prone PCR, FadR, Tetracycline resistance gene References 1. Xiao, Y.; Bowen, C. H.; Liu, D. and Zhang, F., Nature Chemical Biology 12, 339(2016).

137


S6503 Molecular Characterization of a Thermostable Phospholipase A2 from Sciscionella marina

Seung-Yeon KWON1, Yeeun PARK1, Hyo-Ran LEE1, Jeong-Hye YOON1, Se-Yeun HWANG1, Hee-Jeong CHA1, Sun-Shin CHA2, Jin-Byung PARK1 1Dept. of Food Science and Engineering, Ewha Womans University, Seoul, Korea, 2Dept. of Chemistry and Nanosicence, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Phospholipase A2 (PLA2) is a lipolytic enzyme, which is widely used for the production of lysolecithins and enzymatic degumming of edible oils [1]. However, its catalytic activity and thermal stability are not high enough for the industrial applications. Therefore, new PLA2s were explored in the pool of marine micro-organisms which are known to survive in harsh environments. We discovered a new PLA2 from Sciscionella marina (Sm-PLA2). The X-ray crystallography study revealed that the enzyme is composed of five helixes containing two disulfide bonds and one Ca2+-binding site, which is similar to that of the PLA2 from Streptomyces violaceoruber (Sv-PLA2) [2]. Remarkably, the enzyme, isolated after expression in Escherichia coli [3], was thermostable up to 50oC. This is significantly greater than that of the Sv-PLA2 [2] and commercial porcine pancreatic PLA2. The biotransformation performance was also examined. The biotransformation of 200 g/L soybean lecithin (purity: ca. 77%) led to formation of lysolecithin and fatty acids to a conversion of over 80%. This study will contribute to the industrial application of PLA2s. Keywords : Phospholipase A2, Lysophospholipid, Thermostable PLA2, Calcium-dependent enzyme, Crystal structure References 1. S. Cerminati, L. Paoletti, A. Aguirre, S. Peirú, H. G. Menzella, and M. E. Castelli, Appl. Microbiol.

Biotechnol. 103, 2571-2582 (2019). 2. M. Sugiyama, K. Ohtani, M. Izuhara, T. Koike, K. Suzuki, S. Imamura, and H. Misaki, J. Biol. Chem.

277(22), 20051-20058 (2002). 3. H. J. Lee, A. R. Cho, Y. J. Hwang, J. B. Park, and S. K. Kim, J. Microbiol. Biotechnol. 30, 1244-1251 (2020).

138


S6504 Improvement of Solubility and Activity of Tobacco Etch Virus (TEV) Protease by Multiple Mutations

Heejin NAM1, BeomJeung HWANG2, Deog-Young CHOI3, Sooim SHIN1,4, Moonsung CHOI5,6 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju, Korea, 2Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea, 3InThera INC, Seoul, Korea, 4Department of Bioengineering and Biotechnology, College of Engineering, Chonnam National University, Gwangju, Korea, 5Department of Optometry, College of Energy and Biotechnology, Seoul National University of Science and Technology, Seoul, Korea, 6Convergence Institute of Biomaterials and Bioengineering, Seoul National University of Science and Technology, Seoul, Korea Corresponding Author Email : [email protected], [email protected] TEV protease is a 27 kDa catalytic domain of nuclear inclusion a (NIa) polyprotein from tobacco etch virus (TEV). TEV protease recognizes a specific amino acid sequence, ENLYFQG/S, and cleaves the peptide bond between Q and G/S. The catalytic site of the domain consists to three residues: His46, Asp81, and Cys151. In spite of substrate specificity, it is hard to use directly in experiment due to poor solubility during expression and inhibition of catalytic activity by auto-cleavage. Here, we isolated soluble S219V/N mutant TEV proteases in a background of T17S/N68D/I77V mutation. The activities of as-isolated mutants were measured by SDS-PAGE and by monitoring the fluorescence using fluorescent substrate. As a result, both mutants clearly cut off the substrate, and the activities of both mutants were highly improved compared to the control without mutation. Also, with the background mutation, kcat of S219N mutant was 2-fold faster than that of S219V without the change of Km value. These results indicate that the mutations of this study can enhance the activity of TEV protease. Keywords : TEV protease, multiple mutations, improvement of activity References 1. Nam H, Hwang BJ, Choi DY, Shin S, Choi M. Tobacco etch virus (TEV) protease with multiple mutations

to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity. FEBS Open Bio. 2020 Apr;10(4):619-626.

139


S6505 Immunity-enhancing Effect of Vacuoles Derived from Saccharomyces cerevisiae Through Regulation of Inflammatory Mediators and Cytokines

Su-Min LEE, Ra-Mi PACK, Jiho MIN Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Vacuoles extracted from S. cerevisiae are counterparts of mammalian lysosomes the digestive system of cells, and contain proteolytic enzymes, destroying foreign substances that have been introduced through phagocytosis, and digesting the useless components of the cells themselves. Vacuoles promote the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines, and by activating phagocytes, the immune response is enhanced. Besides, after treatment with TLR4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced, and inducible nitric oxide enzyme (iNOS) protein was also significantly reduced. As a result of confirming the activation of TLR4 using western blot and Immuno-fluorescence (IF), TLR4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Thus, the effect of vacuoles in RAW 264.7 cells confirmed its potential as an immune-enhancing agent by increasing immune mediators and phagocytosis, suggesting that TLR4 is the most representative TLR capable of recognizing vacuoles. Keywords : Vacuole, Saccharomyces cerevisiae, Immune response, Cytokine, immunostimulatory activity

140


S6506 Self- and Co-assemble Behavior of Elastin-like polypeptide (ELP) Diblock Copolymers Containing a Charged Moiety

Jeong-Wan CHOI, Jong-In WON Department of Chemical Engineering, Hongik University, Seoul, Korea Corresponding Author Email : [email protected] Elastin-like polypeptides (ELPs) are stimulus-responsive protein-based biopolymers, and some ELP block copolymers can assemble spherical nanoparticles with thermo-sensitivity. In this study, two different ELP diblock copolymers composed of a hydrophobic and a charged moiety each were synthesized, and the dependence of their physical properties on pH, temperature, and salt concentration was investigated. A series of analyses revealed that hydrophobic core micelles could be generated by self-assembly in response to a change in their surroundings, and that they would not be aggregated due to the repulsive force between the same charged molecules. In addition, we demonstrated that self-assemble behavior was closely dependent on the character of a charged amino acid and that complex coacervate core micelle (C3M) was stably generated when two oppositely charged diblock copolymers were mixed. Keywords : Elastin-like polypeptide (ELP), Charged diblock copolymer, Self-assembly, Co-assembly, Complex Coacervate Core Micelle (C3M)

141


S6507 Possibilities and Challenges for Utilizing Cellulosic Biomass by an Engineered Kluyveromyces marxianus

Deok-Ho KWON1,2,3, Suk-Jin HA1,2,3 1Department of Bioengineering and Technology, College of engineering, Kangwon National University, Chuncheon, Korea, 2Department of Biohealth-machinery convergence engineering, Kangwon National University, Chuncheon, Korea, 3Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Thermotolerant yeast Kluyveromyces marxianus KCTC17694 was developed through a directed evolution and random mutagenesis to achieve an efficient xylose fermenting mutant strain. Xylose consumption, ethanol production and ethanol yield by the mutant K. marxianus 17694-DH1 were 68%, 290% and 130% improved, respectively as compared to those from the parental strain. RNA-Seq based transcriptome analysis showed that alcohol dehydrogenases and glucose transporters were up-regulated while TCA cycle involved enzymes were down-regulated in K. marxianus 17694-DH1. Mutant sugar transporter ScGAL2-N376F was overexpressed in K. marxianus 17694-DH1 for improved xylose utilization. The transformant K. marxianus 17694-DHG1, exhibited a 0.49 g/l/h xylose consumption rate and a 0.09 g/l/h ethanol production rate, representing 151% and 242% improved results, respectively, as compared to those from K. marxianus 17694-DH1. Under the optimized fermentation condition by K. marxianus 17694-DHG1, xylose consumption, ethanol production and ethanol yield increased by 238%, 532% and 89%, respectively, compared to those from wild type K. marxianus KCTC17694. Keywords : Kluyveromyces marxianus, ScGAL2-N376F, Ethanol, cellulosic biomass, directed evolutionary approach, mutation References 1. Deok-Ho Kwon, Jae-Bum Park, Eunsoo Hong and Suk-Jin Ha. Bioprocess Biosyst Eng. 42, 63-70(2019). 2. Deok-Ho Kwon, Joong-Hee Park, Deok Yeol Jeong, Jae-Bum Park, Dong-Min Park, Kyoung-Gon Kang,

Seo-Young Choi, Soo Rin Kim and Suk-Jin Ha. KSBB. 34, p243-247(2019). 3. Deok-Ho Kwon, Saet-Byeol Kim, Jae-Bum Park, Suk-Jin Ha. J Microbiol Biotechnol. 30(12):1944-

1949(2020).

142


S6508 Comparative Genomics Determines Strain-dependent Secondary Metabolite Production in Streptomyces venezuelae Strains

Woori KIM1, Namil LEE1, Soonkyu HWANG1, Yongjae LEE1, Jihun KIM1, Hyeseong KIM1, Suhyung CHO1, Bernhard PALSSON2,3,4, Byung-Kwan CHO1,4,5 1Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 3Department of Pediatrics, University of California San Diego, La Jolla, CA, USA, 4Novo Nordisk Foun-dation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark, 5Intelligent Synthetic Biology Center, Daejeon, Korea Corresponding Author Email : [email protected] Streptomyces venezuelae is well known to produce various secondary metabolites (SMs). Although many strains have been classified as S. venezuelae species, genomic differences and diversity in SM production between the strains have never been compared. Here, through completion and comparison of high-quality genome sequences of three S. venezuelae strains harboring chloramphenicol and jadomycin biosynthetic gene clusters (BGCs), we revealed that the three strains share more than 85% of total genes and most of the SMBGCs. Despite such conservation, the strains produced different amounts of chloramphenicol and jadomycin, indicating differential regulation of SM production at the strain level. By comparing the two BGCs among the three strains, we found sequence variations in many genes, the non-coding RNA coding regions, and binding sites of regulators. We anticipate that these genome sequences of closely related strains would serve as useful resources for understanding the complex secondary metabolism. ** Supported by Bio & Medical Technology Development Program (2018M3A9F3079664 to B.-K.C.) through the NRF, funded by the MSIT, and the Novo Nordisk Foundation (NNF10CC1016517) Keywords : Comparative genomics, Streptomyces venezuelae, chloramphenicol, jadomycin, biosynthetic gene cluster

143


S6509 Development of an Efficient Brown Macroalgae Bioprocess Using a Novel Microbial Host; Vibrio sp. dhg

Sung Hwa WOO1, Hyun Gyu LIM1, Dong Hun KWAK2, Sungwoo PARK3, Snag Woo SEO3, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea, 3School of Chemical and Biological Engineering, Institute of Chemical Process, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Although brown macroalgae have enormous potential as an alternative feedstock, their utilization by conventional microbial platforms has been limited due to the inability to metabolize one of the major carbohydrates, alginate. Here, we isolated Vibrio sp. dhg, a novel, fast-growing bacterium that can efficiently assimilate alginate. Based on the systematic characterization of the genomic information of Vibrio sp. dhg, we were able to develop a genetic toolbox for its engineering, and successfully demonstrated its ability to rapidly produce a broad spectrum of chemicals (ethanol, 2,3-butanediol, and lycopene) from brown macroalgae sugar mixtures with high productivity and yield. Collectively, the Vibrio sp. dhg strain is a powerful platform for the conversion of brown macroalgae sugars whose usage will dramatically accelerate the production of value-added biochemicals. Keywords : Vibrio, brown macroalgae, alginate , mannitol, ethanol, 2,3-butanediol, lycopene, metabolic engineering

144


S6510 Metabolic Engineering of Klebsiella oxytoca for High Level Production of 2,3-Butanediol

Ji Won CHA, Ki Jun JEONG Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] With economic and environmental benefits, much research has been focused on conversion of biomass-derived carbohydrates to value-added products [1, 2]. Klebsiella oxytoca is a promising host for the production of 2,3-Butanediol (2,3-BDO) because of its ability to consume a variety of carbon sources [3]. However, K. oxytoca exhibits inefficient utilization of mixed sugars in presence of glucose due to carbon catabolite repression. Here, introduction of xylose symporter and adaptive laboratory evolution (ALE) were performed to achieve the higher consumption rate of xylose, which lead to higher cell growth rate, sugar consumption rate, and 2,3-BDO productivity. Several mutations caused by ALE were identified by whole genome resequencing. Also, it was found that central carbon metabolism was upregulated through transcriptome analysis. To increase 2,3-BDO production, metabolic engineering strategies were employed to the ALE strain. Finally, the engineered strain showed high production of 2,3-BDO via efficient consumption of mixed sugars derived from lignocellulosic biomass (LCB). Taken together, the engineered K. oxytoca is a potential industrial strain for 2,3-BDO production from LCB. Keywords : Klebsiella oxytoca, adaptive laboratory evolution, lignocellulosic biomass hydrolysate, metabolic engineering, 2,3-Butanediol References 1. F. Zhu, C. Wang, K.-Y. San and G. N. Bennett, Ind. Microbiol. Biotechnol. 47(2), 223-232 (2020). 2. A. Karnaouri, G. Asimakopoulou, K. G. Kalogiannis, A. Lappas and E. Topakas, Biomass Bioenergy. 140,

105672 (2020). 3. Y. J. Kim, H. W. Joo, J. Park, D. K. Kim, K. J. Jeong and Y. K. Chang, Biotechnol. Prog. 31(6), 1669-1675

(2015).

145


S6511 Synthetic Protein Quality Control to Enhance Full-length Translation in Bacteria

Jina YANG1,2, Yong Hee HAN3, Jongwon IM1, Sang Woo SEO1,2,3,4,5 1School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 2Institute of Chemical Processes, Seoul National University, Seoul, Korea, 3Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, Korea, 4Bio-MAX Institute, Seoul National University, Seoul, Korea, 5Institute of Engineering Research, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated mRNAs, inevitably generating non-functional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA–RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein, and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6-fold to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant proteins production but also efficiencies of metabolic pathways. Keywords : protein quality control, ProQC, RNA circularization, RNA–RNA hybridization, selective translation References 1. J. Yang, Y. H. Han, J. Im and S. W. Seo, Nat. Chem. Biol. (2021).

146


S6512 Fine-tuning System for the Bacteria Gene Expression

Seung-Woon JUNG1, Jinho YEOM1, Dokyun NA1, Sang Yup LEE2, Seung Min YOO1 1School of Integrative Engineering, Chung-Ang University, Seoul, Korea, 2Department of Chemical and Biomolecular Engineering (BK21 program) and BioProcess Engineering Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Fine-tuning gene expression is one of the most important things in both synthetic biology and metabolic engineering. The optimization of the gene expression levels to construct a genetic circuit or microbial cell factories is a major challenge. We created the synthetic toolboxes for fine-tuning gene expression regardless of the coding sequence in Escherichia coli. Firstly, a leader sequence was designed using the computational model(1) to diminish the structural effects of the coding sequence on the secondary structure of the translation initiation region of mRNA. The optimized leader sequence and the 24 expression toolboxes were combined, and their gene expression fine-tuning abilities were evaluated using four different fluorescence proteins. To show the practical applications of the tunable gene expression system, a synthetic genetic circuit was designed for exhibiting three different states of fluorescence depending on the protein expression levels. Also, the expression levels of key metabolic enzymes were modulated using the tunable gene expression system and as a result, the production of metabolites was enhanced without extensive efforts. Furthermore, this system has been applied and verified to the other strain Pseudomonas putida to prove its general practicality. Consequently, this system can be usefully adapted for applied biological engineering, especially in synthetic biology and metabolic engineering. Keywords : Gene expression, Translation regulation, Fine-tuning, Synthetic biology, Metabolic engineering References 1. Yoo, S. M., Jung, S. W., Yeom, J., Lee, S. Y., & Na, D. (2020). Tunable Gene Expression System

Independent of Downstream Coding Sequence. ACS Synthetic Biology, 9(11), 2998-3007.

147


[S6-6]

부문위원회 및 학생세션 VI

148


S6601 Using the Interaction Between Staufen 1 and ncRNAs to Predict DNMTis Sensitivity in Individual AML Patients

Yongsuk KU1, Joo-Hwan PARK2,3, Ryeongeun CHO1, Yongki LEE1, Hyoung-Min PARK4, MinA KIM1, Kyunghoon HUR1, Soo Young BYUN5, Jun LIU2,3, Young-suk LEE6, David SHUM5, Dong-Yeop SHIN2,3, Youngil KOH2,3, Je-Yoel CHO4, Sung-Soo YOON2,3, Junshik HONG2,3, Yoosik KIM1 1Department of Chemical and Biomolecular Engineering and KAIST Institute for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 2Department of Internal Medicine, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Korea, 3Cancer Research Institute, Seoul National University Hospital, Seoul, Korea, 4Department of Biochemistry, BK21 Plus and Research Institute for Veterinary Science, School of Veterinary Medicine, Seoul National University, Seoul, Korea, 5Screening Discovery Platform, Translation Research Division, Institut Pasteur Korea, Seongnam, Korea, 6Department of Bio and Brain Engineering, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] DNA-methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used clinically to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERVs), which can induce immune response by acting as cellular double-stranded RNAs (dsRNAs). Yet, the post-transcriptional regulation of ERV dsRNAs remains uninvestigated. Here, we find that the viral mimicry and subsequent cell death in response to decitabine require the dsRNA-binding protein Staufen1 (Stau1). We show that Stau1 directly binds to ERV RNAs and stabilizes them in a genome-wide manner. Furthermore, Stau1-mediated stabilization requires a long non-coding RNA TINCR, which enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that MDS and AML patients with lower Stau1 and TINCR expressions exhibit inferior treatment outcomes to DNMTi therapy. Overall, our study reveals the post-transcriptional regulatory mechanism of ERVs and identifies the Stau1-TINCR complex as a potential target for predicting the efficacy of DNMTis and other drugs that rely on dsRNAs. Keywords : Staufen1, double-stranded RNA binding protein, Endogenous retrovirus, TINCR, ncRNA

149


S6602 Emulsion-based Synthesis of Semi-permeable Protocells Composed of Poly(N-isopropylacrylamide)-albumin Hybrid Amphiphiles

Jinmin KIM, Seung Soo OH Materials Science & Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] By allowing transport of certain molecules, semi-permeable polymer membranes have been important to diverse applications. Because of the size-selective permeability, they can be useful for development of biosensors in clinical diagnosis; body fluids, contains not only small analytes but also many other large molecules, of which potential fouling impairs the sensing performance. If the membranes are not permeable to the large molecules, encapsulated molecular probes would maintain the capability. In this work, we propose the use of protein-polymer conjugates to build semi-permeable proteinosomes that can obstruct passage of plasma proteins. For the protein-polymer conjugates, we chose an albumin, the major protein in blood. In constructing the proteino-somes, we invented a self-assembly method using the amphiphilic structure of the conjugates comprised of hydrophobic PNIPAm and hydrophilic albumin protein. Based on oil-in-water emulsion, we successfully synthesized the proteinosomes. encapsulating aptasensors into the semi-permeable proteinosomes, we demon-strate that the resulting protocells can serve as high-performance biosensors, even in undiluted blood and serum. Keywords : Proteinosome, Aptasensor, Serum albumin, Oil-in-water emulsion References 1. Dzieciol, Alicja J., and Stephen Mann. Chemical Society Reviews 41.1 (2012): 79-85. 2. Huang, Xin, et al. Nature communications 4.1 (2013): 1-9.

150


S6603 Hypoxia Responsive Nanoparticles-based Drug Delivery System Capable of Combinational Chemo-photodynamic Therapy

Hoomin LEE1, Debasish KUMAR2, Kwanwoo KIM1, Suheon KIM1, Eunsu KIM1, Sun Chul KANG2, Vivek K. BAJPAI3, Yun Suk HUH1 1Department of Biological Sciences and Bioengineering, Nano Bio High-Tech Materials Research Center, Inha University, Incheon, Korea, 2Department of Biotechnology, Daegu University, Gyeongsan, Korea, 3Department of Energy and Materials Engineering, Dongguk University, Seoul, Korea Corresponding Author Email : [email protected] Hypoxia due to improper supply of oxygen is characteristic of various diseases, including cancer and vascular disease. Hypoxia condition makes cancer tissues resistant to photodynamic therapy and some chemotherapy. Therefore, hypoxia is a problem that we must overcome when using a drug delivery system, and it will also be a useful tool. In this work, we developed HSA-based nanoparticles, RGD-PEG/Ce6-Azo/PTX-HSA nanoparticles (RGD-PEG/Ce6-Azo/PTX-HNPs), in which the chemotherapeutic agent PTX and photosensitizer Ce6 were co-incorporated for targeted chemo-photodynamic combination cancer therapy. Azo bonds break when exposed to a hypoxia environment, releasing Ce6. We confirmed that these nanoparticles release Chlorin e6 effectively in hypoxia condition, and through this, it was confirmed that PDT effect and chemotherapeutic anticancer effect are increased. Keywords : Hypoxia, TME (tumor microenviroment), Chemotherapy, Photodynamic therapy

151


S6604 Thiol Dependent Phase Transition of Liquid Like Protein Condensates to Iron-protein Complexes During the Formation of Byssus

Hyungbin KIM, Dong Soo HWANG Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Mussel is attached to a hard substrate underwater as a sessile marine animal. To adhere to the substrate underwater, it produces proteinaceous byssus that is an extensible and tough biomaterial. The byssal proteins have been widely investigated by many researchers. DOPA(3,4-Dihydroxylphenylalanine), which is modified from tyrosine, is the key amino acid for its adhesion because the catechol moiety of DOPA can participate in the various chemical reactions such as metal coordination, and cross-linking to other amino acids. But, DOPA is easily oxidized. Therefore, it has been an important issue how DOPA maintains a reductive state. It has been investigated that DOPA can be not oxidized by using redox coupling of thiol group in cysteine. That is, mussel foot protein-1 (mfp-1), which contains 16mol% of DOPA can be coordinated to iron due to reversible sacrificial cross-links of cysteine-rich proteins such as mfp-16,17,18. Liquid liquid phase separation (LLPS) is a key phenomenon for secreting the adhesive proteins to be dense and stable. In mussel adhesion, LLPS have been discovered in mfp-1 and 3. The phase separation of mfp-1 is driven by cation-pi interaction triggered by salt especially. However, it is elusive that how the physical properties of the protein droplet are changed during the formation of byssus. Therefore, this work present how the physical properties of mfp-1 protein condensates are changed dependent on thiol during the byssus fabrication. Keywords : mussel, adhesive protein, mfp-1, liquid liquid phase separation(LLPS), thiol, coacervation, DOPA References 1. S. Kim et al, Acs Nano 11, 6764-6772 (2017). 2. T. Priemel, E. Degtyar, M. N. Dean, M. J. Harrington, Nat Commun 8, 14539 (2017). 3. E. Valois, C. Hoffman, D. G. Demartini, J. H. Waite, Langmuir 35, 15985-15991 (2019). 4. F. Jehle et al., Nat Commun 11, 862 (2020). 5. T. Priemel et al., P Natl Acad Sci USA 117, 7613-7621 (2020).

152


S6605 Development of Selective Therapeutic Agent for Acute Myeloid Leukemia Based on Engineered Vacuole

Wooil CHOI, Jiho MIN Graduate school of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Common therapy for cancer treatments are performed through radiotherapy or chemotherapy. These therapies are non-specific therapies with significant side effects. Selective therapeutics is one of spotlighted field in pharmaceutical industry. This research is concerned for studying to improve efficiency for drug delivery by target selectivity, suitability and economical production of drug delivery system based on yeast-derived vacuole. The budding yeast, Saccharomyces cerevisiae, is one of the best studied eukaryotic models. S. cerevisiae has prominent vacuole correspond to lysosome of mammalian cell. In this study, targeting peptide could bind HL-60 cell specifically was expressed on vacuolar outer membrane. The vacuoles with targeting peptide showed enhanced efficacy to HL-60 cells and economic feasibility. This study demonstrates that yeast derived vacuoles with nano-sizing and targeting ability can be used potential bio-based drug delivery system for cancer therapy. Also, this vacuole system provides potential strategies and platform for disease selective treatment through simple manipulation of vacuole. Keywords : yeast engineering, vacuole, drug delivery carrier, cancer therapy, AML References 1. Jihee Yoon, Simranjeet Singh Sekhon, Yang-Hoon Kim and Jiho Min, Mol Cell Biochem, 417 (2016). 2. Vipul Gufrati, Miriam Lee, Young-Joon Ko, Sangeun Lee, Daejin Kim, Hyungjun Kim, Sukmo Kang,

Soyoung Lee, Jinjoo Kim, Hyungsu Jeon, Sun Chang Kim, Youngsoo Jun and Sangyoung Jon, Proc Natl Acad Sci U S A, 113 (2016).

3. Christer Paul, Jan Liliemark, Ulf Tidefelt, GÖsta Gahrton and Curt Peterson, Ther Drug Monit, 11 (1989).

153


S6606 Angular-based Quantitative Assay in 3D Paper-based Analytical Device

Dong-Ho KIM, Seong-Geun JEONG, Chang-Soo LEE Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Three-dimensional Paper-based analytical devices (3D PADs) represent the remarkable potential to overcome lateral flow test or dipstick. However, recent 3D PADs still need a peripheral device such as a camera for quantitative assay. Here, we demonstrate a paper-based technique for straightforward, quantitative assay using the angle of color development. First, we fabricate 3D PADs for angular-based assay using a wax printer and laminator. 3D PAD fabrication process is achieved in two steps: the printing process and the heating process. Second, we demonstrate the angular-based assay on the 3D PAD. To investigate functionalization, we introduce a sample solution, containing bovine serum albumin (BSA), on the sample pad. Tetrabromophenol blue (TBPB), pre-absorbed on assay zone, react with BSA in the fluid. The color of TBPB changes from yellow to blue under Citric acid buffer solution (CBS). In addition, we confirm that the angle of the colored line, where the color changes from yellow to blue, depends on the concentration of BSA. This indicates this angular-based measure-ment allows to perform straightforward, quantitative assay without using an image analyzer. Keywords : Paper-based device, Quantitative assay, Albumin assay References 1. S.G. Jeong and C. S. Lee , Lab Chip 15, 1188-1194 (2015).

154


S6607 Catechol-functionalized Polysiloxane for Underwater Adhesives

Dohoon LEE, Dong Soo HWANG Division of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] The key chemical functionality present in the mfps is the postranslationally modified amino acid 3,4-dihydroxy-L-phenylalanine (DOPA). The catecholic moiety of DOPA forms strong and reversible coordination complexes with metal ions and metal oxides or undergoes covalent crosslinking with neighboring chemical moieties. Many different types of DOPA-based wet adhesives have demonstrated effective wet adhesion performance. However, most efforts using synthetic catechol-functionalized polymers have been in trouble by the oxidative instability of synthesis polymers. the orthogonal reactivity of TPFPB allows the facile one-step transformation of eugenol into a reactive, silyl-protected DOPA mimic. The silyl protecting groups are key to maintaining the stability of the catechol unit throughout the thiol–ene polymer functionalization, processing, and subsequent cross-linking reactions. Overcomes this instability, thereby enabling the preparation of mushroom structure for demanding underwater applications. Keywords : DOPA, Underwater Adhesive, Polysiloxane References 1. J. Heo, T. Kang, S.G. Jang, D.S. Hwang, J.M. Spruell, K.L. Killops, J.H. Waite, C.J. Hawker, Improved

Performance of Protected Catecholic Polysiloxanes for Bioinspired Wet Adhesion to Surface Oxides, Journal of the American Chemical Society 134(49) (2012) 20139-20145.

2. D. Lee, H. Bae, J. Ahn, T. Kang, D.G. Seo, D.S. Hwang, Catechol-thiol-based dental adhesive inspired by underwater mussel adhesion, Acta Biomater. 103 (2020) 92-101.

3. H. Yi, S.H. Lee, H. Ko, D. Lee, W.G. Bae, T.I. Kim, D.S. Hwang, H.E. Jeong, Ultra-Adaptable and Wearable Photonic Skin Based on a Shape-Memory, Responsive Cellulose Derivative, Adv. Funct. Mater. 29(34) (2019) 12.

4. L.M. Campos, I. Meinel, R.G. Guino, M. Schierhorn, N. Gupta, G.D. Stucky, C.J. Hawker, Highly Versatile and Robust Materials for Soft Imprint Lithography Based on Thiol-ene Click Chemistry, Adv. Mater. 20(19) (2008) 3728.

155


S6608 Bioengineering of PD-1-decorated Nanocages Targeting Tumor Draining Lymph Node for Promoting T Cell Activation

Hyo Dong SUNG1, Gi Beom KIM2, Gi Hoon NAM2, Eun Jung LEE1, In San KIM2, Na yeon KIM1 1Department of Chemical Engineering, Kyungpook National University, Daegu, Korea, 2Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea Corresponding Author Email : [email protected] Targeted delivery of an immunomodulatory molecule to the lymph nodes is an attractive approach to improve anti-cancer immunotherapeutic efficacy. In this study, to improve the therapeutic efficacy of the PD-1 blockade-based therapy, we designed nanocages by surface engineering to decorate a programmed cell death protein 1 (PD-1) that is capable of binding against programmed death-ligand 1 and 2 (PD-Ls). This nanocage-mediated multivalent interaction remarkably raises the binding avidity and improves the antagonistic activity, compared to soluble PD-1. Also, upon the desirable size of the nanocages for an optimal tumor-draining lymph node (TDLN)-targeting, we observed rapid draining and an increased accumulation into the TDLNs. Moreover, the interfering of PD-1/PD-Ls axis with the ultra-high affinity in the tumor microenvironment (effector phase) and the TDLNs (cognitive phase) significantly enhance the dendritic cell-mediated tumor-specific T cell activations. As a result, this successfully inhibits the tumor growth and induces complete tumor eradication in some mice. so, The delivery of immunomodulatory molecules with nanocages can be a efficient strategy to anti-tumor immunity. Keywords : Nanocages, Surface engineering, PD-1/PD-Ls blockade, Tumor-draining lymph node, Drug delivery

156


S6609 2D Graphene Oxide is Associated with Induction of Loss in Pluripotency and Triggering of Early Differentiation in Human Induced Pluripotent Stem Cells

Jiwoong HEO1, Jaewon CHOI2, Hee Ho PARK2,3, Jinkee HONG1 1Department of Chemical and Biomolecular Engineering, College of Engineering, Yonsei University, Seoul, Korea, 2Interdisciplinary Program in Biohealth-Machinery Convergence Engineering, Kangwon National University, Chuncheon, Korea, 3Department of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Recently, the dangers of micro/nano-sized plastics and carbon particles have received enormous attention. In this study, we tested the cytotoxicity of 2D graphene oxide (GO) on human induced pluripotent stem cells (hiPSCs). When the 2D graphene oxide was cultured with hiPSCs, it was confirmed that graphene oxide penetrated into the cells. When hiPSCs differentiated into cardiomyocytes in the presence of GO, early triggering of differentiation and up-regulation of the cardiomyocyte marker NKX2.5 were observed. Moreover, the increased expression of caspase 3 and intracellular ROS level were confirmed. Through this, our results demonstrate that GO induces cytotoxicity on stem cell development. Keywords : hiPSC, Cytotoxicity, EB, Apoptosis, ROS References 1. Xue, W., Xue, J., Shirmohammadi, F., Sioutas, C., Lolinco, A., Hasson, A., & Kleeman, M. J., Atmos

Environ. 222, 117088 (2020). 2. Lammel, T., Boisseaux, P., Fernández-Cruz, M. L., & Navas, J. M. Part Fibre Toxicol. 10(1), 27 (2013). 3. Balafkan, N., Mostafavi, S., Schubert, M., Siller, R., Liang, K. X., Sullivan, G., & Bindoff, L. A. Scientific

reports 10(1), 1-14.

157


S6610 Biomimetic Isolation of Affinity Peptides for Electrochemical Detection of Influenza A Virus H5N1 Antigen Ji Hong KIM1, Chae Hwan CHO1, Jae Hwan SHIN1, Da Hyeon NAM1, Hyo Jeong YANG1, Moon Seop HYUN2, Tae Jung PARK3, Jong Pil PARK1 1Department of Food Science and Technology, Chung-Ang University, Anseong, Korea, 2National NanoFab Center (NNFC), Daejeon, Korea, 3Department of Chemistry, Institute of Interdisciplinary Convergence Research, Research Institute of Chem-Bio Diagnostic Technology, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] The influenza A virus H5N1(A/H5N1) caused contagious diseases to both of poultry and human with high fatality rate. Therefore it needs to develop early, fast, sensitive and specific diagnosis method. In this study, we have developed an electrochemical biosensor coated with affinity peptides, which can detect the A/H5N1 antigen, hemagglutinin (HA). Using phage display, particular peptide-displaying phage particles (GHPHYNNPSLQL) which had specific affinity to HA were identified. Based on the molecular structure studies, newly designed six peptides (IFA BP1 - IFA BP6) were chemically synthesized and their biding interaction with HA were observed by using square wave voltammetry (SWV). Isothermal calorimetry (ITC) and surface plasmon resonance (SPR) were also carried out to know more about biomolecular interaction of IFA BP1 peptide sensor. Furthermore, our electrochemical sensor could successfully detect HA spiked in sample of human plasma compared with commercial ELISA method. These results suggest that our developed affinity peptide-based sensor system could be potentially applied diagnostic tool for rapid and effective detection of the A/H5N1 antigen. Keywords : Influenza A virus H5N1, Hemagglutinin, Phage display, Affinity peptide, Electrochemical peptide sensor References 1. J. H. Kim, B. Koh, D. G, Ahn, S. J. Lee, T. J. Park and J. P. Park, Bioelectrochemistry, 137, 107670 (2021). 2. C. H. Cho, J. H. Kim, D. K. Song, T. J. Park and J. P. Park, Biosensor and Bioelectronics, 142, 111482

(2019). 3. S. Hassanpour, B. Baradaran, M. Hejazi, M. Hasanzadeh, A. Mokhtarzadeh and M. Guardia, Trends in

Analytical Chemistry, 98, 201-215 (2018).

158


S6611 Identification of a VOC Biomarker for Early Diabetes Detection Using a Cell-based Disease-model System and Development of Olfactory Receptor-based Nanobiosensor

Seongyeon CHO1, Manki SON2, Dong-guk CHO3, Seunghun HONG3, Tai Hyun PARK1,2,4,5, Hwi jin KO4,5

1School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 2Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Korea, 3Department of Physics and Astronomy, Institute of Applied Physics, Seoul National University, Seoul, Korea, 4BIO-MAX, Seoul National University, Seoul, Korea, 5Institute of Chemical Processes, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Type 2 diabetes is a severe metabolic disease and has become an important issue in preventing serious complications, so it is necessary to identify biomarkers for an early and accurate diagnosis of diabetes. Herein, to identify the biomarker in in vitro system, muscle and adipose cells were exposed to palmitic acid to generate insulin-resistant cells. Volatile organic compounds (VOCs) were derived from both head space of insulin resistant and normal cells and analyzed using SPME-GC/MS. Among the detected VOCs, 17-octadecynoic acid (17-ODYA) was selected as a biomarker for the detection of insulin resistance. 17-ODYA can cause inflammation, and many recent studies have reported that inflammation triggers insulin resistance in muscle and adipose tissues. An olfactory receptor, OR51A4, that specifically bind to 17-ODYA was screened and expressed on the surface of HEK-293 cells to generate nanovesicles. Nanobiosensor was fabricated using carbon nanotube field-effect transistors and nanovesicles for early diagnosis of diabetes. The results showed that the nano-biosensor was able to detect 10 fM of 17-ODTA in a dose-dependent manner and selectively distinguish it from biomarkers in several other diseases. This nanobiosensor is expected to be a promising tool for early diagnosis of diabetes. Keywords : Diabetes, Biomarker, Volatile organic compound, Olfactory receptor, Nanovesicle, Nanobiosensor References 1. H. J. Ko et al., Diabetes 58, 2536 (2009). 2. P. Li et al., Nat. Med. 21, 239 (2015). 3. J. H. Lim et al., Adv. Healthc. Mater. 3, 360 (2014).

159


S6612 Enzymatic Synthesis of Soluble Melanin Nanoparticle Under Acidic Conditions Using Tyrosinase

Hyun KIM1, Uk-Jae LEE1, Hanbit SONG1, Jeongchan LEE1, Byung-Gee KIM1,2,3,4 1School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 2Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea, 3Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea, 4Institute for Sustainable Development(ISD), Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Melanin, a biocompatible heterogeneous polymer, has recently been applied in the biomaterial field due to the ability of metal chelation, paramagnetic, free radical generation, and scavenging. Most of the melanins synthesized by enzymes, including isolated from natural organisms, are insoluble. Hence, a further application is limited without additional purification steps. For the general application of melanin, the chemical synthesis method has been regularly used owing to the simple process compared to enzymatic synthesis. But the chemical method can cause environmental issues, greener synthesis method is still needed. In this study, we synthesized soluble melanin under acidic conditions using a one-step tyrosinase reaction. The enzymatically synthesized melanin is examined by mass spectrometer, UV and IR spectrometry, scanning electron microscopy method. Structure and physicochemical analysis revealed that acidic conditions could successfully reduce an uncontrolled crosslinking between amine and quinone and thus inhibit the formation of the agglomerate. As a result, we showed potential applications on enzymatically synthesized soluble melanin as a biomedical material. Keywords : Melanin, Soluble, Tyrosinase, Acidic condition References 1. Hyeoncheol Francis Son, Sang-Hyuk Lee, Seul Hoo Lee, Hyun Kim, Hwaseok Hong, Uk-Jae Lee, Pyung-

Gang Lee, Byung-Gee Kim, and Kyung-Jin Kim, ACS catal. 8, 11, 10375-10382 (2018). 2. Florian Ponzio, Julien Barthes, Jeróme Bour, Marc Michel, Philippe Bertani, Joseph Hemmerle, Marco

d’Ischia, and Vincent Bal, Chem. Mater. 28, 13, 4697-4705 (2016).

160


[S6-7]

부문위원회 및 학생세션 VII

161


S6701 The Interrelationship Between Entering the Haematococcus lacustris Biomass Growth Stage and Algal Phycosphere Bacteria Community Structure Changes

Sang-Ah LEE1,2, Minsik KIM1, Chi-Yong AHN1,2 1Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Department of Environmental Biotechnology, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Haematococcus lacustris NIES144 could be a one of the anti-oxidant feedstock such as carotenoid pigment (astaxanthin). However, the slow growth of this alga is the major obstacle for producing industrial scale of astaxnathin (Lee et al., 2019). To solve this weak point, algal growth promoting bacterial strains that were co-existed in the algal phycosphere were identified by NGS. This study focused on defining algal growth positive bacteria community modules, and major abundant taxa that had a positive correlation with algal growth were isolated (Sphingomonas hankookenesis, Paenarthrobacter ureafaciens and three species of Microbacterium sp.). Among the isolated bacteria, S. hankoonenesis was the key-role strain for algal growth. Through this study, algal growth promoting effect was verified by positive correlation bacteria with algal growth. For the astaxanthin industrialization, this metagenomic data analysis will give solving the slow growth of Haemato-coccus lacustris some insight. Keywords : NGS, Haematococcus, bacterial community

162


S6702 Chemically Modified Phage Sensor for Sensitive Ovomucoid Detection

Jae Hwan SHIN, Ji Hong KIM, Chae Hwan CHO, Da Hyeon NAM, Hyo Jeong YANG, Jong Pil PARK Department of Food Science and Technology, Chung-Ang University, Anseong, Korea Corresponding Author Email : [email protected] Ovomucoid is one of the allergenic protein derived from egg or egg-based food resources. A typical symptom of food allergy is anaphylaxis, which can threaten the patient’s life in severe cases. Therefore, it is important to accurately and sensitively detect allergens in foods using reliable detection system over conventional methods. To achieve this purpose, we used biopanning of polyvalent phage display to screen affinity peptides capable of binding to ovomucoid. Throughout for rounds of panning, potential affinity peptides were successfully identified and their binding affinities were measured by ELISA assay. After selection of affinity peptides, phage-displayed peptides were chemically modified and immobilized on the gold sensor layer. Finally, the performance of the phage-based sensor was evaluated by square wave voltammetry (SWV). Further detailed results (selection, characterization of binding affinity) will be presented in this talk. Keywords : Chemically modified M13 phage, Ovomucoid, ELISA assay, Square wave voltammetry (SWV) References 1. C. H. Cho, J. H. Kim, D. K. Song, T. J. Park and J. P. Park, Biosensors and Bioelectronics. 142, 111482

(2019). 2. A. S. Torralba, Y. P. Tamarit, S. Morais and A. Maquieira, Trends in Analytical Chemistry. 132, 116050

(2020).

163


S6703 Enhanced Production of Lycopene from Waste Biomass by Metabolic Engineering of Radiation-resistant Deinococcus radiodurans R1 and UV-C irradiation

Chang Keun KANG, Sun-Wook JEONG, Yong Jun CHOI School of Environmental Engineering, University of Seoul, Seoul, Korea Corresponding Author Email : [email protected] Cost-effective production of value-added chemicals is of great interest for achieving a sustainable industry. We accomplished high-level production of lycopene from waste-derived substrates, corn steep liquor (CSL) and glycerol, using metabolically engineered Deinococcus radiodurans R1. To establish lycopene-producing D. radiodurans R1, the crtLm gene encoding lycopene cyclase was deleted to block the conversion of lycopene. Then, the crtB gene encoding phytoene synthase and the dxs gene encoding 1-deoxy-D-xylulose 5-phosphate synthase were overexpressed to overcome rate limiting steps and concentrate the carbon flux toward lycopene. The engineered strain produced 722.2 mg/L (203.5 mg/g DCW) of lycopene from 35.7 g/L glycerol and 25 g/L CSL. To further improve lycopene production, UV-C was irradiated on the engineered strain during cultivation. Lycopene content of the cells was increased by 95.5%, after 12 h of UV-C irradiation using 15W lamp, compared to that obtained without UV-C irradiation. This study shows that lycopene can be produced from waste biomass in a cost-effective and reproducible manner using radiation-resistant D. radiodurans R1. Keywords : Lycopene, Cost-effective production, Deinococcus radiodurans R1, Metabolic engineering, Waste biomass, UV-C radiation

164


S6704 Novel Phage Display Method & Screening and Identification of Selective Binding Peptides to Vitamin-related Biocomplex

Taehwan KIM, Jaewoong LEE, Jiho MIN Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Vitamin D has recently been attracting attention in relation to immunity enhancement. Vitamin D is naturally synthesized in vivo, but it is prone to deficiency. This vitamin D detection is mainly done by detecting the complex forms in the body. Vitamin D is in a complex form with Vitamin D Binding Protein (VDBP) rather than moving alone in the blood. In this experiment, we tried to find a peptide sequence that can specifically screen the Vitamin D Binding Protein Complex (VDBP-Complex) and vitamin D binding protein by using a method modified from the existing phage display technique. We performed bio-panning to screen and classify specific binding phages that selectively attaches to the target substances. And, a number of individual phages from each strain were analyzed their DNA sequences. Then, the peptide sequence from the specific binding phage was analyzed, and after confirming the prominent sequences among them, an affinity test was performed. As a result, that sequences had more the binding affinity ability about 2.2 times to VDBP-Complex than the control. Keywords : Phage display, Vitamin D detection, Vitamin D binding protein, Vitamin D binding protein complex, Vitamin D

165


S6705 Synthesis and Characterization of Synechocystis sp. PCC 6803 Ferredoxin Variants

yeeun CHA Hongik University, Seoul, Korea Corresponding Author Email : [email protected] Metalloproteins exhibit a wide range of electrochemical and redox properties depending on the internal structure surrounding the metal atom(s). Such properties are employed in a wide range of activities, from promoting various oxidation and reduction reactions to carrying out electron transfer inside cells. Among metalloproteins, ferredoxin is a metalloprotein that mediates electron transport across multiple organisms in cells. Synechocystis sp. PCC 6803 ferredoxin (SynFd), which is mutated in this study, participates in the energy generation mechanism within photosynthesis by helping the regeneration of NADPH. We modified the structure of SynFd through genetic modification, The method replaced 6 amino acids at the positively charged position among amino acids within 5 amps of the cluster with negatively charged amino acids, Asparatic acid and Glutamin Acid, respectively. This substituted protein can be used for biochemical synthesis by measuring the rate of electron transfer within the cell (or in a biosystem) of the ferredoxin variant through a cytochrome c assay and an electrochemical system. In particular, the high-energy electrons transferred using ferredoxin applied in the photosynthetic approach are used to generate H2 directly, and only water and sunlight are required to produce this photosynthetic hydrogen. Therefore, the strategy of increasing hydrogen production efficiency through ferredoxine mutations, which are generally inexpensive and widely available, has the potential to be an attractive and carbon-neutral alternative to producing high value-added energy. Keywords : metalloprotein, photosynthesis, protein, electrochemical, electron transfer, cyanobacteria, synechocystis

166


S6706 Anti-oral Biofilm Agent Using Organelles and Target-specific Binding Peptides

Jaewoong LEE Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University Jeonju, Korea Corresponding Author Email : [email protected] Most oral diseases are caused by biofilms that consist of multispecies communities. However, the interactions of the community in health and caries pathogenesis are not well understood. To combat oral biofilm using surface-modified lysosomes which can specifically bind to targets, S. mutans and 2 oral Bacteria were selected for this study. At first, phage display was performed to find to separate specific binding phages to each oral pathogens. Also, 20 individual phages in each round from all strains were analyzed in their DNA sequences. As a result, 4 DNA sequences were conspicuous, and it was synthesized to verify the affinity to each target. The binding affinity results have shown that 3 synthesized peptides specifically bind to each target. And peptide stability test was conducted in several conditions. Also, Lysosome's anti-microbial activity was confirmed before modifying its surface. As a result, the organelles containing the specific peptides were designed to defeat oral biofilm. Furthermore, designed organelles will be checked for their anti-biofilm activity. Keywords : Streptococcus mutans, oral biofilm, phage display

167


S6707 Development of Enhanced Bioconversion System for Tagatose Production in Corynebacterium glutamicum

Eun Jung JEON1, Young Mi LEE2, Eun Jung CHOI2, Ki Jun JEONG1 1Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea, 2Institute of Biotechnology, CJ CheilJedang, Suwon, Korea Corresponding Author Email : [email protected] Tagatose is a ketohexose monosaccharide that is present in small amounts in natural fruits. It is a representative rare sugar that has 1/3 of the calories of sugar and has a low GI (Glycemic index), which is 5% of sugar, but has a similar physical properties and sweetness of sugar and use as a prebiotic. As a method of tagatose production, there is chemical and enzymatic isomerization using D-galactose as a main raw material. However, galactose not easy to use because price volatility is large due to unstable raw milk supply and demand. On the other hand, fructose obtained by using glucose can be used as an alternative with a stable price. In this study, we developed Corynebacterium glutamicum -based whole cell biocatalyst platform for the bioconversion of fructose to tagatose in one-step enzyme reaction. For the expression of enzyme, we optimized the expression systems including plasmid copy number and promoter strength, and with the optimized system, the bioconversion yield was incredibly increased. Keywords : Corynebacterium glutamicum, Tagatose, High copy plasmid References 1. Khuwijitjaru et al.Science, Engineering and Health Studies (2018). 2. Shen, Jing, et al. Journal of agricultural and food chemistry 67.22 (2019).

168


S6708 Cellulose Nanocrystals Coated with a Tannic Acid-Fe3+ Complex as a Significant Medium Component for Enhancing Mass-transfer performance in CH4 Microbial Biotransformation

Eungsu KANG1, Hwa Heon JE2, Hyunsuk CHOI1, Dongsu HWANG2, Yoo Seong CHOI1 1Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 2Division of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] Microbial CH4 biotransformation has become sustainable technologies for producing useful chemicals such as methanol, succinate, and 2-3-butanediol. However, the insufficient supply of CH4, due to mass transfer limitation and low solubility, still needs to be overcome to produce value-added chemicals in CH4 gas fermentation efficiently. Here, we successfully prepared cellulose nanocrystals coated with a tannic acid-Fe3+ complex (TA-Fe3+CNCs) as a medium component to enhance gas-liquid mass-transfer performance. TA-Fe3+CNCs were well suspended in aqueous culture media, stabilized gas bubbles without coalescence, and increased the gas solubility by 20% and the KLa value at a rapid inlet gas flow rate. Remarkably, the metabolite production ability such as methanol, succinate, and formate was improved with the cell growth rate of Methylomonas sp. DH-1, as model CH4-utilizing bacteria, in the TA-Fe3+CNCs nanofluid. These results showed that TA-Fe3+CNCs could be utilized as a significant medium component for CH4 microbial biotransformation Keywords : Cellulose nanocrystals, Nanofluid, Mass transfer enhancement, CH4 microbial biotransformation

169


S6709 Development of Oxygen Transfer Rate (OTR) Prediction Model in CHO Cell Shake Flask Cultures

hanchul KO, Jong Youn BAIK Biological Sciences and Bioengineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Shake flasks are widely used as small-scale mammalian culture vessels during cell line development and bioprocess optimization for the production of biopharmaceuticals. However, the lack of information about culture parameters in shake flasks, such as pH and dissolved oxygen, often leads to oxygen limitation that can adversely affect cell growth and productivity during cell culture, thereby challenging cell line development and scale-up processes. In this work, we report an oxygen transfer rate(OTR) prediction model of shake flasks using Chinese hamster ovary(CHO) cells to reduce oxygen limitation in shake flasks. First, we varied shaking speed and culture volume to measure maximum OTR(OTRmax) and created an OTRmax regression function. Then, we estimated theoretical maximum oxygen-supplied cell density with the known oxygen uptake rates of CHO cells and the OTRmax values. Lastly, we tested if decreasing culture volume or increasing shaking speed could mitigate the adverse effect of oxygen limitation. In conclusion, we believe this OTR prediction model will provide infor-mation about the oxygen availability in shake flasks, presumably assisting to achieve better culture performances. Keywords : Oxygen limitation, Shake flask, Oxygen transfer rate, Chinese hamster ovary (CHO) cell

170


S6710 The Co-repression of Multiple Genes Using Synthetic sRNAs

Jinho YEOM, Jong Seong PARK, Seung Min YOO School of Integrative Engineering, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Efficient control over gene expression is essential in fundamental and applied biological studies, but it is still a major challenge because of the difficulties in regulating multiple gene expression. To overcome this limitation, we report the development and application of a synthetic fusion sRNA (fsRNA)-based system for rapid and efficient multiple target gene knockdown in engineered Escherichia coli. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold. The repression capacity of fsRNA developed was demonstrated by the construction of sRNAs to endogenous genes in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. fsRNA was applied to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. These examples demonstrate that the developed fsRNA-based system enables the rapid development of chemical overproducers without causing cell growth defects and limiting cellular resources. ** This work was supported by an NRF grant funded by the Ministry of Science and ICT (NRF-2019R1 A2C1088504). Keywords : Synthetic RNA, Co-repression, multi-target References 1. D. Na, S.M. Yoo, H. Chung, H. Park, J.H. Park, S.Y. Lee, Nat. Biotechnol. 31, 170-174 (2013).

171


S6711 Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

Seulgi KANG1,2, Yoseb SONG1,2, Sangrak JIN1,2, Jongoh SHIN1,2, Jiyun BAE1,2, Hyeonsik LEE1,2, Suhyung CHO1,2, Byung-Kwan CHO1,2,3 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Intelligent Synthetic Biology Center, Daejeon, Korea Corresponding Author Email : [email protected] Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of syngas, for autotrophic growth in order to produce biomass and metabolites via the Wood–Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution for growth improvement of Eubacterium limosum ATCC 8486 under CO conditions. As a result, the growth rate of evolved population was significantly increased to 1.44 folds. To identify the causal mutations related to growth improvement, we performed whole genome resequencing of each population. Interestingly, we found a key mutation in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding gene. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth fitness of the single clone was comparable to those of the evolved populations and wild type strain under CO conditions. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486. ** This work was supported by the Intelligent Synthetic Biology Centre of the Global Frontier Project (2011-0031957 to B.-K.C.) and the C1 Gas Refinery Program (2018M3D3A1A01055733 to B.-K.C.) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT). Keywords : Acetogens, Carbon monoxide, Adaptive laboratory evolution, CODH/ACS, acsA

172


[S6-8]

부문위원회 및 학생세션 VIII

173


S6801 Development of Transgene Integration Site Identification Technique Using Repeat Sequences of Chinese Hamster Ovary Cells

Sang Yeop LEE, Jong Youn BAIK Department of Biological Sciences and Bioengineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] As therapeutic monoclonal antibodies (mAbs) have become major medicine in the global market, the importance of cell line development (CLD) process for developing recombinant CHO (rCHO) cells producing mAbs has also been highlighted. Random integration, a traditional CLD strategy by which transgenes encoding therapeutic products are randomly incorporated in the genome, often causes positional effects that can exhibit clonal variability in terms of cell growth, productivity, and product quality. Therefore, cost-effective, quick method for identifying gene integration site is desirable. In this study, we propose the Alu polymerase chain reaction (Alu PCR) method that searches the location of transgenes using short interspersed nuclear elements (SINEs). To optimize the Alu PCR, we tested three different types of SINEs and modulated the molar ratio of PCR primers containing uracil. Then, we applied Alu PCR to detect the integration sites of transgene as well as the locus of cytochrome c oxidase gene as a control in the rCHO genome. In conclusion, we prove that Alu PCR can be used to effectively analyze transgene integration site compared to other existing genomics-based strategies. Keywords : Chinese hamster ovary (CHO), Short interspersed nuclear elements (SINEs), Random integration, Alu sequence, Transgene integration site References 1. Grilo, António L., and A. Mantalaris. The increasingly human and profitable monoclonal antibody market

(2019). Trends in biotechnology 37.1: 9-16. 2. Noh, Soo Min, Madhavi Sathyamurthy, and Gyun Min Lee. Development of recombinant Chinese hamster

ovary cell lines for therapeutic protein production (2013). Current opinion in chemical engineering 2.4: 391-397.

174


S6802 Melanin Color Reduction by Lysosomal Fraction and Glutathione Peroxidase (GPX) of Saccharomyces cerevisiae

Gyeongchan JEON, Jiho MIN Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] The melanin compound, a pigment which blocks harmful ultraviolet radiation, can make skin dark and unclean, and can cause hyperpigmentation, like melasma. All eukaryotes have lysosomes that contain hydrolytic enzymes such as protease to degrade waste materials and cellular fragments. In this study, we found that melanin color reduction activity of lysosomal fraction, and that some antioxidant enzymes, especially glutathione peroxidase (GPX), of the lysosomal fraction played an important role to decolorize melanin compounds. A overexpress of GPX2 among the GPX present in Saccharomyces cerevisiae increase both melanin-decolorize activity and peroxidase activity of Lysosomal fraction. In addition, melanin synthesis inhibition, cytotoxicity, artificial tissue, in vivo tests were performed, and in artificial tissue test, the enzymes reduced melanin color, and the result is a evidence of the enzyme's corneous tissue penetration potential. These results provide insights into the regulation of LOE from S. cerevisiae as new cosmetic materials to reduce the color intensity of melanin. Keywords : Melanin, Lysosome, Saccharomyces cerevisiae, Glutathione peroxidase (GPX), Antioxidant enzyme

175


S6803 Identifying Settling Signaling Pathway and EPS Adhesion Protein of Pyropia yezoensis

Sinyang KIM, Dong Soo HWANG Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Macroalgal forest does role as primary producer, habitats of marine juveniles and it had been seriously destroyed by ocean acidification, pollution and eutrophication since the industrial revolution. Putting an artificial macroalgal plant underwater covered with macroalgal spores can be a solution but most spores cannot survive on it or flush away by the wave. Hence, understanding a settling and germling mechanism of the algal spore in molecular level is necessary to give more successive way of building an artificial marine forest. From that point of view, we tried to identify a specific mechanism of settling signaling pathway triggered by mechanical stimuli from the settling substrate using Pyropia yezoensis’ carpospores. The spores showed different morphological aspect on different substrate stiffness which indicates that the algal spore can recognize mechanical level from the environment. We are going to suggest a specific model signal pathway regarding with settling and germling stage of the algal spores by transcriptome analysis, analyzing gene expression changes by time and knockdown assay Keywords : Macroalgae, Algal spores, Marine afforestation, Signaling pathway

176


S6804 A Green Decontamination Strategy Through Selective Biomineralization of Algicidal Microorganisms for Increased Astaxanthin Production from Haematococcus pluvialis at Large Scale

Byung Sun YU, Sang Jun SIM Department of chemical and biological engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected] The aim of this work is to cultivate astaxanthin producing Haematococcus pluvialis using axenic mixotrophic culture system. The biggest problem facing humanity at present is climate change, glacier melting and ocean acidification due to the increase of carbon dioxide. To solve this problems, biological conversion process is required. However, the culture time of Haematococcus pluvialis was at least 60day. We can use mixotrophic culture system for decreasing culture time. But bacteria, fungi and golden algae grow together. The contamination factors attack the Haematococcus pluvialis and the cell was inhibited or die. In this study, the biomass was obtained by using microalgae for the reduction of large amount of carbon dioxide, and calcium carbonate was obtained through biomineralization of contamination factors during the induction stage. The contamination factors have carbonic anhydrase enzyme, which catalyze the biomineralization at pH 7.7 to 8. The zeta potential of Haematococcus pluvialis was so low that biomineralization was not occur effectively. Using this system, we can reduce the culture time up to 40day. Consequently, we can culture Haematococcus pluvialis using axenic mixotrophic culture system in short period. Keywords : Haematococcus pluvialis, Bacteria, Fungi, Biomineralization, Decontamination, Calcite, Large scale

177


S6805 The Effect of Lysozyme on LC-MS/MS Analysis of Biofouling Proteins in Wastewater Treatment

Minhee LEE1, Heejung JUNG1, Seungju CHOI2, Dongsoo HWANG1,3, Seoktae KANG2 1Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2Department of Civil and Environmental Engineering, KAIST, Daejeon, Korea, 3Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] In wastewater treatment, bacterial biofilm makes the matrix that causes biofouling on the membrane. Although the identification of biofouling components using LC-MS/MS has been studied, the biofilm proteins were found at a low proportion because of complexity of proteins and bacterial communities. In the LC-MS/MS, the glycoproteins become a hindrance for protein identification. There are the enzymes, PNGase F, which works by cleaving specific glycans. The lysozyme, one of the enzymatic method, catalyzes the cleavage of glycans between N-acetylglucosamine and N-acetylmuramic acid which are peptidoglycan components of bacterial cell wall. In this study, a single species, E. coli, was treated with reverse osmosis (RO) membrane which were used to collect the biofouling inducing biofilm. The proteins from E. coli extracellular polymeric substances (EPS) on membrane were identified depending on lysozyme treatment through LC-MS/MS. The EPS glycans were removed by the enzyme and the proteins were identified using PEAKS studio software. The effect of lysozyme on E. coli EPS were demonstrated by confirming the number of identified peptides and protein functions. Keywords : Biofilm, Lysozyme, LC-MS/MS, Protein identification, EPS References 1. T. Seviour, N. Derlon, M. S. Dueholm, H.-C. Flemming, E. Girbal-Neuhauser, H. Horn, S. Kjelleberg, M.

C. M. van Loosdrecht, T. Lotti, M. F. Malpei, R. Nerenberg, T. R. Neu, E. Paul, H. Yu and Y. Lin, Water Res. 151, 1-7 (2019).

2. Z. Zhou, F. Meng, X. He, SR. Chae, Y. An, X. Jia, Environ. Sci. Technol. 49 (2), 1068-1077 (2015). 3. S. Sun, P. Shah, ST. Eshghi, W. Yang, N. Trikannad, S. Yang, L. Chen, P. Aiyetan, N. Höti, Z. Zhang, DW.

Chan, H. Zhang, Nat Biotechnol. 34, 84-88 (2016).

178


S6806 Acetogenic Bacteria Utilize Light-driven Electrons as an Energy Source for Autotrophic Growth

Sangrak JIN1,2, Yale JEON3, Min Soo JEON3, Jongoh SHIN1,2, Yoseb SONG1,2, Seulgi KANG1,2, Jiyun BAE1,2, Suhyung CHO1,2, Jung-Kul LEE4, Dong Rip KIM3, Byung-Kwan CHO1,2,5 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Innovative Biomaterials Research Center, KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Department of Mechanical Engineering, Hanyang University, Seoul, Korea, 4Department of Chemical Engineering, Konkuk University, Seoul, Korea, 5Intelligent Synthetic Biology Center, Daejeon, Korea Corresponding Author Email : [email protected] Acetogenic bacteria utilize cellular redox energy to convert CO2 to acetate using the Wood–Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as inorganic materials such as photoresponsive semiconductors. Here, we developed a nanoparticle-microbe hybrid system, in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) were displayed on the cell surface of the industrial acetogen, Clostridium autoethanogenum. The hybrid system converts CO2 into acetate without the requirement of additional energy sources such as H2 and only utilizes light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum utilizes electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicated that genes encoding the metal ion or flavin binding proteins were highly upregulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles. This work was supported by the Intelligent Synthetic Biology Center of the Global Frontier Project (2011-0031957 to B.-K.C.) and the C1 Gas Refinery Program (2018M3D3A1A01055733 to B.-K.C.) through the National Research Foundation of Korea funded by the Ministry of Science and ICT. Keywords : Acetogenic bacteria, Artificial photosynthesis, Cadmium sulfide nanoparticle References 1. S. Jin et al., PNAS, 118(9) (2021).

179


S6807 Alginate-shell and Polydopamine-coated PDMS Frame Inhibit Contraction of Collagen Hydrogel

Seulha KIM1, Jeong Ah KIM2, Tai Hyun PARK1,3 1School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea, 2Center for Scientific Instrumentation, Korea Basic Science Institute, Cheongju, Korea, 3BioMAX/N-Bio Institute, Institute of BioEngineerig, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Collagen is the most abundant protein in mammals. However, it is difficult to use collagen hydrogel as a cell culture platform in vitro, because severe hydrogel contraction occurs. This has disadvantage in experiments because uncontrollable changes appear in the cell microenvironment. Hydrogel properties like cell distribution, adhesive ligand density, diffusivity, stiffness, etc. are critical factors that affect cell behavior and differentiation tendency. In this study, two strategies were used to minimize the contraction of collagen. The vertical contraction was inhibited by alginate shell on the outside, and horizontal contraction was prevented with a polydopamine-coated PDMS frame. These two methods complementarily prevented the contraction of the collagen gel even in long-term culture. In the freely contracted collagen hydrogel, cell differentiation behavior between osteogenesis and adipogenesis was analyzed based on YAP protein localization. This platform is expected to expand the in vitro application of collagen hydrogel. Keywords : Collagen, Hydrogel, Alginate, Polydopamine, Mechanotransduction References 1. V. W. Tang, Mol Biol Cell, 31, 1823 (2020). 2. S. E. Park, A. Georgescu, J. M. Oh, K. W. Kwon, D. Huh, ACS Appl Mater Interfaces, 11, 23919 (2019).

180


S6808 Additional Hydrophobic Nature of Arginine Tunes LLPS Properties with Temperature Changes

Yuri HONG1, Thomas CASEY2, Song-I HAN2,3, Dong Soo HWANG1,4 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2Department of Chemistry and Biochemistry, University of California-Santa Barbara (UCSB), Santa Barbara, CA, USA, 3Department of Chemical Engineering, University of California-Santa Barbara (UCSB), Santa Barbara, CA, USA, 4Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Biomolecular condensates composed of proteins can be formed separately from the liquid cytoplasm by liquid-liquid phase separation (LLPS). The driving forces for LLPS and the physical properties of protein droplets are governed by the amino acid compositions of the constituent proteins. Of the amino acids, the role of arginine vs lysine in LLPS has drawn attention because arginine is prevalent in the phase separation of RNA-binding proteins and is a more potent driver of LLPS compared to other positively charged amino acids, such as lysine. However, what properties of arginine favorably influence LLPS and the physical properties of condensates is still unclear. Here, we demonstrate that arginine can contribute to the hydrophobicity of LLPS through its sp2-hybridized guanidinium group and that hydrophobicity is critical for the physical properties of condensates and temperature-dependent phase behavior. Our findings may provide new insights into the LLPS of stress granule proteins in response to temperature changes in the context of the additional hydrophobic nature of arginine. Keywords : arginine, guanidinium group, hydrophobicity, coacervation, liquid liquid phase separation References 1. Boeynaems S, et al., Trends Cell Biol. 28(6), 420 (2018). 2. Brangwynne Clifford P, Tompa P, & Pappu Rohit V, Nat Phys. 11, 899 (2015). 3. Cooper RJ, Heiles S, DiTucci MJ, & Williams ER, The Journal of Physical Chemistry A. 118(30), 5657

(2014). 4. Ukmar-Godec T, et al., Nat Commun. 10(1), 2909 (2019).

181


S6809 Development of Respiratoid for Supplying Oxygen to Engineered Cells by Cellular Respiration

Ji Yun KIM1, Seok Chan PARK1, Min Su KIM1, Seon Mi JANG1, Jae Kyung PARK1, Dong Yun LEE1,2 1Department of Bioengineering, College of Engineering, and BK FOUR Biopharmaceutical Innovation Leader for Education and Research Group, Hanyang University, Seoul, Korea, 2Institute of Nano Science & Technology (INST), Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] The viability of tissue-engineered cells is damaged by hypoxia due to rare or no microvessel. Here, we newly developed a spontaneous respiration biomaterial, i.e., Respiratoid, to help cellular respiration so that tissue-engineered cells could survive without microvessel. The Respiratoid could convert carbon dioxide into oxygen during cellular respiration. To this end, the intact chloroplast in plat leaf was introduced to biocompatible alginate via a chloroplast-transit-peptide (CTP) that can ‘anchor’ the outer membrane of chloroplast while maintaining the function of chloroplast. The CTP-conjugated alginate was optimized to make a hydrogel for tissue engineering. After physical characterization of the hydrogel, the cytotoxicity and degree of oxygen/carbon dioxide balance of the Respiratoid were evaluated in vitro and in vivo. Collectively, newly synthesized Respiratoid material could be used for successful tissue engineering. Keywords : Respiratoid, Chloroplast, Alginate, Hydrogel, Cell capsule, Hypoxia, Respiration References 1. Pedraza E et al., PNAS 2012;109(11):4245-4250. 2. Gholipourmalekabadi M et al., Trends in Biotechnology 2016;34(12):1010-1020. 3. Thagun C et al., Advanced Science 2019:1902064. 4. Jarvis P et al., Science 1998;282:100-103. 5. Lee H-Y et al., Biomaterials 2015;53:583-591.

182


S6810 Molecular and Electrochemical Approaches for Studying IL-33 Binding Peptides Identified by Phage Display

Chae Hwan CHO1, Se-Young SON1, Ji Hong KIM2, Jae Hwan SHIN1, Jeong Kyu BANG3, Young Ho JEON1, Jong Pil PARK2 1Department of Food Science and Technology, Chung-Ang University, Anseong, Korea, 2College of Pharmacy, Korea University, Sejong, Korea, 3Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Korea Corresponding Author Email : [email protected] Recently, allergic diseases have been great attention according to increase the urbanization worldwide and it was emerged as a global health concerns. Allergy is caused by the complex biological reactions. Among those, IL-33/ST2 signaling is considered as one of the major mechanism in allergic inflammatory diseases. Interleukin 33 (IL33), which is member of the IL-1 cytokine family and commonly expressed in many cell types, can be lead to increase the inflammation reactions through binding to the ST2 receptors. There are some of early detection methods, however, it need to develop more advanced monitoring and detecting systems over conventional methods. In this work, we tried to identify the IL33 specific peptides using phage display technique and evaluated the binding interactions using ELISA and SWV. Furthermore, the binding interaction between IL33/ST2 and affinity peptide was characterized by ITC, SWV and NMR. Keywords : Affinity peptides, Interleukin 33, Allergic diseases, Phage display, IL33/ST2 signaling

183


S6811 Fast Antimicrobial Susceptibility Testing Against Bacteria Using Droplet-based Microfluidic Device

Jae Seong KIM1, Byungjin LEE1, Heon-Ho JEONG2, Chang-Soo LEE1 1Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 2Department of Chemical and Biomolecular Engineering, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Microfluidic approach stands in important position as an analytical tool in biological area due to its ability of high-throughput screening. Here, we present a microfluidic device for antimicrobial susceptibility testing(AST) against bacteria in a single device. Not only integrating several flow-focusing channels but also controlling the flow resistance of two aqueous phases, we could produce the cell-encapsulated droplets having linear concentration gradient of antibiotics. Using this strategy, we tested gentamicin against E.coli O157:H7 to determine the minimum inhibitory concentration(MIC). Growth in droplets were measured by counting the number of cells through fluorescence microscopy. E.coli O157:H7 which produces green fluorescence protein(GFP) facilitated cell counting. Screening the growth profile of cells in droplets having different concentration of gentamicin resulted in MIC determination in only 4-6 h using our device. This results show that our platform has the great potential to be used for AST with other pairs of bacteria (i.e. gram-positive bacteria) and antibiotics. Keywords : Microfluidics, Concentration gradient, Antibiotics, gram-negative bacteria, Parallel Droplet Generation

184


3분 스피치 및 포스터발표

185


PSP01 Isolation of Cabbage Exosome-like Nanovesicles and Investigation of Their Biological Activities in Human Cells

Su Jin KANG1, Jae Young YOU1, Won Jong RHEE1,2 1Department of Bioengineering and Nano-bioengineering, Incheon National University, Incheon, Korea, 2Division of Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Apoptosis is a programmed cell death that occurs in various stresses. Excessive apoptosis can cause serious illnesses such as neurodegenerative diseases. Thus, it is necessary to an investigate material to suppress excessive apoptosis. Extracellular vesicles (EVs) are nano-sized particles derived from living organisms including prokaryotes, eukaryotes and plant. They participated in cell communications through transferring DNA, RNA, protein from originating cells. Although EVs isolated from stem cells are utilized in various diseases recently, however, large quantities of EVs are needed for in vivo applications. Therefore, investigating EVs from a natural source is required. In this study, cabbage and red cabbage were used as nanovesicle sources owing to their easy cultivation. The isolated cabbage exosome-like nanovesicles (Cabex) and red cabbage exosome-like nanovesicles (Rabex) were supplemented to human cell culture media, and there was no cytotoxicity effect. In addition, both Cabex and Rabex promoted cell proliferation and, interestingly, suppressed apoptosis in human cells. In conclusion, Cabex and Rabex can be novel natural components to protect apoptosis and offer novel anti-apoptosis strategy where the apoptosis suppression is required. Keywords : Extracellular vesicle, Exosome-like nanovesicles, Cabbage, Red cabbage, Isolation, Apoptosis References 1. R. Bonafede, J. Brandi, M. Manfredi, I. Scambi, L. Schiaffino, F. Merigo, E. Turano, B. Bonetti, E.

Marengo, D. Cecconi, R. Mariotti, Cells. 8, 1087-1109 (2019). 2. S. Ju, J. Mu, T. Dokland, X. Zhuang, Q. Wang, H. Jiang, X. Xiang, Z. Deng, B.Wang, L. Zhang, M. Roth,

R. Welti, J. Mobley, Y. Jun, D. Miller, H. Zhang, Mol Ther. 21, 1345-1357 (2013). 3. S. Cho, H. C. Yang, and W. J. Rhee, Process Biochem. 22, 197-203 (2020).

186


PSP03 Magnetic Nanoparticles Empowered to Genetically Manipulate Natural Killer (NK) Cells

Ha Eun SHIN1, Kwang-Soo KIM2, Jun-Hyeok HAN1, Dong Keun HAN2, Kyung-Soon PARK2, Wooram PARK1 1Biomedical-Chemical Engineering, The Catholic University of Korea, Bucheon, Korea, 2Biomedical Science, CHA University, Pocheon, Korea Corresponding Author Email : [email protected], [email protected], [email protected] Natural killer (NK) cells are considered immunotherapeutic effectors in the fight aginst cancers. However, genetic materials' transfer remains a serious problem due to the low efficiency of transfection in NK cells. Here, we report that magnetic nanoparticles (MNPs) with a cationic polymer layer can genetically engineer NK cells and follow them in vivo through magnetic resonance (MR) and fluorescent imaging. These nanoparticles were dispersible in water, PBS, and cell culture medium. Also, MNPs quickly formed gene-complexes due to immobilized cationic polymer by a polydopamine. The MNPs-mediated transfection preserved the functional phenotype of NK cells. Interestingly, these nanoparticles produced EGFR-chimeric antigen receptor (CAR) NK cells, which had a significant cytotoxicity in breast cancer cells. The EGFR-CAR NK cells modified by MNPs also encompassed an anti-tumor effect in xenograft mice model and the high accumulation into the tumor. The MNPs with a cationic polymer layer could be an innovative platform for enhanced cancer immunotherapy. Keywords : Gene therapy, Nanoparticles, Natural killer (NK) cells, Cell tracking, Immune cell engineering

187


PSP05 Ligand-assisted Enzyme-accelerated Signal Enhancement for Targeted Cancer Cell Imaging via Polydopamine Deposition

Yeon-Ju LEE, Hea-Jin KIM, Miso KIM, Soohyun HAN, Sooyeon CHOI, Young-Pil KIM Department of Life Science, Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] Cancer cell-targeting imaging is prerequisite for diagnosis and therapy. Despite the recent advances in imaging technologies, identification of cancer cells using affinity-based ligands (peptides or aptamers) is often challenging, due to the limited brightness of fluorescence signal especially at low expression of target receptors. Herein, we propose a ligand-assisted enzyme-accelerated signal enhancement (LA-EASE) technology to image cancer cells, in which an aptamer or a peptide can be used as a cell-targeting ligand and polydopamine (PDA) as a signal amplifier. When biotinylated ligands are treated with their target cancer cells, the LA-EASE reaction is achieved by the sequential reaction of streptavidin-conjugated horseradish peroxidase (HRP) and HRP-accelerated dopamine oxidation, thus leading to the fast deposition of PDA in the vicinity of cancer cells. Additionally, by preferentially binding the amine-coated quantum dots to the PDA deposition area, cancer cells exhibit a strong fluorescence signal. We demonstrated this hypothesis using a receptor-targeted aptamer or peptide in prostate cancer cells (PC-3) or breast cancer cells (MCF-7), respectively. Surprisingly, the LA-EASE method enabled the remarkable fluorescence signal amplification in two model cancer cells with high specificity, which was approximately 100-fold higher than that by a conventional fluorescent imaging using dye-labeled aptamer or peptide. These findings suggest that the designed LA-EASE technology will be useful for targeting cancer cells and tissues as a universal imaging platform with high sensitivity and usability. Keywords : Fluorescence imaging, Dopamine, Aptamer, Peptide, Cancer cells, Signal enhancement References 1. Li J., et al. Natural Biomedical Engineering. 1, 0082 (2017). 2. Wang Y., Luo Y., Bing T., Chen Z., Lu M., et al. PLoS ONE. 9(6): e100243 (2014).

188


PSP06 Single-step Extracellular Vesicle Concentration Method Using Super Absorbent Polymer Beads

Yoo Min HAM1, Hee Cheol YANG1, Jeong Ah KIM2,3, Won Jong RHEE1,4 1Department of Bioengineering and Nano-Bioengineering, Incheon National University, Incheon, Korea, 2Center for Scientific Instrumentation, Korea Basic Science Institute, Daejeon, Korea, 3Department of Bio-Analytical Science, University of Science and Technology, Daejeon, Korea, 4Division of Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Extracellular vesicles (EVs) contain useful biomarkers for disease diagnosis and are promising biomaterials for therapeutic applications. Accordingly, an efficient concentration method is necessary for the large-scale production of EVs from bulk liquid samples, including culture medium, for their clinical purpose. However, current EV concentration methods, including ultrafiltration, are limited with respect to cost, efficiency, and centrifugation time. In this study, we developed the simple and efficient EV concentration method using super absorbent polymer (SAP) beads. SAP beads absorb small molecules, including water, via nano-sized channels but expel and thereby concentrate EVs. Consequently, the beads drastically enrich EVs without affecting EV characteristics and increase the purity of the concentrated EV solution by absorbing protein impurities. To further demonstrate the versatility of the method, we showed that SAP beads successfully enrich EVs in the culture medium, enabling better isolation performance than ultrafiltration. We believe the newly developed method in this study will facilitate the use of EVs as prominent biomaterials for disease diagnosis and therapy. Keywords : Extracellular vesicles, Isolation, Extracellular vesicle concentration, Super absorbent polymer References 1. H. C. Yang, Y. M. Ham, J. A. Kim, and W. J. Rhee, J of Extracellular Vesicles. 10, e12074 (2021). 2. X. Xie, J. Bahnemann, S. Wang, Y. Yang, and M. R. Hoffmann, Sci Rep. 6, 20516 (2016). 3. B. J. Benedikter, F. G. Bouwman, T. Vajen, A. C. A. Heinzmann, G. Grauls, E. C. Mariman, E. F. M.

Wouters, P. H. Savelkoul, C. Lopez-Iglesias, R. R. Koenen, G. G. U. Rohde, and F. R. M. Stassen, Sci Rep. 7, 15297 (2017).

189


PSP07 Formate Dehydrogenase as a Promising Biocatalyst for CO2 Utilization

Kil-Hwan KIM1,2, Gwon Woo PARK1, Myounghoon MOON1, Joon-Pyo LEE1, Jin-Suk LEE1, Won-Heong LEE3, Kyoungseon MIN1 1Gwangju Bio/Energy Research Center, Korea Institute of Energy Research (KIER), Gwangju, Korea, 2Interdisciplinary Program for Agriculture & Life Science, Chonnam National University, Gwangju, Korea, 3Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] The mitigation of carbon dioxide (CO2) is a significant issue and thus CO2 utilization has become sustainable technologies for producing useful chemicals and fuels. Among the chemicals obtained from CO2, formate is of our interest due to its versatility in in various industries such as energy storage. It has been reported that certain formate dehydrogenases (FDHs) can play a role as a CO2 reductase to catalyze CO2 reduction to formate and thus we explore the new FDHs as an efficient CO2 reductase through the genome mining. The newly discovered FDHs were successfully expressed in Escherichia coli and showed the catalytic activity for both CO2 reduction and formate oxidation. The optimum pH for CO2 reduction and formate oxidation were optimized at pH 7 and pH 9, respectively, probably due to the direct hydride transfer mechanism. In addition, kinetic parameters were determined. The newly discovered FDHs can contribute to construct a feasible FDH-driven CO2 refinery system. Keywords : CO2 reductase, CO2 reduction, Formate References 1. K. Min, Y.-S. Park, G.W. Park, J.-p. Lee, M. Moon, C.H. Ko, J.-S. Lee, Bioresour. Technol. 305 (2020),

123155.

190


PSP08 Biofilm Removal Efficiency of Porphyromonas gingivalis Using Cold Atmospheric Pressure Plasma for Peri-implantitis Treatment

Chang-Min LEE1, Young-Il JEONG1, Min-Suk KOOK2, Byung-Hoon KIM1 1Department of Dental Materials, College of Dentistry, Chosun University, Gwangju, Korea, 2Department of Maxillofacial Surgery, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected], [email protected] Peri-implantitis has emerging role in implant failure with an increased treatment of dental implant in clinical application. Cold atmospheric pressure plasma (CAP) produces reactive oxygen and nitrogen species (ROS/RNS) and has sterilization effects for the implant surfaces [1]. We designed an equipment of argon/oxygen CAP for treatment of peri-implantitis. Peri-implantitis treatment efficacy of CAP was evaluated with the removal of Porphyromonas gingivalis (P. gingivalis) biofilm on SLA titanium disk in vitro. Biofilm removal efficiency on the titanium disk was evaluated with crystal violet assay, colony-forming units (CFU) assay and changes of biofilm morphology. ROS generation in aqueous solution was evaluated with 2',7'-dichlorofluorescin diacetate and singlet oxygen sensor green, respectively. ROS generation in aqueous solution was affected by CAP treatment times and powers, i.e. higher the power and the longer the treatment time induced higher ROS in the aqueous solution. Especially, the aqueous ROS level was increased as the mixing ratio of oxygen gas increased. Furthermore, removal efficacy of P. gingivalis biofilm was correlated with CAP treatment, plasma power and quantity of generated ROS in crystal violet and CFU assay. In morphological observation with confocal microscope, P. gingivalis biofilm was also decreased by treatment with CAP. In conclusion, CAP can be used as a simpler and more effective non-surgical option for peri-implantitis. Acknowledgement. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C2008540). Keywords : P. gingivalis, Peri-implantitis, Cold atmospheric pressure plasma, Biofilm Removal Efficiency References 1. H. Yamazaki, T. Ohshima, Y. Tsubota, H. Yamaguchi, J.A. Jayawardena, and Y. Nishimura. Dent. Mater. J.

30, 384 (2011).

191


PSP10 Antimicrobial Activity of Isoprene Produced by Rhodobacter sphaeroides Against Gram-negative and Gram-positive Bacteria

Jaeyoung YU1, Subin LEE2, Jiho MIN1 1Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea, 2Department of Bioprocess Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] The main components of essential oils, isoprenoids (also known as terpenoids), are used as ingredients in pharmaceuticals, food flavors and biofuels, and have great industrial value. In addition, studies have been reported focusing on the antimicrobial activity of terpenes. This study was intended to confirm the antimicrobial activity and mechanism of isoprene released from Rhodobacter sphaeroides. We developed a co-culture system that can be cultured by sharing the atmosphere, and evaluated the antimicrobial activity of isoprene against Gram-negative and Gram-positive bacteria. In addition, we established the most optimal production conditions for production of isoprene from R. sphaeroides through Purge & Trap GC/MS and LC/MS/MS. And to understand the antimicrobial mechanism of isoprene, Field Emission Scanning Electron Microscopy (FE-SEM) was used to evaluate changes in the extracellular membrane and structure. As a result, isoprene showed higher antimicrobial activity against all the bacteria tested, especially Gram-positive bacteria. FE-SEM images indicate that the antimicrobial activity mechanism of isoprene is applied by involvement in the extracellular membrane or the cell wall. Keywords : Rhodobacter sphaeroides, Isoprene, Antimicrobial activity, Co-culture, Terpene

192


PSP11 Development of Histamine Inhibitors Using Yeast/Vacuoles-surface-modified Histamine Binding Protein (HBP)

Hyeweon JANG, Hyojin CHOI, Jiho MIN Department of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Histamine is an immune mediator and helps to accelerate the immune response when an inflammatory reaction occurs, but when it is secreted excessively, an excessive inflammatory reaction occurs, which may cause allergies. So, we devised an inhibitor that relieves the excessive inflammatory response by inhibiting the histamine molecule. Histamine-binding protein (HBP) is a substance secreted from ticks that suppresses the host's inflammatory response and helps facilitate smooth blood absorption. HBP was expressed on the yeast cell wall and on the yeast vacuole surface. Western blot and immunofluorescence were performed to confirm these expressions. Also, the inhibitory ability was confirmed after reacting directly with histamine solution and HBP expression by LC-MS. Phagocytosis assay was performed with RAW 264.7 to indirectly observe the histamine inhibitory effect by HBP expressions. To confirm the signal pathway of the inflammation response of HBP-expressed yeast vacuoles, the expression levels of signal proteins for the inflammatory response stimulated by LPS in RAW 264.7 were confirmed. Keywords : Histamine, Molecular interaction, Recombinant Yeast, Anti-inflammation response

193


PSP12 Genome Architecture and Transcriptome Analysis of Marine Cyanobacteria Synechocystis sp. PCC 7338

Yujin JEONG1, Seong-Joo HONG2,3, Sang-Hyeok CHO1, Seonghoon YOON2,3, Hookeun LEE4, Hyung-Kyoon CHOI5, Dong-Myung KIM6, Choul-Gyun LEE2,3, Suhyung CHO1,7, Byung-Kwan CHO1,7,8 1Department of Biological Sciences, Korea advanced institute of Science and Technology, Daejeon, Korea, 2Department of Biological Engineering, Inha University, Incheon, Korea, 3Department of Biological Sciences and Bioengineering, Inha University, Incheon, Korea, 4Institute of Pharmaceutical Research, College of Pharmacy, Gachon University, Incheon, Korea, 5College of Pharmacy, Chung-Ang University, Seoul, Korea, 6Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 7Innovative Biomaterials Center, KI for the BioCentury, Korea advanced institute of Science and Technology, Daejeon, Korea, 8Intelligent Synthetic Biology Center, Daejeon, Korea Corresponding Author Email : [email protected] Cyanobacteria producing various bio-products are considered promising microbial cell factories. The marine cyanobacterium Synechocystis sp. PCC 7338 has the advantage of growing in seawater, rather than requiring arable land or freshwater. However, how this marine cyanobacterium grows under the high salt stress condition remains unknown. Here, we determined the 3.70 Mbp complete genome of Synechocystis sp. PCC 7338. Differential RNA-seq and Term-seq data provided genome-wide information on genetic regulatory elements. Comparison with freshwater Synechocystis sp. strains revealed that Synechocystis sp. PCC 7338 genome encodes additional genes, which facilitate the adaptation to high salt and high osmotic pressure. A cyclic electron transport through photosystem I actively used by Synechocystis sp. PCC 7338 satisfied the demand for ATP under high salt environment. Our comprehensive analyses provide valuable information to discover the various genomic functions and regulations available for engineering Synechocystis sp. PCC 7338. [Supported by the C1 Gas Refinery Program (2018M3D3A1A01055733 to B.-K.C.) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT)] Keywords : Cyanobacteria, Synechocystis, Marine cyanobacteria, Genome, Transcriptome, Photosynthesis

194


PSP13 Transcriptome and Translatome Changes of Synechocystis sp. PCC 6803 in Response to Photosynthetic Inhibitory Conditions

Sang-Hyeok CHO1, Yujin JEONG1, Seong-Joo HONG2, Hookeun LEE3, Hyung-Kyoon CHOI4, Dong-Myung KIM5, Jaemin SEONG1, Gahyeon KIM1, Choul-Gyun LEE2, Suhyung CHO1,6, Byung-Kwan CHO1,6 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Department of Biological Engineering, Inha University, Incheon, Korea, 3Institute of Pharmaceutical Research, College of Pharmacy, Gachon University, Incheon, Korea, 4College of Pharmacy, Chung-Ang University, Seoul, Korea, 5Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 6Innovative Biomaterials Center, KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Cyanobacteria are able to produce diverse value-added biochemicals from CO2 and light. To utilize the potency, it is critical to understand the photosynthetic mechanism under various environmental conditions. Here, we investigated the inhibitory mechanisms of photosynthesis under high light and low temperature stress in Synechocystis sp. PCC 6803. Under each stress condition, the transcript abundance and translation efficiency were measured using RNA-seq and Ribo-seq, and the transcription unit architecture was constructed by transcription start sites and transcript 3'-end positions obtained from TSS-seq and Term-seq. Our results suggested that the mode of photosynthesis inhibition differed between the two stress conditions. The poor translation of photosystem I resulted from ribosome stalling at the untranslated regions, affecting the overall photosynthesis under low temperature. Our multi-omics analysis with transcription unit provides foundational information on photosynthesis for future industrial strain development. ** Supported by the Korea Bio Grand Challenge (2018M3A9H3024759 to B.-K.C.) through the NRF. Keywords : Cyanobacteria, Synechocystis, Photosynthesis, Transcriptome, Translatome

195


PSP14 Optimization for Bio-milking of Ectoine in an Engineered Methylomicrobium alcaliphilum 20Z from Methane

Hanyu CHAI1, Sukhyeong CHO2, Yun Seo LEE2, Jeong Geol NA1,2, Jinwon LEE1,2 1Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Korea, 2C1 Gas Refinery R&D Center, Sogang University, Seoul, Korea Corresponding Author Email : [email protected] Ectoine is one of the compatible solute produced by halophilic and halotolerant microorganisms, which not only balances the osmotic pressure of cells, but also protect enzymes, DNA and cell membrane under high stress conditions. In this study, we tried to find optimal conditions for ectoine bio-milking to improve ectoine productivity in the engineered Methylomicrobium alcaliphium 20Z ΔectDΔectR using methane as a sole carbon source. In the first step, cells were grown in the growth medium containing 3% NaCl and transferred hyperosmotic conditions to accumulate intracellular ectoine. In the second step, ectoine accumulated M. alcaliphilum 20Z ΔectDΔectR was subjected into hypo-osmotic conditions to secrete the intracellular ectoine without cell bursting. The cultivation condition and intracellular ectoine accumulation conditions were optimized with respect growth phase and medium salinity to reach the highest synthesis. The second step was optimized to approach maximal ectoine excretion with simultaneous minimal cell death caused by the osmotic shock. As a result, extracellular ectoine up to 109.4±3.331 mg/L was attained by bio-milking under optimized conditions in engineered M. alcaliphilum 20Z using methane. Keywords : Ectoine, Bio-milking, Methylobacterium alcaliphilum 20Z, Osmotic pressure, Methane, Optimization

196


PSP15 Effects of in vitro Yeast Vacuoles on Adipocytes, 3T3-L1 Development and Differentiation

Suyeon CHOI, Jiho MIN Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Vacuoles are organelles within cells that contain various hydrolytic enzymes in small lipid pockets and involved in intracellular signaling. In this experiment, it was confirmed that vacuoles extracted from S. cerevisiae suppress the differentiation of 3T3-L1 adipocytes. It was confirmed by Oil Red O staining and Trighlyceride Quantification Assay that the lipid accumulation in the dose-dependently differentiated adipocytes was reduced at a concentration of less than 0.02μm/ml of vacuoles compared with GW9662 as a differentiation inhibitor. It was found that the expression of peroxisome proliferators-activated receptor γ(PPARγ) and cytidine-cytidine-adenosine-adenosine-thymidine(C/EBPa) was markedly decreased in adipocytes treated with vacuoles at the level of transcription messenger RNA by western blot. Also, the vacuoles extracted from recombinant yeast with GFP fluorescence were treated on the cells and observed with a fluorescence microscope, confirming that the vacuoles permeated into the 3T3-L1 preadipocytes. Therefore, it is suggested that yeast-derived vacuoles can be an important source of inhibiting obesity-related inflammation and preventing adipocyte differentiation. Keywords : vacuoles, S. cerevisiae, 3T3-L1, differentiate, adipocytes

197


PSP16 Optimization of 3-hydroxypropionic Acid Production by an Engineered Kluyveromyces marxianus XXX

Kyoung-Gon KANG1,2, Jae-Bum PARK2, Deok-Ho KWON1,2,3, Suk-Jin HA1,2,3 1Department of Biohealth-machinery convergence engineering, Kangwon National University, Chuncheon, Korea, 2Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Korea, 3Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Kluyveromyces marxianus, known as thermo-resistant yeast was studied to produce 3-hydroxypropionic acid (3-HP) used as a raw material for bio-polymer production. As 3-HP is a high value added product from biomass, it has advantages of being biodegradable and harmless as an eco-friendly material. Previously, the K. marxianus JBP2 strain was engineered to produce xylitol at high temperature by random integration of mKmXYL1 gene originated from the K. marxianus 36907-FMEL1 strain. In this study, malonyl-CoA reductase (MCR) gene from Chloroflexus aurantiacus was overexpressed to K. marxianus JBP2 strain for 3-HP production. In addition, acetyl-CoA carboxylase 1 (ACC1) gene from K. marxianus 17555 was overexpressed into K. marxianus JBP2_MCR strain to isolate K. marxianus XXX strain. Finally, K. marxianus XXX produced 5.79 ± 0.60 g/L 3-HP at 24 h, 30oC and 100 rpm which is corresponding to 0.24± 0.2 g/L·h productivity. At 200 rpm, 5.63 ± 0.90 g/L 3-HP was produced in 12 h which is corresponding to 0.47± 0.07 g/L·h productivity. Keywords : 3-hydroxypropionic acid , Kluyveromyces marxianus, biodegradable References 1. Jae-Bum Park, Jin-Seong Kim, Deok-Ho Kweon, Dae-Hyuk Kweon, Jin-Ho Seo & Suk-Jin Ha Appl.

Biochem. Biotechnol. 189, pages459-470(2019). 2. Jae-Bum Park, Kyoung-Gon Kang, Deok-Ho Kwon, Suk-Jin Ha KSBB Journal 35(4), 2020.12, 337-341.

198


PSP17 Effect of Extraction Methods of Aurea helianthus Flowers on Antioxidant and Anti-wrinkle Activities

Nain KIM, Moon-Hee CHOI, Seung-Hwa YANG, Khlood ALHARBY, Hyun-Jae SHIN Department of Chemical Engineering, Chosun University, Gwangju, Korea Corresponding Author Email : [email protected] Aurea helianthus (AH) is traditionally used in China for various medicinal purposes. The bioactive constituents from aerial parts of the AH leaf and stem as well as flowers were widely studied by several researchers showing anti-inflammation, immune-modulatory, antioxidant, and whitening effects. However, the effects of the AH flower on the biological activities have not been thoroughly investigated. This study reported the possible utilization of AH flower extracts (AHF) with different extraction methods and validated antioxidant and anti-wrinkle activities. The AHF was treated with hot water, ultra-sonic cold water, 70% ethanol, respectively. Total phenolic, flavonoid, and hydroxyproline content were estimated. Also, the antioxidant activity of extracts was determined with free radical scavenging (DPPH-2,2-diphenyl-1-picrylhydrazyl) and radical scavenging assay (ABTS 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). The highest phenolic and flavonoid contents were found in the 70% ethanol extracts (72.75 COE mg /g extract and 3,091.64 NAR mg /g extract, respectively). Interestingly, hydroxyproline content was recorded maximum in the cold-water extract. Ethanol extracts (70%) and ultra-sonic cold-water extracts showed the strongest antioxidant activities. Also, the HPLC profile shows that estrogen content was observed maximum in ethanol extracts. These results suggest that AHF extract provides promising antioxidant and anti-wrinkle effects and can be used as a potential natural cosmetic substance in cosmetic industries in the near future. Keywords : Aurea helianthus, medicine plant, extraction methods, extracts, polyphenol, antioxidant activity, anti-wrinkle effect References 1. Y. Kim, S. Lee, J. H. Ryu, K. D. Yoon and S. S. Shin, Biosci. Biotechnol. Biochem. 82(11), 1871-1879

(2018). 2. H. J. Kim, C. G. Park, R. Varghese, J. Y. Lee, Y. Kim and G. H. Sung,Saudi J. Biol. Sci. 24(8), 1943-1947

(2017). 3. Y. Park, K. Lee, C. Lee, A. Song, J. Kim, B. Kim and S. Lee, Sci. Rep. 10(1), 1-10 (2020).

199


PSP19 Functional and Nanostructured Carbon Materials for Supercapacitor Electrode from Bacterial Cellulose Produced from Wasted Glycerol

Sae Hee LEE, Amith ABRAHAM, Alan CHRISTIAN S. LIM, Jeong Gil SEO, Byoung-In SANG Department of Chemical Engineering, Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] Bacterial cellulose (BC), an eco-friendly nano-biomaterial, has attracted extensive attention due to its unique and robust physical characteristics such as excellent chemical stability and high purity and crystallinity, etc. These fascinating features make BC an ideal precursor for the production of highly versatile three-dimensional nano-carbon materials serving as a tunable and flexible scaffold materials. The low yield and high production cost are the challenges for the industrial production of BC. The present study used a cheap carbon source, wasted glycerol from the biodiesel industry, for BC production by Komagataeibacter sucrofermentans. BC was produced with high yield (6.5 g/L) and superior properties. The crystalline domains of BC increased up to 92.2% after removing the amorphous regions by acid hydrolysis. The properties of derived nano carbon materials were evaluated for the electrode application of supercapacitor battery after carbonization under high temperature. This study proposes a novel route for the valorization of waste resources to bio-nanomaterial and follows functional carbon nano-material production and flexible electrodes from BC for various energy systems. Keywords : Bacterial cellulose, Nano-carbon materials, Waste glycerol, Carbonization References 1. H. Zhu, F. Shen, W. Luo, S. Zhu, M. Zhao, B. Natarajan, J. Dai, L. Zhou, X. Ji, R. S. Yassar, T. Li, L. Hu,

Nano Energy, 33, 37-44 (2017).

200


PSP20 Pretreatment of Vacuum-fried Vegetable Makes Different Physical Properties of Fried Chips

Da-Song KIM, Hyun-Jae SHIN Department of Chemical Engineering, Graduate School of Chosun University, Gwangju, Korea Corresponding Author Email : [email protected] The vacuum frying technology is used in snack industries such as potato chips, but is not broadly used in processing agricultural products because of installation cost of the process. Recently, vacuum frying process performing at a pressure lower than atmospheric pressure is used to make novel snacks such as premium fruits and vegetable chips. Vacuum fried chips have low oil and acrylamide contents compared to deep fried ones, so its demand is increasing according to well-being trend. The vacuum fried chips have differences in physical properties depending on the pretreatment before frying, and product quality is strongly dependent on pretreatment methods. In this study, sweet potatoes, purple sweet potatoes, onions, and garlics were investigated with various pretreatments to measure their physical properties such as crispiness. The pretreatment methods used were pre-drying, blanching, osmotic dehydration, anti-browning agent and hydrocolloids coating. The physical properties of vacuum fried chips were measured for color, oil content, crispiness, and hardness. The results of this study can be used to produce premium- grade sweet potatoes, purple sweet potatoes, onions, and garlic-based agricultural products with high palatability and low acrylamide contents. Keywords : vacuum fried chips, vacuum frying, pretreatment, agricultural products, physical properties References 1. X. Zhang, M. Zhang, and B. Adhikari, Trends Food Sci. Technol. 98, 68-81 (2020). 2. A. O. Oladejo, H. Ma, W. Qu, C. Zhou, B. Wu, B. B. Uzoejinwa, D. Onwude, and X. Yang, J. Sci. Food

Agric. 98, 456-466 (2018). 3. J. Garayo, and R. Moreira, J. Food Eng. 55, 181-191 (2002).

201


PSP23 Development of a Strong Anaerobic Reporter System Based Halotag Protein for Screening Strong Novel Promoters in Hexanoic Acid-producing Clostridium sp. JS66

Linh NGUYEN KHANH, Ki-Yeon KIM, Youngsoon UM Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea Corresponding Author Email : [email protected] Enhancing the production of target substances using a strong promoter is one of the key strategies in developing genetically engineered strains. However, a common fluorescent reporter system to screen a strong promoter in aerobes has faced a big limitation for anaerobes like Clostridium strains. Here we report the evaluation of an alternative strong fluorescent reporter for promoter screening in a hexanoic acid-producing Clostridium sp. JS66 using the Halotag protein, which is a modified haloalkane dehalogenase covalently binding to synthesis ligands to produce strong fluorescent signals. Based on the transcription data on Clostridium sp. JS66 during syngas fermentation, 3 promoters with the highest transcription levels were chosen. The fluorescent signals using Halotag-based reporter with 3 promoters were compared with the endogenous thl promoter of C. acetobutylicum ATCC824 commonly used as a strong promoter for Clostridium strains. The labelling results were compared with transcriptome to check the correlation between fluorescence levels and transcription levels. The findings will facilitate future work to develop anaerobes for synthetic valuable biological products. Keywords : Clostridium, Halotag, Promoter screening, flourescent reporter system

202


PSP24 Adsorption of Micropollutants Using Fermented Waste Saccharomyces cerevisiae

Se-Been MUN, Se-Ra JIN, Chul-Woong CHO Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Fermentation waste i.e., Saccharomyces cerevisiae disposal problems in the bio-industry are serious. Therefore, its recycling method should be developed. As one of solutions, it can be used to remove micropollutants as an adsorbent[1]. Therefore, we estimated the adsorption affinity of S. cerevisiae for micropollutants. For the experiment, the fermentation waste was washed with distilled water with no more pretreatment and then freeze-dried As target compounds, we selected several kinds of organic micropollutants in cationic, anionic, and neutral forms. The experimental results showed that the S. cerevisiae adsorbed cationic and neutral micro-pollutants at the natural pH 6.5, but it has no remarkable adsorption with anions. In order to extend the application to uninvestigated compounds, we developed quantitative structure-activity relationship model i.e. linear free energy relationship[2]. These studeis clearly exhibited that the fermentation waste can be used as adsorbent for removal of organic micropollutants. It is believed that this result will help recycle fermentation waste and remove ionic and neutral micropollutants. Keywords : adsorption, micropollutants, modeling, fermentation waste References 1. Cho, C.W., et al., Prediction of organic pollutant removal using Corynebacterium glutamicum fermentation

waste. Environmental Research, 2021. 192. 2. Abraham, M.H. and W.E. Acree, The transfer of neutral molecules, ions and ionic species from water to wet

octanol. Physical Chemistry Chemical Physics, 2010. 12(40): p. 13182-13188.

203


PSP25 Process Variable Optimization of Kenaf Through Torrefaction Based on Physical and Chemical Properties

Seongrae LIM1, Heesun YOON1, Gahee KIM2, Byunghwan UM3 1Department of Chemical Engineering, Hankyong National University, Anseong, Korea, 2Interagency Convergence on New Biomass Industry, Hankyong National University, Anseong, Korea, 3Department of Food Biotechnology and Chemical Engineering and Interagency Convergence on New Biomass Industry, Hankyong National University, Anseong, Korea Corresponding Author Email : [email protected] Torrefaction is a promising technique as a pretreatment of biomass. Since Kenaf (Hibiscus cannabinus) is a herbaceous biomass, energy densification is a compulsory task prior to its use. Cellulose and hemicellulose fractions in Kenaf can be degraded through torrefaction, which leads to increase in heating value and decrease in bulk density. In this work, Kenaf was torrefied at 220, 260, 300 and 340 for 10min. Each conditions were named light (220), mild (260), severe (300) and extra-severe (340). Severe and extra-severe conditions showed notable difference in yields and heating values of 81.56%-19.15MJ/kg and 50.54%-23.75MJ/kg, respectively. Also, moisture and ash contents of torrefied Kenaf were measured since these two parameters are major constituents for herbaceous biomass prior to its use. Consequently, physical and chemical properties of Kenaf changed through torrefaction, which implies designing optimal torrefaction parameter is critical to achieve desired product. Keywords : Torrefaction, Kenaf, Herbaceous biomass, Process variable References 1. Y. Joshi, M. D. Marcello and W. de Jong, J. Anal. Appl. Pyrol. 115, 353 (2015). 2. S. V. Vassilev, D. Baxter, L. K. Andersen and C. G. Vassileva, Fuel 105, 40 (2013). 3. M. Phanphanich and S. Mani, Bioresour. Technol. 102, 1246 (2011).

204


PSP26 Development of Prediction Models for Adsorption Affinities Between Micropollutants and Agricultural Wastes

Bogyeon CHO1, Chul-Woong CHO1,2 1Department of Integrative Food Bioscience and Biotechnology, Chonnam National University, Gwangju, Korea, 2Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Water pollution due to micropollutants and an increase of agricultural waste are serious environmental issues. To solve the issues, we applied the agricultural waste to remove organic micropollutants. For this study, we selected three types of agricultural-derived biowastes i.e., peels of orange, mandarin, and banana, and they were only water washed, grinded, and sieved with pore size 106 µm. The experimental results revealed that the wastes can be used to adsorb organic micropollutants. Especially, they have significant adsorption affinity for cationic compounds, while low adsorption for neutral compounds and ignorable for anions. To understand the adsorption phenomena in molecular level and to predict the adsorption affinity between biowaste and micropollutants, we developed linear free energy relationship (LFER) models. The developed LFER models have high predictabilities with R2 values of 0.80 ~ 0.90. From the modeling, we characterized important molecular interaction potentials contributing to the adsorption and provide an possibility to extend the application to uninvestigated micropollutants. These results exhibited that the agricultural wastes can apply to efficiently remove environmentally issued organic micropollutants in neutral and cationic forms. Keywords : Micropollutants, Absorption, Agricultural wastes, Recycling, Modeling

205


PSP27 Research on a Proper Deep Learning Algorithm for Automated Detection of Coagulative Necrosis in SD Rat

Hyun-Ji KIM1,2, Ji-Hee HWANG1, Heejin PARK1, Byoung-Seok LEE1, Yong-Bum KIM1, Jun HER3, Sang-Yeop JUN3, Jong-Hyun PARK3, Jaeku LEE3, Hwa-Young SON2, Jae-Woo CHO1 1Toxicologic Pathology Research Group, Department of Advanced Toxicology Research, Korea Institute of Toxicology, Daejeon, Korea, 2College of Veterinary Medicine, Chungnam National University, Daejeon, Korea, 3LAC Inc., Seoul, Korea Corresponding Author Email : [email protected], [email protected] The development of artificial intelligence (AI) has led to applications in related academic fields. AI’s capacity for detecting trained images can be applied extensively to tissue abnormality, as well as image analysis. We applied four deep learning-based algorithms including Mask R-CNN, DeepLab, YOLO, and SSD to detect coagulative necrosis in the liver which is critical toxicological evidence in a non-clinical study. The coagulative necrosis was induced by intraperitoneal injection of acetaminophen in SD rats. The necrosis images for the label were confirmed by board-certified veterinary pathologists, and a total of 2,100 labels was used for the data set. All the steps were operated by Tensorflow 2.1.0, and the mean average precision (mAP) was used to calculate the model’s performance. As a result, accuracy rates were followed up by in order of Mask R-CNN (100%), DeepLab (99.52%), SSD (98.1%), and last YOLO (78.1%). Considering the result, the accuracy gained from the test was encouraging, and Mask R-CNN might be an appropriate algorithm for the precise detection of necrosis. Keywords : Deep learning, Liver coagulative necrosis, Non-clinical study

206


PSP28 Label-free Classification of Acute Myeloid Leukemia Cells Through Optical Diffraction Tomography and Deep Learning

Young Seo KIM1,2, Yong-ki LEE2,3, DongHun RYU1,2, Yoosik KIM2,3, YongKeun PARK1,2,4 1Department of Physics, KAIST, Daejeon, Korea, 2KAIST Institute for Health Science and Technology (KIHST), KAIST, Daejeon, Korea, 3Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea, 4TomoCube, Inc., Daejeon, Korea Corresponding Author Email : [email protected] / [email protected] Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by abnormal proliferation of immature myeloblast. Over the past few years, medical experts established clinical guidelines for the diagnosis and treatment of AML. The current diagnostic workup of AML is mainly performed through peripheral blood and bone marrow examination, including cytochemistry, immunophenotypic, and cytogenetic analyses. However, the labeling process is mandatory for most procedures, which makes the diagnosis more time-consuming and laborious. Here, we propose a novel computational method for the diagnosis of AML through optical diffraction tomography (ODT) and deep learning. ODT measures the 3D refractive index (RI) distribution of a single cell. We trained a 3D convolutional neural network (CNN), inspired by FishNet, which extracts unique features from the RI voxels of unlabeled AML cells. A total of six AML cell lines were classified via the trained deep learning algorithm, resulting in high accuracy (> 95%). Thus, we envision that this computational framework will remedy the shortcomings of the conventional diagnosis procedure. Keywords : Acute myeloid leukemia, Diagnosis, Optical diffraction tomography, Convolutional neural network, Deep learning

207


PSP30 Metabolic Engineering of Yarrowia lipolytica for Production of Ricinoleic Acid by Redirection of Lipid Flux Towards Phospholipids

Kwanghyun PARK, Ji-Sook HAHN School of Chemical and Biological Engineering, Institute of Chemical processes, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Ricinoleic acid (RA), which makes up the majority of castor oil, is an unusual hydroxylated fatty acid used in a variety of oleochemical industries, especially to produce bioplastics as a petrochemical replacement. An oleaginous yeast Yarrowia lipolytica can accumulate lipids more than 20% of dry cell weight (DCW), which is advantageous as a host for the production of various lipid products. In this study, we constructed RA-producing Y. lipolytica strain by overexpressing CpFAH12 gene from Claviceps purpurea encoding Δ12 hydroxylase. CpFAH12 hydroxylates oleic acid esterified on the sn2 position of phosphatidylcholine, generating RA. To improve RA production, we deleted FAD2 gene encoding Δ12 desaturase, a competing enzyme producing linoleic acid from oleic acid, and genes involved in beta-oxidation of fatty acids. Furthermore, when the lipid flux was redirected towards phospholipids by additional genetic modifications, the RA content increased to over 75 mg/gDCW, constituting more than 50 % of total fatty acids. Keywords : Ricinoleic acid, Yarrowia lipolytica, metabolic engineering

208


PSP31 Development of a Synechocystis sp. PCC 6803 Cell-free Protein Synthesis System

So Jeong LEE1, Seong-Joo HONG2, Choul-Gyun LEE2, Dong-Myung KIM1 1Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 2Institute of Industrial Biotechnology, Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Synechocystis sp. PCC 6803 is one of the most extensively studied cyanobacterial species, and is expected to be used as a platform strain for producing a wide range of valuable compounds. Although many molecular biology tools have been developed to genetically engineer this organism, the discipline of cell-based genetic engineering is constrained by the intrinsic requirements for vector construction, gene transfer, and cell cultivation, which limit the throughput of the strain development. In recent years, cell-free systems have emerged as a powerful platform to speed up the engineering of microorganisms. Unlike the cell-based methods relying on the growing cells, cell-free synthesis systems harnesses cell extract that retains most of the cellular enzymes. Due to the lack of physical barriers, the cell-free systems allow direct deployment of the genes of interest to the extract, enabling rapid prototyping and analysis of genetic modifications. In this study, a cell-free synthesis system was successfully constructed from Synechocystis sp. PCC 6803. As a result of extensive optimization experiments using superfolder green fluorescence protein as a model, we were able to prepare a cell-free synthesis system that has translational activity enough to produce 15 μg/mL of recombinant proteins. Keywords : Cell-free synthesis, Cyanobacteria, Synthetic biology References 1. Y. Yu, L. You, D. Liu, W. Hollinshead, Y. J. Tang, F. Zhang, Mar. Drugs. 11, 2894 (2013). 2. K. H. Lee, D. M. Kim, FEMS Microbiol. Lett. 365, fny174 (2018).

209


PSP32 Improving Succinate Production Using Methanotroph by N-source Control

Jae-Hwan JO1,2, Jeong-Ho PARK1,2, Min-Sik KIM3 1Gwangju Bio/Energy R&D Center, Korea Institute of Energy Research, Gwangju, Korea, 2Interdisciplinary Program of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea, 3Energy Resources Upcycling Research Laboratory, Korea Institute of Energy Research, Daejeon, Korea Corresponding Author Email : [email protected] Methane is considered as a next generation feedstock due to its cheap price and abundant amount. Methanotroph, which metabolize methane as a carbon source, have been steadily developed as an industrial bio-catalyst. In this study, we produced succinic acid using Methylomonas sp. DH-1. Succinic acid is used in various industries such as cosmetics, pharmaceuticals, agriculture, and food. Methylomonas sp. DH-1 CA strain was obtained by genetic engineering, where succinate dehydrogenase (sdhB) genes was replaced with glyoxylate shunt genes from E. coli to increase succinic acid production. By culturing CA strain repeatedly, a mutant strain (CA40) with high gas consumption rate and succinic acid titer was obtained. CA40 strain showed a high maximum methane consumption rate (7.12mmol/L/h) compared with CA strain (4.78mmol/L/h) and increasing succinic acid titer 1.66-fold (381.4mg/L) compared with CA strain (230.7mg/L). By culturing CA and CA40 strain in 3L CSTR fermenter, CA40 strain consumed most of provided nitrate within 30 hours culture while CA strains did not use up the nitrate. So, we performed nitrate fed-batch culture to prevent N-source deficiency during cultivation. By feeding nitrate after N-source depletion, succinic acid titer was 1.41-fold increased (536.8mg/L) compared with nitrate deficiency condition (381.4mg/L). This study showed that repeating subculture in NMS media and the addition of nitrate to the NMS media eventually contributed to the increase of the succinate titer of Methanotroph. Keywords : Methanotroph, Succinate, N-Source, Fed-batch fermentation

210


PSP33 Metabolic Engineering of Yarrowia lipolytica to Produce Shinorine, a Sunscreen Material

Hyunbin JIN, Chaeyeon JIN, Sojeong KIM School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Mycosporine-like amino acids (MAAs), are bio-based sunscreen materials, which occur naturally in various marine species and other microorganisms. Shinorine is a well-known MAA which absorbs UV radiation, especially UV-A. In this study, we produced shinorine in oleaginous yeast Yarrowia lipolytica by introducing cyanobacterial shinorine biosynthesis genes. Sedoheptulose 7-phosphate (S7P), a starting material of shinorine biosynthesis, is one of the pentose phosphate pathway (PPP) intermediates. Because xylose is assimilated via PPP, we increased carbon flux into the PPP by using xylose as a carbon source after integrating the xylose assimilation genes into the genome. Glycerol is one of the most preferred substrates of Y. lipolytica. Therefore, we further improved shinorine production by using glycerol as a co-substrate with xylose. Keywords : sunscreen, Yarrowia lipolytica, pentose phosphate pathway, shinorine, xylose, glycerol, MAAs

211


PSP34 Integrated Multi-omics Analysis of Differentially Expressed Genes During Growth in Streptomyces griseus

Soonkyu HWANG1,2, Namil LEE1,2, Donghui CHOE1,2, Yongjae LEE1,2, Woori KIM1,2, Ji Hun KIM1,2, Yujin JEONG1,2, Suhyung CHO1,2, Bernhard PALSSON3,4,5, Byung-Kwan CHO1,2,6,7 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 4Department of Pediatrics, University of California San Diego, La Jolla, CA, USA, 5Novo Nordisk Foundation Center for Biosustainability, 2800 Kongens Lyngby, Denmark, 6Intelligent Synthetic Biology Center, Daejeon, Korea, 7Innovative Biomaterials Research Center, KAIST Institutes, Korea Advanced Institute of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Streptomyces have been noticed for the ability to produce secondary metabolites. The secondary metabolism is regulated by complex regulatory systems including the metabolic shift from primary metabolism to secondary metabolism. Despite much effort to understand well-orchestrated gene expressions during Streptomyces growth, system-level information remains scarce. In this study, four multi-omics data of model strain S. griseus NBRC 13350, RNA-seq, ribosome profiling, dRNA-seq, and term-seq, were integrated and analyzed. As a result, different gene functions were enriched according to their transcription patterns, and potential governing sigma factors and transcription factors were found. Also, potential regulatory elements related to 3'-end of transcripts, codon usage, and RNA structures were elucidated. This comprehensive genetic information will provide a foundational resource for rational engineering of Streptomyces. ** This work was supported by the Novo Nordisk Foundation (NNF10CC1016517 to BOP), and the Bio & Medical Technology Development Program (2018M3A9F3079664 to B-KC) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT). Keywords : Streptomyces, differential gene expression, regulatory elements, sigma factor, transcription factor, transcript 3'-end positions, codon usage

212


포스터발표

213


나노바이오공학

(Nano-Bioengineering)

214


P0001 Magnetic Particle-b ased Aptamer Selection for the Detection of Various Analytes

Myeongjune GO, Seung-Woon JUNG, Seung Min YOO School of Integrative Engineering, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Aptamers are oligonucleotide that can bind with specific target covering from small molecules to cell. Aptamers are easily synthesized, modified, stable, and biocompatible. Owing to these distinct advantages, many aptamer applications in diagnosis, therapeutics, and drug delivery carrier have been studied. Aptamers are selected by systematic evolution of ligands by exponential enrichment (SELEX) and the method is diverse with how to separate aptamer candidate from random oligonucleotide pool. In this study, aptamers are selected by simple SELEX by using streptavidin-coated magnetic bead for the detection of violacein, adenosylcobalamin, and melittin. Streptavidin-coated bead was functionalized with biotinylated short length DNAs (B-DNA) which have sequences complementary to random library, forming B-DNA:aptamer duplex. The sequential binding event of target chemicals and aptamers leads a release of aptamers from duplex, following their separation by magnet. This method is expected to provide simple and rapid aptamer selection targeting various kinds of target. ** This work was supported by an NRF grant funded by the Ministry of Science and ICT (NRF-2019R1A2C1088504) Keywords : aptamer, SELEX, magnetic bead, detection References 1. R. Nutiu, Y. Li, Angew. Chem. Int. Ed., 44, 1061-1065 (2005).

215


P0002 Highly Sensitive Gold Nanourchin for Rapid Bovine Coronavirus Detection

Ruth STEPHANIE, Sung Hyun KIM, Min Woo KIM, Chan Yeong PARK, Tae Jung PARK Department of Chemistry, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Bovine coronavirus (BCoV) is a type of pneumoenteric virus that causes respiratory and enteric infections for cattle and wild ruminants. This is a serious threat not only for livestock farms but also to the public health as they are the main source of raw materials for the industry of food and dairy products. This makes the mitigation for bovine diseases becoming important, which requires rapid, stable, and most importantly accurate detection system to prevent production loss. To fulfil those requirements, gold nanoparticles (AuNP) are usually exploited for biosensing detection system – a system known for its simplicity and rapidity. As an effort to increase the sensitivity of the biosensor, enlargement of the surface area of AuNP is preferred, which can be achieved by modifying the shape into nanourchins. Hence, this study demonstrates the synthesis, optimization, and development of gold nanourchins to be furtherly implemented as the active material for BCoV detection. Keywords : bovine coronavirus, gold nanoparticle, electrochemical biosensor, signal enhancement, gold nanourchin References 1. L. J. Saif, Vet. Clin. N. Am.-Food Anim. Pract., 26(2), 349-36 (2010). 2. I.G. Theodorou, Z.A. Jawad, Q. Jiang, E.O. Aboagye, A.E Porter, M.P. Ryan, F. Xie, Chem. Mater., 29(16),

6916-6926 (2017).

216


P0003 Rosette-shaped Graphitic Carbon Nitride as a Highly Efficient Peroxidase Mimic for Glucose Biosensing

Thinh Viet DANG1, Yun Suk HUH2, Moon Il KIM1 1Department of BioNano Technology, Gachon University, Seongnam, Korea, 2Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Graphitic carbon nitride (GCN) has attracted much attention owing to its excellent photocatalytic and fluorescent properties [1]. Herein, we have developed rosette-shaped GCN as a promising alternative to natural peroxidase, through a rapid reaction between melamine and cyanuric acid for 10 min at 35oC, followed by thermal calcination for 4 h. Importantly, rosette-shaped GCN exhibited an excellent peroxidase-like activity, which is ~10-fold higher than that of conventional bulk-GCN, presumably due to its large surface area and high porosity for increasing the number of catalytic events. Rosette-GCN also possessed the peroxidase activity in a wide range of pH including physiological conditions, which is rare to be found from other peroxidase mimics. By exploiting this unique property of rosette-GCN, glucose was successfully determined as low as 1.2 µM using fluorescence-based reaction with the catalytic action of glucose oxidase (GOx) at neutral pH conditions. In the presence of glucose, GOx catalysed its oxidation to produce H2O2, which is subsequently consumed by peroxidase-mimicking rosette-GCN to convert Amplex UltraRed (AUR) into highly fluorescent product [2]. Deploying this strategy, glucose was successfully determined with excellent selectivity and sensitivity with high detection precisions. This rosette-GCN would be highly beneficial for simple and sensitive detection of biologically important target molecules, hence, be widely utilized for sensing applications. Keywords : Graphitic carbon nitride, Peroxidase mimic, Glucose detection, Fluorescent biosensor References 1. X. Wang, L. Qin, M. Lin, H. Xing and H. Wei, Anal. Chem. 91, 10648 (2019). 2. N. S. Heo, H. P. Song, S. M. Lee, H. J. Cho, H. J. Kim, Y. S. Huh and M. I. Kim, Microchim. Acta 187(5),

286 (2020).

217


P0004 Continuous Synthesis of DMPC/DHPC Bicelle Using Hydrodynamic Focusing Micro-mixer and Analysis of Its Physicochemical Properties

Bong Su KANG1, Sung Hak CHOI2, Kee Sung KIN3, Ho Sup JUNG2, Moon Kyu KAWK1 1School of Mechanical Engineering, Kyungpook National University, Daegu, Korea, 2Center for Food and Bioconvergence, Department of Food Science and Biotechnology, Seoul National University, Seoul, Korea, 3Research Inst. of Advanced. Materials, Collage of Engineering, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected], [email protected] Phospholipids are the amphiphilic material that self-assembled in the aqueous solution and compose the bilayered membrane. Bicelle is a disk-like shape self-assembly that comprises a long-chain lipid bilayer domain and a short-chain lipid rim. Due to its structural characteristics, bicelle has outstanding stability compare to other lipid based assemblies. Generally, bicelles are prepared by lipid thin-film method, which compose of complicated processes, so it is necessary to develop a more efficient production process to increase the application of bicelle. In this study, we suggest a continuous synthesis of bicelles by using hydrodynamic microfluidic mixer. The hydrodynamic focusing mixer is a widely known platform, and lots of studies tried to fabricate the self-assembled bio-membrane using this mixer. Various types of microfluidic mixers were fabricated through microfabrication processes, including photolithography and soft lithography. DMPC (14:0 PC) and DHPC (06:0 PC) were applied to investigate the continuous productivities. The size and the physicochemical properties of assemblies were investigated to evaluate the quality of synthesized assemblies. Keywords : MEMS, Microfluidic chip, Self-assembly, Phospholipid, Bicelle References 1. Zook, J. M.; Vreeland, W. N., Effects of temperature, acyl chain length, and flow-rate ratio on liposome

formation and size in a microfluidic hydrodynamic focusing device. Soft Matter 2010, 6, (6), 1352-1360.

218


P0005 Towards Reliable Marine Toxin Detection: Discovering Saxitoxin-specific Bioreceptor via Phage-display Technique

So Yeon KWEON, Chan Yeong PARK, Ruth STEPHANIE, Sung Hyun KIM, Yeong Hyeock KIM, Tae Jung PARK Department of Chemistry, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Marine neurotoxins are natural products primarily generated from phytoplankton and several types of invertebrates and fish. Their classification as neurotoxins stems from their ability to interact with voltage-gated sodium, potassium, and calcium channels, modulating the flux of these ions into various cell types, hence disrupting the neural system.1) Among various kinds of marine neurotoxins, saxitoxin (STX) poses as a serious threat due to its high toxicity and wide distribution that will impact public health significantly. Hitherto, the internationally accepted method, standardized by the Association of Analytical Communities, for STX quantitative analysis is mouse bioassay.2) However, this method is hindered by its ethical issues, in addition to also being costly and highly laborious. As a way to overcome the problem, this study is aimed to develop STX-specific bioreceptors via phage display technology. For this technique, the haptenization of STX was carried out by immobilizing them on the bovine serum albumin through formaldehyde-mediated crosslinking. This is compulsory to enhance the immunogenic effects. The prepared hapten is characterized with SDS-PAGE and MALDI-TOF, then applied for the phage display to discover the STX-specific bioreceptor, so that it can be used furtherly for STX-detection in varying platforms. Keywords : Saxitoxin, Hapten, Phage display, AuNPs, Fluorescence-based detection References 1. Cusick, Kathleen D., Gary S. Sayler, Marine drugs 11.4, 991 (2013). 2. Gao, Shunxiang, Xin Zheng, Jihong Wu, Sensors and Actuators B: Chemical 246, 169 (2017).

219


P0006 Screening of Aptamers Against Bst Polymerase for Increasing Reverse Transcriptase Activity

Do-Young KIM1, Woo-Ri SHIN1, Simranjeet Singh SEKHON1, Sang Yong KIM2, Ji-Hyang WEE2, Ji-Young AHN1, Yang-Hoon KIM1 1Major in Microbiology School of Biological Sciences College of Natural Sciences, Chungbuk National University, Cheongju, Korea, 2Department of Food Science and Biotechnology, Shin Ansan University, Ansan, Korea Corresponding Author Email : [email protected] Loop Mediated Isothermal Amplification(LAMP) is a PCR method that amplifies the target gene using 6 primers. In contrast to the general PCR methods, LAMP does not require a thermal cycler. Bst polymerase is used as enzymes for LAMP methods, that has a weak activity of Reverse Transcriptase(RT). In this study, we developed aptamers against bst polymerase for increasing RT activity using SELEX(Systematic Evolution of Ligands by exponential Enrichment). We confirmed the eluted aptamers concentration for screening of the efficiency of bst aptamer candidates using Nano-drop spectrophotometer. After the SELEX round, the bst-aptamer complex will be measured affinity value using SPR and we will predict the 3-D structure using MOE software. This study can provide a rapid and simple diagnostic methods for disease that are mediated by RNA virus. ** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1010860). ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235). Keywords : Aptamer, Bst polymerase, LAMP, Increasing activity, SELEX

220


P0007 Development of Cholesterol Detection Based on the Bienzyme Nanoparticle by Electrochemical Measurement

Min Woo KIM, Chan Yeong PARK, Tae Jung PARK Department of Chemistry, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] As an essential component for the cellular membrane and nutrients, cholesterol carries out the pivotal roles in normal body functioning. The high level of cholesterol in the blood (> 5 mM) not only induces hypercholesterolemia, which is the accumulation of cholesterol on the arterial vessel wall, but also promotes the development of the cardiovascular disease, type 2 diabetes, and several neurological diseases, thus, managing the level of cholesterol is an important issue. To monitor the cholesterol level, enzyme-based electrochemical sensors were designed. There has been a focus on ChOx-based electrochemical methods. However, the enzyme-based electrochemical sensor has some limitations such as immobilization of enzyme, denaturation, degradation, and the loss of its activity onto the metal electrode. To resolve these problems, in this study, enzyme nanoparticles, the HRP, and obtained ChOx were utilized to synthesize the bienzyme nanoparticle (BENP) with different ratios to enhance mutual synergistic sensing activity. The resulting BENP showed promising selectivity and the developed BENP-based cholesterol electrochemical sensor is shown to have a low detection limit (0.18 mg/dL), high selectivity, and long-term stability (> 25 days). Keywords : Cholesterol detection, Enzyme nanoparticles, Nanobio-sensor, Electrochemistry References 1. Gomathi, P., Ragupathy, D., Choi, J.H., Yeum, J.H., Lee, S.C., Kim, J.C., Lee, S.H., Do Ghim, H., Sens.

Actuator B-Chem., 153(1), 44-49 (2011). 2. Aggarwal, V., Malik, J., Prashant, A., Jaiwal, P.K., Pundir, C.S., Anal. Biochem., 500, 6-11 (2016).

221


P0008 Effective Treatment for Cervical Cancer Through Folic Acid Modified Bovine Serum Albumin Nanoparticles Combining Chemo-photodynamic Therapy

Suheon KIM1, Hoomin LEE1, Shruti SHUKLA2, Vivek K. BAJPAI3, Young-Kyu HANC3, Yun Suk HUH1 1Department of Biological Engineering, NanoBio High-Tech Materials Research Center, Inha University, Incheon, Korea, 2Department of Food Science and Technology, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonipat, Haryana, India, 3Department of Energy and Materials Engineering, Dongguk University-Seoul, Seoul, Korea Corresponding Author Email : [email protected] The Doxorubicin (DOX) of chemotherapy has side effects and Chlorin e6 (Ce6) of photodynamic therapy is not effective due to low solubility. We developed the folic acid modified bovine serum albumin-based nanoparticles loaded DOX and Ce6 (FA-Ce6/DOX/BNPs) to solve the problem. Combined chemo and photodynamic therapy exhibited sphere morphology and a negative zeta potential. Confocal microscopy analysis further validated cellular uptake of FA-Ce6/DOX/BNPs by HeLa cells. Moreover, FA-Ce6/DOX/BNPs potentiated mitochondrial reactive oxygen species (ROS) production in HeLa cells under 671-nm laser exposure, leading to activation of key regulator proteins of apoptosis. Furthermore, we evidenced by gene/protein expression level of apoptotic/anti-apoptotic biomarkers by real time quantitative PCR(RT-qPCR) and western blot analysis. FA-Ce6/DOX/BNPs use in chemo-photodynamic combination therapy with FA as a target cancer cell receptor may provide more effective anti-cancer effects with decreased side effects in cervical cancer. Keywords : chemotherapy, photodynamic therapy, BNPs References 1. H. Jeong, M. Huh, S.J. Lee, H. Koo, I.C. Kwon, S.Y. Jeong, K. Kim, Theranostics. 1 (2011) 230-239. 2. W. Park, S. Cho, J. Han, H. Shin, K. Na, B. Lee, D.-H. Kim, Biomater. Sci. (2018) 79-90.

222


P0009 Co-encapsulation of Multiple Enzymes and Cofactors into Nanoparticles

Seoungkyun KIM, Kiyoon KWON, Giyoong TAE, Inchan KWON School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] Oxidoreductases have been used for synthesis of value-added chemicals via redox reactions due to its selectivity, reaction rate and mild reaction condition. However, these enzymes often require cofactors, which are expensive and small molecules making them difficult to reuse in the immobilized enzyme processes. To overcome this hurdle, there have been attempts to immobilize and regenerate cofactors. However, it is still difficult to build a sustainable enzyme process in a typical industrial continuous or repeated batch processes because cofactors are removed from reaction mixtures along with the reaction products. In this study, we investigated co-encapsulation of enzymes and cofactors using nanoparticles. Keywords : Oxidoreductase, Cofactor, immobilization, Repeated batch

223


P0010 Fabrication of Electrochemical Aptasensor that Ag+ Ion Intercalated-DNA 3Way Junction on Micro-gap for Interferon Gamma Detection in Human Serum

Seungwoo NOH, Taek LEE Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] IFN-γ is a cytokine and has antiviral and antimicrobial properties. To detect the IFN-γ, author fabricated a electrochemical biosensor using Ag+ intercalated multi-functional DNA 3-way junction (MF-3WJ) as a bioprobe. The signal of the biosensor through the MF-3WJ-based SWV method showed a linear range of 1 pg/ml to 10 ng/ml in 10% diluted human serum, and the limit of detection was 0.42 pg/ml. Acknowledgements This research was funded and conducted under「the Competency Development Program for Industry Specialists」of the Korean Ministry of Trade, Industry and Energy (MOTIE), operated by Korea Institute for Advancement of Technology (KIAT). (No. P0002397, HRD program for Industrial Convergence of Wearable Smart Devices) and by Korea Environment Industry&Technology Institute(KEITI) through the program for the management of aquatic ecosystem health, funded by Korea Ministry of Environment(MOE).(2020003030001), and by the Industrial Core Technology Development Program(20009121, Development of early diagnostic system of peritoneal fibrosis by multiplex detection of exosomal nucleic acids and protein markers) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) Keywords : IFN-γ detection, DNA 3-way junction, electrochemical biosensor

224


P0011 Development of Bioelectronic Nose Using Human Olfactory Receptor-embedded Nanodiscs for Diagnosing Gastric Cancer

Dongseok MOON, Tai Hyun PARK School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] The gastric cancer was the fifth frequently diagnosed cancer and the third main cause of death associated with cancer. Many researchers analysed breath from gastric cancer patients and found some carboxylic acids regarded as gastric cancer biomarkers, such as propionic acid, butyric acid, pentanoic acid and hexanoic acid. Nanodisc is a suitable platform to reconstitution of transmembrane proteins because of its stable structure mimicking the native membrane environment. In this research, we purified human olfactory receptors from Escherichia coli and reconstituted the olfactory receptors for native structure of receptors into nanodisc form. Human olfactory receptors (hORs) were selected for detecting the gastric cancer biomarkers which have a carboxylic gourp, propionic acid (hOR51E1, hOR51E2, hOR52D1), butyric acid (hOR51E1, hOR52D1), pentanoic acid (hOR51E1) and hexanoic acid (hOR51E1). In luciferase assay system, we confirmed the three olfactory receptors had different binding affinities to gastric cancer biomarkers respectively. The olfactory receptor-embedded nanodiscs were immobilized on graphene-based field-effect transistor (GFET) for development of bioelectronics nose diagnosing gastric cancer. We expect nanodisc-immobilized bioelectornic nose could sensitively and selectively diagnose the gastric cancer by patterns of olfactory receptors. Keywords : Human olfactory receptor, Nanodisc, Gastric cancer, Pattern, Bioelectronic nose References 1. F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre and A. Jemal, CA. Cancer J. Clin. 68, 394-

424 (2018). 2. J. Oh, H. Yang, G. E. Jeong, D. Moon, O. S. Kwon, S. Phyo, J. Lee, H. S. Song, T. H. Park and J. Jang,

Anal. Chem. 91, 12181-12190 (2019). 3. M. E. Adam, M. Fehervari, P. R. Boshier, S. Chin, G. Lin, A. Romano, S. Kumar and G. B. Hanna, Anal.

Chem. 91, 3740-3746 (2019).

225


P0012 Intein Spliced Di-nanoperforator with Higher and Broad-spectrum Anti-influenza Activity

Seokoh MOON, Mi Soo KIM, Dae-Hyuk KWEON Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] Nanodisc is a nano-sized discoidal lipid bilayer made of phospholipids and membrane scaffold proteins (MSPs). In previous studies, we showed that the nano-perforators, receptor embedded nanodiscs, inhibited influenza A virus infection by entrapping viral RNPs in endolysosome. While GD1a and synthetic receptor composed of a2,6-sialic acid inserted nano-perforators were able to inhibit a2,3- and a2,6-sialic acid targeting viruses respectively, applying two different receptors to single mono-perforators was not effective to inhibit infection of multiple virus strains simultaneously. Therefore, two mono-perforators conjugated di-nanoperforator was devised to target broad spectrum of influenza viruses with higher antiviral activities. For assembly of di-nanoperforator, split intein domains were fused to each N- or C- terminal of MSP and intein ligated mono-perforators were assembled well with those MSPs. Di-perforators were well established by trans-splicing between split intein ligated nano-perforators and total ganglioside inserted homo di-nanoperforators showed over 6 times higher cytophatic effects inhibition than mono-perforators in same disc concentration. The GD1a (a2,3-sialic acid) and synthetic receptor (a2,6-sialic acid) inserted hetero di-nanoperforators were able to inhibit 4 strains of influenza A viruses at the same time. Moreover, the bifunctionality of hetero di-perforators derived increased antiviral activities than mono-perforators. Our results suggest the di-nanoperforator as enhanced virucide for broad-spectrum virus stains. Keywords : influenza virus, nanoparticle, membrane fusion, viral infection, trans-splicing

226


P0013 Fabrication of Electrochemical Biosensor for H1N1 Detection

Jeongah PARK Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] The recent epidemic has raised the importance of early diagnosis of the virus. In order to design a biosensor capable of detecting H1N1(Influenza A virus subtype), This sensor used a multifunctional aptamer that selectively binds only to H1N1. It also introduced carboxyl-molybdenum disulfide to increase sensitivity. H1N1 was confirmed by electrochemical impedance spectroscopy. We also confirmed that H1N1 could be detected from 100 nM to 10 pM and the detection limit was 10 pM. This sensor is inexpensive and has a wide range of applications. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07049407) by Korea Environment Industry&Technology Institute(KEITI) through the program for the management of aquatic ecosystem health, funded by Korea Ministry of Environment(MOE).(2020003030001) by the Industrial Core Technology Development Program(20009121, Development of early diagnostic system of peritoneal fibrosis by multiplex detection of exosomal nucleic acids and protein markers) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) Keywords : Electrochemical Biosensor, DNA 4WJ, Influenza Virus, H1N1, Molybdenum Disulfide Nanoparticle

227


P0014 Monitoring of Multiple Metabolites in Bacterial Communication Using Electrochemical Sensor

Jong Seong PARK1, Mijeong KANG2, Yeonggyu JO1, Chae Won MUN2, Ho Sang JUNG2, Dong-Ho KIM2, Sung-Gyu PARK2, Seung Min YOO1 1School of Integrative Engineering, Chung-Ang University, Seoul, Korea, 2Advanced Nano-Surface Department, Korea Institute of Materials Science (KIMS), Changwon, Korea Corresponding Author Email : [email protected] The monitoring of metabolite secretion by bacterial communication has broad applications in research as well as in the diagnosis and monitoring of infections. To achieve this with great sensitivity, in the present study, we report a strategy to maximize the electrochemical performance of nanostructured electrodes. We utilize the nanoscale space of a Au nanostructured electrode to structurally facilitate the cycling reactions of the communication-related metabolites of bacteria and significantly amplify their electrochemical signals. As applications, we functionalize Au nanostructured electrodes to monitor the secretion of the multiple signaling metabolite from Pseudomonas aeruginosa over the growth and detect such metabolites in the P. aeruginosa-mixed body fluid simulants without any sample pretreatments. This electrochemical sensor enables capturing the secretion of a small amount of signaling metabolites, which is not achievable by conventional detection methods. ** This work was supported by an NRF grant funded by the Ministry of Science and ICT (NRF-2019R1A2C1088504) Keywords : metabolite, diagnosis, nanostructure, electrode, pseudomonas aeruginosa, sensor, electrochemical, signal References 1. Sismaet, H.J., Goluch, E.D. 2018. Electrochemical probes of microbial community behavior. Annu. Rev.

Anal. Chem. 11, 441-461.

228


P0015 Photolithographic Technology for the Shape Modulation of Micropatterns with Single Photomask

Sung Eun SEO, Oh Seok KWON Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Corresponding Author Email : [email protected] Numerous researches about photolithography process were invented and presented the ability to fabricate various micro/nanostructures by using direct laser writing, E-beam lithography, and nanoimprint lithography. Conventionally, functional polymers are patterned with various photolithographic techniques into ordered microstructures utilizing many kinds of confinement. In our research, we produced well-controlled micro-patterns with desired shapes over a large area with high throughput. With our research results, the access to numerous varieties of micropatterns was enabled and the range can be expandable to two dimensional (2D) and three dimensional (3D) multiscale architectures. Keywords : MEMS, photoresist, photolithography, exposure parameter

229


P0016 The Enzyme-ball Gold Nanocluster Based Fluorescent Probe for Sensitive Detection of Hydrogen Peroxide

Ji-Ae SONG, Yeong Joong KIM, Byung-Keun OH Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, Korea Corresponding Author Email : [email protected] In this study, our group developed highly sensitive fluorescence sensor based on enzyme-ball functionalized gold nanoclusters (EB-Au NCs) for detection of hydrogen peroxide (H2O2). The H2O2 can induce damage of cell ingredient, mutation and death of marine organism by leading to oxidation stress. The EB-Au NCs are consisted of protein stabilized gold nanoclusters such as bovine serum albumin stabilized gold nanoclusters (BSA-Au NCs) and horseradish peroxidase stabilized gold nanoclusters (HRP-Au NCs). The synthesized EB-Au NCs indicate about 100 nm size and red fluorescence emission at 720 nm when the EB-Au NCs was excited at 248 nm. As the concentration of H2O2 increases, the fluorescence intensity of EB-Au NCs decrease because of electron transfer between EB-Au NCs and H2O2 and their powerful enzyme reaction as catalyst. For these reasons, comparing BSA-Au NCs and HRP-Au NCs, EB-Au NCs have high fluorescence quenching effect, so that it can measure H2O2 at the very low concentration of 10 nM. Also, by comparing quenching level against several different components of marine environment, it can be confirmed that EB-AuNCs have a good selectivity for H2O2. Keywords : Hydrogen peroxide, Enzyme-ball, Gold nanocluster, Fluorescence sensor References 1. Jianping Xie, Yuangang Zheng, and Jackie Y. Ying, Journal of the American Chemical Society(2009), 131,

888-889. 2. Fang Wen, Yanhua Dong, Lu Feng, Song Wang, Sichun Zhang, andXinrong Zhang, Analytical

Chemistry(2011), 83, 1193-1196.

230


P0017 In vitro Inhibition of Pancreatic Cancer Growth by Dual-functional Aptamer Complex

Woo-Ri SHIN1, Hee-Young CHO1, Sang Yong KIM2, Ji-Hyang WEE2, Yang-Hoon KIM1 1School of Biological Sciences College of Natural Sciences, Chungbuk National University, Cheongju, Korea, 2Department of Food Science and Biotechnology, Shin Ansan University, Ansan, Korea Corresponding Author Email : [email protected] Pancreatic cancer is the 4th leading cause of cancer-death in the world. It is hard to diagnose early and are not easily treated once they spread to other organs. The aptamer that have been treated with pancreatic cancer, will be targeted ALPPL2 expressed on the surface of pancreatic cancer cell and downregulated the CTHRC1 function by blocking the Smad. We selected the aptamer against CTHRC1/ALPPL2 protein. Then, we analyzed the binding structure of the protein-aptamer complex, which is confirmed the RNA aptamer bind to the active site of the protein. Aptamers of each binding to CTHRC1/ALPPL2 were conjugated to produce an aptamer complex with two functions that specifically bind to the surface and inhibit the growth of pancreatic cancer cell. This dual-functional aptamer complex was has high affinity to each target protein active site. ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2019R1I1A1A01062365). ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235) Keywords : Pancreatic cancer, Dual-functional Aptamer, inhibit the cancer cell growth

231


P0018 Nanoparticle Using Coating Technique for Universal Oral Delivery

Yu-Rim AHN1,2, Ji-Hye KIM1,2, Ji-Yeon PARK2, Chi-Yeon SONG2, Hee-Won AN2, Hyun-Ouk KIM1,2 1Department of Biohealth-machinery convergence engineering, Kangwon National University, Chuncheon, Korea, 2Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Vaccination is widely regarded as the only exit strategy for infectious diseases currently spreading around the world. Vaccines are important as the only way to defend our bodies from infectious diseases. Accordingly, it is necessary to study a delivery system that can increase the efficiency of a vaccine to prevent a threatening infectious disease. In this study, we produced Actisomes, a vaccine formulation for oral administration that has its own driving force according to the in vivo environment using nanostructures. We confirmed the synthesis and effectiveness of ActiSomes using DLS, TEM and CLSM. ActiSomes has a driving force by generating hydrogen gas in the intestinal environment, as well as releasing an inherent vaccine. Therefore, ActiSomes is a vaccine formulation for oral administration and can lead to an efficient immune response by resolving a feeling of resistance to pain and maximizing the activation of mucosal immunity. Furthermore, our study can be developed as an efficient vaccine delivery platform using in vivo environment. Keywords : Nanoparticles, Vaccine, immunity

232


P0019 Cancer Immunotherapy Platform Using Human Ferritin Protein Nanoparticle

Chul Joo YOON, Yonghwan SEOL, Young Eun JANG, Ok Jeong MOON, Hyehyun KIM, Sojin JEONG, Yeonhwa YU, Yoobin CHOI, Yusik KIM, Wonkyung AHN Korea University, Seoul, Korea Corresponding Author Email : [email protected] Immunotherapy has emerged as a powerful clinical strategy for treating various types of cancer. However, clinical data shows row response rate and some immune related adverse events(iRAEs). The engineering of the nanoparticle system can be a functionalized platform to tackle the limitations. Here, we designed the engineering strategies on structure-guided surface modifications of self-assembled human ferritin heavy chain (huHF nanoparticles (NPs) to enhance the delivery of tumor antigens (TAs) and immune checkpoint blockade (ICB) for induction of robust TA-specific anti-tumor immune. The engineered huHF NPs to deliver TAs can induce maturation of dendritic cell for the cargo antigens without help of additional adjuvant. The T cell responses are further enhanced by blocking immune checkpoint signaling in tumor tissue with the other huHF NPs contained PD-L1 binding domain of PD-1 on surface. The combined treatment with two surface modified NPs triggered long-term memory protection against tumor rechallenge after elimination of the initial tumors. Thus, our new method based on huHF NPs platform make it easy to develop immunotherapy drugs and improve clinical outcomes of immunotherapy. Keywords : Protein, Nanoparticle, Immunotherapy

233


P0020 Development of Gold Nanocluster Embedded Dual-enzyme Nanoparticles and Their Applications in an Enzyme-based High-sensitive Electrochemical Sensor Myeong-Jun LEE Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, Korea Corresponding Author Email : [email protected] Enzyme based electrochemical-biosensors have been used because of their high target-specific reaction. However, enzyme-based electrochemical sensors have low conductivity of the electrode because protein work as an insulator, so that it is difficult to put a large number of enzymes on the surface of the electrode for utilizing enzyme reaction. To overcome this problem, our group synthesis of dual-enzyme gold nanocluster ball (DENCB) encapsulated organic-inorganic materials made of glucose oxidase gold nanocluster (GOx-AuNC), horseradish peroxide stabilized gold nanocluster (HRP-AuNC) and bovine serum albumin stabilized gold nanocluster (BSA-AuNC). The synthesized nanoparticle enables the combination of a two-enzyme cascade reaction step in one step, in which the GOx-AuNC component of the nanoparticle oxidizes glucose to generate H2O2, which then reacts with the adjacent HRP-AuNC component on the nanoparticle. As the effect of AuNC, these nanoparticles have a good conductivity compared to bare protein. DENCB is applied for glucose electrochemical sensor as a working electrode, which was produced by coating the particles on a gold plate. In this research, DEBNC based glucose electrochemical sensor displays a good electrochemical signal for the detection of glucose and exhibits high sensitivity which can detect the very low concentration of glucose range from 5 to 320 nM. Also, by analyzing other materials and serums together with glucose, it can be confirmed that the sensor has a good selectivity about glucose. Keywords : Gold nanocluster, Dual-enzyme nanoparticle, Electrochemical, Glucose References 1. Sun, J., Ge, J., Liu, W., Lan, M., Zhang, H., Wang, P., Wang, Y, Niu, Z., Nanoscale, 6, 255-262 (2014). 2. Hu, C., Yang, D.-P., Wang, Z., Huang, P., Wang, X., Chen, D., Cui, D., Yang, M., Jia, N. Biosens.

Bioelectron. 41, 656-662 (2013).

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P0021 Novel Washing-free and Label-free Electrochemical Detection Method for Target DNA Utilizing Peroxidase Mimicking DNAzyme

Sang Mo LEE1, Sujeong SHIN1,2, Jaemin KIM1, Sunghyeon KIM1, Jong Chan CHOE1, Ho Don JEONG1, Ki Soo PARK3, Hyun Gyu PARK1 1Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 2Division of Safety Analysis, National Agricultural Products Quality Management Service, Ministry of Agriculture, Food and Rural Affairs, Gimcheon, Korea, 3Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, Korea Corresponding Author Email : [email protected] Herein, we developed a new washing-free and label-free electrochemical strategy to detect target DNA by utilizing the peroxidase mimicking activity of DNAzyme. The key components of this method are DNAzyme-incorporated probes, which are designed to form G-quadruplex structure and possess peroxidase mimicking activity only in the presence of target DNA. The peroxidase mimicking activity of G-quadruplex structure, triggered by the hybridization of DNA probes and target DNA, promotes the precipitation reaction of 4-chloronaphthol (4-CN) on the working electrode. As a result, the electrochemical reaction between the redox materials and the surface of electrode is hampered by the precipitates produced on the electrode surface, leading to a significant increase of the impedance signal. Based on this ingenious electrochemical strategy, the target DNA from sexually transmitted disease (STD) pathogens such as Chlamydia trachomatis, Trichomonas vaginalis, and Herpes simplex virus type 2 in real patient samples were successfully detected, showing its practical capability in clinical diagnostic fields. Keywords : peroxidase mimicking DNAzyme, electrochemical sensor, sexually transmitted disease References 1. W. Yun, F. Li, X. Liu, N. Li, L. Chen, and L. Yang, Microchim. Acta. 185, 457 (2018). 2. Y., Park, C. Y. Lee, K. S. Park, and H. G. Park, Chem. Eur. J. 23, 17379-17383 (2017). 3. S. H. Hwang, J. H. Kim, J. Park, and K. S. Park, Anaylst 145, 6307-6312 (2020).

235


P0022 Lyophilized Visually Readable Loop-mediated Isothermal Nucleic Acid Amplification Test for Detection Mayaro Virus

Seon Hyung LEE1, Keon Young PARK2, Sung-Gun KIM3, Ji-Young AHN1 1Department of Microbiology, Chungbuk National University, Cheong-Ju, Korea, 2Research Institute, Pepgene inc., Seong-Nam, Korea, 3Department of Biomedical science, U1 University, Young-Dong, Korea Corresponding Author Email : [email protected] LAMP is a method of amplifying a target gene in isothermal using Bst polymerase and six primers (each pair of outer, inner and loop primers). LAMP is economical detection method because it is fast, sensitive, specific and requires no expensive equipment. For field diagnosis, LAMP LAMP can greatly shorten the time required by using methods such as freeze-drying of LAMP mixture and colorimetric reagents. We tried to detect the mayaro virus using this amplification method with many advantages. Therefore, we designed 6 LAMP primers within the Mayaro virus-specific site, and established LAMP amplification conditions. In order to minimize the detection process and time, a LAMP mixture excluding template and polymerase was premixed and lyophilized. It was confirmed that the amplification efficiency of the lyophilized LAMP mixture did not decrease when compared with the LAMP mixture before lyophilization. The results of LAMP amplification were confirmed using two colorimetric reagents, SYBR GreenI and Leucocrystal Violet, and conditions for visual detection using colorimetric reagents were established. The detection of the mayaro virus using LAMP established in this study significantly simplifies the process other than amplification, confirming the possibility that it can be used as a rapid diagnosis method in the field. Keywords : LAMP, Mayaro virus, colorimetric detection, LAMP premix References 1. C Carter, K Akrami, D Hall, D Smith, E AronoffSpencer. J Virol Methods, 244, 32-38 (2017). 2. Ma Y, Zhang B, Wang M, Ou Y, Wang J, Li S , Biomed Res Int. 2016, 2906484 (2016).

236


P0023 Crystal Structure of PAT Protein-aptamer Complex by Crystallography

Jin-Pyo LEE1, Woo-Ri SHIN1, Simranjeet Singh SEKHON1, Sung Min WOO2, Ji-Young AHN1, Yang-Hoon KIM1 1Major in Microbiology School of Biological Sciences College of Natural Sciences, Chungbuk National University, Cheongju, Korea, 2Department of Food Science and Biotechnology, Shin Ansan University, Ansan, Korea Corresponding Author Email : [email protected] Recently, issues have been raised regarding the impact of GM crops on biodiversity and environment such as plants and soil. Detection systems for controlling GM crops are important. Aptamers are oligonucleotides that bind to a target molecule with high specificity. We selected the aptamer that specifically binds the herbicide-resistant protein Phosphinothricin N-acetyltransferase (PAT), using the SELEX method. PAT protein was crystallized to confirm interactions through structural analysis. The Bar gene was cloned in pET-21a and expressed by E.coli BL21 (DE3). The protein was purified by three-step chromatography: Affinity, Ion-exchange and Gel filtration chromatography. Sitting drop vapor diffusion was used for crystallization at 291K temperature. Analyzing the structure of protein-aptamer complex, can improve the specificity of aptamer to the target. ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235). ** This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2020R1A2C1009463) Keywords : PAT protein, Aptamer, Crystallography, GM crops, Crystal structure

237


P0024 A Label-free, Ultrasensitive Fluorometric Method for Detecting Ribonuclease H Activity Utilizing Light-up Aptamer

Jinhwan LEE, Junhyeok YOON, Rakyeom KIM, Sungjun YOON, Shin Chan KANG Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] We developed a new label-free and ultrasensitive fluorometric method to detect ribonuclease H (RNase H) based on light-up aptamer. The RNA/DNA chimeric hairpin (HP) promoter is composed of a hairpin structure which has RNA/DNA duplex in its stem. Therefore, target RNase H can recognize and hydrolyze RNA strand in the chimeric HP promoter, thereby releasing T7 promoter sequence which is originally blocked by RNA region in the chimeric HP promoter stem. Then it can be hybridized to its complementary sequence in the broccoli template and complete double-stranded T7 promoter sequence is produced. The downstream region of the broccoli template is then transcribed by T7 RNA polymerase and a large number of broccoli aptamers are produced. They can bind to its pair fluorogenic dye, (Z)-4-(3,5-Difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI) and emit intense fluorescence signal. By using the strategy, we successfully determined RNase H activity as low as 0.000156 U/ml, with excellent selectivity compared to other types of nucleases and DNA repair enzyme. Finally, the practical applicability of this assay system was demonstrated by successfully detecting RNase H activity in complex cellular extract condition. Keywords : Ribonuclease H, Broccoli aptamer, T7 RNA polymerase

238


P0025 Single Step Multiplexed Detection of Extracellular Vesicle miRNA and Surface Proteins Using Flow Cytometer for Cancer Diagnosis

HaNeul LEE1, Hee Choel YANG1, Won Jong RHEE1,2 1Department of Bioengineering and Nano-bioengineering, Incheon National University, Incheon, Korea, 2Division of Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Extracellular vesicles (EVs) have biomarkers that provide disease information with high accuracy, including microRNAs (miRNAs) and proteins. Recently, there are many cancer diagnosis methods using EV biomarker detection, including polymerase chain reaction for miRNA detection and enzyme-linked immunosorbent assay for protein detection. However, cancer is heterogeneous, thus multiplexed detection is required to achieve accurate cancer diagnosis. Recently, simultaneous multiplexed detection of EV biomarkers was developed using antibody and molecular beacon (MB) for EV protein and miRNA detection. However, the method requires high number of EVs because of EV loss during wash steps. Therefore, we developed a new method for simultaneous multiplex detection of EVs proteins and miRNAs using flow cytometry without washing steps. Using a flow cytometry, the high fluorescence signals of protein and miRNA in cancer cell-derived EVs were observed as compared to those in normal cell-derived EVs. The method does not require any addition steps to remove unbound detection probes. Overall, multiplexed detection of EVs miRNAs and proteins using flow cytometry can be developed as a simple and rapid cancer diagnosis method. Keywords : Extracellular vesicles, Cancer, Simultaneous multiplexed detection, Molecular beacon, Flow cytometer References 1. H. C. Yang, and W. J. Rhee, J. Clin. Med. 10, 319 (2021). 2. S. Cho, H. C. Yang, and W. J. Rhee, Biosens. Bioelectron. 146, 11749 (2019). 3. J. H. Lee, M. H. Kwon, J. A. Kim, and W. J. Rhee, Artif. Cells Nanomed. Biotechnol. 46, (Suppl. 3) S52-

S63 (2018). 4. J. H. Lee, J. A. Kim, S. Jeong, and W. J. Rhee, Biosens. Bioelectron. 86, 202-210 (2016). 5. J. H. Lee, J. A. Kim, M. H. Kwon, J. Y. Kang, and W. J. Rhee, Biomaterials. 54, 116-125 (2015).

239


P0026 High Resolution Patterning of Gold Binding Peptide Fused Glucose Dehydrogenase Alpha-Gamma Complex (GDHγα) on Nanopatterned Electrode Surface Fabricated via E-beam Lithography

Hyeryeong LEE, Basit SHARIF, In Seop CHANG School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] In this study, using the FAD-dependent glucose dehydrogenase (FAD-GDH) as model enzyme, it is aimed to control the enzymatic orientation of the enzyme via site-specific fusion of gold binding peptide in enzyme. Most importantly, positioning of synthetized fusion protein will be regulated at nano-level, using nano-patterned electrode fabricated via e-beam lithography technique. For this, 1) the synthetic glucose dehydrogenase (GDH), consisting of the site-specific expression of a gold binding peptide (GBP) on the α-subunit of GDH which enables close proximity between enzymatic active and electrode surface as well as DET of enzyme-electrode interface will be used; 2) the number of GBP repeats fused to FAD-GDH would be optimized in terms of enzymatic catalytic activity and binding affinity; 3) The nano-patterned electrode would be fabricated via e-beam lithography for its unit pattern to have diameter similar to that of target enzyme; 3) the fusion protein will be immobilized on the nano-patterned electrode surface and the binding morphology of enzymatic nanopatterns would be investigated. Keywords : Glucose dehydrogenase, enzyme electrode, binding specificity, enzyme nano-patterning, e-beam lithography, gold binding peptide

240


P0027 NIR-responsive Biodegradable Hyaluronic Acid-based Microneedle for Photothermally-accelerated Transdermal Drug Delivery

Hwanju JEONG1, Yeji JANG1, Gayoung LEE2, Seungki LEE1, Seungyeon HAN1, Yun Jung HEO2, Inhee CHOI1 1Department of Life Science, University of Seoul, Seoul, Korea, 2Department of Mechanical Engineering, Kyunghee University, Yongin, Korea Corresponding Author Email : [email protected] Microneedle (MN) enables the painless and sustainable drug delivery to target the specific skin area compared with traditional drug delivery method such as oral delivery or invasive injection. [1] However, there are still challenging issues in use of MN for delivery system because of its insufficient efficiency and controllability of molecular delivery. Moreover, conventional non-dissolving MN involves the inconvenience of removing it after termination of usages. Herein, we suggest the near infrared (NIR)-responsive hybrid MN composed of biodegradable hyaluronic acid (HA) and plasmonic gold nanoparticles (GNPs). The hybrid MN is manufactured through a reproducible casting method using the mixture of NIR-responsive GNPs, HA solution, and drugs. The obtained MN is easily dissolvable by water, that initiates drug release. The GNPs integrated in the MN generate local heat via photothermal conversion effect resulting in acceleration of drug release. As a proof-of concept demonstration, the drug delivery performance of the MN is tested with the cell and pig skin. We expect the proposed stimuli-responsive MN is very useful in various molecular delivery applications. Keywords : Microneedle, Photothermal conversion, Transdermal drug delivery References 1. Nandagopal, MS Giri, et al., Overview of microneedle system: a third generation transdermal drug delivery

approach(2014), Microsystem technologies 20(7), 1249-1272.

241


P0028 Magnetic Force-based Cell-substrate Tension Regulation for Osteogenesis of Human Mesenchymal Stem Cells

Sungwoo CHO1, Min Ju SHON2, Boram SON1, Tae-Young YOON2, Tai Hyun PARK1 1School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea, 2School of Biological Sciences and Institute for Molecular Biology and Genetics, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Cells can respond to the physical cues, derived from the surrounding microenvironment [1-3]. Nowadays, various studies have been conducted in the respect of the fact that physical properties, such as stiffness and elasticity of the substrate in which cells grow are influencing the differentiation of stem cells [1-5]. In this study, we hypothesized that the key modulator of previous studies is the force, tension, applied to the cell. By incorporating magnetic nanoparticles (MNPs) into the cells, a system that could controls the magnetic force through changing the strength of the external magnetic field was established, resulting in the followings. First, the amount of MNPs introduced into the cell and its cytotoxicity were quantified, showing no cytotoxicity in the range of the amount used in the present study. Second, the expression levels of osteogenic marker genes (COL-1, OPN and OCN) were statistically significantly increased in proportion to the applied magnetic force. Third, ALP staining, ARS staining and osteogenic marker protein (YAP, RUNX2, OPN and OCN) staining showed the upregulated osteogenic function and protein expression of tension-applied group. Finally, the magnetic force was quantified through the Magnetic-tweezer. Through this study, we could find the fundamental key factor of stem cell differentiation by physical stimulation and lead to enhanced osteogenic differentiation. Keywords : Human mesenchymal stem cell, Tension, Osteogenic differentiation, Magnetic nanoparticles, Magnetic tweezer References 1. J. H. Wen et al., Nat. Mater. 13(10), 979-987 (2014). 2. S. Kumar., Nat. Mater. 13(10), 918-920 (2014). 3. H. Taylor-Weiner, N. Ravi, and A. J. Engler., J. Cell. Sci. 128(10), 1961-1968 (2015). 4. S. D. Sommerfeld, and J. H. Elisseeff., Cell Stem Cell. 18.2, 166-167 (2016). 5. K. H. Vining, and D. J. Mooney., Rev. Mol. Cell Biol. 18.12, 728 (2017).

242


P0029 Determining Antibacterial Activity of Fe or Zn Doped Carbon Quantum Dots

Seunghyeon JO1, Songhee LEE2, Sooim SHIN1,2 1Department of Bioengineering and Biotechnology, College of Engineering, Chonnam National University, Gwangju, Korea, 2Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] With increased interests in developing new antibiotics which can overcome antibiotic-resistance, carbon quantum dots(CQDs) are considered to be an alternative solution because of its abundance and low cost. CQDs are nanocarbons small in the sizes(below 10nm) and can play a role of nanoparticles which enhance the binding ability of metals to cellular membranes or cell walls of bacteria. In this study, we have chosen Fe and Zn doped CQDs in chemical library, and determined whether it has antibacterial activity by observing visible growth inhibition using paper discs. The minimum inhibition concentration(MIC) was determined by growth curve of S. aureus and E. coli. They are found to have antibacterial activity against the both bacterium. Keywords : Antibacterial Activity, Carbon Quantum Dots, Antibiotic, Growth Curve, MIC, Nanoparticle, Paper disc

243


P0030 Development of Nanodisc-based Nanotransformer Augmenting Antiviral Efficacy of Anti-hemagglutinin Stem Antibodies

Jaehyeon HWANG, Younghun JUNG, Seokoh MOON, Hyunseok OH, Soomin KIM, Kyeong Won KIM, Jeong Hyeon YOON, Dae-Hyuk KWEON Department of Integrative Biotechnology, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] Several studies have been conducted on neutralizing antibodies (NAbs) against influenza A viruses causing loss of life and economic damage. However, these virustatic antibodies lacking virucidal activity showed limited antviral potency. Here, we develop a nanodisc providing the immunoglobulins with virucidal efficacy by rupturing the viral envelope, resulting in enhancing the antiviral effects. Mechanistically, the nanodiscs carrying Fc-binding peptides bind to NAbs targeting stem region of hemagglutinin are co-endocytosed into host cells. The nanodisc-based nanotransformer (NT) perforate the viral membranes at low pH in the endosome. Administration of the NT, especially with pegylated nanotransformer, reduce morbidity and mortality compared with the NAbs in mouse models. Our results suggest a new class adjuvant of applicable to other neutralizing antibodies and convalescent plasma therapy that confer virucidal potency by antibody-dependent perforation. In conclusion, the membrane nanostructure gives another prospective for concepts of antibody conjugated molecules. Keywords : Neutralizing antibodies, Nanodiscs, Antibody-drug conjugates, Influenza viruses References 1. Choe, W., Durgannavar, T. A., & Chung, S. J. Fc-Binding Ligands of Immunoglobulin G: An Overview of

High Affinity Proteins and Peptides. (2016). Materials (Basel), 9(12). 2. Kong, B., et al., Virucidal nano-perforator of viral membrane trapping viral RNAs in the endosome. (2019).

Nat Commun, 10(1), 185. 3. Shriver, Z., Trevejo, J. M., & Sasisekharan, R. Antibody-Based Strategies to Prevent and Treat Influenza.

(2015). Front Immunol, 6, 315.

244


P0031 Manufacturing Cellulose Nanocrystals (CNC) and Determination of Its Morphologies During the Purification Process

Hye Jee KANG, Bo Som LEE, Mi Dan KANG, Ga Young KIM, Nur ISTIANAH, Mariam AMINU, Young Hoon JUNG School of Food Science and Biotechnology, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] The production of cellulose nanocrystals (CNC) normally requires complex purification steps after acid pretreatment, which include dilutions, neutralization, and concentration. However, only a few reports have been given about changes in the cellulose properties during the purification process. In this research, we analyzed the morphologies, structural properties, and the crystallinity of cellulose using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD), respectively to observe changes during the purification process. The crystallinity was again compared with the crystalline cellulose-binding protein, CtCBD3. The treated cellulose nanoparticles had rod-like appearance with the length of 130-166 nm, the width of 11-15 nm, and the aspect ratio of 11–13. Despite the complexity and harshness of the process, significant changes in the properties of cellulose were not observed, but the increase in the amount of crystalline surface area in accordance to each process. The crystallinity difference in CNC surface might bring out the outmost dispersion ability of CNC. As a result, this study gives information on the changes in cellulose properties during the CNC purification process. Keywords : cellulose nanocrystals, Fourier transform infrared, X-ray diffraction, CtCBD3

245


P0032 Development of a SARS-CoV-2-specific Biosensor for Antigen Detection Using scFv-Fc Fusion Proteins

Mi Jeong KIM1,2, Hye-Yeon KIM1,2, Sun Cheol PARK1,2, Wooyoung KIM1,2,3, Woon hee BAEK1,2,4, Edmond Changkyun PARK1,2,4, Seung Il KIM1,2,4 1Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Korea, 2Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Korea, 3Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon, Korea, 4University of Science & Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a global pandemic, development of a cheap, rapid, accurate, and easy-to-use diagnostic test is necessary if we are to mount an immediate response to this emerging threat. Here, we report the development of a specific lateral flow immunoassay (LFIA)-based biosensor for COVID-19. We used phage display technology to generate four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies. The scFv-Fc antibodies bind specifically and with high affinity to the SARS-CoV-2 NP antigen, but not to NPs of other coronaviruses. Using these scFv-Fc antibodies, we screened three diagnostic antibody pairs for use on a cellulose nanobead (CNB)- based LFIA platform. The detection limits of the best scFv-Fc antibody pair, 12H1 as the capture probe and 12H8 as the CNB-conjugated detection probe, were 2 ng antigen protein and 2.5 × 104 pfu cultured virus. This LFIA platform detected only SARS-CoV-2 NP, not NPs from MERS-CoV, SARS-CoV, or influenza H1N1. Thus, we have successfully developed a SARS-CoV-2 NP-specific rapid diagnostic test, which is expected to be a simple and rapid diagnostic test for COVID-19. Keywords : COVID-19, SARS-CoV-2, Lateral flow immunoassay, Rapid diagnostic test, Antibody References 1. Kim et al. (2021) Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc

fusion proteins., Biosens Bioelectron.1;175:112868.

246


P0033 Development of New Interfacing Compounds for Ultra-stable Biosensors

Jinyeong KIM, Oh Seok KWON Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Corresponding Author Email : [email protected] It is necessary to develop interfacing compounds for immobilizing the bioprobes, including an antibody, aptamer, natural receptors and so on, on the platform of biosensors which are consisted of 2D nanostructure. There are various 2D nanomaterials in the literatures. In particular, a graphene has high-conductivity, so the graphene-integrated biosensors have been constructed to enhance their sensitivity and selectivity. However, the surface of the graphene is highly stable owing to their non-covalent electron pairs, so that their surface modification is challenge. In this study, we demonstrated a new technology for graphene interfacing technologies. Interfacing chemical compounds such as DAN and PDI have appeared particularly relevant for the stabilization and functionalization of the surface of graphene. Through the physiochemical stability of DAN, we synthesized novel PDI compounds connecting the functional groups as an amine, acid, and azide at the end of the molecule, which were able to stably link with bio-receptors as well as improve sensor performance and reproducibility. Keywords : Interfacing chemistry, Surface Functionalization, graphene, FET, Biosensors

247


P0034 Anti-biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-resistant Planktonic Bacteria Cells and Biofilm

Jin-Young KIM, Seong-Cheol PARK Department of chemical Engineering, Sunchon National University, Suncheon, Korea Corresponding Author Email : [email protected] Biofilm consists of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPSs) containing polysaccharides, extracellular DNA, proteins, and lipids. The emergence of multidrug-resistant microorganisms in clinical therapeutics is a global healthcare concern. Antimicrobial peptides present an attractive possibility to solve these problems. The current study showed that the de novo designed three AMPs have an inhibiting effect on biofilm formation and disrupting activity for preformed biofilms in drug-susceptible and drug-resistant P. aeruginosa and S. aureus strains. Moreover, all peptides reduced the biofilm by detaching the carbohydrates, nucleic acids, and lipids of the EPS but did not detach the proteins. Future studies should focus on the use of PS1-2 as treatment for antibiotic resistant bacteria because it shows good efficacy without the risk of cytotoxicity (NRF-2019R1I1A3A01062547). Keywords : Biofilm inhibition, Synthetic antimicrobial peptide, Drug-resistant bacteria, Biofilm degradation

248


P0035 Targeting and Anti-cancer Activity of Conjugated Exosome in Breast Cancer

Hyeonseok KIM1, Mi-Kyeong JANG2 1Department of Polymer science and Engineering, Sunchon National University, Suncheon, Korea, 2Department of Chemical Engineering, Sunchon National University, Suncheon, Korea Corresponding Author Email : [email protected] Cancer, most cause of death in global, was able to distribute many site of human body. Especially, breast cancer had many obstacles for complete treatment because of expressing three types of receptor on their surface. Therefore, among developed various strategies of cancer-therapy, we investigated breast cancer-targeting exosomes that conjugated with curcumin possessing similar chemical structure of estrogen for binding to estrogen receptor expressed on cell membrane. Exosome, approximately 50 - 150 nm size particles that release by variety of cell types and consist of lipid bilayer involving various soluble factors such as proteins, DNA and RNA is promising drug delivery vehicles due to low immunogenicity, high biocompatibility, stable storage. One of the crucial role of exosomes is the target ability to recipient cell for transfer the factor to communicate between cells even if autologous cell. In this study, exosomes extracted from MCF-7 and SK-BR-3 (breast cancer cell line), conjugated with curcumin for targeting estrogen receptor positive breast cancer and delivered entrapped doxorubicin, a conventional anticancer reagent for anti-cancer activity. We elucidated cell selectivity of targeting ligand conjugated exosomes in estrogen receptor positive breast cancer whereas observed low toxicity in normal cell line. Our results possess potential as a notable and novel drug delivery system for cancer therapy. (NRF - 2020R1A2C1013069) Keywords : exosome, drug delivery system, anti-cancer effects, targeting lignad

249


P0036 High Performance Cyt c/graphene FET Platform for Detection of H2O2

Seon Joo PARK, Oh Seok KWON Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Corresponding Author Email : [email protected] Hydrogen peroxide (H2O2), a representative reactive oxygen species (ROS), plays various roles in physiological processes, including cellular signaling, where it regulates cell growth, immune activation, and apoptosis. Moreover, high-level in vivo reactive oxygen species (ROS) can damage many biomolecules via oxidative stress and play vital roles in the pathogenesis of several bodily disorders, such as neurological disorders, atheros-clerosis, cancer, and Alzheimer’s disease. Therefore, rapid and sensitive detection of H2O2 is very important for trace ROS. Herein, we present a highly sensitive graphene field-effect transistor (G-FET) sensor integrated with cytochrome C for H2O2 detection. It shows a detection limit of 100 fM and rapid response time (<1 s). Moreover, our sensor platform was able to specifically discriminate H2O2 from a series of interfering substances, such as dopamine, ascorbic acid, glucose, uric acid and glutamate. Keywords : Hydrogen peroxide (H2O2), graphene field-effect transistor (G-FET), cytochrome c (Cyt c)

250


P0037 Smart Homing Action of Vitamin D3 in Ovarian Cancer

Seong-Cheol PARK, Mi-Kyeong JANG Department of Chemical Engineering, Sunchon National University, Suncheon, Korea Corresponding Author Email : [email protected] Despite advances in tumor diagnosis and chemotherapy development, cancer has still maintained a top cause of disease-related human death worldwide. The strategies for specific tumor tissue targeting have been considered as an important factor in the field of drug or gene delivery system. The aim of this study is to develop gene delivery system with targeting effect into ovarian cancer cell as introducing cholecalciferol (vitamin D3) binding with vitamin D receptor, which is over-expressed onto ovarian cancer cell surface. Polyethylene glycol-cholecalciferol (PC) was prepared to design the gene carrier for targeting ovarian cancer, followed by conjugation of PC material onto polyglutamic acid (PGA). The targeted gene delivery of polymer was proved a specific transfection capacity between L929 and SK-OV3 cells and its homing ability was confirmed in SK-OV3-xenograft mouse model (NRF-2020R1A2C1013069). Keywords : Targeted gene delivery, ovarian cancer, vitamin D3

251


P0038 Discovering Okadaic Acid-specific Bioreceptors via Phage Display, and Constructing the Fluorometric Assay of Okadaic Acid

Chan Yeong PARK, So Yeon KWEON, Ruth STEPHANIE, Min Woo KIM, Tae Jung PARK Department of Chemistry, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] One of the most prevailing marine environmental issues is harmful algae blooms leading to marine biotoxins production. Okadaic acid (OA), one of the most dangerous foodborne toxic substances produced by toxicogenic dinoflagellates, commonly of the Dinophysis and Prorocentrum genera, is the representative toxin of diarrhetic shellfish poisons. This syndrome is responsible for gastrointestinal disturbances, such as diarrhea, vomiting, and abdominal pain. Conventionally, OA detection and monitoring relies on mouse bioassays that possess not only ethical but also technical problems, such as poor sensitivity. Due to that reason, as of 1 January 2015, the method is now prohibited. Another internationally accepted method for their detection is HPLC-fluorescence detection method. While the method offers better sensitivity, it requires lengthy procedures and complex data interpretation. Herein, we have developed okadaic acid-specific bindable bioreceptors through the phage display technology for overcoming the above problems. For this technique, we present the haptenization of okadaic acid by conjugating with bovine serum albumin (BSA). To effectively immobilize the OA on the BSA, DCC/NHS crosslinking method was used. The discovered bioreceptor was then applied for the fluorescence detection system with graphene oxide quantum dots as a quencher. Keywords : Marine toxin, Okadaic acid, Hapten, Phage display, Graphene oxide quantum dots, Fluorometric detection References 1. M. Campàs, J.L. Marty, Anal. Chim. Acta, 605(1), 87-93 (2007). 2. N.M. Llamas, et. al, Anal. Bioanal. Chem., 389(2), 581-587 (2007).

252


P0039 Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Using Graphene-based Biosensor

Giwan SEO1,2, Mi Jeong KIM1,2, Sang-Yeop LEE1,2, Hayoung LEE1,2,3, Edmond Changkyun PARK1,2,3, Seung Il KIM1,2,3 1Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Korea, 2Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Korea, 3Department of Bio-Analysis Science, University of Science & Technology, Daejeon, Korea Corresponding Author Email : [email protected] Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because no specific drugs for COVID-19 are yet available, early diagnosis and management are still crucial for containing the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2. The sensor was produced by graphene-based FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was determined using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a sensitive immunological diagnostic method for COVID-19 that requires no labeling. Keywords : COVID-19, SARS-CoV-2, Graphene, Biosensors

253


P0040 Microfluidics Mixer Platform for Efficient Fluorescent Labeling of Microplastics

Hyun Ho LEE, Seongcheol SHIN, Seungju OH, Hyeyon HUR, Jongmin YANG, Dhanashri DESAI, Gillhwan KIM, Chorong KIM Department of Chemical Engineering, Myongji University, Yongin, Korea Corresponding Author Email : [email protected] Microplastics are small plastic particles less than 1 mm, and their environmental problems are caused by their increased accumulations throughout the food chains. In this study, we fabricate microfluidic mixer device using polydimethylsiloxane (PDMS) by SU-8 photolithography or by laser ablation method for efficient fluorescent labeling of individual microplastics inside of serpentine microfluidic channel. It shows that mixing channel is an efficient platform to fluorescently label the microplastics with a short time scale. Keywords : microfluidics, microplastic, fluorescent labeling, PDMS

254


P0041 Zika Virus Detection Using Reverse Transcription-loop-mediated Isothermal Amplification and Lateral Flow Assays

Gna AHN1, SeonHyung LEE1, Yun Hee BAEK2, Min-Suk SONG2, Yang-Hoon KIM1, Ji-Young AHN1 1School of Biological Sciences, Chungbuk National University, Cheongju, Korea, 2College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea Corresponding Author Email : [email protected] The global spread of Zika virus has become a pressing need for field-ready diagnostics. Here we demonstrated for detecting ZIKV through reverse transcription loop-mediated isothermal amplification (RT-LAMP). ZIKV RT-LAMP were investigated and optimized to ensure proper use of direct-use biological samples (PBS, urine, and plasma). Our ZIKV RT-LAMP could provide detection of the ZIKV RNA in 30 min without off-target amplification at 63 °C. Specificity tests were conducted using BtsI restriction enzyme digitization. Furthermore, we demonstrate the feasibility of ZIKV RT-LAMP by testing the end-point discrimination in various sample matrices. This les us to combine LFA for the direct detection of ZIKV RT-LAMP products. LFA reaction was performed and visualized by hybridization of AuNPs:polyadenylated (polyA10)-ZIKV probe with LAMP amplicons. The resultant ZIKV RT-LAMP products were rapidly and simply assayed by the LFA in less than 5 min without any preparation step for generating the LAMP-probe hybridization, thus providing a powerful strategy for the field-ready ZIKV diagnosis. Keywords : ZIKA, LAMP, Lateral flow assay References 1. Choi, J. R., Hu, J., Gong, Y., Feng, S., Wan Abas, W. A., Pingguan-Murphy, B., & Xu, F. (2016). An

integrated lateral flow assay for effective DNA amplification and detection at the point of care. Analyst, 141(10), 2930-2939.

2. Control, E. C. f. D. P. a. (2016). Rapid risk assessment. Zika virus disease epidemic: potential association with microcephaly and Guillain-Barré syndrome. Second update, 8 February 2016. European Centre for Disease Prevention and Control, Stockholm.

255


P0042 Bifunctional Au Nanoparticles with Metal-enhanced Fluorescence for Highly Sensitive and Facile Detection of Proteolytic Enzyme

Joohyun AN1, Taehyeong HA1, Jin-Ha CHOI2, Jeong-Woo CHOI1 1Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, Korea, 2Department of Chemical Engineering, Jeonbuk University, Jeonju, Korea Corresponding Author Email : [email protected] Caspase-3 is a proteolytic enzyme that plays an essential role in cell apoptosis. Since the expression levels of caspase-3 in cells are low, it is important to use sensitive biosensor for the detection of proteolytic enzymes. In this study, we have developed bifunctional Au nanoparticles (AuNPs) to create highly sensitive metal-enhanced-fluorescence (MEF)-based biosensor for detection of caspase-3. In this biosensor, both single-stranded DNA (ssDNA) and a peptide are linking between AuNPs and the fluorophore. Once a caspase-3 cuts the peptide, the ssDNA is able to maintain an optimal distance for the MEF and thus the fluorescence signal has significantly increased. The proposed biosensor demonstrated the highly sensitive detection of caspase-3 to range from 10 pg/mL to 10 ng/mL, and caspase-3-related preapoptotic cell detection was successfully performed from with high sensitivity (limit of detection : 10 pg/mL). The suggested biosensor is a time efficient, facile and one-step technique for the real-time analysis of intracellular proteolytic enzymes. It can be a competitive method for detection of caspase-3 as well as can be applied to the early diagnosis of several diseases such as cancer and neurodegenerative diseases. ** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2019R1A2C3002300) and by the Ministry of Education (No.2016R1A6A1A03012845). Keywords : Metal-enhanced fluorescence (MEF), Caspase-3, Au nanoparticle (AuNP), Nanobiosensor, Proteolytic enzyme References 1. J.-H. Choi, and J.-W. Choi, Nano Lett., 20 (10), 2020.

256


P0043 Fabrication of Localized Surface Plasmon Resonance Sensor Including Peptide Receptor for Detecting Bifenthrin

Jingon YOO1, Min Ji LEE1, Chae Hwan CHO2, Eun-Seon LEE3, Mi-Wha OH3, Jong Pil PARK2, Yun Suk HUH1 1Department of Biological Engineering, Inha University, Incheon, Korea, 2Department of Food Science and Technology, Chung-Ang University, Anseong, Korea, 3National Institute of Animal Science, Rural Development Administration, Wanju, Korea Corresponding Author Email : [email protected] In 2017, egg contamination scandal was occurred by pesticides in Europe and South Korea. One of the main reasons was bifenthrin. Bifenthrin is a pyrethroid pesticide, widely used for agriculture. United States Environmental Protection Agency (US EPA) classified bifenthrin as a Category C, which means possible human carcinogen. We demonstrated a biosensor for selective detection of bifenthrin in samples. Briefly, this is a plasmonic sensor applying the self-assembly of the gold nanoparticles, was used to conduct density deposition on a transparent glass substrate in order to achieve a regulated diameter of gold nano particle monolayer, which produced longitudinal wavelength extinction shifts through the localized surface plasmon resonance (LSPR) signal. The developed peptides conjugated the LSPR sensing chips are selected with phage display technique. We also use enzyme-linked immunosorbent assay (ELISA) to figure out the binding affinity of peptide attach to bifenthrin. Keywords : bifenthrin, biosensor, LSPR, phage display, gold nanoparticle References 1. Yang, Y., Wu, N. & Wang, C, Environ. Chem. Lett. 16, 1377-1391 (2018).

257


P0044 Aptamer-based Field-effect Transistor for Diagnosis of Highly Pathogenic Avian Influenza Virus

Myoungro LEE, Taek LEE, Jae-Hyuk AHN Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] Early diagnosis of highly pathogenic avian influenza virus (AIV) subtype H5N1 is important for preventing and controlling global epidemics. We report the first use of aptamerized field-effect transistors (FETs) as unlabeled sensors for AIV detection in chicken serum. The signal from the Aptamer-based FET biosensor increased linearly with a detection limit of 5.9 pM as the log of HA protein concentrations increased in the linear range of 10 pM to 10 nM. Acknowledgments This research was funded and conducted under「the Competency Development Program for Industry Specialists」of the Korean Ministry of Trade, Industry and Energy (MOTIE), operated by Korea Institute for Advancement of Technology (KIAT). (No. P0002397, HRD program for Industrial Convergence of Wearable Smart Devices) and by Korea Environment Industry&Technology Institute(KEITI) through the program for the management of aquatic ecosystem health, funded by Korea Ministry of Environment(MOE).(2020003030001), and by the Industrial Core Technology Development Program(20009121, Development of early diagnostic system of peritoneal fibrosis by multiplex detection of exosomal nucleic acids and protein markers) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) Keywords : H5N1 detection, DNA 4-way junction, FET biosensor

258


P0045 Competitive Lateral Flow Assay for Highly Sensitive and Selective Detection of Histamine in Fish

Min Ji LEE1, Hyang Sook CHUN2, Yun Suk HUH1 1Department of Biological Engineering, Inha University, Incheon, Korea, 2Food Toxicology Laboratory, School of Food Science and Technology, Chung-Ang University, Anseong, Korea Corresponding Author Email : [email protected] Histamine is one of the important organic compounds, but it is well known factor to cause an allergy and inflammation-like food poisoning. Therefore, detection of the histamine in food is an important issue for maintaining food safety. In this study, we successfully detect the histamine using lateral flow assay (LFA) which is a rapid, sensitive, cost-effectiveness and highly popular point-of-care (POC) technique. The assay used a specific antibody for histamine as receptor and gold nanoparticles as probe for detection of histamine in fish through competitive LFA strips. With this method, 50 ppm of histamine can be detected in less than 10 min in fish with simple pretreatment of samples. Moreover, Specificity was also evaluated against histamine and histamine analogues. We expect that our LFA sensor was significantly faster than the conventional analysis such as HPLC, ELISA, GC/MS, etc. and possesses the potential for rapid POC testing for food safety monitoring of histamine. Keywords : LFA, Histamine, AuNPs, Competitive, On-site References 1. M. Gagic, E. Jamroz, S. Krizkova, V. Milosavljevic, P. Kopel, V. Adam, J. Agric. Food CHEM. 67(3), 773-

783 (2019).

259


P0046 Gold Nanoparticle Based Lateral Flow Immunoassay for the Rapid and Sensitive Detection of Ractopamine in Pork

Min Ji LEE1, Eun-Seon LEE2, Mi-Wha OH2, Yun Suk HUH1 1Department of Biological Engineering, Inha University, Incheon, Korea, 2National Institute of Animal Science, Rural Development Administration, Wanju, Korea Corresponding Author Email : [email protected] This research is the development of rapid lateral flow immunoassay (LFIA) strip based on colloidal gold for the detection of ractopamine. Ractopamine hydrochloride is the family of β-agonists, which are widely used as growth promotant to increase feed efficiency and weight gain of livestock production. Despite these advantages, ractopamine is narrowly approved in many countries due to its potential risks for human. Many analytical methods for the monitor of ractopamine in livestock depend on instrument-based analysis such as HPLC, LS/MS, GC/MS, and fluorescence. Even though these methods have high selectivity, sensitivity, and accuracy, are need to high cost, sophisticated technique, and complicated sample pretreatment procedure. LFIA technique, which is used in this study, gives many advantages such as the cost-effectiveness, rapid detection, and simplicity. As a result, this sensor is able to not only rapid detect of ractopamine, but also detect sensitively in feeds and animal tissues. LFIA strip of this research is expected as an analysis method for early detection of ractopamine due to the simplicity and speed of the detection process. Keywords : LFIA, Ractopamine, AuNPs, On-site, Detection References 1. B. Dong, S. Zhao, H. Li, K. Wen, Y. Ke, J. Shen, S. Zhang, W. Shi, Z. Wang, Food Chem. 271, 9-17 (2019).

260


P0047 Chitin Nanofiber Composite Materials for Medical Application

Seung Hyeon LEE, Dong Soo HWANG Department of Environment Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Chitin, the second most abundant natural materials, is well known to have guided bone regeneration characteristics, and its nanostructures are well known to mimic extra cellular matrix such as cell adhesion, proliferation, migration. However, chitin also promotes adhesion, proliferation of non-osteogenic cell. To prevent this problem and to fabricate a better guide bone regeneration membrane, Chitin nanofiber based composites materials were made. This material is a double-sided material, made by coating chitin nanofiber with MPC(2-Methacryloyloxyethyl phosphorylcholine), which is well known as a cell membrane biomimetic material. One side facing the bone is composed of chitin nanofiber membrane promotes bone regeneration and the other side facing the soft tissues is composed of MPC/TSMA(Trimethoxysilyl propyl methacrylate) copolymer has anti-fouling effect and suppresses the formation of fibroblasts. The mechanical properties of the membrane were also improved, and the in vitro cell test and ALP activity test of each chitin nanofiber and MPC layer showed the potential of chitin nanofiber/MPC as a composite medical material. Keywords : Chitin nanofiber, MPC, Guide bone regeneration References 1. R.Jayakumar, M.Prabaharan, S.V.Nair and H.Tamura, Biotechnol. Adv. 28, 142(2010). 2. J.-B. Zeng, Y.-S. He, S.-L. Li and Y.-Z Wang, Biomacromolecules 13, 1(2012). 3. Y. Zubery, A. Goldlust, A. Alves and E. Nir, J. Periodontol. 78, 112(2007). 4. N. D. Sanandiya, S. Lee, S. Rho, H. Lee, I. S. Kim and D. S. Hwang, Carbohydr. Polym. 208, 77(2019). 5. S. Choy, D. V. Lam, S. M. Lee and D. S. Hwang, ACS Appl. Mater. Interfaces. 11, 38440(2019). 6. S. Islam, M. R. Bhuian and M. Islam, J. Polym. Environ. 25, 854(2017).

261


P0048 Effects of Swimming Exercise on Autoimmunity Induced via IL-6 Activation

Jong-Kook LEE Department of Chemical Engineering, Suncheon National University, Suncheon, Korea Corresponding Author Email : [email protected] Regular exercise (RE) exerts various beneficial effects such as improving muscle function and enhancing autoimmune responses. To prove their effects, mice performed regular swimming exercise (RSE) for 10 and 15 min/day, 5 days/week for 4 weeks (adaptation, 5 min/day, 5 days/week; after Pseudomonas aeruginosa 3241 (DRPA 3241) infection, 10 min/day, 5 days/week). RSE training reduced visceral adipose tissue mass, activated bowel movements, and enhanced brain cell metabolism. The myokine interlukin-6 (IL-6) that stimulates or activates systemic immunity crosstalk was released from the skeletal muscles such as the tibialis anterior and gastrocnemius during RSE. Further, RSE enhanced redox activity via the production of β-defense 1 antimicrobial peptide (AMP) with thioredoxin 1 (Trx 1) against oxidative stress in the skeletal muscle, intestine, and brain by IL-6 activation. We hypothesized that RSE could induce antimicrobial activity against DRPA 3241 treated with β-defense 1 AMP. We evaluated two mouse groups: non-infected group of without RSE, 10 or 15 min RSE and DRPA 3241 infection without RSE, 10 or 15 min RSE. The RSE mouse group showed reduced visceral adipose tissue mass, an effect activated by lipolytic protein when IL-6 was increased in the skeletal muscle. Moreover, antimicrobial activity, a type of systemic immunity crosstalk activated by β-defensin AMP, was higher in the DRPA 3241-infected RSE mouse group than in the DRPA 3241-infected non-RSE mouse group. Thus, IL-6 is required for RSE to reduce visceral adipose tissue mass and is involved in the activation of potentially important systemic immunity crosstalk. Keywords : Regular swimming exercise, Myokine, Peptide, Lipolytic protein

262


P0049 Graphene-based FET Aptasensor for Real-time Monitoring of Geosmin for Determination of Water Contamination Degree

Seong Gi LIM, Sung Eun SEO, Oh Seok KWON Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Corresponding Author Email : [email protected] In this study, we introduce an aptamer-fabricated graphene-based field effect transistor (FET) sensor to estimate pollution degree of water. The micropatterned two-dimensional (2D) graphene oxide was fabricated with aptamer that is selectively sensitive toward geosmin, the indicator of water defilement. The fabrication of graphene oxide was successfully verified by two different measures. The decrement of the slopes of the current-voltage curve (i-V) shows attachments of biomolecules on the surface, which work as insulating agents. After surface treatment with aptamers combined with fluorescent agents, the electrode surface showed radiation when untreated graphene stayed in the dark. The sensor device detected geosmin linearly from 0.01 nM to 1 μM with detection limit of 0.01 nM. Applicability of the manufactured sensor platform was demonstrated by applying river water to the system, and geosmin in low concentration as 10 nM could be determined. Keywords : geosmin, field-effect transistor, aptasensor, water contamination References 1. Nakatsuka, N., Yang, K., Abendroth, J.M., Cheung, K.M., Xu, X., Yang, H., Zhao, C., Zhu, B., Rim, Y.S.,

Yang, Y., Weiss, P.S., Stojanovic, M.N., Andrews, A.M., Science 362, 319-324. (2018). 2. Park, S.J., Lee, S.H., Yang, H., Park, C.S., Lee, C.S., Kwon, O.S., Park, T.H., Jang, J., ACS Appl. Mater.

Interfaces. 8 (42), 28897-28903 (2016). 3. Park, S.J., Yang, H., Lee, S.H., Song, H.S., Park, C.S., Bae, J., Kwon, O.S., Park, T.H., Jang, J., ACS Nano

11, 5950-5959 (2017).

263


P0050 Control of Flow Rate with Superabsorbent Polymer for the Sensitivity Enhancement of Lateral Flow Assay

Woojin JEONG1, Taeyeong YOU1, Sun Min KIM2, Tae-Joon JEON1 1Department of Biological Engineering, Inha University, Incheon, Korea, 2Department of Mechanical Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected], [email protected] Lateral flow assay (LFA) is widely used point-of-care testing device. Characteristics of LFA are satisfied with affordable, user-friendly, equipment-free. However, since LFA has a relatively low sensitivity, many studies are being conducted to improve the sensitivity. To solve this disadvantage, this research proposed the Super-absorbent polymer (SAP) for the sensitivity enhancement. SAP is hydrophilic, high swelling capacity, and good strength of the swollen gel. LFA is basically using the capillary force for the detection of targets. SAP is integrated into nitrocellulose membrane to slow the capillary flow rate of nanomaterials. Through this, the viscosity of liquid in nitrocellulose membrane is increased. Experimental and mathematical simulations were conducted for optimization of the enhancement of sensitivity. To evaluate the effect of sensitivity, the different SAP concentrations were simulated in LFA. Then the result of sensitivity was resulted with ImageJ. As a result, this LFA with SAP shows three times fold enhancement sensitivity than other LFA platforms for detection of human immunoglobulin G. Keywords : Lateral flow assay, Point-of-care testing, Superabsorbent polymer, flow control References 1. Jung W, Han J, Choi JW, Ahn CH. Microelectron Eng 132, 46(2015). 2. K. Kabiri, H. Omidian, S.A. Hashemi, M.J. Zohuriaan-Mehr. European polymer journal. 39, 1341(2003). 3. C. Parolo, M. Medina-Sanchez, A de la Escosura-Muniz. Lab chip. 13, 386 (2013). 4. X. He, Z. Liu, Y. Yang. ACS Sensors, 4, 1691(2019).

264


P0051 Recombinant TRPV1 as a Potential Material for Detecting Several Agonists with FET Sensor

Siyoung HA, Oh Seok KWON Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Corresponding Author Email : [email protected] The Transient Receptor Potential Vanilloid 1 (TRPV1) channel is an appear target for the treatment of pain with a unique expression profile in marginal nociceptors and the ability to show various activation. Several agonists of endogenous and exogenous nature have been described for ion channel. TRPV1 initially identified as the receptor for interaction with capsaicin, temperature greater than 43 °C, acidic conditions and leads to a painful, burning sensation. Based on this, the objective of this study was to test the antigenic potential of the recom-binant TRPV1 protein for subsequent use in diagnostic tests. The TRPV1 expressed in the Escherichia coli strain BL21 was conformed from experimentally using ELISA and Western blotting and then the recombinant TRPV1 were applied with the surface of receptor to detect target material and improve sensing performance. Finally, we demonstration of the liquid-ion gated system based GFET biosensor for detection of TRPV1 receptor’s agonists, with highly sensitivity, selectivity and rapid responses in real-time. Keywords : FET sensor, biosensor, graphene, receptor, TRPV1

265


P0052 Highly Sensitive Surface-enhanced Raman Spectroscopy (SERS) Platform Based on Core/double Shell (Ag/polymer/Ag) Nanohorn Fabricated for Proteolytic Biosensor

Taehyeong HA1, Jin-Ha CHOI2, Jeong-Woo CHOI1 1Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, Korea, 2Department of Chemical Engineering, Jeonbuk University, Jeonju, Korea Corresponding Author Email : [email protected] Sensitive detection platforms capable of early diagnosis of various diseases has been developed but improve-ments are still needed to reduce reaction time and cost. In this study, we established a surface-enhanced Raman spectroscopy (SERS)-based ultrasensitive platform by using SERS-active hollow polypyrrole (hPPy) nanohorn and peptide-functionalized gold nanoparticles (AuNPs). To create a SERS-active platform, we deposited silver on the inside and outside of the hPPy nanohorn surface; this resulted in the formation of core/double-shells Ag/polymer/Ag nanostructures. In addition, peptide-functionalized AuNPs with Raman dye was pre-immobilized on the Ag/hPPy/Ag nanohorn to enhance the signals and skip further steps for the biomolecular detection. Using this SERS-sensitive novel core/double-shell nanohorn platform, we enhanced Raman signal and improved the sensitivity of caspase-3 associated with several diseases, and achieved a wide detection range from 10 pg/ml to 10 µg/ml. We demonstrate that the highly sensitive and simple detection of this nanohorn platform can be potential to be used for sensitive detection of proteolytic biomarkers and early diagnosis of several diseases. ** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2019R1A2C3002300) and by the Ministry of Education (No.2016R1A6A1A03012845). Keywords : Hollow polypyrrole (hPPy) nanohorn, Surface-enhanced Raman spectroscopy (SERS), Proteolytic biosensor, Au nanoparticles, Caspase-3 References 1. J.-H. Choi, and J.-W. Choi, Applied Surface Science, 506, 144669 (2020).

266


P0053 Production of Basic Fibroblast Growth Factor Protein Nanoparticles Using Human Serum Albumin

Seyoung HONG, Jisoo JANG, Hee Ho PARK Department of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Basic fibroblast growth factor (bFGF) is one of the therapeutic growth factors used for skin regeneration [1] and stem cell culture [2]. Recently, the delivery of bFGF has been studied as a strategy for scar treatment [3]. However, it is known that the stability of the bFGF is relatively short with a half-life about 8 h [4]. In this study, bFGF protein nanoparticles were generated to overcome the short half-life of bFGF. Previously, HSA was used as an ingredient for generation of protein nanoparticles and as a delivery platform due to the advantages of high biocompatibility, ease of preparation and sustained release of carrier proteins. Herein, we generated HSA-bFGF nanoparticles by desolvation method. The HSA-bFGF nanoparticles were spherical in shape and uniform with size of 200 nm, and confirmed controlled release of bFGF. However, further research is required for its efficacy in tissue regeneration. Keywords : Nanoparticle, bFGF particle, Desolvation, HSA nanoparticle, Protein delivery References 1. U. Park, M. S. Lee, J. Jeon, S. Lee, M. P. Hwang, Y. Wang, H. S. Yang, and K. Kim, Acta Biomater. 90:

179-191 (2019). 2. U. Han, H. H. Park, Y. J. Kim, T. H. Park, J. H. Park, & J. Hong, ACS Appl. Mater. Interfaces. 9(30),

25087-25097 (2017). 3. H. Shi, C. Lin, B. Lin, Z. Wang, H. Zhang, 1 F. Wu, Y. Cheng, L. Xiang, D. Guo, X. Luo, G. Zhang, X. Fu,

S. Bellusci, X. Li, and J. Xiao, PLoS One. 8(4): e59966 (2013). 4. S. Lotz, S. Goderie, N. Tokas, S. E. Hirsch, F. Ahmad, B. Corneo, S. Le, A. Banerjee, R. S. Kane, J. H.

Stern, S. Temple, & C. A. Fasano, PLoS ONE. 8(2), e56289 (2013).

267


P0054 Field-effect Transistor Based Portable Microfluidic Device for Real-time Monitoring of Pathogenic Bacteria

Kyung Ho KIM Infectious Disease Research center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Corresponding Author Email : [email protected] The infection from food-borne bacteria in the food and drink etc is a critical issue over the world. For the detection of pathogenic bacteria, various technologies such as Gram-stain, cultures, Enzyme-linked immunosorbent assay (ELISA), lateral flow assay (LFA), polymerase chain reaction, developed, however, which still suffered in many components, high-cost, low-performance, time-consuming. Herein, we demon-strated a portable device consisted of a microfluidic-introduced graphene-based field-effect (FET). The portable sensor platform showed high-performance of 10 cfu/mL LOD, < 5 s rapid detection, and excellent selectivity in mixture bacteria. Those results, this development technology of portable FET sensor platform can be used for real-time monitoring in the field and can be utilized for the diagnosis of other types of diseases such as virus. Keywords : Portable biosensor, Field-effect tansistor, Microfluidic, Real-time monitoring, Disease diagnosis

268


P0055 A Water Soluble, Wearable, and Flexible Silk Film Substrate Based Cortisol Aptasensor

Jai Eun AN, Oh Seok KWON Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Corresponding Author Email : [email protected] In this study, an ultra-light wearable biosensor detecting an important biomarker was manufactured on naturally abundant silk substrate possessing high performance and low toxicity under environmentally benign conditions. The core sensing medium material was produced by electrospinning of polymer nanofiber and subsequent vapor deposition of conducting polymer layer. An electrode for the sensor was patterned effectively on silk substrate using simple lithographic method and fundamental electrical behavior of the sensor was monitored for practical application. The complementary advantages of the silk substrate such as thermal stability and easy removal are also demonstrated. The critical sensing mechanism was dependent on the sensitive and specific interaction between target molecules and aptamer, which was introduced on the surface of the sensing medium material. It was demonstrated that the sensor showed an excellent detection limit (10 pM) and selectivity to a stress hormone, cortisol under the presence of competing molecules. The practical usability of the silk-based sensor was demonstrated by the introduction of the sensor to swab and human skin, which showed comparable performance as the pristine sensor. This study is a solid and sound example to support the usefulness of the silk as a substrate for wearable and disposable biosensor. This methodology can be expanded to the accurate diagnosis of several diseases associated with the cortisol concentration change. Keywords : cortisol, aptasensor, silk film, wearable, flexible

269


단백질/효소공학

(Protein and Enzyme Engineering)

270


P0101 Site-directed Immobilization of the Catalytic Subunit of Mo-Cu Carbon Monoxide Dehydrogenase (CODH) Fused with Gold-binding Peptide on Au Electrode Surface

Stacy REGINALD, Minji KIM, Hyeryeong LEE, Eunmi LEE, In Seop CHANG School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] In this work, we extended the generic approach for the site-directed immobilization of enzymes based on gold-binding peptide affinity of engineered enzymes fused with gold-binding peptide (gbp) (LKAHLPPSRLPS) to the Au electrode surface. The catalytic subunit of the native CO oxidizing enzyme, CO dehydrogenase (CODH) and few CODH variants consisting of gbp fused at N- or/and C-terminus are carefully designed and constructed to assess the changes in biocatalytic activity, the binding kinetics, and bio-electrocatalytic properties. Atomic force microscopy (AFM) was used to analyze the assembly characteristics of the synthetic proteins while their binding kinetics on to Au surface was quantified using surface plasmon resonance (SPR). SEM-EDS is used for elemental analysis of the enzyme immobilized on the Au substrates. Electrochemical test including cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS) are employed to evaluate the bio-electrocatalytic reaction, electroactive enzyme surface and electron transfer rates at the enzyme-electrode interface. Keywords : Site-directed immobilization, Carbon monoxide dehydrogenase (CODH), Enzyme bioelectrocatalysis References 1. Y. S. Lee, S. Baek,, H. Lee, S. S. Reginald, Y. Kim, H. Kang, I.-G. Choi, I. S. Chang, ACS Appl. Mater. &

Interfaces.10 (2018). 2. H. Lee, Y. S. Lee, S. S. Reginald, S. Baek, E. M. Lee, I.-G. Choi, I. S. Chang, Biosens. Bioelectron.,165

(2020). 3. F. A. Al-Lolage, P. N. Bartlett, S. Gounel, P. Staigre, P. N. Mano, ACS Catalysis. 9 (2019), 2068-207.

271


P0102 Improvement of Formate Dehydrogenase Expression in Methylobacterium extorquens AM1 by 5’ UTR Engineering

Uyen Thu PHAN, Byeong Wook JEON, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Formate dehydrogenase 1 (MeFDH1) from Methylobacterium extorquens AM1 – the key responsible enzyme for formate production from carbon dioxide (1) – is homologously overexpressed in M. extorquens AM1 cells by broad-host-range pCM110 plasmid. This expression system contains a universal ribosome binding site (RBS) from gene 10 of bacteriophage T7 which has been reported to give high translation efficiency in E. coli (2). While functional in recombinant M. extorquens AM1, the expression level is limited and requires further improvement. As 5’ UTR (untranslated regions) are known to strongly influence the efficiency of translation (3), modifying the 5’ UTR sequence is expected to alter the translation level of MeFDH1 relative to all other translated coding sequences inside the cells. Modified 5’ UTR sequences were directly derived from native genes that encode highly expressed proteins in M. extorquens AM1 under methanol and succinate growth conditions (4). A software called UTR Designer (5) was used to predict the expression level of MeFDH1 when given the 5’ UTR and coding sequence (reverse engineering). The 16s rRNA sequence was fixed as ‘‘UUUCCUCCA’’. Translation efficiencies were presented as enzyme volumetric yields. As expected, all four selected sequences showed relatively enhanced expression in comparison with universal RBS. In conclusion, the newly introduced 5’UTR appeared to be the most potential as prominently high-activity elements for maximizing translational efficiency of MeFDH1, while universal RBS would not have been an optimal choice for M. extorquens AM1 expression system. Keywords : formate dehydrogenase, Methylobacterium extorquens AM1, 5’ UTR engineering, translation efficiency

272


P0103 Quantification of 2’-Fucosyllactose via Ligand-Dependent Thermal Stabilization of a BoGT6a

Nayeon KIM, Jinkyeong YANG, Seung-Joo KIM, Minju KIM, Youngseo PARK, Dae-Hyuk KWEON Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] 2’-Fucosyllactose is one of the major components of human milk oligosaccharides (HMOs) and has beneficial effects on human health. Since numerous studies have shown that the 2’-FL deficiency of breast milk influences infants health, the quantification method of 2’-FL has been required to forestall their diseases. However, so far, cost-ineffective equipment such as HPLC and MS has played a role as the only way to measure the amount of 2’-FL. Here, we present a general strategy for generating a 2’-FL biosensor through ligand-dependent protein stabilization. Generally, protein-ligand complexes have shown the structural stability than free state protein as they form hydrogen bonds. One of the candidate proteins for 2’-FL binding, Bacteriodes ovatus Glyco-syltransferase 6 (BoGT6a) has shown its structural robustness when heated at 50, 65 for 2 hours after bound to 2’-FL. We designed a mCherry fusion BoGT6a in order to detect the amount of the stabilized protein through fluorescence intensity. When steamed at 100 for 15 minutes, 2’-FL treated fusion BoGT6a showed the intensity of fluorescence by an amount proportional to the concentration of 2’-FL. Even boiling for 5-7 minutes, mCherry fusion BoGT6a maintains its solubility after treated with 1g/L 2’-FL which is in a range of the average amount of 2’-FL in breast milk. The approach we take suggests that carbohydrate biosensors could be easily developed through the binding affinity of protein to a ligand. Keywords : 2’-Fucosyllactose, Ligand dependent protein stabilization, Protein thermal stability, BoGT6a References 1. Majewski, M., Ruiz-Carmona, S., & Barril, X. (2019). An investigation of structural stability in protein-

ligand complexes reveals the balance between order and disorder. Communications Chemistry, 2(1), 1-8. 2. Pham, T. T., Stinson, B., Thiyagarajan, N., Lizotte-Waniewski, M., Brew, K., & Acharya, K. R. (2014).

Structures of complexes of a metal-independent glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-acetylgalactosamine (UDP-GalNAc) and its hydrolysis products. Journal of Biological Chemistry, 289(12), 8041-8050.

3. Bode, L. (2012). Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology, 22(9), 1147-1162.

4. Shin, J., Park, M., Kim, C., Kim, H., Park, Y., Ban, C., ... & Park, Y. C. (2020). Development of fluorescent Escherichia coli for a whole-cell sensor of 2ʹ-fucosyllactose. Scientific reports, 10(1), 1-8.

5. Cimmperman, P., Baranauskine, L., Jachimoviciute, S., Jachno, J., Torresan, J., Michalioviene, V., … Matulis, D. (2008). A quantitative model of Thermal stabilization and destabilization of proteins by Ligands. Biophysical Journal, 95(7), 3222-3231. doi:10.1529/biophysj.108.134973.

273


P0104 A Novel Two-step Chemoenzymatic Pathway for the Asymmetric Synthesis of γ-valerolactone from Levulinic Acid

Yurim KIM1, Jiyeoung JEONG1, Gyulim OH1, Dohoon LEE2, Young Joo YEON1 1Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Korea, 2Green Chemistry and Materials Group, Korea Institute of Industrial Technology (KITECH), Cheonan, Korea Corresponding Author Email : [email protected] A chemoenzymatic reaction pathway was developed for the conversion of levulinic acid to enantiomerically pure γ-valerolactone which can be applied in green solvent, fuel, perfume and flavor industries. Levulinic acid can be obtained from cheap, abundant and environmentally friendly cellulosic biomass. Currently, there have been limitations in producing (R)-γ-valerolactone with high yield and enhanced enantiopurity from free from of levulinic acid. In this study, levulinic acid was transformed into (R)-γ-valerolactone through 4-hydroxyvaleric acid in two steps. In the first-stage reaction, engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) was used for efficient catalytic conversion of levulinic acid to (R)-4-hydroxyvaleric acid. In the second-stage reaction, 1% sulfuric acid was used to lactonize (R)-4-hydroxyvaleric acid to (R)-γ-valerolactone. In conclusion, (R)-γ-valerolactone with approximately 100% yield and >99% enantiomeric excess could be produced from the free form of levulinic acid. Keywords : Chemoenzymatic synthesis, (R)-γ-valerolactone, Levulinic acid, 3-hydroxybutyrate dehydrogenase References 1. D. Lee and Y. J. Yeon, Process Biochemistry. 90, 113-117 (2020).

274


P0105 Soluble Expression of Recombinant Dengue Viral Non-structural Protein 1 in E. coli

Eunjeong KIM, Sooim SHIN Department of Biotechnology and Bioengineering, College of Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Non-structural protein 1 (NS1) of dengue virus is one of the factors that develop the diseases of severe dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF) during the viral pathogenesis. However, the mechanism of NS1 in the progression of the disease is barely understood. Besides, efficient processes for production of recombinant NS1 as a soluble form have not been established. In this study, we constructed a recombinant NS1 expression system to acquire soluble and proper size (40 kDa) of NS1 protein with good yield. We genetically engineered 6xHis-tagged NS1 gene within pET11a and expressed it in Escherichia coli system, which is widely used and easily scaled-up. Notably, we fused the periplasm targeting signal sequence of amicyanin protein that is encoded by mauC gene from Paracoccus denitrificans, at the end of the N-terminus of NS1 and confirmed expression of NS1 protein in periplasm in E-coli system. Our work will further help to find the role of NS1. Keywords : Dengue virus, NS1, Escherichia coli, Targeting periplasmic expression

275


P0106 Reconstruction of Protein Structure Prediction Algorithm Using Deep Learning

Jaehyung KIM, Donghyuk KIM School of Energy and Chemical Engineering, UNIST, Ulsan, Korea Corresponding Author Email : [email protected] In recent years, many protein structure prediction algorithms were deposited on Open source community, Github. However, Alphafold,[1] which performed the most outstanding result has open-sourced a portion. Its algorithm would be roughly divided into three parts: Feature generation from an amino acid sequence, Prediction of distance and its torsion angle, Stabilize its three-dimensional structure. Deepmind, the research group who deposited Alphafold, open-sourced prediction of distance and torsion angle. In this research, we focused on the reconstruction of feature generation, which is the most significant part of the algorithm. By using PSI-BLAST[2] and HHblits[3] as a multiple sequence alignment tool, we can derive its MSA files rapidly and accurately. Also, through deposited distance and torsion prediction code, using torsion and distance data, we can calculate its three-dimensional structure into PDB format files using PyRosetta[4]. We just used a simple free energy stabilization pipeline which is deposited on Github to compare real and predicted model. This study will suggest an improvement in the understanding function of proteins, showing a huge development of protein engineering. Keywords : Alphafold, Protein Structure, Deep Learning, Machine Learning References 1. Senior, A.W., Evans, R., Jumper, J. et al. Improved protein structure prediction using potentials form deep

learning . Nature 577, 706-710 (2020). 2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-

BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389-402 (1997).

3. Remmert, M., Biegert, A., Hauser, A. et al. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment . Nat Methods 9, 173-175 (2012).

4. Chaudhury S, Lyskov S, Gray JJ. PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta . Bioinformatics 26(5): 689-91 (2010).

276


P0107 Paper Chip Format Kit for the Detection of Tyrosine Level in Urine Using Surface Displayed Tyrosinase

Junehyung KIM1,2, Yura JEONG1,2, Chanjin PARK1,2 1Department of Chemical Engineering, College of Engineering, Dong-A University, Busan, Korea, 2Center for Sliver-Targeted Biomaterials, Brain Busan 21 Plus Program, Graduate School, Dong-A University, Busan, Korea Corresponding Author Email : [email protected] Simple and cheap diagnostic kit development is one of the important aims of pharmaceutical developers and companies focused on public health improvement. The Bacillus subtilis spore surface-display technique is a genetic engineering method that is used to develop new-generation diagnostic kits applicable for the early detection of various types of diseases. In this study, we developed a novel simple, rapid, and inexpensive diagnostic paper-based kit to detect tyrosine in urine samples of humans and animals that is applicable for home or laboratory use for the early diagnosis of Tyrosinemia. The Bacillus subtilis spore-displayed tyrosinase system developed by genetic engineering methods was used to prepare a paper-based kit to detect tyrosine in urine samples of different groups of patients (i.e., patients with diabetes, diabetes with chronic kidney disease, and chronic kidney disease) for the detection of tyrosine during the acute disease phase. To confirm the sensitivity and specificity of the kit, tyrosine was also detected in urine samples using conventional liquid chromatography/ mass spectroscopy. Different concentrations of tyrosine (0.1 Nm-1 mM) were detected in urine samples based on visible changes of color from bright brownish-gray to dark brownish-gray within 1 h. The kit could screen samples to distinguish the three groups of patients based on formation of a broad spectrum of colors reflecting the concentration of tyrosine. Keywords : Biosensor, Tyrosinase, Urine Detection

277


P0108 Effective Method to Extract Human Norovirus VP1 from an Inclusion Body Expressed from E. coli

Heejin NAM1, Moonsung CHOI2, Sooim SHIN1,3 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju, Korea, 2Department of Optometry, College of Energy and Biotechnology, Seoul National University of Science and Technology, Seoul, Korea, 3Department of Biotechnology and Bioengi-neering, College of Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected], [email protected] Norovirus is a leading cause of acute gastroenteritis. It is a non-enveloped virus containing a single-stranded positive-sense RNA genome. The capsid mainly consists to major structural protein called VP1. VP1 divides to two domains: S domain for viral assembly, and P domain acting as an antigen. To develop a virus-like particle (VLP) vaccine against norovirus, full-length VP1 possessing P domain and S domain is required. VLP, which is a viral particle without viral genome, was developed as an effective and safe vaccine. However, it is rarely commercialized due to low productivity and lack of expression systems. Here, we constructed the effective method to isolate norovirus VP1 from inclusion body for VLP development. VP1 was expressed as an inclusion body in E. coli and extracted in a soluble form by solubilization using high concentration of urea. All components of buffers were optimized considering the chemical compatibilities of Ni-NTA.[Ss1] The solubilized VP1 was purified using Ni-NTA and dialyzed by urea-gradient. Finally, the extracted VP1 was obtained in a clear solution which indicates its proper folding. We expected that these methods can contribute to diverse VLP production Keywords : Human norovirus VP1, protein solubilization using urea, urea gradient dialysis

278


P0109 Further Engineering of Aldehyde Dehydrogenase for Improving the Catalytic Activity Toward 3-HPA via 3-HP Selection Device and 96-well Microplate-based Screening

Ye Seop PARK1, Abdul NASIR1, Sunjoo CHOI1, Joo Yeon SEOK2, Gyoo Yeol JUNG2,3, Tae Hyeon YOO1,4 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 3Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 4Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] 3-Hydroxypropionic acid (3-HP) is one of the key building blocks which is a precursor of diverse high-added value chemicals and can be used as an additive or preservative agent in the food industry. The molecule can be produced by a simple biological pathway: dehydration of glycerol to 3-hydroxypropanal (3-HPA) by glycerol dehydratase and then oxidation to 3-HP by aldehyde dehydrogenase. Despite successful production of 3-HP through this pathway, several problems including the toxicity of the 3-HPA hamper the 3-HP production by industrial permissive level. To solve the issues, we had engineered one of aldehyde dehydrogenase, α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirilum brasilense for enhancing the catalytic activity toward 3-HPA using 96well microplate-based screening or transcriptional factor-based selection device. For screening better mutants with respect to catalytic activity toward 3-HPA than other KGSADH mutants, we analyzed amino acids of the mutants which have been already screened. From the analysis, we have made a new library that includes several residues that might be important for the catalytic activity with respect to 3-HPA and screened the library via a 3-HP selection system followed by NADH absorbance-based 96well plate screening. Finally, we got the final mutant which has 4.65-fold higher activity than WT. We also found that the mutant has a high preference toward propionaldehyde which has a similar structure with 3-HPA except the -OH group. Nowadays, we are going to analyze modeled structures of WT and the mutant docked with 3-HPA by MD simulation to understand the effects of mutations and conduct several in vivo analyzes for checking the production of 3-HP. Keywords : directed evolution, aldehyde dehydrogenase, 3-HP References 1. Park, Y.S., et al., Sci. Rep. 7(1), 1-12 (2017). 2. Seok, J.Y., et al., Metab. Eng. 47, 113-120 (2018).

279


P0110 Site-specific Conjugation of IgG via a Facile Photocrosslinking Reaction

Jisoo PARK1, Yeaji LEE1, Tae Hyeon YOO1,2 1Department of Molecular Science and Technology, Ajou university, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou university, Suwon, Korea Corresponding Author Email : [email protected] Antibody-based conjugates have become an essential component in a variety of applications from immune-assays especially to drug conjugates because of their proven efficacy and selectivity to target cancer cell. Herein, we describe a peptide-directed photo-crosslinking reaction as a novel site-specific conjugation method of IgG using an Fc-binding peptide and a photoreactive amino acid analogue for generation homogeneous IgG-toxin conjugates. Keywords : Site-specific conjugation of IgG, Immunotoxin, Fc-binding peptide, Photo-reactive amino acid

280


P0111 Conserved Sequence-based Recombination of Homologous Sugar Isomerases from Diverse Species Reveals Genetic Traits for Environmental Adaptation

Jae-Yoon SUNG1, Keunyoung YOO1, Yunhye JOO1, Sun-Mi SHIN2, Sang-Jae LEE3, Immanuel DHANASINGH4, Ngoc Kim Quyen HOANG4, Thịnh-Phát CAO4, Seong-Bo KIM5, Sung Haeng LEE4, Dong-Woo LEE1 1Department of Biotechnology, Yonsei University, Seoul, Korea, 2School of Applied Biosciences, Kyungpook National University, Daegu, Korea, 3Major in Food Biotechnology, Silla University, Busan, Korea, 4Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju, Korea, 5Department of Bio-Living Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Homologous recombination can produce stable progenitors with combined properties from parental sources. Molecular breeding by DNA shuffling can readily generate recombined genes, resulting in evolved enzymes with various intrinsic properties. Here we obtained a variety of functional chimeras from five homologous genes encoding hexameric L-arabinose isomerases (AIs) with a wide range of temperature and pH optima for catalysis through conserved sequence-aided self-assembly of DNA fragments. Out of theoretically 625 variants, we selected 78 functionally active chimeras with distinct temperature and pH optima, differing from those of parental enzymes. The fitness landscape of their activities and the nonsynonymous to synonymous nucleotide substitution rates revealed the region-specific signatures contributing to the intrinsic properties of AIs. Subsequent characterization of several chimeric and mutant enzymes confirmed that the proposed adaptive traits contribute to the temperature and pH dependence of AIs through intra-subunit salt bridges and electrostatic interactions. These evolutionary strategies for environmental adaptation are also found in naturally occurring AI homologs. Therefore, these results suggest that conserved sequence-based recombination can readily provide an unprecedented repertoire of homologous enzymes, implying the rapid adaptive evolution of homologous enzymes fitting into harsh environments. Keywords : recombination, self-assembly, chimeras, environmental adaptation, sugar isomerase

281


P0112 Developing the New Concept of Anti-gene Doping Assay Using CRISPR System

Joon-Yeop YI1,2,3, Minyoung KIM1, Byung-Gee KIM2,3,4,5,6, Junghyun SON1, Changmin SUNG1 1Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea, 2Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea, 3Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea, 4School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 5Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea, 6Institute for Sustainable Development (ISD), Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Doping is prohibited by the World Anti-Doping Agency (WADA) primarily to protect the health of athletes and for the sake of fairness in competition. With the rapid development of genetic engineering techniques for the treatment of genetic diseases, unethical attempts to use genetic techniques to improve sports performance have also arisen. To prevent this, WADA has restricted the use of genes to improve motor performance, non-therapeutic trials of cells that contain genetic elements, and genetic modification which have been listed as prohibited substances. But to date, there has been no known method for detecting gene doping except for RT–PCR doping test. Herein, we propose a CRISPR-Cas9 based gene doping detection system, using Cas9 for detecting cDNA of human erythropoietin (hEPO) as a target gene. With this concept, two sites among four exon-exon junctions in hEPO cDNA were simultaneously targeted by single-guide RNA (sgRNA) and cleaved by Cas9. As a result, the cleaved fragments could be identified on agarose electrophoresis gel with a detection limit of 1.25 nM. We expect that more sophisticated detection methods combined with Cas9-based assay can be a new standard assay. Keywords : CRISPR-Cas9, gene doping control, erythropoietin, sports References 1. Yi et al., Drug Test. Anal. DOI: 10.1002/dta.2980 (2021).

282


P0113 The SufS-SufU Complex Mediating Extracellular Cysteine-cystine Recycling in Fervidobacterium islandicum AW-1 is Associated with Keratin Degradation

Hyeon-Su JIN1, Yena LEE2, Immanuel DHANASINGH3, Jae Yoon SUNG1, Jae Won LA1, Eun Mi LEE4, Yujin KANG5, Do Yup LEE4, Sung Haeng LEE3, Dong-Woo LEE1,2 1Department of Biotechnology, Yonsei University, Seoul, Korea, 2Department of Bio-industrial Engineering, Yonsei University, Seoul, Korea, 3Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju, Korea, 4Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Sciences, Seoul National University, Seoul, Korea, 5Department of Bio and Fermentation Convergence Technology, BK21 PLUS Program, Kookmin University, Seoul, Korea Corresponding Author Email : [email protected] Most extremophilic anaerobes possess an ancient sulfur formation (Suf) system for Fe-S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe-S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. The feather-degrading bacterium has complete Suf-like machinery that is highly expressed in cells grown on native feathers. Detailed differential expression analysis and physicochemical analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Remarkably, metabolite profiling of extracellular and intracellular levels of amino acids revealed that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS–SufU complex may contribute to sulfur availability for cellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions. Keywords : SufS-SufU complex, cysteine and cystine recycling, F. islandicum AW-1

283


P0114 Study of Adsorption Phenomena of Recombinant Lignin Peroxidase Isozymes from Phanerochaete chrysosporium on Solid Lignin Surface

Trang Vu Thien NGUYEN, Seunghyun HAN, Hyeryeong GYE, Jooyoung PARK, Heeyeon BAIK, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] In vivo lignin degradation is composed of a series of complicated process started with the adsorption of lignin modifying enzymes onto lignin surface. Although enzymatic adsorption is prerequisite condition for the enzymatic lignin depolymerization, the adsorption of lignin modifying enzymes on lignin has not been studied so far. In this work, the adsorption behaviors of eight lignin peroxidase isozymes from Phanerochaete chrysosporium (PcLiPs) on poplar milled wood lignin (MWL) were investigated. The findings indicated the adsorption of PcLiPs on MWL is a pH-dependent step, which suggested the contribution of electrostatic interactions between PcLiPs and MWL. The adsorption kinetics of PcLiPs performed at pH 3.0 – 3.5 and 25oC showed that the equilibrium state was reached within 2h. Furthermore, the adsorption isotherms data were obtained under the equilibrium state and fitted to the Langmuir adsorption model, which clearly showed that a few PcLiP has exceptionally higher adsorption capability. Overall, our work suggested that specific PcLiP bearing higher adsorption capacity might play a significant role in the lignin degradation and strongly emphasized the importance of further investigations into the adsorption of lignin modifying enzymes for the efficiently enzymatic delignification process. Keywords : adsorption, lignin peroxidase, Phanerochaete chrysosporium, lignin

284


P0115 Cytochrome P450 2F2 (CYP2F2) Negatively Regulates Browning in 3T3-L1 White Adipocytes

Trang Thi Huyen DANG, MinJi CHOI, Huong Giang PHAM, Jong Won YUN Department of Biotechnology, Daegu University, Gyeongsan, Korea Corresponding Author Email : [email protected] Cytochromes P450 (CYPs) are a multigene superfamily of constitutively expressed and inducible enzymes responsible for the detoxification of many endogenous and exogenous compounds and for the metabolism of numerous medications. The CYP2F2 subfamily is preferentially expressed in the respiratory tract, but its functional role in adipocytes has never been explored. We found that CYP2F2 was highly expressed during the differentiation of the C3H10T1/2 murine mesenchymal stem cells to adipocytes and here we have explored its functional role in adipocytes. The expression of thermogenic marker proteins (PGC-1α, PRDM16, and UCP1) and beige-fat specific genes were significantly increased in Cyp2f2 deficient 3T3-L1 adipocytes. Moreover, Cyp2f2 silencing led to reduced adipogenesis and lipogenesis, and enhanced lipid catabolism through the increased expression of lipolytic and fatty acid oxidative enzymes. A mechanistic study to identify molecular signals for CYP2F-mediated negative regulation in the browning of white adipocytes revealed that CYP2F2 impairs the β3-adrenergic receptor activation as well as its downstream regulators (PKA, p38 MAPK, and ATF2). This data provides evidence that CYP2F2 is a negative regulator of lipid catabolism and browning in white adipocytes, suggesting that inhibitors of CYP2F2 could be potential drugs for the treatment of obesity with a focus on enhancing energy expenditure. Keywords : CYP2F2 , Browning, Obestiy References 1. M. Kotlyar, S. W. Carson, Int. J. Clin. Pharmacol. Ther. 37,8-19 (1999). 2. D. W. Nebert, K. Wikvall, W. L. Miller, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 368, 20120431 (2013).

285


P0116 Loss of ADAMTS15 Promotes Browning in 3T3-L1 White Adipocytes via Activation of β3-adrenergic Receptor

Min Ji CHOI, Jong Won YUN Department of Biotechnology, Daegu University, Gyeongsan, Korea Corresponding Author Email : [email protected] ADAMTSs belong to the superfamily of secreted metalloendopeptidases, some of which are reported to be closely associated with obesity. However, the role of ADAMTS15 is not well characterized in adipocytes. This study investigates the effect of Adamts15 deficiency on lipid metabolism in 3T3-L1 adipocytes, with a focus on the role of browning of white adipocytes. Quantitative real-time PCR, immunoblot analysis, immune-fluorescence and staining methods were applied to evaluate the effects of ADAMTS15 on other target proteins and genes involved in lipid metabolism, after silencing Adamts15 by applying the siRNA technique. Our results demonstrate that ADAMTS15 is expressed in white adipocytes, and the deficiency of Adamts15 promotes browning of white adipocytes by enhancing the expression of brown adipocyte-specific genes and proteins. In addition, silencing of Adamts15 upregulates lipid metabolic activity in white adipocytes, by increasing expression of mitochondrial biogenesis as well as lipolytic and fat oxidative marker proteins, and reducing adipogenic factors. Moreover, mechanistic studies revealed that depletion of Adamts15 induces browning via activation of the β3AR-PKA-CREB signaling pathway. Taken together, our data unveiled a previously unknown mechanism of ADAMTS15 in the regulation of lipids, and the significance of this protein as a pharma-cotherapeutic target to treat obesity-related metabolic disorders. Keywords : ADAMTS15 , UCP1, Browning References 1. T. J. Mead and S. S. Apte, Matrix Biol. 71-72, 225-239 (2018). 2. S. I. M. Porter and D. R. Edwards, Biochem. J. 386, 15-27 (2005).

286


P0117 Carboxylesterase 3 Promotes Differentiation of Osteoblasts via Smad 1/5/9 Pathway

Sulagna MUKHERJEE, Jong Won YUN Department of Biotechnology, Daegu University, Gyeongsan, Korea Corresponding Author Email : [email protected] Recently, much attention has been paid to a potential biochemical cross-talk between the metabolism of the bone and adipose tissue termed “bone-adipose axis.” A cross-linking study to understand the various interacting proteins involved in bone-adipose axis could provide identification of novel targets for development of drugs. Although Ces3 has been known as a major lipolytic enzyme predominant in liver and adipocytes, recent reports identified its presence in skeletal muscles. We here explored functional roles of Ces3 in osteoblasts and mesenchymal stem cells differentiating into osteoblast linage. We investigated the physiological function of Ces3 by stable gene knockdown of Ces3 and exogenous Ces3 induction. Current data support that Ces3 is highly expressed in osteoblasts and promotes proliferation of the cells by increasing the expressions of osteogenic marker proteins and genes. Our mechanistic studies for the first time revealed that Ces3 interacts with BMP11 proteins for regulation of the osteoblasts differentiation and activates the ALK2 and BMP type II receptor proteins following Smad 1/5/9 signaling pathways. In conclusion, current data unveiled a previously unknown mechanism in the regulation of Ces3 and BMP11 in bone-adipose axis, shedding light on Ces3 as a pharmacotherapeutic target to treat metabolic disorders. Keywords : Ces3 , BMP11, osteoblast References 1. J. Lian and R. Lehner, Protein Cell. 9, 178-195 (2018). 2. A. D. Berendsen and B. R. Olsen, Cell Mol. Life Sci. 71, 493-497 (2014).

287


P0118 Production And Higher Yield Approach of Soluble Form of Recombinant Lignin Peroxidases (Pclips) in Pichia pastoris For Industrial Applications

Vikky RAJULAPATI, Seunghyun HAN, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Lignin is the amplest sustainable aromatic polymer on earth. Lignin plays a key role in plants, provide the protect matrix to cellulose microfibrils of cell wall and helps to transport system in plants [1]. Its conversion to the value-added products from biomass waste research is increasing nowadays. Lignin degradation occurs oxidation by lignin peroxidases (EC 1.11.1.14, LiPs) in presence of H2O2. LiPs are the first class of enzymes to degrade lignin. The lignin peroxidases have various applications such as biorefinery, textile, energy, bioreme-diation, cosmetology and dermatology industries. LiPs are majorly secreted by white rot basidiomycete Phanerochaete chrysosporium, (PcLiPs). PcLiP isoenzymes have huge demand for industrial applications [2]. The production of PcLiPs have been showed low expression, yield of protein, incorrect fold and forming inclusion bodies in E. coli expression system. The recovery of LiPs using the unfolding and refolding by urea, which leads the expensive, time consumption process and ultimately the low production yield [3]. To overcome these, the present research approach, the PcLiP isoenzymes expression and production in eukaryotic system using different forms of PPICZ and PPICZα vectors through various Pichia pastoris strains. P. pastoris is widely used for production of heterologous proteins, due to posttranslational modifications, proper folding and high cell density [4]. P. pastoris is a suitable system for express and production of high-level secretion, PcLiP isoenzymes for industry applications. Keywords : lignin peroxidases, Pichia pastoris, Phanerochaete chrysosporium References 1. M. Tien and T. K. Kirk, Sci. 221(4611), 661-663 (1983). 2. R. L. Crawford, John Wiley and Sons. (1981). 3. W. A. Doyle and A. T. Smith, Biochem. J. 315(1), 15-19 (1996). 4. J. L. Cereghino and J. M.Cregg, FEMS Microbiol. Rev. 24, 45-66 (2000).

288


P0119 Repeated Usage of Bacillus subtilis Spore Displayed Tyrosinase for the Biotransformation of Phloretin

Junehyung KIM1,2, Yura JEONG1,2, Chanjin PARK1,2 1Department of Chemical Engineering, College of Engineering, Dong-A University, Busan, Korea, 2Center for Sliver-Targeted Biomaterials, Brain Busan 21 Plus Program, Graduate School, Dong-A University, Busan, Korea Corresponding Author Email : [email protected] Using Bacillus subtilis spore displayed tyrosinase from BM3 biotransformation of phloretin, one of the dihydrochalcone found in apple bark, was tried. After HPLC analysis, biotransformed product was identified and it was further analyzed using LC/MSMS and 1H-NMR to be revealed as 3’-hydroxyphloretin. 100 % conversion of 1 mM phloretin was obtained after 7 hr incubation at 37°C. To exploit the inherent stability of Bacillus spore, which is used as immobilizing vehicle for the tyrosinase, repeated usage of spore displayed tyrosinase was tried. The spores were collected by centrifugation and the reaction was re-performed in the same way as in the initial experiment, and the biotransformation proceeded through 7 recycling cycles without showing any decrease of activity. Keywords : Bacillus subtilis spore, biotransformation, phloretin, tyrosinase

289


P0120 Pseudovirus Assay : Screening Antiviral Drugs in a Simple Way

Jeonghui MOON Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] Viruses have been affected worldwide for a long time. For example, currently most emerging SARS-CoV-2 has been caused many confirmed people and death. To treat these viruses, many antiviral drug researches are proceeding. However, it is forbidden to handle highly pathogenic viruses in low BSL research institutes. To solve this problem, we want to set-up ‘pseudovirus assay’ system. Pseudovirus is a type of virus-like particle (VLP), containing interest virus’s receptor binding protein. It cannot generate progeny containing receptor binding protein, therefore, it has no more replication cycle and thus safe to use instead of highly pathogenic real virus. In this study, HIV gag, pol genes for packaging lentiviral vectors, and modified viral envelope protein plasmids and transfer plasmids were used for high-titer with fluorescence-luminescence dual reporter pseudovirus system. 72 hours after pseudoviruses infect target cells, reporter mCherry and luciferase genes were transferred and expressed in the cell. Thus virus infectivity can be estimated through fluorescence and luminescence values using spectrometer. When antiviral drugs were treated together, the fluorescence value was seen decreased in inverse proportion to the concentration of the drug. Using this high-titer with dual reporter pseudovirus system, it is expected that efficacy of anti-viral drug candidates, targeting high risk viruses, can be confirmed in a simple way in low BSL institutes. Keywords : Pseudovirus assay, Virus, Coronavirus, Anti-viral drug, Anti-coronaviral drug

290


P0121 Site-specific Enzymatic Methylation of Epigallocatechin Gallate (EGCG) by Engineered Bacterial O-methyltransferase

Gyeong-Guk PARK1, Heewon NHO2, Uk-Jae LEE2, Joo-Hyun SEO3, Byung-Gee KIM1,2,4,5,6 1Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea, 2School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 3Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul, Korea, 4Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea, 5Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea, 6Institute for Sustainable Development(ISD), Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Tea shows various health effects including antioxidant, anti-inflammation, anti-cancer, and other therapeutic treatments. The (-)-epigallocatechin-3-gallate (EGCG) is the major catechin compound and active ingredient in green tea. EGCG is unstable and quickly oxidized in the pH range of blood. Additionally, due to low membrane permeability, the high dose of EGCG should be taken orally to expect such bioactive effects. The methylation of EGCG is the solution to overcome physiological disadvantages. Especially, EGCG''3Me is absorbed through the gastrointestinal tract more than EGCG. furthermore, its therapeutic effect is also increased. We found bacterial o-methyltransferases which transfer the methyl group of S-adenosyl methionine (SAM) to the 3'' hydroxyl group of EGCG. From the homology model and docking simulation, we hypothesized the substrate binding site and key residues for EGCG methylation. By further enzyme engineering, we can construct and evaluate mutant variants that structurally are modified for higher activity and regiospecific methylation on EGCG. Keywords : EGCG, o-methyltransferase, enzyme engineering References 1. Maeda-Yamamoto, Mari, Kaori Ema, and Ikuo Shibuichi, Cytotechnology 55.2-3: 135 (2007). 2. Dai, Wenzhong, et al., Journal of Functional Foods 65: 103732 (2020). 3. Wang, Fang-Yuan, et al., Journal of Pharmaceutical Analysis (2020).

291


P0122 Enhancement of Heme and Protein Interactions for Higher Thermodynamic Stability of Lignin Peroxidase

Joo Yeong PARK, Sung Heuck KANG, Trang Vu Thien NGUYEN, Seung Hyun HAN, Hye Ryeong GYE, Hee Yeon BAEK, Yong Hwan KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Soybean peroxidase(SBP) is known as an enzyme that has extremely high thermodynamic stability among plant peroxidase. SBP has a Tm of 83.5 °C in neutral pH. However, as a result of removing heme from SBP, Tm is significantly decreased to 38 °C. This means that interactions between heme and neighboring residues seem to play a big role in high thermodynamic stability of SBP. Based on this experimental observations, the interactions of heme and neighboring residues of LiP were investigated by overlapping 3D structures of SBP and LiP. In-silico MD simulation was performed to confirm candidates that make newly formed interactions with heme. Also, minimization energy was calculated for investigating correlation between the Gibbs free energy and Tm. Through this approach, higher thermodynamic stability of protein and application in enzyme industry are expected. Keywords : Lignin peroxidase, thermodynamic stability, heme

292


P0123 Homogeneous Assay Based on Proximity Proteolysis Reaction for Detection of Proteins and Small Molecules

Hyeon Ji PARK1, Minji GWON1, Tae Hyeon YOO1,2 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou University Suwon, Korea Corresponding Author Email : [email protected] Various types of molecules, including nucleic acids, proteins, and small molecules, serve as biomarkers of interest in clinical and diagnostic fields. Although numerous approaches have been proposed for the development of quantification assays, there is a limit to develop into a generalized platform because most detection methods depend on the analyte properties and require different detection strategies for each molecule. Therefore, a simple, rapid, and sensitive biomarker-sensing method is currently needed for early and reliable diagnosis as well as for developing point-of-care testing. Here, we report a proximity proteolysis reaction (PPR)-based homogeneous assay to develop quantitative detection method for a variety of biomarkers. Binders were devised to recognize molecules of interest and induce colocalization of protease and zymogen at an analyte. Physical closeness between two protein induce PPR, resulting in production of detectable signal. Single-stranded DNAs were utilized as linkers, which allow tethering of enzymes and binders by simple mixing. The developed sensing platforms based on this reaction accomplished the one-step detection of analytes at picomolar concentrations in an hour. Antibodies, aptamers, and antigens including proteins and small molecules showed acceptability as a binder and replacement of the binder facilitates the detection of a wide range of molecules. Owing to the simple and rapid features of this method, this reaction could be a promising approach to establish a universal detection method. Keywords : Biosensing, colorimetric, homogeneous assay References 1. Broza, Y. Y. Chem Rev., 119, 11761-11817 (2019). 2. Hyeon Ji Park, Tae Hyeon Yoo, Chem. ACS Sens., 3, 2066-2070 (2018).

293


P0124 Enzymatic Treatment of Brown Rice for Production of Ferulic Acid

So-Hee SHIN1, Jeong-Ah YOON1, Myoung-Dong KIM1,2 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Korea, 2Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Vanillin is a value-added flavor compound in the food industry. Ferulic acid is a phenolic compound used as a substrate for enzymatic conversion into vanillin by ferulic acid decarboxylase (E.C. 4.1.1.102) and vanillin synthase (E.C. 4.1.2.41). This study aimed to establish optimum pretreatment conditions for the production of ferulic acid from brown rice. Ferulic acid was not detected when laccase was treated on the ground brown rice. However, ferulic acid was produced by approximately 0.5 mg/L when the ferulate esterase was used. The gelatinized brown rice showed three times higher ferulic acid contents than ungelatinized brown rice. The optimum enzyme concentration was 40 U, and the optimum temperature for the enzymatic reaction was 40°C. Keywords : Ferulic acid, Vanillin, Ferulate esterase, Pretreatment, Brown rice

294


P0125 Production of 5-HT7 Receptor-embedded Nanodiscs

Woong Ryeol LEE, Dongseok MOON, Tai Hyun PARK School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] G protein-coupled receptors (GPCRs) constitute the largest receptor family in the human genome, regulating many physiological and pathological processes. Naturally, 34% of all marketed drugs target GPCRs. The serotonin-7 (5-HT7) receptor is one of the most highly targeted GPCRs in the market, with over 20 distinct approved agents. The 5-HT7 receptor regulates many different functions such as circadian rhythm, depression, learning, memory, nociception, etc. However, the receptor is still yet to be fully exploited for therapeutic purposes, one of the reasons being a lack of sensitive tools to properly identify possible ligands. By employing the nanodisc platform, which mimics the native structure of the receptor in a cell, the 5-HT7 receptor can be reconstituted to be used in selective and sensitive biosensors for the detection of 5-HT7 receptor ligands. We expressed the 5-HT7 receptor from E. coli and reconstituted it in nanodiscs consisting of 5-HT7 receptor, membrane scaffold protein, and phospholipid bilayer to mimic the membrane environment. Altogether, the 5-HT7 receptor nanodisc represents an attractive item for use in biosensors. Keywords : serotonin-7 (5-HT7) receptor, nanodisc, GPCR

295


P0126 Linking Two 30Kc19α Monomers with a Peptide Linker for Efficient Dimerization and Cell-penetration

Haein LEE1, Hyeong Seok LEE2, Seong-Hoon KIM2, Soo Hee RYU2, Tai Hyun PARK1 1School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea, 2UPPTHERA R&D center, UPPTHERA Inc., Incheon, Korea Corresponding Author Email : [email protected] When a macromolecular drug is effectively delivered into cells, it is possible to develop new drugs for intracellular disease targets. 30Kc19 is a protein originating from silkworm hemolymph, with a cell-penetrating property. The underlying mechanism of cell-penetration of 30Kc19 has not been elucidated, but it has been discovered that 30Kc19 tends to form dimers at the surface of the cell membrane and that the dimerization is essential for cell-penetration. In the case of large cargo delivery or in vivo system delivery, the formation of 30Kc19 dimer could be difficult. 30Kc19α, the α-helix domain of 30Kc19, is smaller in size and has higher cell penetrating efficiency than 30Kc19. Herein, we developed 30Kc19α-Linker-30Kc19α by linking two 30Kc19α monomers with a peptide linker to increase dimerization efficiency, thereby improving cell-penetration. Optimization of the peptide linker was preceded. After that, it was confirmed that 30Kc19α-Linker-30Kc19α delivered the cargo protein (GFP) into cells with higher efficiency than the conventional 30Kc19α. Therefore, we propose that 30Kc19α-Linker-30Kc19α would be applied to intracellular drug delivery. Keywords : cell-penetrating protein, dimerization, peptide linker, 30Kc19α References 1. X. Chen et al., Adv. Drug Deliv. Rev. 65, 1357-1369 (2013). 2. J. Ryu et al., Biotechnol. J. 11, 1443-1451 (2016). 3. H. H. Park et al., Biotechnol. J. 9, 1582-1593 (2014).

296


P0127 Development of Enzyme Immobilization Process Using Nanoflower for Anti-virus Activity Enhancement

Woonsuk LEE, Geug Tae KIM, Sung Ho HA Department of Chemical Engineering, Hannam University, Daejeon, Korea Corresponding Author Email : [email protected] Enzymes having higher reactivity than conventional chemical catalysts are diverse and specific to each reaction and have high reaction rates and conversion rates. However, most of the free lipases lose their activities when they expose to heat, strong acids, strong alkalis, and organic solvents, Free lipases have low stability and difficulty in reusing after completion of the reaction. In order to solve this problem, an enzymatic reaction is performed in an organic solvent, but exposure to the environment may cause environmental pollution. Thus, an ionic liquid (IL) instead of an organic solvent was used to increase the stability and activity of the enzyme. In addition, the nanoflower immobilization method was selected as a method to immobilize enzymes because it provides a large surface area and a fast reaction response rate which affect anti-virus activity. In this study, Candida rugosa lipase was immobilized in nanoflower. Candida rugosa lipase was selected by comparing 6 types of free lipase. The immobilization rate of Candida rugosa lipase in nanoflower was more than 90%. The enzyme immobilized in the nanoflower with IL showed higher activity and reusability compared to immobilized enzyme in the nanoflower without IL. The activity of the immobilized enzyme in nanoflower was maintained up to 77% for the non-IL and 82% for the used one even after 7 reuses, respectively. Keywords : Lipase, Nanoflower, Ionic Liquids, Activity

297


P0128 Development of Human Immunoglobulin E Quantitative Method Using Personal Glucose Meter

Hyogu HAN, Junehyun PARK, Jun Ki AHN Human Convergence Technology Group, Korea Institute of Industrial Technology, Ansan, Korea Corresponding Author Email : [email protected] Common diseases such as atopic diseases, including bronchial asthma, allergic rhinitis, and atopic dermatitis, which occur with a prevalence of 30-40% worldwide are very important for great global public health. Atopic diseases are generally accompanied by elevated levels of human immunoglobulin E (IgE) in the serum. Thus, atopic disease can be diagnosed by quantification of human IgE in the serum, but conventional methods such as ELISA(enzyme-linked immunosorbent assay) have limitations that require expensive and bulky equipment. In this study, we developed a method for quantification of Human IgE with a personal glucose meter(PGM), a portable analysis device, using labeling particles conjugated with anti-human IgE antibody–alkaline phosphatase to silica nanoparticles and cascade enzyme reaction of hexokinase with pyruvate kinase. Keywords : Human IgE, personal glucose meter, nanoparticle References 1. Ahn, J. K., Kim, H. Y., Lee, C. Y., Park, K. S. & Park, H. G., J. Biol. Eng. 13(2019).

298


미생물공학 및 생물공정공학

(Microbial and Bioprocess Engineering)

299


P0201 Experimental Promoter Identification of a Representative Food-borne Pathogen Salmonella Enterica Subsp. Enterica Serovar Typhimurium LT2 with Near Single Base-pair Resolution

Hoa THI LE1, Assiya TAIZHANOVA2, Ye GAO3,4, Linh KHANH NONG1, Joon Young PARK1, Eun-Jin LE5, Bernhard O. PALSSON4, Donghyuk KIM1,6,7 1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2Department of Genetic Engineering and Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Korea, 3Division of Biological Sciences, University of California, San Diego, La Jolla, USA, 4Department of Bioengineering, University of California San Diego, La Jolla, USA, 5Department of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea, 6School of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 7Korean Genomics Industrialization and Commercialization Center, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Promoter is a fundamental DNA sequence, where general transcription factors and the polymerase assemble, plays as an “on” switch initiate transcription of a single RNA. Especially, in pathogen bacteria, understanding the active promoter is required for expressing pathogenicity-related genes. Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne pathogen. Despite being frequently used as the reference strain for Salmonella, genome-wide DNA regulatory elements in Salmonella remain unclear. In this study, we investigated a combined experimental and computational analysis for evaluating the regions in near single base-pair resolution where transcriptional regulatory elements bind to promoter under the basic condition without any stresses. In other words, we reconstructed the fundamental of genome-wide transcription factors and their correlation. At the same time, we expanded our search to sRNA which is a potential regulatory role on mRNA targets. For the representative strain S. Typhimurium strain LT2, ChIP-exo was first time adopted to accurately determine the binding sites of catalytic RNA polymerase subunit RpoB, and alternative sigma factors during the exponential phase. Integrating ChIP-exo and RNAseq, we have raised a massive binding site of RNA polymerase and RpoD (1,304 and 1,752 binding sites). However, RpoS and RpoE binding sites were less than previous studies because those sigma factors were highly active in specific stress conditions. Furthermore, a total of 156 overlapping regions bound by RpoD and RpoD were identified, along with 160 overlapping sites between RpoD and RpoS being discovered. We analyzed 100 sRNAs, in which 19 sRNAs were new findings in S. Typhimurium strain LT2. The majority of sRNAs genes, as well as most virulence genes, were significantly expressed during the exponential phase. This approach provided novel insights into promoter regions and their functionality, hence, would expedite new treatment against S. Typhimurium strain LT2 and other pathogens on the genome-scale level. Keywords : pathognenicity, promoters, intergenic binding sites, omic data

300


P0202 Development of a Biological Carbon Monoxide Conversion Process Using Clostridium ljungdahlii

Hye Won JO1, Chanyoung SEO1, Seon Jung KIM2, Dong-heon LEE2, Yongbae JEONG2, Hyun Woo KIM3, Yoo Seong CHOI4, Tae Wan KIM1,2, Seung Hwan LEE1,2 1Interdisciplinary Program for Bioenergy & Biomaterials, Chonnam National University, Gwangju, Korea, 2Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju, Korea, 3ProAbtech, Gwangju, Korea, 4Department of Chemical Engineering & Applied Chemistry, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] In this study, we developed the nanomaterial-assisted biological CO conversion process of Clostridium ljungdahlii. From a series of batch cultivations of C. ljungdahlii with CO, it was observed that cell growth was strongly inhibited by high concentration of CO. Therefore, to lower the CO toxicity, we investigated two nanomaterials that could help the microorganism grow well under high concentration of CO. When the cells were grown on CO of 1.0 bar partial pressure in the medium containing 1,000 ppm of tannin based nanomaterial A, CO uptake by cells increased by 409% compared with control experiment in which no nanomaterial was included. In accordance with increase of CO uptake, acetate and ethanol production were observed to increase by 190% and 236%, respectively. This study shows the potential of nanomaterial-assisted microbial process for the production of high value-added substances from industrial by-product gases containing CO. Keywords : Carbon monoxide, microbial conversion, Clostridium ljungdahlii, nanomaterial

301


P0203 Ring Opening Polymerization of Lactide Using Immobilized Burkholderia cepacia Lipase on Titanium Dioxide (TiO2) Support

Hye Won JO1, Kwang Yeol PARK2, Young Woo SHIN3, Hyeon Gyu CHOI3, Chang Hyun KO2, Seung Hwan LEE1,3 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, Chonnam National University, Gwangju, Korea, 2School of Chemical Engineering, Chonnam National University, Gwangju, Korea, 3Depart-ment of Biotechnology and Bioengineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Poly lactic acid (PLA) is a bio-derived polyester, which is produced from the chemical and/or biological synthesis of biomonomer (lactic acid) or microbial fermentation of biomass. Because of its biodegradability, biocompatibility, lack of toxicity and some characteristics similar to conventional plastics, PLA attracts great attention in medical, pharmaceutical, textile, and packaging field. In the process of producing PLA, using immobilized biocatalysts is helpful, due to its eco-friendliness and mild reaction conditions compared with metal catalysts. The purpose of this study is to produce PLA by ring opening polymerization of lactide using the immobilized lipase on TiO2 support. Lipase was immobilized on titanium dioxide by impregnation method. We investigated the effect of temperature, pH, and solvent on the molecular weight, PDI, and conversion. Keywords : PLA, Immobilized biocatalyst, Bioconversion, Ring opening polymerization

302


P0204 Bioconversion of Trehalose from Maltose Using Trehalose Synthase Immobilized on the Surface of Escherichia coli

Kyung Min KIM1, Seong Jung KIM2, Je Ha OH2, Ju Yeon LEE2, Seung Hwan LEE1,2 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, Chonnam National University, Gwangju, Korea, 2Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Trehalose is a naturally occurring non-reducing disaccharide containing two 1,1-linked glucose and can serve as carbon and energy storage source and as a biocompatible stress protectant in various organisms such as bacteria, fungi, insects, and higher plants. Much attention has been concentrated on the application of trehalose as additives, stabilizer, preservatives, and sweeteners in the fields of food, cosmetic, and pharmaceutical industry. For the biosynthesis of trehalose, three different metabolic routes are elucidated. Among these, trehalose synthase (TreS, EC 5.4.99.16) catalyzes the reversible interconversion of trehalose (glycosyl-a,a-1,1-glucose) and maltose (glucosyl-a1-4-glucose), which is economically feasible process from the industrial point of view. In this poster, surface display of trehalose synthase from Pseudomnas putida KT2440 on the E. coli using outer membrane protein as an anchoring motif and optimization of reaction conditions for the production of trehalose will be presented. Keywords : Trehalose, Cell surface display, Bioconversion, Trehalose synthase

303


P0205 Effect of ZVI Productivity of 1,3-propanediol Klebsiella Pneumoniae L17 from Glycerol

Da Seul KONG, Changman KIM, Jaerhim YU, Jung Rae KIM School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] For efficient production of 1,3-Propanediol(1,3-PDO), sustainable and non-toxic regeneration of NADH is of great importance. ZVI (Zero-Valent Iron) can provide reducing equivalent for 1,3-PDO synthesis from glycerol as an electron donor. Klebsiella pneumoniae has a 1,3-PDO production pathway from glycerol and has been extensively investigated as exoelectrogens. In this study, we attempt to produce 1,3-PDO from glycerol by using an electrochemically active strain, K. pneumoniae L17, and ZVI as an electron donor. As a result, the production of 1,3-PDO using ZVI has increased significantly to 24.23 ±1.33 mol/l. These results implicate that ZVI can regulate the bioconversion of electroactive strain such as K. pneumoniae L17, therefore improve glycerol conversion into value-added platform chemicals. Keywords : Zero-valent iron, Klebsiella Pneumoniae L17 , glycerol , 1,3-Propanediol

304


P0206 Identification of Halophilic Bacteria Containing Class I PHA Synthase to Produce Polyhydroxyalkanoates

Youjung KONG1, Haeng-Geun CHA1, HyeJi JIN1, YuBin JEON1, Jeong Chan JOO2, Yung-Hun YANG3, See-Hyoung PARK1, Kyungmoon PARK1 1Department of Biological and Chemical Engineering, Hongik University, Sejong, Korea, 2Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea, 3Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, Korea Corresponding Author Email : [email protected] To produce polyhydroxyalkanoates (PHA), degradable biopolymers, in halophilic bacteria, a Class I PHA synthase (phaC) is necessary in the metabolic pathway. In the previous study, we had designed the primers and determined the PCR conditions to select the bacteria which can produce PHA. In this study, several microorganisms from jeotgal and sea sand were examined by using the established PCR conditions and 5 strains showing the desired size of the PCR band, phaC, were selected. These microorganisms were cultivated using marine 8% (adding NaCl to 80g/L) medium to confirm the potential of PHA production. The tested microorganisms showed the PHB production ability, and the best microorganism from shrimp jeotgal showed about 63% productivity compared to H. halophila. The strains were identified by 16s rRNA sequence analysis and registered in NCBI database. Keywords : Halophilic bacteria, PHA, phaC, Culture collection, 16s rRNA References 1. Riddhi Mahansaria, Jayanta Debabrata Choudhury & Joydeep Mukherjee (2015) Polymerase chain reaction-

based screening method applicable universally to environmental haloarchaea and halobacteria for identifying polyhydroxyalkanoate producers among them. Extremophiles 19, 1041-1054.

305


P0207 Effect of Enzyme Type and Molar Ratio on Enzymatic Synthesis of Naringin Oleate

Kyeonga KIM, Jonghwa LEE, Seungmee LEE, Jemin SON, Suhwan JI, Chulhwan PARK Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] Flavonoids present in plants are phenolic secondary metabolites with antioxidant and anti-inflammatory effects reducing the probability of cardiovascular disease and diabetes. Naringin is a flavonoid that exists in citrus fruits. It has a chemical structure in which two sugar are combined in naringenin. Naringin is used in various products such as cosmetics and supplements due to its excellent antioxidant and cholesterol reduction effects. However, naringin is limited to commercial utilization due to its low stability and solubility in lipophilic systems. To overcome this problem, esterification can be employed. In this study, naringin oleate was synthesized using naringin and oleic acid. The optimal synthesis conditions of naringin oleate were derived through one factor at a time (OFAT) method by controlling the reaction parameters (immobilized enzyme and substrate concentration etc.). Keywords : Flavonoid, Naringin, Oleic acid, Lipase, Naringin oleate, Antioxidant, Optimization References 1. A. N. Panche, A. D. Diwan, S. R. Chandra, Flavonoids: an overview(2016), Journal of Nutritional Science,

5(47), 1-15.

306


P0208 Development of Low-cost Medium for Syngas Fermentation by Clostridium autoethanogenum

Taehwan KIM, Jiung YUN, Byung-keun OH Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Korea Corresponding Author Email : [email protected] Syngas fermentation, which changes waste gas into organic product such as ethanol and acetate through acetogens, is an environment-friendly, low cost and commercial process. During syngas fermentation process, fermentation medium is one the most critical factors due to its nutrient components and role as reaction platform. Therefore, it is significant to develop low cost medium to commercialize syngas fermentation. In standard media, yeast extract, works as nitrogen source, takes big proportion of the total media solution. In this study, the various concentrations of other nitrogen sources, such as vegetable extract and maltose extract, were used to replace yeast extract in standard medium (yeast extract 5 g/L, YE high medium) to retrench the cost of fermentation media. The results showed developed cell growth rate and ethanol concentration in VE high medium (vegetable extract 5 g/L) in 100 mL vial using syngas (15% CO, 45% H2 and 40% N2) by Clostridium autoethanogenum, and based on this conclusion, we preformed continuous fermentation in a 2.5 L bioreactor in YE high medium and VE high medium, respectively. In VE high medium, the maximum growth rate was 7.27%, and production rate of ethanol was 20.45% higher than those in YE high medium. In other words, production capacity was almost maintained without other side effects. Furthermore, the cost was reduced to 36.36% compared with YE high medium. As a result, vegetable extract has potential to replace yeast extract and reduce the cost in syngas fermentation. Keywords : nitrogen source, vegetable extract, syngas fermentation, malt extract, clostridium autoethanogenum

307


P0209 Investigation on Ultrasonic Extraction Mechanism of Paclitaxel from Taxus chinensis

Hak-Gyun KIM, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] Experimental studies were carried out for the ultrasonic cavitation bubble- and gas bubble-assisted extraction of paclitaxel from Taxus chinensis while varying parameters such as ultrasonic power, gas flow rate, and contact time. Extraction efficiency has been dramatically improved, and the time required for extraction has been reduced compared to conventional methods. As a result of investigating the mechanism of extraction in which cavitation and gas bubbles were introduced, it was found that the bubble itself contributes to the improvement of extraction efficiency. In addition, the extraction behavior was quantitatively investigated by calculating the effective diffusion coefficient and mass transfer coefficient of paclitaxel. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : Paclitaxel, Extraction, Ultrasonic cavitation bubble, Gas bubble, Mechanism

308


P0210 Improvement of Syngas Consumption by Clostridium sp. JS66 During Mixotrophic Fermentation with Mannose

Kyoung Won MUN1,2, Ki-Yeon KIM1, Deu Rim YUN1,2, Kyoung Heon KIM2, Youngsoon UM1,3 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea, 2Department of Biotechnology, Korea University, Seoul, Korea, 3Clean Energy and Chemical Engineering, Korea University of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected], [email protected] Clostridium sp. JS66 is a strict anaerobic acetogen producing C2, C4, and C6 acids/alcohols using syngas (consisting of CO, CO2, and H2) and sugars as well. However, the limitation of sugar fermentation is CO2 generation, resulting in low carbon recovery to metabolites. In this study, we first evaluated the consumption of CO2 and H2 evolved during the heterotrophic fermentation by JS66 with various sugars (glucose, mannose, arabinose, and sucrose). Interestingly, mannose was simultaneously consumed with CO2 and H2, while CO2 and H2 evolved during glucose fermentation were sequentially consumed after the complete consumption of glucose. Afterward, mixotrophic fermentation was conducted by supplying both syngas and each of glucose or mannose as a sugar. As a result, the total consumption of H2 and CO during mixotrophic fermentation containing mannose was 1.45 fold higher than glucose. In addition, CO consumption rate in the mannose-based mixotrophic fermentation was higher than the glucose-based one by 1.34 fold. These results imply that mannose can be used as an effective sugar source for mixotrophy by maintaining Wood-Ljungdahl metabolism active even in the presence of sugar. Keywords : Clostridium sp. JS66, Syngas, Mannose, Mixotrophic fermentation

309


P0211 Kinetics, Mechanism, and Thermodynamics Studies of Ultrasound-assisted Acetone/Pentane Precipitation of Paclitaxel from Taxus chinensis

Hye-Su MIN, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] In this study, the purification efficiency of paclitaxel was remarkably improved through ultrasonic acetone–pentane fractional precipitation. The time taken for precipitation was also shortened by a maximum of 192-fold (at 5 °C, 180–250 W) with ultrasound as compared to that of the conventional method. The precipitation was completed within 30 min regardless of temperature (5–25 °C), thereby improving the operating conditions (precipitation time and temperature) as compared to the conventional method. In addition, the rate constant in the fractional precipitation with ultrasound (80–250 W) was also increased by 8- to 13-fold (at 5 °C) as compared to the conventional method. It was found that the cavitation bubble itself acts as a nucleation site, which accelerates nucleation and thereby improves fractional precipitation efficiency. The standard enthalpy change and standard entropy change were negative, while the standard Gibbs free energy change was positive. These results indicate that the precipitation process was exothermic, irreversible, and non-spontaneous. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : Paclitaxel, Ultrasound, Acetone/Pentane Precipitation, Kinetics, Thermodynamics

310


P0212 Effect of Gas Bubbles on the Ultrasonic Fractional Precipitation Efficiency of Paclitaxel

Youngju PARK, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] In this study, a fractional precipitation technique of paclitaxel using ultrasonic cavitation bubbles and gas bubbles is presented. Precipitation efficiency has been dramatically improved, and the time required for precipitation has been reduced compared to conventional methods. In particular, the synergistic effect of mixing cavitation bubbles and gas bubbles during fractional precipitation was confirmed. As a result of investigating the mechanism of fractional precipitation in which cavitation and gas bubbles were introduced, it was found that the bubble itself acts as a nucleation site, resulting in faster nucleation and thereby improving precipitation efficiency. In addition, the precipitation characteristics were quantitatively identified through the determination of kinetic and thermodynamic parameters. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : Paclitaxel, Fractional precipitation, Cavitation bubble, Gas bubble, Synergistic effect

311


P0213 Kinetics, Mechanism, Isotherm and Thermodynamic Studies of Ultrasonic Adsorption of Paclitaxel onto Sylopute

Junho PARK, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] The ultrasonic adsorption characteristics of paclitaxel from extracts of Taxus chinensis cell cultures by the commercial adsorbent Sylopute were investigated using different parameters such as ultrasonic power, initial paclitaxel concentration, adsorption temperature, and contact time. The experimental data were fitted to four adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich in order to study the adsorption mechanisms and characteristics. Also, kinetic analysis was performed using the pseudo-first-order, pseudo-second-order, and intraparticle diffusion models. Furthermore, the thermodynamic parameters, activation parameters, isosteric heat of adsorption, and activation energy were quantitatively examined in order to evaluate the feasibility and nature of the adsorption process. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : Paclitaxel, Sylopute, Ultrasonic adsorption, Adsorption isotherm, Kinetics, Thermodynamics

312


P0214 Effect of Culture Parameters on Production of hEGF

Jemin SON1, Noseong PARK1, Yesol BEAK1, Kyeonga KIM1, Seungmee LEE1, Hah Young YOO2, Chulhwan PARK1 1Department of Chemical Engineering, Kwangwoon University, Seoul, Korea, 2Department of Biotechnology, Sangmyung University, Seoul, Korea Corresponding Author Email : [email protected] The human epidermal growth factor (hEGF) has been in interest for its wound healing effects by promoting cell proliferation. This allows hEGF to be applied in fields such as cosmetic and pharmaceutical. Depending on the efficacy, the need increased and studies on hEGF production were underway. In the progress of the studies, Escherichia coli received the most attention because it is easy to genetically engineer and can produce proteins at a low cost. But E.coli produces the hEGF intracellular, so that it needs steps for cell destruction and refolding. In this study, engineered Pichia pastoris by a pPinkα-HC vector, designed for extracellular secretion, was used to produce the hEGF. To establish the hEGF production condition, effects of essential factors for the hEGF production were investigated. Keywords : Human epidermal growth factor (hEGF), Pichia pastoris, Optimization, response surface methodology (RSM) References 1. S. Eissazadeh, H. Moeini, M.G. Dezfouli, S. Heidary, R. Nelofer, M.P. Abdullah, Brazilian J. Microbiol. 48,

286–293 (2017).

313


P0215 Identification and Verification of Starch Degradation Activities Using Fungal Strains Isolated from Various Nuruks

Seong-Hwan YEOM1, Deok-Ho KWON1,2,3, Suk-Jin HA1,2,3 1Department of Biohealth-machinery convergence engineering, Kangwon National University, Chuncheon, Korea, 2Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Korea, 3Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Nuruk is a Korean traditional fermentation starter made by natural inoculation of various microbes in a high temperature and humid environment with grains such as rice and wheat. When making Nuruk, many microorganisms such as yeast, fungi and lactic acid bacteria were growing. Among them, fungi are well known to degrade starch and it becomes an important raw material for alcohol fermentation when the starch of grains is saccharified to produce glucose. Yeast is fermented with alcohol from glucose and after fermentation, it becomes traditional Korean alcoholic beverage, Makgeolli. In this study, various fungi were isolated from 13 Korean traditional Nuruks and identified using sequencing of internal transcribed spacer (ITS) region. As a result of sequencing analysis, 13 fungi were identified (4 Syncephalastrum sp., 4 Aspergillus sp., 2 Rhizomucor sp., 2 Mucor sp., 1 Rhizopus sp). For the verification of starch degradation activities from 13 fungi, enzymatic assay was performed using culture broth as crude enzyme and starch as a substrate at various reaction conditions. Keywords : Nuruk, starch degradation, glucose References 1. Jang, S. W., Kim, J. S., Park, J. B., Jung, J. H., Park, C. S., Shin, W. C., & Ha, S. J. Food Sci Biotechnol.

24, 1805-1810 (2015).

314


P0216 Enhancement of Phycobiliproteins Production in Engineered Synechocystis sp. PCC 6803 by Box-behnken Design with Stress of Glutamate, NaCl and Light Intensity

Seonghoon YOON, Mi-Jin KANG, Hanbin RYU, Seong-Joo HONG, Choul-Gyun LEE Department of Biological Sciences and Bioengineering, Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Phycobiliproteins (PBPs) are one of the most feasible pigments for human health, cosmetics and diagnostic markers which are produced by cyanobacteria or red algae. A cyanobacterium, Synechocystis sp. PCC 6803, also produces PBPs which are C-phycocyanin and allophycocyanin. B-phycoerythrin (B-PE) is a kind of the PBPs which Synechocystis do not produce. We inserted the gene related to B-PE to Synechocystis sp. PCC 6803 to enhance PBPs accumulation and photosynthetic efficiency. Stress of glutamate, NaCl and light intensity are known as factors to PBPs accumulation. Optimal condition of the factors to enhance PBPs production in engineered Synechocystis sp. PCC 6803 were investigated by statistical method, Box-behnken design (BBD). The cells were cultured in test tubes working volume 70 ml at 30 and 0.1 vvm of air was supplied. High concentration of glutamate and NaCl under low light intensity improve PBPs contents. On the other hand, high light intensity and low NaCl concentration improve cell growth. The results of BBD were validated to large scale photobioreactors. This study could be used for further microalgal engineering to produce PBPs from Synechocystis sp. PCC 6803. Keywords : Phycobiliprotein, Synechocystis, B-Phycoerythrin, Box-behnken design References 1. Hong, S. J., & Lee, C. G. Statistical optimization of culture media for production of phycobiliprotein by

Synechocystis sp. PCC 6701. Biotechnology and Bioprocess Engineering, 13(4), 491-498 (2008). 2. Zavřel, T., Chmelík, D., Sinetova, M. A., & Červený, J. Spectrophotometric determination of phyco-

biliprotein content in cyanobacterium synechocystis. JoVE (Journal of Visualized Experiments), (139), e58076 (2018).

3. Manirafasha, E., Murwanashyaka, T., Ndikubwimana, T., Ahmed, N. R., Liu, J., Lu, Y., ... & Jing, K. Enhancement of cell growth and phycocyanin production in Arthrospira (Spirulina) platensis by metabolic stress and nitrate fed-batch. Bioresource technology, 255, 293-301 (2018).

315


P0217 Surface Display on Outer Membrane Particles Mediated Differential Fusion Site of Antigen 43

Hyo-won YOON, Gna AHN, Yang-Hoon KIM, Ji-Young AHN Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju, Korea Corresponding Author Email : [email protected] The cell envelope of Gram negative bacteria such as Escherichia coli is composed of outer membrane(OM), periplasm, inner membrane(IM). In particular, the OM is an important structure to expose protein of bacterial surface. Because of a simple structure of Ag 43 that was comprised a signal peptide, passenger, and translocator domains, Antigen 43 (Ag43) system, an autotransporter protein, can be chosen for exposure of protein on the bacterial surface. We constructed modified Ag 43 vector for expression of eGFP to identify surface display on outer membrane and confirmed a protein expression. We confirmed surface display based on the structural difference of the Ag43 and mainly used β-barrel domain deleted different sites at C-terminus of Ag43 and these were confirmed to eGFP expression. Finally, OM particles were made by the isolation method of OM fraction using lysozyme to confirm the exposed proteins. These performed to evaluate the exposed protein for applications of OM particles. This exposed platform is able to use as application of functional products and liposome like particles. Keywords : Antigen 43(Ag 43), Surface display, Outer membrane paricles References 1. M. Park, G. Yoo, J.H. Bong, J. Jose, M.J. Kang and J.C. Pyun, Biochim Biophys Acta Biomembr. 1848(3),

842 (2015). 2. G.Y. Lee, J.H. Bong, J. Jung, M.J. Kang, J. Jose and J.C. Pyun, Biosens Bioelectron. 156, 112110 (2020). 3. B. Ramesh, V. G. Sendra, P. C. Cirino and N. Varadarajan, J Biol Chem. 287(46), 38580 (2012).

316


P0218 Development of Water Pretreatment Process for Pre-purification of Paclitaxel from Taxus chinensis

Myeonggi LEE, in-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] In this study, a water pre-treatment method for the separation and purification of an anticancer agent paclitaxel from plant cell cultures was developed. When the methanol extract obtained by biomass extraction was pre-treated with water, a high yield of high-purity paclitaxel was observed within a short period of time. The main process parameters (crude extract/water ratio, pretreatment time) in the water pretreatment process were optimized. In addition, the impurity removal behavior was quantitatively investigated through high-performance liquid chromatography analysis. Thus, the efficiency of the separation and purification process for paclitaxel was improved dramatically by water pretreatment, particularly in terms of the reduction of operating times and the simplification of processes. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : Paclitaxel, Water pretreatment, Purification, Process, Development

317


P0219 Effect of Enzyme Type and Molar Ratio on Enzymatic Synthesis of Naringin Acetate

Seungmee LEE1, Yesol BAEK1, Jemin SON1, Kyeonga KIM1, Hyeseon LEE1, Jong-Min OH2, Chulhwan PARK1 1Department of Chemical Engineering, Kwangwoon University, Seoul, Korea, 2Department of Electronic Materials Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] Flavonoids, natural antioxidants found in plants, have biological and pharmacological activities. Naringin is a typical citrus flavonoid with strong antioxidant, antibacterial and anti-cancer effects. It can be used as preventative and treatment for obesity, diabetes, high blood pressure, and metabolic syndrome. However, naringin is difficult to use in various industries due to its low solubility and stability. To improve the problem, researchers have suggested acylation of naringin using lipase. Naringin ester, acylated flavonoid, has improved physical and chemical properties. In this study, synthesis of naringin acetate was performed and variables that affect synthesis (enzyme type and molar ratio etc.) were studied to figure out optimal synthesis conditions. When acetyl anhydride or vinyl acetate were used as acyl donors, the conversion was obtained over 80%. Keywords : Flavonoid, Naringin, Acyl donor, Flavonoid ester, Naringin acetate, Acylation, Lipase, Optimization References 1. de Araújo, M., Franco, Y., Messias, M., Longato, G., Pamphile, J., Carvalho, P., Biocatalytic synthesis of

flavonoid esters by lipases and their biological benefits(2017), Planta Medica, 83(01/02), 7-22.

318


P0220 Separation and Purification of Diamines from Microbial Fermentation Broth

Jong An LEE1,2,3, Jung Ho AHN1,2,3, Inho KIM1, Sheng LI1, Sang Yup LEE1,2,3,4 1Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, Daejeon, Korea, 2Metabolic and Biomolecular Engineering National Research Laboratory and Institute for the BioCentury, KAIST, Daejeon, Korea, 3Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon, Korea, 4BioInformatics Research Center and BioProcess Engi-neering Research Center KAIST, Daejeon, Korea Corresponding Author Email : [email protected] 1,3-diaminopropane (1,3-DAP), 1,4-diaminobutane (1,4-DAB), and 1,5-diaminopentane (1,5-DAP) are important chemicals due to their wide industrial use. Over the last decade, several studies on bio-based production of diamines by metabolically engineered microorganisms have been published. However, development of efficient methods for the recovery of 1,3-DAP, 1,4-DAB, and 1,5-DAP from fermentation broth has not been reported. In this study, an effective process for the separation and purification of the diamines from fermentation broth without using highly flammable or toxic solvents was developed. The optimal process resulted in separation of extremely pure 1,3-DAP, 1,4-DAB, and 1,5-DAP with yields of 87±3%, 86±4%, and 81±2%, respectively. The strategy reported here could be similarly applicable in developing downstream processes to recover and purify other diamines and related chemicals from fermentation broth. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation of Korea. Keywords : 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, purification, fermentation broth References 1. J. A. Lee, J. H. Ahn, I. Kim, S. Li, and S. Y. Lee, Chem. Eng. Sci. 196, 324-332 (2019).

319


P0221 Synthesis, Characterization, and Application of Fully Biobased and Biodegradable Nylon-4,4 and Nylon-5,4

Jong An LEE1,2,3, Jung Ho AHN1,2,3, Inho KIM1,2,3, Sheng LI1, Sang Yup LEE1,2,3,4 1Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, Daejeon, Korea, 2Metabolic and Biomolecular Engineering National Research Laboratory and Institute for the BioCentury, KAIST, Daejeon, Korea, 3Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon, Korea, 4BioInformatics Research Center and BioProcess Engineering Research Center KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Nylons are widely used polyamides and the production of fully biobased nylons has been of great interest but not yet realized. Here, we report synthesis and characterization of bionylon-4,4 and -5,4, two rarely studied nylons, using succinic acid, 1,4-diaminobutane, and 1,5-diaminopentane produced by fermentation of metabolically engineered bacteria. The synthesized bionylons exhibit excellent thermotolerance and water absorptivity. Bionylons are also successfully used as a matting agent to reduce surface gloss of ASA. Furthermore, bionylons are found to be biodegradable, providing further environmental benefit upon disposal. The strategy for the synthesis of fully biobased nylons comprising fermentative monomer production and purification, polymerization, characterization, and application reported here can serve as a guideline for synthesizing other fully biobased polymers from renewable resources. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT, through the National Research Foundation (NRF) of Korea. Keywords : bionylon, biodegradation, 1,4-diaminobutane, 1,5-diaminopentane, succinic acid, succinic acid, matting agent References 1. J. A. Lee, J. H. Ahn, I. Kim, S. Li, and S. Y. Lee, ACS Sustain. Chem. Eng. 8(14), 5604-5614 (2020).

320


P0222 Improved Drying Method for Removal of Residual Solvent from (+)-Dihydromyricetin by Pre-treatment with Ethanol

Hee-Gun LEE, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] In this study, a drying method using ethanol pretreatment was developed that can effectively remove residual solvent from (+)-dihydromyricetin. Rotary evaporation with ethanol pretreatment was sufficient to remove residual acetone below the ICH limit (5,000 ppm for acetone). In addition, residual ethanol met the ICH-specified value (5,000 ppm) after simple rotary evaporation alone with pre-treatment with water, and residual water also met the specified value (<4%) for active pharmaceutical ingredients. At all the drying temperature (35, 45, and 55 °C), a large amount of the residual solvent was initially removed during the drying, and the drying efficiency increased when increasing the drying temperature. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : (+)-Dihydromyricetin, Residual solvent, Removal, Pre-treatment, Drying

321


P0223 Screening and Characterization of the Mutant Lactobacillus plantarum GNS300 for Exopolysaccharide Production

Jae-Youn JUNG1,2, Deok-Ho KWON1,2,3, Yoo-Jin LEE4, Young-Keun SONG4, Suk-Jin HA1,2,3 1Department of Biohealth-machinery convergence engineering, Kangwon National University, Chuncheon, Korea, 2Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Korea, 3Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea, 4Prod.Dev.& Marketing Team, The Garden of Naturalsolution Co., Ltd, Osan, Korea Corresponding Author Email : [email protected] The exopolysaccharide made by lactic acid bacteria (LAB) is a biological polymer produced to withstand the harsh environment in the human gut. Therefore, it is considered a safe biology material. It also has been shown the potential for the cosmetic and pharmaceutical industries as a natural alternative to chemical additives. However, the amount of exopolysaccharide produced by wild-type LAB is very poor generally. Therefore, it is necessary to improve exopolysaccharide production through strain development. Random mutagenesis was performed to improve the production capacity of exopolysaccharide using Lactobacillus plantarum. Among more than 600 mutants, the mutant L. plantarum GNS300 was isolated showing improved crude exopoly-saccharide production through the total sugar quantification method. The mutant L. plantarum GNS300 produced 2.82 g/L of crude exopolysaccharide which was 180% improved crude exopolysaccharide production. Through the optimization of fermentation conditions using a bioreactor, 2.93 g/L of crude exopolysaccharide was produced from 20 g/L of glucose at 35oC, 500 rpm, and 0.1 vvm conditions for 12 h. Keywords : Lactobacillus plantarum, Exopolysaccharide, Random mutagenesis References 1. Tallon, R., Bressollier, P., & Urdaci, M. C. RES MICROBIOL. 154. 705-712 (2003).

322


P0224 Effective Production of Low Molecular Weight Oligosaccharides by Using Ulvan Lyase and Its Possibility for Medical Use

YeWon JO, Jeong Eun HYEON Department of Food Science and Biotechnology, College of Knowledge-Based Services Engineering, Sungshin Women's University, Seoul, Korea Corresponding Author Email : [email protected] Polysaccharides derived from marine algae are used in various biomedical research and medical treatment applications. These polysaccharides are biologically active compounds that require various studies. The biological activity of green algal polysaccharides is affected by its molecular weight. Compared to high molecular weight ulvans, low molecular weight ulvans exhibit more reactivity, higher solubility and easier absorption. Therefore, a low molecular weight ulvan depolymerization method is required to obtain useful substances. In this study, we did several designs to enzymatically depolymerize ulvan. First, green algae were macerated with 2% cellulase and incubated at 55, and lyophilized ulvan polysaccharide was extracted by adding 1% hydrogen peroxide. And then, ulvan lyase, which depolymerizes ulvan polysaccharides, was constructed by dockerin-cohesin interaction as enzyme complex. This ulvan depolymerization method is utilized for efficient low molecular weight ulvanproduction. it can be used to develop pharmaceutical technology using various properties of low molecular weight ulvan such as anticoagulation, antioxidant, and antitumor. Keywords : green algae, ulvan lyase, ulvan depolymerization, ulvan derived oligosaccharides, dockerin-cohesin References 1. Qingqing Li , et al., J Appl Phycol. 30, 2017-2027 (2018). 2. Venkat Rao Konasani , et al., Algal Research. 36, 106-114 (2018). 3. Jian Gao , et al., Mar Drugs. 17, 568 (2019).

323


P0225 Multifunctional Oligosaccharide Production Through Efficient Enzymatic Degradation of Porphyran by Constructing Dockerin-cohesin Based Artificial Complex

JooHee HAN, JeongEun HYEON Department of Food Science and Biotechnology, College of Knowledge - Based Services Engineering, Sungshin Women's University, Seoul, Korea Corresponding Author Email : [email protected] Porphyran, a polysaccharide composed of red algae, is a source of new biomass raw materials and a multifunctional oligosaccharide material with various physiological activities. The breakdown of porphyrans into oligosaccharide is a process that provides excellent and promising alternative resources, which is obtained by the glycolysis using various porphyranolytic enzymes in several stages. In this study, porphyran was extracted by grinding, ethanol reaction, sterilization, extraction, heat treatment and drying method from porphyra Yezoensis, and this porphyran was used as a substrate for the study. In addition, we have established a method for efficient hydrolysis through an enzyme complex obtained through dockerin-cohesin interaction that degrades natural algal polysaccharides. The dockerin-cohesin interaction is designed by genetically binding dockerin module to the end of an existing enzyme, and then attaching cohesin module to obtain a protein complex. The designed protein complex has been shown to further increase its activity on the substrate, which can be considered as a useful way to obtain efficient oligosaccharides or monosaccharides through hydrolysis of red algae. Keywords : Porphyran, Dockerin-Cohesin, New biomass raw materials, Multifunctional oligosaccharide material References 1. Jeong Eun Hyeon, et al., Process Biochemistry. 47 (2012). 2. Dae Hee Kang, et al., Bioresour Technol. 250 (2018). 3. Yuying Zhang, et al., J Agric Food Chem. 68 (2020).

324


P0226 Kinetics and Mechanism Studies of Ultrasonic Fractional Precipitation of (+)-Dihydromyricetin

Jieun HONG, Jin-Hyun KIM Dept. of Chemical Engineering, Kongju National University, Cheonan, Korea Corresponding Author Email : [email protected] AbstractThe purification efficiency of (+)-dihydromyricetin, a bioactive plant flavonoid, was remarkably improved through ultrasound-based fractional precipitation. Precipitation efficiency has been dramatically improved, and the time required for precipitation has been reduced compared to conventional methods. As a result of investigating the mechanism of fractional precipitation in which ultrasonic cavitation and gas bubbles were introduced, it was found that the bubble itself contributes to the improvement of precipitation efficiency. In addition, the ultrasonic fractional precipitation behavior was quantitatively investigated by calculating the precipitate size and diffusion coefficient of (+)-dihydromyricetin. ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2018R1D1A3A03000683). Keywords : (+)-Dihydromyricetin, Fractional precipitation, Cavitation bubble, Gas bubble, Mechanism

325


P0227 Flow Cytometric Assessment of Extracellular Polysaccharides in Chlamydomonas asymmetrica

Lisa LANGE1, Jaehyeok HEO2, Christoph LINDENBERGER3, Alexander JAHN4, Man-gi CHO2 1Department of Chemical Engineering, Bergakademie Freiberg, Freiberg, Germany, 2Department of Global Biotechnology, Dongseo University, Busan, Korea, 3Department of Environmental Technology, OTH Amberg-Weiden, Amberg, Germany, 4Department of Biotechnology, LSTME Busan, Busan, Korea Corresponding Author Email : [email protected] The cyanobacterium Chlamydomonas asymmetrica produces extracellular polysaccharides (EPS), which can be present in attached (capsular) or detached (colloidal) form. Quantification and distinction is challenging due to diverse character of EPS, but rapid assessment is of interest due to medical and commercial potential e.g. for their antiviral uses. Here we report on a method for the use of flowcytometry in conjunction with the fluorescent nucleic acid stain Sytox green for the assessment of capsular EPS in a bubble column culture of C. asymmetrica. The cyanobacterium was cultivated in AF6 media under 10.36 µmol/m2s photon flux at 30°C. A correction factor function for cell density-based fluorescence quenching was determined and various methods for capsular EPS detachment evaluated. A correlation between culture age and flowcytometric fluorescence signal intensity was observed, suggesting accumulation of nucleic acid in the capsular EPS attached to the cell wall of C. asymmetrica, as confirmed by fluorescence microscopy. Results suggest that the described method is a valid approach for quantification of capsular eps, but that colloidal eps in C. asymmetrica is significantly more abundant than its capsular variety. Keywords : EPS, Chlamydomonas asymmetrica, flow cytometry

326


P0228 Photodynamic Therapy of Oral Squamous Cell Carcinoma Using Reactive Oxygen Species-responsive Nanophotosensitizers Composed of Phenyl Boronic Acid Pinacol Ester-conjugated Chitosan Conjugates

Chang-Young KIM, Chang-Min LEE, Eun-Byul KOOK, Byung-Hoon KIM, Young-Il JEONG Department of Dental Materials, College of Dentistry, Chosun University, Gwangju, Korea Corresponding Author Email : [email protected], [email protected] Photosensitizers for photodynamic therapy (PDT) of cancer generate reactive oxygen species (ROS) under light irradiation and, therefore, they have minimal side-effects in the normal cells or tissues [1]. However, photosensitizers are dispersed freely in the whole body by systemic administration and then causes phototoxicity problem in the skin. This issue requires that patients protect from sun-light for a long time. For this study, chitosan was conjugated with phenyl boronic acid pinacol ester (PBAP) and chlorin e6 (Ce6)-incorporated nanophotosensitizers were fabricated for ROS-sensitive delivery of photosensitizers against cancer. Ce6-incorporated chitosan-PBAP nanophotosensitizers (Ce6-ChitoPBAP) were evaluated with SCC-15 oral squamous carcinoma cells (OSCC). Ce6-incorporated nanophotosensitizers showed spherical shapes in TEM observation with less than 300 nm diameter. Ce6 was released from nanophotosensitizers with ROS-sensitive behavior, i.e. Ce6 release rate was faster in the presence of hydrogen peroxide. Ce6-incorporated nanophoto-sensitizers showed higher phototoxicity against SCC-15 OSCC compared to Ce6 itself while they have negligible toxicity against cancer cells and normal cells in the absence of light. In conclusion, Ce6-incorporated nanophotosensitizers can be considered as a suitable option for PDT of OSCC. Acknowledgement: This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C1102850). Keywords : Reactive oxygen species, nanophotosensitizers, oral squamous carcinoma, drug delivery References 1. P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S. O. Gollnick, S. M. Hahn, M. R.

Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B. C. Wilson, J. Golab. CA Cancer J. Clin. 61, 250 (2011).

327


P0229 A New Approach for Computing Microalgae Culture Growth Inside a Photobioreactor

Sun-Hwa JUNG1, Christopher MCHARDY2, Cornelia RAUH2,3,4, Alexander JAHN3, Giovanni LUZI3, Antonio DELGADO3,4, Christoph Peter LINDENBERGER1 1Department of Mechanical Engineering and Environmental Engineering, OTH Amberg-Weiden, Kaiser-Wilhelm-Ring 23, Amberg, Germany, 2Department of Food Biotechnology and Food Process Engineering, Technical University of Berlin, Berlin, Germany, 3LSTME Busan Branch, Busan, Korea, 4Institute of Fluid Mechanics, University of Erlangen-Nuremberg, Cauerstraße 4, Erlangen, Germany Corresponding Author Email : [email protected] Microalgae are regarded as one of the most promising potential choices to overcome the increasing demand for food and energy resources in the 21st century. These organisms are usually cultivated in open or closed photobioreactors (PBR) and utilize the energy of light for growth and maintenance. Over the decades, many mathematical models with varying degrees of complexity have been proposed to compute the growth of algae inside PBR, since they represent the basis for the optimization of process conditions and reactor designs. The majority of these models are based on the fact that light intensity decreases inside a PBR as a result of the absorption and scattering effects of the biomass. We propose a new method to predict the apparent growth inside photobioreactors, based on a biomass inhibition concept rather than the traditional way of computing the light attenuation inside a PBR. The resulting equation presents an asymmetric sigmoid relation between biomass concentration and apparent growth rate and contains parameters that solely depend on the reactor radius and incident light intensity. The parameters have been calibrated using the numerical solutions of the growth rate obtained with a classical Type II model. We utilize the new equation to predict the growth behavior of three phototrophic organisms in three reactors of different sizes. The proposed method simplifies the calculation of the growth of phototrophic organisms in cylindrical reactors and it does not require any specific numerical software. Keywords : Algae Cultivation, Numerical Methods, Growth Kinetic, Inhibitory Effects

328


P0230 Numerical and Experimental Investigation of Fluid Flow in Bubble Column Reactors

Giovanni LUZI1, Christopher MCHARDY2, Bastian EYSEL2, Christoph LINDENBERGER3, Cornelia RAUH1,2,4, Antonio DELGADO1,4 1LSTME Busan Branch, Busan, Korea, 2Department of Food Biotechnology and Food Process Engineering, Technical University of Berlin, Berlin, Germany, 3Department of Mechanical Engineering and Environmental Engineering, OTH Amberg, Kaiser-Wilhelm-Ring 3, Amberg, Germany, 4Institute of Fluid Mechanics, University of Erlangen-Nuremberg, Cauerstraße 4, Erlangen, Germany Corresponding Author Email : [email protected] Bubble columns are gas-liquid multiphase contactors where the gas is introduced from the sparger at the bottom of a column filled with liquid. These reactors are widely used in chemical, metallurgical, petrochemical industries as well as in biotechnological processes. In recent years the computational power has increased significantly, and computational fluid dynamics (CFD) models have been developed and improved to simulate the two-phase flow typical of bubble column reactors. Numerical simulations are highly requested and desirable since they can in principle offer a valid predictive tool compared to expensive experiments. However, the results of numerical simulations need to be validated with experimental results to prove their validity. In this regard, we utilized the open-source software OpenFOAM® to simulate the gas-liquid flow in the monodisperse regime of a bubble column reactor with a cylindrical cross-section equipped with a dip-tube sparger. The profiles of the time-averaged axial liquid velocity obtained numerically are compared with those measured experimentally with a Particle Image Velocimetry (PIV) system at five different heights of the column. The numerical results are found to be in good agreement with the experimental data, providing that the value of the turbulent Schmidt number is chosen correctly. This proves the ability of numerical models to capture the highly dynamic flow motion typical of bubble column reactors and accurately predict the liquid characteristics in gas-liquid systems. Keywords : Two-Phase Flows, Numerical Simulations, Particle Image Velocimetry , Bubble Column

329


P0231 Optimization of the Liquid Spawn Composition to Obtain the Mushroom Mycelium of Selected Mushroom Species

Jegadeesh RAMAN, Hyun-Jae SHIN Department of Biochemical & Polymer Engineering, Chosun University, Gwangju, Korea Corresponding Author Email : [email protected] Mycelia of edible and medicinal mushrooms are grown on sawdust and liquid media to form a thick mat resembling leather, so-called mushroom leather. Production of high dense liquid spawn at laboratory scale could significantly suppress contamination to mushroom-based leather production. The present study aimed to investigate the potential of producing liquid spawn of selected mushroom species (Fomitella fraxinea, Wolfiporia extensa, and Agaricus sp.). We optimized the mycelial radial growth on different commercial media, temperature, and pH; among them, YMA (Yeast Malt Extract), 27 - 28°C, and 6 - 5.5 pH shown fast radial growth. Besides, submerged fermentation in a 1 L Scott bottle bioreactor under controlled conditions and evaluate its ability to colonize pine sawdust and rice bran substrate for mycelia production. The liquid spawn cultivation medium contains 10 g L−1 of dextrose, 5 g L−1 of peptone, yeast, and malt extract each 3 g L−1, at 28 ± 2 °C with an agitation speed of 150 rpm and oxygen partial pressure of 30 - 40%. Maximum biomass was obtained from Agaricus sp. and F. fraxinea was achieved after 16 days of fermentation. The mycelium's texture and density produced with the liquid spawn were superior to that cultivated in the solid spawn. The developing liquid spawn could be practically applied in mushroom-based leather production. Keywords : Mushroom, Liquid spawn, Radial growth, Mycelia, Mushroom-based leather References 1. W. R. Zhang, S. R. Liu, Y. B. Kuang and S. Z. Zheng, Mycobiology 47(1), 97-104 (2019). 2. G. Kawai, H. Kobayashi, Y. Fukushima and K. Ohsaki, Mycoscience 37, 201-207 (1996). 3. F. Antunes, S. Marcal, O. Taofiq, A. Morais, A. C. Freitas, I. Ferreira and M. Pintado, Molecules 25(11),

2672 (2020).

330


P0232 Epidemiology of Acute Diarrheal Disease in Chungcheongnam-do, 2019-2020

Kangbum LEE, Dogyeong KWON, Bumju SEO, Hanna JEON, Mina PARK, Hyeonggak KANG, Jongdae KIM Chungcheongnam-do Health & Environmental Research Institute, Hongseong, Korea Corresponding Author Email : [email protected] Acute gastroenteritis is one of the most common diseases worldwide. The aim of this work was to evaluate the epidemiological surveillance of viral acute diarrheal disease in children and adults, 650 fecal specimens using real-time RT-PCR for a period of two years, between January 2019 to December 2020 Chungcheongnam-do. For this study, 650 fecal specimens obtained through the acute gastroenteritis were collected by laboratory surveillance project with pediatric hospitals in Chungcheongnam-do and examined for norovirus, group A rotavirus, sapovirus, enteric adenovirus and astrovirus. A total of 108 cases (detection rate; 16.6%) samples of virus detection of acute gastroenteritis. Overall, norovirus (10.9%, 71/650) were the most common viruses found, followed by rotavirus (3.6%, 24/650), sapovirus (1.4%, 9/650), astrovirus (0.9%, 6/650), and enteric adenovirus was not detected. By age, it was detected at the age of under 5 years, and the highest was 67.5%. Norovirus, a pathogen that is generally understood to exhibit winter season, measured highest rates in January (22.5%) and February (26.7%). Norovirus GII was identified in 98.5% of detected norovirus and the most prevalent genotypes were GII.3 As the conclusion, the analysis results obtained from this surveillance study in acute diarrheal disease will be used as a scientific basis for policy making of management, also a continuous it is required for estimation the prevalence of different acute gastroenteritis causing pathogens. Keywords : Epidemiology, Acute Diarrheal, real-time RT-PCR

331


P0233 Effect of Symbiotic Bacteria on the Growth of Filamentous Cyanobacteria (Trichormus variabilis)

Mirye PARK, Chang Soo LEE Protist Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources, Sangju, Korea Corresponding Author Email : [email protected] Co-cultured bacteria community was analyzed using Illumina Miseq sequencing in our previous study after the cryopreservation of Trichormus variabilis. During the experiment, we have isolated two bacterial species. One was Gemmobacter sp. which was least frequently found bacteria and the other was Sphingomonas koreensis which was one of the most abundantly found in the culture of T. variabilis. In this study, we detected the effects of two bacterial species on the growth of T. variabilis. In 40 ml of BG11 broth media at culture flask, 103 cells/ml of initial T. variabilis cells was co-cultured with 104, 105, 106 cells/ml of each bacterium. After 7 day of culture under 20 temperature, 35% humidity, 12h:12h light-dark photoperiod, 20 μmol m-2s-1 irradiance and 200 rpm agitation, cells aggregation of T. variabilis was found under optical microscopic observation in the co-culture group with Gemmobacter. Thus, Gemmobacter sp. was regarded to affect the growth of T. variabili by the attachment and aggregation of T. variabilis. Keywords : co-cultured, cyanobacteria, Gemmobacter sp., Sphingomonas koreensis, Trichormus variabilis

332


P0234 Time-resolved Combined Analysis of Transcriptome and Proteome of the Pathogenicity-activated Plant Pathogen Xanthomonas oryzae pv. oryzae

Seunghwan KIM, Chang-Muk LEE, Seong-ho AHN, Daseul LEE Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Admini-stration, Wanju, Korea Corresponding Author Email : [email protected] Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen responsible for causing bacterial blight in rice. The immediate alterations in Xoo upon initial contact with rice are essential for pathogenesis at the infection site. We studied time-resolved genome-wide gene expression in pathogenicity-activated Xoo cells at the transcriptome and proteome levels. The early response genes of Xoo include genes related to cell motility, inorganic ion transport, and effectors, which aid Xoo cells to invade leaf tissues, obtain scarce cofactors, and evade immune responses in rice. The alteration of gene expression is initiated as early as few minutes after the initial interaction and changes with time. The time-resolved comparison of the transcriptome and the proteome shows time differences between transcriptional and translational expression peaks in many genes, although the overall expression pattern of mRNAs and proteins is conserved. The discrepancy suggests an important role of translational regulation in Xoo at the early stages of pathogenesis. The gene expression analysis using time-resolved transcriptome and proteome provides the unprecedent valuable information regarding Xoo pathogenesis during the initial stages of Xoo-rice interaction. Keywords : Xanthomonas oryzae pv. oryzae, Xoo-rice interactions, proteome, transcriptome, time-resolved gene expression, pathogenicity

333


P0235 Bacterial Community Composition Changes by Light Limitation in Large-scale Cyanobacterial Cultivation

Taehui YANG, Ja-young CHO, Changsoo LEE, Eui-jin KIM Prokaryote Research Team, Nakdonggang National Institute of Biological Resource, Sangju, Korea Corresponding Author Email : [email protected] Large-scale cultivation of mixed cyanobacteria Anabaena sp. and Microcystis sp. in different light conditions was conducted for verifying the cyanobacterial cell growth and the abundance of cyanobacteria-associated bacteria in a greenhouse system. Cyanobacteria cell growth, environmental and chemical parameters were measured for analyzing the interaction between cyanobacteria and its symbiotic bacteria. During the cultivation period, the difference in cell growth pattern was observed between control (natural light) and light-limited groups (reduction of blue, green, and blue/green light, respectively). The variations of bacterial communities were observed in each experimental group separately. In the exponential phase of cyanobacteria cell growth, Acidovorax sp. was the dominant Family in control, green and blue/green group, nevertheless, Family Sphingobium sp. was dominant in the blue group. Family Mycobacterium sp. getting increased in the stationery and death phase in light-limited groups, while the control group was not. Also, there are differences in the consuming pattern of nitrogen and phosphorus which indicated the possibility of interaction between cyanobacteria and its symbiotic bacteria. Keywords : cyanobacteria, bacterial community, light limitation

334


P0236 Metagenomic Analysis of Wastewater in Industrial Estate by Shotgun Sequencing

You-Jung JUNG1, Sang-Hoon LEE2, Hyeokjun YOON1, Garam JEONG1, Chae Won LEE1, Hee-Deung PARK2, Moonsuk HUR1 1National Institute of Biological Resources, Environmental Research Complex, Incheon, Korea, 2School of Civil, Environmental and Architectural Engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected], [email protected] The aim of this study to confilm the characteristics of useful prokaryotes present in the extreamly contaminated environment, such as industrial wastewater and functional genetic information of extremophile living in contaminated environment. Metagenomic taxonomic and functional gene analysis were profiled using shotgun metagenome analysis (Illumina NovaSeq). We then analyzed functional gene results using KEGG database. As a result, 25 species were identified, and the dominant species were identified as Alicyclipylus denitrificans, which are characterized by cyclohexanlo-degrading and nitrate-reducing. 47% of the genes involved in metabolism were found and genes for biosynthesis of antibiotics and vitamins were found. And then we identified related genes of xylene, dioxine and styrene degradation. These metagenomic results will contribute to securing useful genetic resources and understanding the environment at the gene level. Keywords : Shotgun metagenomics , functional gene, industrial wastewater References 1. Mechichi T, Erko S, Georg F Alicycliphilus denitrificans gen. nov., a cyclohexanlo-degrading, nitrate-

reducing beta-proteobacterium. Int J Sys Evol Microbiol. 55(1): 147-199 (2003). 2. Chalita M, Ha SM, Kim YO, Oh HS, Yoon SH, Chun J. Improved metagenomic taxonomic profiling using

a curated core gene-based bacterial database reveals unrecognized species in the genus Streptococcus. Pathogens. 9(3) (2020).

335


P0237 A Photo-selection Strategy for High Production of Astaxanthin in Haematococcus pluvialis

Ho Seok KWAK Department of Food Science and Engineering, Dongyang Mirae University, Seoul, Korea Corresponding Author Email : [email protected] Haematococcus pluvialis is known as the richest natural source of astaxanthin (3–5% in dry cell weight). Moreover, the astaxanthin from the H. pluvialis extracts predominantly consists of 3S, 3'S stereoisomer which is the most effective isomer for human application compared to the others. However, H. pluvialis is so sensitive that it is difficult to cultivate and produce astaxanthin in large quantities. And there is problem that the productivity of astaxanthin is low because the cultivation period is longer than 4 weeks. Therefore, in this research, we developed a selection strategy for astaxanthin hyperproducing H. pluvialis mutants using photoaxis to enhance the astaxanthin productivity. Randomly mutagenized cells were put in a microfluidic chip to analyze phototactic performances and the correlation with photosynthetic efficiency. Based on this positive relationship, we simply isolated cells showing fast phototactic responses from a mixture of 10,000 mutants. Among them, HM21 mutant strain showed 172.2 ± 4.12 mg L−1 of the highest astaxanthin production, which is 1.44-times higher than that of wild-type. This wide-scope screening method expedites both upstream and downstream astaxanthin quantification, making it a useful tool for isolating microalgae with high astaxanthin production. Our approach provides a powerful screening tool for rapid improvement of microalgal strains to enhance photosynthetic productivity. Keywords : Haematococcus pluvialis, Microfluidics, Phototaxis

336


P0238 LC-MS/MS-based Metabolite Identification for the Protein Hydrolysates of Porcine Whole Blood and Its Application for the Microbial Culture Media

Seonghun KIM1,2, Hoy Kon JEON1 1Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Korea, 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology(UST), Daejeon, Korea Corresponding Author Email : [email protected] Animal bloods are agricultural byproducts in meat processing industries. Although the tons of blood wastes is not utilized and disposed, it could be a potential bioresource for bioconversion into higher value products. In this study, we identified the metabolic compounds in the enzymatic hydrolysate of porcine whole blood for the application of microbial culture media. Porcine whole blood was hydrolyzed by endo- and exo-protease cocktails. After the enzymatic hydrolysis, the crude hydrolysate was formulated to dried powder form to analyze the content of soluble compounds. A total of 287 metabolites were identified through liquid chromatography‐mass spectrometry/mass spectrometry (LC-MS) analysis under cationic and anionic modes. Overall, 82 metabolites, including the twenty essential amino acids, were quantitatively analyzed. Other metabolites involved in glycolysis/ glyconeogenesis, TCA cycle, amino acid biosynthesis, and glutamate metabolism were also detected in the enzymatic blood hydrolysate. Based on these analysis, the blood lysate was used as a nitrogen source in the culture media preparation for two GRAS strains, Lactobacillus plantarum and Bacillus sp. B-100. These bacteria showed the normal growth patterns in the culture media supplemented with the blood lysate. In addition, Bacillus sp. B-100 also displayed successfully its anti-fungal activities toward pathogenic fungi strains, Hytophthora capsici, Colletotrichum gloeosporioides, Fusarium fujikuroi, and Cylindrocarpon destructans, like the comparable bioactivityies of the strain cultivated in the defined medium. This analytical data could contribute the potential utilization of a blood byproduct as the supplementary compounds in the microbial culture medium. Acknowledgement: This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET), funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(1545021966) Keywords : Porcine whole blood, LC-MS, Metabolite analysis, enzymatic blood hydrolysate, Microbial culture media, essential amino acids, GRAS strain, Latobacillus plantarum, Bacillus sp., protease cocktail References 1. Fu et al. Food Res. Int. 121, 28 (2019). 2. Zheng et al. BioMed Res. Int. 2013, 278927 (2013).

337


P0239 Characterization and Production of Prodigiosin Analogues Having Anticancer Activity from Methylocystis sp. JR2

Jaewon RYU1, Si Wouk KIM2 1Department of Environment Bio Research, Eco P&G co. Suwon University, Hwaseong, Korea, 2Department of Environmental Engineering, Chosun University, Gwangju, Korea Corresponding Author Email : [email protected] A novel methanotroph, isolated from the rice paddy field soil, was identified as Methylocystis sp. JR2 by 16s rDNA gene sequencing. The strain produced a pink pigment in the NMS medium containing low phosphate concentration (≤0.1mM). Maximal absorption peaks and color of the pigment were changed by the solvents and pHs. Pigment was purified and analyzed by LC-MS/MS with m/z 338.7, 352.6 and 366.7. These values were the typical characteristics of the prodigiosin analogues consisting of 2-methyl-3-hexyl, 2-methyl-3-heptyl and 2-methyl-3-octyl prodiginine, respectively. Maximal productivities of these analogues were 62.9 and 8.1 mg/L for 2-methyl-3-heptyl prodiginine and 2-methyl-3-octyl prodiginine, respectively. To our best knowledge, these were the maximum concentrations obtained from methanotrophs. Whole genome sequencing of the isolate revealed that the genome consisted of a total of five contigs of 4,362,918 bp with GC content of 63.39%. The prodigiosin-related genes were located in contig2 in an operon. The anticancer efficacy of the extracted pigment demonstrated that the IC50 values were 15.6, 500, 250 and 250 / in prostate cancer cell line LNCap and PC3, breast cancer cell line MDA-MB-231, and lung cancer cell line NCI-H460, respectively. These results showed that the anticancer activity of the pigment was similar to the typical prodigiosin. Keywords : Methanotroph, Methylocystis sp., Prodigiosin

338


P0240 Effect of Carbon Sources for Enhanced Cyclosporin A (CyA) Production by Tolypocladium inflatum ATCC 34921 in Submerged Culture

Eun Jeong KIM1, Kyung Rae KIM1, Hyeong Ryeol KIM1, Hah Young YOO2, Ja Hyun LEE3, Chulhwan PARK4, Seung Wook KIM1 1Department of Chemical and Biological Engineering, Korea University, Seoul, Korea, 2Department of Biotechnology, Sangmyung University, Seoul, Korea, 3Department of Food Science and Engineering, Dongyang Mirae University, Seoul, Korea, 4Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] Cyclosporin A (CyA) is a secondary metabolite produced from various species of fungi which has been used to suppressing organ transplant infection, immune disease as an immunosuppressive drug. Tolypocaladium inflatum is known as one of the fungi that produce lots of CyA. Many studies on T. inflatum culture have been conducted to increase CyA production. However, low productivity still needs to be solved in submerged culture. In this study, the effect of various carbon sources (Glucose, Galactose, Mannose, Fructose, Xylose, Arabinose, Glycerol, Sucrose, Maltose, and Cellobiose) was investigated for maximizing CyA production using T. Inflatum ATCC 34921 in submerged culture. It was found that glucose and galactose were most effective as carbon sources in CyA production. Moreover, mixed glucose and galactose (1:1) show a synergy effect on cell growth and metabolite production respectively with maximum CyA production of about 161.84 mg/L. Keywords : Cyclosporin A, Tolypocaladium inflatum, Carbon source, submerged culture References 1. S. A. Survase, L. D. Kagliwal, U. S. Annapure, R. S. Singhal, Biotechnology Advances, 29, 418-435 (2011). 2. H. Liu, D. Huang, L. jin, C. Wang, S. Liang, J. Wen, Microbial Cell Factories, 16, 160 (2017).

339


P0241 Effect of Culture Media on a Newly Isolated Freshwater Microalga Parachlorella sp. for Enhanced Production of Fatty Acids

Z-Hun KIM1, Hanwool PARK2, Choul-Gyun LEE2 1Microbial Research Department, Nakdonggang National Institute of Biological Resources, Sangju, Korea, 2Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Parachlorella is known as efficient fatty acid producers that could be used for biofuels, feeds, fertilizers, etc. Microalgae show various responses to culture conditions even within the same species. In this study, growth and fatty acid composition of a newly isolated Parachlorella sp. from Nakdonggang river of Korea by different culture media were investigated. The microalga was cultivated in 400 mL bubble column photobioreactors using BG-11, BBM, TAP, and Modified TAP (MTAP) media. It was shown that using BBM medium led to higher fatty acid accumulation(34%) while using TAP medium led to higher biomass productivity(0.34 g/L/day). Composition of TAP medium was modified to have the N:P ratio of BBM while also varying concentrations of N and P to improve fatty acid productivity. One of the modified TAP, MTAP-1 (104.8 mgN/L, 135.2 mgP/L, N:P ratio = 0.77), showed highest fatty acid concentration of 0.69±0.04 g/L while those of TAP and BBM were 0.48±0.06 g/L and 0.40±0.02 g/L, respectively. The results showed that microalgal fatty acid productivity could be enhanced by changing the N:P ratio and their concentrations. Keywords : Parachlorella, fatty acid, culture media References 1. Fu, W., O. Gudmundsson, A.M. Feist, G. Herjolfsson, S. Brynjolfsson, B.Ø. Palsson, Maximizing biomass

productivity and cell density of Chlorella vulgaris by using light-emitting diode-based photobioreactor (2012), J. Biotechnol. 161(3): 242-249.

340


P0242 Effect of Butyric Acid Produced by E. coli Nissle 1917 on the Treatment of Ulcerative Colitis

Young-Tae PARK, Taejung KIM, Young-Joo KIM, Jungyeob HAM Korea Institute of Science and Technology Natural Products Research Institute, Gangneung, Korea Corresponding Author Email : [email protected] Butyric acid belongs to short-chain fatty acids (SCFAs) and is the most plentiful metabolite derived from bacterial fermentation of carbohydrates such as dietary fibers in the intestine. Faecalibacterium prausnitzii (FP) is one of the most abundant bacteria in the human intestinal microbiota and regarded as a major effector in human intestinal health because of its anti-inflammatory effects. It produces butyric acid which has beneficial effect on human gut health. However, the extreme oxygen sensitivity has been a major obstacle to cultivate and study physiological characteristics of this organism. The genes encoding Butyryl-CoA dehydrogenase (BCD) and Butyryl-CoA: Acetate-CoA-transferase (BUT) in FP were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health. According to the results of butyric acid production in wild-type E. coli expressing BCD, BUT or both, BCD was shown to be essential, while BUT was dispensable, for the production of butyric acid. The anti-inflammatory effects of butyric acid production were tested by administrating these strains into DSS-induced colitis model mice. Keywords : E.coli Nissle1917, Butyric acid, Butyryl-CoA dehydrogenase(BCD), Sugar preference, DSS-colitis model, Anti-inflammatory References 1. Souza ÉL, Elian SD, Paula LM, Garcia CC, Vieira AT, Teixeira MM, J. Med. Microbiol. 65, 201 (2016). 2. Kiesler P, Fuss IJ, Strober W,CMGH.1,154 (2015). 3. Arribas B, Rodríguez‐Cabezas M, Camuesco D, Comalada M, Bailón E, Utrilla P, Br. J. Pharmacol.

157,1024 (2009). 4. Ouwerkerk JP, Aalvink S, Belzer C, de Vos WM, ) Int J Syst Evol Microbiol, 66,4614 (2016).

341


P0243 Characteristics of Starmerella Vaccinii Isolated from Dianthus chinensis

Jung-Un KIM1, So-Hee SHIN1, Jeong-Ah YOON1, Myoung-Dong KIM1,2 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Korea, 2Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Two hundred fifty-eight yeast strains were isolated from wildflowers in Chuncheon, Gangwon Province, Republic of Korea. MBY/L7303 strain isolated from Dianthus chinensis showed the highest growth in the medium containing galactose as a carbon source. This strain was identified as Stamerellera vaccinia, and its 26S rRNA gene sequence was registered to the GenBank(MW465704). Compared with the previously reported control, the S. vaccinii MBY/L7303 utilized more various non-fermentable sugars such as cellobiose, xylose, and sucrose. However, it did not produce ethanol. The strain grew to a maximum cell density (OD600) of 21.93±0.09 in the cellobiose-supplemented medium. The highest specific growth rate was 0.41±0.00 1/h at pH 5.0 and 30. Keywords : Starmerella vaccinii, Dianthus chinensis, wildflowers, yeast, carbon utilization

342


P0244 Optimization of Fermentation and Separation Process for 1,3-Propanediol Production

Woo-Shik SHIN, Jaehoon CHO Korea Institute of Industrial Technology (KITECH), Cheonan, Korea Corresponding Author Email : [email protected] 1,3-propanediol (1,3-PDO) is an organic compound with many commercial applications. Conventionally, PDO is produced from crude oil products. However, DuPont are investing in the biological production of PDO using renewable feedstocks instead of petrochemicals such as corn. PDO has numerous applications from polymers to cosmetics, foods, lubricants, and medicines. Recently, PDO has come under spotlight as a cosmetic moisturizer is much better moisturizing power than petrochemical products such as propylene. In this study we investigated the effect of various fermentation process factors including the optimization of culture medium to produce PDO. Also, we examined performed optimizing the electrodialysis among separation process in lab-scale for the production of PDO as a raw material for cosmetics derived from crude glycerol (a by-product of biodiesel production). Keywords : 1,3-propanediol, fermentation, electrodialysis, crude glycerol

343


P0245 Effect of Culture Conditions on Hexanol Production from Syngas by Clostridium carboxidivorans P7

Hyun Ju OH, Gyeongtaek GONG, Ja Kyong KO, Sun-Mi LEE, Youngsoon UM Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea Corresponding Author Email : [email protected] Biological production of hexanol has been drawing attention to replace petroleum-derived hexanol which is widely used in chemical synthesis and plastic industry. Syngas is a promising carbon source for bio-hexanol production, providing enough reducing equivalents for chain elongation and alcohol synthesis. Acetogen bacteria mainly produce C2 compounds such as acetate and ethanol from syngas through Wood-Ljungdahl pathway. Among acetogen bacteria, Clostridium carboxidivorans P7 has been known to produce C6 compounds from syngas. In this study, the effect of gas fermentation conditions was investigated to improve hexanol production by C. carboxidivorans P7. When temperature was lowered from 37 to 30 degrees, hexanol production increased by 1.5 fold. The production of hexanol was improved to 1.22g/L by refilling the headspace of culture with syngas to continuously provide syngas. Interestingly, when 2 g/L of ethanol was added, 1.32 g/L of ethanol was consumed and 2.34 g/L of hexanol was produced. Further research on culture condition optimization and genetic modification would be required to improve hexanol production. Keywords : clostridium, C6 alcohol, syngas

344


P0246 Efficient Agar Plate-based Screening Method to Isolate New Lactobionic Acid-producing Microorganisms

Yu-Ri OH1, Hee Jeong HAN1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Lactobionic acid (LBA) has recently emerged as an important substance used in various industries such as cosmetics, foods, and pharmaceuticals. In this study, we developed a simple, efficient and high-throughput method to screen LBA-producing microorganisms. We first prepared an agar plate to isolate LBA-producing microorganisms by utilizing the property of LBA to solubilize colloidal calcium carbonate (CaCO3), resulting in formation of a clear halo around colonies on the nutrient broth agar plate containing CaCO3. Subsequently, we confirmed the LBA production of the isolated microorganisms by high-performance liquid chromatography (HPLC) analysis. Among approximately 560 colonies from soil samples in Ulsan, Korea, we found 3 colonies forming the clear halo around them on the prepared LBA-screening agar plate and all these strains could produce LBA from lactose by analyzing their culture supernatants using HPLC. Phylogenetic analysis by comparing their 16S rRNA nucleotide sequences revealed that these strains were identified as Pseudomonas spp., and Alcaligenes faecalis. Among these bacteria, A. faecalis was the new LBA-producing bacterium which was reported for the first time in this study. From the above results, the LBA-screening method which we devised in present work was highly effective to isolate and identify new LBA-producing microorganisms. Keywords : Lactobionic acid, Agar plate-based screening, Lactobionic acid-producing microorganisms

345


P0247 Value-added Conversion of Biodiesel into the Versatile Biosurfactant Sophorolipid Using Starmerella bombicola

Yu-Ri OH1, Jeong-Hun KIM1, Juyoung HWANG1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] In this study, we tested whether biodiesel could be used as a feedstock for the microbial synthesis of sophorolipid, a high value-added chemical. Sophorolipid was successfully produced from Starmerrella bombicola using biodiesel as a hydrophobic substrate. Fed-batch fermentation was carried out for the high-level production. Compared to flask culture, the sophorolipid production concentration significantly increased from 58.1 g/L to 224.2 g/L (an approximate increase of 386%) using fed-batch fermentation, which is the highest value ever reported using fatty acid methyl esters and fatty acid ethyl esters as hydrophobic substrates. Different from rapeseed oil-based sophorolipid (ROSL), the biodiesel-based sophorolipid (BDSL) contained a new type of sophorolipid called esterified sophorolipid (approximately 19.8%). The BDSL demonstrated better surface-active properties, lower surface tension (34.2 vs. 35.8 mN/m, respectively), and a decreased critical micelle concentration (25.1 vs. 26.3 mg/L, respectively) compared to the ROSL. Given these results, the BDSL is expected to be used in various industrial fields where vegetable oil-based sophorolipids, the commercialized forms of sophorolipids, have been used. To our knowledge, this is the first report to describe the conversion of biodiesel for the production of a high value-added chemical. Keywords : Sophorolipid, High-level production, Biodiesel, Starmerrella bombicola

346


P0248 Homologous Expression of Quinoprotein Glucose Dehydrogenase in Pseudomonas taetrolens to Enhance Lactobionic Acid Productivity

Yu-Ri OH1, Seung Soo LEE1, Young-Ah JANG1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] This is the first study on improving lactobionic acid (LBA) production capacity in Pseudomonas taetrolens by genetic engineering. First, quinoprotein glucose dehydrogenase (GDH) was identified as the lactose-oxidizing enzyme of P. taetrolens. Of the two types of GDH genes in P. taetrolens, membrane-bound (GDH1) and soluble (GDH2), only GDH1 showed lactose-oxidizing activity. Next, the genetic tool system for P. taetrolens was developed based on the pDSK519 plasmid for the first time, and GDH1 gene was homologously expressed in P. taetrolens. Recombinant expression of the GDH1 gene enhanced intracellular lactoseoxidizing activity and LBA production of P. taetrolens in flask culture. In batch fermentation of the recombinant P. taetrolens using a 5 L bioreactor, the LBA productivity of the recombinant P. taetrolens was approximately 17% higher (8.70 g/(L h)) than that of the wild type (7.41 g/(L h)). The LBA productivity in this study is the highest ever reported using bacteria as production strains for LBA. Keywords : Lactobionic acid, Quinoproteion glucose dehydrogenase, Homologous expression, Whey, Pseudomonas taetrolens

347


P0249 Genetically Engineered Pseudomonas taetrolens as a Whole-cell Biocatalyst for Efficient Production of Lactobionic Acid

Yu-Ri OH1, Young-Ah JANG1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Lactobionic acid (LBA) has been widely used in the food, pharmaceutical, and cosmetic industries. Pseudomonas taetrolens is an efficient LBA-producing bacterium. To improve the LBA-production ability of P. taetrolens, we modified the strain by genetic engineering. We performed homologous expression of the quinoprotein glucose dehydrogenase gene in P. taetrolens and measured the intracellular lactose-oxidizing activity and LBA production titer. In flask cultures at 12 h of incubation, the intracellular lactose oxidizing activity (0.159 U/g dry weight cell) and LBA production titer (77.2 g/L) of the recombinant P. taetrolens were approximately 118 % and 69 % higher than those (0.073 U/g dry weight cell and 45.8 g/L, respectively) of wild-type P. taetrolens. Using this recombinant strain as a whole-cell biocatalyst (WCB), the effects of reaction parameters, such as reaction temperature, cell density, and cell harvest time, were investigated on LBA production. Under optimized reaction conditions, the LBA production titer, yield, and productivity of WCB were 200 g/L, 95.6 %, and 16.7 g/L/h, respectively. Compared with our previous study, LBA production titer, yield, and productivity, which are key factors for industrial LBA production, were significantly improved by fermentation of wild-type P. taetrolens. Moreover, the reaction for LBA production could be performed up to seven times without a significant reduction in productivity, implying that this WCB was rather robust. Our results suggest that the utilization of whole-cell biocatalysis using recombinant P. taetrolens provides a potential solution to achieve economically feasible production of LBA Keywords : Lactobionic acid, Quinoprotein glucose dehydrogenase, Homologous expression, Whole cell biocatalysis, Pseudomonas taetrolens

348


P0250 Purification and Characterization of a Lactose-oxidizing Enzyme from Pseudomonas taetrolens Capable of Producing Valuable Lactobionic Acid

Yu-Ri OH1, Seung Soo LEE1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] The LBA productivity in this study is the highest ever reported using bacteria as production strains for LBA. In this study, we successfully purified a novel lactose-oxidizing enzyme in Pseudomonas taetrolens for the first time. The purified enzyme was identified as malate:quinone oxidoreductase (MQO, EC 1.1.5.4), which showed the malate-oxidizing activity converting malate into oxaloacetate. We characterized the enzymatic properties of this interesting MQO from P. taetrolens, such as the substrate specificity toward various saccharides and the effects of temperature, pH, and metal ions on the activity and stability of MQO. MQO exhibited unique substrate specificity, as it only oxidized disaccharides with reducing-end glucosyl residues, such as lactose, but not monosaccharides. Using the high oxidizing activity of MQO toward lactose, we successfully produced lactobionic acid (LBA), a valuable organic acid used in the cosmetic, food, and pharmaceutical industries, from lactose in Escherichia coli in which the quinoprotein glucose dehydrogenase gene was inactivated, the LBA nonproducing strain, by heterologously expressing MQO with pyrroloquinoline quinone. At 37 h cultivation in a 300 mL flask culture, the LBA production, yield, and productivity of the recombinant E. coli strain were 23 g/L, 100%, and 0.62 g/L/h, respectively. This study is the first to reveal the lactose-oxidizing activity of MQO, which could be used for producing LBA in heterologous bacteria. Keywords : Malate:quinone oxidoreductase, Pseudomonas taetrolens, Lactobionic acid, Lactose-oxidizing activity, Enzyme purification and characterization

349


P0251 Draft Genome of Wickerhamomyces anomalus MBY2396 Isolated from Nuruk

Jeong-Ah YOON1, Myoung-Dong KIM1,2 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Korea, 2Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Wickerhamomyces anomalus is a yeast known to produce aroma components such as ethyl acetate and 2-phenylethanol. W.anomalus MBY2396 showing osmotolerance to KCl, NaCl, sorbitol, sucrose, glucose, and fructose was isolated from Nuruk. The draft genome of W.anomalus MBY2396 was assembled de novo from long-sequence PacBio sequel system reads by using CANU (v1.7). With a total length of 14,638,111 bp, the draft genome consists of 22 contigs with N50 of 1,476,932 bp and GC content of 34.5%. The draft genome sequence data of W.anomalus MBY2396 is available in GenBank under the accession nos. PRJNA706047, and SAMN18115211 for Genome, Bioproject, and Biosample, respectively. Keywords : Wickerhamomyces anomalus, Nuruk, draft genome, assembly

350


P0252 Growth-promoting Effect of Alginate Oligosaccharides on Rhodobacter sphaeroides

Nguyen Thi My LE, Ae-Young MO, Do-Young HWANG, Yu-Nyeong CHOI, Sang-Hoon HA, Seung-Moon PARK Division of Biotechnology, College of Environmental & Bioresource Science, Jeonbuk National University, Iksan, Korea Corresponding Author Email : [email protected] In this study, effects of an alginate oligosaccharide mixture (AOS) to Rhodobacter sphaeroides were investigated. The growth of R. sphaeroides was differently promoted by AOS in various concentration. The maximum effect of AOS to this bacterium was obtained at 0.5 % supplemented in LB medium. Particularly, growth-promoting by AOS is strain-specificity to R. sphaeroides but not in other provieded bacteria, to named but not a few, Escherichia coli, Lysinibacillus xylanilyticus, Pantoea rwandensis sp., Chryseobacterium gregarium P 461/12. The R. sphaeroides growth under AOS effect is different at various modes in which white and blue light productively showed in development of the cell profileferation. Furthermore, studies at molecular level, the determination of transcript expression via qPCR reaction of fbp2 gene that encoding for Fructose-1,6-bisphosphatase class 1 and 2, RSP-0557 gene that encoding for a hypothetical protein, DUF1127 domain and RegB gene transcript that encoding for sensor histidine kinase in R.sphaeroides under AOS 0.5 % effect resulted an acceleration of R. sphaeroides growth under AOS 0 % in comparison with control strain. These results suggest that AOS is useful for improving the growth of R. sphaeroides that is a promise for symbiotic to gastrointestinal tract of human and animal. Keywords : Rhodobacter sphaeroides, Alginate oligosaccharide, qPCR, RSP-0557, RegB

351


P0253 Comparison of Gut Microbiota from Jeju Black-Swine Fed Different Feed Additives Containing Rhodobacter sphaeroides, Lactobacillus and Alginate Oligosaccharide

Nguyen Thi My LE, Ae-Young MO, Do Young HWANG, Yu Nyeong CHOI, Sang-Hoon HA, Seung-Moon PARK Division of Biotechnology, College of Environmental & Bioresource Science, Jeonbuk National University, Iksan, Korea Corresponding Author Email : [email protected] Jeju Black-Swine is a remarkable model with tenderness, supercilious taste and high-quality meat, which has been used as livestock animal. In this experiment, we determined changes in microbiome community of fecal samples from sixteen pigs in feedback to alternative feeding additives composing of Rhodobacter sphaeroides, Lactobacillus, AOS and negative control. Through 16S rRNA gene sequencing high-throughput approaches, 486 different OTUs were achieved. A vennDiagram visualization with desired clusters is revealed 397 unique OTUs confirmed through all samples with 50 candidates were considered as core signature. There were 16 phylum, 20 class, 37 order, 63 family and 147 genera shared via all samples. Firmicutes (52.85%), Bacteroidetes (29.58%), Spirochaetes (12.18%) and Proteobacteria (4.26%) were obtained as four most predominant phyla. Alpha-diversity analysis showed no significant differences between treatment and control groups. Whereas in Firmicutes was statistically decreased and Spirochaetes was increased in R. sphaeroides fed group compared to control and others. In addition, beta-diversity measures by MDS and a statistical analysis using Volcano plot indicated the effective changes in taxonomic composition that could be useful for next findings. Our results provided an insight in important responses of microbiome changes to R. sphaeroides, Lactobacillus, AOS supplementation, it could be promised of potential applications for probiotic and prebiotic. Keywords : Rhodobacter sphaeroides, Alginate oligosaccharide, Lactobacillus, 16S rRNA, microbiome, alpha-diversity, beta-diversity

352


P0254 Enhancement of CO2-derived Polyhydroxybutyrate (PHB) Production by Cyanobacterium Synechococcus elongatus UTEX 2973 Using Flue Gas SoYoung LEE, HyeJin ROH, SangJun SIM Korea University, Seoul, Korea Corresponding Author Email : [email protected] Plastic usage has increased around the world and processing used plastics generates greenhouse gases such as CO2 which is the main cause of global warming. So we need to develop bioplastics as a substitute of petroleum-based plastics. Among the various bioplastics, Polyhydroxybutyrate(PHB) is promising because it is similar to Polyproplyene(PP), the main component of conventional plastics. Cyanobacteria accumulate PHB as an energy source using CO2 and a novel PHB-producing cyanobacterial strain was developed by harnessing fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 with introduction of heterologous phaCAB genes. Under photoautotrophic condition, the engineered strain produced 420 mg L-1 (16.7% of dry cell weight) with the highest specific productivity of 75.2 mg L-1 d-1. When compared with a native PHB producer Synechocystis PCC 6803 under nitrogen deprivation, the engineered strain exhibited 2.4-fold higher PHB productivity. This study can provide a promising solution to address petroleum-based plastic waste and contribute to CO2 mitigation. Keywords : Polyhydroxybutyrate, Strain Engineering, Bioplastic, Carbon capture and utilization References 1. Abernathy, M.H., Yu, J., Ma, F., Liberton, M., Ungerer, J., Hollinshead, W.D., Gopalakrishnan, S., He, L.,

Maranas, C.D., Pakrasi, H.B., 2017. Deciphering cyanobacterial phenotypes for fast photoautotrophic growth via isotopically nonstationary metabolic flux analysis. Biotechnol. Biofuels 10 (1), 1-13.

2. Balaji, Sundaramoorthy, Gopi, Kadiyala, Muthuvelan, Bhaskaran, 2013. A review on production of poly β hydroxybutyrates from cyanobacteria for the production of bio plastics. Algal Res. 2 (3), 278-285.

3. Beck, Christian, Knoop, Henning, Axmann, Ilka M, Steuer, Ralf, 2012. The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms. BMC Genom. 13 (1), 56.

4. Brandl, H., Gross, R.A., Lenz, R.W., Fuller, R.C., 1988. Pseudomonas oleovorans as a source of poly (β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl. Environ. Microbiol. 54 (8), 1977-1982.

5. Chaogang, W., Zhangli, H., Anping, L., Baohui, J., 2010. Biosynthesis of poly-3- hydroxybutyrate (phb) in the transgenic green alga chlamydomonas reinhardtii 1. J. Phycol. 46 (2), 396-402.

353


P0255 Metabolic Engineering of Methylomicrobium alcaliphilum 20Z for Enhanced Production of Ectoine from Methane

Yun Seo LEE1, Sukhyeong CHO1, Hanyu CHAI2, Young Joo YEON3, Jinwon LEE1,2 1C1 Gas Refinery R&D Center, Sogang University, Seoul, Korea, 2Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Korea, 3Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Korea Corresponding Author Email : [email protected] Methylomicrobium alcaliphilum 20Z is a methanotrophic bacteria, can utilize methane as a sole carbon source. Recently, methanotrphs has been attracting attention as a tool of metabolic engineering for methane-based biotechnology. In this study, we have constructed M. alcaliphilum 20Z mutant to enhance ectoine production by deletion of ectoine production inhibitory genes. First, the native plasmid of M. alcaliphilum 20Z was removed by knocking out a locus in the native plasmid containing repB gene, resulting in the transformation efficiency and endogenous plasmid stability was remarkably increased. Second, ectD gene and ectR gene which are inhibitory genes of ectoine production was deleted in mutant strain, M. alcaliphilum 20ZDP. Finally, hydroxyectoine, a representative by-product in the ectoine biosynthesis pathway, was not produced at all and the final ectoine yield was enhanced in the M. alcaliphilum 20ZDPectDectR. Keywords : Methylomicrobium alcaliphilum 20Z, Ectoine, Native plasmid, Metabolic engineering

354


P0256 Enhancement of Microalgal Biomass Productivity by Using Expandable Transparent Film Bag Photobioreactor and Real-time Monitoring System

Jeong Seop LEE Korea University, Seoul, Korea Corresponding Author Email : [email protected] The aim of this work was to develop a photobioreactor which has high areal productivity for increasing biomass and lipid production in microalgal cultivation. To accomplish economic microalgae mass production, lower investment cost is essential. We developed low cost film bag reactor. Film bag reactor has advantage of cost, but its smaller volumetric capacity that comes from weak mechanical strength is main limitation. To overcome this problem, additional bulkheads were introduced to increase column size safely. In addition, spacer was devised to link film bags for single large volume reactor. By using this advanced polymer bag reactor, areal productivity of biofuel was greatly increased. Thus, we expect that economic mass production of microalgal fuel becomes feasible. Keywords : photobioreactor, microalgae, CCUS References 1. Y. Chisti, Trends Biotechnol., 26, 126 (2008). 2. H. S. Kim, A. R. Guzman, H. R. Thapa, T. P. Devarenne and D. E. Ingber, Annu Rev Biomed Eng., 3, 335

(2001).

355


P0257 Study on Growth of Extremophile Galdieria sulphuraria for Stable Phycocyanin Production

Jin-Kyu LIM1,2, Ha-Eun YANG1,2, Won-Kun PARK1 1Dept. of chemistry and energy engineering, Sangmyung University, Seoul, Korea, 2Dept. of bio engineering, Sangmyung University, Seoul, Korea Corresponding Author Email : [email protected] Outdoor algal cultivation is the ideal system that is able to maximize the benefits from photosynthesis because of the utilization of sunlight and the CO2 fixation. However, outdoor algal cultivation has been failed in the industrial scale because of less productivity caused by the realistic limitation of stressful outdoor conditions and it makes most of the ventures companies have closed or changed their target product. Extremophile Galdieria sulphuraria is acidophilic and thermophilic microalgae, so it can stand various stresses occurred at outdoor. Also, it contains phycocyanin which is antioxidant available for high value nutrients market. However, there have been few researches on the variation of the phycocyanin content during the cell development and growth, thus, we hope to build the economic cultivation and harvesting strategies for future microalgal industry. In order to do that, phototrophic and mixotrophic cultivation were tested in a flask scale. For both conditions, biomass yield, photosynthesis efficiency, and phycocyanin content were examined. Furthermore, we’ve checked the content of phycocyanin in wet cells during the cultivation period. We believed that this research would be a stepping stones for solving the problems related to productivity and economic feasibility. Keywords : extremophile, cultivation, phycocyanin, phototrophic, mixotrophic

356


P0258 Parameter-integrated Model for Conceptual Design and Kinetic Simulation of Microbial Syngas Conversion Process

Nulee JANG, In Seop CHANG Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] Syngas fermentation is being highlighted as promising route to alternate thermochemical platform. Low gas-liquid mass transfer (GLMT) of bioreactor makes it difficult to supply substrate gas to liquid phase, severely limiting metabolic rate. GLMT performance is determined by complicate relationships among reactor design, operating condition, liquid property and microbial traits. The accuracy of design work is likely to decrease with typical models. In this work, the parameter-integrated model was proposed for conceptual design and sophisticated simulation of gas-circulated bubble column. Productivity was more enhanced when mass transfer coefficient and gas supply rate simultaneously increased rather than increasing only one of the two under the simulated conditions. Change in metabolic rate accompanied variation of biomass productivity, which affected microbial nutrients requirements and operating cost. Change in nutrient requirement influenced salting-out and bubble coalescence. In addition, interactions among various parameters could be simulated. This model and methodology will be applied as a core part for design work, economic, environmental impact, and life cycle assessment. Keywords : Syngas fermentation, Modeling, Simulation, Process design, Bubble column, Gas-liquid mass transfer References 1. N. Jang, M. Yasin, S. Park, R. W. Lovitt and I. S. Chang, Bioresour. Technol., 239, 387-393 (2017). 2. N. Jang, M. Yasin, H. Kang, Y. Lee, G. Park, S. Park and I. S. Chang, Bioresour. Technol., 263, 375-384

(2018). 3. M. Yasin, N. Jang, M. Lee, H. Kang, M. Aslam, A. A. Bazmi and I. S. Chang, Bioresour. Technol. Rep., 7,

100207 (2019). 4. N. Jang, Ph.D. dissertation, GIST library 436665 (2021).

357


P0259 Medium Chain Length Polyhydroxyalkanoates (PHA) Production from Alkane (n-octane, n-decane, n-dodecane) by Pseudomonas species

Jong-Min JEON, So-Jin PARK, Eun-Su HONG, Jeong-Jun YOON Green & Sustainable Materials R&D Department, Korea Institute of Industrial Technology (KITECH), Cheonan, Korea Corresponding Author Email : [email protected] Polyhydroxyalkanoate (PHA) is bio-degradable plastics to replace petroleum based, and which made from renewable biomass has interest. In particular, medium chain length PHAs (mcl-PHA), which contains C6 to C14 carbon chain. Mcl-PHA has more elastic properties than short chain length, is used as a material for medical or paint coating. In this study, PHA was produced by culturing Pseudomonas species and using alkane (n-octane, n-decane, and n-dodecane) as carbon sources. Alkane mixture was used to determine optimal concentration and ratio of PHA production. As an initial approach, strain were selected among five Pseudomonas species that produce PHA using alkanes as carbon sources. As a result, Pseudomonas species produced 1.7g/L of cell dry weight and 0.9g/L of PHA under the condition of 20% of alkane as sole carbon source. Mixture analysis revealed optimal condition for PHA production when the n-octane, n-decane, and n-dodecane ratio is 9.15, 6.44 and 4.29%, result in 2.39g/L of PHA and 52% of PHA content. In addition, in the production of PHA monomers, it was confirmed that n-Octane is relatively better to utilize than other alkane. The obtained equation between the composition of mixed alkane and monomer composition of produced mcl-PHA suggest that has relationship of carbon source and produced PHA properties which can be controlled. ** This study was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT, MSIT) (NRF- 2020R1A2C2102381) Keywords : Pseudomonas, mcl-PHA, alkane

358


P0260 Fermentation of Triticale Silages at Different Moistures with Effective Lactic Acid Bacteria

Ki Choon CHOI, Ilavenil SOUNDHARRAJAN, Palaniselvam KUPPUSAMY Grassland and Forage Division, National Institute of Animal Science, Rural Development Administration, Cheonan, Korea Corresponding Author Email : [email protected] Preservation and enhancement of nutrients in plant-based feeds are considered a cost-effective strategy in animal health performance. In the current study, we isolated naturally occurring lactic acid bacteria (Pediococcus pentosaceus, Lactobacillus Plantarum, Lactobacillus rhamnosus) and analyzed its fermentation efficiency on the silage production at low and high moistures triticale by the ensiling method. The addition of LAB when the ensiling process significantly reduced the silages' pH compared to non-inoculated silages at both moistures. Dominant LAB and lower yeast and mould counts were noted in the inoculated silages than the non-inoculated. Silages inoculated with LAB showed a higher amount of lactic acid with lower concertation of acetic acid. Reduced or nil butyric acid production was noted in silages inoculated with different LAB compared to control silages. Interestingly, high-quality silages were produced by the addition of LAB mixtures than single culture treatment, in the means of high lactic acid content with lower acetic acid and butyric acid level. Overall data suggested that the isolated LAB strains have potential effects on the fermentation of triticale silages at different moistures; mainly mixed cultures could be used as additives to improve the quality of silages and preserve the native form of nutrients by controlling undesirable microbial growth via dominating LAB populations Keywords : Lactic acid bacteria, triticale,silage, fermentation References 1. AOAC. 1990. Official method of analysis. 15thed. Washington, DC. 2. Ilavenil, S., Kim, D.H., Srisesharam, S., Kuppusamy, P., Park, H.S., Yoon, Y.H., Kim, W.H., Song, Y.G.,

Choi, K.C. 2017. Application of customised bacterial inoculants for grass haylage production and its effectiveness on nutrient composition and fermentation quality of haylage. 3 Biotech, 7(5), 321.

359


P0261 Understanding of Cyclic Voltammogram of Electron Transfer System via Flavin and Flavocytochrome of Shewanella oneidensis MR-1

Serah CHOI, In Seop CHANG Gwangju Institute of Science and Technology, Gwangju, Korea Corresponding Author Email : [email protected] Electroactive microorganisms form a biofilm on the electrode of the bioelectrochemical system and exchange electrons with the electrode through the electron transfer system. Shewanella oneidensis MR-1 is one of the representatives of electrochemically active microorganisms. It has several kinds of electron transport system; for example, it uses flavin, a mediator secreted out of the outer cell membrane, to transfer electrons without physical contact, and direct electrons through flavocytochrome in which flavin is combined as a cofactor of redox proteins on the outer membrane. In this study, based on the differential pulse voltammetry, which can clearly distinguish electron transport systems, the electron transport systems corresponding to the oxidation and reduction peaks of the cyclic voltammetry were specified. As the biofilm of S oneidensis MR-1 was developed at the anode of the bioelectrochemical system, it was confirmed that a reduction reaction is predominant for electron transfer via flavin, and oxidation reaction is predominant for electron transfer via flavocytochrome on cyclic voltammogram. Keywords : Microbial electrochemical system, Biofilm, Electron transfer system References 1. A. J. McCormick, P. Bombelli, R. W. Bradley, R. Thorne, T. Wenzel and C. J. Howe, Energy Environ. Sci.,

8.4, 1092-1109 (2015). 2. X. Chuai, Y. Jangir and M. Y. El-Naggar, Electrochim. Acta, 198, 49-55 (2016).

360


P0262 Optimization of Culture for the Production of Recombinant Urate Oxidase with Non-natural Amino Acids

Jeong-Haeng CHO1, Jong-il CHOI2 1ProAbtech Co., Ltd., Gwangju, Korea, 2Department of Biotechnology and Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] In this study, in order to increase the half-life in the body, the optimization of the culture medium and expression regulation for the production of recombinant urease into which non-natural amino acids were introduced was performed. As a result of comparing the initial three basic mediums, the highest cell growth and expression of the recombinant protein in the B medium were confirmed, and after that, the composition of the medium maximizing the activity of the recombinant urate enzyme was confirmed using the reaction surface analysis method. Keywords : recombinant urate oxidase, non-natural amino acids, recombinant Escherichia coli, response surface method

361


P0263 Isolation and Characterization of a New Superior Lactobionic Acid-producing Bacterium, Enterobacter cloacae, from Soil

Hee Jeong HAN1, Yu-Ri OH1, Gyeong Tae EOM1,2 1Bio-based Chemistry Research Center, Korea Research Institute of Chemical Technology (KRICT), Ulsan, Korea, 2Advanced Materials and Chemical Engineering, University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Lactobionic acid (LBA) is a specialty aldonic acid which has been broadly employed in various industries including foods, cosmetics, and pharmaceuticals. In this study, we isolated a new excellent LBA-producing bacterium from soil, designated as strain KRICT-1, and phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain was identified as Enterobacter cloacae. Previously, we successfully produced LBA using Pseudomonas taetrolens, one of the most efficient LBA-producing bacterium. In a flask culture at 30 °C, E. cloacae exhibited approximately 1.08-fold higher LBA productivity (8.37 g/L/h) than that (7.75 g/L/h) of P. taetrolens which was cultivated at 25 °C, the growth temperature which P. taetrolens showed the highest LBA production ability, showing same LBA production titer (209.3 g/L) and yield (100%) using 200 g/L lactose as a substrate, indicating that E. cloacae KRICT-1 was a more efficient LBA-producing bacterium than P. taetrolens. Moreover, the LBA productivity of E. cloacae KRICT-1 improved to 8.72 g/L/h at 35 °C and maintained a high LBA productivity (7.14 g/L/h) even at 40 °C. Because the lactose solubility at 40 °C is higher than that at 25 °C, more lactose can be solubilized in the culture medium for LBA production at 40 °C. Thus, the high-titer production of LBA over 209.3 g/L can be possible if E. cloacae KRICT-1 is cultivated with lactose at 40 °C. When E. cloacae KRICT-1 was cultivated at 35 °C in 5-L fermenter, the LBA productivity (9.97 g/L/h) further improved compared with that (8.72 g/L/h) obtained in flask culture. After whole-genome sequencing of E. cloacae KRICT-1, we found that two quinoprotein glucose dehydrogenases from this strain could produce LBA from lactose by recombinantly expressing these genes in Escherichia coli. Our present results clearly demonstrated that E. cloacae KRICT-1 can be a new promising host for industrial LBA production. Keywords : Lactobionic acid, Enterobacter cloacae, Pyrroloquinoline quinone, Quinoprotein glucose dehydrogenase

362


P0264 Optimization of Cultivation of Bacillus velezensis with Antibacterial Activity Against Plant Pathogen Xanthomonas euvesicatoria

Min-Kyoung SEO1,2, Hyun-Joo YANG1, Jong-Kook JUNG1, Heon-Woo KU1, Hyo-Song NAM1 1Research Center for Environmentally Friendly Agricultural Life Sciences, Jeonnam Bioindustry Foundation, Gokseong, Korea, 2School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Xanthomonas euvesicatoria (Xeu) was the causative agent of bacterial spot on pepper. Bacillus velezensis was antimicrobial activity X. euvesicatoria (Xeu) and antifungal activities Botrytis cinerea, Rizoctonia solani. The purpose of this study was to develop an economical culture medium for the optimal cell growth and endospore forming by submerged fermentation of B. velezensis. The optimal carbon and nitrogen sources were determined as sucrose and soy bean flour, respectively. Bacillus velezensis was mass cultivated at 37°C, pH 7.0 for 36 h in 500 L submerged fermenter using the optimal medium. The results showed that bacterial cells of 5.0 x 109 cfu/ml and sporulation yield of 90%. The functional metabolites including antimicrobial agents ware also investigated from cell-free supernatant of Bacillus velezensis. Keywords : Bacillus velezensis, Mass cultivation, Bacterial spot on pepper, Antifungal activity References 1. Min-seong Kyeon, Soo-hyeong Son, Young-hee Noh, Yong-eon Kim, Hyok-in Lee, Jae-soon Cha., Xantho-

monas euvesicatoria causes bacterial spot disease on pepper plant in Korea(2016)., The Plant Pathology Journal, 32(5), 431-440.

2. Myeong Hoon Joo and Ji Yeon Kim, Optimization of medium components for the production of crude bioserfactant by Bacillus subtilis JK-1(2011)., J.Appl.Biol.Chem, 54(1), 7-14.

363


P0265 Optimization of Fermentation Process for Improved Production of Free Fatty Acid by an Engineered E. coli

Tae Seok KIM1, Hyun Wook JUNG2, Sung Kuk LEE1,2 1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Oleochemicals, the substitute of petrochemicals, are on the rise as sustainable and bio-based chemicals as recognizing importance of environment costs. Among them, fatty acids and their chemical derivatives abound in our living environment. Here, we have developed engineered fatty acid overproducing strain by enhance of mainstream of fatty acid synthesis pathway. Through several researches, our existing developed fatty acid overproducing strain has not sufficiently optimized in our culture system especially in fed batch culture condition where culture environment has a huge impact on productivity of cells. For identification of engineered system impacts on our fatty acid overproducing strain., multivariate analysis of free fatty acid overproducing by restoration approach is conducted. From the results, we have identified FadR and MMC overexpression have a significant effect over free fatty acid production of engineered strain. Finally, we have adjusted culture condition in fed batch culture. The fatty acid overproducing strain performed higher fatty acid production up to 30g/l in a 1.5-liter bioreactor fermentation under the nitrogen limiting condition. Keywords : FadR, long chain fatty acid, fed batch, nitrogen limitation References 1. Xiao, Y., Bowen, C., Liu, D., et al. Exploiting nongenetic cell-to-cell variation for enhanced biosynthesis.

Nat Chem Biol. 12, 339-344 (2016). 2. Shin KS, Lee SK. Introduction of an acetyl-CoA carboxylation bypass into Escherichia coli for enhanced

free fatty acid production. Bioresource Technology. 245(Part B), 1627-1633 (2017).

364


바이오화학 및 화장품 생물공학

(Bio-based Chemicals and

Cosmetic Bioengineering)

365


P0301 L-proline Based DES Preparation and Application for Glycine gracilis Skvortsov Extraction

Hye Jung KANG, Junseong PARK Department of Engineering Chemistry, Chungbuk National University, Cheongju, Korea Corresponding Author Email : [email protected] A new type of green solvent, deep eutectic solvent (DES), is attracting considerable interest as an alternative to organic solvents. DES is a mixture of hydrogen bond receptors and hydrogen bond donors that form hydrogen bonds, resulting in lower melting points than individual component eutectic systems. In this study, the eutectic temperature and ratio of DES based on L-proline were determined and confirmed these physical characteristics. When comparing Glycine gracilis Skvortsov with Glycine Max, it was confirmed that epicatechin was present only in Glycine gracilis Skvortsov, and the most effective extraction method was selected by ultrasonic extraction, microwave extraction, and agitation extraction of Glycine gracilis Skvortsov. DES efficiency was estimated by extracting Glycine gracilis Skvortsov extract and measuring 7 types of isoflavone and antioxidant activity. As a result, DES was confirmed to be a suitable solvent for selective extraction of natural products. ** This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT)(No.NRF-2019R1F1A1058645) Keywords : L-proline, deep eutectic solvent, Glycine gracilis Skvortsov

366


P0302 Influence of Oriental Processing on the Functional Components and Antioxidant Activity of Saururus chinensis

HeeKyoung GO, JunSeong PARK Department of Engineering Chemistry, Chungbuk National University, Cheongju, Korea Corresponding Author Email : [email protected] The leaves of Saururus chinensis contain a large amount of phenolic compound such as quercetin, quercitrin. oriental processing is a traditional processing technology of herbal medicine. In this study, Saururus chinensis were extracted by four different methods including original, distilled water (D.W) steam, ethanol (EtOH) steam and roasting. As a result of HPLC analysis, quercitrin, miquelianin, and quercetin were confirmed, and quercetin was found only by roasting. The total polyphenol content was higher in the leaves than in stem, and the highest content was confirmed at 13.43 mg gallic acid equivalent (GAE)/g in leaf D.W steam and 5.51 mgGAE/g in stem roasting. DPPH free radical scavenging activity in leaves roasting method measured the highest activity at 68.24%, in stems EtOH steam measured the highest activity at 44.24%. ABTS free radical scavenging activity, both leaves and stems were the highest at 46.39% and 19.97%. When using oriental Processing, it has better efficacy and activity than the original product. ** Acknowledgement: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT)(No.NRF-2019R1F1A1058645) Keywords : Saururus chinensis, Oriental Processing, Phenolic compound, Flavonoid, Antioxidant

367


P0303 Inhibitory Effects of Dryopteris erythrosora Extract on Tyrosinase Activity and Melanin Production in B16F10 Melanoma Cells

Yeon-Su KOO, Taejin PARK, Ji Han SIM, Min-Seon KIM, Seung-Young KIM Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea Corresponding Author Email : [email protected] In this study, we investigated the effect of Dryopteris erythrosora extract on anti-oxidation and tyrosinase activity and melanin production as natural products of functional cosmetics. Melanin contents of B16F10 melanoma cells were decreased by 10%, 21%, and 24% in a dose-dependent manner, upon Dryopteris erythrosora extract treatment at 50, 100, and 200 μg/mL, respectively. Concentration of 200 μg/mL of Dryopteris erythrosora extract especially induced greater decreases in tyrosinase activity. As a result, Dryopteris erythrosora extract showed a 70% reduction in concentration of 200 μg/mL. Dryopteris erythrosora extract suppressed expression of tyrosinase, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. Concentration of 200 μg/mL of Dryopteris erythrosora extract especially induced greater decreases in tyosinase activity and melanogenic enzyme protein expressions. This results indicate that Dryopteris erythrosora extract inhibits melanin synthesis and tyrosinase activity, and Dryopteris erythrosora extract may be an ideal candidate as a skin whitening agent. Keywords : Dryopteris erythrosora, anti-oxidation, tyrosinase activity, whitening agent, microphthalmia-associated transcription factor References 1. H. Ando and Y. Niki, M. Ito, K. Akiyama, M. S. Matsui, D. B. Yarosh, M. Ichihashi. Investigative

Dermatology. 132, 1222-1229 (2012). 2. M. Ishikawa, I. Kawase, F. Ishii. Biological and Pharmaceutical Bulletin. 30, 2031-2036 (2007). 3. J. Y. Jang and H. N. Kim, Y. R. Kim, B. W. Kim, Y. H. Choi, B. T. Choi. Life Science. 20, 1617-1624

(2010). 4. Y. G. Lee, J. Lee, N. Y. Lee, N. K. Kim, D. W. Jung, W. Wang, Y. Kim, H. G. Kim, T. N. Nguyen, H. Park,

N. I. Baek. Applied Biological Chemistry. 60, 5-12 (2017). 5. M. Zhang, J. Cao, X. Dai, X. Chen, Q. Wang. IJPR. 11, 991-997 (2012).

368


P0304 Development of a PCR Tube-adopted Electrochemical Biosensor

Wonjae KIM, Minjeong KIM, Nguyen Anh Nhung TRAN, Taekyoung LEE, Minsub CHUNG Department of Chemical Engineering, Hongik University, Seoul, Korea Corresponding Author Email : [email protected] The COVID-19 pandemic has passed a year. The current diagnosis method of using a fluorescent marker probe has prevented many patients from being tested on time due to processing cost, expensive fluorescent probes, and large instruments. Electrochemical analysis methods are being studied as an alternative to Fluorescence analysis. Electrochemical analysis requires electrodes, but inserting thin electrodes into PCR tubes causes problems such as solution evaporation through electrode gaps, temperature disturbance, and hindering DNA amplification. In this study, we devise a design electrode guide that facilitates electrochemical measurement during the PCR amplification and investigate the effect of electrode material. A tight PCR tubes were designed by 3D printing to prevent evaporation caused by electrode gaps and enable the insertion of electrodes. Electrode materials in contact with PCR solutions were studied. The performance of the designed electrode guides and electrodes has been studied for EIS analysis and electrophoresis. Keywords : biosensor, pcr, Electrochemical analysis

369


P0305 Toward Sustainable Biodegradable Plastics: Process Design and Integrative Analyses

Hyunwoo KIM, Wangyun WON Department of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Lactic acid (LA) is an important bio-based monomer for the synthesis of polylactic acid (PLA), which is biodegradable and biocompatible plastic replacing conventional non-biodegradable plastic. In this study, we present the integrated process for the production of LA from both of lignocellulosic biomass (Strategy A) and biomass-derived sugar (Strategy B). Further, we conduct integrative analyses including techno-economic, pioneer plant, uncertainty analyses, and life-cycle assessment to investigate the comprehensive economic feasibility and environmental sustainability for the proposed process. To minimize utility consumption, heat exchanger network is designed via pinch analysis. From the techno-economic analysis, the minimum selling prices (MSPs) of LA for the Strategies A and B are determined to be $1,498/ton and $1,491/ton, respectively, which are lower than current market price of LA ($1,526/ton). Given the unexpected expenses during the construction via pioneer plant analysis, it is observed the MSPs of Strategies A and B can be increased to $2,340/ton and $1,641/ton, respectively. Furthermore, uncertainty analysis using Monte-Carlo simulation is performed to quantify the risk and uncertainty in biomass-derived LA production. Through the life-cycle assessment, we evaluate the sustainability by quantifying the environmental impact of the proposed process. Keywords : Process design, Biodegradable , Economics, Sustainability

370


P0306 Development of Cosmetic Materials Using Grape Skin Extract and Chamaecyparis Obtusa Distillate Extract

Eun Min SHIN, Ju Yeon KIM, Si Eun PARK, Eun Saem LEE, Eun Chae HWANG, Chang-Joon KIM Department of Chemical Engineering, Gyeongsang National University, Jinju, Korea Corresponding Author Email : [email protected] Grape skins include high amount of bioactive substances including anthocyanin-family compounds, which are considered to be highly effective in antioxidants and anticancer properties. Unfortunately, grape skins have been discarded. Chamaecyparis obtusa distillate contains various terpenes and it is phytoncide with strong bactericidal power. It has recently attracted attention as one of good cosmetics materials. In this study, the antioxidant and whitening activity of grape skin extract, Chamaecyparis obtusa distillate extract, and a mixture of two substances was investigated. The mixture of dried grape skin powder and distilled water was stirred for one hour. Solids were removed by filtration to obtain the extract. The contents of total phenolic compounds and flavonoids in the extract and the recovery were measured for solution with different ratio of powder and distilled water. Antioxidant activity (DPPH radical scavenging activity) and whitening activity (tyrosinase inhibition) were measured by varying the concentration of the extract. Grape skin extract exhibited high DPPH radical scavenging activity (70.5%) whereas its tyrosinase activity inhibition was relatively low (21.1%). On the other hand, the antioxidant activity of Chamaecyparis obtusa extract was very low (1.6%) but its whitening activity was very high (86.5%). Grape skin extract (46 mg/mL) and hamaecyparis obtusa distillate extract were mixed in different ratio. The DPPH radical scavenging activity and tyrosinase activity inhibition of 10% of the Chamaecyparis obtusa mixture were 74% and 49%, while they were 54% and 77%, respectively for 30%-Chamaecyparis obtusa mixture. The result suggests that two types of extract mixture can be used as good materials for functional cosmetics. Keywords : Grape skin, Chamaecyparis Obtusa distillate, Antioxidant activity, Whitening activity, Cosmetics References 1. In Ae Lee and Hyo Jung Kim, Ji-Sun Lim, Ji Jeon Seo, Lesley Ann Quintos, Chan Ho Jang, Jong-Sang

Kim, Cancer Prev Res. 14. 77-83 (2009). 2. Keuk-Jun Kim and Joung Hee Kim, Syng-Ook Lee, Kook Bae Do, Won Dae Ji, Sun Gun Kim, Young Doo

Back, Korean J Clin Lab Sci. 50, 37-43 (2018).

371


P0307 Co-production of Biofuels and Biochemicals from Lignocellulosic Biomass: Process Synthesis and Analysis

Byeongchan AHN, Bomin CHOE, Wangyun WON Department of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] As environmental pollution becomes more serious, the utilization of lignocellulosic biomass is becoming important rapidly. In this paper, we propose an integrated process for biofuels (butene oligomer; BO) and biochemicals (1,3-butadiene; 1,3-BD and adipic acid; ADA) co-production by combining the separation process into the catalytic conversion process. 1,3-BD and ADA are often used to the polymer synthesis such as synthetic rubber and nylon 6,6 respectively, and those of market size are sufficiently tremendous. To mitigate required energy consumption, we carried out heat integration using pinch analysis. Through techno-economic analysis, we determine the minimum selling price (MSP) of BO is $3.33/GGE. Moreover, we compare the base case (proposed process) and alternative case, where lignin is not utilized as ADA. The result indicates that the absence of lignin utilization leads to a significant increase in MSP ($6.06/GGE). We also quantify and compare the environmental impact of biomass-derived BO and petroleum-derived gasoline via life-cycle assessment. Keywords : global warming, lignin utilization, biorefinery, adipic acid, biomass, biofuel, sustainable, 1,3-butadiene

372


P0308 Co-production of 1,4-Pentanediol and Adipic Acid from Lignocellulosic Biomass: Techno-economic and Life-cycle Analyses

Min-Young OH, Wangyun WON Department of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Global warming issues have been mainly caused by the undiscriminating use of fossil-derived resources, such as plastic. Lignocellulosic biomass can be produced to adipic acid (ADA), a source of nylon 6.6, and 1,4-pentanediol (1,4-PDO), a biodegradable plastic monomer. This study suggests the co-production strategy, which produces ADA and 1,4-PDO by lignin separation and catalytic conversion processes. In the suggested process, lignocellulosic biomass derivatives, cellulose and hemicellulose, are concurrently converted to gamma-valerolactone (GVL) and split into ADA and 1,4-PDO productions. The heat exchanger network is designed via pinch analysis to reduce utility requirements. The techno-economic and life-cycle assessments are performed to evaluate the economic and sustainability of the base case (proposed process) as well as alternative cases. The results represent that the case producing the most ADA leads to an optimal minimum selling price of $1,325/ton-ADA and has insignificant impacts on the environment. This co-production strategy can provide flexibility to respond to the global market and national environmental policy. Keywords : renewable, MSP, LCA, TEA, corn stover

373


P0309 Screening of Single-stranded DNA Aptamers for Specific Parathyroid Hormone Aptamers for Specific Parathyroid Hormone

IN-HWAN OH1, Woo-Ri SHIN1, Simranjeet SINGH SEKHON1, Sung Min WOO2, Ji-Young AHN1, Yang-Hoon KIM1 1Major in Microbiology School of Biological Sciences College of Natural Sciences Chungbuk National University, Cheongju, Korea, 2Department of Food Science and Biotechnology, Shin Ansan University, Ansan, Korea Corresponding Author Email : [email protected] Parathyroid hormone (PTH), secreted from the parathyroid gland, is a peptide composed of 84 amino acids and plays a key role in regulating the blood calcium level. In order to determine the exact cause in the diagnosis of various diseases caused by disorders of calcium metabolism, antibodies are mainly used to measure PTH in the blood from the patient. However, due to the very short half-life of the parathyroid hormone and the characteristics that exist in various forms, there is a problem that a false positive reaction occurs in tests using antibodies. Therefore, there is a need to develop other methods capable of clearly detecting parathyroid hormone. In this study, aptamer specific for PTH was selected through SELEX process and SPR analysis. Western blot analysis showed that the selected aptamer specifically binds PTH. ** This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2020R1A2C1009463). ** This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2020R1A6A1A06046235). Keywords : Parathyroid hormone (PTH), Aptamer, SELEX References 1. Sekhon, Simranjeet Singh, et al. Nanoscale 9.22 (2017): 7464-7475. 2. Hocher, Berthold, et al. PloS one 7.7 (2012): e40242.

374


P0310 Conversion of Lignocellulosic Biomass to Biofuel and Biochemicals: Process Design and Analyses

Bomin CHOE, Wangyun WON Department of Chemical Engineering, Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Today as increasing of the use of fossil fuel that is a limited energy source, the alternative energy sources are needed. Lignocellulosic biomass is an alternative energy source, which has a potential to meet the future energy requirement and lead environmental health limited the CO2 emission. To enhance the economics and carbon efficiency, we designed a new process coproducing biofuel (i.e. butene oligomer) and biochemicals (i.e. 1,5-pentandiol and adipic acid). The techno-economic analysis (TEA) for the proposed process was performed alongside a pioneer plant analysis to investigate the feasibility of the proposed process. We performed the sensitivity analysis and uncertainty analysis, and identified the major driver in the economics. Additionally, through the life cycle assessment (LCA), the environment impacts were estimated and the contributors in environment were confirmed. These TEA and LCA results can guide to improve the process design at the viewpoints of economic and environment. Keywords : Lignocellulosic biomass, Process design, Techno-economic analysis, Life cycle assessment

375


P0311 A Novel Promiscuous Antibacterial N-acyl Amino Acid Synthase from Soil Microbiome Chang-Muk LEE, Daseul LEE, Seunghwan KIM, Seong-ho AHN Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju, Korea Corresponding Author Email : [email protected] Bacillus subtilis-overlay screening using soil microbial metagenome identified an antibacterial clone synthesizing antibacterial long-chain N-acyl amino acid synthase (NAS) gene. Sequence analysis of the clone showed an ORF NasYPL was responsible for the production of the NAS. The ORF was 903 bp long, and the deduced amino acid sequence showed the highest 70% identity with a hypothetical protein from Massilia niastensis, indicating a novel gene. Phylogenetic analysis demonstrated that NasYPL belongs to Group 1 NAS. Heterologous expression of the NasYPL gene construct in Pseudomonas strains (P. putida and P. koreensis) conferred antibacterial activities against Listeria monocytogenes, and Staphylococcus epidermidis. Mass spectral analysis of the antibacterial fractions identified long-chain N-acyl tyrosine, N-acyl phenylalanine, and N-acyl leucine (or isoleucine) derivatives linked with fatty acids, indicating promiscuous enzymatic activities of NasYPL. Therefore, NasYPL expression by host-specific manner may provide applicable eco-friendly natural antibiotics to biotechnologically important Pseudomonas strains. Keywords : Antibacterial agent, Lipoamino acid, Metagenome, Microbiome, N-acyl amino acid synthase

376


P0312 Quercetin Glycosides Inhibit Keratinocyte Apoptosis Induced by Diesel Particulate Extract Activating NADPH Oxidase and Neutral Sphingomyelinase

Neung-Ho AHN, Ga Ryun KIM, Youn Kyoung SON, Moonsuk HUR, Hye Yoon PARK National Institute of Biological Resources, Environmental Research Complex, Incheon, Korea Corresponding Author Email : [email protected] Human epidermis is positioned at the interface with the external environment, protecting our bodies against external challenges, including air pollutants. Emerging evidence suggests that diesel particulate extract (DPE), a major component of air pollution, leads to impairment of diverse cellular functions in keratinocytes (KC). In this study, we investigated the cellular mechanism underlying DPE-induced KC apoptosis. We first addressed cell death occurring in KC exposed to DPE, paralleled by increased activation of NADPH oxidases (NOXs) and subsequent ROS generation. Blockade of NOX activation with a flavonoid glycosides attenuated the expected DPE-induced KC apoptosis. Therefore, we sought to investigate whether structurally- and functionally-different natural compounds could reduce DEPs-mediated cellluar apoptosis. Quercetin glycosides from Euphobia supina(ES) significantly increase keratinocyte survival in response to DEPs and also slightly attenuated ceramide production, while dramatically elevated SIP production in cells. Keywords : diesel particulate extract , Euphobia supina, NADPH oxidases, Quercetin glycosides

377


P0313 Fine Particulate Matter from Beijing Induces Oxidative Stress, Apoptosis, and Inflammatory Responses in Keratinocytes

Ilekuttige Priyan Shanura FERNANDO1, Eui Jeong HAN2, Min Ju KIM2, Kirinde Gedara Isuru Sandanuwan KIRINDAGE2, ArachchigeMaheshika Kumari JAYASINGHE2, Ginnae AHN1,2 1Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea, 2Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Fine dust (FD) has recently immerged as a global concern causing numerous health issues via oxidative cell damage. Recently special attention has been paid off for studies on the effects of FD on the skin as a causative agent of chronic skin diseases including skin barrier defects, pruritus, and immunological dysregulations. Herein, the effects of fine dust collected from Beijing, China were investigated for their damaging effects on HaCaT keratinocytes. Based on dose and time response analysis, FD treatment-induced intracellular ROS generation and resulted in cell damage. Intracellular ROS were evaluated by both fluorescence microscopic and FACS analysis. FD exposure-induced mitochondrial depolarization and caused apoptosis as evidenced by apoptotic body formation, and accumulation of sub-G1 hypodiploid cells. The events were proceeding via mitochondria and ER stress-mediated apoptosis pathways. Further, FD elevated inflammatory responses as elicited by the activation of key mediators in the NF-κB pathway including the nuclear translocation of NF-κB p65. Moreover, FD disrupted skin barrier functions involved in the formation of tight junctions and moisturization. Further studies would potentiate the search for drugs that can inhibit FD-induced detrimental skin responses. ** This work has supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1I1A1A0107320311). Keywords : Particulate matter, Oxidative stress, Skin barrier

378


P0314 Anti-melanogenic Effect of Ligustrum japonicum Biorenovate Extract in B16F10 Melanoma Cells

ji-han SIM, tae-jin PARK, kyung-mi LEE, min-sung KANG, hye-hyun HONG, seung-young KIM Department of Pharmaceutical Engineering & Biotechnology, Sun-moon University, Asan, Korea Corresponding Author Email : [email protected] Biorenovation is a microbial enzyme-based structural modification of component compounds in natural products such as plant extracts as well as man-made compounds with the potential benefits of reduced cytotoxicities and improved biological activities compared with its reaction substrates. In this study, we examined the whitening effect of Ligustrum japonicum (LJ) and LJ Biorenovation extract (LJBR) in α-MSH-stimulated B16F10 melanoma cells. In the non-toxic concentrations of 25, 50, and 100 μg/ml, the inhibitory effect of LJ and LJBR on melanin synthesis and tyrosinase activity were measured. LJBR exhibited a stronger inhibitory effect on melanin synthesis and tyrosinase activity relative to its substrate LJ. In addition, the protein expression level of MITF, TRP-1, TRP-2, and tyrosinase, which are known to play a critical important role in melanin synthesis, was measured. As expected, a greater inhibitory effect on melanogenesis was observed in LJBR than in untreated group. These findings strongly suggest that Biorenovation can assist the generation of a novel skin-lightening material. Keywords : Ligustrum japonicum, Biorenovation, tyrosinase, B16F10, Melanin References 1. González S, Fernández-lorente M, Gilaberte-Calzada Y. 1. 26:614-26(2008). 2. Slominiski, A., Tobin, D. J., Shibahara, S., and Wortsman, J. 1. 84: 1155-1228(2004). 3. Miyamura, Y., Coelho, S.G., Wolber, R., Miller, S. A., Wakamatsu, K., Zmudzka, B.Z., Ito, S., Smuda, C.,

Passeron, T., Choi, W., Batzer, J., Yamaguchi, Y., Beer, J. Z., and Hearing, V. J.1. 20: 2-13 (2006). 4. Moon, S. H., Chung, Y. C., Hyun, C. G.1. 8, 140 (2019). 5. Aroca, P., Urabe, K., Kobayashi, T., Tsukamoto, K., Hearing, V. J. 1. 268: 25650-25655(1993) .

379


P0315 Effect of Cnidium japonicum Miq. Crude Extract Against Ultraviolet A-induced Photoaging in Human Dermal Fibroblasts

HyunJin JO1, JungHwan OH2, Fatih KARADENIZ2, HyeRan KIM1, SoYoung PARK1, Youngwan SEO3, Chang-Suk KONG1,2 1Department of Food and Nutrition, Silla University, Busan, Korea, 2Marine Biotechnology Center for Pharma-ceuticals and Foods, Silla University, Busan, Korea, 3Division of Marine Bioscience, Korea Maritime and Ocean University, Busan, Korea Corresponding Author Email : [email protected] As human life expectancy increases along with interest in beauty, health, and appearance, the development of effective cosmetic materials derived from natural products is increasingly gaining attention. Fibroblasts of dermis layer produce collagen and elastin, which regulate the elasticity and integrity of the skin. UVA, one of the causes of exogenous aging, penetrates deep into the dermal layer of the skin, causing generation of excessive reactive oxygen species (ROS). Accumulation of ROS stimulates the expression of MMPs, an enzyme that breaks down the collagen and elastin, resulting in skin aging. In this study, we investigate the anti-photoaging effects of C. japonicum crude extract and its solvent fractions in UVA-irradiated human dermal fibroblasts. The C. japonicum extract and fractions appeared to have cytoprotective properties against UVA-induced cell apoptosis and ROS. Besides, they also inhibited UVA irradiation-induced MMP expression. Based on the results of the present study, C. japonicum was expected to serve as a potential source for cosmeceutical agents for the prevention and treatment of UVA-induced skin damages. Keywords : Anti-photoaging, Cnidium japonicum Miq, Human Dermal Fibroblasts

380


P0316 The Conjugation of PGC-1a-derived Peptide and Phytochemical Enhances Antioxidant and Anti-aging

Kwanwoo KIM1, Hoomin LEE1, Chi-Hu PARK2, Hyoung Shik KIM2, Yun Suk HUH1 1Department of Interdisciplinary Biosystem, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, Korea, 2Natural Bioactive & Anticancer Research Institute, YEPBio Co., Ltd., Suwon, Korea Corresponding Author Email : [email protected] The skin as a largest heterogeneous organ of the human body is vulnerable to premature aging due to external stress. As the skin ages, wrinkles occur due to reduced cell proliferation and the denaturation of the extracellular matrix. Therefore, in this study, a peptide, (galloyl)2-KTPPTTP (Gal2-Pep) was synthesized by combining TPPTTP, and gallic acid (GA). Antioxidant activity was confirmed using DPPH and DCF-DA assays. The reactive oxygen species (ROS) scavenging activity of Gal2-Pep was more stable compared to GA alone; after four weeks at room temperature, its ROS scavenging activity remained higher than 50%. The present research study proved that Gal2-Pep increases the expression of PGC-1a to prevent oxidative stress and proves its potential for use in anti-aging application through decreasing the expression of matrix metalloproteinase-1 (MMP1) and increasing the expression of type I collagen. Keywords : PGC-1a, ROS, Antioxidant, Anti-aging, Mitochondria, Gallic acid, Peptide

381


P0317 Potential Anticancer Effect of Metasequoia glyptostroboides Extract by Inducing Apoptosis Pathway

Eunsu KIM1, Hoomin LEE1, Cheolwoo OH1, Suji KIM1, Debasish KUMAR DEY2, Vivek K. BAJPAI3, Young-Kyu HAN3, Yun Suk HUH1, Hyung Kyo KIM4 1Department of Biological Engineering, NanoBio High-Tech Materials Research Center, Inha University, Incheon, Korea, 2Department of Biotechnology, Daegu University, Gyeongsan, Korea, 3Department of Energy and Materials Engineering, Dongguk University-Seoul, Seoul, Korea, 4Department of Biomaterials Research Center, GENPEAU Corporation, Incheon, Korea Corresponding Author Email : [email protected] Extracts from M. glyptostroboides have been found to have a variety of biological and pharmacological activities, but their effects on cervical cancer have not been addressed. This study was conducted to evaluate the anticancer effect of M. glyptostroboides floral cone extracts prepared with various organic solvents such as n-hexane, dichloromethane (DME), chloroform and ethyl acetate against cervical cancer. Cervical cancer cell line HeLa cells showed higher cytotoxicity than normal cell control, COS-7, under the administration of 50 µg/ml of DME extract. Also, the number of cells at the sub-G1 phase, which indicates apoptotic cells, was increased approximately fourfold upon treatment with the DME and CE extracts compared with that in the negative control. Comprehensive results including GC–MS analysis suggest that DME organic extract derived from the floral cones of M. glyptostroboides induces the apoptosis pathway, which allows the extract of M. glypto-stroboides floral cones to become a potential material for anticancer drugs targeting apoptosis avoidance. Keywords : Metasequoia, Anticancer, Cervical cancer, Apoptosis

382


P0318 Whitening Activities of Brassica oleracea Sprout Biorenovated Extract in B16F10 Melanoma Cells

Jung-Hwan KIM1, Kyung-Mi LEE1, Kyong-wol YANG2, Joon-Ho HWANG2, Kyeong-Hwan KANG2, Seung-Young KIM1 1Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea, 2Jeju Love Co, Ltd, Jeju, Korea Corresponding Author Email : [email protected] Biorenovation is a method of structurally modifying components contained in natural products based on microbial enzymes. Bio-transformed plant crude-extracts are reported to have improved biological efficacy compared with its reaction substrates. In this study, we investigated anti-melanogenic activity of Brassica oleracea sprout (BO) and BO biorenovation extract (BOBR) in α-MSH stimulated B16F10 melanoma cells. BOBR at concentrations between 50 and 200 μg/mL exhibited a stronger suppression of melanin synthesis, tyrosinase activity and the expression of melanogensis-related protein compared to its substrate (BO). In addition, BOBR at 200 μg/mL inhibited the melanogensis effect by α-MSH to the level of non-treated control. These results suggest that BOBR could be a novel skin-lightening source for cosmetics and pharmaceutical ingredients. Keywords : Brassica oleracea sprout, Bio-renovation, B16F10 melanoma cell, tyrosinase, melanin References 1. E. H. Kim, Asian J Beauty Cosmetol. 4(2), 195-204 (2006). 2. S. H. Jeong, Asian J Beauty Cosmetol. 16(4), 599-607 (2018). 3. S. Y. Park and J. D. Kim, The Korean Society of Cosmetics and Cosmetology. 10(2), 157-172 (2020). 4. K. Maeda and M. Fukuda, J. Soc. Cosmet. Chem. 42, 361-368 (1991). 5. Y. H. Han, G. T. Lee, H. S. Soh, Y. S. Lee, I. S. Cho, K. Y. Park and J. W. Lim, Korean Society For

Horticultural Science. 5, 52 (2008).

383


P0319 Cytotoxicity Screening of Extracts from Narcissus tazetta var. chinensis Roem. in Macrophage Cell Lines

Kyung A CHOI National Institute of Medical Welfare, Kangnam University, Yongin, Korea Corresponding Author Email : [email protected] Narcissus tazetta var. chinensis Roem. is one of Amaryllidaceae family. Many species of Narcissus tazetta have been known for anti-inflammatory, detergent, diuretic, emetic, and purgative effects. This study was aimed to determine cytotoxicity activities of extracts from Narcissus tazetta var. chinensis Roem. in macrophage cell lines. At the 200 ug/ml concentration, ethanol extracts from N. Acroplis strongly decreased the survival rate of Raw264.7 macrophage cell line, whereas those from Pimpernel and Signor did not decrease the survival rate of Raw264.7 macrophage cell line in MTT Assay. This suggests that Pimpernel and Signor may have much less cytotoxicity in macrophage cell line. Further studies remain to test its underlying molecular mechanisms. Keywords : Narcissus tazetta, Cytotoxicity, Pimpernel, Signor

384


P0320 Screening of Medicinal Plants as an Anti-cancer Agent in Lung Adenocarcinoma and Skin Melanoma Cells

Neha KAUSHIK1, Hyunji OH1, Hyeonyeong YANG1, Yeasol LIM1, Dohyun LEE1, Miyeon SEO1, Miri KIM1, Gyeryeol PARK1, Sehoon JANG1, Junseok LEE1, Kyung A CHOI2, June Hyun KIM1 1Department of Biotechnology, The University of Suwon, Hwaseong, Korea, 2National Institute of Medical Welfare, Kangnam University, Yongin, Korea Corresponding Author Email : [email protected] Globally, cancer is a deadly disease which severely affects the human population. There is a constant demand for new therapies to treat and prevent this life-threatening disease. Recently, scientific and research interest is drawing its huge attention towards naturally-derived compounds as they are considered to have less toxic side effects compared to existing treatment methods. The plants produce secondary metabolites which are known for their anticancer activities leading to the development of new clinical drugs. The present study aimed to investigate the potential cytotoxicity of ethanol extract of Hibiscus syriacus Cinnamomum loureirii plant parts on human lung adenocarcinoma (A549) and skin melanoma (G361) cells along with normal counterparts. Importantly, biochemical analysis results showed that Cinnamomum loureirii plants extracts have high phenol and flavonoids contents with good antioxidant activity. This plant possesses potent anti-proliferative activity on the malignant A549 and G361 cell lines which are correlated with the production of nitric oxide. Further studies are necessary to detect, isolate, and characterize the phytochemicals existing in the ethanolic extract that could have anticancer potential in the treatment of different cancer types. Keywords : Growth inhibition, Hibiscus syriacus, Cinnamomum loureirii, Lung Adenocarcinoma, Skin Melanoma cell line References 1. S. Kumar and P. Kashyap, J Intercult Ethnopharmacol. 4, 109 (2015). 2. M. Greenwell, and P.K.S.M. Rahman, Int J Pharm Sci Res. 6, 4103 (2015). 3. J.H. Kim, Y.B. Choi, H.J. Lee, Y.H. Kim, J.H. Kim, J.H. Sim, and Y.S. Sohn. Kor. J. Plant Res. 29, 588

(2016). 4. N. Kaushik, H. Yang, S. Jeong, N.K. Kaushik, P. Bhartiya, L. Nhat Nguyen, E.H. Choi, and J.H. Kim, Appl.

Sci. 10, 7543 (2020).

385


P0321 The Effects of Gleditsia japonica Extract Following Biorenovation Melanogenesis-related Genes in B16F10 Melanoma Cells

Ji-Hyeon KIM, Taejin PARK, Seung-Young KIM Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea Corresponding Author Email : [email protected] Biorenovation is a method of modifying the structureof a broad range of substrates such as chemical compoundsand plant extract by microbial enzymes with thepotential benefits of reduced cytotoxicities and enhanced biologicalactivities relative to its parent substrates. The purposeof this study was to generated Gleditsia japonica Miquel leaf(GJ) extract using Biorenovation technology and tested thewhitening properties of its Biorenovation product (GJBR) in α-melanocyte-stimulating hormone (α-MSH) stimulated B16F10melanoma cells. As a result of in vitro tyrosinase activity assay,GJBR showed significant whitening effect than GJ. Especially inB16F10 cells, we found that pre-treatment of GJBR efficientlyprevented extracellular secretion of melanin with dose-dependentmanner. Therefore, it suggests that maybe various componentsthat converted by biorenovation contained in the GJBRaffect multiple factors involved in α-MSH-induced melanogenesisin B16F10 cells. Then together, our results indicate that GJBRcould be a new source for the ingredients of cosmetics. Keywords : Gleditsia japonica, biorenovation, tyrosinase, whitening agent, melanogenesis References 1. M. Iwata and T. Corn, S. Iwata, M. A. Everett, B. B. Fuller. J. Invest. Dermatol. 95: 9-15 (1990). 2. Y. G. Kim. and D. N. Pham, Y. H. Lee, J. J. Jo, E. Y. Choe, Y. H. Lee, S. B. Kim, C. J. Kim. Clean Technol

23: 401-407.23: 401-407 (2017). 3. S. Alaluf and D. Atkins, K. Barrett, M. Blount, N. Carter, A. Heath Pigment Cell Res. 15: 119-26 (2002). 4. H. Ando and Y. Niki, M. Ito, K. Akiyama, M. S. Matsui, D. B. Yarosh, M. Ichihashi J Invest Dermatol.

132: 1229 (2012). 5. Y. A. Jang and J. T. Lee Journal of Life Science. 28: 900-907 (2018).

386


P0322 Anti-inflammatory Effect of Colocasia esculenta Biorenovate Extract in LPS-stimulated RAW 264.7 Cells

Taejin PARK, Ji Han SIM, Hyehyun HONG, Seung-Young KIM Department of Pharmaceutical Engineering & Biotechnology, Sun-moon University, Asan, Korea Corresponding Author Email : [email protected] Biorenovation is a method of modifying the structure of a broad range of substrates such as chemical compounds and plant extract by microbial enzymes with the potential benefits of reduced cytotoxicities and enhanced biological activities relative to its parent substrates. We generated Colocasia esculenta L. Schott root extract using Biorenovation technology and tested the anti-inflammatory properties of its Biorenovation product (CEB) in lipopolysaccharide (LPS)-treated RAW264.7 murine macrophages. CEB inhibited the LPS-induced production of pro-inflammatory markers such as nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) in a concentration dependent manner without no noticeable toxicities in RAW 264.7 cells. Then together, our results indicate that CEB could be a new source for the ingredients of cosmetics and pharmaceuticals. Keywords : Anti-inflammation, Bio-renovation, Colocasia esculenta L. Schott References 1. T. H. Mogensen. Clin Microbiol Rev. 22, 240-273 (2009) 2. S. I. Grivennikov and F. R. Greten, M. Karin. Cell. 140, 883-899 (2010) 3. S. K. Choo, and Y. T. Park, K. W. Yun. Korean Journal of Plant Resources. 15, 293-297 (2002) 4. J. H. Hong. Donguibogam: Principles and Practice of Eastern Medicine. pp. 1195-1196. 5. H. R. Choi and J. S. Park, K. M. Kim, M. S. Kim, K. W. Ko, C. G. Hyun, S. Y. Kim. Ind Eng Chem. 63,

255-261.

387


P0323 Study of Optimized Extraction Conditions for Active Component of Fine Ginseng Root Using Pyrolysis Process

Jin-Young SUNG, In-Seon YUN, Jung-Tae LEE, Sang-Woo NAM, Yong-Min KIM Dept. of Cosmetics and Beauty Biotechnology, Semyung University, Jecheon, Korea Corresponding Author Email : [email protected] This study was conducted to find out the optimal extraction conditions to obtain active component of fine ginseng root using pyrolysis process. According to previous studies, it is known that ginsenoside Re is converted in to ginsenoside Rg2 and Rg2 is converted into Rg6 and F4. So, we focused on ginsenoside Rg2 among active ginsenoside of fine ginseng root. The pyrolysis reactor was manufactured of a screw conveyor type, and the pyrolysis reaction considered temperature and reaction time as variables. It was reacted for 10, 30, 50 minutes at a temperature of 480 to confirm the change in the content of ginsenoside Rg2. As a result of performing the pyrolysis reaction for 10 minutes, there was no change. After 30minutes of reaction, it was confirmed that the content of ginsenoside Rg2 was increased. However, after 50minutes of reaction, it was confirmed that the ginsenoside Rg2 content decreased. Our findings show that ginsenoside Rg2 could be seen to change as soon as it was created, and we confirmed that the content of ginsenoside Rg2 reached their heights at 30minutes of reaction. Keywords : Pyrolysis, Fine Ginseng Root, Ginsenoside Rg2 References 1. Sun-A Lee, Hee-Kyung Jo, Min-Chang Sung, Soon-Hyun Cho, You-Chan Song, Byung-Ok Im and Sung-

Kwon Ko, Changes in the Contents of Prosapogenin in Ginseng Radix Palva(Panax ginseng) Depending on the Extracting Conditions(2012), The Korean Society of Pharmacognosy, 43(2), 152-156.

2. Noriko Yamabe, Kyung-Il Song, Woo-Jung Lee, Im-Ho Han, Ji-Hwan Lee, Jung-Yeob Ham, Su-Nam Kim, Jeong-Hill Park and Ki-Sung Kang, Chemical and Free Radical-scavenging Activity Changes of Ginsenoside Re by Maillard Reaction and Its Possible Use as a Renoprotective Agent(2012), Journal of Ginseng Research, 36(2), 256-262.

3. Eun-Son Hwang, Taek-Hwan Lee, Sang-Yong Park, Tae-Hoo Yi and Sun-Yeou Kim, Enzyme-modified Panax ginseng inhibits UVB-induced skin aging through the regulation of procollagen typeⅠand MMP-1 expression(2014), Food Function, 5(2), 265-274.

4. Demirbas A, Mechanisms of liquefaction and pyrolysis reactions of biomass(2000), Energy Conversion and Management, 41(6), 633-646.

388


P0324 Screening Salt Marsh Plants for Anti-adipogenic Activity

JungHwan OH1, HyunJin JO2, HyunJung LEE2, JiHo YANG2, SoYoung PARK2, YoungWan SEO3, Chang-Suk KONG1,2 1Marine Biotechnology Center for Pharmaceuticals and Foods, Silla University, Busan, Korea, 2Department of Food and Nutrition, Silla University, Busan, Korea, 3Division of Marine Bioscience, Korea Maritime and Ocean University, Busan, Korea Corresponding Author Email : [email protected] Obesity results from a chronic imbalance between energy intake and energy expenditure. This disease has become a main obstacle for a healthy life, since it is a known risk factor for metabolic disorders. Thus, there is intense interest in finding an effective anti-obesity agent derived from natural products. Salt marsh plants have unique physiological mechanisms that help them survive in harsh environments such as sea spry, high salinity, and direct sunlight. This study screened the effect of extracts from ten salt marsh plants including Artemisia scoparia, Artemisia capillaris, Cnidium japonicum, Salsola komarovii, Calystegia soldanella, Demidovia tetragonoides, Carex pumila, Rosa rugosa, Lathyrus japonicus and Corydalis heterocarpa on anti-adipogenic activity in 3T3-L1 cells by measuring lipid accumulation. Among them, A. capillaris, C. japonicum and C. heterocarpa inhibited intracellular triglyceride accumulation more than 40% at the concentration of 25 μg/ml. These results suggested that the A. capillaris, C. japonicum and C. heterocarpa might possess obesity inhibiting components, which may be used as potential sources for anti-obesity functional food agents. Keywords : adipogenesis, Salt marsh plants, 3T3-L1 cells

389


P0325 Melanogenesis Inhibitory Effects of Phryma leptostachya Callus Using Biorenovation in B16F10 Melanoma Cells

Kyung-Mi LEE1, Taejin PARK1, Eun Yee JIE2, Suk Weon KIM2, Dong-Hwan HAN1, Seung-Young KIM1 1Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea, 2Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Korea Corresponding Author Email : [email protected] Bio-renovation is the microorganism-assisted conversion of the structure and improvement of biological efficacies of natural products and synthetic compounds. In this study, we investigated the effect of plant callus on anti-melanogenic effect and compared the activity changes of bio-renovated callus. Phryma leptostachya bio-renovation products at the concentrations of 100, 200, 400 μg/mL suppressed tyrosinase activity and the expression of melanogensis-related protein such as tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2) and microphthalmia-associated transcription factor (MITF) without a loss of cell viability in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells relative to its substrate. Moreover, cell viabilities of PLBR increased over the tested concentrations compared to its substrate (PL). PLBR at 400 μg/mL inhibited the α-MSH-induced melanogensis effect to the level of non-treated control. These results indicate that the structure of various compounds contained in Phryma leptostachya (PL) were modified through a bio-renovation technique and which conferred anti-melanogenic properties to PL. Keywords : Phryma leptostachya, Bio-renovation, Tyrosinase activity, Whitening agent, MITF References 1. M. R. Slominski, M. A. Zmijewski and A. T. Slominski. Exp Dermatol. 24(4), 258–259 (2015). 2. H. S. Song, H. J. Moon, B. E. Park, B. S. Choi, D. J. Lee, J. Y. Lee, C. J. Kim and S. S. Sim. Yakhak Hoeji.

51, 500-507 (2007). 3. M. P. Przemyslaw and G. Maja. Acta biochimica polonica. 53(3), 429-443 (2006). 4. A. Wilczek, H. Kondoh and Y. Mishima. Pigment Cell Research. 9(2), 63–67 (1996). 5. C. Chen, H. Zhu, D. Zhao and J. Deng. Helvetica Chimica Acta. 95(2), 333–338 (2012).

390


P0326 The Whitening Effects of Scolopendra subspinipes Extract and Its Effective Component Using GC/MS

JiEun LEE, YuSeon NOH, SungHwa OH, YongJoo KIM, YoungSu JANG Nano Bio Research Center, Jeonnam Bioindustry Foundation, Jangseong, Korea Corresponding Author Email : [email protected] Currently, there is a great interest in cosmetics using natural resources because the use of synthetic materials is restricted. Therefore, it is important to use insects as natural resources. People are disgusted with insects, but they are used in health foods and functional cosmetics through various studies. In particular, Scolopendra subspinipes mutilans(SSM) has been studied to be effective in skin treatment and care. SSM are studied for antibacterial and antitumor, but the whitening effect of SSM is not known exactly. Therefore, we studied the whitening effect of SSM. This study was performed to investigate the whitening effect using in vitro tyrosinase inhibition assay of solvent(100% ethanol, distilled water) extraction, SFE(Supercritical Fluid Extraction) oil and SFE shell from the SSM. As a result, the 100% ethanol extraction showed the greatest tyrosinase inhibition activity to 53.31% and concentration-dependent. Also, the 100% ethanol extraction of SSM was analyzed by gas chromatography-mass spectrometry (GC-MS). The results demonstrated that 5 components were identified totally in samples, and Glyceryl trilinoleate(G.T) was confirmed as a selective component for whitening effect. The G.T showed the tyrosinase inhibition activity to 45.78% and concentration-dependent. In conclusion, we identified the whitening effect of SSM and G.T through tyrosinase activity. These SSM extractions and G.T will be applied to the ingredients of cosmetics. Keywords : Scolopendra subspinipes mutilans(SSM), tyrosinase activity, whitening effect References 1. Hwang JY, Park TS, Son JH (2013) Whitening effect of extracts and fractions from Diospyros kaki calyx. J

Life Sci 23, 383-388. 2. Kim IW, Lee JH, Kwon YN, Kim SH, Yun EY, Nam SH, Ahn MY, HWANG JS (2014) Inhibitory effect of

melanin synthesis using organic solvent extracts from Scolopendra subspininpes mutilans. J Seric Entomol Sci 52. 1-5

391


P0327 Effect of Biorenovation Product of Brassica napus on the Production of Melanin Production and Tyrosinase Activity in B16F10 Melanoma Cells

Byeong Min CHOI, Taejin PARK, Jung Hwan KIM, Seung-Young KIM Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea Corresponding Author Email : [email protected] Brassica napus (BN) has been reported to have various drug efficacies such as cancer, aging, but there is research on the scientific efficacy of whitening effect. Biorenovation is a method of modifying the structure of a broad range of substrates such as chemical compounds and plant extract by microbial enzymes with the potential benefits of reduced cytotoxicities and enhanced biological activities relative to its parent substrates. we generated BN extract using biorenovation technology and tested the whitening effect properties of its biorenovation product (BNBR) in α-MSH stimulated B16F10 melanoma cells. Our results showed that BNBR significantly inhibited enzymes of melanogenesis such as TRP-1, TRP-2, MITF, tyrosinase in a concentration dependent manner without no noticeable toxicities. Therefore, it suggests that maybe various components that converted by biorenovation contained in the BNBR affect multiple factors involved in α-MSH-induced melanogenesis in B16F10 cells. And our results indicate that BNBR could be a new source for the ingredients of functional cosmetics. Keywords : Brassica napus, Melanin, Tyrosinase, Biorenovation, Whitening agent References 1. S. A. D'Mello and G. J. Finlay, B. C. Baguley. International Journal of Molecular Sciences. 17: 1144

(2016). 2. J. Bonaventure and M. J. Domingues, L. larue. Pigment Cell Melanoma Res. 26: 316–325 (2013). 3. H. R. Choi and J. S. Park, K. M. Kim, M. S. Kim, K. W. Ko, C. G. Hyun, J. W. Ahn, J. H. Seo, S. Y. Kim.

Journal of Industrial and Engineering Chemistry. 63: 255-261 (2018). 4. M. S. Kim and T. J. Park, J. S. Lim, S. Y. Kim. KSBB Journal. 34(1): 49-53 (2019). 5. T. J. Park and J. H. Sim, H. H. Hong, D. H. Han, S. Y. Kim. KSBB Journal. 35(2): 162-168 (2020).

392


P0328 Biological Activities of Lysimachia quelpaertensis Callus Extracts by Plant Cell Culture and Biorenovation Technology

Kristina LAMA, Taeijn PARK, Seung-Young KIM Department of Pharmaceutical Engineering & Biotechnology, Sunmoon University, Asan, Korea Corresponding Author Email : [email protected] Due to the depletion and loss of natural products, various types of materials are being developed. Accordingly, studies using callus using plant tissue culture technology are being conducted. Callus refers to the unorganized flow cell mass of plants. Callus cells usually develop in the wound area of a living plant and protect the wound. For biological research and bioengineering research, pure callus can be induced by sterilizing a part of plant tissue and transferring it to a tissue culture medium. In this study, Biorenovation was used for callus. Biorenovation is a method of modifying the structure of a broad range of substrates such as chemical compounds and plant extract by microbial enzymes with the potential benefits of reduced cytotoxicities and enhanced biological activities relative to its parent substrates. We applied this method to Lysimachia quelpaertensis (LQ) callus to obtain Lysimachia quelpaertensis Biorenovated extracts (LQBR), and compared the whitening activity efficacy of LQ and LQBR. As a result, experiments were conducted at concentrations (12.5, 25, 50 µg/mL) without cytotoxicity in B16F10 melanoma cells treated with stimulants. In addition, as a result of comparing the inhibitory effect of melanin production and tyrosinase activity, a large whitening activity was confirmed in LQBR. And by measuring the expression levels of enzymes and proteins involved in melanin formation, it was demonstrated that LQBR induces more inhibition of protein expression than LQ. These results reveal new possibilities for limited cosmetic materials. Keywords : Lysimachia quelpaertensis, Biorenovation, Calus, B16F10, Melanin References 1. S. T. Oh, H. S. Jung, M. J. Cho, M. J. Song, S. H. Moh, and H. H. Seo, Journal of the Korea Academia-

Industrial cooperation society. 15, 5628-5636 (2014). 2. A. Barbulova, F. Apone, and G. Colucci, Cosmetics. 1, 94-104 (2014). 3. T. J. Park, J. H. Sim, H. H. Hong, D. H. Han, and S. Y. Kim, KSBB. 35(2), 162-168 (2020).

393


P0329 Anti-inflammatory Activity of Distylium racemosum Gall Bio-renovation Product in LPS-induced RAW 264.7 Macrophages

Hyehyun HONG1, kristina LAMA1, Won jae CHI2, seungyoung KIM1 1Department of Pharmaceutical Engineering & Biotechnology, Sun-moon University, Asan, Korea, 2Micro-organism Resources Division, Biological Resources Research Department, National Institute of Biological Resource, Incheon, Korea Corresponding Author Email : [email protected] Bio-renovation is a method of structural modification of organic compounds with potential benefits of reducing toxicity and enhancing biological activity using microbial enzyme catalytic reactions. In this study, we applied this technique to obtain a product which we named Distylium racemosum Biorenovation Product (DRB) from Distylium racemosum extract (DR) and tested its anti-inflammatory effect on RAW264.7 macrophages stimulated by LPS (Lipopoly Saccharide). We confirmed an increase in cell survival in RAW264.7 macro-phages treated with DRB compared to DR. In addition, DRB treatment suppressed the production of pro-inflammatory cytokines in a dose-dependent manner, and concomitantly decreased the protein expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). In conclusion, we developed a novel anti-inflammatory compound using biorenovation and demonstrated its efficacy in cell-based in vitro assays. Keywords : Anti-inflammation, Bio-renovation, Distylium racemosum gall References 1. Taejin Park, Ji Han Sim, Hyehyun Hong, Dong-Hwan Han, and Seung-Young Kim, KSBB Journal. 35(2),

62-168 (2020). 2. S. I. Jang, C. S. Jun, K. C. Kwak, M. S. Bae, J. H. Lee, K. Y. Kim, Y. G. Yun, and G. Y. Chai, Korean J.

Oriental Physiology & Pathology. 20, 455-459 (2006).

394


P0330 Increasing Biomass Production of Zeaxanthin Producing Microalga Chlamydomonas reinhardtii dZL by Central Composite Design Jiho MIN1, Seong-Joo HONG1, Eon-Seon JIN2, Choul-Gyun LEE1 1Department of Biological Sciences and Bioengineering, Department of Biological engineering, Inha University, Incheon, Korea, 2Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea Corresponding Author Email : [email protected] Microalgae synthesize several pigments, among which are useful pigment that are good for human health. Zeaxanthin, one of the useful pigments, can be used as a treatment for age-related macular discoloration, and we used a model microalgae, Chlamydomonas reinhardtii to enhance zeaxanthin productivity. It was mutated into a strain called dZL to accumulate zeaxanthin, and artificially stressed to promote the synthesis of zeaxanthin. The goal of this experiment is to increase zeaxanthin production by increase biomass production. We selected three factors (nitrogen source, phosphate source, light intensity), that affect biomass production, and design Central Composite Design (CCD) experiment, based on previous cultivation condition. In each factors, we used previous condition for central point, removed for negative point, doubled for positive point. Biomass production was 1.98 g/L/7days in control group. But in CCD experiments, surface analysis showed a maximum yield of 3.04 g/L and a maximum yield of 3.40 g/L in one of the experimental group. The experiments are expected to increase biomass production and contribute to medical zeaxanthin production. Keywords : microalgae, zeaxanthin, CCD

395


식품생물공학

(Food Biotechnology)

396


P0401 The Novel Development of AMR Gene Detection Method for Multiple ESBL Variants Consisting of Single-nucleotide Polymorphisms

Seyoung KO1,2, Jaewon LIM1,2, Seung-Min YANG3, Jong Ryeol KIM4, Hae-Young KIM3, Donghyuk KIM1,2,5 1School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 3Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin, Korea, 4School of Engineering, Nazarbayev University, Astana 010000, Republic of Kazakhstan, 5Korean Genomics Industrialization and Commercialization Center, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Increasing antibiotic resistance(AMR) of pathogenic bacteria places a serious burden on public health in industrialized countries, but techniques for controlling AMR of pathogenic bacteria remain limited. In this study, an Extended Spectrum Beta-Lactamase (ESBL) was targeted, and a diagnostic kit was developed that can detect AMR genes composed of Single-Nucleotide Polymorphisms using SYBR green Real-Time PCR assay. A target gene was selected according to the frequency of AMR occurrence based on a result of Whole-Genome sequence analysis. As a result, a total of 70 genes that can cover 95% or more of ESBL Hit were selected, but it was confirmed that the selected target genes had high sequence similarity by subgroup and consisted of SNPs. Therefore, the diagnostic kit was composed of 79 combination primers using a 3'mismatch primer design technique and 13 single detection primers designed to have specificity with low similarity between target genes by calculating the SNP pattern to detect resistance genes for each. The evaluation of this assay was carried out using 73 reference strains. Therefore, we report the development of a specific method using Real-Time PCR assay to detect ESBL resistance with multiple variant by having a fast mutation rate, which is found in pathogenic strains. ** This research was supported by a grant (20162MFDS010) from Ministry of Food and Drug Safety in 2020 and 2021. Keywords : Antimicrobial resistance, AMR, ESBL, Detection Method, Single-Nucleotide Polymorphism References 1. Jia, B., Raphenya, A. R., Alcock, B., Waglechner, N., Guo, P., Tsang, K. K., ... & Doshi, S. (2016). CARD

2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic acids research, gkw1004.

397


P0402 Genome-wide Multi-omics Analysis of Mucin-degrading Bacteria Reveals Their Nutrient-dependent Distinct Metabolic Features in the Human Gut Kyoung Su KIM, Ji-Young LEE, Hyeon-Su JIN, Dong-Woo LEE Department of Biotechnology, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Mucin-degrading (MD) bacteria play a key role in shaping commensal microbiota in the human gut. However, their metabolic features remain elusive. Herein we performed a genome-wide analysis to investigate the physiology and metabolism of MD bacteria in a nutrient-dependent manner. The genomes of Ruminococcus gnavus (RG) and Akkermancia muciniphila (AM) contained 3,345 and 2,246 protein-coding genes, respectively. However, their functional annotations exhibited >60% mismatches with those from different pipeline configurations, which led us to revise the genome annotations of both MD bacteria using the UniProtKB/Swiss-Prot database, followed by additional manual curation. Consequently, we reassigned 25 genes for RG and 47 genes for AM, respectively. The revised genome annotations allowed us to identify the core metabolisms of both MD bacteria together with their distinct MD enzymes. To cross-validate the reconstructed metabolic pathways, we characterized the physiology and fermentation of both MD bacteria grown on various substrates. Our multi-omics analyses integrated with microbial phenotypes provide insight into their metabolic conse-quences of nutrient-dependent phenotypes. Keywords : Mucin-degrading bacteria, Genome annotation, Anaerobic fermentation, Ruminococcus gnavus, Akkermansia muciniphila

398


P0403 Isolation of Extracellular Vesicles from Onion and Investigation of Their Anti-inflammatory Effect in Macrophage

Si Eun KIM1, Jae Young YOU1, Won Jong RHEE1,2 1Department of Bioengineering and Nano-bioengineering, Incheon National University, Incheon, Korea, 2Division of Bioengineering, Incheon National University, Incheon, Korea Corresponding Author Email : [email protected] Inflammation is an immune response to defend against infection. However, the number of patients damaged by extreme immune reactions due to westernization of the diet, stress, and autoimmunity increases. Hence, many anti-inflammatory drugs have been studied. Previous studies have shown that methanol extract of onion had an anti-inflammatory effect. However, methanol extract of onion had low solubility and stability, and it is difficult to deliver into a cell. To solve this problem, we isolated extracellular vesicles (EVs) from onion (Onex) and their anti-inflammatory effects were investigated. Onex was isolated from onion juice using size-exclusion chromatography combined with ultrafiltration. Onex was tested for its cytotoxicity in RAW 264.7 macrophage that no cytotoxicity was observed. Also, Onex significantly suppressed the expression of cytokine and pro-inflammatory proteins when macrophages were treated with lipopolysaccharide. Because EVs are stable and safe materials, Onex can be developed as a novel therapeutic anti-inflammatory biomaterials. Keywords : Extracellular vesicle, onion, inflammation, isolation, macrophage References 1. S. Nasri, M. Anoush and N. Khatami, Afr. J. Pharm. Pharmacol. 6, 1679-1684 (2012). 2. O. P. B. Wiklander, M. Á. Brennan, J. Lötvall, X. O. Breakefield and S. E. Andaloussi, Sci. Transl. Med.

11, 8521 (2019). 3. S. Raimondo, F. Naselli, S. Fontana, F. Monteleone, A. L. Dico, L. Saieva, G. Zito, A. Flugy, M. Manno,

M. A. D. Bella, G. D. Leo and R. Alessandro, Oncotarget. 6, 19514-19527 (2015).

399


P0404 Development of a Ratiometric Fluorescent Sensor for the Detection of Ethanol in Alcoholic Beverages

Hong Dinh DUONG, Juyeon KIM, Jong Il RHEE School of Chemical Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] In this work, a ratiometric fluorescent ethanol sensor was developed to detect the concentrations of ethanol in alcoholic beverages, based on the sensitivity of a complex of resazurin(RA)/resorufin(RO) to ethanol (EtOH) and a reference dye (platinum meso-tetra (pentafluorophenyl) porphyrin (PtP)) doped in polystyrene particles (PS@PtP). RO was produced from the reduction of RA under the catalysis of a hydrotalcite (HT). The RA/RO*HT complex was captured in the so-gel matrix of methyl trimethoxysilane (MTMS), and PS@PtP in matrix of ethyl cellulose. Some redox reaction of EtOH on the membrane of RA/RO*HT could occur as (i) the oxidation of ethanol to acetaldehyde and (ii) the increase of RA reduction, through electron flows followed by EtOH ↔ HT ↔ RA/RO. These possible redox reactions could lead to increase the fluorescence intensity of the ethanol-sensing membrane with increasing ethanol concentrations. The ethanol-sensing membrane had a linear detection range from 1 to 20 vol% EtOH. It also showed high reversibility, good stability and fast response time in this concentration range. This sensitive and stable ethanol sensor was used to measure alcohol contents in some Korean alcoholic beverages. Keywords : Ethanol determination, Resazurin, Resorufin, Hydrotalcite, Fluorescent sensing membrane

400


P0405 Efficient Astaxanthin in Xanthophyllomyces dendrorhous Extraction Method Study for Vegan Seafood Development : Optimal Culture Conditions and Enzyme Reaction Method

YouKyeong LEE, JeongEun HYEON Department of Food Science and Biotechnology, Sungshin Women's University, Seoul, Korea Corresponding Author Email : [email protected] In the food industry, interest in vegan foods and functional foods is continually growing. In the vegan food market, there is also a growing movement to secure the deficiency of nutrients available from animal ingredients. However, most of research is a case that has been developed as a substitute for livestock products such as pork, beef, and chicken. The above study designed the optimal culture conditions of Xanthophyllomyces dendrorhous to harvest natural astaxanthin to be used in the development of vegan seafood , and studied a method of efficiently extracting astaxanthin from X. dendrorhous. In the research of optimal culture conditions, a design is proposed in which shaking culture is performed for at least 4 days to maximum 9 days by irradiating white light. Here for mass culture, the optimal culture period should be adjusted according to the Scale Up level. In the research of efficient extraction method of astaxanthin, the enzyme reaction method showing 0.04966mg/mL is suggested as a more suitable method than the Soy Oil stirring method showing the concentration of 0.00251mg/mL. It is expected that the development of functional raw materials that can be applied to a wider range of seafood products will be possible through research on the basis of vegan seafood using microorganisms. Keywords : Xanthophyllomyces dendrorhous, Astaxanthin, White light irradiation, Optimal culture conditions, Soy oil stirring method, Enzyme reaction method, β-1,3-glucanase References 1. Juan Luis de la Fuente, et al., J Biotechnol. 148, 144 (2010). 2. Zuharlida Tuan Harith, et al., Microorganisms. 8, 430 (2020). 3. Shengzhao Dong, et al., ScientificWorldJournal. 694305 (2014).

401


P0406 A Comparative Study on Encapsulation Using Cyclic Glucans for Enhancing Solubility and Stability of Bioactive Compounds

Ji-Young LEE1,2, Shin-Joung RHO3, Eun-Mi HONG1,2, Yong-Rho KIM1,2,3,4 1Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul, Korea, 2Global Smart farm Convergence Major, Seoul National University, Seoul, Korea, 3Center for Food and Bioconvergence, Seoul National University, Seoul, Korea, 4Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Bioactive compounds derived from nature can give various health benefits when they are used for pharmaceutical, food and cosmetic industries. However, their application is limited because of their low stability, solubility and poor bioavailability. In this study, three bioactive compounds (β-carotene, lycopene, retinol) encapsulated in various cyclic glucans (β-cyclodextrin, cycloamylose) were investigated. We studied the possibility of forming complexes and the protecting effect of them. As a result, we found that beta-carotene was degraded during complexing process in watery environment using UV-spectrophotometer scanning and HPLC. On the other hand, lycopene and retinol were not degraded when they formed complexes. The UV stability of inclusion complex, in the case of β-carotene and retinol, was improved than their free form. However, lycopene-CD complexes seemed more stable than free lycopene while lycopene-CA complexes showed lower stability or no difference. Therefore, the formation of inclusion complex with cyclic glucans depends on their structure, suggesting the possibility of improving its water-solubility and stability. Keywords : cyclic glucan, cyclodextrin, cycloamylose, bioactive compounds, beta-carotene, retinol, lycopene, inclusion complex

402


P0408 Physical Management of Disruptive Foams in Industrial Plants Used in Food Technology and Biotechnology as Well as Technical Chemistry

Antonio DELGADO1,2, Bernhard GATTERNIG1,2,3, Anuhar NESME1, Nikola TOPIC1, Lidia TORRES1, Hans HOHAGEN1, Mohammad MOBARAK1, Julian THüNNESEN1, Andreas BAUR1, Christopher MCHARDY4, Cornelia RAUH2,4, Alexander JAHN2,5, Man-gi CHO2,5 1Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Fluid Mechanics, Erlangen, Germany, 2LSTME, German Engineering Research and Development Center, Busan, Korea, 3Weihenstephan-Triesdorf, University of Applied Sciences, Chair of Process Engineering, Merkendorf, Germany, 4Technical University Berlin, Department of Food Biotechnology and Food Process Engineering, Berlin, Germany, 5Dongseo University, Department of Bio-chemical Engineering, Busan, Korea Corresponding Author Email : [email protected] This contribution elucidates simulative and experimental results of novel physical management methods for the prevention, inhibition, and destruction of isotherms and hot disruptive foams. The latter can result in considerable operational disturbances in industrial plants, causing the cost of personal, material, and economical resources to rise starkly. Technical producers apply chiefly chemical agents for destroying foams that, in turn, cause impurities that are not acceptable for chemical, biotechnological, and food production or wastewater treatment. Controlling foams requires ensuring a negative foam generation balance. Foam prevention occurs when the flow conditions guarantee a high stability of the liquid phase against deformations. This requires controlling inertial and capillary effects in the liquid-gaseous systems. If the stability is very high, then flow domains exist in which bubbles and, thus, foams can´t exist. If such non-existence domains can´t be gained, similar measures permit, at least, a physically-based inhibition of foams. Here, ultrasonic-based destruction by exciting foams in eigenfrequency is described for the first time. Keywords : Disruptive Foams, Industrial plants, Chemical Production, Biotechnological Production, Food Production, Operational Disturbances, Chemical Contamination, Physical Management, Prevention of Foams References 1. P.R. Garrett, Defoaming: Theory and industrial applications. Defoaming: Theory and Industrial

Applications (CRC Press, 2017). doi:10.1201/9781315140827 2. K. Gladbach, A. Delgado and Corneliy Rauh World Journal of Mechanics, 7(2017) 297-322. 3. Ch. McHardy, J. Thünnessen, T. Horneber, J. Kostova, M.A. Hussein, A. Delgado. PAMM 18(1),1 (2018). 4. L. Zoheidi, Ch. Panradl, C. Rauh, and A. Delgado, Journal of Food Process Engineering 40(6) (2017). 5. B. Gatternig, A. Osorio Nesme, L. Almazán, J. Thünnesen, T. Beck, Ch. McHardy, Th. Bernstein, G. Luzi, Th. Stahl,

C. Rauh, A. Delgado. Proc. 2019 World Congress on Advances in Nano, Bio, Robotics, and Energy ANBRE19, 17-21. September 2019, Jeju Island, Korea (2019).

6. J. Thünnesen, B. Gatternig, A. Delgado. In: Proc. 27. GALA-Fachtagung.

403


P0409 Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS) for Identification of Lactiplantibacillus plantarum Group Based on Species-specific Mass Peaks

Eiseul KIM1, Dong-Won CHOO2, Chang-Gyeom KIM3, Hae-Yeong KIM1 1Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea, 2Department of Bioprocessing Technology, Korea Polytechnics, Nonsan, Korea, 3Department of Bioinformatics and Biosystems, Korea Polytechnics, Seongnam, Korea Corresponding Author Email : [email protected] Lactiplantibacillus plantarum group consists of three closely related species of L. plantarum, L. paraplantarum, and L. pentosus. These species are difficult to discriminate by conventional identification methods due to the high similarity of the 16S rRNA gene. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated to distinguish L. plantarum group species. A total of 131 isolates were identified at the species level by the bioTyper database. However, a single colony of some strains was identified as different species with similar score values, showing the limitations of the bioTyper database. To accurately distinguish the three species, the mass spectra obtained by MALDI-TOF MS were analyzed to identify specific mass peaks. Mass peaks at 3,928±1, 6,903±1, and 2,843±1 m/z were specifically found in L. plantarum, L. paraplantarum, and L. pentosus, respectively. These peaks were verified with isolates, and the result was 100% consistent with the specific PCR. Our method demonstrates that a method by species-specific mass peaks allows high-resolution identification for L. plantarum group at the species level. Keywords : Lactiplantibacillus plantarum group, MALDI-TOF MS, Identification, Specific mass peak References 1. E. Kim, S. M. Yang, H. B. Kim, H. Y. Kim. J. Microbiol. Methods. 178, 106064 (2020).

404


P0410 Development of Sauce Using Vegetable Seasoning Ingredients

Ga-yeon KIM, Se-young KWON, Eun-Hee PARK Research and Development Institute, Metascreen Inc., Chuncheon, Korea Corresponding Author Email : [email protected] This study about development of a new sauce by using vegetable ingredients such as red pepper, radish, garlic, and grain materials such as rice and beans. GX(control), GX1, GX2, GX7, and GX10 were prepared by varying the mixing ratio of each vegetable material and grain material, flavor components analyzed using an electronic nose. PCA showed that samples were classified according to the composition of vegetable ratio, and the amount of red pepper and radish was thought to affect the change in taste. Two products (Source A, B) were developed using the finally selected blending ratio, and sensory evaluation was performed for the product using a 7-point scale. The overall acceptability of Source A was 4.47±0.97, which was higher than that of Source B (4.00±1.02). In the results, the flavor composition changes depending on the content of red pepper in sauces. We built a sauce with high preference according to the characteristics of flavor components. Keywords : Source, Flavor component, Sensory evaluation, PCA References 1. SY Jeon, JS Kim, GC Kim, SY Choi, SB Kim, KM Kim. Korean J Food Cook Sci. 33(5). 538~550 (2017)..

405


P0411 Hot Water Extract of Moringa oleifera Ameliorate H2O2-Induced Oxidative Stress and Apoptosis in Vero Cells

Ilekuttige Priyan Shanura FERNANDO1, Eui Jeong HAN2, Min Ju KIM2, Ginnae AHN1,2 1Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea, 2Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Recent studies demonstrate that the dysregulated production of reactive oxygen species (ROS) and the resulting oxidative stress are implicated in the pathogenesis of numerous detrimental cellular responses caused by numerous factors including, xenobiotics, exposure to ionizing radiation, and disease conditions. At present, a special interest is paid towards research on antioxidants for the prevention of oxidative stress and associated health issues. Moringa oleifera is a well-known source of biocompatible antioxidants. The present study discloses the evaluation of the antioxidant activity of M. oleifera hot water extract (MOH) on H2O2 induced

oxidative stress in Vero cells. Per analysis, MOH indicated DPPH and ABTS+ radical scavenging with IC values of 102.52 and 122.55 µg/ml. MOH dose-dependently reduced intracellular ROS generation in H2O2 stimulated Vero cells while increasing the cell viability with the best bioactivity at 125 µg/ml. Further analysis using fluorescence microscopy and flowcytometry supported the above findings. H2O2-induced mitochondrial depolarization and apoptosis were dose-dependently suppressed by MOH. The suppression of apoptosis was mediated via the mitochondrial apoptosis pathway. Further analysis of MOH bioactivity and safety would potentiate its applications in manufacturing functional food. ** Research funded by Small and Medium Business Administration (G26S283817601) Keywords : Moringa oleifera, Oxidative stress, Antioxidants

406


P0412 Inhibitory Effects of Metergoline on Nav1.2 Voltage-dependent Sodium Channel

Junho LEE Department of Biotechnology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. In the present study, we investigated the effects of metergoline on neuronal Nav1.2 voltage-dependent sodium channel activity. The two-microelectrode voltage clamp technique was used to study the regulation of metergoline on Na+ current in Xenopus oocytes expressing cRNA-encoding Nav1.2 α and β1 subunits in the rat brain. In oocytes that expressed neuronal Na+ channels, metergoline induced inhibitory effects on the peak of Na+ currents. The metergoline-induced tonic inhibitions of peak Na+ currents were voltage- and concentration-dependent and reversible. The half maximal inhibitory concentration (IC50) in peak currents of rat brain Na1.2 channels was 4.6 ± 1.3 M. Metergoline treatment produced a 3.6 ± 0.4 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. In addition, metergoline produced a use-dependent blockade of the Na+ channel after high-frequency stimulation, indicating that metergoline could exert an inhibitory effect on the open state of the Na+ channel. Taken together, these results indicate that metergoline might regulate neuronal Nav1.2 voltage-dependent channels that are expressed in Xenopus oocytes. Our study further suggests that metergoline can be an important pharmacological target as a potent inhibitor of neuronal Nav1.2 channels. Keywords : Metergoline, Ergot alkaloid, Antidepressant

407


P0413 Safety Assessment of Four IDCC Probiotic Bacteria Through Phenotypic and Genomic Analysis

Minjee LEE, Won Yeong BANG, O-Hyun BAN, Jungwoo YANG IBS Research Center, Ildong Bioscience, Pyeongtaek, Korea Corresponding Author Email : [email protected] Probiotics play an important role in health benefits on the host. However, they also possess the potential for infectivity or in situ toxin production; thus, requiring a comprehensive assessment of their safety. In this study, we report genomic characteristics of four IDCC probiotics isolated from breast-fed infants feces and fermented foods. Phenotypic assays based on enzyme activities and carbohydrate fermentation profiles represented metabolic features of the strain. Safety evaluation for antimicrobial resistance, biogenic amines production and cytotoxicity to a murine mouse model suggested its safe use as probiotic strains. Our findings on the genetic background of these strains and its potential features provide functional and safe probiotic strains for human consumption. Keywords : Probiotics, Safety assessment, Minimal inhibitory concentration, Genome analysis References 1. O. H. Ban, S. Oh, C. Park, et al, Food Sci. and Nutr. 8, 6269-6274 (2020). 2. B. S. Lee, O. H. Ban, W. Y. Bang, et al, Ann. Microbiol. 71, 10 (2021). 3. M. Shin, O. H. Ban, Y. H. Jung, J. Yang and Y. Kim, Lett. Appl. Microbiol. (2021). 4. W. Y. Bang, S. A. Chae, O. H. Ban, et al, Microbiol. Biotechnol. Lett. 49, 1-6 (2021).

408


P0414 Real-time PCR Method for Detecting Salmonella infantis Using Serovar-specific Genetic Markers Identified by Pangenome Analysis

Seung-Min YANG, Dayoung KIM, Hae-Yeong KIM Institute of Life Sciences and Resources Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea Corresponding Author Email : [email protected] Recently, the prevalence of Salmonella Infantis has increased in human and animal isolates, and a rapid detection method is needed to cope with this. In this study, a specific genetic marker for serovar Infantis was identified by comparative genomic analysis, and the real-time PCR method was developed to rapidly and accurately detect them. Specific genetic marker, such as protein coding genes present in all genomes of serovar Infantis but not in other genomes of Salmonella serovars, was mined from publicly available 555 whole-genome sequences by pangenome analysis. As a result, gene coding a usg protein (accession no. CEI43307.1) was discovered as a specific genetic marker. A primer targeting the usg gene can detect serovar Infantis in a pure and mixed culture of eight serovars, including strains with similar antigenic determinants and strains known for high prevalence. The developed real-time PCR method was applied to 54 Salmonella strains and 32 non-Salmonella pathogenic strains and distinguished with an accuracy of 100%. Our method can diagnose Infantis sensitively, rapidly, and conveniently and can be considered an alternative to conventional serotyping methods. Keywords : Salmonella Infantis, Real-Time PCR, Pangenome, Serovar

409


P0415 Composition Changes According to Early Cutting of Elk Velvet Antlers

Dong-Kyo KIM, Sang-Hoon LEE, Eun-Do LEE, Jinwook LEE, Hee-Jong ROH, Sung-Soo LEE, Kwan-Woo KIM Animal Genetic Resources Research Center, National Institute of Animal Science, Hamyang, Korea Corresponding Author Email : [email protected] This study investigated to measure composition change by part and growth days(40th day, 60th day) of elk velvet antlers. The collected samples were divided tip, upper, middle, base by position. Samples were analyzed moisture, crude protein, crude fat, crude ash, crude fiber, pH, minerals fatty acid and amino acid contents. The content of crude protein and crude fat was high in tip of velvet antler by position, dry matter and crude ash were high in base. Crude protein contents was increase in upper, but decreased in base of antler by growth day. On the other hand, contents of crude fiber and crude ash were increased in base by growth day. pH value was lower at tip, but there was no difference between growth day. In Ca, P and K, there was a significantly difference by growth day in middle of antler. Saturated fatty acids(SFA) were tended to increase by growth day, unsaturated fatty acids(UFA) were tended to decrease. But SFA and UFA are no significantly difference. Most of amino acids were high in 60 days velvet antler, but isoleucine(Ile) was low in 60 days. This result will provide information on the change in the component of antler in case of early cutting. Keywords : Deer, Velvet antler, Growth day, Position, Composition References 1. M. L. Johansen, L.K. Bak, A. Schousboe, P. Iversen, M. Sorensen, S. Keiding, H. Vilstrup, A. Gjedde, P.

Ott and H.S. Wasgepetersen, Neurochemistry International, 50, 1042-1051 (2007). 2. B. T. Jeon, S. H. Moon, S. R. Lee and M. H. Kim, Korean Society for food science of animal resources, 30,

989-996 (2010). 3. S. R. Lee, B. T. Jeon, S. J. Kim, M. H. Kim, S. M. Lee and S. H. Moon, Asian-Australasian journal of

animal sciences, 20 1546-1550 (2007).

410


P0416 Influence of pH and Gel Structure on the Stability of Lutein Loaded Pickering Emulsion Stabilized by Quinoa Starch Granules

Hyejeong SHIN1,2, Shin-Joung RHO3, Yong-Ro KIM1,2,3,4 1Department of Biosystems Engineering, Seoul National University, Seoul, Korea, 2Global Smart farm Convergence Major, Seoul National University, Seoul, Korea, 3Center for Food and Bioconvergence, Seoul National University, Seoul, Korea, 4Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] As a particle stabilizer for Pickering emulsions, starch granules with hydrophobic modification are becoming a promising candidate due to its biodegradable and inexpensive properties. OctenylSuccinate Quinoa starch(OSQ) based Pickering Emulsion especially shows stable novel emulsifying properties for small (0.5-2μm diameter) starch granules compared to other starches and a unimodal particle distribution. This study was performed to investigate the stability of lutein loaded O/W Pickering Emulsion stabilized by OSQ in various pH range (3 - 9) and thermal treatment at 25, 60, 90. At lower pH level, the particle size, charge of emulsion and retention rate of lutein fluctuated relatively little after 15 days storage. As for the thermal treatment, creaming index and particle size of emulsion decreased as treated temperature increased and lutein retention rate increased with emulsion stability, which might be attributed to the structure of emulsion. Increasing temperature increased storage modulus and apparent viscosity, associated with formation of gel-network. These results suggest OSQ can be used effective stabilizer of lipophilic bioactive carrier at proper conditions. Keywords : Pickering emulsion, OSA-Quinoa starch, Stability

411


P0417 Exploration of Domestic Saccharomyces cerevisiae Strains for Grape Juice Fermentation

Ju-Yeong MOON1, Jeong-Ah YOON1, Myoung-Dong KIM1,2 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Korea, 2Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] One hundred fifty Saccharomyces cerevisiae strains were explored for grape juice fermentation. Compared with four ethanol-producing commercial yeasts, S. cerevisiae MBY1704 isolated from traditional fermented food produced 60.45±0.03 g/L of ethanol at 25 for 48 hrs, corresponding to the best ethanol production. The MBY1704 showed the superior synthesis of sweety and fruity flavor components such as ethyl laurate, ethyl octanoate, and phenethyl alcohol compared to commercial yeasts, suggesting its successful application in the wine industry. Keywords : Saccharomyces cerevisiae, Wine, Ethanol production, Flavor components

412


P0418 Evaluation of Beer Fermentation with Yeasts Isolated from Nuruk

Yu-Jeong LEE1, Myoung-Dong KIM1,2 1Department of Food Biotechnology and Environmental Science, Kangwon National University, Chuncheon, Korea, 2Institute of Fermentation and Brewing, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Saccharomyces cerevisiae is a yeast used to ferment beer or bread. As a beer-fermenting yeast, commercial yeast should have a high ability to produce alcohol. S. cerevisiae strains isolated from nuruk were explored for their ethanol production using wort as substrate. Among them, two strains were selected for good ethanol production capability. Analysis of flavor components revealed that the selected strains produced more flavor compounds than commercially available control strains. The selected strains produced 14 types of flavor components and exhibited a sweet and fruity flavor by phenethyl alcohol. MBY/L2795 strain had the highest capability to produce ethanol. This study suggests that the S. cerevisiae MBY/L2795 strain isolated from nuruk might be a suitable workhorse for beer production. Keywords : Saccharomyces cerevisiae, Beer fermentation, Ethanol, Flavor, Phenyethayl alcohol

413


P0419 Effects of 5-Bromo-3,4-dihydroxybenzaldehyde Isolated from Polysiphonia morrowii on Type 1 Allergy Reaction in IgE-mediated Mast Cell and Passive Cutaneous Anaphylaxis Mice Model

Eui Jeong HAN1,2, Min Ju KIM2, Soo-Jin HEO3, Eun-A KIM3, Ilekuttige Priyan Shanura FERNANDO4, Hyeong Nam JEON4, Seo-young PARK4, Ginnae AHN2,4 1Research Center for Healthcare and Biomedical Engineering, Chonnam National University, Yeosu, Korea, 2Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea, 3Jeju International Marine Science Center for Research & Education, Korea Institute of Ocean Science & Technology (KIOST), Jeju, Korea, 4Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] In this study, we investigated the anti-allergic effect of 5-Bromo-3,4-dihydroxybenzaldehyde (BDB) isolated from Polysiphonia morrowii on IgE-mediated allergic reaction in mast cells and passive cutaneous anaphylaxis (PCA) mouse model. The result showed that BDB did not show any cytotoxic effects at the used all concentrations in bone marrow-derived cultured-mast cells (BMCMCs). BDB markedly suppressed the degranulation of BMCMCs by reducing the release of β-hexosaminidase in a dose dependent manner. Also, BDB dose-dependently decreased the binding of IgE with FcεRI known as a receptor of BMCMCs as well as its expression. In addition, BDB reduced the mRNA expression levels and the secretion of inflammatory cytokines by suppressing the activation of nuclear factor (NF)-κB signaling. In further study, the application of BDB significantly reduced the PCA reaction known as a type I allergic reaction in IgE stimulated and BSA-sensitized mouse ear. Taken together, these results suggested that BDB has the anti-allergic effect and might be a natural material for the remedy of allergy. This work (Grants No. 2017R1D1A1B04035921) was supported by National Research Foundation of Korea. Keywords : Polysiphonia morrowii, Bone marrow-derived cultured-mast cells, Type 1 allergy reaction

414


P0420 Effects of apo-9′-fucoxanthinone Isolated from Sargassum horneri Against Polyethylene-induced Oxidative Stress in Human Keratinocytes

Eui Jeong HAN1,2, Min Ju KIM2, Hyeong Nam JEON2, Seo-young PARK2, Ginnae AHN2,3 1Research Center for Healthcare and Biomedical Engineering, Chonnam National University, Yeosu, Korea, 2Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea, 3Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] In this study, we investigated effects of apo-9′-fucoxanthinone (A9FXN) isolated from Sargassum horneri against polyethylene-induced oxidative stress on in HaCaT keratinocytes. First, A9FXN improved the cell viability by reducing the production of reactive oxygen species (ROS) in polyethylene-stimulated HaCaT cells. In addition, A9FXN markedly inhibited the DNA fragmentation and the apoptotic body formation by regulating the expression levels of apoptosis mediated molecules in polyethylene-stimulated HaCaT cells. Moreover, A9FXN suppressed the activation of nuclear factor (NF)-κB p65 by regulating the phosphorylation of IκB-α and NF-κB, whereas activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling. Taken together, these results suggest that A9FXN effectively suppressed the polyethylene-induced oxidative stress by activating the Nrf2/HO-1 signaling in HaCaT keratinocytes. ** This work (Grants No. 2020R1A6A3A0109950011) was supported by National Research Foundation of Korea in 2021. Keywords : Apo-9′-fucoxanthinone, HaCaT keratinocytes, Oxidative stress

415


P0421 Administration of Sargassum horneri Ethanol Extract Improves Atopic Dermatitis in House Dust Mite/DNCB-Stimulated NC/Nga Mice Model

Eui Jeong HAN1,2, Min Ju KIM2, Hyun-Soo KIM3, Seo-Young KIM4, You-Jin JEON5, Youngheun JEE6, Ginnae AHN2,7 1Research Center for Healthcare and Biomedical Engineering, Chonnam National University, Yeosu, Korea, 2Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea, 3National Marine Biodiversity Institute of Korea, Seocheon, Korea, 4Chuncheon Center, Korea Basic Science Institute, Chuncheon, Korea, 5Department of Marine Life Science, Jeju National University, Jeju, Korea, 6Department of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju, Korea, 7Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Here, we investigated the therapeutic effect of Sargassum horneri ethanol extract (SHE) in house dust mite (HDM)/DNCB-stimulated NC/Nga mice, an atopy mice model. The oral administration of SHE attenuated the atopic dermatitis symptoms, including the skin dermatitis severity, the scratching frequency and TEWL in HDM/DNCB-stimulated NC/Nga mice. The significant attenuation of the serum immunoglobulin (Ig)E, IgG1 and IgG2a levels was observed after the administration of SHE. The histological analysis also revealed that SHE inhibited the epidermal hyperplasia and hyperkeratosis with the dermal infiltrations of mast cells and eosinophil. In addition, SHE reduced the expression levels of cytokines (IL-4, IL-5, IL-6, IL-10, IL-13 and IFN-g) and chemokines (RANTES, eotaxin and TARC) by decreasing mRNA expression levels of atopy initiators (IL-25, IL-33 and TSLP) as well as the regulation of the mRNA expression levels of T-bet, GATA-3 and STAT-3 in the affected skin. Moreover, the oral administration of SHE led to the reduction of spleen and lymph node size as decreasing the population of immune cells such as eosinophil, inflammatory monocyte, neutrophil and/or macrophage, as well as the mRNA expression levels of IL-4, IL-13, IFN-γ, and TARC. Taken together, these results suggest that the SHE has beneficial potentials for the treatment of allergic diseases. ** This work (Grants No. M01201920150306) was supported by Korea Institute of Marine Science & Technology Promotion (KIMST). Keywords : Atopic dermatitis, Sargassum horneri, Inflammatory cytokines

416


P0422 (-)-Loliolide Purified From Sargassum horneri Alleviates UVB-induced Skin Barrier Damage in HaCaT Cells

Min Ju KIM1, Eui Jeong HAN1, Ilekuttige Priyan Shanra FERNANDO1, Hyeong Nam JEON1, Seo-Young PARK1, Hyun-Soo KIM2, You-Jin JEON3, Kyounghoon LEE4, Ginnae AHN1,5 1Department of Food Technology and Nutrition, Chonnam National University, Yeosu, Korea, 2National Marine Biodiversity Institute, Seocheon, Korea, 3Department of Marine Life Science, Jeju National University, Jeju, Korea, 4Department of Marine Production Management, Chonnam National University, Yeosu, Korea, 5Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] In this study, we evaluated the protective activity of 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetra hyddobenzo-furan-2(4H)-one (HTT) isolated from Sargassum horneri against ultraviolet B (UVB)-induced cellular damages in HaCaT cells, a human keratinocyte. The results showed that HTT improved the cell viability by reducing the intracellular reactive oxygen species (ROS) generation in UVB-irradiated HaCaT cells. In addition, HTT inhibited apoptosis via the reduction of the sub-G1 cell population and the apoptotic body formation by modulating the expression levels of Bax/Bcl-2, compared to the only UVB-irradiated HaCaT cells. HTT suppressed the activation of nuclear factor (NF)-κB signaling by regulating the phosphorylation of IκB-α and NF-κB p65 as well as the translocation of NF-κB p65 into nucleus. Furthermore, the pre-treatment of HTT led to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling. Interestingly, the protective capacities of HTT against UVB-induced cellular damages were significantly blocked by the inhibition of HO-1 signaling. This suggests that HTT could be used as a potential means of protection against damage caused by UVB irradiation. In addition, HTT improved skin barrier and skin moisturization. These results imply tat HTT has the effect of inhibiting skin barrier damage and moisture reduction in UVB-irradiated HaCaT cells. ** This work (Grants No. M01201920180359) was supported by Korea Institute of Marine Science & Technology Promotion (KIMST). Keywords : Sargassum horneri, Loliolide, UVB, Skin barrier, HaCaT

417


에너지 및 환경생물공학

(Energy and Environmental

Biotechnology)

418


P0501 Selection of Organic Carbon for Cultivation of Anabena Under Mixotrophic Cultivation Mode

Lan-Anh HOANG THI, Duy Trinh NGUYEN, Joonyeob LEE, TaeYoon LEE Department of Environmental Engineering, Pukyong University, Busan, Korea Corresponding Author Email : [email protected] The main objective of this study was to evaluate the effects of acetate on the cultivation of anabena under mixotrophic condition. Four different types of acetates were used for the anebena cultivation. Among them, ethyl ace-tate was found to be the most effective and the growth rates linearly increased as the amount of ethyl acetate increased. When 40 mM of ethyl acetate was used, the highest values of specific growth rate of 0.979 day−1 and maximum biomass productivity of 0.293 g L−1 d−1 were obtained. On the contrary, input of acetic acid and butyl acetate inhibited the growth of anabena. For aeration tests, 0.54 vvm was optimum for anabena cultivation. For a semi-continuous cultivation test, ethyl ace-tate was used after 0.54 vvm test was finished. Lower specific growth rate and maximum biomass productivity were obtained compared to those from batch cultivation tests. However, the greatest maximum concentration of 5.91 g/L was obtained during the semi-continuous cultivation test. Keywords : Anabena azollae, Mixotrophic cultivation, Specific growth rate, Maximum biomass productivity, Semi-continuous cultivation

419


P0502 Mixotrophic Scenedesmus acuminatus Under Semi-continuous Culture System

Lan-Anh HOANG THI, Duy Trinh NGUYEN, Joonyeob LEE, TaeYoon LEE Department of Environmental Engineering, Pukyong University, Busan, Korea Corresponding Author Email : [email protected] The purpose of this study was to determine optimum value of aeration, acetate dosage, and CO2 input for the cultivation of Scenedesmus acuminatus. Highest specific growth rate and maximum biomass productivity was obtained by the aeration of 0.72 vvm and lower specific growth rates and maximum biomass productivity were obtained for other aeration tests. When putting 0.3 M of ammonium acetate in JM medium, the highest specific growth rate and maximum biomass productivity were obtained. CO2 input tests were performed during semi-continuous culturing tests. The highest specific growth rate (0.460 d-1) and maximum biomass productivity (0.936 g L-1 d-1) were obtained after replacing 50% of solution with 0.3 M of acetate solution for CO2 input tests. In aeration tests, the highest specific growth rate (0.381 d-1) and maximum biomass productivity (0.253 g L-1 d-1) were obtained when cultivating it with JM medium, but the specific growth rate and maximum biomass producitivty were significantly decreased when 50% of solution was replaced by acetate containing solution Keywords : Scenedesmus acuminatus, Semi-Continuous Cultivation, Carbon Dioxide, Nitrogen, Phosphate, Acetate

420


P0503 Green Fabrication of Magnetic Iron Nanoparticles Using Extremophilic Bacterium Deinococcus radiodurans R1 Live Cells for the Efficient and Stable Removal of Toxic Arsenic

Chang Keun KANG, Younghoon Kim KIM, Sun-Wook JEONG, Yong Jun CHOI School of Environmental Engineering, University of Seoul, Seoul, Korea Corresponding Author Email : [email protected] Arsenic (As) contamination in water poses a serious threat to public health and living organisms. Although physicochemical arsenic treatment methods have been developed, microbial in vivo bioremediation processes using live cell fabricated nanoparticles have not yet been reported. Here, we report the development of an efficient As removal method using magnetic iron nanoparticles immobilized extremophilic microorganism, Deinococcus radiodurans R1 (DR). First, magnetic iron nanoparticles were successfully fabricated by the DR strain without additional devices and treatments, and characterized by scanning electron microscopy-energy dispersive X-ray spectroscopy, dynamic light scattering, zeta-potential, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy analysis. Second, the maximum removal capacity of the magnetic iron nanoparticle-immobilized DR (DR-FeNPs) for As(V) was evaluated under the optimized conditions. Finally, the removal capacity of DR-FeNPs in the presence of various competitive anions was also investigated to simulate the practical application. More than 98% of As(V) was efficiently removed by DR-FeNPs within 1 h, and the removal efficiency was stably maintained for up to 32 h (98.97%). Furthermore, the possibility of recovery of DR-FeNPs after use was also suggested using magnets as a proof-of-concept. Keywords : Deinococcus radiodurans R1, Magnetic nanoparticle, Arsenic, Adsorption, Bioremediation

421


P0504 Development of Amplifying PS Microplastic Detection Through a Gold Nanorods (AuNRs) Plasmonic Effect

Hyunho LEE, Cholong KIM, Hyeyeon HUR, Seungju OH, Jongmin YANG, Seongcheol SHIN, Dhanashri DESAI, Gillhwan KIM Department of Chemical Engineering, Myongji University, Yongin, Korea Corresponding Author Email : [email protected] Today, waste plastics after humans usage frequently causes serious environmental problems. As large plastics are decomposed into microscale particles, they float under the sea and sea level, causing major problems seriously for marine environment. Eventually, it affects humans detrimentally, who are the ultimate predators. Because concentrated environmental hormones and chemical toxicity can accumulate and affect the human body. In this study, gold nanorods (AuNRs) combined with polystyrene binding peptide (PSBP) and the specifically bonded polystyrene (PS) microplastics are found to amplify peaks through Au NRs to make PS detection more efficiently. Its effect is believed a plasmonic origin that detect microplastics to solve the aforementioned problems. Keywords : microplastic, polystyrene particles, gold nanorods, PSBP, microneedle

422


P0505 Photoautotrophy of Polyhydroxybutyrate (PHB) from CO2 Using Rhodobacter sphaeroides in Bioelectrochemical System

Shuwei LI Department of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] To alleviate the greenhouse effect and climate change, it is of great importance to develop sustainable CO2 capture and utilization. The microbial electrosynthesis (MES) has been highlighted to convert CO2 to value added metabolites and intermediate chemicals by using electrochemically active bacteria as biocatalyst and electricity as reducing power. However, the MES only produced simple organic acids or alcohols from CO2. In this study, we presented a novel bioelectrosynthetic pathway for polyhydroxybutyrate (PHB) production using a photosynthetic bacteria, Rhodobacter sphaeroides. The electrode attached R. sphaeroides directly uptakes electrons from the electrode surface, while the suspended Rhodobacter sphaeroides utilize the electrochemically evolved hydrogen as electron mediator to convert CO2 to polyhydroxybutyrate (PHB). The results show that MES-driven electrons and protons transfer enabled direct conversion of CO2 into higher-value added organic matter such as PHB. The PHB production after 5 days, in suspension (16.68mg/L, 7.44% of DCW) compared to electrode attached cell (11.74mg/L, 22% of DCW). Keywords : Photoautotrophy, polyhydroxybutyrate, bioelectrochemical system

423


P0506 The Effect of Cell Numbers Present in Biofertilizer on the Growth and Health of Wheat

Su Hyeon PARK, Joong Kyun KIM Department of Biotechnology, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] As a means of complete reutilization, the production of biofertilizer from fishery wastewater arouse our interest. Nowadays, hydroponics is also concerned about its use in plant cultivation due to serious Earth’s environment and weather. Considering this situation, production of high-quality biofertilizer is very important. In this study, therefore, we investigated the effect of cell numbers present in the biofertilizer on plant growth. To complete this purpose, mackerel wastewater was degraded at 45 for 36 h by mixed microorganisms isolated from rockworms, and 36-h culture supernatant was used as biofertilizer for wheat hydroponics against control. So far, the reported cell numbers in the commercial biofertilizer has been known as 1.0×108 cells. Based on the above information, various cell numbers were applied to hydroponics of wheat. The 36-h biodegradation revealed 14.47% hydrolysis, and a better result in the growth (length and fresh weight) and the health (chlorophyll and carotenoid) indices of wheat was obtained from higher cell numbers. Keywords : mackerel wastewater, biodegradation , biofertilizer, cell number , hydroponics References 1. Hyun Yi Jung and Joong Kyun Kim, 2020. Complete reutilisation of mixed mackerel and brown seaweed

wastewater as a high-quality biofertiliser in open-flow lettuce hydroponics, Journal of Cleaner Production 247, 119081.

2. Rural Development Administration of South Korea, 2021. Establishing and designating the fertilizer process specification. online at https://www.law.go.kr/admRulLsInfoP.do?admRulSeq=2100000195611#AJAX

424


P0507 Adsorption and Capture of Polyethylene Microplastics by Oleaginous Yeast Rhodosporidium toruloides Strains

Jong Hyuk PARK1,2, Sung Ok HAN1, Gyeongtaek GONG2 1Department of Biotechnology, Korea University, Seoul, Korea, 2Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea Corresponding Author Email : [email protected] / [email protected] In our daily life, the consumption of plastics has gradually increased over the past 50 years, and the amount of microplastics (MPs) has increased accordingly. The inflow of microplastics from various routes leads to adsorption with organic matter and diffusion into the ecosystem. These adsorbents can accumulate in cells and potentially lead to metabolic disorders and cytotoxicity. Therefore, a reduction technology for microplastics is required. Rhodosporidium toruloides (R. toruloides), known as an oleaginous yeast, is a robust host that can efficiently use a wide range of carbon sources. Moreover, R. toruloides is natural producer of lipids and carotenoids. During the growth of yeast suspending with MPs, some R. toruloides strains showed heteroaggre-gation and sedimentation in culture medium. Furthermore, the suspended MPs can be captured using simple centrifugation process. The heteroaggregates’ surface morphology between the MPs and the yeast strains was determined by the SEM (scanning electron microscope). This study presents the possibility of adsorption and capture of microplastics by using microbial strains. Keywords : Micorplastics, Rhodosporidum toruloides, Heteroaggregation, Adsorption, Capture

425


P0508 Metabolic Engineering of Clostridium acetobutylicum for Enhancing Bio-hydrogen Production by Overexpression of FeFe Hydrogenase

Ye-Seung SON, Jong-Min JEON, Jeong-Jun YOON Green and Sustainable Materials R&D Group, Korea Institute of Industrial Technology (KITECH), Cheonan, Korea Corresponding Author Email : [email protected] Clostridium acetobutylicum is an attractive industrial microorganism for bio-hydrogen production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, C. acetobutylicum was metabolically engineered to enhance the hydrogen production. C. acetobutylicum carrying G-6-P dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, higher than the wild type. Moreover, optimization of pH levels and glucose concentration for the CA-hydA strain leads to increase 1.26 folds higher hydrogen productivity in 4 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production. Keywords : Bio-hydrogen, Clostridium acetobutylicum, FeFe hydrogenase, Glucose-6-phosphate dehydrogenase

426


P0509 Polystylene Nanoplastics Detection Based on Localized Surface Plasmon Resonance with Specific Binding Peptide

Seungju OH, Hyeyeon HUR, Jongmin YANG, Dhanashri DESAI, Seongcheol SHIN, Gillhwan KIM, Cholong KIM, Hyun Ho LEE Department of Chemical Engineering, Myongji University, Yongin, Korea Corresponding Author Email : [email protected] Recently, a huge concern has been imposed on hazardous aspects of nanoplastics in terms of environmental issues. The microplastics including nanoplastics as debris are usually smaller than 1 mm in diameter and these microplastics have potentials to bind toxic substances. The toxic microplastics can be concentrated through food chain. Many kinds of sensors for microplastics could have been introduced in response to the environmental hazard posed by the steadily increasing amount of microplastics. In this study, gold (Au) nanoparticles (NPs) based localized surface plasmon resonance (LSPR) system was developed to detect the polystylene(PS) particles in a simple sampling format. Also, the sandwich binding assay that was developed to bind to microplastics peptides increases the sensitivity of detection due to the dual plasmonic effects. The proposed LSPR system will be a novel protocol microplastic detection tool. Keywords : microplastics, polystylene particles, localized surface plasmon resonance(LSPR), gold nanoparticles References 1. Jung, H., Jung, J., Kim, Y. et al, Surface Plasmon Resonance Characteristics of Au Nanoparticles Layered

Sensor Chip for Direct Detection of Stress Hormone Conjugated by Nanoparticles, BioChip J, 12, 249-256 (2018).

427


P0510 Lysine-cyclodipeptide-based Polyamidoamine Microparticles: Balance Between the Efficiency of Copper Ion Removal and Degradation in Water

Surim YOO1, Sungbin JU2, Dong Soo HWANG1,2 1Divisioin of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Toxic heavy metal contamination in an aquatic environment is a major social health issue that causes poisoning and disease. Micro-hydrogel particles consisting of hyperbranched poly-amidoamine (HPAM) has demonstrated as removing ability of heavy metal ions owing to their high absorbance activity, yet vulnerable to hydrolysis which leads to re-polluting by Cu2+ ions release. In this study, we copolymerize lysine diketopiperazine (L-DKP) with EDA and N,N′-methylenebisacrylamide (MBA) via a Michael addition reaction-mediated inverse suspension polymerization to obtain highly efficient Cu2+ absorbing materials with controlled degradation. At optimal L-DKP content, the microparticle exhibits the performance of 159 Cu2+ mg/g but with higher degradation resistance, as only 38 wt% was lost. In contrast to HPAM microparticles and synthetic microbeads such as polystyrene, Cu2+ ions were significantly retained within the working time as well as decomposed within six months. These results provide an industrial, environment-friendly, and long-lasting absorbent for water purification. Keywords : Water purification, Heavy metal ion removal, Microparticle, Polyamidoamine, Diketopiperazine, Degradation References 1. Ju S., Eom Y., Kim S. Y., Hwang S. Y., Hwang D. S., Oh D.X., Park J., Lysine-cyclodipeptide-based

polyamidoamine microparticles: Balance between the efficiency of copper ion removal and degradation in water, Chem. Eng., 123493 (2019).

428


P0511 Fe(III)-doped Activated Biochar Sorbents Triggers Mitochondria Dysfunction with Oxidative Stress in Daphnia magna

Songhee LEE1, Eunhea JHO2, Sooim SHIN1 1Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju, Korea, 2Department of Agricultural and Biological Chemistry, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Sorbent using magnetite(Fe3O4) particles in the form of cartridges is frequently used material for water deconta-mination. However, particles can be released from the cartridge into the environment, so the sorbents can be another source of contaminants. For check the hazard of magnetite sorbent, bamboo-derived biochars and magnetite nanoparticles were prepared and used to synthesis of Fe(III)-doped activated biochar. In this study, we investigated the potential effects of magnetite sorbent-exposure on oxidative stress in Daphnia magna. Level of ROS/RNS, and amount of GSH, activity of SOD and mitochondrial complex I to IV were measured in D. magna raised with different concentrations of Fe(III)-doped sorbents. As a result, groups treated at relatively low/medium concentration of sorbent show significantly increased level of ROS/RNS than control. Moreover, activity/level of antioxidant markers were significantly reduced upon low dose of sorbent exposure. Also, activity of complex III in mitochondria exhibited markedly decreased in concentration dependent manner. It demonstrates that sorbent paralyzes the antioxidant system and also affects the function of mitochondria of D. magna. Keywords : Oxidative stress, Environmental assessment, Daphnia magna, Synthesized sorbents, magnetite

429


P0512 The Effect of POPs on Mitochondria in Zebrafish Organs After the Decomposition of Fat Induced by Caloric Restriction

Dongshin YANG1, Hwayeon LIM2, Sooim SHIN1,2 1Department of Biotechnology and Bioengineering, College of Engineering, Chonnam National University, Gwangju, Korea, 2Interdisciplinary Program of Bioenergy and Biomaterials Graduate School, College of Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Persistent organic pollutants (POPs) are lipid-soluble environmental toxins and accumulated in the human body, especially in adipose tissue. The main outcomes of POPs exposure are metabolic diseases caused by mitochondrial dysfunction. Although the characteristics of POPs deposited in adipose tissue are already known, the effects of the released POPs by the decomposition of fat in adipose tissue on other organs are barely understood. To this end, zebrafish are exposed to POPs within four conditions; control (C), obesity control (OC), POPs exposure with obesity (OP), and caloric restriction after POPs exposure with obesity (OPR). From their relevance with mitochondrial dysfunction, mitochondrial protein amount and the respiratory complex activity was measured in liver, eyes, and brain expected to be main target organs affected by releasing POPs from adipose tissue. The results indicated OPR group significantly enhanced the activity of complex I compared to other groups in every organ, whereas the activity of complex III was inhibited in comparison with the OP group in the liver and eyes. It demonstrated that POPs released from adipose tissue give the discrepancy depending on organs. Keywords : mitochondrial dysfunction, zebrafish, POPs, caloric restriction

430


P0513 Production of Biologically Active Substances from Waste Shrimp Feed as a Sustainable Resource

Se Young JUNG, Joong Kyun KIM Department of Biotechnology, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] Shrimp is one of favorite seafoods, and thus, its production from farms yearly increases according to the increase in the demand for consumption. The feed for shrimp is not completely ingested by the shrimp, resulting in the accumulation of waste feed at the bottom of farms. The accumulated waste causes not only water pollution, but also the outbreak of diseases detrimental to shrimp. Therefore, efficient treatment of the waste feed is necessary. As a means for the reutilization of the waste feed, we tried to obtain biologically active substances from it. To complete this purpose, shrimp-feed powder (<38 μm) was degraded at 45 for 72 h by the mixed microorganisms isolated from rockworms, 72-h culture supernatant was fractionated by molecular weight, and each fraction was further purified by size exclusion chromatography (SEC). Two times SEC revealed that the target fraction (<1 kDa) had antimicrobial activity. As a result, the waste feed could be a sustainable resource for the production of biologically active substances. Keywords : waste shrimp feed, biodegradation, biologically active substances, size exclusion chromatography References 1. Ja Young Cho, Hyun Yi Jung, Joong Kyun Kim, 2019. Biodegraded mackerel wastewater selectively

inhibits harmful algal blooms. Journal of Hazardous Materials, 364, 349-355. 2. Ja Young Cho, Joong Kyun Kim, 2018. Isolation and identification of a novel algicidal peptide from

mackerel muscle hydrolysate. Journal of Chromatography B, 1093-1094, 39-46. 3. Hyunyi Jung, Jayoung Cho, Changhoon Kim, Joong Kyun Kim, 2016. Biodegradation ability of

microorganisms screened from rockworm (Marphysa sanguine) viscera for purification of water in shrimp farm. 한국생물공학회 학술대회, 337-337.

431


P0514 Investigation of Bio-hydrogen Production Using Hydrolysate of Rice Straw Saccharified Under Various Catalyst Conditions

Do-Hyung KIM1, In Seung JO1, Beom Jung KANG1, Byung Hwan UM2, Kyeong Keun OH3, Jun Seok KIM4, Jeong-jun YOON1 1Green and Sustainable Materials R&D Group, Korea Institute of Industrial Technology (KITECH), Cheonan, Korea, 2School of Food Biotechnology and Chemical Engineering, Hankyong National University, Anseong, Korea, 3R&D Center, SugarEn Co., Yongin, Korea, 4Department of Chemical Engineering, Kyonggi University, Suwon, Korea Corresponding Author Email : [email protected] Rice is one of main crops in korea, and about 7 million tons of rice straw are produced annually. Carbohydrate content in rice straw is about 64%. Its hydrolysate refined through extraction process can be used as a substrate for bio-hydrogen production by anaerobic fermentation. In this study, soluble carbohydrate in rice straw was extracted under several catalytic conditions with acid and alkaline. And it used as a substrate to produce bio-hydrogen.The optimal condition of hydrogen production using rice straw hydrolysate as a substrate was hydrolysate saccharified under 1% H2SO4 condition. The amount and yield of bio-hydrogen production at this condition were 0.08 mol/L and 1.14 mol-hydrogen/mol-consumed sugar, respectively. On the other hand, hydrolysate saccharified under NaOH catalytic condition was shown relatively low productivity. However, it was confirmed that hydrogen-producing bacteria consumed large amounts of soluble polysaccharides in the hydrolysate. This means that rice straw can be used as a substrate in bio-hydrogen production. Keywords : Anaerobic fermentation, Bio-hydrogen, Biomass, Rice straw, Saccharification

432


P0515 Separation of Microplastic Particles Using Iron Oxide Magnetic Nanoparticle in Wastewater

Yejin HEO1, Heeho AHN1, Cheol KANG1, Byeong-Gwuan SEO1, JangHeon CHOI1, Eun-Hee LEE2, Seung-Woo LEE1 1Department of Fine Chemistry, Seoul National University of Science and Technology (SEOULTECH), Seoul, Korea, 2Department of Microbiology, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] Microplastics(MPs) have been a significant concern because humans and wild animals can ingest MPs present throughout the aquatic ecosystem. Despite the adverse health effects caused by MP, such as damaging cells, inducing inflammatory and immune reactions, the practical removing procedure of the MPs has not been established yet. Bulk plastic does fragment into smaller pieces when exposed to ultraviolet (UV) radiation, photooxidation, mechanical forces, and other degradation processes. Such fragmentation has resulted in the spread of nano- and micro-plastic worldwide, and this process is not environmentally sustainable. As the pollutant particles' size decreases in water treatment applications, it gets more challenging to remove the MPs in the aquatic system. Also, it takes a lot of time and cost to remove from the marine system. Iron oxide magnetic nanoparticles(IOPs) are widely used as sorbents of pollutants in water and wastewater treatment. The unique magnetic property introduces aggregation of the IOPs along adsorb contaminants present in wastewater. In this study, we conducted magnetic separation by adsorbing various sizes of < 1 μm MPs. Adsorption proceeded rapidly within 1 minute and showed a more than 95 % removal rate with an adsorption capacity of 11 mg/g. After the adsorption process, desorption was also performed by applying external energy showing the recycling of MPs was possible. Keywords : Microplastics(MPs), Ironoxide magnetic nanoparticles(IOPs), Magnetic separation References 1. J. Grbic, B. Nguyen, E. Guo, J. B. You, D. Sinton and C. M. Rochman, Environ. Sci. Technol. Lett. 6(2),

68-72 (2019). 2. R. Narvaez, Oscar M., Goonetilleke, Ashantha, Perez, Leonidas, & Bandala, Erick R. Chemical Engineering

Journal. 414, 128692 (2021).

433


P0516 Degradation of Polyethylene Terephthalate (PET) Through Polyethylene Terephthalate-degrading Enzyme (PETase)

Sumin LEE1, Yejin WON2, Hongryul KIM1, Jaewook MYUNG1 1Dept. of Civil and Environmental Engineering, KAIST, Daejeon, Korea, 2Dept. of Systems Biotechnology, Chung-Ang University, Anseong, Korea Corresponding Author Email : [email protected] For many years, plastic recycling rate has been very low because the cost for plastic recycling cost is higher than for new production. As a result, plastic waste is accumulating at a staggering rate, causing various environ-mental pollution. After the discovery of polyethylene terephthalate (PET)-hydrolytic emzyme, biotechnological strategy to recycle PET has gained interests among stakeholders. Ideonella sakaiensis, a newly discovered bacterium in 2016, has a PET degrading enzyme called IsPETase. In this study, IsPETase has been used to degrade PET for 24 h in aerobic conditions. Each sample with different concentrations of IsPETase (20, 40, 80, and 100 mg/ml) was collected every 12 h and its compositions were analyzed via HPLC. The result indicates that PET was decomposed into terephthalic acid (TPA), which is a monomer in PET synthesis and has a potential to be upcycled into biodegradable plastics. Keywords : PET, recycling, enzyme, TPA

434


P0517 Effect of a Keystone Bacterium, Roseobacter sp., on the Growth of Alexandrium in the Algal-bacterial Community

Chang Soo LEE, Mirye PARK, Kiwoon BAEK, Ahyoung CHOI Nakdonggang National Institute of Biological Resources, Sangju, Korea Corresponding Author Email : [email protected] Alexandrium is one of major agents to cause marine harmful algal blooms and incur issues in public health such as paralytic shellfish poisoning. In this study, we have investigated the effect of keystone bacteria, Roseobacter, on the growth of Alexandrium with an optical microscope and a flow cytometer. After the co-culture with Roseobacter for 8 days, the growth of Alexandrium was promoted and the cell motility of Alexandrium was increased. A significant difference of cell concentration of Alexandrium was found between the co-culture with Roseobacter and contorol group (P-value 0.048). Further analysis using a flow cytometer showed that the change of the algal-bacterial community between the co-culture with Roseobacter and control group. When living cells were observed by fluorescein diacetate staining, the number of living Alexandrium cells was increased when the Roseobacter was added. The result indicated that Roseobacter could influence on the harmful algal growth of Alexandrium by the microbial interaction between microalgae and bacteria. Keywords : Roseobacter, Alexandrium, co-culture, microalgae, bacteria

435


P0518 Simulating Active Sludge Process Using STOAT Simulator

Daechul CHO1, Taewook KIM1, Sunjoo CHO1, Jongki JEON2 1Department of Environmental Engineering, Soonchunhyang University, Asan, Korea, 2Department of Chemical Engineering, Kongju National University, Kongju, Korea Corresponding Author Email : [email protected] Due to population growth and expansion of industrial diversity, the amount of sewage to be treated has been increasing. The sewage treatment plant shall be treated with appropriate practices in line with these changes. However, any flexibles change in the process of the sewage treatment plant in operation can sometimes result in inappropriate sewage treatment. Pilot-scale experiments are very costly because of setup of facilities and manpowered operations. Also, it may be difficult to derive accurate experimental values because reproducibility of water treatment and external conditions cannot be guaranteed. Such problems can be solved using an appropriate sewage treatment simulator. A virtual sewage treatment plant is proposed using STOAT, a free sewage treatment simulator in this study, some key problems were evaluated through many trial experiments in computer. The results of a virtual experiment showed that 240 mg/l of inflow BOD was reduced to 4.11 mg/l on average. we are very certain that the STOAT simulations can provide with valuable predictions in the tested process, an activated sludge process. Keywords : STOAT, WWTP, Activated sludge, Simulation References 1. Gao YN, Liu XZ, Zhang RX, Shan JJ, Sun MQ, Zong ZX, et al. Operation and management of Liaoning

waste water treatment plants by STOAT Simulation, 04019 (2016).

436


P0519 Isolation and Characterization of Carbofuran-degrading Soil Bacteria

Haeseong PARK, Sun Il SEO, Ji-hwan LIM, Gui Hwan HAN, Pyoung Il KIM Center for Industrialization of Agricultural and Livestock Microorganism, Jeongeup, Korea Corresponding Author Email : [email protected] Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranoyl-Nmethylcarbamate) is a carbamate-based chemical pesticide that has contributed greatly to the improvement of agricultural productivity for a long time. However, excessive use and improper management of carbofuran are accelerating soil and water pollution. Recently, bioremediation of polluted farmland has been successfully achieved by means of microbiological approaches. In this study, we isolated three bacterial strains (GHC2-1, GHC2-2 and GHC2-5) which able to degrade carbofuran pesticide-contaminated soils. 16S rDNA analysis revealed that GHC2-1, GHC2-2 and GHC2-5 strains were members of Bacillus, Pseudomonas, and Achromobacter, respectively. Their carbofuran degrading activity were evaluated in liquid culture using different carbofuran concentrations. All three strains degraded carbofuran over 97%. Especially, the GHC2-2 strain degraded 99% of carbofuran in R2A medium with 100 mg L-1 concentration within 5 days. Considering the enhanced carbofuran degrading properties of three strains it is obvious that they will be effectively used for biological remediation of contaminated soil. Keywords : Carbofuran, Enrichment culture, Pesticide-residue degradation

437


P0520 Plant Root Hairs Can Contribute for Soil Holding

Hyung-Taeg CHO, Heeseung CHOI Department of Biological Sciences, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Root hairs form a substantial portion of the root surface area. Compared with their nutritional function, the physical function of root hairs has been poorly characterised. This study investigates the physical role of root hairs of Arabidopsis thaliana seedlings in facilitating the interaction of the root with water and soil and in plant survival upon soil disruption. Five transgenic lines with different root hair lengths were used to assess the physical function of root hairs. Upon soil disruption by water falling from a height (mimicking rainfall), long-haired lines showed much higher survival rates than short-haired lines. The root-pulling test revealed that a greater amount of soil adhered to long-haired roots than to short-haired roots. When seedlings were pulled out and laid on the soil surface, survival rates of long-haired seedlings were higher than those of short-haired seedlings. Moreover, the water holding capacity of roots was much greater among long-haired seedlings than short-haired seedlings. These results suggest that engineering root hairs can contribute for soil protection from erosion and seedling survival upon soil disruption. Keywords : root hair, soil, soil holding, soil erosion

438


P0521 Growth Medium-dependent Algicidal Activity of Strain S8 Against a Harmful Algal Bloom, Microcystis aeruginosa

Ja Young CHO, Taehui YANG, Eui-jin KIM, Chang Soo LEE Microbial Research Department, Nakdonggang National Institute of Biological Resources, Sangju, Korea Corresponding Author Email : [email protected] The cyanotoxins produced by cyanobacteria cause a serious threat to aquatic animals and human health. Algicidal bacteria have been reported as one of the biological treatments to control cyanobacterial harmful algal blooms(CyanoHABs). Previously, we isolated and identified bacterium S8 that reduces chlorophyll-a levels and accumulates reactive oxygen species against M. aeruginosa. In addition, the algal cell viability was reduced by bacterial treatment. The metabolites of microorganisms can vary depending on the culture conditions, such as nutrition and temperature. Consequently, various culture supernatant was treated with harmful algae in order to improve the algal activity. The medium used in the experiment were nutrient broth, reasoner's 2A broth, marine broth 2216 and tryptic soy broth. When bacterium S8 was cultured in tryptic soy broth, the algicidal activity was highest. We used different concentrations of glucose and phosphate based on tryptic soy broth. For high algicidal activity, a concentration lower than 2 g/l of glucose and 8 g/l of tryptone were required. The results can be used to study algicidal activity using bacterium S8. It is necessary to cultivate the microorganisms under more culture conditions for large-scale and field applications. ** This work was supported by a grant from the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment(MOE) of the Republic of Korea (NNIBR202102102). Keywords : Algicidal bacteria, Harmful algal blooms, Cyanobacteria, Biological treatment

439


P0522 Fibrobacterium geumgangensis JA-25 Gen. Nov., Isolated from Freshwater

Sanghwa PARK1, Ja Young CHO1, Dong-Hyun JUNG1, Seok Won JANG1, Jung Hye EOM1, Seung Won NAM2, Dae Ryul KWON2 1Bacteria Research Team. Freshwater Bacteria Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju, Korea, 2Protist Research Team. Freshwater Bacteria Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju, Korea Corresponding Author Email : [email protected] Gram-stain negative, pink colored and designated strain JA-25T was isolated from sediment of freshwater geumgangensis river, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain JA-25T belongs to the Family Cytophagaceae. Strain JA-25T was closely related to Fibrella aestuarina DSM 22563T(93.6%), Spirosoma migulaei KCTC 52028T(89.4%) and showed less than 90% sequence similarity to other members of the Family Cytophagaceae. The average nucleotide identity values of strain JA-25T with F. aestuarina DSM 22563T was 73.6% and in silico DNA-DNA hybridization values was 20.1 %. The novel strain grew over a temperature range of 15~30°C, at pH values of 6.0-9.0 and in the presence of 0-1%(w/v) NaCl. The major cellular fatty acids were Summed feature 3 (C16:1ω6c/C16:1ω7c), C16:1ω5c, C16:0(>10%). The genomic DNA G+C content was 52.5 mol%. On the basis of the phylogenetic inference, phenotypic and chemotaxonomic data, strain JA-25T should be classified as a novel species of the novel genus Fibrobacterium for which the name Fibrobacterium geumgangensis sp. nov. is proposed. Keywords : Freshwater, Fibrobacterium geumgangensis , Cytophagaceae References 1. M. Filippini , M. Svercel, E. Laczko, A. Kaech , U. Ziegler, H. C. Bagheri,. 61. 184 (2011).

440


P0523 Self-Optimizing Breeding Control of Tenebrio molitor Larvae Based on a NIR Monitoring System

Tobias BECK1, Andreas BAUR1, Bernhard GATTERNIG1,2,3, Antonio DELGADO1,2 1Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Fluid Mechanics, Erlangen, Germany, 2LSTME, German Engineering Research and Development Center, Busan, Korea, 3Weihenstephan-Triesdorf, University of Applied Sciences, Chair of Process Engineering, Merkendorf, Germany Corresponding Author Email : [email protected] The rising population and the increasing demand on food supply and feeding stuff in agriculture trends to search on new sources for ingredients and products. Also, a trending aspect is a more critical view on the climate combability of the product especially for the carbon dioxide and the water footprint during production. A promising field of research is the mast of Tenebrio Molitor also known as mealworm as source for nutritive substance. There are new challenges to applicate existing machine technology and plants and methods in food industry to this new value chain and conditions. Current states of technology provide a wide field of processing technologies for insect-based products. The chemometric analysis of the product composition is currently done offline. These methods are expensive, and they also slow down the process as well. In this project a new method is developed to increase the mast efficiency based non-invasive online monitoring of the breeding progress. The main approach is to combine established measuring techniques in food applications with self-optimizing automation algorithms. The self-optimizing algorithm controls the feeding of the breed and the water supply. During process, the algorithm deals with variation in the insect growth by flexible controlling the addition of food and water to the breed. The developed techniques will be applied in an experimental model for industry-orientated insect breeding. Keywords : Tenebrio Molitor, near-infrared measuring, self-optimizing controlling

441


P0524 Evalution of Environmental Pollution and Biodiversity According to Burning(Korean Traditional Knowledge) of Agriculture Fields, Korea

Minjae KONG, Sungwook JEON, Jinkwan SON, Kwangho KIM National Institute of Agricultural Sciences, RDA, Jeonju, Korea Corresponding Author Email : [email protected] In the past, when there was no means for controlling rice pests, the traditional means of controlling rice pests was burning the rice fields before and after the Lunar New Year. It fell a lot, and the negative effect of burning the rice paddy, such as the disruption of the agro-ecosystem biota, the generation of fine dust, and the cause of the forest fire, is much greater than the positive effect of burning the rice paddy (Garren, 1943; Clement and Touffet, 1981; Kim, 2002) In recent years, the practice of burning rice paddies is prohibited by law. Therefore, in this study, we investigated the changes in arthropods occurring in agricultural land after incineration of rice paddies, and evaluated the effects of incineration on the biodiversity of agroecosystem. Arthropods were investigated before and after incineration of rice paddies and rice paddies in winter to investigate pests, natural enemies, and non-worms. The amount of pest incineration and non-incineration was analyzed. As a result of the density analysis of winter arthropods, the density of overwinthering arthropod was useful insects(80∼97%) > insect pests (5∼17%) > the other arthropod (2∼3%) in order. Keywords : Burning of rice paddy field levee, Arthropod, Biodiversity, Arthorpod density References 1. B. Clement and J. Touffet, Vegetation dynamics in Brittany heathlands after fire, Vegetation, 46, 157-166

(1981). 2. K. H. Garren, Destruction of a fire and its effects in the Nylsvley, Nature Reserves, Sourth African National

Science Report Series, 62, 1-39 (1982). 3. H. S. Kim, Broad-casting Scenario, KTV (2002).

442


P0525 Manufacture of Microalgal Biofilm Using Fluid Flow in Conduit

Dae Geun KIM1, Bo Young KIM2, Dong Eun SHIN2, Hui Ju KIM2 1LED Agri-bio Fusion Technology Research Center, Jeonbuk National University, Iksan, Korea, 2College of Environmental & Bioresource Sciences, Jeonbuk National University, Iksan, Korea Corresponding Author Email : [email protected] Microalgae have the ability to remove nitrogen and phosphorus from the water and produce oxygen through photosynthesis, and are attracting attention for the purpose of water purification in recent years. However, it is difficult to use microalgae for the purpose of purifying water because microalgae are suspended and grown in water as cells of very small size. Biofilm refers to a community in which microorganisms are mixed and has a specific shape, and it is expected to be directly applicable to water purification because it has a specific shape through mixing of microalgae and microorganisms. [1]. Laminar flow is a phenomenon that occurs when water passes through a conduit, and is a phenomenon in which water exerts a physical force on the inner wall of the conduit. In this study, a method of artificially manufacturing a biofilm through laminar flow was developed. In previous studies, it is known that the type of microalgae has the most influence on biofilm formation. However, our research shows that the developed device can induce biofilms even with microalgae such as Haematococcus sp, which generally do not adhere well through changes in flow rate, nutrients, temperature, and light. As a result of the study, it was found that most algae form a biofilm within 10 days, and through these results, it is expected that biofilm induction of desired microalgae will be possible. Keywords : Biofilm, Microalgae, Wastwater treatment References 1. Y. Su, A. Mennerich, B. Urban, Bioresour. Technol., 218, 1249-1252 (2016).

443


P0526 Aluminum Recovery from Water Treatment Sludge by Leaching as a Coagulant for Phosphorus Removal

Tuan Van TRUONG, Heonki KIM, Dong-Jin KIM Department of Environmental Sciences and Biotechnology, Hallym University, Chuncheon, Korea Corresponding Author Email : [email protected] Dried (105C) and calcined (700C) Al sludge (AS) were leached by acid and alkali for Al recovery and used for phosphorus removal as a coagulant. Calcined AS had higher Al and lower COD content than the dried AS. Both acid and alkali leaching recovered more than 90% of the Al from the AS. Response surface method (RSM) of Box−Behnken Design (BBD) was applied to find optimal condition for phosphorus removal with the Al containing leachate. It showed that the leached Al solutions performed the highest phosphorus removal efficiency at pH range of 4 - 6.4 and Al/P molar ratio higher than 2. Phosphorus removal efficiency was more than 98% at optimal condition for all the Al leachate. FTIR and XRD analyses confirmed that the recovered precipitate contained AlPO4 and Al(OH)3. Keywords : Al recovery, calcination, phosphorus removal, water treatment sludge

444


P0527 Climate-based Spatial Prediction of Vespa velutina nigrithorax Distribution in South Korea by Using Ensemble Mapping

Se-hyun KIM1, Dong Eon KIM2, Heejo LEE2, Sunghoon JUNG3, Wang-Hee LEE1,4 1Department of Smart Agriculture Systems, Chungnam National University, Daejeon, Korea, 2Invasive Alien Species Research Team, Division of Ecological Safety, Bureau of Survey and Safety, National Institute of Ecology, Seocheon, Korea, 3Department of Applied Biology, Chungnam National University, Daejeon, Korea, 4Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Vespa velutina nigrithorax is an invasive wasp which has consistently caused problems by threatening local biodiversity. According to recent reports, V. v. nigrithorax is continuously expanding its territory, necessitating the use of species distribution model based on the most recent distribution data for early monitoring of it. In this study, potential areas of V. v. nigrithorax were evaluated for the world and South Korea where suffered from its invasion. The recent distribution data was obtained by cross-checking the public species database, and previous studies. In addition, field survey in South Korea obtained from National Institute of Ecology was included. With the updated distribution data, we developed CLIMEX and MaxEnt models as a function of climatic variables; then, they were incorporated to produce an ensemble map that could enhance the prediction reliability. As a result, temperate and subtropical regions were to be potential habitats, including western Europe, eastern USA, Far East, and several areas in Australia and South America. In particular, South Korea showed high occurrence possibility all over the country, suggesting intensive monitoring would be necessary. Keywords : CLIMEX, ensemble mapping, Maxent, potential habitat, yellow-legged hornet

445


P0528 Pandemic Paradox: A Message for Sustainability of Language and Life

Soo-Yeon KIM1, Hye-Won CHOI2, Duk Jae OH3 1Department of English Language and Literature, Sejong University, Seoul, Korea, 2Department of English Language and Literature, Ewha Womans University, Seoul, Korea, 3Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] Languages and living entities have been argued to share striking resemblance (Kim and Oh 2017). According to generative grammarians, human languages consist of principles that are common to all the languages in the world and parameters, a choice of which determines an individual language; Living entities, likewise, share the genetic information in a cell by means of the biological principle such as the central dogma among species and a choice of parameters differentiates species. Just as information transfer or communication by means of language has been a critical tool for survival of humans, the transfer of genetic information is inevitable for the sustainability of any living species. The COVID-19 pandemic, known to be caused by a zoonotic transfer of the virus from Malayan pangolins (Manis javanica) and/or Rhinolophus affinis bats to humans, can be an example of an unwarranted genetic transfer across species boundaries. Apparently, a radical or forced transfer beyond the species boundary may threat the very existence of the species. In parallel, any attempts of forced transfer between languages as a means of subjugation of one culture over another can be dubbed a “cultural pandemic,” which threatens the cultural identity of the people in hand. It often was destructive to peaceful coexistence between groups of people, as the Japanese colonial policy of liquidating the Korean language clearly showed. Paradoxically though, the “negative” zoonotic transfer can end up enhancing humans’ immunity to the virus and thus, eventually strengthening their tolerance toward external threats. Not surprisingly, human history has also witnessed that transnational, transracial, and transcultural transfers across languages have enriched human thoughts and ideas, contributing to the development of human society. As such, a right balance in boundary-crossing communication, i.e., positive transfers between species and between languages, without infringing upon the uniqueness of the other, is the key to harmonic symbiosis of different individuals, groups of people, and species, and ultimately to the sustainability of societies as well as all living entities. This is the message the viral and cultural pandemics equally teach human beings and the humanity for their sustainability. Keywords : Principle, Parameter, Transfer, Symbiosis, Communication, Pandemic, Identity, Sustainability References 1. Kar, S. and J. Leszczynski. From Animal to Human: Interspecies Analysis Provides a Novel Way of

Ascertaining and Fighting COVID-19, Cell Press 1(2): 1-2 (2020). 2. Kim, S. et al. Multi-disciplinary Consideration for Sustainability of Living Entities and Their Societies.

Proceeding of the 7th International Science Conference 92, Katmandu University, World Science Congress (2017).

3. Kim, S. and D. Oh. A Linguistic Approach to Communication Strategies of Biological Systems. KSBB Journal 32(1): 29-34 (2017).

4. Kim, S. et al. Biological Features in Human Language. Proceedings of AFOB 2020 Regional Symposium, Universitas Gadjah Mada (2020).

5. Searls, D. The Language of Genes. Nature 420: 211-217 (2002).

446


P0529 The Effect of Bio Curtain on the Mitigation of Odorous Gases from Swine Barn

Jung Kon KIM, Kyeong-seok KWON, Jong-Bok KIM, Dong-Jun LEE, Ka-Young YANG, Dong-Hwa JANG Animal Environment Division, National Institute of Animal Science, Rural Development Administration, Wanju, Korea Corresponding Author Email : [email protected] As the production of livestock rise, the concentration of odor emitted from livestock farms has significantly increased. Due to this, complaints about livestock odor have increased considerably, and it has become a social problem. The bio curtain is practiced on many swine farms in the South Korea, mainly for mitigating air quality around the swine farm. The bio curtain, is a commercially available adaptation of a windbreak wall, is designed to cover the fans on a tunnel ventilated swine barn. And it helps livestock producers with dust and odor emission control by using exhaust fans to blow the contaminated air straight up and out of the bio curtain structure. In This study, we present the evaluation of a bio curtain on the mitigation of odorous gases from pig house. The ammonia(NH3) and Hydrogen sulfide(H2S) removal rates of swine barn with bio curtain were 6.5 times and 2.1 times higher than that of swine barn without bio curtain, respectively. Keywords : Livestock odor, bio curtain, ammonia, hydrogen sulfide References 1. J.Wi, S. Lee, E. Kim, M. Lee, J. A. Koziel and H. Ahn, Atmosphere, 10(4), 170(2019). 2. Y. Jang, M. W. Jung, BioMed Research International, 2018(7), 1-7(2018).

447


P0530 Optimization of Experimental Conditions Using Bayesian Optimization

SeoJoung PARK, Donghwa SEO, Donghyuk KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected], [email protected] Bayesian Optimization(BO) is an optimal solution determination algorithm that has the maximum or minimum value of unknown objective function f(x) with undefined shape. It can be used efficiently in the experimental design process such as optimize external conditions. In the situations that experimental conditions with unclear relationships, BO enables effective design by quickly and economically with as few experiments as possible.In this study, we applied BO to optimize the composition of Li battery material using python. For this, LiMnO data was received from Material Projects and optimized the bandgap and density that were most relevant to the performance. It is probabilistically estimated using Gaussian Process and the next experiment point is suggested by Acquisition Function. As a result, the optimal solution was obtained by updating the function values to model. Based on these attempts, it is being applied to find the optimal culture conditions for bioreactors. Also, this study will further promote to bio-energy fields such as synthesis of microbial electrodes. Keywords : Machine Learning, Deep Learning, Optimization, Experimental Condition Control, Automation

448


P0531 The Fungi and Bacteria Assessment for the Drainage Water Reuse of Protected Horticulture Facility, Korea

Jinkwan SON, Taekyung KANG, Minjung PARK, Ryukab LIM, Minjae KONG National Institute of Agricultural Sciences, RDA, Jeonju, Korea Corresponding Author Email : [email protected] Korea's protected horticulture is speedy increase through many automation technologies. One of the green-house technologies, hydroponics cultivation is an easy way to directly inject nutrients into plants. The nutrient solution supplied accordingly is administered up to 20~30% more than the amount required by the plant. This is called drainage water (waste water), and is discharged outside the greenhouse in a state of high concentration N and P. It is negative for the environment(Son et al., 2019), therefore must be recycled. However, if it contains harmful fungi and bacteria, it will need sterilization(Naidu et al., 2019). The concentration of fungi and bacteria detected in tomato, paprika, and strawberry extract was analyzed. These results can be used to determine the scope of sterilization.: 1. The total of 36 total of 12 sites of tomato, paprika, and strawberry were analyzed for the wastewater

discharged from horticulture. 2. The fungi detection of paprika 12 sites analyzed 7families 7gunus 16species. The sum of the fungi detection

concentrations is 160. Bacteria detected 2families 2gunus 2species with a concentration of 38. 3. The fungi detection of Tomato 12 sites analyzed 6families 6gunus 9species. The sum of the fungi detection

concentrations is 36. Bacteria detected 3families 3gunus 5species with a concentration of 36. 4. Fungi detection of Strawberry 12 sites analyzed 8families 8gunus 16species. The sum of the fungi detection

concentrations is 203. Bacteria detected 2families 2gunus 5species with a concentration of 76. 5. In general, the concentration of fungi and bacteria detected was analyzed in the order of Strawberry > Paprika

> Tomato. Major detection species were analyzed in Fusarium, Pseudomonas, and Pythium. These species need to be sterilization to reuse the drain water. This research can be used for water pollution prevention, water conservation, climate change, etc.

** Acknowledge : This study was supported by the 2021 RDA Fellowship Program(Project number PJ014190) of National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea. Keywords : Horticulture, Purification, Ecosystem services, Recycle, Sustainable References 1. Son, J., Choi, D., Kong, M., Yun, S., Park, M., Kang, D, The Water Quality and Purification Load

Assessment of Drain Water of Facility Horticulture Areas(2019), J. Environ. Sci., 28(12), 1199-1208. 2. Naidu, G., Ryu, S., Thiruvenkatachari, R., Choi, Y., Jeong, S., Vigneswaran, S, A critical review on

remediation, reuse, and resource recovery from acid mine drainage(2019), Environ. Pollut., 247, 1110-1124.

449


P0532 Predicting Occurrence Possibility of Haemaphysalis longicornis by Using MaxEnt Model

Sunhee YOON1, Jae-Min JUNG2, Se-Hyeon KIM1, Sunghoon JUNG1,3, Wang-Hee LEE1,2 1Department of Smart Agriculture Systems, Chungnam National University, Daejeon, Korea, 2Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Korea, 3Department of Applied Biology, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Asian longhorned tick (ALT), Haemaphysalis longicornis, is known as the vector of Severe Fever with Thrombocytopenia Syndrome (SFTS). Currently, it has been widely distributed in South Korea, suggesting that early spatial evaluation is necessary for setting up control strategy. In this study, we used MaxEnt which predicted occurrence possibility of a species based on presence‐only species records, and total of 19 bioclimatic variables were inserted for training and testing the model. As a result, we could obtain major variables which affected the distribution of ALT and develop a map coding the regional occurrence possibility in South Korea. To enhance the model reliability by reducing multicollinearity among the bioclimatic variables, it is necessary to select the bioclimatic variables without inter-variable correlation based on the statistical analysis on the variables. Through this work, we expect to provide an initial insight for developing highly accurate species distribution model which is practically useful for monitoring and controlling the ALT. Keywords : bioclimatic variables, Haemaphysalis longicornis, MaxEnt, occurrence possibility, species distribution model

450


P0533 Competitive Evaluation of Coal-fired Flue Gas Applied Wild Type and Mutant Strains of Microalgae

Young Joon SUNG1, Jeong Seop LEE1, Hong-Ki YOON1, Hyunjin KO2, Sang Jun SIM1 1Department of Chemical and Biological Engineering, Korea University, Seoul, Korea, 2Korea Western Power Co., LTD. Corresponding Author Email : [email protected] Microalgae is a sort of algae that intake carbon dioxide from organic or inorganic carbon source and produce various useful substances. Especially, green microalgae strain have high conversion efficiency of carbon dioxide and they produce lipids by specific stresses since its metabolic behavior. In this study, three kinds of environmental adapted microalgae strains were cultivated by direct application of coal-fired flue gas that represented its high concentration of carbon dioxide. The cultivation sits is located in Taean branch of Korea Western Power Co., LTD.(Taean, Chungcheongnam-do,South Korea, 36.9038446,126.2324481). The micro-algal biomass productivity of the species microalgal wild strains and the selected mutant strains derived therefrom were evaluated, and the strains cultured under the same conditions at the same time were evaluated for competition. In addition, a scale-up test of a large scale culture process of a single 1 ton scale was also conducted. The maximum biomass production was the highest at 0.703 g L-1 day-1 in Neochloris oleoabundans UTEX 1185 mutant 13, and the lipid contents were 45.70 %, and the calorific productivity for direct combustion was 3.553 kJ L-1 day-1. In other words, satisfactory results indicating the possibility of direct combustion of microalgal biomass appeared, and incidentally, the effect of immobilizing carbon dioxide of 0.309 g CO2 L-1 day-1 was accompanied. Keywords : microalgae, lipid, coal-fired flue gas References 1. Hong ME, Yu BS, Patel AK, Choi HI, Song S, Sung YJ, et al. Enhanced biomass and lipid production of

Neochloris oleoabundans under high light conditions by anisotropic nature of lightsplitting CaCO3 crystal. Biores Technol. 2019;287:121483.

2. Hong ME, Chang WS, Patel AK, Oh MS, Lee JJ, Sim SJ. Microalgal-based carbon sequestration by converting LNG-fred waste CO2 into red gold astaxanthin: the potential applicability. Energies. 2019; 12(9):1718.

3. Zeng X, Danquah MK, Chen XD, Lu Y. Microalgae bioengineering: from CO2 fxation to biofuel production. Renew Sustain Energy Rev. 2011;15(6):3252-60.

4. Sung YJ, Kim JYH, Bong KW, Sim SJ. Microdroplet photobioreactor for the photoautotrophic culture of microalgal cells. Analyst. 2016;141(3):989-98.

5. Choi YY, Patel AK, Hong ME, Chang WS, Sim SJ. Microalgae bioenergy with carbon capture and storage (BECCS): an emerging sustainable bioprocess for reduced CO2 emission and biofuel production. Bioresour Technol Rep. 2019;7:100270.

451


P0534 Optimization of Alkali Pretreatment to Improve Glucose Recovery from Sicyos angulatus

Kang Hyun LEE1, Seunghee KIM1, Jeongho LEE1, Chulhwan PARK2, Hah Young YOO1 1Department of Biotechnology, Sangmyung University, Seoul, Korea, 2Department of Chemical Engineering, Kwangwoon University, Seoul, Korea Corresponding Author Email : [email protected] Sicyos angulatus is widely spread throughout Asia and Europe, hindering agricultural productivity, and has been designated as an invasive species by the Ministry of Environment in Korea. S. angulatus has the potential to be used as a raw material for biorefinery because it is composed of cellulose, hemicellulose and lignin. The aim of this study is to optimize the NaOH pretreatment of S. angulatus to improve glucose recovery. The chemical compositions of S. angulatus were determined to be 16.4% glucan, 6.2% xylan, 11.8% lignin, 1.1% ash and 64.5% others. The optimum pretreatment conditions were as follows: 2% (w/w) NaOH, 121°C and 10 min. Glucan content (GC) and enzymatic digestibility (ED) of S. angulatus pretreated under the optimum conditions were 46.7% and 55.3%, respectively. Through NaOH pretreatment, GC and ED of S. angulatus were improved by 2.8-fold and 2.5-fold, respectively, compared to the untreated S. angulatus. Keywords : Sicyos angulatus, pretreatment, enzymatic hydrolysis, glucose recovery

452


P0535 Thermochemical Conversion of Defatted Microalgae into Formic Acid

Gwi-Taek JEONG, Sung-Koo KIM Department of Biotechnology, School of Marine and Fisheries Life Science, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] Formic acid (FA) is an important chemical used in diverse industrial applications. Moreover, FA has been considered as a potential eco-friendly medium for H2 storage/transportation. In this work, FA was produced from lipid-extracted residue of Scenedesmus obliquus as the biomass via sulfuric acid-catalyzed thermochemical conversion. 25.82% FA yield was achieved under the condition of 5% biomass, 0.8 M sulfuric acid, at 180 °C for 40 min. The relationship of FA yield and CSF was adequately matched with a non-linear sigmoidal equation. In conclusion, lipid-extracted residue of S. obliquus could be used as a bioresource for the production of valuable bio-based chemicals like FA. Keywords : Scenedesmus obliquus, Formic acid, lipid-extracted residue References 1. GT Jeong and SK Kim, Fuel 283, 118907 (2021).

453


P0536 Production of Levulinic Acid from Defatted Chlorella vulgaris

Gwi-Taek JEONG, Sung-Koo KIM Department of Biotechnology, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] Levulinic acid (LA) is a versatile intermediate that can be converted into a wide range of chemical derivatives for the production of fuels and chemicals. This study carried out the production of LA from the defatted C. vulgaris under HCl-catalyzed thermochemical conversion. The highest 40.72% LA yield was obtained under the conditions of 5% biomass and 0.95 M HCl at 170 °C for 60 min. As a result, the defatted microalge residue represents a potential resource to replace fossil resource. Keywords : C. vulgaris, Levulinic acid, hydrothermal conversion References 1. GT Jeong and Sk Kim, Journal of Environmental Chemical Engineering 9, 105142 (2021).

454


P0537 Influence of Flowrates to a Reverse Electro-dialysis (RED) Stack on Performance and Electrochemistry of a Microbial Reverse Electrodialysis Cell (MRC)

Usman GHANI, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] An MRC is a bioelectrochemical system combining a microbial fuel cell (MFC) with a RED stack to generate electricity from salinity gradient and organic wastewater with simultaneous treatment. Operating an MRC at an optimum flowrate to RED is important because it is closely related with energy production rate and economic feasibility. However, influence of RED flowrates on MRC electrochemistry and power production have not been investigated. For this purpose, four different flowrates of high concentration and low concentration solutions were tested. Maximum power density was highest in 10 mL/min (3.71 W/m2) and optimum current density was highest in 7.5 mL/min (5.36 A/m2). By mere increasing the flowrate to MRC, maximum power and optimum current densities increased by 17.7% and 16.2%. EIS showed that impedances of anode, cathode and full-cell were decreased by 51%, 31% and 19%, respectively. Anode CV showed that peak current density was increased by 25.7%. COD removal and CE were not affected by RED flowrate. Power generation at 7.5 mL/min and 10 mL/min were not so different, but current production was better at 7.5 mL/min. Therefore, considering energy production, the RED flowrate of 7.5 mL/min is a reasonable choice for MRC operation. Keywords : Microbial reverse electrodialysis cell, Reverse electrodialysis stack, RED flowrate, Electro-chemistry, Electrochemical impedance spectroscopy References 1. Post JW, Hamelers HV, Buisman CJ. Energy recovery from controlled mixing salt and fresh water with a

reverse electrodialysis system. Environ Sci Technol 2008;42:5785e90. 2. Ramon GZ, Feinberg BJ, Hoek EM. Membrane-based production of salinity-gradient power. Energy

Environ Sci 2011;4:4423e34.

455


P0538 Improved Structures of Stainless Steel Current Collector Increase Power Generation of Microbial Fuel Cells by Decreasing Cathodic Charge Transfer Impedance

Usman GHANI, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial fuel cell (MFC) is an innovative environmental and energy system that converts organic wastewater into electrical energy. For practical implementation of MFC as a wastewater treatment process, a number of limitations need to be overcome. Improving cathodic performance is one of major challenges, and introduction of a current collector can be an easy and practical solution. In this study, three types of current collectors made of stainless steel (SS) were tested in a single-chamber cubic MFC. The three current collectors had different contact areas to the cathode (P 1.0 cm2; PC 4.3 cm2; PM 6.5 cm2) and increasing the contacting area enhanced the power and current generations and coulombic and energy recoveries by mainly decreasing cathodic charge transfer impedance. Application of the SS mesh to the cathode (PM) improved maximum power density, optimum current density and maximum current density by 8.8%, 3.6% and 6.7%, respectively, comparing with P of no SS mesh. The SS mesh decreased cathodic polarization resistance by up to 16%, and cathodic charge transfer impedance by up to 39%, possibly because the SS mesh enhanced electron transport and oxygen reduction reaction. However, application of the SS mesh had little effect on ohmic impedance. Keywords : Microbial fuel cell, Cathode, Current collector References 1. Logan BE. Peer reviewed: Extracting hydrogen and electricity from renewable resources. Environ. Sci.

Technol. 2004;38:160A-167A. 2. Lovley DR. Bug juice: Harvesting electricity with microorganisms. Nat. Rev. Microbiol. 2006;4:497-508.

456


P0539 Effects of Vertical and Horizontal Configurations of Different Numbers of Brush Anodes on Performance and Electrochemistry of Microbial Fuel Cells

Usman GHANI, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] To maximize wastewater treatment and energy production by microbial fuel cells (MFCs), it is important to design the optimal anode arrangement. In this study, four brushes were tested horizontally or vertically to the cathode as the number of the anodes increased from one to four. In the horizontal configuration, adding the anodes greatly reduce electrode resistance and enhanced cell performance, showing four anodes (H4) was the best. In the vertical configuration, two anodes (V2) showed greatest performance and greatest decrease in anode resistance. Compared with one anode, maximum power increased by 59% in H4 and by 18% in V2; anode polarization resistance decreased by 95% in H4 and by 74% in V2; anode impedance decreased by 91% in H2 and by 73% in V2. Cathode resistance was relatively constant, showing adding anodes had negligible effect on it. Because diffusion resistance increases with increasing distance between an anode and a cathode, the vertical anodes should have different diffusion resistance and performances. In this study, adding more anodes vertically decreased cell performance in V3 and V4. However, in a cyclic voltammetry test, current production was substantially increased when the third and the fourth brush anodes were introduced in the both arrangements. Compared with one anode, current production increased by 200% in H4 and by 205% in V4. It shows that the external electrical input relieved diffusion resistance and increased current generation and that installing anodes away from the cathode is a good strategy to increase current production in a system with external power supply such as microbial electrolysis cell. Based on the results, we suggest the following strategy: i) install multiple anodes horizontally along the cathode; ii) install multiple anodes in the second row horizontally along the cathode; iii) install multiple anodes both horizontally and vertically if there is an external power supply. Keywords : Microbial fuel cell, Brush anodes, Impedance References 1. Al-Mamun, A., Ahmad, W., Baawain, M.S., Khadem, M., Dhar, B.R., 2018. A review of microbial

desalination cell technology: configurations, optimization and applications. J. Clean. Prod. 183, 458e480. 2. Eaktasang, N., Kang, C.S., Ryu, S.J., Suma, Y., Kim, H.S., 2014. Enhanced current production by

electroactive biofilm of sulfate-reducing bacteria in the microbial fuel cell. Environ. Eng. Res. 19 (1), 115-115.

457


P0540 Influence of External Resistance on Electrogenesis, Methanogenesis, and Anode Prokaryotic Communities in Microbial Fuel Cells

Mahasin Ali MOHAMMOD, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] The external resistance (R ext) of microbial fuel cells (MFCs) regulates both the anode availability as an electron acceptor and the electron flux through the circuit. We evaluated the effects of R ext on MFCs using acetate or glucose. The average current densities (I) ranged from 40.5 mA/m2 (9,800 Ω) to 284.5 mA/m2 (150 Ω) for acetate-fed MFCs (acetate-fed reactors [ARs]), with a corresponding anode potential (E an) range of −188 to −4 mV (versus a standard hydrogen electrode [SHE]). For glucose-fed MFCs (glucose-fed reactors [GRs]), I ranged from 40.0 mA/m2 (9,800 Ω) to 273.0 mA/m2 (150 Ω), with a corresponding E an range of −189 to −7 mV. ARs produced higher Coulombic efficiencies and energy efficiencies than GRs over all tested R ext levels because of electron and potential losses from glucose fermentation. Biogas production accounted for 14 to 18% of electron flux in GRs but only 0 to 6% of that in ARs. GRs produced similar levels of methane, regardless of the R ext. However, total methane production in ARs increased as R ext increased, suggesting that E an might influence the competition for substrates between exoelectrogens and methanogens in ARs. An increase of R ext to 9,800 Ω significantly changed the anode bacterial communities for both ARs and GRs, while operating at 970 Ω and 150 Ω had little effect. Deltaproteobacteria and Bacteroidetes were the major groups found in anode communities in ARs and GRs. Betaproteobacteria and Gammaproteobacteria were found only in ARs. Bacilli were abundant only in GRs. The anode-methanogenic communities were dominated by Methanosaetaceae, with significantly lower numbers of Methanomicrobiales. These results show that R ext affects not only the E an and current generation but also the anode biofilm community and methanogenesis. Keywords : Microbial fuel cell, External resistance, Electron acceptor References 1. Aelterman, P., S. Freguia, J. Keller, W. Verstraete, and K. Rabaey. 2008. The anode potential regulates

bacterial activity in microbial fuel cells. Appl. Microbiol. Biotechnol. 78:409-418. 2. Bond, D. R., D. E. Holmes, L. M. Tender, and D. R. Lovley. 2002. Electrodereducing microorganisms that

harvest energy from marine sediments. Science 295:483-485.

458


P0541 Impedance Characteristics and Polarization Behavior of a Microbial Fuel Cell in Response to Short-term Changes in Medium pH

Mahasin Ali MOHAMMOD, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] pH oppositely influences anode and cathode performance in microbial fuel cells. The differential electroche-mical effects at each electrode and the resultant full-cell performance were analyzed in medium pH from 6.0 to 8.0. Potentials changed -60 mV/pH for the anode and -68 mV/pH for the cathode, coincident with thermo-dynamic estimations. Open circuit voltage reached a maximum (741 mV) at pH 7, and maximum power density was highest (712 mW/m2) at pH 6.5 as the cathode performance improved at lower pH. Maximum current density increased and apparent half-saturation potential (EKA) decreased with increasing medium pH due to improved anode performance. An equivalent circuit model composed of two time constant processes accurately fit bioanode impedance data. One of these processes was consistently the rate-limiting step for acetate-oxidizing exoelectrogenesis, with its pH-varying charge transfer resistance R2 ranging from 2- to 321-fold higher than the pH-independent charge transfer resistance R1. The associated capacitance C2 was 2-3 orders of magnitude larger than C1. R2 was lowest near EKA and increased by several orders of magnitude at anode potentials above EKA, while R1 was nearly stable. However, fits deviated slightly at potentials above EKA due to emerging impedance possibly associated with diffusion and excessive potential. Keywords : Microbial fuel cell, pH, Bioanode References 1. Saima Aftab, Afzal Shah, Jan Nisar, Muhammad Naeem Ashiq, Mohammad Salim Akhter, Aamir Hassan

Shah. Marketability Prospects of Microbial Fuel Cells for Sustainable Energy Generation. Energy & Fuels 2020, 34 (8), 9108-9136.

2. Jianting Liu, Liling Wei, Huiqiang Wang, Gongjia Lan, Haijun Yang, Jianquan Shen. Silk gel-based N self-doped porous activated carbon as an efficient electrocatalyst in neutral, alkaline and acidic medium. Fuel 2021, 287, 119485.

459


P0542 Comparison of Anode Bacterial Communities and Performance in Microbial Fuel Cells with Different Electron Donors

Mahasin Ali MOHAMMOD, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial fuel cells (MFCs) harness the electrochemical activity of certain microbes for the production of electricity from reduced compounds. Characterizations of MFC anode biofilms have collectively shown very diverse microbial communities, raising ecological questions about competition and community succession within these anode-reducing communities. Three sets of triplicate, two-chamber MFCs inoculated with anaerobic sludge and differing in energy sources (acetate, lactate, and glucose) were operated to explore these questions. Based on 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE), all anode commu-nities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone in the Bacteroidetes class (99% similarity). Various other Geobacter-like sequences were also enriched in most of the anode biofilms. While the anode communities in replicate reactors for each substrate generally converged to a reproducible community, there were some variations in the relative distribution of these putative anode-reducing Geobacter-like strains. Firmicutes were found only in glucose-fed MFCs, presumably serving the roles of converting complex carbon into simple molecules and scavenging oxygen. The maximum current density in these systems was negatively correlated with internal resistance variations among replicate reactors and, likely, was only minimally affected by anode community differences in these two-chamber MFCs with high internal resistance. Keywords : Microbial fuel cell, Chemical Oxygen Demand, Clone Library, Bacteroidetes References 1. Aelterman P, Rabaey K, Clauwaert P, Verstraete W (2006a) Microbial fuel cells for wastewater treatment.

Water Sci Technol 54(8):9-15. 2. Aelterman P, Rabaey K, Pham HT, Boon N, Verstraete W (2006b) Continuous electricity generation at high

voltages and currents using stacked microbial fuel cells. Environ Sci Technol 40(10): 3388-3394.

460


P0543 Effects of Brush-anode Configurations on Performance and Electrochemistry of Microbial Fuel Cells

Sunghoon SON, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] For practical implementation of MFC, increasing power generation is important because it is closely related with energy production rate and wastewater treatability. However, it is not known which relative arrangement of anode and cathode gives the best performance, and it is necessary to know electrochemical reference point of the brush anode for this. Five different brush-anode configurations were tested in a single-chambered cubic MFC. By merely changing a brush anode configuration, power and current densities were increased by 20% and 30%, respectively. The horizontally-positioned anode configuration (H1) with the closest anode-cathode distance produced the highest power and current. EIS showed that anode impedance and full-cell impedance were decreased by 60% and 49%, respectively. CE and EE were not significantly affected by the anode-cathode distance, but the horizontal type cells showed relatively higher CE, EE and COD removal rate and shorter batch time. The center of a titanium current collector and the center of carbon fibers of a brush-anode were found to be statistically-significant reference points for MFC electrochemistry. Keywords : Microbial fuel cell, Anode configuration, Ion transfer, Impedance References 1. Feng, Y., Yang, Q., Wang, X., Logan, B.E., Treatment of carbon fiber brush anodes for improving power

generation in air–cathode microbial fuel cells (2010), Journal of Power Sources 195(7), 1841-1844. 2. Koo, B., Jung, S.P., Recent Trends of Oxygen Reduction Catalysts in Microbial Fuel Cells: A Review

(2019), Journal of Korean Society of Environmental Engineers 41(11), 657-675.

461


P0544 Comparison of Hydrogen Production and System Performance in a Microbial Electrolysis Cell Containing Cathodes Made of Non-platinum Catalysts and Binders

Sunghoon SON, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial electrolysis cell (MEC) is an innovative electrochemical technology that decomposes organic matter in anode and produces hydrogen in cathode. It is imperative to use a high-performance and a low-cost cathode material to make the application of MEC economically viable. In this study, five different cathodes made of low-cost materials were tested in MECs. The materials included activated carbon (AC) and nickel powder (Ni) as a cathode catalyst; polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) as a catalyst binder; stainless steel mesh (SSM) as a cathode substrate or a cathode itself. Among the tested cathodes, Ni/AC/PTFE obtained the best performance, followed by Ni/AC/PVDF, AC/PVDF, flamed-oxidized SSM (SSM/F) and SSM. Ni/ AC/PTFE exhibited the best performance in hydrogen production rate (HPR, 1.88 L/L d), hydrogen purity (97.5%), coulombic efficiency (124%), energy efficiency (216%), cathodic capacitance (0.9924 F), along with the lowest cathodic impedance (35 Ω). The worst performed SSM showed as follows: 0.57 L/L d of HPR, 71% of hydrogen purity, 36% of coulombic efficiency, 62% of energy efficiency, 0.0008 F of cathodic capacitance and 62 Ω of cathodic impedance. This study shows quantitatively the electrochemical and performance transitions of MEC according to the cathode component changes. Keywords : Microbial electrolysis cell, Hydrogen production, Nickel, Linear sweep voltammetry, Electroche-mical spectroscopy impedance

462


P0545 Comparative Evaluation of Performance and Electrochemistry of Microbial Fuel Cells with Different Anode Structures and Materials

Sunghoon SON, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Various materials and anode structures have been applied to enhance microbial fuel cell (MFC) performance. However, their comparative evaluation of performance and electrochemistry has not yet been investigated in detail under a same condition. In this study, a carbon-cloth anode, an anode-cathode assembly, and a brush anode with two different orientations were tested under a same condition for comparative analyses on their performance and electrochemistry, in order to reveal their unique electrochemical characteristics. The brush anode cells exhibited better performance than the carbon cloth cells. The brush anodes showed 41-72% higher maximum power densities, 18-75% higher maximum current density and 24-32% higher optimum current densities than the carbon cloth anodes. The brush anodes showed 25-43 Ω lower anodic polarization resistance than the carbon cloth anodes. The brush anodes showed 1.6-21.2 Ω lower ohmic impedance, 7.7-10.6 Ω lower charge transfer impedance and 9.3-31.8 Ω lower anodic impedance than the carbon cloth anodes. Anodic ohmic impedance was greatest in the carbon-cloth-anode MFC (21.9 Ω), where loose contact between a carbon cloth and a current collector might cause the high ohmic resistance, and large solution resistance in the cell could diminish anode performance due to slow ion transport. Keywords : Microbial fuel cell, Electrode assembly, Anode configuration, Electrochemistry References 1. Logan, Bruce E., et al. Microbial fuel cells: methodology and technology. (2006), Environmental science &

technology 40.17: 5181-5192. 2. Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy generation (2005), Trends

Biotechnol; 23:291-8.

463


P0546 Reproducibility Evaluation of Linear Sweep Voltammetry Tests of Electrodes in Microbial Electrolysis Cells

Bonyoung KOO, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial electrolysis cells (MECs) are a promising environmental system performing simultaneous organic waste stream treatment and producing valuable products. Electrode is a key component in a microbial electrolysis cell (MEC) and it needs significant improvement for practical implementation of MEC. For effective development of electrode technology, accurate and reproducible analytical methods are very important. Linear sweep voltammetry (LSV) is an essential analytical method for evaluating electrode performance; however, it has not been firmly established yet in the MEC field. In this study, biological brush (BB), abiotic brush (AB), Pt wire (PtW), stainless steel wire (SSW) and mesh (SSM)) were tested to explore the most suitable counter electrode in different medium conditions. Coefficient of variation (Cv) for Imax of LSV was comparatively analyzed. In BB-anode LSV, SSW (0.48%) and SSM (2.17%) showed higher reproducibility as a counter electrode. Reproducibility of anode LSV test was good in stainless steel wire and mesh as counter electrode. In SSM-cathode LSV, BB (1.76%) and PtW (2.01%) produced more reproducible results. In the Ni-AC-SSM-cathode LSV, PtW (3.54%) and BB (8.81%) produced more reproducible results. It shows electrode used in the operation is an appropriate counter electrode in the acetate-added condition. However, in the absence of acetate, PtW (1.24%) and BB (1.71%) produced more reproducible results in SSM cathode and PtW (0.61%) and SSW (1.21%) did in the Ni-AC-SSM-cathode, showing PtW is an appropriate counter electrode. This is because Pt is not electrochemically polarized as a counter electrode in the cathode LSV test. Reproducibility evaluation results in this study suggest that counter electrodes can be set according to the various conditions in anode and cathode LSV test. Keywords : Microbial electrolysis cell, Linear Sweep Voltammetry, Coefficient of variation, Counter electrode References 1. Duce, R.A.; LaRoche, J.; Altieri, K.; Arrigo, K.R.; Baker, A.R.; Capone, D.; Cornell, S.; Dentener, F.;

Galloway, J.; Ganeshram, R.S. Impacts of atmospheric anthropogenic nitrogen on the open ocean (2008), Science, 320, 893-897.

2. Logan, B.E.; Rabaey, K. Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies (2012), Science, 337, 686-690.

464


P0547 Characterization of Impedance and Polarization of Carbon-felt Bioanodes and Activated-carbon Cathodes in a Continuous-flow Microbial Fuel Cell

Bonyoung KOO, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial fuel cell (MFC) is an innovative environmental and energy system that converts biomass energy in wastewater into electrical energy and purify wastewater by using a microbial electrochemical reaction. For the practical implementation of MFC as a next-generation wastewater treatment process, MFC performance should be enhanced far more than the current level. Characterizations of the electrochemistry of MFC is very important in developing bio-electrochemical energy producing wastewater treatment process. Compared to the develop-ment of MFC technology, however, understanding of its electrochemical characterization is still insufficient. The main reason is that its electrochemical analysis is very difficult due to the complex nature of the anode biofilm, which is a key to generating electricity. In this experiment, the influence of the measurement potential of impedance and the scanning rate for polarization curve on the MFC electrochemistry was investigated. The experiment was performed after stabilizing the system for accurate measurement. Unlike the previous batch tests showing the lowest anodic impedance at -400 mV vs. Ag/AgCl, the anodic impedance decreased and the current production increased as the anode potential increased up to +5.7 mV vs. Ag/AgCl in the continuous flow MFC. The polarization curves were produced at two scanning rates (1 and 0.1 mV/s) in a continuous mode, and those electrochemical data were comparatively analyzed. The rapid scan rate has the advantage of being able to collect data in a short time, but the measured data differs from the performance value when operating the MFC with a constant external resistance. When it is difficult to maintain a steady state for a long time in an MFC, it will be possible to produce polarization curves in a short time with a faster scanning rate. When performance analysis is needed, the comparative analysis would be possible among the data at different conditions through extrapo-lation. Keywords : Microbial fuel cell, Carbon Felt, Continuous-flow, Scan rate References 1. Logan, Bruce E. Exoelectrogenic bacteria that power microbial fuel cells. (2009) Nature Reviews

Microbiology 7.5: 375-381. 2. Torres, César I., et al. A kinetic perspective on extracellular electron transfer by anode-respiring bacteria.

(2010) FEMS microbiology reviews 34.1: 3-17.

465


P0548 Addition of Reduced Graphene Oxide to an Activated-carbon Cathode Increases Electrical Power Generation of a Microbial Fuel Cell by Enhancing Cathodic Performance

Bonyoung KOO, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial fuel cells (MFCs) are innovative bio-electrochemical systems (BES) that are being developed for an energy-positive wastewater treatment process that generates electricity from organic waste with the concurrent wastewater treatment. Activated carbon (AC) is an inexpensive catalyst for oxygen reduction in an air cathode of microbial fuel cells (MFCs). In the AC-based cathode, carbon black (CB) is used as a conductive supporting material. In this study, it was hypothesized cathodic performance would increase if reduced graphene oxide (rGO) replaces CB in an optimum ratio. Because rGO has high electrical conductivity and unique shape with flat and large areas, it can be used for improving the electrical conductivity and facilitating electron transfer in the cathodic catalyst layer by intercalation into the catalyst layer in replace of CB. rGO replaced CB in the four different weight ratios. Maximum power density was the best in rGO15 (2,642 mW/m2) followed by rGO5 (2,142 mW/m2). In the optimum external resistance operation, rGO5 and rGO showed similar power (~1,060 mW/m2), higher than the others. Linear sweep voltammetry, cyclic voltammetry, and impedance spectroscopy also showed that the optimal rGO additions improved cathodic performance and reduced cathodic internal resistance. Due to the flatter and wider shape of rGO and 5 times higher electrical conductivity than CB, the rGO addition improved the cathodic performance, but the complete replacement of CB with rGO decreased the cathodic performance due to the increased thickness and the morphological crack. The optimum rGO addition is a simple and effective method for improving cathodic performance. Keywords : Microbial fuel cell, Activated carbon, Reduced graphene oxide, Maximum power density References 1. Logan, Bruce E. Exoelectrogenic bacteria that power microbial fuel cells. (2009), Nature Reviews

Microbiology 7.5: 375-381. 2. Jung, Sokhee Philemon. Practical implementation of microbial fuel cells for bioelectrochemical wastewater

treatment. (2013) J. Korean Soc. Urban Envrion 13.2: 93-100.

466


P0549 Performance Enhancement of a Microbial Fuel Cell by Physico-chemical Treatments of Activated-carbon Catalyst of an Air Cathode

Viet Hoa Huong TRAN, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial Fuel cell (MFC) is an innovative electrochemical system, being developed for an energy-positive wastewater treatment process for a sustainable society. Improvement of electrode technology is very important for the practical use of MFC. In this study, for developing practical cathode catalyst technology, physicchemical modified activated carbon (AC) catalysts and platinum were tested for performance and electrochemical characterization in an MFC under the same condition, potentially to replace expensive platinum catalysts. Comparing with a maximum power density of a platinum-coated cathode (976 mW/m2), a Co-N-C/AC cathode made with activated carbon doped with cobalt and 1,10-phenanthroline at 800 produced 1,526 mW/ m2 in the MFC condition, which was 56% higher than the Pt-coated cathode. A 500AC cathode made with heat-treated activated carbon at 500 produced 1,394 mW/m2 and non-treated activated carbon cathode (Plain AC) produced 1,014 mW/m2. The tested activated-carbon electrodes showed electrochemical performance and power production superior to the Pt-coated cathode. Electrochemical performance of cathodes was increased as more physico-chemical treatments were added to activated carbon catalysts. Cathode impedance results showed that enhanced electrochemical performance was attributed to decrease of cathode charge transfer resistance, possibly due to the physical-chemical modification of activated carbon and the catalyst change. Keywords : Microbial fuel cell, Physicochemical treatment, Activated-carbon catalyst, Air-cathode

467


P0550 Establishing the Stable Cell Performance Time and Anode Maturation Time of a Single Chamber Microbial Fuel Cell

Viet Hoa Huong TRAN, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] In microbial fuel cells (MFCs), to obtain accurate and reproducible experimental results, it is important to determine the ‘anode maturation time’. In this study, four single-chamber MFCs were tested to know when the cell can produce stable and maximum performance. According to the linear sweep voltammetry (LSV) polarization tests, three MFCs could obtain stable maximum power densities after 9 weeks. Average maximum power densities from the 9th to the 17th week were highest in MFC-4 (2990 mW/m2), followed by MFC-2 (2983 mW/m2), MFC-3 (2368 mW/m2) and MFC-1 (837 mW/m2). Polarization resistance showed that MFC-1 had much larger anode resistance (36.6-85.4 Ω) than the other MFCs (1.7-11.6 Ω). Possibly due to the bad inoculation, MFC-1 showed the lowest performance with the highest anode resistance. Anodic cyclic voltammetry (CV) showed that current production increased by time and MFC-1 had much smaller current production (17.09 mA) than the other MFCs (21.73 - 24.44 mA) in the 17th week. Current production enhancement indicated anode biofilm became more mature by time, but overall cell performance did not increase accordingly. However, in the 17th week, anode resistance of MFC-1 was reduced by 47%, resulting in cell performance improvement. This study showed that the stable cell performance of a single chamber MFC with a brush anode was 9 weeks. Nevertheless, anode needed more than 17 weeks to attain the mature state. Keywords : Microbial fuel cell, Stable cell performance time, Anode maturation time References 1. Bond, D.R.; Lovley, D.R, Electricity production by Geobacter sulfurreducens attached to electrodes (2003),

Applied and Environmental Microbiology, 69, 1548-1555. 2. Jung, S.P.; Pandit, S, Chapter 3.1 - Important Factors Influencing Microbial Fuel Cell Performance (2018),

Microbial Electrochemical Technology, Mohan, S.V., Varjani, S., Pandey, A., Eds. Elsevier, 377-406.

468


P0551 Effects of Wire-type and Mesh-type Anode Current Collectors on Performance and Electrochemistry of Microbial Fuel Cells

Viet Hoa Huong TRAN, Sokhee JUNG Department of Environment and Energy Engineering, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Microbial fuel cell (MFC) is an innovative environmental and energy system that converts organic wastewater into electrical energy. For the practical implementation of MFC as a wastewater treatment process, a number of limitations need to be overcome. Improving anode performance is one of the major challenges, and introduction of a current collector can be an easy and practical solution. High-performance electrode materials make it possible to achieve high power generation of MFC systems by reducing internal resistance. Carbon-based materials are generally used for the MFC anode electrode, but their conductivity is much lower than metal materials. In this study, it was hypothesized increasing metal current collector areas improve anodic performance. The performance of the carbon felt anode was compared using titanium wire and stainless steel mesh as a current collector. In the IV polarization test, maximum power density, maximum current density and optimum current density were 33%, 34% and 30% higher in CF-M (2,311 mW/m2, 16,815 mA/m2 and 7,651 mA/m2) than CF-W (1,737 mW/m2, 12,566 mA/m2 and 5,874 mA/m2), respectively. However the stainless steel mesh used as a current collector of CF-M inhibits microbial growth and adhesion on the carbon felt and reduces mass transfer because it decreases the surface area of carbon felt. Therefore, when using stainless steel mesh as a current collector, it should be used in consideration of the problem of microbial adhesion. Keywords : Microbial fuel cell, Stainless steel mesh, Titanium wire, Anodic performance References 1. Logan, B., Cheng, S., Watson, V., Estadt, G., Graphite fiber brush anodes for increased power production in

air-cathode microbial fuel cells (2007). Environ. Sci. Technol. 41, 3341-3346. 2. Hidalgo, D., Tommasi, T., Bocchini, S., Chiolerio, A., Chiodoni, A., Mazzarino, I., Ruggeri, B., Surface

modification of commercial carbon felt used as anode for Microbial Fuel Cells (2016). Energy 99, 193-201.

469


P0552 Climatic Preferences for Argentine ant, Linepithema humile, by Analyzing Long-term Monthly Climatic Data in the Habitat

Jae-Min JUNG1, Dae-Hyeon BYEON1, Jae-Woo SONG1, Se-Hyun KIM2, Sunhee YOON2, Wang-Hee LEE1 1Department of Biosystems Machinery Engineering, Chungnam National University, Daejeon, Korea, 2Department of Smart Agriculture Systems, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Argentine ant has currently spread to many countries, including Europe and Asia, and are causing economic losses as well as negative impacts on the indigenous species. In particular, native ants have been deprived of their habitat because of the invasion of Argentine ant. For this reason, it can be an effective approach to analyze the climate in the regions where Argentine ant established so that the potential areas exposed to its invasion is to be selected in advance. The occurrence data for Argentine ant was obtained from Global Biodiversity Information Facility (GBIF), and the climatic variables (the monthly maximum temperature, minimum tempera-ture and rainfall) located in occurrence coordinates were investigated using a histogram and kernel density function. As a result, it was found that Argentine ant established mainly in the region where the monthly maximum temperature, the monthly minimum temperature, and the monthly rainfall were approximately 21°C, 8°C, and less than 200 mm, respectively. In addition, it was found that Argentine ant would rarely occur high rainfall-areas. As the possibility of Argentine ant invasion is high due to worldwide trades; thus, a basic data to assess its invasive risk might be necessary. In this point of view, this study can provide very initial insight for characterizing the favorable climatic conditions for Argentine ant, and can be applied into evaluating the potential risk areas in South Korea. Keywords : climate, Linepithema humile, invasive ant species, kernel density function, potential risk area

470


P0553 Utilization of Gas Hydrates as an Innovative Leavening Agent for Baked Goods

Bernhard GATTERNIG1,2,3, Timo CLASSEN3, Yvonne RINGELSPACHER3, Antonio DELGADO1,3 1Weihenstephan-Triesdorf, University of Applied Sciences, Chair of Process Engineering, Merkendorf, Germany, 2LSTME, German Engineering Research and Development Center, Busan, Korea, 3Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Fluid Mechanics, Erlangen, Germany Corresponding Author Email : [email protected] Most types of bakery products can structurally be considered as solidified foams, generating the texture and mouthfeel desired by the consumer. Traditional leavening agents such as yeast or chemical agents (sodium bicarbonate, ammonium bicarbonate, or potash) show disadvantages like long, energy intensive leavening periods (for yeast) or deterioration of product quality concerning taste and introduction of potentially harmful substances (aluminium salts, phosphates, acrylamide) for chemical agents. Consequently, the present application investigates the use and technical realization of a novel baking agent based on a physically stabilized solid form of CO2 in the shape of gas hydrates. The composition of the gas hydrates, containing only water and CO2, ensures that food safety is maintained and no negative effects on taste and processability occur even at high dosage. The main objective of this work is to develop a process for the production of selected bakery products using gas hydrate as foaming agent for the first time worldwide. This article describes the adaptation of an existing gas hydrate reactor for the production of gas hydrate and presents experimentally investigated release kinetics of the gas hydrates. Based on these findings, the first steps of a mechanistic-numerical modelling of the leavening effect of gas hydrate are demonstrated. In the future, the manufacturing process of selected products employing gas hydrates will be optimized. The aim is to ensure an ideal integration of the new concept into the baking process and to demonstrate potential savings compared to conventional processes. Keywords : Gas Hydrates, Baked goods production, Numerical modelling

471


의공학 및 바이오제약공학

(Biomedical and

Biopharmaceutical Engineering)

472


P0601 Development of a Loop-mediated Isothermal Amplification (LAMP) Assay for Diagnosing Invasive Candidiasis

Da Hye LIM1, Hyunseul JEE1, Ahran KIM2, Young Lan CHOE2, Dong Hun KWAK2, Jeonghun NAM3, Woong Sik JANG4, Chae Seung LIM2 1Department of Medical Sciences, Graduate School of Medicine, Korea University, Seoul, Korea, 2Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul, Korea, 3Department of Song-Do Bio-Environmental Engineering, Incheon Jaeneung University, Incheon, Korea, 4Department of Emergency Medicine, College of Medicine, Korea University Guro Hospital, Seoul, Korea Corresponding Author Email : [email protected] Candidasis is a syndrome in which candida fungus penetrates into the blood and infects various organs, causing systemic inflammatory reactions. The death rate from candidasis is 40 to 54 percent, and the later the treatment is given, the higher the death rate becomes. Blood culture is used as a standard diagnosis method to diagnose candidasis. However, blood culture is generally takes 2-4 days, showing a low sensitivity. RT-PCR, which is non-culture-based method, has high sensitivity and specificity, but, this method takes about 3 hours, requiring expensive instruments, and specialized technicians. Loop-mediated isothermal amplification (LAMP) is a method for nucleic acid detection with high specificity and sensitivity without the need for specialized equipment and high level of skill. Here, we have developed a candida pan RT-LAMP assay for the detection simultaneously seven species of candida infection. Candida pan RT-LAMP assay detected simultaneously seven species of candida infection in optimized condition. Keywords : Loop-mediated isothermal amplification, Candida, point-of-care test References 1. J. Zirkel, H. Klinker, A. Kuhn, M. Abele-Horn, D. Tappe, D. Turnwald, H. Einsele and W.J. Heinz, Med.

Mycol. 50, 50 (2012). 2. M. Mikulska, M. Bassetti, S. Ratto and C. Viscoli, Mediterr. J. Hematol. Infect. Dis. 3, e2011007 (2011). 3. E.K. Spanakis, G. Aperis and E. Mylonakis, Clin. Infect. Dis. 43, 1060 (2006). 4. H. Yera, B. Sendid, N. Francois, D. Camus and D. Poulain, Eur. J. Clin. Microbiol. Infect. Dis. 20, 864

(2001).

473


P0602 Development of a Multiplex Loop-mediated Isothermal Amplification (LAMP) Assay for On-site Diagnosis of SARS CoV-2

Da Hye LIM1, Hyunseul JEE1, Ahran KIM2, Young Lan CHOE2, Dong Hun KWAK2, Jeonghun NAM3, Woong Sik JANG4, Chae Seung LIM2 1Department of Medical Sciences, Graduate School of Medicine, Korea University, Seoul, Korea, 2Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul, Korea, 3Department of Song-Do Bio-Environmental Engineering, Incheon Jaeneung University, Incheon, Korea, 4Department of Emergency Medicine, College of Medicine, Korea University Guro Hospital, Seoul, Korea Corresponding Author Email : [email protected] SARS-CoV-2 is a respiratory disease that occurred in China, and is an infectious disease caused by beta coronavirus, the same type as SARS and MERS. SARS CoV-2 is still spreading worldwide, and there is an urgent need to conduct rapid diagnosis followed by patient isolation and treatment. Currently, an RT-qPCR-based test is the most representative diagnosis method for SARS CoV-2, but these method require nearly three hours to produce results and skilled technicians are necessary. LAMP is a highly sensitive, low-cost, single-tube technology to detect the target nucleic acid sequences. In this study, we have developed multiplex SARS CoV-2 LAMP primer/probe sets using strand-displaceable probes, based on the region of the RdRP, E and N gene of the aligned sequences of SARS CoV-2 subtypes. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets were evaluated, and the RdRP/N/IC multiplex LAMP assay showed lowest detection limits among five combination. The multiplex RdRP/N/IC RT-LAMP assay showed comparable sensitivities for clinical samples to that of the Allplex™ 2019-nCoV Assay and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit. Keywords : Loop-mediated isothermal amplification, Severe acute respiratory syndrome coronavirus 2, point-of-care test References 1. S.A. Meo, A.M. Alhowikan, T. Al-Khlaiwi, I.M. Meo, D.M. Halepoto, M. Iqbal, A.M. Usmani, W. Hajjar

and N. Ahmed, Eur Rev Med Pharmacol Sci. 24, 2012 (2020). 2. J.F. Chan, S. Yuan, K.H. Kok, K.K. To, H. Chu, J. Yang, F. Xing, J. Liu, C.C. Yip, R.W. Poon and H.W.

Tsoi, Lancet. 395, 514 (2020). 3. A. Tahamtan and A. Ardebili, Expert Rev. Mol. Diagn. 20, 453 (2020). 4. V.M. Corman, O. Landt, N. Kaiser, R. Molenkamp, A. Meijer, D.K. Chu, T. Bleicker, S. Brünink, J.

Schneider, M.L. Schmidt and D.G. Mulders, Euro Surveill. 25, 2000045 (2020). 5. J. Pang, M.X. Wang, I.Y. Ang, S.H. Tan, R.F. Lewis, J.I. Chen, R.A. Gutierrez, S.X. Gwee, P.E. Chua, Q.

Yang and X.Y. Ng, J. Clin. Med. 9, 623 (2020).

474


P0603 Antioxident Activity of the Halophyte Angelica Japonica and Its Inhibitory Effect Against Cancer Migration

HPS JAYAPALA1,2, Youngwan SOE1,3 1Division of Marine Bioscience, Korea Maritime & Ocean University, Busan, Korea, 2Ocean University of Sri Lanka, Colombo, Sri Lanka, 3Korea Research Institute of Marine Science and Technology, Busan, Korea Corresponding Author Email : [email protected] The halophyte Angelica japonica is well-known as a perennial medicinal herb growing in coastal area of Jeju Island of South Korea (1). The aim of this study was to investigate antioxidant activity of Angelica japonica extract and its solvent fractions, and their inhibitory effect against cell migration in HT 1080 cells. The antioxidant effect was determined by estimating authentic ONOO and ONOO- generated from SIN-1 (3-morpholinsydnonimine) in vitro as well as by measuring the degree of occurrence of intracellular reactive oxygen species (ROS) and nitric oxide (NO). The aqueous MeOH and n-BuOH solvent fractions showed relatively good antioxidant activity in the overall antioxidant assay systems. Cell migration for the HT-1080 cells was observed by wound healing assay for crude extract and its solvent fractions. Cell mobility in the HT-1080 cells was strongly inhibited by the 85% aqueous MeOH and n-BuOH fractions. The above activity results were significantly correlated with polyphenol and flavonoid contents of the samples. In conclusion, the halophyte A. Japonica shows its potential to provide natural products that need to prevent oxidative stress-related diseases and cancer metastasis. Keywords : Halophyte, Angelica japonica, antioxidant activity, cell migration, HT-1080 References 1. Plant For A Future . 2013. Retrieved 2020, from Angelica japonica PFAF Plant Database:

https://pfaf.org/User/Plant.aspx?Latin Name=Angelica+japonica

475


P0604 Disposable Microfluidic Concentrator for Image-based Cytometric Analysis of Residual White Blood Cell

Hyunseul JEE1, Dahye LIM1, Woongsik JANG2, Younglan CHOE3, Ahran KIM3, Donghun KWAK3, Chaeseung LIM3, Jeonghun NAM4 1Department of Medical sciences, Graduate school of Medicine, Korea University, Seoul, Korea, 2Department of Emergency Medicine, College of Medicine, Korea Univ. Guro Hospital, Seoul, Korea, 3Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul, Korea, 4Department of Song-Do Bio-Environmental Engineering, Incheon Jaeneung University, Incheon, Korea Corresponding Author Email : [email protected] White blood cells (WBCs) from blood components should be removed to prevent some transfusion reactions. For quality control of blood components, an accurate and easy method to count residual WBCs is required. Recently, a portable microscopic cell counter system was invented to replace conventional methods such as flow cytometry. Lately, in our group, a fundamental study of viscoelastic microfluidics technique concentrating WBCs has been conducted to overcome the low accuracy of the microscopic methods at low concentration levels of WBCs. In this study, we fabricated a plastic microfluidic device for mass production applying previous technology and evaluated the device’s performance using clinical samples. Using RBC concentrates with different concentration levels of WBCs, linearity, %CV, and concentration ratio of the device was evaluated and it was r2=0.991, 21%, and 60-folds, respectively. These results indicate that the performance of the microscopic method is improved compared to before using the device. Therefore, our device can be a simple and mass-productive WBC concentration tool for enhancing accuracy of the portable microscopic method at low concentrations of WBCs. Keywords : Mass Production, Plastic device, Viscoelastic, Residual WBC, Disposable device References 1. Transfusion TKSoB. Guidelines for blood transfusion. 4 ed. O-song, Korea: Division of Human Blood

Safety Surveillance, Korean Centers for Disease Control and Prevention (2016). 2. S. Dzik, G. Moroff, L. Dumont, Transfusion, 40(5), 513-20 (2000). 3. S.Y. Bae, C.H. Lee, J.S. Kim, C.S. Lim, C.K. Lee, K.N. Lee, V. Sang, 92(1), 64-8 (2007). 4. J.H. Nam, W.S. Jang, C.S. Lim, Talanta. 197, 12-9 (2019).

476


P0605 Cultivation and Isolation of Anaerobic Human Gut Microbiota Using Culturomics

Jeong-Hoon KIM1, Gi-Ung KANG2, So-yeon YANG3, Jung-Mo LEE4, Jae-Ho SHIN2, Hong KOH5, Dong-Woo LEE1,4 1Graduate Program in Bio-industrial Engineering, Yonsei University, Seoul, Korea, 2Department of Applied Biosciences, Kyungpook National University, Daegu, Korea, 3Department of Life Science and Biotechnology, Yonsei University, Seoul, Korea, 4Department of Biotechnology, Yonsei University, Seoul, Korea, 5Department of Pediatrics, Yonsei University College of Medicine, Seoul, Korea Corresponding Author Email : [email protected] A dysbiosis in the human gut microbiota can be one of the leading causes of ulcerative colitis (UC). Accordingly, fecal microbiota transplantation (FMT) has been a promising UC treatment because it showed a high success rate of >58.6% (n=29). To investigate whether the microbial composition correlates with the severity of UC, we identified fecal-derived anaerobes to compare the microbial community using a culture-based approach between patients and donors examined. Consequently, we identified 237 anaerobes from the fecal samples of ten donors and five patients. We also performed a microbial community analysis using 16S rRNA gene sequence-based approaches compared to the culturable gut microbes. These results supported the notion that the gut microbial communities of donors differ from those of patients. Although both analyses showed similar community compositions at the phylum levels, the culture-based analysis revealed distinct microbial species between donors and patients. Notably, phylogenomic studies of cultured microorganisms indicate that functional dysbiosis significantly affected the efficacy of FMT for UC patients rather than a shift in the microbial ecosystem. Therefore, the present study suggests that culturomics-based functional analysis can complement the conventional community-based microbiome analysis to develop microbiome-aided therapeutics for UC. Keywords : culturomics, phylogenomics, pangenome, fecal microbiota transplantation, inflammatory bowel disease

477


P0606 A Novel Text Classification Tool for Automated Identification of User-desired Literatures from Online Resources

Hyun Hak KIM1, Joongyeon CHOI1, Soohwan JEONG1, Minsung KO1, Goo JANG2, Se Chan KANG3, Hyunggun KIM1 1Department of Biomechatronic Engineering, Sungkyunkwan University, Suwon, Korea, 2College of Veterinary Medicine, Seoul National University, Yongin, Korea, 3Department of Oriental Medicine Biotechnology, College of Life Science, Kyung Hee University, Seoul, Korea Corresponding Author Email : [email protected] It requires a considerable amount of time and effort for researchers to use conventional online search methods to collect accurate experimental research literature depending on the scarcity of the information and the capability of the researcher. While deep learning is an ideal solution for such a task, it also requires substantial time and cost in preparing for training data. Desired (+) and undesired (-) literatures were defined depending on whether it includes information of “gene knockout experiments using a rat model to develop atherosclerosis”. Ten articles per group were tokenized for classification training and six were used for validation. The Multinomial Naïve Bayesian Classifier was employed to calculate the probability of occurrence for positive or negative classification using each section of the articles. Among the article sections, “Abstract” revealed the best performance of 94.1% accuracy with 90.9% (+) and 95.7% (-) F1-scores. A novel text classification tool was developed for automated accurate identification of user-desired literatures with few training data. This tool can help researchers reduce time and cost for collection of desired research articles online. Keywords : Automated literature search, Text classification, Multinomial Naïve Bayesian References 1. A. Rahman and U. Qamar, IEEE Int. Conf. Software Eng. Serv. Sci. 1, 63-67 (2016). 2. G. Singh, B. Kumar, L. Gaur and A. Tyagi, Int. Conf. Automat. Computat. Tech. Manage. 1, 593-596

(2019).

478


P0607 Optimization of Metallothionein Fusion Protein Complex with Zinc/DNA Nanocluster for the Treatment of Oxidative Stress Induced Diseases

Hongbin KIM1, Su Yeon LIM1, Yudong HONG1, Kwang Suk LIM1,2 1Interdisciplinary Program in Biohealth-machinery convergence engineering, College of ACE, Kangwon National University Chuncheon, Korea, 2Department of Biotechnology and Bioengineering, College of ACE, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Metallothionein(MT) is a small protein composed of 61 amino acids including 20 cysteine residues. MT is over-expressed in various organs exposed to many stress conditions such as heavy metals, hunger, fever or inflammation. MT has been reported that it could protect the cell from oxidative stress by reactive oxygen species(ROS) due to their antioxidant effect. In the previous study, we analyzed the characteristics of the Zinc/DNA nanocluster and suggested its potential as a gene carrier. In particular, we have reported that Zn2+ ions present in DNA could interact with the sulfhydryl groups in cysteines of targeting peptides through the ligand-to-metal charge transfer(LMCT). In this study, we developed a new method to incorporated therapeutic protein into DNA without any chemical conjugations by utilizing a LMCT transition, which occurs between divalent metal ions and the sulfhydryl group in cysteine from metallothionein. We prepared Tat fused MT recombinant fusion protein (Tat-MT) to improve intracellular uptake of MZD complex. The formation of the MZD complex was evaluated and determined morphology and size. The protective effect of Tat-MT/Zinc/DNA(tMZD) was measured in H9c2 cell against oxidative stress. This tMZD complex might be useful combination systems to deliver therapeutic protein and DNA. Keywords : Metallothionein, Oxidative stress, Gene delivery

479


P0608 Biological Evaluation of Single-use Bioreactor System, CELBIC System, for Microcarrier Cell Culture

Seohyun PARK, Jinwoo LEE, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] A typical stationary (2D) cell culture system is not suitable for large-scale culturing of adherent cells due to a limited surface area for cell attachment. It is also difficult to control the culture environment such as DO and pH. To overcome these issues, we used microcarriers that can significantly increase the area in which cells can attach within the specific culture space, and the CELBIC system, single-use bioreactor system, that can control the culture environment such as DO and pH. The CELBIC system operates by multi-directional rocking movement and can be stirred by the waves of the media. In addition, it is a system that can reduce risks of contamination in the cell culture and burdens such as cleaning and sterilization by using single-use bag. In order to apply to the CELBIC system without an impeller, we performed microcarrier cultures using human embryonic kidney cells (HEK293A) with various microcarriers and experimental conditions in Erlenmeyer flasks. Then we evaluated microcarrier cell cultures at working volume of 500 mL and 1 L in single-use bags using the CELBIC system. The Hillex II microcarrier showed the most stable cell growth and maintained cell adhesion to the microcarrier during cell culture in Erlenmeyer flasks. In the case of single-use bag using the CELBIC system, cells were well attached to the microcarriers, but it was difficult to maintain them after continuous agitation for cell culture. The CELBIC system has many advantages in the mass culture of suspension cells, but it was still difficult to apply to the mass culture of adherent cells. Therefore, it is necessary to further develop processes for continuously maintaining the cells attached to the microcarriers for expansion of adherent cells in the CELBIC system. It is also essential to study for biomaterials that can overcome the disadvantages of microcarriers in the CELBIC system. Keywords : Microcarrier cell culture, Single-use bag, Single-use bioreactor system

480


P0609 Imaging of Bones in vivo Using Peptides Specific to Hydroxyapatite

Jinho BANG1,2, Heesun PARK1,3, Sunghyun KIM1 1Center for Convergence Bioceramic Materials, Convergence R&D Division, Korea Institute of Ceramic Engineering and Technology, Cheongju, Korea, 2Department of Industrial Pharmacy, Chungbuk National University, Cheongju, Korea, 3Department of Life Science, Korea University, Seoul, Korea Corresponding Author Email : [email protected] The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~89% retained on hydroxyapatite after one month). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications. Keywords : Hydroxyapatite, bone, imaging, peptide, phage display, fluorescence References 1. Bang, J., Park, H., Yoo, J. et al. Selection and identification of a novel bone-targeting peptide for

biomedical imaging of bone. Sci Rep 10, 10576 (2020). https://doi.org/10.1038/s41598-020-67522-4.

481


P0610 Dengue Virus Antigen NS1 Detection Using Affibody Conjugated with Gold Nanoparticles

Youngwoo SONG1,2, Jinho BANG2, Jaesung WOO1, Sunghyun KIM2 1Department of Life science, Korea University, Seoul, Korea, 2Center for Convergence Bioceramic Materials, Convergence R&D Division, Korea Institute of Ceramic Engineering and Technology (KICET), Cheongju, Korea Corresponding Author Email : [email protected] Infection with dengue virus (DENV) is a serious health issue that causes severe dengue fever and occasionally lethal complications, such as dengue hemorrhagic fever. Therefore, rapid and sensitive DENV detection is important to reduce morbidity and mortality. Here, we screened dengue NS1-specific affibodies and developed anti-NS1 affibody-functionalized AuNPs (anti-NS1 affibody-AuNPs) to improve the detection sensitivity of NS1 antigen in ELISA. First, we screened NS1 antigen-specific affibody molecules (ZNS112, ZNS116, and ZNS146) from the affibody phage library. The affibodies were then expressed and purified from Escherichia coli. Among them, the ZNS112 affibody showed the highest equilibrium binding constant (Kd) of 1 μM and this affibody was functionalized on AuNPs measuring 20 nm in diameter. The developed anti-NS1 affibody-functionalized AuNPs ((ZNS112)2-AuNP) were used as carriers to achieve amplification of the signal in ELISA. (ZNS112)2-AuNP showed good properties, such easy synthesis, high number of affibodies conjugation on AuNPs, and excellent stability under harsh conditions with high salt concentrations and temperature. In addition, this nanoparticle-based enhanced ELISA resulted in a 14.2-fold signal amplification performance for dengue NS1 detection in comparison with affibody-based ELISA. This novel and sensitive method using (ZNS112)2-AuNP may have applications in the detection of DENV in infected patients at an early stage and for the detection of other pathogens in clinical diagnostics. Keywords : Dengue Virus, NS1, Affibody, gold nanoparticle, Detection References 1. Bang, J., Park, H. et al. Sensitive detection of dengue virus NS1 by highly stable affibody-functionalized

gold nanoparticles. New J. Chem., 2018,42, 12607-12614.

482


P0611 Generation of Random Adeno-associated Virus (AAV) Libraries and in vivo Selection of Novel AAV Variants

Seokmin OH, Yoojin KIM, Dongsoo LEE, Songyeon KIM, Seohee KIM, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Directed evolution of AAV through several rounds of in vitro / in vivo selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. AAV libraries used for directed evolution are based on wild-type AAV which has specific tropisms depending on AAV serotype. For directed evolution to be used successfully, it is important to maximize the diversity of AAV libraries and control mutation rate on AAV capsid during the production of the viral libraries. Here, we demonstrate that several AAV serotype based libraries are generated with Error-prone PCR and have high diversity of the libraries. Also, the mutation rate was controlled by manganese concentration. With these AAV libraries, we are conducting in vivo AAV libraries selection on mouse hypoxic-ischemic encephalopathy (HIE) model to improve transduction efficiency compared to parent AAV. Keywords : AAV, Directed evolution, HIE model, vector engineering

483


P0612 TANNylated-AAV for Enhanced Gene Delivery

Zheng Rong LAU, Ginah HAN, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Adeno-associated virus (AAV) has received tremendous attention for developments in gene therapy applica-tions, leading to several approvals of AAV-based gene therapy products such as Glybera, Zolgensma, and Luxturna. However, much can still be done in optimizing the efficacy of AAV as a delivery vehicle for gene therapy. Among the many drawbacks of AAV as a delivery vector, the problem of its relatively low genome packaging capacity of only about 4.7 kb is especially detrimental, resulting in cases of which transgene need to be fragmented and packaged separately prior to administration. Herein, we demonstrate that through rather simple tannic acid treatment onto AAV to form tannic acid-treated AAV (namely TANNylated-AAV), the transduction efficiency is enhanced. Tannic acid is found to have high affinity to proline-rich extracellular matrices (ECM) and collagens, which leads us to hypothesize that TANNylated-AAV can allow for enhanced targeted delivery. We utilized hypoxic ischemic (HIE) brain injury mouse model to observe enhanced AAV transduction towards ECM-rich reactive astrocytes populated glial scar region of infarct area. Keywords : Adeno-associated virus, Gene Delivery, Tannic acid

484


P0613 Biomaterial Conjugated AAV for Drug and Gene Delivery System

Dongsoo LEE, Yoojin KIM, Seokmin OH, Zheng Rong LAU, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] The nervous system consists of the central and peripheral nervous systems, and the central nervous system (CNS) consists of the brain and spinal cord, which control the body. The sensory, motor, and speech disorders are caused by damage to the CNS. Severe damage cause stroke, in which blood vessels in the brain are blocked or ruptured, dementia due to brain cell death or loss of nerve connections, and epilepsy, which causes temporary brain dysfunction due to excessive stimulation of nerve cells. Currently, no definitive treatment for CNS diseases has been developed, and there are many difficulties in delivering drugs or genes to the central nervous system for treatment. In this study, adeno-associated virus(AAV) is used as a drug and gene delivery vector in the CNS by conjugating the biomaterial M, which has the specific binding properties of the central nervous system. AAV is a non-pathological human parvovirus and is attractive for drug and gene delivery because of its safety profile and ability to persist in non-dividing cells for long periods of time. Keywords : AAV, Delivery System, CNS, Biomaterial

485


P0614 Production of Japanese Encephatilis Virus Vaccine Using Plant Expression System

Won-Kyung LEE1, Kiran BHILARE1, Jun-Hye SHIN1,2, Jin-Seo KIM1, Juheon MOON1, Jong-Won KEUM1, Wipa TANGKANANOND3, George LOMONOSSOFF4, Kee-Jong HONG2, Seong-Ryong KIM1,2 1Department of Life Science, Sogang University, Seoul, Korea, 2PhytoMab Co., LTD, Seoul, Korea, 3Department of Biotechnology, Thammasat University, Prathumtani, Thailand, 4Department of Biochemistry, John Innes Centre, Norwich, UK Corresponding Author Email : [email protected] Current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. An alternative vaccine production platform was developed using either Nicotiana benthamiana transient expression system or Oryza sativa cell culutre system. Codon-optimized sequences of multi-epitope peptide (MEP) of JEV with or without signal peptide were cloned into the plant expression vectors, pHREAQ2 or pMYN75. Agrobacterium tumefaciens containing those vectors were used to produce the JEV envelope proteins in either N. benthamiana or O. sativa. Purification of the MEP antigen was established from the N. benthamiana leaf expression. Non-clinical studies such as the efficacy and stability of the JEV antigen is being studied and will be discussed in the meeting. Keywords : Japanese encephalitis virus, multi-epitope peptide , N. benthamiana, vaccine

486


P0615 [Review] Mussel Protein-based Adhesive Therapeutics for Localized Cancer Therapy

Soobeen IM1, Yeonsu JEONG2, Kye Il JOO3, Hyung Joon CHA1,2 1School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 3Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Systemic chemotherapy has been commonly treated after removal of primary tumor to eliminate residual cancer cells and prevent recurrence. However, insufficient local accumulation and systemic toxicity of anticancer drugs restrain effective therapeutic effects in clinical trials, resulting in cancer recurrence and metastasis. Recently, mussel adhesive protein (MAP)-based localized drug delivery platform has been developed to provide prolonged retention and high absorption of drugs in cancer. MAP-based pH- and enzyme-responsive thera-peutics carriers were fabricated and exhibited great surface adhesion on tumor tissue surfaces through superior adhesive properties of MAPs. The unique tumor microenvironments including acidic condition and overex-pressed enzyme induced selective release of anticancer drug and immune checkpoint inhibitor and achieved significant inhibition of tumor growth in vivo. This adhesive nanotherapeutics have great potential as a promising local drug delivery system for highly effective cancer therapy. In this presentation, we will review mussel protein-based adhesive nanotherapeutics as a promising approach for local drug delivery in adjuvant cancer therapy. Keywords : mussel adhesive proteins, adjuvant cancer therapy, adhesive nanoparticles, imuGlu References 1. Yeonsu Jeong, Yunkee Jo, Bumjin Kim, Byeongseon Yang, Kyeil Joo, Hyung Joon Cha, ACS Nano, 12,

8909 (2018). 2. Kye Il Joo, Yeonsu Jeong, Sung-Min Hwang, Mincheol Shin, Jaeyun Lee, Garima Sharma, Haena Lee, Sin-

Hyeog Im, Hyung Joon Cha, Biomaterials, 263, 120380 (2020). 3. Bumjin Kim, Hogyun Cheong, Byeonghee Hwang, Hyung Joon Cha, Angew. Chem, 54, 7318 (2015).

487


P0616 Efficient Activation of Dendritic Cells with Immuno-modulating Functional Nanoparticles in Hydrogel

Jaesung LIM1, Hee Seung SEO1, Jun-Hyeok HAN2, Wooram PARK2, Chun Gwon PARK1 1Biomedical Engineering, Sungkyunkwan University, Suwon, Korea, 2Biomedical-Chemical Engineering, The Catholic University of Korea, Bucheon, Korea Corresponding Author Email : [email protected], [email protected] Modulation of the immunosuppressive tumor microenvironment (TME) is crucial for effective tumor treatment. Herein, we demonstrate an immune-modulating hydrogel containing functional nanoparticles. These nanopar-ticles were synthesized by a heat-up method and coated with CpG oligonucleotide capable of activating the dendritic cells. When the CpG-coated nanoparticles were treated to bone marrow-derived dendritic cells (BMDCs), it upregulated activation marker for DCs such as CD80, CD86, CD40, and MHC2 molecules, suggesting the nanoparticles-mediated maturation of BMDCs. For sustained and local delivery of the prepared CpG-coated nanoparticles to the tumor resection site, the functional nanoparticles were embedded in a hydrogel based on methacrylate-modified hyaluronic acid. Finally, the immune-modulating hydrogel was implanted in a fat pad after the surgical-resected breast tumor model in vivo. The implantation of the immune-modulating hydrogel could be able to effectively activate the DCs in the vicinity of the tumor resection site, resulting in the activation of cytotoxic T cells. We believe this strategy has great potential for effective prevention of tumor recurrence and metastasis. Keywords : Magnetic Nanoparticle, Dendritic Cell, Hydrogel, Tumor Microenvironment, Cancer Immunotherapy References 1. K. S. Kim, J. H. Han, J. H. Park, H. K. Kim, S. H. Choi, G. R. Kim, H. S. Song, H. J. An, D. G. Han, W.R.

Park and K. S. Park, Biomaterials. 221, 119418 (2019). 2. L. Ren and Y. T. Lim, Adv. Funct. Mater, 28, 47 (2018).

488


P0617 Exosomes Derived from Bovine Milk Promotes Hair Regeneration by Activating Wnt/β-catenin Signaling

Yeongji JANG1,2, Hyosuk KIM2, Hyun Kyu SONG1, Sun Hwa KIM2, Yoosoo YANG2 1Department of Life Science, Korea University, Seoul, Korea, 2Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, Korea Corresponding Author Email : [email protected] Exosomes, consisting of nano-sized extracellular vesicles surrounded by a lipid bilayer 30-150 nm in diameter, are released from most cell types. They are found in a variety of biological fluids, in particular, large amount of exosomes are known to be contained in milk. Milk exosomes affect various aspects of cell biology in anti-inflammation and tissue regeneration. Here we focused on the milk exosomes from colostrum, as potential novel treatments for reactivating hair follicle stem cells and in this way enhance hair follicle growth, regeneration and development. Our results showed that milk exosomes can accelerate hair regeneration by activating wnt/β-catenin signaling, which plays an important role in hair regrowth. Further, we also investigated which factors in exosomes have been implicated in hair cycling process. Collectively, based on the contents of exosome from bovine milk, we provide the potential of milk exosomes in treating hair regeneration. Keywords : Milk exosome, Hair regeneration, Wnt/β-catenin

489


P0618 Immune Response of Antigen Adsorbed to Aluminum-based Adjuvant Candidate on MHC-restricted Antigen Presentation

Woo Min HWANG1, Young Ran LEE2, Man Bock GU1 1Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea, 2Center for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering & Technology(KICET), Cheongju, Korea Corresponding Author Email : [email protected] Vaccines are used with adjuvants that elevate the immune response to the antigen. Aluminum-based adjuvants increase the stability of protein antigen adsorption and increase phagocytosis of antigen presenting cells(APCs). In this study, we show that AlOOH has excellent ovalbumin(OVA) adsorption ability, and it was confirmed that the presentation of OVA epitope on the cell surface was increased by phagocytosis of the OVA-loaded AlOOH complex(AlOOH-OVA) by dendritic cells. To analyze the immune response of AlOOH-OVA in mouse model, C57BL/6 mice were injected subcutaneously immunized twice a 2-week interval. Then, serum was collected to analyze the production of OVA specific antibodies. In addition, CD8 T cells were isolated from splenocytes and the function of OVA-specific CD8+ T cells was analyzed. As a result, we confirmed that not only the humoral immune response but also the cellular immune response were increased in the mice injected with AlOOH-OVA than OVA only. These findings suggest that AlOOH can be used as an aluminum-based adjuvant with excellent antigen adsorption efficiency and antigen presentation ability in dendritic cells. Keywords : adjuvants, aluminum oxyhydroxide, immune response

490


P0619 The Regulatory Effect of Alisma Rhizomes and Their Triterpenoids on α3β4 Nicotinic Acetylcholine Receptor Activity

Junho LEE Department of Biotechnology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Alisma Rhizomes is used as a diuretic, hypolipidemic, anti-diabetic and anti-inflammatory agent in traditional East-Asian medicine. In this study, we tested the effect of Alisma Rhizomes on the α3β4 nicotinic acetylcholine (nACh) receptor channel current in Xenopus oocytes. The acetylcholine-induced inward peak current (IACh) was measured with the two-electrode voltage-clamp technique. This experiment shows that the α3β4 nACh receptor cRNA injected into oocytes followed by co-application with Alisma Rhizomes inhibited IACh in a noncompetitive or voltage insensitive condition. The half maximal inhibitory concentration (IC50) of Alisma Rhizomes was 12.5 ± 3.4 ug/ml and the Vmax was 55.4 ± 4.7. Protostane-type triterpenoids are the main active ingredient of Alisma Rhizomes (Alisol A, Alisol B, Alisol B 23-acetate, Alisol C 23-acetate). The respective IC50 values of Alisol A, Alisol B, Alisol B 23-acetate, and Alisol C 23-acetate were 1.7 ± 0.1, 2.8 ± 0.5, 2.6 ± 0.7 and 3.5 ± 0.3 uM in the α3β4 nACh receptor expressed in Xenopus oocytes. Altogether, our research shows that protostane-type triterpenoids may modulate the α3β4 nACh receptors expressed in oocytes in a reversible, concentration dependent and non-competitive manner. Furthermore, this modulation of the nACh receptor activity by protostane-type triterpenoids could underlie the pharmaceutics actions of Alisma rhizome. Keywords : Protostane-type triterpenoids, Nicotinic acetylcholine receptor, Ligand-gated ion channel

491


P0620 Molecular Determinants of α3β4 Nicotinic Acetylcholine Receptors Inhibition by Triterpenoids

Junho LEE Department of Biotechnology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] In the present report, we studied the modulation of triterpenoids on the activity of the human nicotinic acetylcholine receptor type α3β4. Two-electrode voltage clamp experiments were used to test acetylcholine mediated inward current (IACh). Treatment with triterpenoids (dehydroeburicoic acid, 6α-hydroxypolyporenic acid C and pachymic acid) inhibited IACh in a concentration dependent and reversible manner. The IC50 values for pachymic acid, dehydroeburicoic acid, and 6α-hydroxypolyporenic acid C were 14.9, 37.7, and 20.9 μM, respectively. The inhibitory regulation of IACh by each triterpenoid showed in a non-competitive manner on the activity of α3β4 nicotinic acetylcholine receptors. These results show that triterpenoids (pachymic acid, dehydroeburicoic acid, 6α-hydroxypolyporenic acid C) can be used as agents to modulate the activity of nicotinic acetylcholine receptor type α3β4. Furthermore, molecular docking studies of 6α-hydroxypolyporenic acid C on α3β4 nicotinic acetylcholine receptors in silico showed that this molecule interacted predominantly with residues at cavities in the α3 subunit and β4 subunit. This docking assays indicated four potential binding sites for this ligand in the extracellular region at sensor domain of α3β4 nicotinic acetylcholine receptors. In point mutagenesis of those whose Ala substitution, 6α-hydroxypolyporenic acid C potency decreased on W25A of α3 subunit or N109A of β4 subunit in both mutants. The double mutation of W25A of α3 subunit and N109A of β4 subunit was significantly attenuated inhibitory effects by 6α-hydroxypolyporenic acid C. All taken together, this study revealed that molecular basis of α3β4 nicotinic acetylcholine receptors by triterpenoids and provides a novel potent interaction ligand. Keywords : Triterpenoids, Ligand-gated ion channel, nicotinic acetylcholine recepto

492


P0621 Effects of Triterpenoids from Poria cocos Wolf on the Serotonin Type 3A Receptor-mediated Ion Current

Junho LEE Department of Biotechnology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Poria cocos Wolf (P. cocos Wolf) is used to treat chronic gastritis, edema, nephrosis, gastric atony, acute gastroenteric catarrh, dizziness, emesis and vomiting. Triterpenoids are a class of natural compounds produced by P. cocos Wolf that contain acyclic 30-carbon precursors. In this study, we investigated the effect of triterpenoids (PA, Pachymic acid; DA, Dehydroeburicoic acid; HA, 3-hydroxylanosta-7,9(11),24-trien-21-oic acid) on human 5-hydroxytryptamine 3A (5-HT3A) receptor channel activity, which is one of the ligand-gated ion channel families. The two-electrode voltage-clamp technique was used to examine 5-HT3A mediated current. The inhibitory effect of triterpenoids on 5-HT-induced inward current (I5-HT) occurred in a concen-tration dependent and reversible manner. Furthermore, the half-inhibitory concentrations (IC50) of PA, DA and HA were 3.2 ± 0.2, 5.5 ± 0.6 and 1.4 ± 0.2 μM, respectively. This corresponded to an order of potency for the inhibition of I5-HT in oocytes expressing human 5-HT3A receptor of HA > PA > DA. Finally, inhibition of I5-HT by triterpenoids occurred in a non-competitive manner, while inhibition by HA and PA showed more voltage-dependency. Taken together, these results indicate that triterpenoids may regulate the expressed 5-HT3A receptors in Xenopus oocytes. Furthermore, this regulation of the ligand-gated ion channel activity by triterpenoids may be one of the pharmacological actions of Poria cocos Wolf. Keywords : Triterpenoid, Poria cocos Wolf, Serotonin type 3A receptor

493


P0622 Chitosan-alginate-pectin-coated Suspended Liquid-encapsulating (CAPSuLE) Marbles for Therapeutic Molecules Storage and Delivery

Yoojin KIM1, Seokmin OH1, Dongsoo LEE1, Mihyun KIM1, Seohee KIM1, Eunmi KIM2, Jae-Hyung JANG1 1Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea, 2Material Research Division, R&D Unit, AmorePacific Corporation, Yongin, Korea Corresponding Author Email : [email protected] Developing a cutting-edge system ensuring long-lasting functionality of therapeutic molecules and fulfilling diverse delivery modes is challenging. A quasi-spherical triple-layered liquid transporter with multimodal characteristics was fabricated by the collaboration of three concepts: liquid marble, synergistic carbohydrate packing, and complex coacervation. To our knowledge, pectin, a liquid stabilizing substance that can convert rapidly from a transient hydrophobic powder to a hydrophilic gel, has not yet been used as a liquid–air interfacial material. In addition to the structural chirality-mediated packing between pectin and alginate, an electrostatic association driven coacervate layer was generated on the outermost layer to tailor the pectin-coated liquid marble as a therapeutic agent carrier. The resulting system, Chitosan–Alginate–Pectin-coated Suspended-Liquid-Encapsulating (CAPSuLE) marble, showed sufficient mechanical strength to resist the harsh surround-dings and exhibited versatile features: ecofriendly sustainability, responsiveness to multiple external stimuli, coacervate-driven coalescence for linking individual building blocks, and a self-healing ability. The proposed CAPSuLE system can expand the applicability of the liquid marble concept in the field of applied bioscience and biomedical engineering. Keywords : capsule, pectin, liquid marble, coacervate, controlled delivery References 1. Y. Kim, S. Oh, H. Lee, D. Lee, M. Kim, H.S. Baek, W.S. Park, E. Kim and J-H Jang, Biomater. Sci., 9,

1639-1651 (2021).

494


P0623 Amelioration of Angiogenesis, Adipogenesis and Lipogenesis to Attenuate Obesity by Targeting the Associated Promoting Signaling Molecules and Transcriptional Factors with Combination of Theasaponin E1, Stevioside and Rebaudioside A in Obesity Model Cells and Animals

Muhammad Imran KHAN1, Muhammad Zubair KHAN1, Jin Hyuk SHIN1, Tia Sang HAN1, Min Yung KIM2,3, Jong Deog KIM1,2,3 1Department of Biotechnology, Chonnam National University, Yeosu, Korea, 2Department of Refrigeration Engineering, Chonnam National University, Yeosu, Korea, 3Research center on Anti-Obesity and Health Care, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Obesity a metabolic disorder is a general public health problem, associated with other metabolic and cardio-vascular diseases and health complications characterized by increased body weight due to abnormal expansion of adipose tissues with over accumulation of fats. Natural products with adipogenesis regulating potential are highly recommended as therapeutic agent for obesity treatment. In present study we evaluated thesynergistic anti-adipogenic and anti-lipogenic efficacy of standardized formulation of rebaudioside A, sativoside and theasaponin E1 (RASE1). Anti-angiogenesis effects of RASE1was investigated in-vivo with high fats and high carbohydrates diet induced obesity model mice. Orlistat was used as positive control while animals not treated with samples were selected as normal controls. Mice were divided different groups with five mice per group. Adipose tissues, livers and blood were analyzed after dissection of the animals at the end of experiment. Results demonstrated thatRASE1 inhibited. Oral administration of theRASE1 significantly reduced body weight, body fat-pad weights, blood cholesterols and triglycerides level, ALT, AST glucose, insulin, and various adipokines e.g. leptin,IL-10, IL-6, and TNF-α levels in mice fed with high lipids and carbohydrates diets andRASE1 as compared to the corresponding untreated controls. RASE1 suppressed the expression of adipogenic genes and lipid metabolism genes (LPL, aP2, PPARγ, SREBP, leptin and C/EBPα, FAS, and MLYCD,ACC-1 and GPAT,) in adipose tissues and livers of mice. Moreover, the natural products combination RASE1enhance the levels of AMP-activated protein kinase (AMPK) in liver tissue as well as adiponectin in serum and adipose tissue. Collectively, these results revealed that RASE1is a potential therapeutic formulation for treating or preventing obesity. Keywords : Metabolic disorder, synergistic, RASE1

495


P0624 Amelioration of Taupathy via Suppression of Tau Hyperphosphorylation by Reducing the Expression Level of Various Involved Genes and Kinesis with Theasaponin E1, a Therapeutic Target for Alzheimer’s Treatment

Muhammad Imran KHAN1, Muhammad Zubair KHAN1, Jin Hyuk SHIN1, Tae Sang HAN1, Jong Deog KIM1,2 1Department of Biotechnology, Chonnam National University, Yeosu, Korea, 2Research center on Anti-Obesity and Health Care, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Alzheimer disease (AD) is neurodegenerative disorder and principal type of dementia. Formation of extracel-lular senile plaques and cytoplasmic neurofibrillary tangles (NFT) in brain due to the abnormal production and aggregation of amyloid beta (Aß)and tau proteins are the major molecular cause of AD. Paired helical filaments (PHF) are formed due to hyperphosphorylation of tau leads to formation of NFT which accumulate and aggregates causing pathological conditions and eventually cause neuronal death. Tau phospohorylation is controlled byseveral genes and proteins. Several kinesis such as GSK3 beta, CDK5 and CAMII, etc are involved in phosporylation of tau and phasphateses such as PH1A, PH1B etc are involved in dephosphorylation of tau, the imbalance of which leads to hyperphosphorylation and finally NFT. In thisstudy we elucidate the effects of theasponin E1 on the activities andgene’s expression level of kinases and phasphateses involved in the phos-phorylation and dephosphorylation of tau. The therapeutic neuroprotective effects of green tea seed isolated theasaponin E1 was examine using SHY-5Y cell lines. Effect of thaesaponin E1 on the expression level oftau generating and phosphorylation promoting genes were investigated by RT- PCR,ELISA and western blotting.Cells were cultured in EMEM media supplemented with 10% FBS in 6-well plate and incubated at 37°C in a CO2 incubator. Cells were treated with different concentrations of E1andwere processed for checking the enzymatic activities, RNA and proteins extraction.Our results demonstrated that theasaponin E1 significantly reduced Aβ concentration and tau hyperphosphorylation in dose-dependent manner by suppressing expression level of GSK3 β,CDK5, CAMII, MAPK, EPOE4(E4),PICALM . E1 also enhance expression level of PH-1A and TREM2 etc.However, the inhibitory and gene suppressiveeffects werecomparatively lower in case of IL-1ß and BIN1.ELISA and western blotting resultsshown reduction in APP and Aß and p-tau. The overall results revealedthat E1 significantly reduce Aβ and p-tau level hence possesses neuroprotective effects. Keywords : amyloid beta (Aß), amyloid plaques, neurofibrillary tangles

496


P0625 Attenuation of Obesity with Synergistic Therapeutic and Preventive Effects of Natural Products by Suppression of the Associated Promoting Signaling Pathways

Muhammad Imran KHAN1, Jin Hyuk SHIN1, Tia Sun SHIN2,3, Jong Deog KIM1,3 1Department of Biotechnology, Chonnam National University, Yeosu, Korea, 2Department of Food Science and Nutrition, Chonnam National University, Gwangju, Korea, 3Research center on Anti-Obesity and Health Care, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] The aim of the present study was to evaluate anti-obesity activities coupled with anti-angiogenic activities of stevia and green tea extracted compounds in HUVECs and 3T3- L1 cells. Dried plants leaves were extracted with 70% ethanol and pure compounds fractions were obtained by HPLC. The green tea seed extracted saponin and stevia extracted compounds were combined in standardized formulation as RASE1. Antiangiogenic activities were determined by anti-proliferative effect of RASE1on HUVECs. In the presence of various concentrations of RASE1, 3T3-L1 preadipocytes were induced to differentiate. Lipid accumulation in differentiated adipocytes was quantified by Oil-red O staining. RASE1significantly suppressed angiogenesis by inhibiting proliferation and tube formation of HUVECs via down regulating VEGRF 2, PI3K, β‐catenin, NF‐kB, and Akt1 in a dose dependent manner. RASE1 inhibited lipid accumulation by suppression of adipogenesis and lipogenesis promoting signaling proteins, transcriptional factors and genes i.e. PPARγ, CCAAT, C/EBPα, aP2, FAS, and LPL.RASE1 enhanced AMPK activation to p-AMPK in 3T3 cells quantified and expressed by western blotting. RASE1 inhibit lipid accumulation by regulating adipogenesis and lipogenesis related genes and signaling proteins. The anti-obesity activities of RASE1 associated with antiangiogenic activities. Hence RASE1 is a good combination of natural products with anti-obesity potential. Keywords : angiogenesis, antiadipogenic, 3T3-L1

497


P0626 Green Tea Seed Extracted Theasaponin E1 Ameliorates Alzheimer’s Disease by Attenuating Aß Peptide Levels in SweAPP N2a Cells

Muhammad Imran KHAN1, Jin Hyuk SHIN1, Jong Deog KIM1,2 1Department of Biotechnology, Chonnam National University, Yeosu, Korea, 2Research center on Anti-Obesity and Health Care, Chonnam National University, Yeosu, Korea Corresponding Author Email : [email protected] Alzheimer’s disease (AD) is the most frequent type of dementia affecting memory, thinking and behaviour. The major hallmark of the disease is pathological neurodegeneration due to abnormal aggregation of Amyloid beta (Aβ) peptides generated by β- and γ-secretases via amyloidogenicpathway. To evaluate the effects of theasaponin E1 on the inhibition ofAβ producing β- and γ-secretases (BACE1, PS1& NCT) and acetyl-cholinesterase and activation of the non-amyloidogenic APP processing α-secretase (ADAM10). Also, Theasaponin E1 effects on Aβ degrading and clearing proteins neprilysin and insulin degrading enzyme (IDE). The effect of Theasaponin E1 on these crucial enzymes was investigated by RT-PCR, ELISA, western blotting and fluorometric assays using mouse neuroblastoma cells (SweAPP N2a). Theasaponin E1 was extracted and purified from green tea seed extract via HPLC, and N2a cells were treated with different concentrations for 24 hours. Gene and protein expression in the cells were measured to determine the effects of activation and/or inhibition of theasaponin E1 on β- and γ-secretases, neprilysin, and IDE. The results demonstrated that theasaponin E1 significantly reduced Aβ concentration by activation of the activities of α-secretase and neprilysin and expression level of ADAM10 and NEP. The activities of β- and γ-secretase were reduced by theasaponin E1 and BACE1, PS1, and nicastrin were down regulated in a dose-dependent manner. Similarly, theasaponin E1 significantly reduced the activities of acetylcholinesterase. Also reduced the expression level of APP.Overall, from the results it is concluded that green tea seed extracted saponin E1 possess therapeutic significance as a neuroprotective natural product recommended for the treatment of Alzheimer’s disease. Keywords : Alzheimer’s disease, amyloid precursor proteins, amyloidogenic pathway, α-secretase, Theasa-ponin E1, SweAPP N2a cells

498


P0627 Adiponectin Induces the Age-related Salivary Gland Lipid Accumulation

Ji Won KIM Department of Otorhinolaryngology-Head and Neck Surgery, Inha University College of Medicine, Incheon, Korea Corresponding Author Email : [email protected] Background Dry mouth or salivary hypofunction is frequently observed in aged individuals. Enhanced lipid accumulation plays a critical role in cell senescence in the salivary gland (SG). We investigated the molecular mechanisms by which signaling molecules control adiponectin mediated lipogenesis and senescence in SG. Methods We compared the histology and senescence markers among young, old, and far-old aged SG cells. Morpho-logical change examination, senescence-associated β-galactosidase assay (SA-β-gal), expression patterns of 8OHdG, TUNEL assay were performed. We extracted RNA from young, and old aged SG cell and performed microarray and KEGG pathway analysis. Results The histological findings in far-old aged SG demonstrated the presence of more lipocytes, chronic inflame-mation, fibrosis, and foci of lymphocytic infiltration compared with tissue from young-aged SG. In old SG tissue, the expression of cellular senescence was identified in β-galactosidase assay, and apoptosis was examined by TUNEL assay. High 8-OhdG immunostaining reactivity was detected in old SG. In microarray analysis, senescence-associated ADIPOQ and adiponectin expression was checked. Also, immunohistochemical staining and qPCR using adipoQ-siRNA showed adiponectin is associated with age-related salivary gland lipid accumulation. Conclusion Adiponectin might induce the age-related salivary gland lipid accumulation. ADIPOQ could be target molecule for the modulation of cell senescence of SG. Keywords : adiponectin, salivary gland, aging, lipogenesis

499


P0628 Smart Contact Lens with Transparent Silver Nanowires Strain Sensor for Continuous Intraocular Pressure Monitoring

Tae yeon KIM, Sei kwang HAHN Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Continuous intraocular pressure (IOP) monitoring can provide a new paradigm in the management of patients with glaucoma as a facile alternative for the conventional diagnostic methods. However, the low sensitivity and functional instability of IOP sensors has limited the practical applications of smart IOP contact lenses for glaucoma. Here, we have developed a smart contact lens integrated with a transparent silver nanowire (AgNW) IOP strain sensor and wireless circuits for non-invasive, continuous IOP monitoring. After confirming the robust stability of the IOP sensor in the smart contact lens in the presence of tears and repeated eyelid blink model cycles, we were able to monitor IOP changes on polydimethylsiloxane (PDMS) model eyes in vitro. In vivo tests demonstrated that our fully integrated wireless smart contact lens could successfully monitor the change of IOP in living rabbit eyes, which was clearly validated by the conventional invasive tonometer IOP test. Taken together, we could confirm the feasibility of our smart contact lens as a non-invasive platform for IOP monitoring of glaucoma patients. Keywords : silver nanowires, sensor, smart contact lens, intraocular pressure, glaucoma

500


P0629 [Review] Quantum Dots For Killing Multidrug Resistant Bacteria via Photodynamic Therapy

Inho NAM, Hyung Joon CHA Division Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] The emergence of multidrug resistant (MDR) bacteria has evolved in response to extensive use of antibiotics and represents a significant issue in the biomedical field and public health concern. However, a treatment of MDR bacteria has limitations due to time-consuming for isolation and ineffective antibiotics. Photodynamic therapy using a quantum dot (QD) is recently proposed as a promising solution for killing MDR bacteria. The QDs can damage bacteria independently of the antibiotic resistance pattern by generating reactive oxygen species responding to light exposure with great advantages of superior photostability and tunability compared with conventional organic photosensitizer. However, the majority of QDs contains cadmium or other heavy metals which are essential limitaion to the applications in biomedical field. For that reason, researches on cadmium-free and/or carbon-based QDs have been developed. In this review, the innovated strategies for designing biocompatible QDs and their benefits are reviewed for effective MDR bacteria treatment with reduced biosafety concerns. Keywords : Quantum dots, Multidrug-resistance bacteria, Antibiotics , Photodynamic therapy References 1. Courtney. C, Goodman. S and McDaniel, J. Nat. Mat. 15, 529 (2016). 2. Ethel. O, Elnaz. Y, Imad. N, Ivan. P and Elaine. A, J. Nanoscale. 12, 10609(2020). 3. Yanhong. L, Hui. H, Weijing. C and Baodon. M, Mater. Chem. Front. 4. 1586(2020). 4. Q. Xin, H. Shah, A. Nawaz, M. Z. Akram and A. Batool, J. Adv Mat 31. 1804838(2019). 5. Yu. L, Scott. H, Yu. S, Chun. H and Binesh. U, J. Adv. Health. Mat. 22, 2545(2016).

501


P0630 Cell Derived Vesicles as Drug Delivery Carriers and Functional Biomaterials

Jihyeon SONG, Hyejung MOK Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea Corresponding Author Email : [email protected] Cell derived vesicles have been noted as emerging biomaterials as drug carriers, coating materials, and bioactive materials. Previously, cancer cell-derived vesicles were coated on the surface of polymeric micro-particles to increase surface roughness to provide efficient delivery to phagocytic cells e.g. macrophages and dendritic cells. We also prepared nano-sized vesicles from platelets as functional hemostatic biomaterials. Compared to platelets, platelet derived nanovesicles showed efficient hemostatic activity without release of inflammatory cytokines. In addition, we developed platelet derived vesicles from activated-platelets to increase hemostatic activity of vesicles. In particular, nanovesicles from activated platelets formed denser and more prompt fibrin networks than vesicles from non-activated platelets probably due to surface active glycoproteins for hemostasis. Lastly, anticancer drugs were loaded within cancer cell derived vesicles to deliver anticancer drugs to target cancer cell via homotypic interactions. In addition, nanovesicles from cancer cells and platelets exhibited promising biocompatibility, which could serve as functional biomaterials in vitro and in vivo. Keywords : cell derived vesicles, anticancer drug, functional biomaterials

502


P0631 Temperature and Oxidation-sensitive LCST Polymer Gel Loaded with Photosensitizer and Anticancer Drug

Soo Chan PARK, Jin-Chul KIM Department of Biomedical Science & Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Polymer gels have been synthesized to make the drug carrier sensitive to temperature and oxidation. The gel used indocyanin green (ICG) as a photosensitizer and doxorubicin (DOX) as an anticancer drug. First, an LCST block copolymer was prepared through free radical synthesis of HEA (Hydroxyethyl acrylate) and PVS (Phenyl vinyl sulfide). Structure and quantitative analysis was performed through 1H-NMR, and it was observed that it was oxidized by H2O2. Absorbance was measured using a UV-Vis spectrophotometer to observe the change in LCST for each PVS ratio. After that, a gel was prepared using Glutaraldehyde and Ethylene Glycol dimeth-acrylate as a crosslinking agent. Insulation was used to see both the effect of temperature and ROS, and a constant-temperature water bath was used to observe the effect of ROS only, and the release rate according to the NIR irradiation for gel was observed. ICG generates ROS and heat by 808 nm NIR irradiation. Because of PVS has a sulfide group, it can be oxidized in response to ROS. The oxidized sulfide group becomes a sulfoxide or sulfone group and becomes hydrophilic so that increasing LCST. Thus, the swollen gel can release the drug. Keywords : Oxidation Sensitivity, Photosensitizer, LCST, NIR, Drug Delivery

503


P0632 Reverse Phase-HPLC Method Development for Simultaneous Determination of Purity of Collagenase Class I and II

Junghun PARK1, Woo Jong LEE1,2 1Safety System R&D Group, Korea Institute of Industrial Technology (KITECH), Yeongcheon, Korea, 2Connext Co., Ltd, Yeongcheon, Korea Corresponding Author Email : [email protected] Collagenases are enzymes that recognize and break the peptide bonds in collagen. The collagenases are available as process materials for cell separation and raw materials for biopharmaceuticals. The collagenases are classified into class I and II according to their structures and enzymatic abilities [1]. To analyze the purity of the collagenases, reverse phase (RP)-HPLC is mainly used [2]. In general, RP-HPLC method was utilized by a general-purpose protein analysis-based method without analyte-specific optimization process. Therefore, it is very difficult to sensitively identify product-related impurities and it does not work simultaneously. To overcome this limitation, we developed an optimized RP-HPLC method that can simultaneously analyze two collagenase classes for identifying product-related impurities. By using recombinant collagenase class I and II, RP-HPLC method optimization was performed by comparing various conditions such as mobile phase, gradient, column temperature, and injection amounts. As a result, we successfully developed RP-HPLC method for collagenases which shows greatly enhanced purity determination performances, and which can be analyze collagenase class I and II simultaneously. Keywords : Collagenase, Reverse phase-HPLC, Method development, Recombinant protein, Purity References 1. G. H. J. Wolters, et. al, Diabetes, 44(2), 227-233 (1995). 2. H. Brandhorst et. al, Transplantation, 85(3), 456-461 (2008).

504


P0633 Mechanically Supportable Bio-adhesive Liquid-phase Delivery System by Complex Coacervation

Hyun-Woo PARK, Hyo-Woo LEE, Jae-hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Complex coacervation is liquid-liquid phase separation by charge interaction of oppositely charged poly electrolytes or macroions. Because of the characteristic of complex coacervates, it can be injectable to very narrow aperture or crack. So, it can be easily transplanted to area that are difficult to transplant by surgical operation such as brain or spinal cord. However general complex coacervate easily spread out in bio system because of its aqueous property. To solve this challenge, bio-adhesive and mechanically supportable complex coacervate was developed using chitosan catechol and heparin. Chitosan catechol which is positively charged polymer has bio-adhesive property and solidify in neutral pH by crosslinking. And heparin which is negatively charged polymer has affinity to many growth factors so that it can encapsulate many growth factors. Therefore, the complex coacervation system of chitosan catechol and heparin give many conveniences to medical fields. Keywords : Complex coacervation, Bio-adhesive, Injectable, Scaffold

505


P0634 Development of Nano-self-assembly Gene Delivery System Using Cellulose Nanocrystal with Zinc/DNA Nanocluster

Kyoungcheol CHOI1, Chan Hee LEE1, Sugyeong KIM1, Kwang Suk LIM1,2 1Interdisciplinary Program in Biohealth-machinery convergence engineering, College of ACE, Kangwon National University Chuncheon, Korea, 2Department of Biotechnology and Bioengineering, College of ACE, Kangwon National University, Chuncheon, Korea Corresponding Author Email : [email protected] Gene therapy is one of promising therapeutic drugs to treat diabetes, cancer, rheumatism, and incurable diseases through gene transfer such as DNA and RNA. Effective gene delivery into a target cell or tissue is the most important part of gene therapy development. The development of a stable and suitable gene delivery system can significantly reduce the risk profile of clinical gene therapy. Although various non-viral and viral systems have been reported, there are still many limitations including toxicity and immunogenicity in pharmaceutical applications. Cellulose nanocrystals have excellent biocompatibility and properties, so they are used in various industries and research. Recently, it has been reported that CNC can be used in biomaterials, tissue engineering, and drug delivery in the field of biotechnology. To develop the gene delivery system of CNC, we applied zinc/DNA nanocluster systems. In the previous study, we have demonstrated the mechanism of the ZD cluster by endocytosis and determined their transfection efficacy. In this study, we developed a nano-self-assembly gene delivery system using CNC and Z/D nanoclusters. The formation of the CZD complex (CNC with ZD nanocluster) was optimized and detected their morphology and size. The protective effect of CZD complex was evaluated in H9c2 cells against oxidative stress using metallothionein and heat shock protein27 expression vector as DNA. This CZD complex might be useful gene delivery systems to treat various diseases. Keywords : Cellulose nanocrystal, Metal-bound DNA, Gene therapy References 1. K.S Lim, Daniel Y Lee and D.A Bull, Adv. Funct. Mater, 25 (2015). 2. K.S Lim, D.Y Lee and Y.W WON, ACS Macro lett., 6 (2017). 3. Cynthia E. Dunbar and Katherine A. High, Science, 359 (2018). 4. Hao Yin, Rosemary L. Kanasty, Nat Rev Genet, 15 (2014).

506


P0635 Development of Therapeutic for Osteoporosis Treatment

Jeongman AN1, DongYun LEE1, Yong-kyu LEE2 1Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea, 2Department of Chemical & Biological Engineering, Korea National University of Transportation, Chungju, Korea Corresponding Author Email : [email protected] We have designed an oral formulation targeting the bile acid transport system that facilitates a large permeation of rhPTH from the intestine to target sites. In addition, the bile acid transport system possessed both active and passive transport of oral rhPTH formulations using the enterohepatic recirculating system. We have proved that various transport proteins, receptors, physicochemical, and biological factors and mechanisms are involved in this complex recycling route. To date, we have proved the advantages of the bile acid transport system to target delivery of this hormone, the biophysical and biochemical stability of our proposed system. Keywords : osteoporosis, biotechnology, drug and gene delivery

507


P0636 Development of Therapeutic for Diabetes Treatment

Jeongman AN1, DongYun LEE1, Yong-kyu LEE2 1Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea, 2Department of Chemical & Biological Engineering, Korea National University of Transportation, Chungju, Korea Corresponding Author Email : [email protected] We investigated the effect of dose quantity on the therapeutic efficacy of oral GLP1 gene therapy. Using a multimodal gene complex (GLP1/PTCA), we showed glycemic improvement drawn for up to 1 week in three progressives diabetic mouse models. To the best of our knowledge, this is the first report of any dosage form of GLP1 agonist for the most extended period of glucose control using a minimal quantity of genes (＜0.5 mg human equivalent dose). Since Rybelsus® is the only FDA-approved oral GLP1 agonist that needs to be taken with 7 or 14 mg daily, oral GLP1/PTCA may provide the first once or bi-monthly oral treatment option for diabetic patients according to allometric principles. Keywords : diabetes, biotechnology, Drug and gene delivery

508


P0637 Blue Mussel-derived Peptides Lead to Activation of Nrf2 and HO-1 Signaling Pathway to Alleviate H2O2-induced HUVEC Damage

Yunok OH1, Jae-Young JE2 1Convergence Research Center for Smart Healthcare, Kyungsung University, Busan, Korea, 2Department of Marine-Bio Convergence Science, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] Increased oxidative stress is considered as a major cause of endothelial dysfunction, which is linked to the pathogenesis of cardiovascular diseases. Accordingly, this study was undertaken to identify the cytoprotective peptides from blue mussel and to investigate molecular mechanisms underlying the cytoprotective in oxidative stress-mediated endothelial injury. In this study, two peptides with the sequence of FTVN and EPTF were identified and their cytoprotective effects were confirmed. The results showed that the cytoprotective peptides ameliorated H2O2-mediated HUVEC injury through reducing mitochondrial ROS generation and upregulation of Nrf2-mediated antioxidant defense system. Flow cytometry and nuclear staining analysis revealed that BMCH treatment markedly reduced apoptosis in HUVECs. Western blot analysis revealed that FTVN and EPTF pre-treatment significantly prevented H2O2-mediated HUVEC apoptosis by activation of Nrf2 and HO-1 pathway. Taken together, these results suggest that blue mussel -derived peptides may be a promising agent for oxidative stress-related diseases through upregulating cellular antioxidant capacity and downregulating apoptosis. Keywords : antioxidant, endothelial dysfunctiontion, cytoprotective, apoptosis

509


P0638 Enhancing Osteogenic Effect of Gallic Acid-g-chitosan Through Wnt/beta-catenin Signaling Pathway

Yunok OH1, Jae-Young JE2 1Convergence Research Center for Smart Healthcare, Kyungsung University, Busan, Korea, 2Department of Marine-Bio Convergence Science, Pukyong National University, Busan, Korea Corresponding Author Email : [email protected] Chitosan and its derivatives have been considered as good materials for bone tissue regeneration. Generally, antioxidants were implicated as potential reagents to enhance osteogenesis in bone cells. Thus, we first prepared chitosan conjugated with gallic acid (GA) to obtain a natural-compound based antioxidant and investigated the antioxidant effect of Gallic acid-g-chitosan (GAC). GAC clearly showed significantly higher antioxidant activity that the plain chitosan (PC). We further investigated the effect of GAC antioxidant on osteogenic differentiation in murine bone marrow stem cells (mBMMSCs). Next, we investigated the effect of GAC antioxidant on osteogenic differentiation in murine bone marrow stem cells (BMSCs). GAC enhanced phenotype markers of osteogenic differentiation such as alkaline phosphatase (ALP) and mineralization in a dose dependent manner. Molecular mechanism studies revealed that GAC activated Wnt1 and Wnt3a mRNA expressions followed by translocation of β-catenin into the nucleus and increased expression of β-catenin targeted genes, including Runx2, asterisks, and cyclin D1 as well as type I collagen. Taken together, our study showed that GAC antioxidant effectively enhanced the osteogenic effect through Wnt/b-catenin signaling pathway in mBMMSCs. Keywords : chitosan, antioxidant, osteogenesis, Wnt/beta-catenin signaling

510


P0639 A Study on the Eco-friendly Functional Materials from Marine Microalgae

Eun Young YOON, Jaeyeon PARK, Seun Joo MOON, Min Kyoung JUNG, Tae Yeon YIN Advanced Institutes of Convergence Technology, Suwon, Korea Corresponding Author Email : [email protected] The heterotrophic dinoflagellate has been reported to be a typical efficient producer of long-chain essential fatty acids such as, eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3). Two new compounds—a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG)—were isolated from the methanolic extract of the heterotrophic dinoflagellate Oxyrrhis marina cultivated by feeding on dried yeasts. The complete structures of these compounds were determined using NMR spectroscopy and chemical reactions. The trioxilin was found to be derived from DHA, and the polar lipid SQDG was found to contain a docosahexaenoyl substituent. Compounds 1 and 2 significantly suppressed the NO production induced by LPS on RAW264.7 cells without affecting cell viability. Thus, we research for utilizing high value-added products originating from marine plankton and securing original technologies related to mass culture technology. Keywords : High concentration DHA, Marine plankton, Functional raw materials, Eco-friendly materials

511


P0640 The Essential Function of miR-5739 in Embryonic Muscle Development

Ji-Heon LEE, Joon HWANG, Yong-kyu LEE 4D Biomaterials Center, Korea National University of Transportation, Chungju, Korea Corresponding Author Email : [email protected] MicroRNAs (miRNAs) consist of small nucleotides, which act as transcription factors that bind to the 3’ untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of microRNA-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 gene is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. These results demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development. Keywords : Human embryonic stem cells, Mesoderm development, micro RNA, Muscle, Vessel References 1. D. Subramanyam, S. Lamouille, R.L. Judson, J.Y. Liu, N. Bucay, R. Derynck, R. Belloch. Nat Biotechnol.

29 (2011). 2. H.M. Chang, N.J. Martinez, J.E. Thornton, J.P. Hagan, K.D. Nguyen, R.I. Gregory, Nat Commun. 3 (2012). 3. A. Amador-Arjona, F. Cimadamore, C.T. Huang, R. Wright, S. Lewis, F.H. Gage, A.V.Terskikh. Proc Natl

Acad Sci USA. 112 (2015). 4. J.K. Yoo, J. Kim, S.J. Choi, C.H. Kim, D.R. Lee, H.M. Chung, J.K. Kim. Biochem Biophys Res Commun.

415 (2011). 5. D. Srivastava, A.J. Heidersbach. Circ Res. 112 (2013). 6. S.J. Park, J.H. Lee, S.G. Lee, J.E. Lee, J. Seo, J.J. Choi, T.H. Jung, E.B. Chung, H.N. Kim, J. Ju, Y.H. Song,

H.M. Chung, D.R. Lee, S.H. Moon. Stem Cells. 37 (2019). 7. E.N. Poon, B. Hao, D. Guan, M. Jun Li, J. Lu, Y. Yang, B. Wu, S.C. Wu, S.E. Webb, Y. Liang, A.L. Miller,

X. Yao, J. Wang, B. Yan, K.R. Boheler. Cardiovasc Res. 114 (2018). 8. K.A. D'Amour, A.D. Agulnick, S. Eliazer, O.G. Kelly, E. Kroon, E.E. Baetge. Nat Biotechnol 23 (2005). 9. G. Bindea, B. Mlecnik, H. Hackl, P. Charoentong, M. Tosolini, A. Kirilovsky, W.H. Fridman, F. Pages, Z.

Trajanoski, J. Galon. Bioinformatics. 25 (2009).

512


P0641 Antimicrobial Resistance and Molecular Genotyping of Enterococci Strains Isolated from Swine Farms

Shabnam AGHAMORADI, Jae-Young OH, Lee Sang HYEON, Hyeonbin KIM, Jong-Chan CHAE Division of Biotechnology, Jeonbuk National University, Iksan, Korea Corresponding Author Email : [email protected] Enterococci are gastrointestinal commensals that can colonize a variety of environments such as human, animals, soil, water, and food. The emergence of enterococcal resistance to key antimicrobial agents is being emphasized in terms of transmission of antimicrobial resistance and public health. This study was investigated on antimicrobial resistance and molecular genotyping in 433 Enterococcus faecalis (EFS) and 155 E. faecium (EFM) isolates from swine farms. In the EFS strains, the resistance to quinupristin-dalfopristin (Q/D) was the highest at 91.9% (398/433), followed by tetracycline (91.5%), gentamicin (high-level, 66.1%), chloramphenicol (57.0%), ciprofloxacin (51.7%), and linezolid (16.2%). Tetracycline resistance from EFM strains was the highest at 38.1% (59/155), followed by gentamicin (18.1%), ciprofloxacin (16.1%), chloramphenicol (11.6%), nitrofurantoin (11.0%), Q/D (10.3%), and ampicillin (2.6%). Resistance to vancomycin was not detected in all enterococci strains. Sixty-five sequence types (STs) were identified in the EFS strains, and ST1029 was the most (18.0%, 78/433), followed by ST506 (7.6%), ST1019 (7.2%), ST476 (7.2%), ST620 (5.5%), ST585 (5.3%), and ST16 (4.8%). EFS ST1029 and ST620 strains were mainly found in swine farms in two regions, but ST1019, ST585, ST506, ST476, and ST16 strains were distributed in all farms in three regions. Among the STs in EFM, ST1619 was the most dominant (20.7%, 32/155), followed by ST296 (n=11) and ST646 (n=11). The EFM strains belonging to ST1619 were detected only in one farm and were isolated at all breeding stages. Linezolid-resistant E. faecalis (LREFS) strains were detected in pigs at all breeding stages in three regions, of which ST476 and ST585 predominated. In addition, all LREFS strains were optrA-positive and cfr-negative and ST100 strains carrying both optrA and poxtA were identified in one farm. Enterococci isolated from domestic pigs showed high resistance to other antibiotic categories except penicillins and glycopeptides. Furthermore, EFS strains carrying a plasmid-mediated linezolide resistance gene were first identified in this study, and were highly correlated with STs isolated from human patients. It is necessary to clarify the epidemiological association between humans and animals through further studies on the mechanism of resistance. Keywords : enterococci, linezolid-resistant E. faecalis, optrA, poxtA References 1. A. R. Freitas, A. P. Tedim, C. Novais, V. F. Lanza and L. Peixe, Microb. Genom. 6:e000350 (2020). 2. M. J. G. T. Vehreschild, M. Haverkamp, L. M. Biehl, S. Lemmen, and G. Fätkenheuer, Vancomycin-

resistant enterococci (VRE): a reason to isolate?, Infection. 47(1), 7-11(2019).

513


P0642 Melatonin Nanospheres Inhibit the Phosphorylation of p38 MAPK and NF-κB Responsible for the Gut Apoptosis and Autophagy in Induced by V. vulnificus VvpE

Seong-Ryeong LIM, Su-Ji PARK, Sei-Jung LEE Department of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan, Korea Corresponding Author Email : [email protected] Melatonin, a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, has been used as a food additive for the treatment of various diseases, but a functional role of melatonin nanosphere (MN) in the regulation of the autophagic cell death in the gut during bacterial infection has not described yet. In this study, we have investigated the effect of MN prepared from melatonin during autophagic death of colonic epithelial HCT116 cells induced by the VvpE produced by V. vulnificus. Interestingly, MN significantly blocked the apoptotic and autophagic process via inhibition of intracellular reactive oxygen species (ROS) induced by recombinant VvpE. Also, MN significantly inhibited the phosphorylation of p38 mitogen-activated protein kinases and nuclear factor-kappa B responsible for the expression of LC3-II and Beclin-1 in VvpE-treated HCT116 cells. We suggest that MN blocks autophagic cell death induced by VvpE via the inhibition of ROS-mediated signaling events in HCT116 cells. Keywords : Autophagic Cell Death, Melatonin Nanosphere, V. vulnificus References 1. Lee, Y.M.; Park, J.P.; Jung, Y.H.; Lee, H.J.; Kim, J.S.; Choi, G.E.; Han, H.J. and Lee, S.J. J Biomed Sci 27,

21 (2020). 2. Chang, C.C.; Huang, T.Y.; Chen, H.Y.; Huang, T.C.; Lin, L.C.; Chang, Y.J. and Hsia, S.M. Oxid. Med. Cell

Longev. 2018, 9015765 (2018).

514


P0643 Tart Cherry Extract Containing Chlorogenic Acid, Quercetin, and Kaempferol Inhibits the Mitochondrial Apoptotic Cell Death Elicited by Airborne PM10 in Human Epidermal Keratinocytes

Do-Wan KIM, Sei-Jung LEE Department of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan, Korea Corresponding Author Email : [email protected] Tart cherry (Prunus cerasus L.), a medicinal food containing high concentrations of phytochemicals, has a variety of antioxidant activities and health benefits. Here, we investigate the functional effect of tart cherry during apoptotic cell death elicited by airborne particulate matter with a diameter of < 10 μm (PM10) in human epidermal keratinocytes HaCaT cells. The PM10 particles significantly reduced the viability of HaCaT cells. The cytotoxic effect of PM10 was restored upon a treatment with tart cherry (200 μg/mL) containing chlorogenic acid, quercetin, and kaempferol. Tart cherry inhibited the intracellular reactive oxygen species (ROS) responsible for the distinctive activations of the extracellular-signal-regulated kinase (ERK) and p38 MAPK in PM10-treated HaCaT cells. Interestingly, tart cherry significantly restored the expression levels of Bax, Bcl-2, and cleaved caspase-3 as regulated by the phosphorylation of nuclear factor-kappa B (NF-κB). These results indicate that tart cherry is a functional food that blocks the PM10 signaling pathway responsible for mitochon-drial apoptotic cell death in human epidermal keratinocytes. Keywords : Apoptosis, Particulate matter, Tart cherry References 1. Sharpe, J.C.; Arnoult, D. and Youle, R.J. 1644, 107 (2004). 2. Lin, T.; Mak, N.K. and Yang, M.S. Toxicology 247, 145 (2008).

515


P0644 Protective Effect of Astaxanthin Nanosphere Against Autophagic Cell Death of Caco-2 Cells Induced by Vibrio vulnificus VvpE

Eun-Ju KIM, Ye-Young KIM, Sei-Jung LEE Department of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan, Korea Corresponding Author Email : [email protected] Astaxanthin, a kind of xanthophyll carotenoid, has many biological functions, but its bioavailability is limited due to low absorption and hydrophobicity. In this study, we investigated the role of astaxanthin-loaded nanospheres (AN) in the prevention of apoptosis and autophagy in human intestinal epithelial Caco-2 cell induced by Gram-negative bacteria Vibrio vulnificus. Recombinant protein VvpE, produced by V. vulnificus induces the autophagic cell death in Caco-2 cells. The cytoxic effect of VvpE was inhibited by treatment with AN (10 ng/mL), which showed a 100-fold higher than that of astaxanthin. AN inhibited the activation of c-Src responsible for phosphorylation of MAPK mediated by the production of reactive oxygen species (ROS) in VvpE-treated Caco-2 cells. In addition, AN blocked the expression of apoptosis related-proteins (Bax, Bcl-2, cleaved caspase-3) and autophagy related-protein (LC-3, Beclin-1) through the transcriptional activity of nuclear factor-kappa B (NF-κB). These results indicate that AN is a functional substance that prevents the VvpE signaling mechanism of V. vulnificus, which causes gastrointestinal autophagic cell death. Keywords : Apoptosis, Astaxanthin Nanosphere, VvpE References 1. Negroni, A.; Cucchiara, S. and Stronati, L. 2015, 250762 (2015). 2. Song, E.J.; Lee, S.J.; Lim, H.S.; Kim, J.S.; Jang, K.K.; Choi, S.H. and Han, H.J. 6, 27080 (2016).

516


조직공학 및 생체재료공학

(Tissue Engineering and

Biomaterial Engineering)

517


P0701 Vascular Paint Complex Coacervate System Using Gallol Rich Biomaterials

Mihyun KIM, Yoojin KIM, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Vascular diseases according to the development of modern society can be fatal and require rapid treatment. Stent, mechanically widening the blood vessels can cause tissue damage, restenosis, and thrombosis. Vascular Paint as drug delivery platforms can be degraded in vivo to overcome these challenges. Drug/gene delivery to local sites in the blood vessels using complex coacervate system enhance adhesiveness, local delivery and biodegradable. Hyaluronic acid is an immunoneutral polysaccharide that is ubiquitous in the human body and has been in clinical use for biocompatible material. Chitosan is an attractive biomaterial with biodegradability. We will synthesize biocompatible adhesive gallol conjugated biomaterials by synthesizing with ‘Pyro gallol’ and which can show mucoadhesion and adhesiveness in wet environment. AAV (adeno-associated virus) is introduced into the complex coacervate to study biomaterials that local gene delivery, sustained release, and biodegradation in vivo. Keywords : coacervate, biomaterial, chitosan

518


P0702 Raman Spectroscopy-based 3D Analysis of Odontogenic Differentiation of Human Dental Pulp Stem-cell Spheroids

Huijung KIM1, Yoojoong HAN2, Intan Rosalina SUHITO1, Hyungbin SON1, Hyung-Ryong KIM3, Tae-Hyung KIM1 1School of Integrative Engineering, Chung-Ang University, Seoul, Korea, 2R&D division, Nanobase, Inc., Seoul, Korea, 3College of Dentistry, Dankook University, Cheonan, Korea Corresponding Author Email : [email protected], [email protected], [email protected] In this study, we report a non-destructive and label-free 3D Raman mapping-based 3D analytical method that helps identify critical factors responsible for the potential of variability in differentiated stem cell spheroids. Human dental pulp stem cell spheroids with various densities are generated using two different methods and are further undergoing the odontogenic differentiation. Next, cellular differentiation in live 3D spheroids is investigated based on three different Raman peaks, namely, 960 cm-1, 1,156/1,528 cm-1, and 2,935 cm-1, which correspond to hydroxyapatite (HA, odontogenesis marker), β-carotene (precursor of HA), and cellular components (cell reference). By correlating such cell differentiation-related peaks and water/medium peaks, it has been revealed that the diffusion of the medium containing various nutrients and differentiation factors is a crucial factor determining the variations in the 3D differentiation of stem cell spheroids. Considering the challenges associated with high variation in spheroid and organoid differentiation, we conclude that the proposed Raman-based 3D analysis is useful for stem-cell-based regenerative therapy and drug screening. Keywords : Raman spectroscopy, Three-dimensional culture, Stem-cell spheroid, Differentiation, Odontogenesis References 1. Intan Rosalina Suhito, Novi Angeline, Kwang-Ho Lee, Huijung Kim, Chun Gwon Park, Zhengtang Luo ,

Tae-Hyung Kim, A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model(2020), Small, 2002436.

2. Intan Rosalina Suhito, Yoojoong Han, Junhong Min, Hyungbin Son, Tae-Hyung Kim, In situ label-free monitoring of human adipose-derived mesenchymal stem cell differentiation into multiple lineages(2018), Biomaterials, 154, 223-233.

519


P0703 Development of Photo-curable Hyaluronic Acid Hydrogel with Growth Factors for Drug Delivery

SungJun MIN1, Seung Hyeon KIM1, Ho-Jin MOON2, Jae Beum BANG3, Il Keun KWON2 1Department of Dentistry, Graduate School, Kyung Hee University, Seoul, Korea, 2Department of Dental Materials, School of Dentistry, Kyung Hee University, Seoul, Korea, 3Department of Dental Education, School of Dentistry, Kyung Hee University, Seoul, Korea Corresponding Author Email : [email protected] Many methods have been studied for drug delivery systems to regenerate damaged tissues. However, there are some drawbacks such as low efficiency and denaturation of protein. Therefore, photo-immobilization method is suggested to immobilize protein-drug. Photo-immobilization method is simple-reaction and also needs no additional crosslinking reagent. In addition, Immobilization via photo-reaction could stabilizes drugs or growth factors for sustained release. Introduction of photo-reactive azido groups into natural polymers produces materials applicable to the medical field. Since hyaluronic acid is a natural polymer with biodegradable and biocompatibility, it could be used as a biomaterial to immobilize protein-drug. The aim of this study was to prepare photo-curable hyaluronic acid hydrogels for enhanced drug delivery. Photo-reactive hyaluronic acid derivatives (Az-HA) were synthesized to immobilize proteins such as growth factor. It was confirmed that azido group was introduced into hyaluronic acid by FT-IR and 1H-NMR. Mechanical properties of Az- HA hydrogel were examined using rheological analysis. Protein release analysis was conducted to confirm whether the protein drug immobilized with Az-HA derivative was continuously released. Biocompatibility of Az-HA was confirmed by cytotoxicity assay. It was confirmed by migration assay that Az-HA immobilized with fibroblast growth factor (FGF) could be lead superior cell proliferation compared to the control group. As a result, we suggest that the photo-curable hyaluronic acid hydrogels are considered to have an applicability as a biomedical material capable of drug delivery. Keywords : hyaluronic acid, photo-curable, protein immobilization, growth factor References 1. V. Truong-Le, P. Lovalenti and A. M. Abdul-Fattah, Adv. Drug Deliv. Rev., 93, 95-108 (2015). 2. Y. Ito, T. Yamauchi and K. Omura, Kobunshi Ronbunshu, 61, 501-510 (2004). 3. Binata Joddar, Takashi Kitajima and Yoshihiro Ito, Biomaterials, 32, 8404-8415 (2011). 4. F. Z. Volpato, J. Almodóvar, K. Erickson, K. C. Popat, C. Migliaresi and M. J. Kipper, Acta Biomater., 8,

1551-1559 (2012). 5. Francesca Felice, Ylenia Zambito, Ester Belardinelli, Angela Fabiano, Tatiana Santoni and Rossella Di

Stefano, International Journal of Biological Macromolecules, 76, 236-241 (2015).

520


P0704 Enhanced Osteogenic Differentiation of Mesenchymal Stem Cells Using Size-controlled Graphene Oxide

Sora PARK, Yun Ki KIM, Jyongsik JANG, Tai Hyun PARK School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] There is increasing interest in studying stem cell differentiation through cellular physical stimulation which can be translated into cell-recognized tension. It has been known that physical stimulation can direct human mesenchymal stem cell differentiation which called mechanotransduction. Recently, graphene oxide (GO), major derivative of graphene, has been synthesized as promising material which has suitable physico-chemical characteristics for stem cell lineage specification. GO can interact with integrin, the transmembrane receptor protein, through electrostatic, hydrophobic interactions. However, GO used in previous stem cell research has used GO with an irregular morphology and size. Such irregularity of GO causes diverse cellular responses according to lateral sizes of GO. In this study, we fabricated graphite mechanically with narrow size distribution by adjusting the ball-milling time. Then, size-controlled GO sheets were chemically synthesized from ball-milled graphite using modified Hummer’s method. Size distribution of GO were measured by DLS analysis. Dose-dependent cytotoxicity of the size-controlled GO sheets on bone-marrow derived human mesenchymal stem cells (BM-hMSCs) was observed. The interaction between GO sheet and BM-hMSCs was analysed with TEM and SEM. Also, effect of GO with osteogenic differentiation of hMSCs were measured by staining and qRT-PCR. Also, by analyzing the shape and size of the cells through immunostaining, we confirmed that focal adhesion was key component involved in promoting osteogenic differentiation. We suggest that the size-controlled GO sheets would be efficient candidate for enhancement of osteogenic differentiation of hMSCs. Keywords : stem cell differentiation, graphene oxide, mesenchymal stem cell, osteogenesis References 1. S. Even-Ram, Cell 126, 645-647 (2006). 2. S. Hu, J. Nanosci. Nanotechnol. 15, 6327-6341 (2015). 3. R. Podila, J. Biochem. Molecular Toxicology 27, 50-55 (2013). 4. K. Shin, ACS Appl. Mater. Interfaces 6, 5531-5537 (2014).

521


P0705 Microfluidic Model to Investigate Liver and Kidney Interaction in Hypoxia-induced Condition

Jongkwon PARK, Soonjo KWON Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Liver is a lobule structure with the gradient of oxygen by blood flowing central vein, and the area with low oxygen pressure is significantly low despite the high amount of oxygen requirement by hepatocytes [1, 2]. Thus, the liver is vulnerable to hypoxic environment such as hypoxemia or low perfusion, causing hypoxic hepatitis (HH) [3]. Furthermore, HH is associated with renal disease and it can further exacerbate renal disease [4]. Therefore, it is critical to develop a model to investigate liver and kidney interaction in hypoxia-induced conditions. In this study, the microfluidic device is used to keep hypoxia condition and to connect two different tissues. The oxygen gas is controlled with scavenging line with flowing mixture solution of 0.1% w/w sodium sulfite and 10 μM cobalt chloride. Platinum Octaethylporphyrin (PtOEP) film and Image J program were used for monitoring oxygen concentration [5]. The oxygen concentration in cell chamber was dropped to 3% under these conditions. Cell viability were measuring with lactate dehydrogenase (LDH) assay and live-dead cell assay. Liver-specific functions such as albumin secretion and CYP1A1 and kidney-specific functions such as urea or albumin uptake are analyzing. In this study, it is suggested that hypoxia-induced diseases can be influenced on interaction between hepatocytes and renal cells in vitro. ** This study was funded by Natural Research Foundation of Korea, as the Basic Research Laboratory Program (1711119453). Keywords : Hypoxia, liver-kidney interaction, Microfluidic device References 1. Nath, B. and G. Szabo, Hepatology 55(2): p. 622-633 (2012). 2. Jungermann, K. and T. Kietzmann, Hepatology 31(2): p. 255-260 (2000). 3. Waseem, N. and P.-H. Chen, Journal of clinical and translational hepatology 4(3): p. 263. 4. Drolz, A., et al., Annals of intensive care 6(1): p. 1-9 (2016). 5. Wang, L., et al., Lab on a Chip 13(4): p. 695-705 (2013).

522


P0706 Electromagnetic Fields Induce Neural Differentiation of hBM-MSCs and Promote Neurogenesis in Cerebral Ischemia Model

Hee-Jung PARK, Juhye CHOI, Myeong-Hyun NAM, Young-Kwon SEO Department of Medical Biotechnology, Dongguk University, Seoul, Korea Corresponding Author Email : [email protected] Stroke are increasing around the world and are a major cause of death. Recently, the incidence of stroke in middle-aged adults has increased significantly, it is no longer just a disease for the elderly. Electromagnetic field (EMF) have been successfully employed as alternative treatment method in wide field. Our aim in this study was to determine, whether EMF with various frequency has capacity into neural differentiation on hBM-MSCs and effect of EMF exposure after transplantation of hBM-MSCs in mouse ischemia stroke model. We verified cells viability and cytotoxicity analysis using MTT and LDH assay and evaluated neural differentiation on hBM-MSCs exposed to EMF by FACS and immunohistochemical analysis. We confirmed neurogenesis of the cells as expression of neural-related gene and protein. In addition, we identified the effect of EMF exposure and hBM-MSCs on cerebral ischemia though histological staining and motor function test. In conclusion, this present study indicated that EMF with 60 Hz frequency has potential to stimulate neural differentiation of hBM-MSCs in vitro and promote neurogenesis and to enhance functional recovery process on post-stroke after transplantation of EMF treated hBM-MSCs. Keywords : Cerebral ischemia stroke, Electromagnetic fields, Human bone marrow mesenchymal cells, Neural differentiation References 1. A. Avan, H. Digaleh, M. Di Napoli, S. Stranges, R. Behrouz, G. Shojaeianbabaei, A. Amiri, R. Tabrizi, N.

Mokhber, J.D. Spence, J. D., & M.R. Azarpazhooh, BMC medicine, 17(1), 191 (2019). 2. E. Urnukhsaikhan, T. Mishig-Ochir, S.C. Kin, J. K. Park & Y. K. Seo, Applied Biochem. & Biotech.

181(4), 1360-1371 (2017).

523


P0707 Strategy for Enhancing the Life Span of Natural Killer Cells Using Mechanical Stimulation

Myeongkwan SONG, Soonjo KWON Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Adoptive cell therapy, also known as cellular immunotherapy, has recently attracted attention as a new cancer treatment method. Natural killer (NK) cells with the advantage of allogenic transfer are used as a cell therapy strategy along with Tumor-Infiltrating Lymphocyte (TIL), Engineered T Cell Receptor (TCR), and Chimeric Antigen Receptor (CAR) T Cell. However, for clinical applications, NK cells have problems with low ex vivo expansion and short life span. To enhance the life span of the NK cell, we exposed vibrational stimulation to the human NK cell line, KHYG-1. The intensity of vibrational stimulation was calculated as sinusoidal acceleration and was stably controlled from 0.1g to 1.0g. CCK-8 assay was performed to determine whether vibrational stimulation exposed to KHYG-1 affects cell viability. There was no statistically significant decrease in cell viability at all intensities. To evaluate whether vibration stimulation affects the life span, we are evaluating the expression of telomerase related genes (hTERT, TERC, TEP1, PINX1, IL-2, IL-7, IL-15, and IL-21) and telomerase activity. In addition, we are observing the changes in apoptosis compared to the control group by immunostaining (Annexin V, Caspase-3) and western blot (Bcl-2, PD-1). These findings provides as a strategy for improved ex vivo expansion of NK cells and maintaining a long life span. Keywords : cell therapy, NK cell, mechanical stimulation, life span, telomerase References 1. Vivier, E., S. Ugolini, D. Blaise, C. Chabannon, and L. Brossay, Targeting natural killer cells and natural

killer T cells in cancer (2012). Nature Reviews Immunology. 12: 239-252. 2. Zhang, Y., D. L. Wallace, C. M. De Lara, H. Ghattas, B. Asquith, A. Worth, G. E. Griffin, G. P. Taylor, D.

F. Tough, P. C. L. Beverley, and D. C. Macallan, In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection (2007). Immunology. 121: 258-265.

524


P0708 Strategies for Enhancing the Expansion and Half-life of Natural Killer Cells Using 3D Microcarrier Culture

Minseon LEE, Soonjo KWON Department of biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Adoptive cell therapy became one of the most important cell-based immunotherapy. Natural killer (NK) cells have been considered as potential source for adoptive cell therapy among immune cells owing to the advantage of allogenic transfer. However, for clinical applications, strategies for NK cell expansion still should be developed due to limited ex vivo expansion and short half-life of NK cells. In this study, we designed 3D culture using alginate microcarrier as a scale-up strategy for enhancing cell proliferation and half-life for immune-therapy. Cell culture system through alginate scaffold leads to long-term expansion providing benefits for cell growth. Furthermore, NK cells encapsulated in microspheres were protected from tumor microenvironment and following decrease in cytotoxic activity when infiltrated into solid tumor. KHYG-1 cells were capsulated in alginate gel by dropping cell containing 6% alginate solution in 5% calcium chloride (CaCl2) for solidification through centrifuging at 1000 rpm. Generated spherical microcarrier were obtained with diameters ranging from 70μm and 500μm. We are analyzing the effects of microsphere-encapsulated culture on cell proliferation, gene expression, and telomerase activity related to half-life within 14 days of expansion. These findings could suggest the strategy for enhancing ex vivo scale-up expansion and long-term efficiency of NK cell treatment by extending half-life of NK cells, which can be used in combination with genetically engineered immune cells. Keywords : Natural killer cell, NK cell expansion, alginate microcarrier, 3D culture, half-life References 1. Wu, D. et al. ACS Appl. Mater. Interfaces 11, 33716–33724 (2019). 2. Ahn, Y. H. et al. Biomaterials 247, 119960 (2020). 3. Lee, B.-H., Li, B. & Guelcher, S. A. Acta Biomater. 8, 1693–1702 (2012).

525


P0709 [Review] Microgels-based Granular Hydrogels for Biomedical Application

Sang Min LEE, Hyung Joon CHA Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] Hydrogel microparticles (i.e., microgels) are micro-scale hydrogels consist of various natural polymer or protein based precursor. Microgels are formed with water-in-oil emulsions on microfluidics and have uniformity, functional controllability. Because of these properties, microgels are used as building block for 3D bioprinting, cells scaffold or other various biomedical applications. To construct a cell-friendly structure with microgels, physical or chemical interactions between microgels called micro-assemblies are required. In this presentation, we will review granular hydrogels which are one type of physical micro-assemblies. Granular hydrogels are physically constructed by jamming process though vacuum filtration. This jammed microgels have shear-thining, self-healing properties that can suitable for 3D bioprinting ink and can incubate cells inside or outside surface of microgels for cells scaffold. By these features, granular hydrogels are promising platform for biomedical application. Keywords : Hydrogel microparticles, Microgel, Granular hydrogel, Jammed, Microgel -assemblies References 1. C. B. Highly, K. H. Song, A. C. Daly, J. A. Burdick, Advanced Science, 6, 1801076(2019). 2. M. Shin, K.H. Song, J. C. Burrell, D. K. Cullen, J. A. Burdick, Advanced Science, 6, 1901229(2019). 3. B. B. Mendes, A. C. Daly, R. L. Reis, R. M. A. Dominguse, M. E. Gomes, J. A. Burdick, Acta

Biomaterialia, 119, 101-113 (2021).

526


P0710 Ionically Crosslinked Chitosan Based Temperature-responsive Hydrogel Loaded with Bone Filler for Bone Regeneration

Jae Seo LEE1, Seung Yeon LEE2, Hye Jin JEON2, Il Keun KWON2 1Department of Dental Materials, School of Dentistry, Kyung Hee University, Seoul, Korea, 2Department of Dentistry, Graduate School, Kyung Hee University, Seoul, Korea Corresponding Author Email : [email protected] Commonly, there are many cases of bone defects which were occurred by disease or trauma. To rebuild defected bone tissues, bone fillers (BFs), which are relatively inexpensive and readily available, have used as important materials to fill diseased area. However, BFs have serious problem to quickly disperse around tissue area and not continuously locate in the defected area after transplantation. To prevent the problem, hydrogel is used as a carrier to hold BFs in tissue engineering. In this study, we synthesized succinylated chitosan (SCS) based hydrogel which has a high decomposition rate and excellent biocompatibility, and fabricated BFs-loaded SCS (SCS-B). The SCS-B hydrogel showed high physical properties, compressive strength. Also, we confirmed that BFs were well distributed inside SCS hydrogel. Moreover, SCS-B hydrogel exhibited high cell growth rate and bone differentiation rate. After, the surroundings with hydrogel were decomposed to tissues and the BF was remained in the same place for 6 weeks. Overall, this ideal and ecofriendly hydrogel could be used bioactive drug carriers, which can be apply at broad range for bone tissue engineering. Keywords : Hydrogel, Succinylated chitosan, Bone fillers, Drug delivery vehicle, Bone tissue engineering

527


P0711 Magnetically Capturable Adhesive Proteinaceous Microbots as an Orally Administrable Drug Carrier for Targeted Treatment of Esophageal Cancer

Hyun Sun CHOI1, Yun Kee JO2, Hyung Joon CHA1 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Biomedical Convergence Science and Technology, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] In the esophagus, where swallowed fluids and muscular contractions constitute a highly dynamic environment, it has been a challenge to achieve targeted administration of therapeutic drugs to a lesion site. Here, we propose intelligent microparticles with magnetically capturable locomotion and dynamic fluid-resistant adhesive properties, using a bioengineered mussel adhesive protein (MAP). Iron oxide (IO) nanoparticles-embedded MAP microparticles (MAP@IO MPs) are readily localized to a specific region in tubular-structured passageway upon exposure to an external magnetic field and adhere for a prolonged period of time without a magnetic field, even in the esophagus tissue in vivo after oral administration. Moreover, doxorubicin (DOX)-loaded MAP@IO MPs exhibited a sustainable DOX release profile as well as effective anticancer therapeutic activity. Thus, MAP@IO MPs with the magnetically capturable behavior and robust underwater adhesive properties could provide an intelligent modular approach for targeted locoregional therapeutics delivery to a site of action in dynamic fluid-associated tubular organs such as the esophagus. Keywords : Magnetic targeting, Prolonged adhesion, cancer therapy

528


P0712 The Effects of Hypoxia on Liver-specific Functions in Hepatocytes

Seonmyeong CHOO, Soonjo KWON Department of biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] The most important point for maintaining function of the liver is to maintain appropriate oxygen concentration. However, in pathogenesis of several liver disease, hepatocytes are frequently in a hypoxic state. Hypoxia affects not only the angiogenesis, glucose metabolism, but also the survival and proliferation of cells [1]. Hypoxia condition was established using cobalt chloride(100um) which is a PHD inhibitor prevents HIF-1α degradation and sodium sulfate(4mM) which is an oxygen remover in media [2]. The hepatocellular carcinoma cell line (Huh7) was exposed to hypoxia condition for 24 hours. Hypoxia could induce changes in liver functions such as albumin secretion, CYP450, and urea synthesis. After the treatment, Gene expression level of albumin, ARG1, CYP1A1 decreased following exposure of hypoxia condition to the cells. To evaluate the changes in cell metabolism of hepatocytes under hypoxia condition, we are analyzing angiogenesis, glucose metabolism and proliferation markers. This study would understand mechanism of hypoxia-induced diseases in liver and could be used to screen new drugs of hypoxia-induced diseases. ** This study was funded by Natural Research Foundation of Korea, as the Basic Research Laboratory Program (1711119453). Keywords : Hypoxia, liver, liver function References 1. Shih, Shu‐Ching, and Kevin P. Claffey, "Hypoxia‐mediated regulation of gene expression in mammalian

cells." International journal of experimental pathology 79.6 (1998): 347-357. 2. Dubbelboer, Ilse R., et al., "Liver cancer cell lines treated with doxorubicin under Normoxia and hypoxia:

Cell viability and oncologic protein profile." Cancers 11.7 (2019): 1024.

529


P0713 Pregnancy Rate in Korean Black Goats with Natural and Synchronized Estrus After Artificial Insemination with Frozen Semen

Kwan-Woo KIM, Eun-Do LEE, Jinwook LEE, Sung-Soo LEE, Sang-Hoon LEE Animal Genetics Resources Research Center, National Institute of Animal Science, RDA, Hamyang, Korea Corresponding Author Email : [email protected] To enable the crossbreeding of Korean black goats with non-native breeds for small or subsistence farmers, the present study introduced a new estrus synchronization technique with subsequent artificial insemination (AI). In addition, this study determined the pregnancy rate in Korean black goats with natural and synchronized estrus after artificial insemination using frozen semen. Our data highlighted that the percentage of motile sperm from the electro-ejaculated samples declined significantly after freezing and thawing. In addition, sperm motility significantly declined with the sperm incubation period (0 to 120 min at 37°C) in thawed sperm samples. Estradiol (E2) levels remained unchanged at 24 h after withdrawal of controlled internal drug release (CIDR); however, at 48 h and 72 h after CIDR removal, E2 levels increased significantly. These data helped to identify two key time points for AI: 24 h (after CIDR removal), at which time E2 decreases, and 48 h, at which time progesterone increases. Additionally, the group inseminated 48 h after CIDR removal exhibited significantly higher pregnancy and parturition rates (42.9%) than the group inseminated 24 h after CIDR removal (28.6%). In conclusion, these studies indicate that an optimized estrus synchronization process, along with optimized timing of AI, will promote the Korean black goat breeding industry. Keywords : Korean Black Goat, Frozen-Thawed Semen, Artificial Insemination, Estrus Synchronization References 1. Wheaton JE, Carlson KM, Windels HF, Johnston LJ, CIDR: a new progesterone-releasing intravaginal

device for induction of estrus and cycle control in sheep and goats, Anim Reprod Sci, 33, 127-41(1993). 2. Leboeuf B, Restall B, Salamon S, Production and storage of goat semen for artificial insemination, Anim

Reprod Sci, 62, 113-41 (2000).

530


P0714 Efficient in vivo Direct Conversion of Fibroblasts into Cardiomyocytes Using a Nanoparticle-based Gene Carrier

Jongpil KIM Department of Chemistry, Dongguk University, Seoul, Korea Corresponding Author Email : [email protected] The reprogramming of induced cardiomyocytes (iCMs) has shown potential in regenerative medicine. However, in vivo reprogramming of iCMs is significantly inefficient, and novel gene delivery systems are required to more efficiently and safely induce in vivo reprogramming of iCMs for therapeutic applications in heart injury. In this study, we show that cationic gold nanoparticles (AuNPs) loaded with Gata4, Mef2c, and Tbx5 function as nanocarriers for cardiac reprogramming. The AuNP/GMT/PEI nanocomplexes show high reprogramming efficiency in human and mouse somatic cells with low cytotoxicity and direct conversion into iCMs without integrating factors into the genome. Importantly, AuNP/GMT/PEI nanocomplexes led to efficient in vivo conversion into cardiomyocytes after myocardial infarction (MI), resulting in the effective recovery of cardiac function and scar area. Taken together, these results show that the AuNP/GMT/PEI nanocarrier can be used to develop effective therapeutics for heart regeneration in cardiac disease patients. Keywords : reprogramming, heart regeneration, gold nanoparticles

531


P0715 Controlled Delivery According to Porosity of Electrospun Fiber Made of Poly(Vinylpyrrolidone) and Polycaprolactone

Joowon KIM, Yongwook SON, Jae-Hyung JANG Department of Chemical & Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] Many people change the properties of various scaffolds and develop them to be beneficial to the living body. The initial release rate of blending the prepared scaffold and virus/cells/drugs into tissues is fast. To compensate for this, we created controlled delivery by controlling the release kinetics of the cells and drugs that contained in the scaffold. Another problem is the lossless transfer of viruses to be loaded into the scaffold. To solve these problems, the solution was prepared by dissolving Poly(Vinylpyrrolidone) in EtOH solvent to include AAV in the core. The shell uses Polycaprolactone, a biocompatible polymer that is commonly used. This electrospun fiber exposes the PVP from the core to the outside through pores in the PCL. In this article, We examined the possibilities of application to the controlled delivery depending on the situation. Keywords : Electrospinning, Scaffold, Controlled Delivery

532


P0716 Production of Oligo-alginate Using Solution Plasma Process and Evaluation of Its Potentials as a Functional Biomaterial

Ji Min KIM1, Mubarak Ali DAVOODBASHA2,3, Sang-Yul LEE3,4, Jung-Wan KIM1,3 1Dept. of Bioengineering and NanoBio Engineering, Graduate School of Incheon National University, Incheon, Korea, 2School of Life Sciences, B.S.Abdur Rahman Crescent Institute of Science and Technology, Chennai, India, 3Center for Surface Coating and Technology, Korea Aerospace University, Goyang, Korea, 4Department of Material Engineering, Korea Aerospace University, Goyang, Korea Corresponding Author Email : [email protected], [email protected] Solution plasma process(SPP) is an efficient and safe physical method that can be utilized for the development of functional biomaterials. In this study, SPP was employed to degrade alginate without adding any chemicals. Alginate is an acidic hetero-polysaccharide with potentials in biomedicine and foods due to its non-toxicity and biocompatibility. However, its low solubility and high viscosity have limited its application. Alginate solutions(0.1-0.5%) were discharged by plasma at 800 V, 35 kHz for 15-60 min. Viscosity of the solutions decreased remarkably and degradation was confirmed by the DNS, TLC, FT-IR, GPC analyses. The resulting oligo-alginate was antioxidant proved as hydrogen(DPPH), hydroxyl, and superoxide radical scavengers. Oligo-alginates(100 mg∙ml-1) derived from 0.5% alginate by discharging plasma for 30-60 min promoted growth of lettuce significantly. It also enhanced growth of HEK293 cells significantly in vitro. Cell numbers were greatly increased as the concentration of oligo-alginate increased from 5 to 1,000 µg∙ml-1 with no toxicity against the cells. Therefore, oligo-alginate with potentials as biomaterials could be prepared readily by SPP. Keywords : Solution plasma process, oligo-alginate, biomaterials References 1. Takai, O., 2008, Solution plasma processing (SPP), Pure Appl. Chem., 80(9):2003-2011. 2. Watthanaphanit A., et al., 2013, Effect of polymer concentration on the depolymerization of sodium

alginate by the solution plasma process, Polymer Degradation and Stability, 98(5):1072-1080. 3. Nagasawa, N., et al., 2000, Radiation-induced degradation of sodium alginate, Polymer Degradation and

Stability, 69(3):279-285. 4. Luan, L. Q., et al., 2012, Preparation of Oligoalginate Plant Growth Promoter by γ Irradiation of Alginate

Solution Containing Hydrogen Peroxide, J. Agric. Food Chem. 60 (7):1737-1741. 5. Kelishomi, Z. H., et al., 2016, Antioxidant activity of low molecular weight alginate produced by therm al

treatment, Food Chem. 196:897-902. 6. Subrata, B., et al., Development and characterization of alginate coated low molecularweight chitosan

nanoparticles as new carriers for oral vaccinedelivery in mice, 2015, Carbohydrate Polymers., 121:403-410. 7. Honghyun Park., et al., Alginate hydrogels modified with low molecular weight hyaluronate for cartilage

regeneration, 2017, Carbohydrate Polymers., 162:100-107. 8. Manon F. Pritchard., et al., A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal

Microcolony Formation and Enhances Antibiotic Effectiveness, 2017, Antimicrobial Agents and Chemo-therapy., 61(9): e00762-17.

533


P0717 Nanoparticle as a Prodrug of Bile Acid to Improve Bone Regeneration and Osteogenic Differentiation of Mesenchymal Stem Cells by Scavenging Hydrogen Peroxide

Hyoeun PARK1, Yoshie ARAI1, Byoung Ju KIM1, Alvin BELLO2, Inho BEAK1, Anoop PUTHIYOTH DAYANANDAN1, Jiseong KIM1, Hyejong CHOI1, Minju LEE1, Sunjun LEE1, Soo-Hong LEE1 1Department of Medical Biotechnology, Dongguk University, Seoul, Korea, 2School of Integrative Engineering, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Ursodeoxycholic acid (UDCA) is a type of bile acid as helps regulate cholesterol homeostasis as well as has antioxidant and anti-inflammatory effects. Furthermore, in our previous study, we discovered that repeated administration of hydrophilic bile acids enhance in vivo bone regeneration in calvarial defect mouse model. However, repeated injections of bile acid cause various side effects. Nanoparticles are used because they sustained release and increase drug stability. For these reasons, we designed a polymeric prodrug of UDCA nanoparticles (PUDCA) using peroxalate ester linkages to induce the long-term release of UDCA and scavenge hydrogen peroxide. In this study, we analyze effect of PUDCA in adipo-, osteogenic differentiation of human mesenchymal stem cells (hMSCs). As well, we performed in vivo experiment using long bone defect model in rat to assess the effect of PUDCA. As the results, PUDCA is remarkably entered the hMSCs. The PUDCA promotes the osteogenic differentiation, but reduces the adipogenic differentiation of hMSCs compared with UDCA. In addition, we observed that PUDCA significantly decreases the ROS level during ostoegenic induction. In vivo experiments have shown that bone regeneration is enhanced in PUDCA-treated group compared with UDCA group. Through the results, we introduce PUDCA as new medicament for influential bone tissue regeneration by scavenging the hydrogen peroxide and promoting the osteogenic differentiation of MSCs. Keywords : bile acid, Ursodeoxycholic acid (UDCA), Nanoparticle, bone regeneration, osteogenic differen-tiation, mesenchymal stem cells

534


P0718 Characterization of the CD200+ Skeletal Stem Cells for Bone Formation via Endochondral Ossification

InHo BAEK, Soo-Hong LEE, Byoung Ju KIM, Yoshie ARAI, Hyoeun PARK, Anoop PUTHIYOTH, Ji Seong KIM, Hye Jong CHOI, Min ju LEE, Sunjun LEE Department of Medical Biotechnology, Dongguk University, Goyang, Korea Corresponding Author Email : [email protected] Skeletal stem/progenitor cells regulate bone growth and homeostasis by differentiating into various types of cells including osteoblast, osteoclast and chondrocytes. Recently, accumulating evidence supports the idea that stem and progenitor cells play important roles for repairing bone defect. In mammals, skeletal growth occurs at the growth plate, however, the function of the Skeletal Stem Cells (SSCs) is not well understood. Our study suggests that SSCs have important contributions to bone formation via endochondral ossification. Also, it demonstrated stem-like properties including ability to self-renew and to give rise to proliferative chondrocytes in growth plate. Here, we examined osteogenic capacity for bone repair according to age at 4, 6, 8 weeks Sprague Dawley (SD) rat from the growth plate tissue. SSCs were isolated from collagenase-digested tibial and femoral growth plate tissue from each SD rat. The isolated SSCs were plated as monolayer with a density of 2 X 105 cells per both leg of rat and grown for 10 days in DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin (P/S). We sorted CD200 (OX-2 membrane glycoprotein) positive cells in isolated SSCs. The characterization of CD200+ SSCs were confirmed by the expression of osteogenic and chondrogenic markers via fluorescent activating cell sorting (FACS), Real Time polymerase chain reaction (qPCR), and Western Blotting (WB). Interestingly, CD200+ SSCs demonstrated high expression of osteogenic markers Runt-related transcription factor 2 (RUNX2), Osteopontin (OPN), Type 1 collagen (Col I), and chondrogenic markers SPY-Box Transcription Factor 9 (SOX9), Aggrecan (ACAN), Type 2 collagen (Col II) compared with chondrocytes. Our results indicate that CD200+ SSCs have correlative osteogenic and chondrogenic potentials compared to each origin cells in vitro. These findings might imply that CD200+ SSCs is a better cell source for spontaneous bone formation via endochondral ossification process and can promote a better, and a high-quality bone regeneration. Keywords : Skeletal stem cells, Endochondral ossification, Bone formation References 1. E. Mackie, Y. Ahmed, L. Tatarczuch, K.-S. Chen, M. Mirams, The international journal of biochemistry &

cell biology .40, 46-62 (2008).

535


P0719 A Study on Electrospun Block 3D Structure Construction Through Co-axial Electrospinning and the Potential of Application in Gene Therapy

Hyowoo LEE, Hyunwoo PARK, Jae-Hyung JANG Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : j-jang @ yonsei.ac.kr In tissue engineering, there are many attempts to engineer virtually every human tissue. Engineering techniques generally require the biomimetic scaffold, which serves as a three-dimensional template for biomedical application. In this research, the goal is to construct a three-dimensional human organoid structure with an Electrospun Block fabricated in this lab. The Electrospun Block was developed using electrospun core-shell nanofibers with characteristics that mimic the topography of the extracellular matrix (ECM). This study also focuses on the moldable electrospinning method and its potential to be utilized in the biomedical field. Keywords : coaxial electrospinning, 3D scaffold, Electrospun Block

536


P0720 Epitranscriptomic N 6-Methyladenosine Modification Is Required for Direct Lineage Reprogramming into Neurons

Won JUN Department of Nanobiomedicine, Dankook University, Cheonan, Korea Corresponding Author Email : [email protected] N6-methyladenosine (m6A), a conserved epitranscriptomic modification of eukaryotic mRNA (mRNA), plays a critical role in a variety of biological processes. Here, we report that m6A modification plays a key role in governing direct lineage reprogramming into induced neuronal cells (iNs). We found that m6A modification is required for the remodeling of specific mRNAs required for the neuronal direct conversion. Inhibition of m6A methylation by Mettl3 knockdown decreased the efficiency of direct lineage reprogramming, whereas increased m6A methylation by Mettl3 overexpression increased the efficiency of iN generation. Moreover, we found that transcription factor Btg2 is a functional target of m6A modification for efficient iN generation. Taken together, our results suggest the importance of establishing epitranscriptomic remodeling for the cell fate conversion into iNs. Keywords : direct lineage reprogramming, N6-methyladenosine, nduced neuronal cells

537


P0721 Supramolecular Injectable Hyaluronate Hydrogels for Cartilage Tissue Regeneration

Sang Hoon JEONG, Sei Kwang HAHN Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Mesenchymal stem cells (MSCs) have been widely investigated to repair damaged cartilage tissues for the treatment of arthritis. Despite these great efforts, the difficulty in the spatiotemporal control of delivered cells has limited the further clinical development with rapid clearance. Here, we developed injectable hyaluronate (HA) hydrogels to encapsulate MSCs for controlled cartilage tissue regeneration based on the supramolecular chemistry between β-cyclodextrin (β-CD) modified HA (HA-CD) and adamantane (Ad) modified HA (HA-Ad). The spatiotemporally controlled delivery of MSCs from the supramolecular HA hydrogels resulted in the statistically significant chondrogenic differentiation and extracellular matrix (ECM) deposition in vitro and in vivo. Taken together, supramolecular HA hydrogels might be successfully harnessed as an injectable delivery system of MSCs for cartilage tissue regeneration and other tissue engineering applications. Keywords : Mesenchymal stem cell, Hyaluronate, Supramolecuar hydrogel, Cell therapy, Cartilage tissue engineering References 1. S. H. Jeong, M. Kim, T. Y. Kim, H. Kim, J. H. Ju and S. K. Hahn, ACS Appl. Bio Mater. 3, 5040 (2020).

538


P0722 Modification of Magnesium Hydroxide Particles into PLGA Microspheres for Acid Neutralization-mediated Anti-inflammation

Jieun HAN1, Joon-Kyu KIM1, Kyoung-Won KO2, Chun Gwon PARK3, Dong Keun HAN2, Wooram PARK1 1Biomedical-Chemical Engineering, The Catholic University of Korea, Bucheon, Korea, 2Biomedical Science, CHA University, Pocheon, Korea, 3Biomedical Engineering, Sungkyunkwan University, Suwon, Korea Corresponding Author Email : [email protected] Poly(lactic-co-glycolic acid) (PLGA) microspheres have been widely used in that they can deliver various active pharmaceutical ingredients and enable sustained release of them. However, the degradation of PLGA produces acidic by-products that cause tissue inflammation limiting its biocompatibility. Therefore, magnesium hydroxide (MH), an alkaline ceramic, was hydrophobically modified by capping ricinoleic acid (RA) and used as a potential additive for acid-neutralizing effect. The RA capped MH (RA-MH) was encapsulated in PLGA microspheres and had a loading content of more than four times that of pristine MH. Thus, PLGA microspheres encapsulated with RA-MH had better acid neutralization effect than the control. In an in vitro cell experiment, we have confirmed that the RA-MH-encapsulated PLGA microspheres showed excellent biocompatibility with anti-inflammatory effects. We expect that PLGA microspheres containing RA-MH can be applied to drug delivery systems and tissue engineering for various diseases treatment. Keywords : Biodegradable polymers, Polymeric microspheres, Magnesium hydroxide, Acid neutralization, Anti-inflammation

539


P0723 Enhancing Osteogenic Differentiation in Bone Defect Using an in situ Graphene Oxide Incorporated Injectable Hydrogel

Sang Jin LEE1,2, Haram NAH3, Dong Nyoung HEO1, Su A PARK4, Shin-Young PARK5, Il Keun KWON1 1Department of Dental Materials, School of Dentistry, Kyung Hee University, Seoul, Korea, 2Richard and Loan Hill Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, USA, 3Department of Dentistry, Graduate School, Kyung Hee University, Seoul, Korea, 4Department of Nature-Inspired Nanoconver-gence Systems, Korea Institute of Machinery and Materials, Daejeon, Korea, 5Program of Dental Education and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Graphene oxide (GO) incorporated hydrogels have received great attention and have shown excellent potential for use in the field of bone tissue engineering due to their unique osteogenic functionalities. However, current hydrogel systems are limited in their ability to provide an appropriate amount of GO to the lesion area. To overcome this issue, we designed a GO incorporated glycol chitosan (gC)-hyaluronic acid (HA) injectable hydrogel system via a simple oxidation technique. Through oxidation, aldehyde groups were introduced onto the HA. Blending this with gC allowed for the formation of an aqueous hydrogel matrix. Physico-chemical characterization demonstrated that the gC/oHA/GO hydrogel matrix exhibited robust mechanical properties and stability. The in vitro and in vivo results showed that GO incorporated injectable hydrogels enhanced bone tissue regeneration as compared to control injectable hydrogels. Therefore, our results indicate that our injectable hydrogel system could be used for delivering GO. This material may serve as an excellent tissue scaffold for use in treating bone defects. Keywords : Graphene oxide, Injectable hydrogel, Bone tissue engineering

540


P0724 Resilin Motif-incorporated Mussel Adhesive Protein-based Hydrogel for Potential Use in Intervertebral Disc

Seung Kyeum CHO1, Jin Young YUN2, Yun Jung YANG3, Hyung Joon CHA1,2 1Division Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea, 2Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 3Department of Biological Engineering, Inha University, Incheon, Korea Corresponding Author Email : [email protected] Back pain has been a major problem and the reasons of this symptom are mainly caused by the degeneration of the intervertebral disc. Disc degeneration may be from the degeneration of adjacent tissues and give the risk of spinal stenosis. Resilin found in the cuticle regions of many insects has an elastic and rubber like property letting it have an outstanding function to prevent sudden transfer of forces in the presence of water by distributing energy, similar to the nature of bone-tendon insertion site which makes it an interesting material for tissue engineering applications. Mussel adhesive proteins (MAPs) are promising protein-based bioadhesive for underwater adhesion. We developed the fusion protein Res-MAP (Resilin-MAP) that might be able to replace the nucleus pulpous, which uptake the repeated and sustained stimulation, and to quickly adhere and fix between the intervertebral discs. We successfully expressed and purified Res-MAP, fabricated it to elastic hydrogel, and tested its mechanical properties. Keywords : resilin, MAP, intervertebral disc, nucleus pulpous, resilience, adhesion References 1. Guokui Qin, Xiao Hu, Peggy Cebe, David L. Kaplan. Nature Communications, 1003 (2012). 2. Christopher M. Elvin, Andrew G. Carr, Mickey G. Huson, Jane M. Maxwell, Roger D. Pearson, Tony

Vuocolo, Nancy E. Liyou, Darren C. C. Wong, David J. Merritt, Nicholas E. Dixon, Nature 437, 999-1002 (2005).

3. Cha, H. J., Hwang, D. S. and Lim, S. Biotechnol, 631-638 (2008). 4. Jeon, E. Y., Hwang, B. H., Yang, Y. J., Kim, B. J., Choi, B. H., Jung, G. Y. and Cha, H. J., Biomaterials 67,

11-19 (2015). 5. Kim, B. J., Oh, D. X., Kim, S., Seo, J. H., Hwang, D. S., Masic, A., Han, D. K. and Cha, H. J.,

Biomacromolecules 15(5), 1579-1585 (2014).

541


합성생물공학 및 대사공학

(Synthetic Biology and

Metabolic Engineering)

542


P0801 Dsembler (DNA Assembly Designer): A Software Tool for Minimizing InDel Errors in DNA Assembly

Aporva GUPTA1,2, A-young PARK1,3, Eugene RHA1, Seong-Kun BAK1,2, Wonjae SEONG1,2, Dae-Hee LEE1,2, Seong Keun KIM1, Haseong KIM1,2, Seung Goo LEE1,2 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology, Daejeon, Korea, 3Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] The declining costs of gene synthesis combined with increased dependence on computational systems, has contributed in explosive research on synthetic genomes around the world. Current established genome assembly protocols depend upon short oligonucleotides assembled by Polymerase Chain Assembly (PCA). To ensure accurate assembly of genes, designing oligonucleotides using the right criteria is essential. Here, we introduce a public, web-based application, Dsembler (DNA Assembly Designer) that considers various thermodynamic and physical conditions of oligomers, such as melting temperature, GC content, repeats, and thymine at the 3’ end, that often result in insertions or deletions in an assembled gene; oligonucleotides that minimize such errors are selected. This algorithm was developed on Python. Dsembler and a popular tool by John Hopkins, GeneDesign, were used to assemble various M13 bacteriophage fragments (520bp-4020bp). Dsembler sequences displayed a lower error rate and higher frequency of error-free colonies. In the future, we expect to incorporate an Artificial Intelligence model to help generate errorless genome assemblies. Keywords : Genome Synthesis, Computational Biology, Python, Oligomer Design

543


P0802 An Efficient DNA Assembly Approach with Computationally Designed Oligomers

A-young PARK1,2, Aporva GUPTA1,3, Eugene RHA1, Seong-Kun BAK1,3, Wonjae SEONG1,3, Dae-Hee LEE1,3, Seong Keun KIM1, Haseong KIM1,3, Seung-Goo LEE1,3 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea, 3Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] The growing popularity of Synthetic Genomics, has increased the demand for fast, accurate, and cost effective genome synthesis. Today, the emergence of various reliable DNA assembly technologies has allowed for the complete synthesis of viral, bacterial, and yeast eukaryotic genomes. Here, we present an economical, and streamlined gene synthesis approach, assisted by computationally designed and microchip-synthesized oligonucleotides. The described protocol incorporates key assembly steps: oligomer design, error-removal, and DNA assembly of long fragments. As a proof of concept, M13 bacteriophage genome (6.4kb) was constructed in Escherichia coli using an error prone microchip synthesized oligo pool without pre-purification. By designing optimal oligomers and reducing mismatch errors, the minimum error rate was brought down to under 1 error per kbp. The optimal oligomer design was constructed by our web-based tool, Dsembler, which helps minimizing possible assembly errors. We anticipate that Dsembler along with oligo-microchip approach enables to achieve cost-effective and fast DNA assembly on genome-scale. Keywords : Genome Assembly, Oligomer Design, Gene Synthesis, Long-read Sequencing, Oligo Pools, Nanopore Sequencing

544


P0803 Combinatorial DNA Part Assembly for Large-scale Pathway Library Construction

Seong-Kun BAK1,2, Wonjae SEONG1,2, A-young PARK1,3, Eugene RHA1, Hyewon LEE1, Kilkoang KWON1, Haseong KIM1,2, Seung-Goo LEE1,2 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology, Daejeon, Korea, 3Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] In multi-gene metabolic pathways, the expression levels of each gene can substantially influence the overall performance of the synthetic system. It is well known that optimal performance is achieved by the appropriate expression ratio of the genes. Numerous techniques have been established to identify the optimal ratio of gene expressions by investigating a broad spectrum of their expressions. However, due to the exponential increment of combinations as the number of genes increases in a pathway, traditional assembly techniques are difficult to cover all the possible combinations. This study presents a combinatorial DNA part assembly approach by modifying golden-braid assembly and mutagenesis PCR. While golden-braid uses two enzymes and two vectors, our approach enables to continuously assemble DNA modules from scratch parts to long pathway infinitely using one enzyme and one vector. We confirmed the part combinatorial ratios are constantly remained in each assembly step using the long-read sequencing technique. We anticipated that this approach is helpful to optimize multi-gene pathway systems by constructing a large-scale pathway library with a high-throughput screening system. Keywords : Combinatorial DNA assembly, Oxford Nanopore long-read sequencing, Golden-braid assembly References 1. Alejandro Sarrion-Perdigones, Erica Elvira Falconi, Sara I. Zandalinas, Paloma Jua´rez, Asun Ferna´ndez-

del-Carmen, Antonio Granell, Diego Orzaez. PLoS ONE. 6(7): e21622 (2011). 2. Miten Jain, Hugh E. Olsen, Benedict Paten & Mark Akeson.Genome Biology. 17:239 (2016). 3. Registry of Standard Biological Parts[http://parts.igem.org]

545


P0804 High-level Production of 4-Hydroxyvalerate from Levulinic Acid via Whole-cell Biotransformation Decoupled from Cell Metabolism

Doyun KIM1, Sung Kuk LEE1,2 1Department of Biomedical Engineering, Ulsan National Institute of Science and Technology(UNIST), Ulsan, Korea, 2School of Energy & Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] γ-Hydroxyvalerate (4HV) is an important monomer that has been used to produce various valuable polymers and products. In this study, an engineered 3-hydroxybutyrate dehydrogenase that can convert levulinic acid (LA) into 4HV was co-expressed with a cofactor (NADH) regeneration system mediated by an NAD+-dependent formate dehydrogenase (CbFDH) in the Escherichia coli strain, MG1655. The resulting strain produced 23-fold more 4HV in a shake flask. The 4HV was highly produced at low aeration condition suggesting that ATP is not a limiting factor. All of these factors are considered beneficial characteristics for the production of target compounds, especially at an industrial scale. Under optimized conditions in a 5 L fermenter, the titer, produc-tivity, and molar conversion efficiency for 4HV reached 100 g/L, 4.2 g/L/h, and 92%, respectively. Our system could prove to be a promising method for the large-scale production of 4HV from LA at low-cost and using a renewable biomass source. Keywords : Levulinic acid, 4-hydroxyvaleric acid, Escherichia coli, Biotransformation References 1. Yeon, Y. J., Park, H. Y., Yoo, Y. J., Bioresour. Technol. 134, 377-380 (2013).

546


P0805 Application of Embden-Meyerhof-Parnas Pathway Disrupted Escherichia coli for NADPH-dependent Bio-production

Kyung Hyun CHO1, Ye Eun KIM1, Chang Hee KIM2, Young Shin RYU1, Yuchan KIM2, Sung Kuk LEE1,2 1Department of Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2Department of Biomedical Engineering UNIST, Ulsan, Korea Corresponding Author Email : [email protected] In nature, Escherichia coli utilize glucose mainly via Embden-Meyerhof-Parnas pathway (EMPP) while some others such as Pseudomonas catabolize glucose via Entner-Doudoroff pathway (EDP) and pentose phosphate pathway (PPP). A redirection of glycolytic flux can provide energy and reducing power for growth and valuable production. However, loss of genes encoding phosphofructokinase in E. coli showed a significant growth deficiency. Here, we applied adaptive laboratory evolution for an EMPP-disrupted E. coli strain to recover its growth rate on glucose as a sole carbon source. By combining genotypic- and phenotypic- analysis, the mutations that contributed cell fitness recovery were characterized. The evolved strain was further applied for 3-hydroxypropionic acid (3-HP) production and lycopene production which requires high amount of NADPH and increased production rate of both 3-HP and lycopene compared to unevolved strain. Keywords : Adaptive laboratory evolution, Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, Pentose phosphate pathway, 3-Hydroxypropionic acid References 1. C. P. Long, J. E. Gonzalez, A. M. Feist, B. O. Palsson, and M. R. Antoniewicz, PNAS. 115, 222-227 (2018).

547


P0806 Engineering Bacteroides thetaiotaomicron as a Delivery Vehicle for Secreted Therapeutic Proteins

Taehyun KIM1,2, Seong Keun KIM1, Seung-Gyun WOO1,2, Min-Jeong CHOI1, Seung-Goo LEE1,2, Dae-Hee LEE1,2 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon, Korea Corresponding Author Email : [email protected] Bacteroides thetaiotaomicron is one of the most dominant species in the human gut, and it possesses many probiotic characteristics such as metabolite secretion, xenosiderophore, and outer membrane vesicles. Recently, B. thetaiotaomicron has attracted attention as an emerging chassis for microbiome engineering, including diagnostics and treatment of disease-associated symptoms in the gut. However, molecular tools and information lack to engineer B. thetaiotaomicron for therapeutic applications. In this study, we engineer B. thetaiotaomicron as a delivery vehicle for therapeutic proteins. We first cultivated B. thetaiotaomicron under various growth conditions and searched novel signal peptide sequences from its secretomes. Three major proteins secreted into the culture media were identified by peptide mass fingerprinting. These secreted proteins of B. thetaiotaomicron contain a signal peptide (BtSP) that transports their cargos across the cytoplasmic membrane via a Sec-dependent pathway. We then probed the feasibility of whether the BtSP efficiently transports bio-active cargo proteins. Anti-inflammatory proteins fused with the BtSP at their N-terminal were expressed in B. thetaiotaomicron. The fusion proteins were successfully excreted into the culture media. Overall, we established a Bacteroides secretion system that will serve as a versatile tool for the delivery of therapeutic proteins from the engineered live bacteria. Keywords : Bacteroides thetaiotaomicron, Microbiome engineering, Secretion, Anti-inflammatory protein References 1. J.L Catlett et al, mSystems 5, e000252 (2020). 2. W. Zhu et al. Cell Host Microbe 27, 3 (2020). 3. L. Durant et al. Microbiome 8, 88 (2020).

548


P0807 Development of Pseudomonas asiatica as a Host for the Production of 3-hydroxypropionic Acid from Glycerol

Thi Trinh NGUYEN1,2, Suman LAMA1, Satish Kumar AINALA3, Mugesh SANKARANARAYANAN2,4, Ashish Singh CHAUHAN2, Jung Rae KIM2, Sunghoon PARK1,2 1School of Energy and Chemical Engineering, UNIST, Ulsan, Korea, 2School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea, 3NOROO Bio R&D Center, NOROO Holdings Co., Ltd, Suwon, Korea, 4Department of Biotechnology, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Chennai, India Corresponding Author Email : [email protected] Pseudomonas asiatica C1, which could grow on glucose and aerobically synthesize coenzyme B12, was isolated and developed as a microbial cell factory for the production of 3-hydroxypropionic acid (3-HP) from glycerol. Three heterologous enzymes, glycerol dehydratase (GDHt), GDHt reactivase (GdrAB) and aldehyde dehydro-genase (ALDH), constituting the 3-HP synthesis pathway, were introduced, and three putative dehydrogenases, responsible for 3-HP degradation, were disrupted [1, 2]. In addition, the transcriptional repressor glpR and the glycerol kinase glpK were removed to increase glycerol import while eliminating the catabolic use of glycerol. Furthermore, the global regulatory protein encoded by crc and several putative oxidoreductases (PDORs) were disrupted. One resulting strain, when grown on glucose, could produce 3-HP at ~700 mM in 48 h in a fed-batch bioreactor experiment, with the molar yield >0.99 on glycerol without much by-products. This study demonstrates that P. asiatica C1 is a promising host for production of 3-HP from glycerol. Keywords : Pseudomonas asiatica C1, 3-hydroxypropionic acid, glycerol, glycerol dehydratase, aldehyde dehydrogenase References 1. Zhou, S., Ashok, S., Ko, Y., Kim, D.M., Park, S., 2014. Development of a deletion mutant of Pseudomonas

denitrificans that does not degrade 3-hydroxypropionic acid. Appl. Microbiol. Biotechnol. 98, 4389-4398. 2. Zhou, S., Catherine, C., Rathnasingh, C., Somasundar, A., Park, S., 2013. Production of 3-hydroxypropionic

acid from glycerol by recombinant Pseudomonas denitrificans. Biotechnol Bioeng. 110, 3177-3187.

549


P0808 Adding Acetate into a Substrate Profile of an Efficient Glucose/Xylose Co-fermenting Saccharomyces cerevisiae for Lignocellulosic Biorefinery

Tseveendorj ENKH-AMGALAN1,2, Ja Kyong KO1, Gyeongtaek GONG1, Youngsoon UM1,2,3, Sun-Mi LEE1,2,3 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea, 2Clean Energy and Chemical Engineering, University of Science and Technology (UST), Daejeon, Korea, 3Green School (Graduate School of Energy and Environment), Korea University, Seoul, Korea Corresponding Author Email : [email protected] Acetate is one of the major inhibitors in lignocellulosic hydrolysates. Thus, converting acetate into a target product would maximize the conversion yield by relieving inhibitory effects and supplying an additional carbon source. Here, we developed an acetate utilizing strain capable of glucose/xylose co-fermentation. Specifically, we improved the acetate tolerance of xylose utilizing strain (XUSE) through adaptive laboratory evolution (ALE). Through ALE, acetate tolerant XAE strain was generated and it showed 4-fold increase in ethanol yield (0.448 g/g vs 0.117 g/g for XAE vs XUSE) during xylose fermentation in the presence of acetate. An acetate pathway was then introduced into the GPD2 locus of XAE generating XAE-A1 strain. XAE-A1 utilized 0.81 g/L acetate during mixed-sugar fermentation possibly due to the redirected NADH supply from glycerol synthesis. When sugar cane hydrolysate was fermented, XAE-A1 produced ethanol with a maximum yield of 0.512 g/g supported by additional carbon supply from acetate. When XAE-A1 strain was further engineered to harbor PHB production pathway, the engineered strain showed efficient PHB production performance with increased titer and yields in the presence of acetate. This study showed the inhibitory compound of acetate could be used as an additional carbon source to improve overall yield in lignocellulosic biorefinery. Keywords : Saccharomyces cerevisiae, Hydrolysate fermentation, PHB, lignocellulose, acetate utilization, biorefinery References 1. Ja Kyong Ko, Tseveendorj Enkh‐Amgalan, Gyeongtaek Gong, Youngsoon Um, Sun‐Mi Lee, Improved

bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid (2019), GCB Bioenergy, 12(1), 90-100.

2. Phuong Hoang Nguyen Tran, Ja Kyong Ko, Gyeongtaek Gong, Youngsoon Um, Sun-Mi Lee, Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocel-lulosic biorefinery (2020), Biotechnology for biofuels, 13(1), 12.

550


P0809 Development of Dynamic Regulation Switch for Supply of Redox Cofactors Necessary for the Production of 3-Hydroxypropionic Acid

Yuchan KIM1, Sung Kuk LEE1,2 1Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] 3-Hydroxypropionic acid (3-HP) is a platform chemical that can be used as the precursor for the production of valuable compounds, including acrylic acid and 1,3-propandiol. Using glucose as the substrate, Escherichia coli can synthesize 3-HP from malonyl-CoA, by heterologous expression of malonyl-CoA reductase, the enzyme derived from Chloroflexus aurantiacus. However, requirement of additional redox cofactors is limiting factor of this pathway. Excess amounts of redox cofactors are necessary for the production, though it causes growth defection once it is accumulated in the cell. In this study, dynamic regulation system was designed for reflux of metabolic pathways, to overcome the bottlenecks of both redox cofactor requirement and toxicity with accumulation. Final product-inducible system was applied for expression of the genes needed for reflux of metabolic pathway. This allows decoupling of growth and production stage, using substrate for the growth at the growth stage, whereas using substrate for the production of 3-HP in the production phase, with the provision of larger amounts of redox cofactors necessary for the heterologous enzyme required for the production of 3-HP. Keywords : 3-hydroxypropionic acid, dynamic regulation, redox cofactors, Escherichia coli References 1. C. Rathnasingh, S. M. Raj, Y. Lee, C. Catherine, S. Ashok, and S. Park, J. Biotechnol. 157(4), 633-640

(2012).

551


P0810 Identification of the Operon of Genes Related to Carbon Monoxide Consumption in Eubacterium limosum KIST612

Byeongchan KANG, Soyoung OH, Ji-Yeon KIM, Hyunsoo KANG, In Seop CHANG School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Corresponding Author Email : [email protected] Transcription in prokaryotes proceeds as the related gene units consist of a single operon. These operons induce bacteria to form favorable conditions for a given environment by regulating the level of transcription according to the substrate or specific environmental conditions. Eubacterium limosum KIST612 is a Gram-positive, obligately anaerobic, and acetogenic bacteria. This strain can use carbon monoxide as the sole energy source and carbon source. In E. limosum KIST612, it was confirmed that the transcription level of genes is regulated depending on the substrate or environmental conditions. This mechanism is called transcriptional regulation. This pattern of transcriptional regulation has also been confirmed in genes for consuming carbon monoxide, a representative substrate that E. limosum KIST612 can use. To investigate the regulation of the level of trans-cription of a gene related to carbon monoxide consumption, we analyze the operon unit of the related gene by co-transcription analysis and identify the transcription regulator. Keywords : Eubacterium limosum KIST612, Operon, Carbon monoxide

552


P0811 Enhanced Secretion of Heme via Membrane Modification of Engineered Corynebacterium glutamicum

Young Jin KO, Minhye KIM, Joonhee CHANG, Hae Jin CHOI, Sung Ok HAN Department of Biotechnology, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Heme is applied as an organic iron supplement and main ingredient in the food industry as well as pharma-ceutical in medical fields. Here, we developed heme-secreting Corynebacterium glutamicum at high level by modifying cell membrane components. First, CRISPR-based recombineering was performed for the disruption of genes encoding heme binding membrane proteins. Membrane-bound heme and binding/unbinding rate were 1.20- and 1.26-fold lower in the engineered strain knocking out three heme-binding proteins (KOP strain) than the wild-type C. glutamicum, respectively. When these proteins were knocked out in the heme-overproducing strain, resulting in the KOPHM strain, heme secretion was also increased 2.22-fold. Second, reducing fatty acid content on the membrane was performed by treating the cell wall inhibitor in the cultivation. Interestingly, ETB-added cells displayed decreased binding/unbinding rate and membrane-bound heme. The KOP strain treated by ETB showed the lowest binding/unbinding rate and membrane-bound heme. Finally, The ETB-treated cultivation of the KOPHM strain showed a 1.75-fold increase in heme secretion compared to the normal cultivation condition. Thus, these biotechnological strategies enable the efficient heme secretion in C. glutamicum. Keywords : Heme, Membrane engineering, Corynebacterium glutamicum, CRISPR References 1. Y.J. Ko, Y.C. Joo, J.E. Hyeon, E. Lee, M. Lee, J. Seok, S.W. Kim, C. Park, S.O. Han, Sci. Rep. 8:14460

(2018).

553


P0812 Lycopene Production Enhancement in Escherichia coli by Expression of Key MEP Pathway Enzymes from Vibrio sp. dhg

Min Jae KIM1, Myung Hyun NOH1, Sung Hwa WOO1, Hyun Gyu LIM1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] Microbial fermentation is a promising method that can overcome major bottlenecks in conventional methods of lycopene production, such as low yields and poor quality. To further improve lycopene production, it is important to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are essential in lycopene production using MEP pathway. Here, we demonstrated the superior activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. We found that Dxs and IspA in Vibrio sp. dhg showed 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression increased the specific lycopene production by 1.88-fold. Our findings could be widely used to increase production of lycopene and other carotenoids. Keywords : Lycopene, Vibrio sp. dhg, Dxs, IspA

554


P0813 Improving Itaconic Acid Production with Engineered Escherichia coli Consuming Acetate

Min Jae KIM1, Hyun Gyu LIM1, Myung Hyun NOH1, Sung Hwa WOO1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Korea Corresponding Author Email : [email protected] The Utilization of cheap carbon sources is both crucial to production cost and economic feasibility. In the previous study, the production of itaconate from acetate was constructed with a rational metabolic engineering strategy, proving the potential of acetate as a promising carbon source. In this study, we introduced a combinatorial approach to further increase the production of itaconate. Initially, the itaconate-specific screening device could be constructed using a transcriptional regulator and selection marker. Thus, Adaptive Laboratory Evolution (ALE), utilizing natural selection toward microorganisms under selection pressure, was introduced to itaconate-producing strain. The evolved strain could produce 1.06 g/L of itaconate, a 1.24-fold increase in comparison with parental strain and the yield was significantly enhanced by 7.16-fold. The rational approach will be applied to maximize the itaconate production and the mutant will be additionally analyzed with WGS for validation. Keywords : Acetate, Itaconate, ALE References 1. Jo, Noh, and Lim et al, Microb Cell Fact. 18, 57 (2019).

555


P0814 Development of Metabolically Engineered Corynebacterium glutamicum Synthesizing L-Carnosine Regarded as a Treatment of Metabolic Syndrome

Minhye KIM, Young Jin KO, Sung Ok HAN Korea University, Seoul, Korea Corresponding Author Email : [email protected] L-carnosine has been regarded as a new chemotherapeutic because of its distinguishing functions. Biosynthesis of L-carnosine has been required to replace chemical process. Here, we firstly developed metabolically engineered Corynebacteirum glutamicum producing L-carnosine. First, pentose phosphate pathway (PPP) was reinforced by introducing H36 promoter and attenuating flux from glucose to pyruvate. Second, histidine and β-alanine synthetic pathways were strengthened by overexpressing genes. Third, a mammalian’s carnosine synthetic pathway was introduced. Enhanced PPP increased the flux to PRPP, thus, the PPP enhanced strain produced 2.0-fold more increased L-carnosine production than that of control. Additionally enhanced histidine and β-alanine pathways resulted in a 2.4-fold and a 7.2-fold increase of L-carnosine production compared to PPP enhanced strain. The final stain which is consolidated all modified pathways accumulated 145.89 mg/L of L-carnosine. Finally, the effects of antioxidant and anti-glycation were confirmed in synthesized L-carnosine. Consequently, we suggested a new L-carnosine biosynthetic process and showed its potential as a treatment of metabolic syndrome. Keywords : L-carnosine, L-histidine, β-alanine, Pentose phosphate pathway, Metabolic engineering, Coryne-bacterium glutamicum

556


P0815 Role of YieP and YohJK for 3-Hydroxypropionic Acid Tolerance Improvement and Effect in Klebsiella pneumoniae

Huichang RYU1, Thuan Phu NGUYEN-VO1,2, Sunghoon PARK1,2 1School of Energy and Chemical Engineering, UNIST, Ulsan, Korea, 2School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] The 3-hydroxypropionic acid (3-HP) tolerance in Klebsiella pneumoniae was tested by growing the cell with glycerol as carbon source in presence of varied initial 3-HP addition, which was determined based on the 3-HP tolerance level of E. coli. The transcription factor, yieP, was studied carefully and it has been observed as gene regulating yohJK, the putative 3-HP exporter encoding gene in E. coli [1, 2]. We examined the homologous gene in K. pneumoniae which has similar genomic identity compared with the yieP and yohJK in E. coli by using sequence alignment and found that there are one and two identical genes for yieP and yohJK, respectively. We tested the deletion mutant of K. pneumoniae J2B for yieP, yohJK1, and yohJK2 to check the possibility of same functional role from the gene. The yieP deletion mutant strain showed much better growth profile than wild type when the initial 3-HP concentration is higher than 300mM up to 500mM; the yohJK1 deletion mutant along with yieP deletion showed highly reduced tolerance compared with yieP single deletion, but yohJK2 deletion mutant showed similar growth profile as yiep single gene deletion mutant. Also, we constructed the new K. pneumonia recombinant strains which overexpress YohJK1 and YohJK2 individually. The over-expression of YohJK1 showed the improvement of 3-HP tolerance clearly compared with wild type and YohJK2 overexpression recombinant strains. The effect of 3-HP tolerance improvement toward 3-HP production in K. pneumonia was also tested in the flask scale experiment. We used the K. pneumoniae J2B ΔdhaT ΔyqhD ΔldhA strain with overexpression of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH) and set up the cultivation following the condition previously reported [3]. We could confirm that the additional yiep deletion mutant strain showed slight improvement of 3-HP production when the initial 3-HP concentration was from 200mM up to 400mM. These results strongly suggest the possibility of genes, yieP and yohJK1 in K. Pneumo-niae, as global regulator and 3-HP exporter encoding gene, respectively. Also we can highly expect the applicability of this result to improve the 3-HP production along with the 3-HP tolerance improvement. Keywords : Klebsiella pneumoniae, 3-hydroxypropionic acid, yieP, yohJK, glycerol References 1. Nguyen-Vo, T. P., Liang, Y., Sankaranarayanan, M., Seol, E., Chun, A., Ashok, S., Chauhan, A. S., Kim, J.

& Park, S., Development of 3-hydroxypropionic-acid -tolerant strain of Escherichia coli W and role of minor global regulator yieP. Metab. Eng. 53, 48-58 (2019).

2. Nguyen-Vo, T. P., Ko, S., Ryu, H., Kim, J., Kim, D. & Park, S., Systems evaluation reveals novel transporter YohJK renders 3-hydroxypropionate tolerance in Escherichia coli. Scientific Reports 10, 19064 (2020).

3. Ashok, S., Sankaranarayanan, M., Ko, Y., Jae, K., Ainala, S. K., Kumar, V. & Park, S., Production of 3-Hydroxypropionic Acid From Glycerol by Recombinant Klebsiella pneumonia dhaT yqhD Which Can Produce Vitamin B12 Naturally. Biotechnol BioEng. 110 (2), 511-524 (2013).

557


P0816 Comparative Analysis of RpoD-family Regulon Between Salmonella and Escherichia coli Under the Heat-shock Condition

Joon Young PARK1, Ye GAO2, Donghyuk KIM1,3 1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 3School of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] The heat-stress is the most fundamental stress to bacteria within their natural environs, and bacteria have various mechanisms that adapt to the stress. The RpoH is one of RpoD-family sigma factor and is known to regulate the expression of heat-shock proteins and various genes under heat-shock conditions. In this study, Escherichia coli and Salmonella, the representative Gram-negative pathogenic bacteria, were comparatively analyzed through genome-wide analysis. ChIP-exo (Chromatin immunoprecipitation-exonucleases) was used to identify binding sites of sigma factors on whole-genome. From E. coli, 2,505 RpoD binding sites and 129 RpoH binding sites were found, and 82 identical binding sites were identified. Additionally, 2,439 RpoD binding sites and 115 RpoH binding sites were found, and 84 identical binding sites were revealed in Salmonella. Moreover, transcript analysis was performed through RNA-seq, differentially expressed genes were found when compared to 37 °C under heat shock (42 °C). Through these data, the binding pattern of RpoD and RpoH under heat shock condition and the expression pattern of gene clusters called sigmulon which controlled by sigma factors were identified. Keywords : Escherichia coli, Salmonella, Heat-shock, Sigma factors, ChIP-exo, RNA-seq

558


P0817 Genome-scale Model Reconstruction of the Metabolic Network in terms of Producing Various Antibiotics in Streptomyces venezuelae ATCC 15439

Ina BANG1, Woori KIM2, Byung-kwan CHO2, Donghyuk KIM1 1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea, 2Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Streptomyces belonging to Gram-positive bacteria are organisms with a unique life cycle. They are included in bacteria but grow by creating a filamentous form that resembles a fungus. It also has a linear form of the chromosome. Above all, they have the potential to produce various bioactive secondary metabolites, especially antibiotics. We specifically analyzed its genome by focusing on the Streptomyces venezuelae ATCC 15439 strain. First, clusters of orthologous groups were analyzed for whole genomes to confirm the distribution of functions. Second, we identified 36 clusters and 949 related genes through the secondary metabolite genome mining. Finally, we reconstructed the Streptomyces venezuelae ATCC 15439 draft genome-scale model. The model reconstruction is based on related literature, previous models for Streptomyces coelicolor which is the most widely used in genetic studies, publicly available databases like BIGG, KEGG, and The Seed. This draft model includes 1146 genes, 2730 reactions, and 2332 metabolites. It contains 103 genes related to the secondary metabolite pathway. This is the first model for this strain, which will be able to greatly help its metabolic pathway and flux prediction. Keywords : Metabolic modeling, in silico modeling, Streptomyces

559


P0818 Directed Evolution of Aldehyde Dehydrogenase for 3-Hydroxypropionic Acid Production Using Synthetic High-throughput Selection Device

Dongyeop BAEK1, Joo Yeon SEOK2, Hyun Gyu LIM1, Sunghoon PARK3, Tae Hyeon YOO4, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, POSTECH, Pohang, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea, 3School of Energy and Chemical Engineering, UNIST, Ulsan, Korea, 4Department of Molecular Science and Technology, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] 3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP from glycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has been revealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield and productivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a synthetic selection device using a 3-HP responsive transcription factor and developed a selection approach for 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a 2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. Our approach provides the simple and efficient tool to engineer the pathway enzymes in metabolic engineering. Keywords : 3-hydroxypropionic acid, aldehyde dehydrogenase, directed evolution, high-throughput selection device References 1. J. Y. Seok and J. Yang et al., Metab. Eng., 47, 113-120 (2018).

560


P0819 Metabolic Engineering of Corynebacterium glutamicum for the Production of 5-Hydroxyvaleric Acid

Yu Jung SOHN1, Minsoo KANG2,3, Kei-Anne BARITUGO1, Jina SON1, Kyoung Hee KANG2, Mi Hee RYU2, Siseon LEE4, Mingi SOHN2, Ye Jean JUNG2,3, Kyungmoon PARK3, Si Jae PARK1, Jeong Chan JOO4, Hee Taek KIM5 1Department of Chemical Engineering and Materials Science, Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul, Korea, 2Research Center for Bio-based Chemicals, Division of Specialty and Bio-based Chemical Technology, Korea Research Institute of Chemical Technology, Daejeon, Korea, 3Department of Biological and Chemical Engineering, Hongik University, Sejong, Korea, 4Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea, 5Department of Food Science and Technology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] We report fermentative production of 5-hydroxyvaleric acid (5-HV), an important C5 platform chemical that can work as a versatile building block, from glucose as a sole carbon source using metabolically engineered Corynebacterium glutamicum. First, an artificial 5-HV biosynthesis pathway that initiates with the first three reaction steps of an L-lysine catabolic pathway encoded by Pseudomonas putida davTBA genes along with a subsequent intracellular reduction step was constructed in C. glutamicum. To efficiently obtain 5-HV, various aldehyde reductase candidates which might mediate intracellular reduction of glutarate semialdehyde to 5-HV were examined: CpnD from Clostridium aminovalericum, Gbd from Ralstonia eutropha, ButA from C. glutamicum, and YihU, YahK, and YqhD from Escherichia coli. Through the flask and batch cultivations, and YahK from E. coli was determined to be the most suitable aldehyde reductase for 5-HV production. Further enhancement in 5-HV production was achieved by suppressing glutaric acid by-production through the chromosomal deletion of an endogenous gabD gene responsible for the conversion of glutarate semialdehyde into glutaric acid. The final engineered C. glutamicum expressing davTBA genes and the yahK gene along with disruption of an endogenous gabD gene could produce 52.1 g/L of 5-HV with the yield of 0.33 g/g glucose in fed-batch fermentation. Keywords : 5-hydroxyvaleric acid, Corynebacterium glutamicum, L-Lysine References 1. Y. J. Sohn, M. Kang, K. A. Baritugo, J. Son, K. H. Kang, M. H. Ryu, S. Lee, M. Sohn, Y. J. Jung, K. Park,

S. J. Park, J. C. Joo, H. T. Kim, ACS Sustainable Chem. Eng. 9(6), 2523-2533 (2021).

561


P0820 High-yield Biosynthesis of Protocatechuate Through Fine Tuning of Biosynthetic Pathway

Jinho YEOM, Seung Min YOO School of Integrative Engineering, Chung-Ang University, Seoul, Korea Corresponding Author Email : [email protected] Protocatechuate (PCA) is promising building block for synthesizing polymer and plastics. It also has pharma-ceutical activities. PCA is naturally present in many plants, but it is impractical to directly extract from natural resources due to low yields. Many efforts have been made over the past years to produce PCA from microbial strains (1), but previous studies either create auxotrophic strain or lack a sufficiently diverse and precise regulation gene expression involved in PCA biosynthesis. We demonstrated an efficient production method via precise tuning of gene expression that related to PCA biosynthesis in E. coli. We used qsuB and precisely controlled the expression level using tunable gene regulation system (2). To decrease precursor consumption, the expression levels of of aroE and ydiB, involved in shikimate pathway, were modulated by synthetic sRNAs with different strength (3). Furthermore, several gene modules containing enzymes involved in shikimate pathway and expected to increase the intracellular production of PCA were overexpressed. ** This work was supported by an NRF grant funded by the Ministry of Science and ICT (NRF-2019 R1A2C1088504) Keywords : Metabolic engineering, Protocatechuate, fine-tuning, benzoate References 1. S. Pugh, R. McKenna, M. Osman, B. Thompson, D.R. Nielsen, Process Biochemistry 49 (11), 1843-1850

(2014). 2. S.M. Yoo, S.W. Jung, J. Yeom, S.Y. Lee, D. Na, ACS Synth. Biol. 9 (11), 2998-3007 (2020). 3. D. Na, S.M. Yoo, H. Chung, H. Park, J.H. Park, S.Y. Lee, Nat. Biotechnol. 31, 170-174 (2013).

562


P0821 Markerless Gene Deletion and Integration for the Expression of Heterologous Biosynthetic Gene Clusters in Pseudomonas putida Strain

Hye eun YU1, Kyeong Rok CHOI1, Jae sung CHOI1, In Jin CHOI1, Dahyeon PARK1, Sang yup LEE1,2 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark Corresponding Author Email : [email protected] Pseudomonas putida has been widely used for the production of valuable natural products. Gene knockout tools for P. putida have been studied, however, integration strategy of heterologous genes into chromosomal DNA of P. putida needs to be developed to the efficient methods. Current methods depend on time consuming homo-logous recombination method and transposon mediated random insertions. here we report a RecET recom-bination system for markerless integration of heterologous genes in the P. putida genomic DNA. The capacity and efficiency of the recombination system were demonstrated by gene deletion of various genetic loci on the P. putida chromosome with 0.6 to 101.7kb DNA length. The RecET recombineering system was studied here results in successful integration of biosynthetic gene clusters for protein, polyketide, isoprenoid, and amino acid derivatives. This recombineering system was optimized through application of Cre/lox system and completed by plasmid curing systems. The developed system here will be applicable in metabolic engineering of P. putida for efficient gene deletion and integration. Keywords : genetic engineering, engineering tools, metabolic engineering, markerless

563


P0822 Systems Metabolic Engineering of Corynebacterium glutamicum for the Efficient Production of Glutaric Acid

Hye eun YU1, Tae hee HAN1, Gi bae KIM1, Sang yup LEE2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea Corresponding Author Email : [email protected] Recently, there has been increased demand for C5 platform chemicals such as glutaric acid that is widely used building block for the synthesis of polyesters and polyamides. Here we report the systemic engineered microbial producers for efficient glutaric acid in L-lysine overproducing Corynebacterium glutamicum BE strain. From the previous study, an optimal metabolic biosynthetic pathway with Pseudomonas putida L-lysine monooxy-genase and 5- aminovaleramid amidohydrolase genes and 4- aminoburyrate aminotransferase and succinate semialdehyde dehydrogenase genes from C. glutamicum was introduced into the C. glutamicum BE strain. By genome wide analyses simulation, 11 target genes for increasing the L-lysine were identified and expressed at desired levels. Moreover, ynfM gene was identified and overexpressed to further enhance glutaric acid production. Optimized fermentation conditions also could result in efficient production of glutaric acid. Keywords : Glutaric acid, coryne, lysine, metabolic engineering

564


P0823 Development of Escherichia coli Strain for Extracellular Free Haem Production

Hye eun YU1, Xin Rui ZHAO1, Kyeong Rok CHOI1, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2BioProcess Engineering Research Center, KAIST, Daejeon, Korea, 3BioInformatics Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Haem is porphyrin derivative prosthetic group for hemoproteins and it plays important roles in almost all organisms for transporting oxygen, transferring electrons to supply energy. For these reasons, haem has been used in healthcare and dietary supplement industries. Escherichia coli has been developed to produce free haem, but none of studies was able to produce extracellular free haem. Here we report secretory production of free haem by engineered E. coli strains through the C5 pathway and construction of optimized downstream pathway of the haem biosynthesis. Strains with deletion of ldhA, pta and yfeX genes, which are responsible for lactate and acetate formation and encoding putative haem degradation enzyme, produced 7.88mg l-1 of total haem with 1.26mg l-1 of extracellular haem in flask cultivation. Fed batch cultivation of the engineered strain overexpressing haem exporter CcmABC enhanced production and secretion of haem 73.4(63.5% of total haem) and 151.4(63.3%) mg l-1 from glucose only and glucose with L-glutamate respectively. These strategies could be applied for efficient microbial production of free haem for pharmaceutical uses and food industries. Keywords : metabolic engineering, Escherichia coli, haem, heme, free heme

565


P0824 Metabolic Engineering of Microorganisms Using Repurposed Type III Polyketide Synthase as Malonyl CoA Biosensor

Hye eun YU1,2, Dongsoo YANG1,2, Won Jun KIM1,2, Seung Min YOO1,3, Jong Hyun CHOI4, Shin Hee HA1, Mun Hee LEE1,4, Sang Yup LEE1,2,3,5 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3BioProcess Engineering Research Center and BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 4Applied Microbiology Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Korea, 5Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark Corresponding Author Email : [email protected] Malonyl CoA is an important building block for the production of diverse value-added chemicals including natural products, but competition of target compounds limits the intracellular malonyl-CoA availability. For high throughput screening of targets that that increase the malonyl-CoA pool, diverse malonyl CoA sensors have been developed. However, there have limitations, including narrow applicable strain range, only in Escherichia coli and Saccharomyces cerevisiae and requirements of signal transduction multi-steps. Here, we report novel colorimetric malonyl-CoA biosensor for industrially important bacteria: Corynebacterium glutamicum, Pseudomonas putida, and Escherichia coli. RppA is a type III polyketide synthase producing colored metabolites flaviolin that was developed as malonyl-CoA biosensor in E. coli. Increased malonyl-CoA accumulating cells were detected by the colorimetric screening of enhanced red colored cells. Other type III polyketide synthases were able to be repurposed as malonyl-CoA biosensors. To screen the target, 1,858 synthetic small regulatory RNA library was synthesized and knockdown targets were used to produce two types of polyketide (7-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. By knocking out these genes alone or in combination, the engineered strain could produce methylsalicylic acid, aloesone, resveratrol, and naringenin in high concentration. In conclusion, the RppA biosensor is expected to screen the strains capable of producing malonyl-CoA derived products efficiently. Keywords : biosensors, malonyl-CoA, metabolic engineering, systems metabolic engineering, Escherichia coli

566


P0825 Production of Polyhydroxyalkanoate Copolymer Using Methylorubrum extorquens AM1 from Formate

Jihee YOON, Min-Kyu OH Department of Chemical and Biological Engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Formate is a promising environmentally friendly and sustainable feedstock synthesized from syngas or carbon dioxide. Methylorubrum extorquens is a type II methylotroph that can use formate as a carbon source. It accumulates polyhydroxyalkanoates (PHAs) inside the cell, mainly producing poly-3-hydroxybutyrate (PHB), a degradable biopolymer. Owing to its high melting point and stiff nature, however, mechanical property improvement is warranted in the form of copolymerization. To produce the PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the endogenous gene phaC was deleted and the pathway genes bktB, phaJ1, and phaC2, with broader substrate specificities, were heterologously expressed. To improve the incorporation of 3-hydroxyvalerate (3HV), the expression level of bktB was improved by untranslated region (UTR) engineering, and the endogenous gene phaA was deleted. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, when propionate and butyrate were supplemented, PHBVs with 3HV portions of up to 70.6% were produced. This study shows that a PHBV copolymer with a high proportion of 3HV can be synthesized using formate, a C1 carbon source, through metabolic engineering and supplementation with short-chain fatty acids. Keywords : PHBV copolymer, formate, short-chain fatty acid, Methylorubrum extorquens AM1, methylotroph, metabolic engineering

567


P0826 The N-terminal Incorporation of Unnatural Amino Acid with Bacterial Translation System Engineering

Sangwoo LEE1, Byeong Sung LEE1, Woon Jong CHOI1, Tae Hyeon YOO1,2 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] Addition of unnatural amino acids to protein used in various purposes. By orthogonal translation systems, unnatural amino acids are incorporated to target proteins. In particular N-terminal position of protein modifi-cation, unnatual amino acid can be incorporated without affecting protein structure. But fomal orthognal translational pair, Methanococcus jannaschii tyrosyl tRNA and aminoacyl-tRNA synthetase only recoginzes amber codons in elogation position. In this study, focusing on differences of initiator tRNA and elongator tRNA, we engineered Mj tRNA to interact formyltransferase and initiation factor 2 which participate in the assembly of aminoacylated initiator tRNA and new tRNA pair system incorporated unnatural amino acid at N-terminal position of target protein in overexpression of formyltrasferase. The engineered Mj tRNA / aminoacyl-tRNA synthetase pair could be used for protein engineering of incorporation of unnatural amino acid to N-terminal position. Keywords : Unnatural amino acid, Orthogonal translational system, Initiator tRNA engineering

568


P0827 Metabolic Engineering of Escherichia coli for the Production of Benzoic Acid from Renewable Source

Zi Wei LUO, Youngjoon LEE, Sang Yup LEE Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess Engineering Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Benzoic acid (BA) is one of the important platform aromatic compounds in chemical industry. Also, it is widely used as food preservatives in its salt forms. However, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the biosynthesis of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. Fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries [NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557] from the Ministry of Science and ICT through the National Research Foundation of Korea.] Keywords : Metabolic Engineering, Escherichia coli, Benzoic acid

569


P0828 Metabolic Engineering for Microbial Production of a Grape Flavor Compound, Methyl Anthranilate

Zi Wei LUO, Jae Sung CHO, Youngjoon LEE, Sang Yup LEE Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess Engineering Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Methyl anthranilate (MANT) is widely used compound for grape scent and flavor. However its manufacturing process requires petroleum-based processes. Here, we report the direct fermentative production of MANT from glucose by metabolically engineered Escherichia coli and Corynebacterium glutamicum strains harboring a synthetic plant-derived metabolic pathway. Fed-batch fermentation of the engineered E. coli and C. glutamicum strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L of MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) Keywords : Methyl anthranilate, Escherichia coli, metabolic engineering

570


P0829 Microbial Biosynthesis of Aromatic Polymers from Metabolically Engineered Escherichia coli Strains

Jung Eun YANG1, Si Jae PARK2, Won Jun KIM3, Hyeong Jun KIM4, Bumjoon KIM4, Hyuk LEE5, Jihoon SHIN6, Youngjoon LEE7, Sang Yup LEE7 1World Institute of Kimchi, Gwangju, Korea, 2Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea, 3Hanwha chemical, Daejeon, Korea, 4Polymer and Nano Electronics Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, KAIST, Daejeon, Korea, 5Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon, Korea, 6Center for Bio-based Chemistry, Green Chemistry & Engineering Division, Korea Research Institute of Chemical Technology, Daejeon, Korea, 7Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess Engineering Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Engineered Escherichia coli strains developed in this study is able to produce aromatic polyesters from glucose through one-step fermentation. Clostridium difficile isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase and evolved polyhydroxyalkanoate synthase genes are expressed in a engineered D-phenyllactate-producer strain. Poly(3-hydroxybutryate-co-D-phenyllactate) copolymers, with different molar compositions are produced through expression of Cupriavidus necator β-ketothiolase and reductase genes. This is the first attempt on the one-step fermentative production of aromatic polyesters from renewable resources. ** This work was supported by the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) and also by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation of Korea. Keywords : Escherichia coli, Polyhydroxyalkanoate, Metabolic engineering

571


P0830 A Simple Assay Method for the Inhibitory Neurotransmitter Gamma-aminobutyric Acid (GABA) Through Complementary Cell-free Translational Assay

Youjin LEE, Hye Jin LIM, So Jung LEE, Dong-Myung KIM Department of Chemical engineering and Applied Chemistry, Chungnam National University, Daejeon, Korea Corresponding Author Email : [email protected] Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system (CNS) and is involved in the modulation of synaptic transmission. It is known to be effective in improving brain blood flow, increasing oxygen supply, relieving stress, and insomnia, and accordingly, the importance of measuring GABA is increasing. Presently, high-performance liquid chromatography (HPLC) and gas chromatography represent the gold standards of GABA assay, often in association with chemical derivatization with 1-fluoro-2,4-dintrophenyl-5-alaninamide, o-phthalaldehyde, naphthalene-2,3-dicaboxaldehyde, phenyl isothiocyanate, and dabsyl chloride can enhance sensitivity and quantifiability of detection [1, 2 ,3]. Whereas the importance of routine monitoring of GABA is increasing, these existing methods commonly suffer expensive and time-consuming, requirements of highly sophisticated equipment [4]. In this study, we developed a method that wires the enzyme reactions of GABA conversion with the cell-free protein synthesis to generate highly amplified and readily measurable biological signals in response to trace amounts of GABA in biological samples. If a cell-free protein is synthesized using a reaction solution lacking a specific amino acid, protein synthesis will not occur. If you hang the reaction solution in which alanine was produced through transamination reaction, protein will be produced. For example, through the transamination reaction, GABA and pyruvate can be converted to alanine and succinate-semialdehyde. With the amount of alanine converted through the transamination reaction, the concentration of GABA can be measured with complementary cell-free protein synthesis (CCFPS). 1. Screening of transaminase for the detection of GABA by CCFPS. 2. Purification and optimizing purified transaminase reaction concentration and time. 3. Detecting GABA through CCFPS reaction programed with the gene of sfGFP. Keywords : GABA, cell-free protein synthesis, transaminase References 1. Y. Lu, H. Zhang, X. Meng, L. Wang, X. Guo. .Anal. Lett. 43, 2663 (2010). 2. L. Tan, F. Liu, N. Luo, J. Li . Relat. Technol. 29,45 (2006). 3. Roopkumar Sangubotla, Jongsung Kim. Applied Surface Science. 490,61 (2019). 4. V.H.C. Castro, C.L.L. Valenzuela, J.C.S. Sánchez, K.P. Peña, S.J.L. Pérez, J.O. Ibarra, A.M.Curr.

Neuropharmacol. 12,490 (2014).

572


P0831 Enhanced Succinic Acid Production by Mannheimia succiniciproducens Employing Optimal Malate Dehydrogenase

Jung Ho AHN1,2,3, Hogyun SEO4,5, Woojin PARK4,6, Jihye SEOK4,6, Jong An LEE1,2,3, Won Jun KIM1,2,3, Gi Bae KIM1,2,3, Kyung-Jin KIM4,6, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon, Korea, 3Bioinformatics Research Center and BioProcess Engineering Research Center, KAIST, Daejeon, Korea, 4School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daegu, Korea, 5Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, Korea, 6KNU Institute for Microorganisms, Kyungpook National University, Daegu, Korea Corresponding Author Email : [email protected] Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, while M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g·L-1 of SA with the maximum productivity of 21.3 g·L-1·h-1, demonstrating the importance of enzyme optimization in strain development. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT (MSIT) through the National Research Foundation (NRF) of Korea. This work was further supported by the C1 Gas Refinery Program (NRF-2016M3D3A1A01913250) from MSIT through NRF. Keywords : Mannheimia succiniciproducens, succinic acid, malate dehydrogenase, Corynebacterium glutamicum References 1. J. H. Ahn, H. Seo, W. Park, J. Seok, J. A. Lee, W. J. Kim, G. B. Kim, K. -J. Kim, and S. Y. Lee, Nat.

Comm. 11, 1970 (2020).

573


P0832 Development of an Engineered Escherichia coli Strain Grows Solely on CO2 and Formic Acid

Junho BANG1,2, Chang Hun HWANG1,2, Jung Ho AHN1,2, Jong An LEE1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon, Korea, 3BioInformatics Research Center and BioProcess Engineering Research Center KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Developing synthetic autotroph or formatotroph has been attractive issue as a solution for the environmental problems and conversion of useless C1 compounds to value-added chemicals. The development of Escherichia coli strains capable of growing solely on CO2 and FA was already reported. However, the final cell densities were too low (less than OD600 of 1). Here we report the development of an E. coli strain capable of growing solely on CO2 and FA to relative high cell density (more than OD600 of 11) by introducing the synthetic CO2 and formic acid assimilation pathway, expressing two formate dehydrogenase genes, fine-tuning metabolic fluxes and optimizing the levels of cytochrome bo3 and bd-I ubiquinol oxidase. This E. coli strain is expected to be utilized as a useful platform strain that growing solely on CO2 and FA and producing valuable chemicals only through C1 compounds assimilation. ** This work was supported by the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2016M3D3A1A01913250). Keywords : Escherichia coli, carbon dioxide, formic acid, C1 gas refinery, formatotroph References 1. J. Bang, C. H. Hwang, J. H. Ahn, J. A. Lee, and S. Y. Lee, Nat. Microbiol. 5, 1459-1463 (2020).

574


P0833 Escherichia coli as a Model System to Study the Mitochondrial Mutations in Biliary Atresia

Ji-Young LEE1, Hong KOH2, Dong-Woo LEE1 1Department of Biotechnology, Yonsei University, Seoul, Korea, 2Department of Pediatrics, Yonsei University College of Medicine, Severance Children’s Hospital, Severance Pediatric Liver Research Group, Seoul, Korea Corresponding Author Email : [email protected] Biliary atresia (BA) is a hepatic disease characterized as blockage of bile flow caused by the absence of extrahepatic ducts. Even after a successful Kasai surgery, a considerable number of patients exhibit hepato-cellular dysfunction due to mitochondrial mutations. Previously, we identified the BA-specific SNP-like mitochondrial mutations in the ND2 and ND4 subunits of the complex I. To investigate the effect of those mutations on the structure and function of complex I, we generated Escherichia coli mutant strains lacking the ND2 and ND4 subunits as microbial counterparts, respectively. The ΔnuoN and ΔnuoM mutants exhibited severe growth defects under anaerobic conditions, whereas they, including Δndh, a knock-out strain of NuoA-N isozyme, showed a similar pattern to WT under aerobic conditions. To examine the effect of BA-specific mutations on bacterial growths, we generated several E. coli mutants and characterized their growth patterns. Remarkably, some mutant strains showed growth defects due to a lack of peripheral alternative respiratory chains similar to those of ΔnuoN and ΔnuoM under anaerobic conditions, indicating that the mutations hinder the cellular growth through the impaired catalytic function of complex I. Therefore, we suggest that these E. coli mutant strains can be used as a model system to study the effect of mitochondrial mutations in complex I. Keywords : Biliary Atresia, Mitochondrial Complex I, Escherichia coli References 1. Koh, Hong, et al. Scientific reports 8.1 (2018): 1-14.

575


P0834 Metabolic Engineering of Cupriavidus necator H16 for De Novo Biosynthesis of Resveratrol from Carbon Dioxide

Yong Jae JANG1,2, Kyoung Heon KIM2, Ja Kyong KO1 1Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea, 2Department of Biotechnology, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Resveratrol is a natural polyphenolic compound produced by various plants. Due to its health benefit and increasing demand, efforts aided by synthetic biology and metabolic engineering have made the microbial production of plant-derived natural products to a greater extent. A lithoautotrophic bacterium, Cupriavidus necator, was used as a microbial platform for producing resveratrol from different carbon sources (e.g., fructose and carbon dioxide). Since this microbial platform is genetically tractable and has a high-flux carbon storage ability, it has been engineered to produce a variety of valuable compounds. De novo biosynthesis of resveratrol in C. necator was consisted of tyrosine ammonia lyase (TAL), 4-coumarate-coA ligase (4CL), and stilbene synthase (STS) with the supply of L-tyrosine. The intracellular malonyl-CoA availability, which is the major bottleneck of the phenylpropanoid pathway, was improved by overexpressing acetyl-CoA carboxylase (ACC) and deleting PHB pathway. As a result, >3 mg/L of resveratrol was produced from fructose by using this engineered strain. With further improvement of metabolic capabilities of the recombinant strain, we will show lithoautotrophic production of resveratrol from carbon dioxide in a hybrid microbial electrosynthetic system. Keywords : Resveratrol, Cupriavidus necator, Metabolic engineering

576


P0835 Precise Rebalancing the Precursor by Fine-control of gapA Expression for the Biosynthesis of Isoprenoids in Escherichia coli

Kumyoung JEUNG1, Juyoung JUNG2, Jae Hyung LIM3, Joo Yeon SEOK1, Gyoo Yeol JUNG1,3 1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea, 2Department of Life Sciences, POSTECH, Pohang, Korea, 3Department of chemical engineering, POSTECH, Pohang, Korea Corresponding Author Email : POSTECH, Pohang, Korea Isoprenoids form one of the most diverse group of natural products. Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3 phosphate and pyruvate to divert carbon flux toward the products of interest. In this study, we demonstrated that precursor rebalancing is one of the critical steps for the biosynthesis of isoprenoids in Escherichia coli via the DXP pathway. First, we constructed a lycopene-overproducing E. coli strain by the amplification of the native DXP pathway using synthetic promoters and redesigned 5’-untranslated regions (5’-UTR). Next, fine-controlled precursor balancing was investigated by tuning PpsA or GAPDH. The results showed that tuning-down of gapA improved the specific lycopene by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids. Finally our strategy could be broadly utilized in the field of metabolic engineering and synthetic biology to increase the production of numerous high-value isoprenoids. Keywords : isoprenoid, 5'-UTR, gapA, metabolic engineering, synthetic biology, precursor balancing

577


P0836 Improved Yield of Recombinant Protein via Flagella Regulator Deletion in Escherichia coli

Jae-Ho HAN, Min-Kyu OH Department of Chemical & Biological Engineering, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADPC ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC and ptsG deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced green fluorescent protein yield per glucose consumption increased 1.81-fold in the flhC mutant strain. Thus, our study demonstrates that high-yield production of the recombinant protein was achieved with reduced flagella formation. Keywords : Flagella, Metabolic flux analysis, 13C-MFA, ATP, NADPH, E. coli, Escherichia coli

578


P0837 Microbial Production of Fatty Acids and Fuels by Metabolically Engineered Rhodococcus Opacus Strains

Hye Mi KIM1, Tong Un CHAE1,2, So Young CHOI1,2, Won Jun KIM1,2, Ji Hye HYUN1,2, Sang Yup LEE1,2,3 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3BioInformatics Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] Production of free fatty acids(FFAs) and the derivatives from renewable non-food biomass by microbial fermentation is getting great attention. We developed metabolically engineered Rhodococcus opacus strains producing FFAs, fatty acid ethyl esters(FAEEs) and long-chain hydrocarbons (LCHCs). Culture conditions were optimized to produce and accumulate 82.9g/L triacylglycerols (TAGs) in R.opacus from glucose. Then, The strains were metabolically engineered to hydrolyze TAGs by in vivo expression of lipases and produced 50.2g/L FFAs, and also 21.3g/L FAEEs and 5.2g/L LCHCs by further metabolic engineering such as deleting acyl-coenzyme A (CoA) synthetases or dehydrogenases and overexpressing several genes. The strategies for metabolic engineering and the engineered strains developed in this study will be helpful to establish oleaginous biorefinery platforms for the sustainable production of chemicals and fuels. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation(NRF) of Korea(NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) Keywords : Metabolic engineering, Synthetic biology, Lipids, oleaginous bacterium, Rhodococcus opacus, free fatty acids

579


P0838 Biosynthesis of Biodegradable Polymer, Poly(d‐lactate‐co‐glycolate‐co‐4‐hydroxybutyrate) for Biomedical Applications

So Young CHOI1,2, Tong Un CHAE1,2,3, Jihoon SHIN4, Jung Ae IM1,2,5, Ji Hye HYUN1,2, Sang yup LEE1,2,3,5 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Applied Science Research Institute, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 4Center for Bio‐based Chemistry, Green Chemistry and Engineering Division, Korea Research Institute of Chemical Technology, Daejeon, Korea, 5BioProcess Engineering Research Center and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea Corresponding Author Email : [email protected] Poly(d‐lactate‐co‐glycolate‐co‐4‐hydroxybutyrate) [poly(d‐LA‐co‐GA‐co‐4HB)] and poly(d‐lactate‐co‐glycolate‐ co‐4‐hydroxybutyrate‐co‐d‐2‐hydroxybutyrate) [poly(d‐LA‐co‐GA‐co‐4HB‐co‐d‐2HB)] are getting great atten-tion for applications as new biomedical polymers. Here, we report the increased production of such polymers with metabolic engineering of Escherichia coli. The resulting engineered E. coli strain successfully produced poly(d‐LA‐co‐GA‐co‐4HB‐co‐d‐2HB) and poly(d‐LA‐co‐GA‐co‐4HB) from glucose and xylose. By controlling the expression levels of the heterologous genes and performing fed‐batch cultivations, the polymer content and titer was increased to 65.76wt% and 6.19g/L, and also the monomer fractions in the polymers could be controlled. Particularly, the 4HB‐rich polymers showed viscous and sticky properties suggesting that they may be used as medical adhesives for potential biomedical applications. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation(NRF) of Korea(NRF‐2012M1A2A2026556 and NRF‐2012M1A2A2026557) Keywords : 4-hydroxybutyrate, polyhydroxyalkanoate, metabolic engineering, glycolate, lactate, poly(D-lactate-co-glycolate-co-4-hydroxybutyrate)

580


P0839 Development of a Deep Learning-based Tool DeepTFactor for Transcription Factors Prediction

Gi Bae KIM1,2,3,4,5,6, Ye GAO7,8,9, Bernhard O. PALSSON8,9,10, Ji Hye HYUN1,2, Sang Yup LEE1,2,3,4,5,6 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 4KAIST Institute for Artificial Intelligence, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 5BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 6BioInformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 7Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA, 8Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 9Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, CA, USA, 10Novo Nordisk Foundation Center for Biosustainability, Kongens Lyngby, Denmark Corresponding Author Email : [email protected] A transcription factor (TF) is sequence-specific DNA-binding protein that plays major role in transcription initiation. We developed DeepTFactor, a deep learning-based tool for the prediction of TFs, employing convolutional neural network that has three subnetworks in parallel. It was successful in predicting TFs of both eukaryotic and prokaryotic origins. Analyzing the gradients of prediction score with respect to input suggested that DeepTFactor detects DNA-binding domains and other latent features for TF prediction. It predicted 332 candidate TFs of Escherichia coli, and three were experimentally validated by identifying genome-wide binding sites with ChIP-exo experiments. We also provided DeepTFactor as a stand-alone program and it predicted 4,674,808 TFs from 73,873,012 protein sequences in 48,346 genomes. DeepTFactor can be used as a valuable tool for understanding the regulatory systems of organisms. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries(NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation(NRF) of Korea. Keywords : deep learning, transcription factor, transcription regulation, y-ome, ChIP-exo References 1. G. B. Kim, Y. Gao, B. O. Palsson and S.Y. Lee, PNAS. 118(2) (2021).

581


P0840 High‐level 3‐Hydroxypropionic Acid Production by Metabolically Engineered Escherichia coli Using Glycerol

Je Woong KIM1,2,3, Yoo-Sung KO1,2,3, Tong Un CHAE1,2,3, Ji Hye HYUN1,2, Sang Yup LEE1,2,3,4 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3BioProcess Engineering Research Center, KAIST, Daejeon, Korea, 4BioInformatics Research Center, KAIST, Daejeon, Korea Corresponding Author Email : [email protected] As climate change is a critical environmental problem, the conventional petrochemical‐based processes are being altered to environmentally-friendly bio-based processes to produce chemicals. Here, 3‐hydroxypropionic acid (3‐HP), which is an industrially valuable three carbon (C3) chemical, was highly produced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. Glycerol-dependent 3-HP biosynthetic pathway was constructed with the introduction of heterologous genes and subsequent screening of these genes combinations. The conditions of fed‐batch fermentation were optimized to further increase 3‐HP production with several strategies. The final engineered E. coli strain with dissolved oxygen control and continuous feeding strategy produced 76.2 g/L of 3‐HP with the yield of 0.457 g/g glycerol and productivity of 1.89 g/L/h. ** The work on was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A 2026557) and by Hanwha Chemical). Keywords : 1,3-hydroxypropionic acid, 1,3-propanediol, Escherichia coli, metabolic engineering, glycerol, crude glycerol References 1. J.W. Kim, Y.S. Ko, T.U. Chae and S.Y. Lee, Biotechnol. Bioeng, 117( 7), 2139–2152 (2020).

582


P0841 Enhanced Production of L-serine in Escherichia coli Through Synthetic Protein Scaffold of SerB, SerC, and EamA

Ashokkumar KUMARAVEL, Jae Hoon JEONG, Kim-Ngan T. TRAN, Soon Ho HONG Department of Chemical Engineering, University of Ulsan, Ulsan, Korea Corresponding Author Email : [email protected] While L-Serine has wide applications and great market potential, its application is limited because of its high cost and low yields. Bacterial cell factories have many advantages for producing L-serine from renewable carbon resources. To achieve L-serine production with high efficiency, three enzymes (SerB, SerC, and EamA) were physically re-localized by using a scaffold system GBD:SH3:PDZ. Such strategy was highly effective in improving the production of L-serine in Escherichia coli. The highest concentration of L-serine harvested was at 3.8 g/l after 48 h in strain E. coli with the scaffold GBD:SH3:PDZ at ratio of 1:2:1, reaching a high yield of 0.24 mol/mol glucose. Keywords : L-Serine, Protein scaffold system, Bioproduction, Metabolic engineering

583


P0842 Effects of Triterpenoid Alisol-F on Human 5-hydroxytryptamine 3A and Nicotinic Acetylcholine Receptor Channel Activity

Junho LEE Department of Biotechnology, Chonnam National University, Gwangju, Korea Corresponding Author Email : [email protected] Alisma Rhizome is a known tradition medication, which has been used for its diuretic, hypolipidemic, anti-diabetic, and anti-inflammatory purposes for thousands of years. The primary compounds of Alisma Rhizome are protostane type triterpenes, such as Alisols A, B or C. We previously demonstrated that Alisol derivatives (Alisols A, B, and C) have inhibitory effects on 5-hydroxytryptamine 3A (5-HT3A) currents.1 In this study, we tested the effects of a new triterpene, Alisol-F, on human 5-HT3A and nicotinic acetylcholine (nACh) receptor channel currents by using Xenopus oocytes expressing these channels. Co-application of Alisol-F inhibited 5-HT3A and nACh receptor-mediated inward peak currents. The inhibitory effect of Alisol-F on 5-HT and ACh-induced inward peak currents occurred in a reversible and concentration-dependent manner. The half maximal inhibitory concentrations (IC50) of Alisol-F were 79.4 ± 11.0 and 21.2 ± 6.0 uM for the 5-HT3A and nACh receptors, respectively. In addition, the inhibition of I5-HT and IACh by Alisol-F occurred noncompetitive and voltage insensitive manner. Taken together, these results show that Alisol-F may regulate 5-HT3A and nACh receptors channel expressed in Xenopus oocytes. Keywords : Alisma Rhizome, Ligand-gated ion channel, Nicotinic acetylcholine receptor

584


P0843 Othorlog Algorithm for Detecting sRNA in the Shigella flexneri Serotype 2a Strain 2457T

Khoi NHAT PHAM, Joon Young PARK, Linh KHANH NONG, Donghyuk KIM Schools of Energy & Chemical Engineering (ECHE), UNIST, Ulsan, Korea Corresponding Author Email : [email protected] The sRNAs has known as the small non-coding RNA, divided into cis-acting RNA as expressed from opposite their target gene and trans-acting sRNA with the expression in intergenic/or untranslated region [1]. In bacteria, small RNA has a crucial role in the expression of outer membrane proteins, iron homeostasis, quorum sensing, and notable bacterial virulence. Shigella sflexneri Serotype 2a Strain 2457T belongs to the Shigella genus, which is well-known as cause dysentery [2]. Cause of the small 50-500 bp size, the detection of sRNA has been challenged to the researcher for now. In the NCBI database, the sRNA of S. flexneri 2a str. 2457T have not been fully annotated. This research developed an Orthorlog algorithm that was applied for identifying sRNA in S. flexneri 2a str. 2457T from known sRNA in reference, closed relationship, Escherichia coli MG1655. The validation of candidate sRNAs was carried out by ChIP-exo and RNA-seq dataset. The results located 109 sRNA in S. flexneri 2a str. 2457T, only 14 sRNA detected based on the Ortholog algorithm compared with Rfam-infernal and NCBI database. Based on the ChIP-exo dataset, 8/14 sRNAs bind to RpoD (σ70) and RpoN (β core of RNA polymerase), 1/14 sRNA regulated by RpoE (σ24) and 2/14 sRNAs bound to RpoN(σ54) and RpoS (σ38), respectively. Two virulence sRNA C0362, ryeC specified and validated with RpoD, RpoN ChIP-exo, and RNA-seq data. Its function possibly controls iron uptake and survival acid-stress condition. This research introduced the Ortholog algorithm to detect sRNA based on closed relationship strains. Keywords : small RNA, Ortholog algorithm, S. flexneri 2a str. 2457T, ChIP-exo, RNA-seq References 1. Bloch. S. et al. Toxins (Basel). 9(6), (2017). 2. Wei. J. et al. Infect Immun. 71(5), 2775-86 (2003).

585


P0844 Deep-learning Based Investigation of ChIP-Exo Reads (DICER) to Analyze RpoN Regulon of Enterobacteria

Kabimoldayev ILYAS, Khanh Linh NONG, Donghyuk KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea Corresponding Author Email : [email protected] Chromatin immunoprecipitation (ChIP)-exo has emerged to be an advance modification of ChIP-seq. It provides a single base-pair (bp) resolution of genome-wide protein binding location hence, facilitates accurate binding mode prediction. While peak calling algorithms such as MACE have been developed for ChIP-exo analysis, application of MACE on bacterial dataset often yields large proportion of false-positive peaks. MACE outputs require manual post-processing by human experts, which can be tricky and extremely time consuming. Deep-learning based Investigation of ChIP-Exo Reads (DICER) was developed using convolution neural network. The first step of DICER reduces noise in the ChIP-exo data, allowing its second step to perform the final peak calling. The software analyzes each 40 bp region of genome, and classifies it as peak or non-peak based on its signal-to-noise ratio. In this study, curated ChIP-exo data for sigma factor RpoN in Escherichia coli K-12 MG1655 was used as training set. With RpoN ChIP-exo dataset of 10 different Enterobacteria strains, not only DICER predicted more peaks with RpoN binding motif than MACE, it also detected much lesser peaks without motifs. Combined analysis of RpoN binding sites identified by DICER and RNA-seq data revealed the presence of pervasive promoters, and anti-sense transcription start sites associated with RpoN binding sites. Keywords : Deep learning, ChIP-exo, peak calling

586


P0845 Mechanism of 3-Hydroxypropionic Acid Tolerance by Deletion of a Less-characterized Transcription Factor yieP in Escherichia coli W

Thuan NGUYEN-VO1,2, Seyoung KO1, Huichang RYU1, Donghyuk KIM1, Jung Rae KIM1, Sunghoon PARK1,2 1School of Energy and Chemical Engineering, UNIST, Ulsan, Korea, 2School of Chemical and Biomolecular Engineering, Pusan National University, Busan, Korea Corresponding Author Email : [email protected] Toxicity of organic acids such as 3-hydroxypropionic acid (3-HP), propionic acid, lactic acid, is an important issue that limits microbial production of them at industrial levels (~ 1M). As a case study to tackle this problem, adaptive laboratory evolution has been successfully adopted to evolve Escherichia coli W for tolerance against 3-HP (1). Our previous study showed that the deletion of a less-characterised transcription factor (YieP) mainly responsible for the tolerance against 3-HP. In this current study, we exploited physiological and transcrip-tomics study (RNA-seq along with ChiP-exo) to study the mechanism that the deletion of yieP confers 3-HP tolerance. The growing experiment in several organic acids (C2-C4) suggested that yieP deletion conferred specific-anion tolerance toward 3-HP but not other organic acids. The transcriptomics along with deletion experiments suggested that deletion of yieP improved 3-HP tolerance via expression of two genes encoding inner membrane proteins yohJ and yohK. The role of YohJ/K was further studied using 3-HP responsive biosensor. Our results suggested that yohJK encode for (a) novel 3-HP exporter(s). Keywords : adaptive laboratory evolution, 3-hydroxypropionic acid, acid tolerance, transporter, yieP, transcrip-tion factor, transcriptomics References 1. Nguyen-Vo et al., Met. Eng.. 53, 48 (2019).

587


P0846 Improved Ribose Productivity by Random Mutation of Bacillus subtilis

Seung-Joo KIM1, Yong-Hwan LEE1, Kyeong-Won KIM1, Jin-Kyeong YANG1, Na-Yeon KIM1, Yong-Cheol PARK2, Dae-Hyuk KWEON1 1Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea, 2Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul, Korea Corresponding Author Email : [email protected] Ribose is a value-added five-carbon sugar used in the production of riboflavin and inosine monophosphate, and used in energy drinks and supplements. Ribose is biosynthesized through the pentose phosphate pathway from glucose. To increase the productivity of ribose, we randomly mutated Bacillus subtilis using 2-Aminopurine. In a 96 well plate, the amount of ribose production of mutant was measured using the FLIPRbs biosensor that measures the amount of ribose through Fluorescence Resonance Energy Transfer (FRET) binding to ribose. Seventeen strains with high levels of ribose production were screened upon they produced 18.2 g/L of ribose in the flask fermentation within 72 h while WT strain produced 12.5 g/L ribose in the same culture time. However, we found serious colony-to-colony variation of ribose problem. We considered these phenomena happened wild type endospores remained inside B. subtilis. When we continued subculture to remove the endospore from wild type. The productivity of mutant could be stabilized. Keywords : Ribose, Glucose, Bacillus subtilis, Pentose phosphate pathway, Endospore, Metabolic engineering References 1. Y. Park, H. Lee, C. Kim, J. Seo, Effects of oxygen supply and mixed sugar concentration on D-ribose

production by a transketolase-deficient Bacillus subtilis SPK1, J. Microbiol. Biotechnol. 23 (2013) 560. 2. L. Wu, Z. Li, Q. Ye, Enhanced d-ribose biosynthesis in batch culture of a transketolase-deficient Bacillus

subtilis strain by citrate, J. Ind. Microbiol. Biotechnol. 36 (2009) 1289-1296. 3. Y.-C. Park, S.-G. Kim, K. Park, K.H. Lee, J.-H. Seo, Fed-batch production of d-ribose from sugar mixtures

by transketolase-deficient Bacillus subtilis SPK1, Appl. Microbiol. Biotechnol. 66 (2004) 297-302. 4. Y.C. Park, J.H. Choi, G.N. Bennett, J.H. Seo, Characterization of d-ribose biosynthesis in Bacillus subtilis

JY200 deficient in transketolase gene, J. Biotechnol. 121 (2006) 508-516.

588


P0847 Hexanoic Acid Production in Escherichia coli by Fine-tuning of atoB Expression

Seungjin KIM1, Seong Gyeong KIM1, Sungho JANG1, Gyoo Yeol JUNG1,2 1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea, 2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea Corresponding Author Email : [email protected] We demonstrate that rebalancing of metabolic fluxes at acetyl-CoA can substantially improve the titer and productivity of hexanoic acid. First, a hexanoic acid-producing E. coli strain was constructed by expressing the genes coding β-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Mega-sphaera sp. MH in a butyric acid producer strain. Expression of mhact coding ACT increased hexanoic acid production by 47% compared to that with an E. coli thioesterase, TesB. Next, metabolic flux was optimized at the acetyl-CoA branch point by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Five different synthetic 5′-untranslated regions (5′-UTRs) were designed for atoB using UTR Designer to modulate the expression level of the gene. The flux-optimized variant produced 528 mg/L hexanoic acid in 36 h, and this titer represents an 8.7-fold increase compared to that of the non-optimized parental strain. Keywords : hexanoic acid, atoB, UTR

589


P0848 Adenylation Domain Engineering and Culture Condition Optimization for Improved Production of Nonribosomal Peptide

Eunji KIM, Young Eun DU, Dong-Chan OH, Yeo Joon YOON Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] The novel cyclic nonribosomal peptides (NRPs), ohmyungsamycins A and B produced by marine Streptomyces strains, are synthesized by a NRP synthetase (NRPS). Ohmyungsamycin A particularly exhibits more potent activity against Mycobacterium tuberculosis and human cancer cells than ohmyungsamycin B. The substrate promiscuous adenylation (A) domain in the second module of OMS synthetase recruits either L-Val or L-Ile to synthesize ohmyungsamycins A and B, respectively. Engineering of the substrate-coding residues of this A domain increased ohmyungsamycin A production by 1.2-fold, coupled with a drastic decrease in ohmyung-samycin B production. Furthermore, the culture conditions (sea salt concentration, inoculum size, and the supply of amino acids to serve as building blocks for ohmyungsamycin) were optimized for ohmyungsamycin production in the wild-type strain. Finally, cultivation of the A2-domain-engineered strain under the optimized culture conditions resulted in up to 3.8-fold increases in ohmyungsamycin A yields and an 8.4-fold decrease in ohmyungsamycin B production compared to the wild-type strain under the initial culture conditions. These results demonstrate that a combination of substrate specificity manipulation of the A domain and the simple optimization of culture conditions can be a useful strategy to improve the yields of the desired NRP product and can be applied for many other cases where similar NRP congeners are synthesized by a single NRPS. Keywords : Nonribosomal peptide synthetase, Adenylation domain engineering, Site-directed mutagenesis, Culture condition optimization

590


P0849 Enzymatic Synthesis of Isepamicin Analogs with Improved Antibacterial Activity Against Isepamicin-resistant Pathogens

Yeon Hee BAN, Joong Ho JEONG, Yeo Joon YOON Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Aminoglycosides (AGs), one of the oldest classes of antibiotic agents, have strong antibacterial activity against Gram-positive and Gram-negative bacterial pathogens because they interfere with protein biosynthesis by acting on the bacterial ribosome. However, serious antimicrobial resistance has emerged as a result of the long-term and wide clinical usage of AGs. The development of new AG antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6′)-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6′-N-acylated isepamicin (ISP) analogs, 6′-N-acetyl/-propionyl/-malonyl ISPs. They were all active against ISP-resistant Gram-negative bacteria tested, and 6′-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated that the AME-mediated regiospecific modification provides efficient access to unnaturally acylated AGs with improved pharmacological potential. ** This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2019R1A2B5B03069338: Y.J.Y.; 2021R1C1C2006260: Y.H.B.). Keywords : isepamicin analogs, enzymatic synthesis, 6′-N-acylation, antibacterial activity References 1. Y. H. Ban, M. C. Song, H. J. Kim, H, Lee, J. B. Wi, J. W. Park, D. G. Lee, and Y. J. Yoon, Biomolecules

10, 893 (2020).

591


P0850 Cell Factory Design and Culture Process Optimization for Muconic Acid Biosynthesis in C. glutamicum ATCC 13032

Woo-Shik SHIN, Jaehoon CHO, Ji Woon CHUNG Korea Institute of Industrial Technology (KITECH), Cheonan, Korea Corresponding Author Email : [email protected] Corynebacterium glutamicum, which is a gram-positive bacterium has been widely used in biorefinery industry. Cis,cis-muconic acid (MA) has been widely used for the production of industrial chemicals such as adipic acid (AA), terephthalic acid and pharmaceuticals. MA was also produced using the Pseudomonas putida or P. sp strains. We studied to produce MA from glucose through shikimate pathway in C. glutamicum. Here, we have generated the engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshi-kimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. Also, to improve the MA productivity, a Non-PTS system was developed. Finally strain P28 produced 4.01 (g/L) of CA, which was 14% more than the parental strain. Keywords : Corynebacterium glutamicum, cis,cis-muconic acid, phosphotransferase system, carbon flux

592


P0851 Systems Evaluation Reveals Characterization of Methylorubrum extorquens AM1 with Improved Formate Tolerance

Gyu Min LEE, Donghyuk KIM School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Korea Corresponding Author Email : [email protected] Formate is one of attractive microbial feedstocks. The most important advantage is that it has no safety concerns. Although Methylorubrum extorquens AM1 can convert formate to carbon dioxide by FDH, it showed slower growth than methanol. In this study, adaptive laboratory evolution (ALE) was used to improve formate tolerance. First, formate resistance of wild-type strain was checked on 1g/L to 14g/L at 1g/L intervals using sodium formate. As a result, wild-type strain eventually did not grow in 13g/L sodium formate during 72 h. Since wild-type strain showed big difference in growth at 5g/L sodium formate after 48h, ALE was started from the concentration. The strain was cultured at 28 for 48h and after undergoing 5 serial transfers, the concentration was increased at intervals of 2g/L. Finally, we obtained the population of mutant that grow in 13g/L sodium formate. After preculture, streaking was done and each single colony candidate was patched. At 13 g/L sodium formate medium, all four candidates showed improved growth curves than wild-type strain. However, the final OD was about 0.06, which is not a significant result. A way to further improve the mutants will be required. Keywords : Methylorubrum extorquens, Adaptive laboratory evolution, formate

593


P0852 Microbial Production of Poly(2-hydroxyisovalerate-co-lactate) Through Metabolic Engineering

Jung Eun YANG1, Je Woong KIM1, Young Hoon OH2, So Young CHOI1, Hyuk LEE3, A-Reum PARK3, Jihoon SHIN2, Si Jae PARK4, Youngjoon LEE1, Sang Yup LEE1 1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus Program), BioProcess Engineering Research Center, KAIST, Daejeon, Korea, 2Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon, Korea, 3Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon, Korea, 4Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul, Korea Corresponding Author Email : [email protected] Development of systems metabolic engineering have enabled the production of non-natural polyhydroxy-alkanoates (PHAs), containing 2-hydroxyacids such as lactate (LA) and 2-hydroxybutyrate (2HB). Here, an Escherichia coli strain was genetically engineered to produce 2-hydroxyisovalerate (2HIV), and therebt biosynthesize poly(13.2 mol% 2HIV-co-7.5 mol% 2HB-co-42.5 mol% 3HB-co-36.8 mol% LA). Increased 2HIV fraction was achieved by further deletion of genes encoding enzymes involved in competing pathways and byproduct formation. The engineered strain produce poly(20 mol% 2HIV-co-80 mol% LA), with the polymer content of 9.6% w/w. These results suggest novel PHAs containing 2HIV can be produced by engineering branched-chain amino acid metabolism. ** This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A 2026556 and NRF-2012M1A2A2026557) and NRF grant funded by the MSIP (NRF-2016R1A2B4008707). Keywords : Metabolic Engineering, Escherichia coli, Polyhydroxyalkanoate

594


P0853 Effects of Overexpression of Endogenous NADP-dependent Malic Enzyme on Biomass and Lipid Production in Nannochloropsis salina

Seungjib JEON1,2,3, Hyun Gi KOH2,4, Jun Muk CHO1,2, Joon-Chul LEE3, Nam Kyu KANG2,4, Yong Keun CHANG1,2 1Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Korea, 2Advanced Biomass R&D Center, Daejeon, Korea, 3Human Convergence Technology Group, Korea Institute of Industrial Technology (KITECH), Ansan, Korea, 4Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Corresponding Author Email : [email protected] Microalgae are promising alternative feedstocks that can be used to produce biofuels and replace conventional fossil fuels. In order to increase the lipid production of microalgae, strain development via genetic engineering is required. In this study, we tried to increase carbon flux into fatty acid synthesis by overexpressing the NADP-dependent malic enzyme, NsME1, in Nannochloropsis salina. We found that the biomass and FAME contents were increased in NsME1 overexpressing transformants, such that the FAME yield of the top-producing NsME1 transformant was 53% higher than the WT. To understand the effects of NsME1 on lipid production, we analyzed the total carbon concentration and NADPH/NADP ratio, which were found to be enhanced in the transformants. We also investigated mRNA expression levels of genes involved in C4-like CCM and fatty acid synthesis by qRT-PCR, and confirmed their positive contribution to fatty acid production. Taken together, our results demonstrate that overexpression of NsME1 could simultaneously improve the carbon concentration and reducing power in cells, thereby increasing the lipid and FAME yields of N. salina. Keywords : Microalgae, Nannochloropsis salina, Malic enzyme, Carbon concentrating mechanism

595


P0854 Improved Itaconic Acid Production by Employing Protein Scaffold Between GltA, AcnA, and CadA in Recombinant Escherichia coli

Jae Hoon JEONG, Ashokkumar KUMARAVEL, Kim-Ngan T TRAN, Soon Ho HONG Department of Chemical Engineering, University of Ulsan, Ulsan, Korea Corresponding Author Email : [email protected] Itaconic acid is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein-protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/l of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30oC, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/l. Keywords : Protein scaffold, Bioproduction, Metabolic engineering, Itaconic acid

596


P0855 Biosynthesis of Nonimmunosuppressive ProlylFK506 Derivatives with Neurotrophic Activity

Hang Su CHO1, Heon Joo LEE1, Haein YOON2, Eunji CHEONG1, Yeo Joon YOON2 1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea, 2Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] FK506 (tacrolimus) has diverse biological activities such as immunosuppressive, antifungal, neuroprotective, and neuroregenerative activities. This clinically important drug is used to prevent allograft rejection of organ transplants. In order to separate the neurotrophic activity from its immunosuppressive activity, new FK506 derivatives, 9-deoxo-36,37-dihydro-prolylFK506 and 9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506, were developed by combined modification of C1/C9/C31 and C36-C37 positions. The proline replacement in 9-deoxo-36,37-dihydroFK506 and 9-deoxo-31-O-demethyl-36,37-dihydroFK506 showed further reduction of immunosuppressive activity (more than 120-fold), compared with previous derivatives. Compared to FK506, they reduced immunosuppressive activity by ~1.2 × 105- and 2.2 × 105-fold, while retained neurite outgrowth activity. In addition, these derivatives exhibited significant increase of synaptic transmission, suggesting that substitution of pipecolate with proline has a great effect on reduction of strong immunosuppressive activity of FK506 with enhanced neurotrophic activity. Keywords : FK506 derivatives, proline substitution, immunosuppressive activity, neurotrophic activity References 1. Jung, J. A., Lee, H. J., Song, M. C., Hwangbo, A., Beom, J. Y., Lee, S. J., Park, D. J., Oh, J. H., Ha, S.,

Cheong, E., Yoon, Y. J. J. Nat. Prod. online published (2021).

597


P0856 Metabolic Rewiring of Bypass Pathways for Enhancing Isopropanol Production in Corynebacterium glutamicum

Joy CHA, Young Jin KO, Myeong-Eun LEE, Sung Ok HAN Department of Biotechnology, Korea University, Seoul, Korea Corresponding Author Email : [email protected] Isopropanol (IPA) is a secondary alcohol mainly used in pharmaceuticals, bioenergy, and chemical products, making it a promising component as a bio-substitution for petroleum. In our previous study, we constructed an isopropanol-producing Corynebacterium glutamicum, by introducing an IPA biosynthesis pathway. Nonetheless, production yield was low. Thus, in this study, we conducted a push and pull engineering in C. glutamicum. First, an isoprenoid bypass was integrated to push the flux toward isopropanol biosynthesis. However, the introduction of the bypass did not increase isopropanol production. Second, four different secondary alcohol dehydrogenase genes derived from various bacterial and yeast origins were introduced into C. glutamicum to pull out isopropanol production. These genes were screened through enzyme activity assays and further verified by GC-FID. The enzyme, called CbS, showed the highest specific activity among the other enzymes. Consequently, when the CbS enzyme was overexpressed, isopropanol was successfully produced. This indicates that the utilization of bypass and heterologous gene expression in C. glutamicum is a metabolically worth strategy. Keywords : Isopropanol, Isoprenoid bypass, Secondary alcohol dehydrogenase, Metabolic engineering, Corynebacterium glutamicum References 1. H. J. Lee, J. Son, S. J. Sim and H. M. Woo, Plant Biotechnol. J. 18(9), 1860-1868 (2020).

598


P0857 RiboRid: a Low Cost, Advanced, and Ultra-efficient Method to Remove Ribosomal RNA for Bacterial Transcriptomics

Donghui CHOE1,2, Richard SZUBIN3, Saugat POUDEL3, Anand SASTRY3, Yoseb SONG1,2, Yongjae LEE1,2, Minjeong KANG1,2, Eojin YOO1,2, Ui-Gi KIM1, Suhyung CHO1,2, Bernhard PALSSON3,4, Byung-Kwan CHO1,2 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Innovative Biomaterials Research Center, KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 3Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 4Department of Pediatrics, University of California San Diego, La Jolla, CA, USA Corresponding Author Email : [email protected] (Bernhard Palsson), [email protected] (Byung-Kwan Cho) RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcript 3′ ends information (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of the transcriptomic studies is to remove ribosomal RNAs (rRNA) that comprise more than 90% of total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA. The method implemented experimental considerations of minimizing non-specific degradation of mRNA and capable of depleting pre-rRNAs that often comprises a large portion of RNA even after rRNA depletion. We demonstrated the successful removal of rRNA up to removal efficiency of 99.99% for various transcriptome studies including RNA-Seq, Term-Seq, and ribosome profiling, with the cost approximately $10 per sample. The method is expected to be a robust means in large-scale high-throughput bacterial transcriptomic studies. ** This study is supported by the Korea Bio Grand Challenge (2018M3A9H3024759 to B.-K.C.) and the Basic Science Research Program (2018R1A1A3A04079196 to S.C.) through the National Research Foundation of Korea (NRF). Keywords : rRNA depletion, Transcriptome, RNA-seq

599


P0858 Complete Mitochondrial Genome of the Orange-spotted Trevally, Carangoides bajad (Perciformes, Carangidae) and a Comparative Analysis with Other Carangidae Species

Ha Yeun SONG, Jiyoung WOO, Dae-Sung LEE Department of Genetic Resources, National Marine Biodiversity Institute of Korea, Seocheon, Korea Corresponding Author Email : [email protected] The orange-spotted trevally, Carangoides bajad, (Perciformes, Carangidae), is an inshore marine fish that inhabits the Indo-West Pacific. Carangids are an important food source in the commercial fisheries industry in Southeast Asia. The C. bajad is listed as Least Concern in IUCN Red List due to fishing and harvesting aquatic resources. The complete mitochondrial genome of the C. bajad, which belongs to the family Carangidae was determined. The complete mitochondrial genome has a length of 16,556 bp and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. C. bajad has a mitochondrial gene arrangement that is typical of vertebrates. Phylogenetic analysis using mitochondrial genomes of 13 related species revealed that C. bajad formed a well-supported monophyletic group with the other Carangidae species. This mitochondrial genome could be helpful for developing conservation strategy. Keywords : Mitochondrial genome, Perciformes, Carangidae, Carangoides bajad, Conservation

600


항체공학 및 세포배양공학

(Antibody Engineering and Cell

Culture Engineering)

601


P0901 Unfolded Protein Response Monitoring System for Intuitive Identification of ER Stress in CHO Cells

Minji KYEONG1, Jae Seong LEE1,2 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] As the biopharmaceutical industry expands, improving the production of therapeutic proteins using CHO cells is important. However, excessive and complicated protein production causes protein misfolding, triggering ER stress. When the ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of ER. However, when the cells fail to control prolonged ER stress, UPR turns into an inducer of apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and desired quality. In this study, we developed a fluorescence-based UPR monitoring system in CHO cells. We integrated mGFP into the endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor. The mGFP expression level changed according to the ER stress induced by chemical treatment in the engineered cell line. Using this monitoring cell line, we have generated stable antibody-expressing CHO cell lines while continuously monitoring the endogenous UPR level. Taken together, these data demonstrated the utility of this UPR monitoring system and its potential for applications in CHO cell line development. Keywords : BiP, Chinese hamster ovary cells, Endogenous gene tagging, ER stress, High producing cell line, Monitoring system, Unfolded Protein Response

602


P0902 Strategies and Development of DMSO-free Cryopreservation Formulation for Mammalian Cells

Beom Jin KIM, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] A cell may have difficulty preserving and maintaining cells for a long time due to the possibility of genetic or generational mutations during the incubation period. Therefore, most biopharmaceutical and cell therapy companies perform cryopreservation of cells at extremely low temperatures. However, during the freezing process, cells undergo physical stress, such as osmotic imbalance, dehydration, and the formation of intracel-lular ice crystals. Cryoprotective agents(CPAs) are used to solve these problems. The most commonly used CPA is Dimethyl-Sulfoxide(DMSO). However, DMSO itself is not only toxic to cells but can also induce some cells (e.g., HL-60 promyeloblast cells) to differentiate. For this reason, several freeze-preserving badge products without DMSO have been released and require the development of more diverse products. We plan two strategies in this work to develop a cryopreserving formulation without DMSO. First of all, the strategy is to identify CPAs proven through previous studies, select them through classification and individual screening, and second, the strategy is to set the standard through analysis of several products that have already been released. CPAs are about four types of classifications; cellular permeability chemical and amino acid, sugar, and polymer. Through this experiment, we checked how much of each candidate material had the ability to freeze and preserve and selected effective candidate materials. the following checked the product charac-teristics, osmotic pressure, component analysis, freeze preservation capability, for some products that have already been released and referred to the setting of standards for our composition development. Ultimately, if cryopreservation formulation excluding DMSO with various problems is developed, cell therapy can be simplified in the form of 'ready-to-use', and the demand for DMSO-free cryopreservation media used in cell therapy production is expected to expand further. Keywords : Cryoperservation, Cryoprotective agents (CPAs), Dimethyl-sulfoxide (DMSO) References 1. Bhattacharya, Sankha. (2018). Cryopretectants and Their Usage in Cryopreservation Process. 10.5772/

intechopen.80477. 2. C Tarella. Cancer Res. 1982 Feb;42(2):445-9. 3. Sankha Bhattacharya. (2016). asian journal of pharmaceutics. vol 10, No 3. 154-159.

603


P0903 Artificial Host Spot Included UCOE to Enable Efficient Knock-in and High and Stable Transgene Expression in Mammalian Cells

Seulmi KIM1, Jae Seong LEE1,2 1Department of Molecular Science and Technology, Ajou University, Suwon, Korea, 2Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea Corresponding Author Email : [email protected] CRISPR/Cas9-mediated targeted gene integration (TI) has been used for the generation of recombinant mammalian cell lines with predictable transgene expression. To fully implement the TI-mediated cell line development, it is critical to identify hot spots rendering high transgene expression and high knock-in (KI) efficiency, but it is still cumbersome. In this study we developed an artificial KI site which can be used as hot spots in different genomic loci. UCOE (Ubiquitous Chromatin Opening Element) was employed because of its ability to open chromatin and to enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated in CHO and HEK293 cells, followed by TI of EGFP to the artificial UCOE KI site. The CHO-K1 random pool harboring 3’2.2kb A2UCOE showed a 2-fold increase in the EGFP expression level and KI efficiency and much lower CV compared to the control without UCOE, while UCOE-KI HEK293T random pools showed limited effects but much higher indel efficiency. Thus, this work demonstrates the possibility of artificial UCOE KI sites that could streamline screening of high-production targeted integrants by mitigating selection of genomic hot spots. Keywords : Chinese hamster ovary (CHO), CRISPR/Cas9, Hot spot, Knock-in, UCOE

604


P0904 Evaluation of Biological Performance in a Newly Developed Single-use Bioreactor System, CELBIC System, for Large Scale Culture of Recombinant CHO Cell

Hyunwoo KIM, Junghyun CHO, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] The usage of single-use bioreactors for cell culture has been increasing continuously, as the bio-industry of biopharmaceuticals (proteins, antibodies, cell therapies and gene therapies products) has expanded dramatically during recent decades. The SUBs (single-use bioreactors) have several advantages compared with the conventional stainless steel based bioreactors, such as flexibility in scale, low risk of contamination, reduced labors for cleaning/sterilization, and validation/regulation issues. In this study, a newly developed cell culture system, CELBIC system, driven by multi-directional rocking movement is introduced and evaluated its physical and biological performances as a cell culture device. The recombinant cell line derived from CHO-S cell was cultured for producing IFN-beta(interferon beta) of 50 liters working volume in batch culture. The CELBIC system was controlled for making orbitally shaken motion with 10 ° tilt angle and 20 rpm. ELISA and western blot were used for analysis of IFN-beta concentration. From these experiments, the maximum viable cell density was 8.81 x 106 cells/mL in the CELBIC system and this result was the highest value among all groups of experiment. The maximum viable cell density and the IVCD(Integral of Viable Cell Density) were increased up to 1.42 folds and 1.47 folds respectively compared to a control group. In summary, since the CELBIC system driven by orbital movement provides sufficient and excellent conditions for cell cultures, it can be an attractive candidate for suspension cell culture device to produce biopharmaceutics, in particular, antibodies or therapeutic proteins from recombinant CHO cells. Keywords : Animal cell culture, Large scale culture, Single-use bioreactor

605


P0905 Comparative Analysis of Exosomes Between Adherent and Microcarrier Culture of Adipose Derived Stem Cells

So Yeon PARK1, Jae Woo PARK2, Youn-Je PARK1 1Department of Food Science and Technology, Kongju National University, Yesan, Korea, 2Viblioteca Co. Ltd., Ilsan, Korea Corresponding Author Email : [email protected] Stem cells have been used in clinical purpose, and exosomes isolated from stem cells are also developed as therapeutics or drug carrier. Due to mass production system for stem cells or exosomes getting more important, 3 dimensional dynamic culture using microcarrier have been applied to produce more cells and exosomes. In this study, exosomes from stem cell using microcarrier culture was comparatively analyzed with those using adherent culture. Adipose derived stem cells(ADSC) were cultured for 24 days in adherent culture and in microcarrier culture, respectively. As a result of nanoparticle tracking analyzer(NTA), exosome pattern was better in middle period of both culture, which are 5, 6, 7 passages of adherent culture and 9, 12, 15, 18 days of microcarrier culture. Average size of exosome was slightly larger in microcarrier culture than in adherent culture. Total yield of exosome from microcarrier culture was about 2 times of adherent culture, and exosome purity was also much higher in microcarrier system than adherent culture. These results suggest that microcarrier culture system could be useful for efficient production of exosomes with high purity and high yield from ADSCs. Keywords : exosome, Adipose derived stem cell, microcarrier culture

606


P0906 The Optimization of Media Components to Improve Cell Growth and Productivity in CHO-S Cells

Ji Yun PARK, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] The culture of CHO cells requires media to support cell growth and production. Such media must contain essential nutrients including amino acids, energy source, lipid, vitamin, and inorganic salts. Especially, amino acids and energy sources have been recognized as the most crucial components to support cellular functions and production. In this study, we optimized the concentration of amino acids and energy source in in-house media, SJ-PFM2.0 by referring to commercial medium. Through comparison with commercial medium and SJ-PFM2.0, the concentration of amino acids in SJ-PFM2.0 are then adjusted to obtain higher cell growth and productivity. In addition, diverse energy sources were screened to determine the most effective one to CHO cell culture, and we applied it to SJ-PFM2.0. we evaluated cell growth, metabolism, and productivity of CHO-S in enhanced SJ-PFM2.0. Finally, by optimizing the concentration of amino acids and energy source, further enhancement of cell growth and productivity could be achieved. This study suggested that the optimization of amino acids and energy sources is an essential step for improving CHO cell culture media, and it can be used to develop media to achieve high cell growth and productivity of recombinant protein. Keywords : Cell culture media, Amino acid, Energy source, Biotechnology, Media formulation References 1. Ritacco, F. V., Wu, Y., and Khetan, A. (2018). Cell culture media for recombinant protein expression in

Chinese hamster ovary (CHO) cells: history, key components, and optimization strategies. Biotechnol. Prog. 34, 1407-1426.

2. A. Salazar, M. Keusgen, J. Von Hagen, Amino Acids 2016, 48, 1161.

607


P0907 Expression of a Fusion Gene Encoding Escherichia coli Heat Labile Toxin and the Foot and Mouth Disease Virus VP1 Protein in Saccharomyces cerevisiae for FMD Vaccine Development

My Tieu Ngoc LE1, Ngoc-Luong NGUYEN2, Hae-Ryeong YOON1, Jaehyeon LEE1, Beom-Tae KIM3, Dae-Hyuk KIM1,2 1Department of Bioactive Material Sciences and Institute of Bioactive Materials, Jeonbuk National University, Jeonju, Korea, 2Department of Molecular Biology and the Institute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju, Korea, 3Research Center of Bioactive Materials, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Vaccination is extremely effective at fighting infectious diseases, especially viral diseases, but subunit vaccines are poorly immunogenic. To solve this problem, novel adjuvants have been explored. Our study focused on producing an oral vaccine against Foot and Mouth Disease (FMD) virus, using Saccharomyces cerevisiae as the expression host to express a gene encoding for Escherichia coli heat-labile enterotoxin subunit B (LTB) fused with a yeast codon optimized gene encoding for VP1 antigen through flexible linkers. The fusion genes, termed LTB-VP1 were cloned into a yeast episomal vector, yEpGPD-TER, and the recombinant plasmids were transformed into S. cerevisiae 2805 strain. Transformation was confirmed by colony PCR and E. coli back transformation. Expression was examined at both transcription and translation levels, through Northern blot and Western blot, respectively. Northern blot showed the transcription of the target genes, however, Western blot detected only weak signal of the target proteins, indicated that the protein product of the cloned gene was successfully expressed. However, further studies on optimization of promoters, terminators and gene codons are required. Keywords : Novel adjuvants, oral vaccine, Foot and Mouth Disease, fusion genes

608


P0908 Development of an Oral Dengue Tetravalent Vaccine in Saccharomyces cerevisiae by Immune Complex Approach

Ngoc-Luong NGUYEN1, Kum-Kang SO1, Heewon SEO2, Yo-Han KO1, Jeesun CHUN1, Yong-Suk JANG1,2, Dae-Hyuk KIM1,2 1Department of Molecular Biology and the Institute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju, Korea, 2Department of Bioactive Material Sciences and Institute of Bioactive Materials, Jeonbuk National University, Jeonju, Korea Corresponding Author Email : [email protected] Immune complex based vaccines are old ideas that have recently gained traction due to their superiority over traditional vaccines, particularly in their ability to induce strong cellular immune response. In this study, we developed an immune complex dengue tetravalent vaccine by expressing immune complexes containing a consensus Dengue envelope domain III fused to modified murine IgG fragment crystallizable domains (scEDIII-PIGS) in the Baker’s yeast. The IgG Fc contains 10 amino acid of the C1 domain, the hinge region, C2 and C3 and the mu chain from IgM at the C terminal in order to facilitate pentameric and hexameric immune complex formation. Western blot of protein A chromatography elution fraction showed bands corresponding to multimeric scEDIII-PIGS, indicated that immune complexes have successfully expressed. Recombinant yeasts expressing these immune complexes were fed to Balb/c mice in the forms of cell free protein extract or whole cell suspension to test their oral vaccine efficacy. Mice were fed four times in one-week intervals. One week after the final feeding, anti-sera and fecal extract were collected and tested for the presence of specific IgG and IgA, respectively, against purified scEDIII. Keywords : Dengue vaccine, immune comples, Saccharomyces cerevisiae, fragment crystallizable

609


P0909 Development of a New IGF-1 Detection Method Using Quenchbody

Jeongsu YOO1,2, Hee-Jin JEONG3, Yung-Hun YANG2, Minyoung KIM1, Hophil MIN1, Junghyun SON1, Changmin SUNG1 1Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea, 2 Department of Microbial Engineering, Konkuk University, Seoul, Korea, 3Department of Biological and Chemical Engineering, Hongik University, Sejong, Korea Corresponding Author Email : [email protected] Human growth hormone (hGH), one of the major anti-doping substances, has been abused to improve athletic performance ever since it was considered a prohibited substance. Recently, as one of the anti-doping analysis methods for hGH, a hGH biomarker analysis method has been proposed. One of the hGH biomarkers, insulin-like growth factor-1 (IGF-1), is detected using the Immunoradiometric assay (IRMA) technique according to the World Anti-Doping Agency (WADA) guidelines. However, the IRMA method has disadvantages in terms of the number of trained personnel required to use radioactive materials and the stability of the experiment. In this study, we are conducting research to apply a fluorescence-based antibody, quenchbody, to detect IGF-1 more quickly and conveniently. In detail, 0.3 mg/L of anti-IGF-1 scFv could be obtained through the MBP fusion technique and its binding affinity against IGF-1 has been confirmed by indirect ELISA. Next, we plan to attach a fluorescent dye using a single-chain variable fragment (scFv) expressed in E. coli and evaluate its reactivity. Keywords : Single chain variable fragment, Quenchbody, Maleimide-thiol reaction, IGF-1, bio-doping References 1. Abe. R et al., J. Am. Chem. Soc. 133, 43 (2011). 2. Andres. H et al., US Patent 10,301,383 (2019).

610


P0910 Development of Gut-mucus Chip for Intestinal Absorption Study

Seung Yeon LEE1, Yeon Jae KANG1, Bum Sang KIM1, Dong Hyun KIM2, Jong Hwan SUNG1 1Department of Chemical Engineering, Hongik University, Seoul, Korea, 2School of Electrical and Electronic Engineering, Yonsei University, Seoul, Korea Corresponding Author Email : [email protected] The intestinal mucosa acts as a barrier to protect the intestinal cells from foreign substances and interferes with drug delivery. The Caco-2 based cell culture model is a standard in vitro model system for testing drug uptake. However, it is difficult to say that it reflects the actual delivery of drugs in the intestine because it does not consider the effect of the mucous membrane. Here, we developed a microfluidic embedded chip using Caco-2 cells coated with mucin protein. It was observed that the coated mucin layer was maintained under the flow conditions. In addition, the role of the mucus layer as a barrier in the flow environment was confirmed through the study of the absorption of the fluorescent material according to the presence of mucin. And the intestinal mucosa adhesion rate of particles was compared by applying non-intestinal mucosa-adhering polymer particles and intestinal mucosa-adhering polymer particles to this system. Caco-2 cells coated with mucus provide a more physiologically realistic intestinal epithelial environment to study absorption processes. It is hoped that the mucin-coated gut chip could potentially be used as a new, more accurate in vitro model of the gut. Keywords : Microfluidics, Gut-on-a-chip, Mucin, Intestinal mucus References 1. Sung, J.H., Y. Wang, and M.L. Shuler, APL Bioeng, 2019. 3(2): p. 021501. 2. Jihwan Oh & Bumsang Kim, Korea-Australia Rheology Journal 32, 41-46(2020).

611


P0911 Development of High-throughput Assay Using High-sensitive Quenchbody Against Procollagen Type III N-terminal Peptide

Joon-Yeop YI1,2,3, Hee-Jin JEONG4, Byung-Gee KIM2,3,5,6,7, Minyoung KIM1, Junghyun SON1, Changmin SUNG1 1Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea, 2Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea, 3Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea, 4Department of Biological and Chemical Engineering, Hongik University, Sejong, Korea, 5School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea, 6Bio-MAX/N-Bio Institute, Seoul National University, Seoul, Korea, 7Institute for Sustainable Development (ISD), Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Procollagen type III N-terminal peptide (P-III-NP) is a biomarker of growth hormone which is on the prohibited substances list by the World Anti-Doping Agency (WADA). The recently developed P-III-NP analysis method is the radio-immunoassay (RIA) detection method that has issues with long analysis time, radiation safety, and expensive equipment. Thus, the development of an analysis method that can overcome these shortcomings is required. To this end, we tried to develop a fluorescence-based antibody sensor called “Quenchbody” and a high-throughput anti-doping method. In this study, the quenchbody was synthesized by maleimide-thiol chemistry with ATTO- and TAMRA-maleimide dye. For the antibody, 5 mg/L of N-terminus cysteine-tagged anti-PIIINP scFv was produced in E. coli. As the result, dose-dependent fluorescence response was confirmed and the limit of detection (LOD) was 1.64 nM in the TAMRA-labeled quenchbody. Developed immunoassay with 2 nM of quenchbody could be performed within 30 minutes from the experimental preparations to validations. Keywords : Quenchbody, Fluorescence-based antibody, high-throughput assay References 1. E.R. Burchardt et al., US patent. US 7,541,149 B1. (2009). 2. J. Dong et al., Biotechnol Bioeng. 117(5), 1259-1269 (2020).

612


P0912 Development of Fluorescent Nanobodies for Simple and Rapid Anti-recombinant Human Growth Hormone (rhGH) Doping Test

Jaehoon JUNG1,2, Hee-Jin JEONG3, Yung-Hun YANG2, Minyoung KIM1, Junghyun SON1, Changmin SUNG1 1Doping Control Center, Korea Institute of Science and Technology, Seoul, Korea, 2Department of Microbial Engineering, Konkuk University, Seoul, Korea, 3Department of Biological and Chemical Engineering, Hongik University, Sejong, Korea Corresponding Author Email : [email protected] Recombinant human growth hormone (rhGH) known as somatropin has been used for a drug abuse in sport doping, so World Anti-Doping Agency (WADA) banned rhGH. Taking the rhGH changes the ratio between native hGH and rhGH, therefore, a doping test can be performed by detecting this change. But this method not only takes a long time but also complicated. In this study, to complement these disadvantages, we aimed to develop a simple and high-throughput anti-hGH doping method using fluorescence-based quenchbody. In detail, a nanobody, known as VHH, which is consist of the smallest antigen binding domain derived from heavy chain-only antibody (HCAb) of camelids is expressed in E.coli and is labeled with maleimide dye. In this study, we designed a Q-body based on NbGH04 and NbGH06, which are known to have high binding affinity for rhGH. The antigen binding reaction of NbGH04 and NbGH06 with rhGH was evaluated by enzyme-linked immunosorbent assay (ELISA). Next, we plan to attach a fluorescent dye using a VHH expressed in E. coli and evaluate its reactivity. Keywords : Nanobody, Antibody, VHH, Doping test, Human growth hormone, recombinant human growth hormone, Quenchbody References 1. Abbady. AQ et al., Gen. Comp. Endocrinol. 204 (2014). 2. Abe. R et at., J. Am. Chem. Soc. 133, 43 (2011). 3. Pardon. E et al., Nat. Protoc. 9, 3 (2014).

613


P0913 Effect of Various Serum Supplements in Culture Medium on the in vitro Development of Goat Embryos

Kwan-Woo KIM, Eun-Do LEE, Dong-Kyo KIM, Jinwook LEE, Sung-Soo LEE, Sang-Hoon LEE Animal Genetics Resources Research Center, National Institute of Animal Science, RDA, Hamyang, Korea Corresponding Author Email : [email protected] Attempting to improve goat embryo production, this study examined the effects of fetal bovine serum (FBS), goat blood serum (gBS), and polyvinyl alcohol (PVA) on the in vitro development and embryo quality of goats. In vitro fertilized embryo culture mediums were supplemented individually with FBS, gBS, and PVA, all at 10%, to determine their effects on embryo development efficiency and blastocyst quality. The results showed that the non-serum supplementation group (control) presented a significantly lower cleavage rate and lower blastocyst formation. By contrast, the gBS and PVA supplementation groups exhibited a significant increase in the cleavage rate and better blastocyst formation than the control and FBS supplementation groups. Further-more, the TUNEL assay performed to confirm blastocyst quality indicated the same pattern as the embryo development experiment. In summary, supplementation with gBS or PVA enhanced the in vitro development of goats more efficiently than supplementation with FBS or non-serum. On the other hand, considering the risk of an unidentified factor in gBS, PVA appears to be safer and more efficient for use in the in vitro development of goat embryos. Keywords : TUNEL Assay, Serum, In Vitro Culture, Goat, Embryo, Blastocyst References 1. DelCollado M, Saraiva NZ, Lopes FL, Gaspar RC, Padilha LC, Costa RR, Rossi GF, Vantini R, Garcia JM,

Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos(2015), Reprod. Fertil. Dev., 28(11), 1721-1732.

2. Yoon JD, Hwang SU, Kim M, Jeon Y, Hyun SH, Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro(2019), Biol. Reprod., 101(1), 63-75.

614


P0914 Development of Hyperosmotic Resistant CHO Cell Line to Enhance Recombinant Protein Productivity by Using Aldose Reductase Gene (AKR1B1)

Yun Ju NA, Soo Ah JEONG, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] When exposed to extraordinarily high osmolality, the cells undergo numerous perturbations, such as increased DNA strand breaks, cell cycle arrest and elevated reactive oxygen species (ROS). However, when the osmotic stress increases, the cells spontaneously accumulate organic osmolytes such as sorbitol as a self-defense mechanism. Sorbitol is synthesized from glucose, catalyzed by aldose reductase (AR), and it is metabolized to fructose by sorbitol dehydrogenase. The AR serves as an osmoregulatory role because its transcription level is increased in order to maintain intracellular osmolality under hyperosmotic condition. During fed-batch which is widely used to enhance the productivity of the therapeutic proteins in the biopharmaceutical industry, there would be issues caused by the high osmotic pressure in the cell culture environments such as cell growth inhibition and the cell death. The increase of the high osmotic pressure resistance might help to solve these problems. In this study, we generated a recombinant cell line that produces the AR and confirmed that the AR producing cells have hyperosmotic resistance. The AR producing cell line has higher growth in normal media and hyperosmotic media. Also, the AR producing cell line has higher productivity in normal media and hyperosmotic media. These results may contribute to improving the productivity of the therapeutic proteins during fed-batch. Keywords : Cell line, aldose reductase, Hyperosmotic resistant References 1. Maurice B.Burg, Joan D.Ferraris. Cellular Response to Hyperosmotic Stress Physiol Reb. 87:144-1474

(2007).

615


P0915 Enhancing Viability of CHO Cells Using 30Kc19-30Kc6α

Tai Hyun PARK1,2, Yeong Kyu PARK2 1Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Korea, 2The School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Most of medicines and drugs were developed as chemicals in the past while new drugs developed today are mostly recombinant proteins and antibodies for treatments of diseases. This transition in trend of developing new drugs is mainly due to Chinese hamster ovary (CHO) cells since recombinant proteins and antibodies can be easily produced from CHO cells. However, productivity of CHO cells is generally lower than that of E. coli. Thus, development of CHO cell with high productivity has become major goal in pharmaceutical industries. One of the methods that enhances productivity of CHO cells is to increase their viability through apoptosis inhibition since viability decreases remarkably in late apoptotic stages. Here, we report an anti-apoptotic protein, 30Kc6α which can inhibit the release of cytochrome-c by blocking bax on the mitochondria membranes. In order to increase soluble expression in E. coli, 30Kc6α is fused with 30Kc19, which has the cell-penetration ability as well. In this study, it was found that 30Kc6α was expressed as soluble protein when fused with 30Kc19. 30Kc19-30Kc6α was also able to penetrate into CHO cells and increase their viability. Altogether, our data provide evidence that 30Kc19-30Kc6α has high potential to increase the recombinant protein or antibody productivity of CHO cells by enhancing their viability. Keywords : CHO, CPP, viability References 1. Park, H.H., Sohn, Y.S., Yeo, J.W., Park, J.H., Lee, H.J., Ryu, J.N., Rhee, W.J., and Park, T.H., Process

Biochemistry. 49, 1516-1526 (2014). 2. Park, H.H., Choi, J.S., Lee, H.J., Ryu, J.N., Park, J.H., Rhee, W.J., and Park, T.H., Process Biochemistry.

50, 973-980 (2015).

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P0916 Selection of Isolation Method of Exosomes from 3D Culture Media of Adipose Derived Stem Cells

Jiwon YANG1, Ji Su LEE1, Jae Woo PARK2, Youn-Je PARK1 1Department of Food Science and Technology, Kongju National University, Yesan, Korea, 2Viblioteca Co. Ltd., Ilsan, Korea Corresponding Author Email : [email protected] Exosomes are small membrane vesicles of endocytic origin, and contain various molecules such as proteins, nucleic acids, lipids and some metabolites. Exosomes play an important role in inter-cellular communication and are developed as a drug delivery system or a therapeutic agent. So it has become important to isolate exosomes efficiently and with high yield. In this study, eight methods for exosome isolation were comparatively analyzed from culture media of adipose derived stem cells(ADSC). ADSCs were 3-dimensionally cultured using microcarrier. Different exosome isolation methods were analyzed with combination of Exoquick reagent, 100 kD filtration, 0.45 um or 0.2 um filtration, ultracentrifugation and so on. Isolated exosomes were characterized with nanoparticle tracking analyzer(NTA), TEM, western blot and total protein assay. As a result, ultracentrifugation after centrifugal pretreatment was selected as the best exosome isolation method. Total protein content of exosome isolated by selected method was lower than those by other methods, however total nanoparticle number and purity was highest, and exosome marker was strongest among different methods. Keywords : exosome, 3D culture, adipose derived stem cell

617


P0917 Construction of in vitro scFv Expression Production System for On-site Diagnostic Kit

Hyuck Jin KWON1, Jinyoung KIM1, Gyeseo PARK2, Myung-Hee KWON2, Mi-Gi LEE1 1Biocenter, Gyeonggido Business & Science Accelerator, Suwon, Korea, 2 Department of Biomedical Sciences, Ajou University, Suwon, Korea Corresponding Author Email : [email protected], [email protected] Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens. Specific monoclonal and recombinant antibodies are available for many plant pathogens and have contributed to the high sensitivity detection. Currently, monoclonal or polyclonal antibodies are widely used for the diagnosis of phytopathogenic viruses. Considering the scale of farm in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody for plant virus detection was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. This study demonstrated that scFv is a suitable alternate for diagnosis of plant virus on pilot scale production. Keywords : scFv, Escherichia coli, On-site Diagnostic Kit, plant virus detection , pilot scale production References 1. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, et al. Science 242:423. 2. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, et al. Proc Natl Acad Sci

USA 85:5879.

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P0918 Antibody-based Targeting of Cell Surface GRP94 Specifically Inhibits Cetuximab-resistant Colorectal Cancer Growth

Sukmook LEE Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul, Korea Corresponding Author Email : [email protected] Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. Cetuximab, a human/mouse chimeric monoclonal antibody, is effective in a limited number of CRC patients because of cetuximab resistance. This study aimed to identify novel therapeutic targets in cetuximab-resistant CRC in order to improve clinical outcomes. Through phage display technology, we isolated a fully human antibody strongly binding to the cetuximab-resistant HCT116 cell surface and identified the target antigen as glucose-regulated protein 94 (GRP94) using proteomic analysis. Short interfering RNA-mediated GRP94 knockdown showed that GRP94 plays a key role in HCT116 cell growth. In vitro functional studies revealed that the GRP94-blocking antibody we developed strongly inhibits the growth of various cetuximab-resistant CRC cell lines. We also demonstrated that GRP94 immunoglobulin G monotherapy significantly reduces HCT116 cell growth more potently compared to cetuximab, without severe toxicity in vivo. Therefore, cell surface GRP94 might be a potential novel therapeutic target in cetuximab-resistant CRC, and antibody-based targeting of GRP94 might be an effective strategy to suppress GRP94-expressing cetuximab-resistant CRC. Keywords : cetuximab resistance, GRP94, human antibody, colorectal cancer

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P0919 Using Ultrasound-guided Ovum Pick-up Technology to Improve Elk Embryo Production Efficiency

Eun-Do LEE, Sang-Hoon LEE, Dong-Kyo KIM, Jinwook LEE, Sung-Soo LEE, Kwan-Woo KIM Animal Genetics Resources Research Center, National Institute of Animal Science, RDA, Hamyang, Korea Corresponding Author Email : [email protected] To improve and increase the utilization of female elk, this study examined the possibility of collecting their embryos during the non-breeding season using ultrasound-guided ovum pick-up (OPU) technology. Once follicles in the ovaries of an elk were observed, the embryos were recovered through the inhalation of follicles using an OPU ultrasound probe. A total of 125 embryos were recovered from 195 follicles, resulting in a recovery rate of 64.4%. Morphological evaluation of the embryos retrieved revealed that 21.6% of embryos were in grade A, 13.6% in grade B, 13.6% in grade C, and 51.2% in grade D. The developmental efficiency of the recovered embryos was also investigated using in vitro maturation, in vitro fertilization, and in vitro culture. After cultivating 61 embryos through in vitro fertilization, 44 embryos were found to be fertilized, a fertilization rate of 72.1%. Fourteen embryos developed to the blastocyst stage, indicating a 22.9% development rate. This study confirmed that the recovery of fertilized embryos from a seasonal breeder such as elk is possible via the ultrasound-guided OPU method. If the efficiency of in vitro fertilization and in vitro culture can be improved through further research, it will help improve the efficiency of elk embryo production through the trans-plantation of fertilized embryos. Keywords : Elk, Deer, In Vitro Culture, Ovum Pick-Up, Embryo References 1. Berg DK, Asher GW. New developments reproductive technologies in deer. Theriogenology. 59(1), 189-

205 (2003). 2. Locatelli Y, Hendriks A, Vallet JC, Baril G, Duffard N, Bon N, Ortiz K, Scala C, Maurel MC, Mermillod P,

Legendre X. Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction. Theriogenology. 78(9),2039-2049 (2012).

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P0920 A Proteomics Approach for the Identification of Valuable Antibodies from Human Serum

Gyu-Min LIM, Hee-Jin JEONG, Byung-Gee KIM Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea Corresponding Author Email : [email protected] Next Generation Sequencings are enabling analysis of antibody repertoires in humans at an unprecedented level . However NGS antibody repertoire data does not represent functional antibody repertoires. Because B cell genomic level repertoire diversity > 10⁷ is much bigger than Functional antibody 1000 ~ 100000 diversity. Moreover, genomic level expression mRNA does not represent protein level expression which is working blocks in all living things. So Integration of NGS data with proteomics data enables discovering of significant antibodies in human specimen. To find valuable antibody, IgG purification from human specimen (serum) is important. Whole antibody SDS page gel and antibody fragments SDS page gel show successful IgG purification from serum. Before starting antibody proteomics , we completed High resolution Q-tof mass spectrometry set up . We set up peptide mapping analysis which is used for sequence identification. After that, we set up proteomics analysis. We identify over 900 protein group and 4000 proteins from human intestine cell sample . Using PLGS program, we quantifying protein expression level. Conclusionally, we complete set up of high resolution mass spectrometry analysis. From now on, we will conduct an antibody analysis based on previous set-up. Keywords : proteomics, antibody, mass spectrometry References 1. Ute Distler, nprot. 2016.042 (2016). 2. Daniel R. Boutz, ACS analytical chemistry. 20;86(10):4758-66 (2014).

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P0921 UTR(Untranslated Region) Engineering to Improve Recombinant Interferon-β Productivity in CHO Cells Under Low Temperature

Yuntae HWANG, Jieun KIM, Duk Jae OH Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul, Korea Corresponding Author Email : [email protected] CHO cell culture under mild hypothermia is extensively used to enhance recombinant protein productivity efficiently. Under mild hypothermic condition, the well-characterized Cold Shock Proteins(CSPs), Cold Inducible RNA binding Protein(CIRP) and RNA Binding Motif3(RBM3) are highly expressed in mammalian cells. They are known as RNA Binding Proteins (RBP) interacting with specific mRNA and regulating their expression level by controlling mRNA stability and translation efficiency. In this study, we developed the expression vector containing the UTR elements, which are reported to increase gene expression by binding CIRP or RBM3, to improve the recombinant human interferon-β productivity in CHO cell cultures under low temperature. The results showed that the specific 3’UTR elements of thioredoxin (TXN) increase the IFN-β production in CHO-K1. Also, the specific 3’UTR element of eIF5A(eukaryotic initiation factor5A) increases the production in CHO-S. These results suggested that engineering UTR of expression vectors could be a useful strategy to increase therapeutic protein production. Moreover, Further enhancement of production could be achieved in CHO cells under low temperature by optimized UTR elements. Keywords : Untranslated region, CHO cell culture , Mild hypothermia, Cold Shock Proteins References 1. Masterton, R.J. and C.M. Smales, Pharm. Bioprocess, p. 49-61 (2014). 2. Al-Fageeh, M.B., Biotechnol Bioeng, 93(5): p. 829-35 (2006). 3. Zhong, P. and H. Huang, Future Sci OA. p. FSO246 (2017).

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2021 한국생물공학회 춘계학술발표대회 및 국제심포지엄