AbstractWhen beginning research in synthetic biology, the go-to organism is the bacterium E. coli. Our research demonstrates the utility of a methanogenic archaeon, Methanococcus maripaludis, as an alternative platform. Our two primary focuses in this study are: 1) improving the genetic tools for synthetic biology available in archaea and 2) demonstrating the utility of archaea in synthetic biology through production of geraniol. Previously, a synthesized gene encoding geraniol synthase was expressed in M. maripaludis, and geraniol production was quantified as 0.2% of lipid dry weight using GC/MS. In order to optimize geraniol production, we are experimenting a combination of chemical, biological and computational approaches that include: an improved geraniol extraction procedure resulting in better recovery, a fluorescent protein monitor for protein expression and regulation, a synthetic ribosome-binding-site library enabling variable levels of expression, and a metabolic model of the lipid biosynthesis pathway subjected to flux balance analysis.

Modeling & Flux Balance Analysis

Human Practices

Future Work

Acknowledgements

References

Geraniol Production

Modeling & Flux Balance Analysis

Protein Expression/Quantification Tool

Ribosome Binding Site Library

Methanococcus maripaludisM. maripaludis is a methanogenic archaeon native to salt marshes. It utilizes CO2 and H2 or formate through a process called methanogenesis, a form of anaerobic respiration that results in the production of methane. M. maripaludis’ relatively simple genome, cheap substrates and mesophilic living conditions make it an ideal organism for synthetic biology. M. maripaludiscreates isoprenoid lipids to make up its membrane, and these lipids can be precursors to high value chemicals.

�Geraniol is an acyclic monoterpene-alcohol�Has many uses in fragrances and flavorings�Has a higher energy density than ethanol and a

heat of combustion similar to diesel�Can be used as a botanical insect repellent�Has been shown to inhibit the growth of

pancreatic, prostate, and colonic cancers

Idea: Successful transformation of a plasmid containing a gene for geraniol synthase (GS) will enable a methanogen to synthesize geraniol.

Step 1: A vector (Registry #Bba_k1138000) containing a codon optimized GS gene was transformed into M. maripaludis, thereby extending its natural isoprenoid pathway (Fig 3). Cultures containing this plasmid were tested for geraniol production.

Step 2: Geraniol was extracted from GS expressing cultures. These extractions were concentrated under a stream of N2 then evaluated by Gas Chromatography/Mass Spectrometry (GC/MS).

Results: The chromatograph shown in Fig. 4 is the result of running an extraction from a GS expressing M. maripaludis sample. Mass Spec confirmed the peak here to be the presence of geraniol ((trans)-3,7-Dimethyl-2,6-octadien-1-ol). A wild type M. maripaludis culture was also extracted and run through GC/MS and no peak was present. We were able to calculate the production of geraniol using our construct as 0.2% of lipid dry weight by plotting the concentration of various standards against the integral area of the peak created and obtaining a linear relationship.

Fig. 4 Geraniol was shown to be present in the extracellular content of a GS expressing Methanococcusculture. The chromatographs are partnered with their respective mass specanalyses, confirming these peaks at identical retention times to be caused by the presence of geraniol.

Fig. 3 Geraniol is synthesized from the compound geranyl pyrophosphate which occurs naturally in Methanococcus along the isoprenoid lipid pathway. A Methanococcus cell that successfully expresses GS will be able to catalyze the reaction producing geraniol.

Idea: Increase geraniol yield by mapping the entire lipid biosynthesis pathway in M. maripaludis, performing a genome-scale flux balance analysis and developing strategies for the overproduction of isoprenoid compounds.

Results: We designed a metabolic model for isoprenoidbiosynthesis based upon the complete pathway using the Biocyc, KEGG, and SEED databases (Fig. 5). We obtained the wild type M. maripaludis model file [2], and made the modifications described in Fig. 6. We utilized MATLAB software in the COBRA toolbox to run simulations on our modified model to obtain the results shown in Fig. 7.

In progress: Continue running simulations in MATLAB to optimize production of geraniol be it through knock-out strains, up/down regulation of particular genes, beneficial environmental conditions, etc.

Outlook: Expand upon the isoprenoid biosynthesis model by including regulatory elements upon each reaction. Develop strategies for the overproduction of isoprenoid compounds.

The vector below has been optimized for expression in M. maripaludisand uses the red fluorescent protein, mCherry, as a fluorescent reporter. The use of BioBrick� standards allows researchers to insert any gene of interest that will be covalently linked to mCherry.

�Improve upon geraniol extraction methods from M. maripaludiscultures�Combine knowledge accumulated in this study to further optimize geraniol production, such as using high strength RBS sequences for better expression of GS, and making mutants/changing cultivating conditions that may increase metabolic flux towards geraniol.�Further extension of isoprenoid pathway to geranyl acetate and possible other high-value chemicals.

[1] Whitman, William B. "Methanococcus maripaludis strain C5, strain C6 and strain C7." Methanococcus maripaludis C6. Doe Joint Genome Institute, n.d. Web. 8 Oct. 2012. http://genome.jgi-psf.org/metm6/metm6.home.html.

[2] Goyal, Nishu, W. Widiastuti, IA Karimi, and Z. Zhou. "A Genome-scale Metabolic Model of Methanococcus Maripaludis S2 for CO2 Capture and Conversion to Methane." Europe PubMed Central, 2014. Web. 29 Oct. 2014. http://europepmc.org/abstract/MED/24553424.

*Dr. William B. Whitman may be contacted at Whitman@uga.edu*Peyton Smith may be contacted at Smithpa@uga.edu

�President’s Venture Fund�Office of the Vice President for Research�1Departments of Microbiology and 2College of Engineering�Department of Biochemistry and Molecular Biology, and Genetics�Franklin College Student Activity Fee Allocation Committee�UGA Alumni Association�3UGA iGEM Club, past and present

Idea: The ribosome binding sites of archaea are not well characterized. A ribosome binding site (RBS) with higher affinity for ribosomes will result in increased translation efficiency.

The pMEV4-mCherry vector mentioned above (Fig. 8) will allow us to characterize a library of RBS for use in methanogens. Primers were designed for a mutation on each single base along a 12 base-pair mutation region. This region includes the RBS, spacer, and first base of the start codon (Fig 9). Additionally, two primers were designed from M. maripaludis 16S rRNA data to create a ‘perfect� and ‘negative� RBS.

Fig. 8 pMEV4-mCherry vector. The red fluorescent protein, mCherry, was codon optimized (shown in red) and cloned into the vector. The

restriction sites just downstream of mCherry allow us to covalently link a protein of interest to mCherry. This vector contains selective marker

genes for replication in both E. coli and M. maripaludis.

Fig. 5 Isoprenoid biosynthesis pathway in M. maripaludis. This pathway begins with the product of autotrophic CO2 fixation, acetyl-CoA, and proceeds through the mevalonatepathway. No experimental evidence has been shown that Methanococcus ultilizes the

MEP/DOXP pathway

Fig. 1 Methanococcus maripaludis [1] Fig. 2 M. maripaludis is an obligate anaerobe, so we must use an anaerobic chamber to handle cultures

Inspire: Local Outreach Projects�Provoking interest in the sciences to

local students is an important aspect of UGA iGEM. We have visited East Athens Community Center and Middle Georgia Regional Library to engage the students in fun, hands-on experiments.

Inform: Raising Awareness of Synthetic Biology�UGA iGEM presented at the

Bioenergy Systems Research Institute retreat to demonstrate the utility of synthetic biology in the bioenergy industry. We were awarded best undergraduate presentation.�UGA iGEM presented at the 9th

Annual Georgia Environmental Conference where we spoke to many environmental leaders in the state about how we use synthetic biology to develop greener solutions. We were awarded the GEC Scholarship for best undergraduate project.

Fig. 9 The region in this circuit labeled �Mutation Region� is where we will make mutations upon each single base for every nucleotide different from the

native RBS. This figure (not to scale) shows the native RBS sequence

Methanococcus: The New Archaea-type for Synthetic BiologyEstablishing Methanogenic Archaea as an Alternative Platform in Synthetic Biology

Peyton Smith1,3*, Rebecca Buchanan1,3, Mengyin Cheng2,3, Kevin Moriles 1,3, Rachit Jain2,3, Narendran Sekar2,3, Sirisha Naga2,3 , Zhe Lyu1,3, William Whitman1,3*, Yajun Yan2,3

University of Georgia, Athens, Georgia 30602

Metabolite Metabolite Description Metabolite Formula

grl geraniol C10H18O

grl_e geraniol ext C10H18O

Rxn Name Rxn Description Formula

R620 Geraniol Formation grpd + h₂o � ppi + grlR621 Geraniol Formation grl � grl_eR622 Geraniol Exchange grl �

Objective Function

Max Biomass(mmol/gDCW/hr)

Max Geraniol (mmol/gDCW/hr)

Constraint CO₂ (10) CO₂ (10) and Biomass (0.04)CO₂ -10 -10H₂ -7.469 -7.346NH₄ -36.534 -36.454CH₄ 8.322 8.248H₂O 20.7 20.655Biomass 0.0413 0.04Geraniol 0 0.0127

pMEV4-mCherry

Results: We developed a novel protocol for the characterization of fluorescence in M. maripaludis. Using this method, we were able to thoroughly characterize three sequences (BBa_K1383[000-002]); the native RBS and the theoretical �perfect� and �negative� RBS�s. Fig. 10 shows the visual appearance of mCherry for these sequences, and Fig. 11 illustrates the quantitative data received by running triplicates of our samples in a plate-reader.

In progress: Revive and characterize the entire library of variants.

Fig 10

Fig 11

Educate: Teaching Fundamental Biology�UGA iGEM has taught

classes at Clarke Middle School on the role of bacteria and how they impact our daily lives.

Fig. 6 Modifications made to the existing wild type Methanococcus maripaludis model [2].

Fig. 7 Results from running MATLAB simulations with a COBRA toolbox.

The supporting figure shown here are the modifications we made to the wild type M. maripaludis model file, which is elaborated more on in the Modeling & Flux Balance Analysis section shown to the right.