Engineering Saccharomyces cerevisiae for the production of

Engineering Saccharomyces cerevisiae for the production of 1,2-propanediol using glycerol

as a carbon and energy source

by

Zia-ul Islam

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in

Biochemical Engineering

Approved Dissertation Committee

Prof. Dr. Elke Nevoigt Prof. of Molecular Biotechnology Jacobs University Bremen, Germany

Prof. Dr. Matthias Ullrich Prof. of Microbiology Jacobs University Bremen, Germany

Prof. Dr. Hector Marcelo Fernandez Lahore Prof. of Biochemical Engineering Jacobs University Bremen, Germany

Dr. Mhairi McIntyre Workman Associate Manager, Novo Nordisk, Denmark

Date of Defense: December 9, 2016

Department of Life Sciences & Chemistry Jacobs University Bremen, Germany

Statutory Declaration I, Zia-ul Islam hereby declare, under penalty of perjury, that I am aware of the

consequences of a deliberately or negligently wrongly submitted affidavit, in particular

the punitive provisions of § 156 and § 161 of the Criminal Code (up to 1-year

imprisonment or a fine at delivering a negligent or 3 years or a fine at a knowingly false

affidavit).

Furthermore, I declare that I have written this PhD thesis independently, unless where

clearly stated otherwise. I have used only the sources, the data and the support that I have

clearly mentioned.

This PhD thesis has not been submitted for the conferral of a degree elsewhere.

_________________________ ____________________________ Place Date

Signature

To my beloved parents Aziz-ur-Rehman and Saeeda Bebi, without their support, encouragement and sacrifices this would not have been possible to come all the way through my education career.

I

Acknowledgements This work is an outcome of many people’s contributions in different ways. In this part, I

would like to acknowledge most, if not all of them.

First and foremost, I owe my gratitude to my supervisor Prof. Dr. Elke Nevoigt, for giving

me the opportunity to work in her lab, providing guidance, teaching, and financial and

moral support. Besides a good scientist, I witnessed her as an amazing human being.

I would like to thank my thesis reviewers, Prof. Dr. Matthias Ullrich and

Prof. Dr. Fernandez Lahore, who agreed to be the members of my dissertation committee.

I am very grateful to Dr. Mhairi Workman for kindly agreeing to be on my dissertation

committee and devoting her time to travel all the way from Copenhagen.

My heartiest gratitude goes to Dr. Mathias Klein, who was not only my second supervisor,

but also a great friend. He guided and supported me throughout my Ph.D. journey. Thanks

to him for his long, fruitful discussions, scientific quarrels, help and making my social life

in Bremen enjoyable.

I would like to acknowledge the very friendly and supportive environment that was

provided to me by my former and current lab friends and colleagues, Steve, Miguel

(roomie), Max, Solvejg, Pingwei, Joeline, Raquel, Salman, Joseph, Sid, and Andrea. I

enjoyed working with all of you.

I would like to thank my Pakistani friends in Bremen, that includes Salim, Anas, Jalal, Saad,

Omer, Bali, Ahmad, Farid, Samaria, Muneeb and Sohaib who provided me with the desi

environment.

I owe my sincere thanks to my wonderful host family, Heinz and Sabine for their affection,

love, and care during my stay for almost 7 years in Germany. They gave me an opportunity

to explore the German culture and showed me different places in Germany. I felt blessed

to have a second family thousand miles away from home, as they were always beside me

just like my birth parents when I was in trouble.

I am deeply indebted to my dearest friend Kateryna Dmytrychenko for being such a

wonderful friend, for her understanding, and unwavering support.

Last but not least, my profound gratitude goes to my family. Their love and continuous

support dominated the feeling of being thousands of miles away from them. Words cannot

describe the love I have for them. Thank you very much to my lovely and sweet sisters Dr.

Mahpara, Dr. Maria, and my very very and very loving brother Dr. Zia-ur-Rehman. Thanks

to my dear sister-in-law Dr. Saeeda and my brother-in-law Dr. Murad for their support

and also for being such a fantastic addition to our family.

II

Table of Contents

ACKNOWLEDGEMENTS ...................................................................................................................................................... I

ABSTRACT ............................................................................................................................................................................ IV

LIST OF ABBREVIATIONS ................................................................................................................................................. V

LIST OF FIGURES ................................................................................................................................................................ VI

LIST OF TABLES .............................................................................................................................................................. VIII

LIST OF PUBLICATIONS ................................................................................................................................................... IX

CONFERENCES/ WORKSHOP ATTENDED ................................................................................................................... X

INTRODUCTION | CHAPTER 1 ......................................................................................................................................... 1

1.1 1,2-PROPANEDIOL (1,2-PDO): A CHEMICAL IN DEMAND ..................................................................................... 2 Structure and properties of 1,2-PDO .................................................................................................................... 2 Applications of 1,2-PDO ............................................................................................................................................. 3 Chemical routes for 1,2-PDO production ........................................................................................................... 5 Microbial production of 1,2-PDO ........................................................................................................................... 6

1.2 GLYCEROL AS A CARBON SOURCE FOR MICROBIAL 1,2-PDO PRODUCTION ........................................................ 11 The need for glycerol valorization ..................................................................................................................... 11 Glycerol utilization pathways in yeast .............................................................................................................. 13 Previous work carried out in Nevoigt’s group (our laboratory) regarding the replacement

of the G3P pathway for glycerol catabolism by the DHA pathway in CBS 6412-13A .................. 17 1.3 THE POTENTIAL OF S. CEREVISIAE AS A PRODUCTION HOST ................................................................................. 17

PROJECT OBJECTIVES | CHAPTER 2 ........................................................................................................................... 19

MATERIALS & METHODS | CHAPTER 3 .................................................................................................................... 22

3.1 MATERIALS ............................................................................................................................................................. 23 Chemicals ....................................................................................................................................................................... 23 Kits .................................................................................................................................................................................... 23 Plasmids ......................................................................................................................................................................... 24 Primers ........................................................................................................................................................................... 26 Microorganisms used in this study .................................................................................................................... 29 Media ............................................................................................................................................................................... 30

3.2 METHODS ............................................................................................................................................................... 31 Culturing conditions ................................................................................................................................................. 31 Molecular biology techniques............................................................................................................................... 32 Methods used for genetic engineering of S. cerevisiae............................................................................... 37 Culturing condition for 1,2-PDO production and determination of metabolite

concentration by HPLC ............................................................................................................................................ 40

RESULTS | CHAPTER 4 .................................................................................................................................................... 42

4.1 EXPRESSION OF HETEROLOGOUS GLYCEROL FACILITATORS IN S. CEREVISIAE (CBS 6412-13A) FOR IMPROVEMENT OF GLYCEROL UTILIZATION .......................................................................................................... 43

Glycerol growth performance of selected non-conventional yeast species in comparison to CBS 6412-13A ........................................................................................................................................................ 43

Comparison of heterologous expression of Fps1p homologues from selected non-conventional yeasts in CBS 6412-13A .............................................................................................................. 46

4.2 MODULAR METABOLIC ENGINEERING OF S. CEREVISIAE FOR 1,2-PDO PRODUCTION FROM GLYCEROL AS A CARBON SOURCE .............................................................................................................................................. 49

Description of metabolic engineering modules implemented in S. cerevisiae for 1,2-PDO production from glycerol ........................................................................................................................................ 49

Genetic engineering of S. cerevisiae for implementation of modules aiming at efficient 1,2-PDO production from glycerol ..................................................................................................................... 52

4.3 EFFECT OF THE CULTIVATION MEDIUM ON 1,2-PDO PRODUCTION ................................................................... 64

III

4.4 EFFECTS OF THE DIFFERENT METABOLIC ENGINEERING MODULES ON PHYSIOLOGY AND 1,2-PDO PRODUCTION OF THE STRAINS IN YG MEDIUM ..................................................................................................... 68

The implementation of the heterologous 1,2-PDO pathway in combination with the glycerol facilitator only resulted in the production of trace amounts of 1,2-PDO ....................... 68

Tpi1p down-regulation in the strain PDO-FPS slightly increased the 1,2-PDO production ..... 68 Replacement of the native FAD-dependent glycerol catabolic pathway (G3P) by the NAD+-

dependent pathway (DHA) in PDO-FPS strain slightly enhanced 1,2-PDO production ............. 69 The combination of Tpi1p down-regulation and glycerol catabolic pathway replacement

remarkably increased the 1,2-PDO production ............................................................................................ 70 4.5 EFFECTS OF A SECOND COPY OF THE EXPRESSION CASSETTES FOR ECMGSA AND/OR OPGDH ENCODING FOR ENZYMES OPERATING IN CRUCIAL STEPS OF 1,2-PDO PATHWAY .............................................................. 72

Genomic integration of a second copy of EcmgsA and Opgdh in strain PDO-FPS-Tpi1pdown-DHA ........................................................................................................................................ 72

Characterization of PDO-FPS-Tpi1pdown-DHA derivatives with a second copy of EcmgsA and/or Opgdh ............................................................................................................................................................... 75

4.6 A SO FAR UNKNOWN METABOLITE WITH THE RETENTION TIME OF ACETOL ACCUMULATES IN THE ENGINEERED STRAINS ............................................................................................................................................. 78

DISCUSSION & FUTURE PROSPECTS | CHAPTER 5 ............................................................................................... 80

5.1 DISCUSSION ............................................................................................................................................................ 81 Possible bottlenecks that hinder the efficient glycerol utilization in S. cerevisiae ........................ 81 Potential limitations for further increasing 1,2-PDO production in the generated

S. cerevisiae strains .................................................................................................................................................... 82 Differences in the strain background and cultivation conditions might have caused the

comparably low 1,2-PDO production in the strain solely expressing the heterologous 1,2-PDO pathway ....................................................................................................................................................... 84

The replacement of the native FAD-dependent glycerol catabolic pathway with an NAD+-dependent pathway enhances ethanol formation ....................................................................................... 86

Accumulation of unknown metabolite with the retention time of acetol ......................................... 87 5.2 FUTURE PROSPECTS ............................................................................................................................................... 88

General recommendations ..................................................................................................................................... 88 Alleviating the allosteric inhibition of methylglyoxal synthase ............................................................ 88 Exploration of alternative methylglyoxal synthases .................................................................................. 89 Methylglyoxal is toxic for the cells; its detoxification pathways also causes competition to

1,2-PDO pathway for carbon flux ........................................................................................................................ 90

REFERENCES ...................................................................................................................................................................... 92

IV

Abstract

Biodiesel production via transesterification generates approx. 10% (w/w) glycerol, which

is considered as a glut and needs to be valorized. The production of 1,2-propanediol

(1,2-PDO) could prove to be one interesting valorization route. 1,2-PDO is an important

commodity chemical used in food, drug, and cosmetic industries. Beside these

applications, its frequent use as a less-toxic (compared to ethylene glycol) antifreeze, as a

favorable solvent in photographic industries, in polyester resin preparation, and in

preparation of biodegradable plastics makes it a chemical in high demand.

S. cerevisiae is one of the favored platforms for metabolic engineering and a popular

microbial host to produce high-value chemicals. In this study we attempt engineering

S. cerevisiae for 1,2-PDO production from glycerol. As a starting point we used a

previously selected S. cerevisiae strain (CBS 6412-13A) naturally able to grow on glycerol

with μmax of ∼0.13 h-1. First, the glycerol growth of CBS6412-13A on glycerol was further

improved by ∼39%, reaching to a μmax of ∼0.18 h-1 by expressing a heterologous glycerol

facilitator (Fps1p from Cyberlindnera jadinii).

In order to produce 1,2-PDO in the latter strain, three heterologous 1,2-PDO pathway

enzymes were co-expressed. As the 1,2-PDO pathway enzyme need redox equivalent in

the form of NADH, the native glycerol catabolic pathway was replaced by an NADH

generating pathway. To further enhance the 1,2-PDO production in such an engineered

strain, the triose phosphate isomerase that competes with 1,2-PDO pathway for

dihydroxyacetone phosphate was down-regulated by replacing the native TPI1 promoter

by the weak promoter TEFmut2. Additionally, the key genes encoding the heterologous

1,2-PDO pathway enzymes were overexpressed by introducing their second copies into

the genome. The reduction of Tpi1p activity in combination with NADH-generating

glycerol catabolic pathway and overexpression of key 1,2-PDO enzymes resulted in a

remarkable improvement of 1,2-PDO production from glycerol and a titer of ∼3.7 g/L was

achieved. This concentration of 1,2-PDO is the highest ever achieved (∼70% more than

previously reported) in engineered S. cerevisiae using glycerol as a carbon source.

V

List of abbreviations

1,2-PDO ATP

1,2-propanediol adenosine-5’-triphosphate

bp ble Cas9 CRISPR CSM CjFPS1

base pair phleomycin resistance gene CRISPR associated protein Clustered Regulatory Interspaced Short Palindromic Repeats complete supplementation mixture C. jadinii FPS1

DAK dihydroxyacetone kinase DHA dihydroxyacetone DHAP dihydroxyacetone phosphate DNA deoxyribonucleic acid dNTP EcmgsA EcgldA EcyqhD

deoxynucleoside triphosphate E. coli mgsA E. coli gldA E. coli yqhD

FAD flavin adenine dinucleotide (oxidized form) FADH2 EDTA G418 gRNA KpFPS1

flavin adenine dinucleotide (reduced form) ethylenediaminetetraacetate geneticin guide RNA K. pastoris FPS1

Kb KanMx4

kilo base pair kanamycin resistance gene

LB Luria-Bertani medium L-G3P L-glycerol 3-phosphate LiAc lithium acetate nat1 nourseothricin resistance gene NAD+ nicotinamide adenine dinucleotide (oxidized form) NADH nicotinamide adenine dinucleotide (reduced form) OD600nm optical density at 600 nm Pagdh O. parapolymorpha gdh PCI phenol/chloroform/isoamyl-alcohol PCR polymerase chain reaction PEG polyethylene glycol PtFPS2 rpm

P. tannophilus FPS2 rotation per minute

SM synthetic medium YlFPS1 Y. lipolytica FPS1 μmax maximum specific growth rate

VI

List of Figures

Figure 1. Structure of 1,2-PDO molecule showing two distinct parts, the hydroxyl groups and the hydrocarbon backbone ............................................................................... 2

Figure 2. Known microbial pathways for 1,2-PDO formation from different substrates ........................................................................................................................................................... 7

Figure 3. Biodiesel production for the years 2000 to 2015 and predicted production during 2016 to 2020 as estimated by the OECD-FAO (Organization for Economic Co-operation and Development, Food and Agriculture Organization) according to www.agri-outlook.org ................................................................................................................................ 12

Figure 4. Two glycerol catabolic pathways, i.e. the phosphorylative (via L-G3P) andthe oxidative pathway (via DHA) have been characterized in yeasts.. ............................ 15

Figure 5. Two-step genetic modification process via GIN11 counter-selectable system ............................................................................................................................................................... 39

Figure 6. Growth performance in synthetic glycerol medium (initial pH was set to 4) of four selected non-conventional yeast species in comparison to the S. cerevisiae strain CBS 6412-13A .......................................................................................................... 45

Figure 7. Growth performance of CBS 6412-13A (CBS) expressing Fps1 homologues from C. jadinii DSM 2361 (CBS CjFPS1), K. pastoris X-33 (CBS KpFPS1), P. tannophilus CBS 4044 (CBS PtFPS2) and Y. lipolytica IBT 446 (CBS YlFPS1) in comparison to wild-type strain CBS 6412-13A ......................................................................................................................... 47

Figure 8. Glycerol growth performance of the strain CBS 6412-13A wild-type strain (CBS) and derivatives of this strain expressing Fps1 homologues from four non-conventional yeast species .................................................................................................... 48

Figure 9. Modular metabolic engineering for 1,2-PDO production from glycerol in S. cerevisiae CBS 6412-13A ................................................................................................. 51

Figure 10. Simplified flowchart how strains (CBS 6412-13A derivatives) used in this study were constructed ..................................................................................................... 52

Figure 11. Construction of PDO-FPS strain. ...................................................................................... 55

Figure 12. Construction of PDO-FPS-Tpi1pdown strain. ................................................................ 57

Figure 13. Construction of PDO-FPS-DHA strain ............................................................................ 60

Figure 14. Construction of PDO-FPS-Tpi1pdown-DHA strain ...................................................... 62

Figure 15. Impact of growth medium on 1,2-PDO production of one selected S. cerevisiae strain engineered in the current study. ...................................................................... 67

VII

Figure 16. Physiological characterization of S. cerevisiae strains (CBS 6412-13A derivatives) engineered for 1,2-PDO production from glycerol. ............................................................................................................................................................ 71

Figure 17. Construction of strain PDO-FPS-Tpi1pdown-DHA with second copies of Opgdh and/or EcmgsA ........................................................................................................................... 74

Figure 18. Physiological characterization of PDO-FPS-Tpi1pdown-DHA derivatives expressing a second copy of EcmgsA and/or Opgdh ............................................... 77

Figure 19. Time courses of the concentration of the acetol-like metabolite in the genetically engineered S. cerevisiae strains generated for 1,2-PDO production from glycerol in this study ................................................................................................................................... 79

VIII

List of Tables

Table 1. Applications of 1,2-PDO .............................................................................................................. 3

Table 2. 1,2-PDO concentrations produced from various substrates by bacterial wild-type strains. .......................................................................................................................... 8

Table 3. 1,2-PDO concentrations achieved with genetically engineered bacteria using different substrates. .......................................................................................................... 9

Table 4. Summary of genetically engineered yeast generated so far for the production of 1,2-PDO ............................................................................................................................... 11

Table 5. Kits used in this study. ............................................................................................................. 23

Table 6. Plasmids used in this study. ................................................................................................... 24

Table 7. Primers used in this study. ..................................................................................................... 26

Table 8. Microorganisms used in this study. .................................................................................... 29

Table 9. Media used in this study. ......................................................................................................... 30

Table 10. Summary of maximum specific growth rates (µmax) and lag phases of four selected yeast species and the S. cerevisiae strain CBS 6412-13A in synthetic medium containing 6% v/v glycerol at initial pH of 4 .................................................................................................................................................. 46

Table 11. Summary of the most important genetically engineered CBS 6412-13A derivatives for analyzing the effects of the suggested metabolic engineering Modules aiming at for 1,2-PDO production using glycerol as carbon source .............................................................................................................. 63

Table 12. Summary of the constructed derivatives of strain PDO-FPS-Tpi1pdown-DHA for elucidation of the effects of overexpressing enzymes (encoded by EcmgsA and Opgdh) that operate in crucial steps of the 1,2-PDO pathway ............................................................................ 75

IX

List of publications

I. Islam Z, Klein M, Nevoigt E. Enhanced 1,2-PDO production in Saccharomyces-

cerevisiae using glycerol as carbon and energy source.

Manuscript in preparation.

II. Klein M, Islam Z, Knudsen PB, Carrillo M, Swinnen S, Workman M, Nevoigt E

(2016). The expression of glycerol facilitators from various yeast species improves

growth on glycerol of Saccharomyces cerevisiae. Metab Eng Commun 3: 252-257.

III. Klein M, Carrillo M, Xiberras J, Islam Z, Swinnen S, Nevoigt E (2016). Towards the

exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based

bioprocesses. Metab Eng 38: 464-472.

X

Conferences/ workshop attended

I. PYFF6 - 6th Conference on Physiology of Yeasts and Filamentous Fungi, July 11-14, 2016, Lisbon, Portugal.

II. Bremen Life Sciences Meeting May 29-30, 2015, Bremen, Germany.

III. Bio-Prodict 3DM workshop November 5-7, 2014, Bio-Prodict BV, Nijmegen, The Netherlands.

IV. MOLIFE Retreat, March 25-26, 2013, Bad Bevensen, Germany.

1

Introduction | Chapter 1

INTRODUCTION | CHAPTER 1

2

1.1 1,2-propanediol (1,2-PDO): a chemical in demand

Structure and properties of 1,2-PDO

1,2-propanediol (propylene glycol or propane-1,2-diol, CAS Registry Number 57-55-6) is

an important commodity chemical with approximately 3 billion lb/year global demand

with a variety of applications (see 1.1.2). Physically, 1,2-PDO is a clear, colorless and

odorless liquid with faintly sweet taste and possesses a syrup-like consistency. It acts as

an excellent solvent, stabilizes and connects insoluble fluids (emulsifier) i.e. holds and

equally dissolves active ingredients in a certain medium, which otherwise would not mix.

It also serves as a medium for the active ingredients (excipient), absorb/holds

water/moisture (hygroscopic), reduces the freezing point, increases the boiling point and

proves to be highly stable with high flash and boiling points. GRAS (generally recognized

as safe) status has been assigned to 1,2-PDO for use in food, cosmetics, and medicines by

U.S. Food and Drug Administration (FDA).

The chemical structure of 1,2-PDO reveals the presence of a stereogenic center at the C2

carbon atom and therefore two enantiomers ((R)-1,2-PDO and (S)-1,2-PDO) exist.

Commodity 1,2-PDO is a racemic mixture of both forms. 1,2-PDO is amphiphile due to the

presence of hydroxyl groups and the hydrocarbon backbone. These two parts confer both

hydrophilicity and lipophilicity to this molecule as shown in Fig. 1.

C

C

CO

O

H

H

H

H

H

H

H

H

Likes oily substances

Likes water

Figure 1. Structure 1 of 1,2-PDO molecule showing two distinct parts, the hydroxyl groups and the hydrocarbon backbone.

1 Figure adapted from www.Propylene-glycol.com.

Lipophilic

Hydrophilic


3

Applications of 1,2-PDO

1,2-PDO possess many essential combinatory properties that perform various important

functions in different products. For instance, being chemically neutral at standard

conditions, 1,2-PDO does not react with other substances and hence offers an outstanding

possibility for combining chemicals with contrasting solubility to obtain single

homogenous fluids, as in perfumes 2 . Table 1 summarizes a number of important

applications and the specific role of 1,2-PDO in each product.

Table 1. Applications of 1,2-PDO

APPLICATION FUNCTION

Aircrafts and automotive de-icing fluids

1,2-PDO can reduce the freezing point of water to -60°C. This freezing point reduction helps in keeping the wings of the aircraft ice-free. In addition, 1,2-PDO is used in automotive windscreen deicers.

Pharmaceuticals

The main function of 1,2-PDO in pharmaceuticals is to solubilize the desired ingredients in a formulation and provide homogenous distribution so that the product application always delivers the exactly prescribed amount of curing agent.

Food industry

1,2-PDO (pharmaceutical grade) is used as a food additive under the E-number 1520. It acts as a humectant to keep the food moist.

Paints & coatings

A substantial contribution to the protective power of paints and varnishes is imparted by 1,2-PDO, helping in preservation and protection of buildings against weathering and also maintaining the quality and sharpness of floors in high-traffic areas.

Plastics resins

1,2-PDO is subjectable to a trouble-free processing of high performance, unsaturated polyester resins such as reinforced plastic laminates for marine construction bath & kitchenware, pipes, and blades for windmills, etc. 1,2-PDO also works as starter element for basic materials (polyols) e.g. dipropylene glycol phthalate; used as plasticizers for polyvinyl chloride (PVC) resins.

2 The Dow Chemical Company (www.dow.com).


4

Cosmetics and toiletries

1,2-PDO is one of the essential ingredients in more than 4000 cosmetic products due to its role as viscosity decreasing agent and solvent.

Dust suppression

1,2-PDO is ideal for use as a dust suppression agent, due to its hygroscopic nature, low vapor pressure, and low toxicity. Housekeeping issues, such as dust formation and danger of dust explosions in mining and cement grinding applications, make dust suppression inevitable.

Dyes and inks 1,2-PDO is an excellent compatibilizer and solvent for most of the dyes and inks used in modern high-speed printing.

Lubricants

1,2-PDO has an affinity for oils and has a low volatility and a high boiling point that make it an essential component of textile lubricant formulations. The FDA has approved its indirect usage as a surface lubricant in the part metal manufacture.

Natural Gas Dehydration

1,2-PDO acts as an antifreeze agent that is necessary to avoid condensation in pipelines/forming hydrocarbon due to the presence of water content in natural gas during its recovery.

Heat transfer Fluids

In engine coolants, 1,2-PDO is used to reduce the freezing point of the liquid, to avoid overheating of the engines. In burst protection fluids, 1,2-PDO, due to its high boiling point, lowers vapor pressure.

Animal feed

1,2-PDO as a humectant that helps dogs in the digestion of food by keeping it soft, moist and chewy. Besides its use as a moisturizer, it is also used as a direct energy source for cattle.3

3 References: www.propylene-glycol.com www.dow.com www.cefic.org www.shell.com


5

Chemical routes for 1,2-PDO production

Adolphe Wurtz (1859) was the first who demonstrated that 1,2-PDO can be formed by

hydrolysis of propylene glycol diacetate. The first commercial production of

1,2-PDO by Carbide and Carbon Chemicals Corporation in 1931 was based on the

chlorohydrin process in which propylene oxide was formed followed by its hydrolysis to

1,2-PDO. In the 1930’s, Du Pont Corporation also produced 1,2-PDO as a by-product

during high-pressure coconut oil hydrogenation. Dow Chemical Corporation (in 1942),

and Wyandotte Chemical Corporation (in 1948), also produced 1,2-PDO by several other

processes (Martin and Murphy 2000).

Propylene oxide is the primary intermediate in any chemical 1,2-PDO production process

at industrial level. There are two main routes used for the production of propylene oxide.

One method is named as chlorohydrin process, where the propylene is converted to

propylene chlorohydride, which is then converted to propylene oxide. In the second

process, isobutane or ethylbenzene are converted to an alkyl hyperoxide first, and then

this alkyl hyperoxide reacts with propylene to form propylene oxide. All commercial 1,2-

PDO production routes known so far, require high temperature, high pressure, and the

non-catalytic hydrolysis of propylene oxide. For instance, hydration reactor conditions

require a temperature of 120-190°C at pressures up to 2170 kPa accompanied by a large

excess of water. After the reaction, multi-effect evaporators and drying towers are used

to remove water, followed by purification of 1,2-PDO using high vacuum distillation.

Most of these current commercial processes for the production of 1,2-PDO are based on

non-renewable petroleum resources. Besides the use of non-renewable petroleum

resources for 1,2-PDO production, there are also reports (D’Hondt et al., 2008; Yuan et al.,

2010) that describe the chemical conversion of glycerol to 1,2-PDO. There are at least

seven companies or entities that are working towards catalyst-based chemical production

of 1,2-PDO from biodiesel-derived crude glycerol using proprietary methods. These are

Cargill/Ashland (joint venture), Archer Daniels Midland (ADM), Pacific Northwest

National Laboratory, Virent Technologies, Huntsman, and Dow Chemicals4. The catalytic

production of 1,2-PDO from glycerol is considered a more promising approach instead of

4http://thinking.nexant.com/sites/default/files/report/field_attachment_abstract/200803/0607S4_abs.pdf


6

the hydration of petro-based propylene oxide (Musolino et al., 2011). Different studies

have been reported in the literature that describes the use of mixed catalysts for glycerol

hydrogenolysis. It includes the use of Ru/C (or Rh/SiO2) + Amberlyst (Miyazawa et al.,

2007), Rh/C+H2WO4 (Chaminand et al., 2004) and Pt/SiO2–Al2O3 (Gandarias et al., 2010).

However, the glycerol conversion rate was low and mainly acidic medium was used to

test noble metals. Besides the use of acidic medium, the glycerol hydrogenolysis was also

attempted in a basic medium in combination with mixed catalysts such as Pt/C + NaOH

(Maris et al., 2007), and Ru/TiO2 + NaOH (Feng et al., 2007). Although the use of NaOH

improved glycerol conversion, it also resulted in by products such as ethylene glycol and

methanol. A 91.0% glycerol conversion rate with 98.7% selectivity was achieved by using

Rh-promoted Cu/solid-base catalysts (Xia et al., 2012). However, it is obvious from these

described chemical conversion methods, the use of chemical catalysts is inevitable. These

reactions are also accompanied with high temperatures and poses serious threats to the

environment and is economically not viable.

Microbial production of 1,2-PDO

Besides chemical production, 1,2-PDO can also be generated in a biological way since

metabolic pathways for its production do exist in nature, particularly in certain

microorganisms. Identifying, improving and harnessing the 1,2-PDO formation

capabilities of these organisms will potentially offer new eco-friendly solutions as

opposed to current petrochemical-based and energy consuming processes for 1,2-PDO

production.

Known metabolic pathways leading to 1,2-PDO formation

In microorganisms, several routes have been reported for the formation of

1,2-PDO (Fig. 2). In the first route, deoxy sugars like fucose or rhamnose are used by

bacteria such as S. typhimurium as the main carbon and energy source. However, the use

of expensive deoxy sugars is not feasible in commercial processes aiming at 1,2-PDO

production.

A second metabolic route to 1,2-PDO using methylglyoxal (MG) as an intermediate (Fig. 2)

has been described in Thermoanaerobacterium thermosaccharolyticum, E. coli and

Clostridium sphenoids when cells grow with glucose or glycerol as a carbon source. In


7

these pathways, the glycolytic dihydroxyacetone phosphate (DHAP) is first converted to

MG by methylglyoxal synthase. The subsequent metabolism of MG to 1,2-PDO proceeds

either via acetol (hydroxyacetone) or lactaldehyde. In this pathway, methylglyoxal

synthase seems to be the most important enzyme as it connects the glycolytic

intermediate DHAP to the rest of 1,2-PDO pathway (Fig. 2). However, the 1,2-PDO

production pathway is not the only pathway diverting from DHAP, but both glycolysis or

gluconeogenesis (depending on the carbon source) also compete for this intermediate

(Fakas et al., 2009). The key enzyme that acts on DHAP is triose-phosphate isomerase

(encoded by TPI1).

It has also been discovered that Lactobacillus buchneri is able to convert L-lactic acid to

1,2-PDO under anoxic conditions (Elferink et al., 2001). However, the enzymes

responsible for these conversions have not been characterized yet. While the conversion

of R-lactate to R-lactaldehyde has recently been explored by BRAIN AG (Germany) and

patented (Koch et al., 2012).

Figure 2. Known microbial pathways for 1,2-PDO formation from different substrates. Abbreviation: DHAP (Dihydroxyacetone phosphate).


8

Unlike eukaryotes, the majority of prokaryotes contain MG synthase (Fig. 2) allowing

these organisms to naturally produce 1,2-PDO. Wild-type microorganisms (prokaryotes)

that are natural 1,2-PDO-producer and have been reported in the literature are

summarized in Table 2. Among the detected 1,2-PDO concentrations, the by far highest

level of 1,2-PDO (9 g/L) has been reported for a strain of the species

Thermoanerobacterium thermosaccharolyticum when grown in glucose medium.

In eukaryotes, the native production of small amounts of 1,2-PDO from glucose/glycerol

as e.g. reported by Murata et al. (1985) is still debatable. The enzymatic activity of the

crucial enzyme methylglyoxal synthase (MG synthase) in S. cerevisiae has been vaguely

described by Murata et al. (1985) but never explicitly confirmed in any later study and

hence has been considered doubtful among the scientific community. Nevertheless, a

small amount of non-enzymatic formation of MG and S-D-lactoylglutathione has also been

observed in yeast during the sporulation period by Shigematsu et al. (1992).

Table 2. 1,2-PDO concentrations produced from various substrates by bacterial wild-type strains.


9

Genetic engineering of bacteria for the production of 1,2-PDO

Several attempts have been made to genetically engineer bacteria such as E. coli and

C. glutamicum for higher 1,2-PDO production (Table 3). However, no single attempt was

found so far to meet the demands for industrial 1,2-PDO production. One reason could be

that bacterial growth is inhibited by methylglyoxal since methylglyoxal accumulation in

cells has been proven to be bactericidal (Ferguson et al., 1998).

Genetic engineering of yeast for 1,2-PDO production

Wild-type S. cerevisiae does not seem to have the ability to produce 1,2-PDO, in fact no

significant amount of 1,2-PDO have been reported in cultures of non-engineered

S. cerevisiae as mentioned above. This is most probably due to the absence or lacking

activity of the first crucial enzyme (methylglyoxal synthase) of 1,2-PDO pathway (Fig. 2)

Table 3. 1,2-PDO concentrations achieved with genetically engineered bacteria using different substrates.


10

in this organism. In fact, no bacterial methylglyoxal synthase homologues can be found in

the genome of S. cerevisiae.

A few engineering studies with the goal of heterologous 1,2-PDO production have been

carried out in Saccharomyces cerevisiae (S. cerevisiae) and Komagataella pastoris

(K. pastoris) strains (Table 4). However, the obtained 1,2-PDO concentrations did not

exceed those reached with engineered E. coli (Table 3). For instance, a detectable amount

of 1,2-PDO from glucose in S. cerevisiae (0.24 g/L) has been reported by the sole

expression of E. coli’s mgsA gene (Hoffman 1999). This result suggests the existence of

some (partly unknown) native oxidoreductase activities in S. cerevisiae that naturally

convert MG into acetol and/or lactaldehyde and then to 1,2-PDO. These native

oxidoreductases (potential candidates are Adh4p, Gre2p, or Bdh1p) don’t have high

substrate specificity and usually can accept more than one substrate (Inoue and Kimura

1995). Among the approaches listed in Table 4, there is even one where the production of

1,2-PDO in S. cerevisiae has been attempted by Jung et al. (2011) starting from glycerol,

an another interesting attractive carbon source (see section 1.2). Interestingly, this

resulted in the highest concentration of 1,2-PDO achieved in yeast so far (2.18 g/L).

In more detail, the highest titer and yield of 1,2-PDO in S. cerevisiae was obtained with the

strain where the 1,2-PDO pathway genes i.e. EcmgsA and EcgldA were co-expressed with

the gdh encoding glycerol dehydrogenase from Ogataea parapolymorpha (Opgdh) along

with the overexpression of native GUP1 (glycerol transporter). According to the authors,

the Opgdh encoding glycerol dehydrogenase is supposedly catalyzing the conversion of

glycerol to DHA (Fig. 4), while the GUP1 was previously reported to encode a glycerol

transporter (Holst et al., 2000). The authors also noticed that the introduction of E. coli

mgsA and gldA genes had a positive impact on glycerol growth of S. cerevisiae, possibly

due to a better redox balance in the cytosol. Nevertheless, the implementation of

heterologous 1,2-PDO pathway in S. cerevisiae still results in a redox-imbalance when the

entire pathway from glycerol to 1,2-PDO is considered as the glycerol dehydrogenase

(encoded by gldA) needs NADH.


11

Table 4. Summary of genetically engineered yeast generated so far for the production of 1,2-PDO. The genetic modifications are shown as well as the 1,2-PDO concentrations obtained from the respective substrates.

1.2 Glycerol as a carbon source for microbial 1,2-PDO production

The need for glycerol valorization

The increase in demand for petroleum fuel, its ephemeral status, and increased

greenhouse gas emission during the last few decades compelled biofuel industries to

grow. Implementation of new laws will eventually supersede the fossil fuels by renewable

ones. For instance, according to the European Union Directive EP 2003/30/ES, by 2020,

energy production from renewable sources should be 20% and fuels should contain 10%

of renewable bio-components. Among others, the production of biodiesel by

transesterification of vegetable and animal fats is one solution. Biodiesel production

generates approx. 10% (w/w) glycerol as a by-product (Durnin et al., 2009). A significant

growth in the world biodiesel industry has been observed in the last decade and will even

(tpi1∆)

(Jung and Lee, 2011)

(Jung et al., 2008)

(Hoffman, 1999)

)

(Lee and DaSilva, 2006)

)

(Jeon et al., 1999)

)

(Expressing glycerol-

dehydrogenase from Ogataea-

polymorpha and overexpression of

glycerol transporter (GUP1).

(Anaerobic conditions)

(3 copies of gene) (3 copies of gene)

(Barbier et al., 2011)

Genes heterologously expressed or overexpressed

Substrate 1,2-PDO conc.(g/L)

Host *DHAP **MG Acetol 1,2-PDO Lactaldehyde 1,2-PDO

S. c

ere

vis

iae

mgsA (E.coli)

gldA (E. coli)

Glycerol 2.18

mgsA (E.coli)

gldA (E. coli)

Glucose 1.11

mgsA (E.coli)

gldA (E. coli) Glucose 0.52

mgsA (E.coli)

dhaD (Citrobacter freundii)

Glucose 0.45

mgsA (E. coli)

gldA (E. coli)

Glucose 0.14○ g/g of cells

K. p

ast

ori

s

mgsA (E.coli)

gldA (Pichia ofunaensis) Glycerol 0.11

*DHAP-Dihydroxyacetone phosphate **MG-Methylglyoxal ○ Titer not available


12

continue (Fig. 3). Thus, the available amount of the by-product crude glycerol is also

expected to increase.

=Forecast

Figure 3. Biodiesel production for the years 2000 to 2015 and predicted production during 2016 to 2020 as estimated by the OECD-FAO (Organization for Economic Co-operation and Development, Food and

Agriculture Organization) according to www.agri-outlook.org.

Increased generation of crude glycerol calls for suitable valorization routes. In fact, this

will not only reduce the glut but will also contribute to the viability of biodiesel industry

(Yang et al., 2012). One possible route towards crude glycerol valorization is its use as a

carbon source for growing microorganisms used in industrial biotechnology. In the

context of de novo biosynthesis processes, the use of glycerol as a substrate or co-

substrate is also encouraged due to its higher reducing power compared to glucose or

other sugars. In fact, the use of glycerol results in higher maximum theoretical yields when

it comes to the production of reduced compounds whose biosyntheic pathways require

more electrons than delivered during sugar catabolism. Several avenues concerning

microbial valorization of glycerol have already been established. The most notable

application is certainly the commercial production of 1,3-proapnediol by Clostridium

butyricum (Wilkens et al., 2012). Other applications include single cell oil and oxalic acid

0

5

10

15

20

25

30

35

40

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

20

13

20

14

20

15

20

16

20

17

20

18

20

19

20

20

Bio

die

sel

pro

du

ced

(th

ou

san

d t

on

s)


13

production in Aspergillus niger (André et al., 2009), eicosapentaenoic acid in Pythium

irregular (Athalye et al., 2009), and citric acid in Yarrowia lipolytica (Rywinska and

Rymowicz 2009).

Glycerol utilization pathways in yeast

A number of yeast species are able to utilize glycerol as a carbon source, even though

there are huge differences with regard to the efficiency of glycerol dissimilation (Gancedo

et al., 1968; André et al., 2010; Chatzifragkou et al., 2011). For instance, Lages et al. (1999)

screened 42 yeast species for growth on glycerol, out of which Puccinia anomola and

Cyberlindnera jadinii grew exceptionally well and achieved growth rates of 0.29 h-1 and

0.32 h-1, respectively. A different study hypothesized that; the membrane of C. jadinii (syn:

C. utilis) is 105 times more permeable to glycerol than S. cerevisiae (Gancedo et al., 1968).

The subject was recently reviewed in detail by Klein et al (2016 c).

The first step of glycerol utilization is its transport into the cell. Both passive and active

mechanisms have been considered for glycerol import. While active transport requires

energy, passive transport (facilitated transport) does not. Passive transport of glycerol

(i.e. influx and efflux) is thought to be mediated by yeast Fps1p, which is a channel protein

belonging to the family of major intrinsic proteins (MIP). An Fps1p homologue has also

been discovered in the species S. cerevisiae (Luyten et al., 1995; Van Aelst et al., 1991).

Interestingly, facilitated diffusion is not the main mechanism of uptake during growth of

S. cerevisiae on glycerol (Swinnen et al., 2013) as it will be elaborated below. Rather,

Fps1p has been shown to be mainly responsible for glycerol export in S. cerevisiae during

hypo-osmotic shock (Tamás et al., 1999). Although FPS1 homologues have been detected

within the genome sequences of several non-conventional yeast species that utilize

glycerol much better than S. cerevisiae, the quantitative participation of passive glycerol

import during glycerol dissimilation in these yeasts is not yet clear.

The major mechanism of glycerol uptake in S. cerevisiae during growth on glycerol is

active transport via a membrane protein encoded by STL1. Stl1p mediates the symport of

glycerol and protons (Kayingo et al., 2009; Ferreira et al., 2005). Genes encoding for Stl1p

homologues have also been identified in the genomes of many other yeast species

including those which grow well in glycerol. It can be concluded that two general types of

transport mechanisms/proteins seem to be involved in the utilization of glycerol by


14

yeasts in general. Interestingly, it has been recently shown that the function of Stl1p in

S. cerevisiae can be replaced by Pachysolen tannophilus Fps2p (Liu et al., 2013).

Once glycerol has entered the cells, it is either first phosphorylated or catabolized

oxidatively (Fakas et al., 2009) as shown in Fig. 4. The phosphorylative pathway

(hereinafter referred to as G3P pathway) is well-known in S. cerevisiae, where glycerol is

first converted to L-glycerol-3-phosphate (L-G3P) by glycerol kinase encoded GUT1

(Pavlik et al., 1993). L-G3P is then oxidized to dihydroxyacetone phosphate (DHAP) by

GUT2 encoded mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase

(Rønnow and Kielland-Brandt 2004).

In the oxidative pathway (hereinafter referred to as DHA pathway), glycerol is first

converted to dihydroxyacetone and then to dihydroxyacetone phosphate by NAD+-

dependent glycerol dehydrogenase and dihydroxyacetone kinase, respectively. This latter

pathway does not seem to be active during growth of S. cerevisiae on glycerol however,

the DHA pathway is the only operating pathway in some methylotrophic yeasts such as

Hansenula ofunaensis (Tani and Yamada 1987). Both the oxidative and the

phosphorylative pathway coexist in some other yeast species such as in the

methylotrophic yeast Ogataea parapolymorpha (Tani and Yamada 1987). Further

metabolism of dihydroxyacetone phosphate obtained from either pathway is carried out

by the glycolytic or the gluconeogenic pathway (Fakas et al., 2009).


15

Figure 4. Two glycerol catabolic pathways, i.e. the phosphorylative (via L-G3P) and the oxidative pathway

(via DHA) have been characterized in yeasts. Fps1p mediates the entry of glycerol into the yeast cell via

facilitated diffusion, while the active transport of glycerol is carried out by Stl1p. The phosphorylative (via

L-G3P) pathway seems to be only active in S. cerevisiae (Pavlik et al., 1993; Rønnow and Kielland-Brandt

2004). The ‘DHA pathway’ is known to operate in several methylotrophic yeasts such as Hansenula

ofunaensis and Ogataea parapolymorpha either alone or together with the phosphorylative (via L-G3P)

pathway, respectively (Tani and Yamada 1987). Abbreviation: Sc, S. cerevisiae.

Several strains of S. cerevisiae have been identified and characterized with regard to their

ability to use glycerol as a carbon source (Barnett et al., 2000). Although a great

intraspecies diversity has been reported by the authors, there is no comprehensive

quantitative evaluation with regard to e.g. µmax or duration of lag phase available in the

literature. Furthermore, all studies carried out so far have been conducted in the presence

of media supplements such as amino acids and nucleic bases or even more complex

compounds such as yeast extract. It has been demonstrated by several authors that many


16

commonly used S. cerevisiae strains do not grow at all in synthetic glycerol medium

without such a supplementation (Merico et al., 2011; Ochoa-Estopier et al., 2011; Swinnen

et al., 2013). In fact, there exists a synergistic relationship between different supplements

(amino acids and nucleic bases) in growth media and the glycerol consumption of

commonly used laboratory strains of S. cerevisiae (Swinnen et al., 2013).

Several approaches have been published to improve the growth on glycerol of S. cerevisiae

strains in the absence of medium supplements. For example, adaptive evolution strategies

were applied by Merico et al. (2011) and Ochoa-Estopier et al. (2011) As a result, the

evolved strains reached a μmax of approximately 0.2 h-1.

In parallel, Nevoigt and co-workers (our laboratory) screened 52 S. cerevisiae strains for

growth on glycerol as a sole carbon source i.e. without any further medium

supplementation (Swinnen et al., 2013). It was shown that several isolates could grow

with µmax up to 0.15 h-1 even though long lag phases were observed. However, the well-

characterized laboratory strains such as CEN.PK113-7D, W303-1A, and S288c are not able

to grow at all under these conditions. In the same study, a S. cerevisiae strain (CBS 6412 –

for information about this strain the reader is referred to Hubmann et al., 2013) was

identified, which has the ability to grow up to a μmax of ~0.10 h-1. Among all tested strains,

CBS 6412 had the shortest lag phase of ~27 hours. This led to the fastest accumulation of

biomass when compared to all other strains. Later, a meiotic haploid segregant called

CBS 6412-13A, was obtained as it is easy to perform tractable genetic manipulation in it

compared to in polyploid parent. The CBS 6412-13A showed even slightly better growth

on glycerol compared to its diploid parent. In fact, the haploid CBS 6412-13A grew with a

μmax of ~0.13 h-1 and a lag phase of ~16 hours in synthetic glycerol medium. In this study,

the strain CBS 6412-13A was chosen as the baseline strain for all metabolic engineering

steps aiming at 1,2-PDO production from glycerol.


17

Previous work carried out in Nevoigt’s group (our laboratory) regarding the replacement of the G3P pathway for glycerol catabolism by the DHA pathway in CBS 6412-13A

As mentioned in the chapter 1.2.2, the G3P pathway is the major (if not the only) glycerol

catabolic pathway in S. cerevisiae. This has even been confirmed to be valid in the strain

CBS 6412-13A. In fact, the deletion of either GUT1 or GUT2 completely abolished growth

on glycerol (Swinnen et al., 2013). In the context of exploiting glycerol’s reducing power

for the production of reduced compounds such as 1,2-PDO, it is important to know that

the native glycerol catabolic pathway (G3P pathway) cannot fulfill this function, as the

respective electrons are lost via Gut2/FADH directly to the mitochondrial respiratory

chain. Therefore, the G3P pathway (Fig. 4) has been recently replaced by the NAD+-

dependent DHA pathway (Fig. 4) in the strain CBS 6412-13A (Klein et al., 2016 b) by

expression of Opgdh gene encoding glycerol dehydrogenase from Ogataea-

parapolymorpha for oxidizing glycerol to dihydroxyacetone (DHA) (Fig.4). while the

subsequent phosphorylation of DHA was enhanced by the overexpression of the native

DAK1 gene encoding a glycerol kinase.

The knowledge about the genetic modifications required for functional glycerol catabolic

pathway replacement in CBS 6412-13A i.e. the deletion of GUT1, the expression of O.

polymorpha gdh and overexpression of S. cerevisiae DAK1, were used in the current work

as it was necessary to provide redox equivalents (NADH) for the 1,2-PDO pathway

engineering in CBS 6412-13A.

1.3 The potential of S. cerevisiae as a production host

S. cerevisiae has become a valuable model microorganism in fundamental and applied

research due to its historic involvement in wine making, brewing and baking industry

(Barnett 2003). Among all eukaryotic organisms, S. cerevisiae was the first whose genome

was completely sequenced (Goffeau et al., 1996). S. cerevisiae is a superior natural

producer of ethanol which explains the industrial-scale commercialization of this process

in current bioethanol production (Porro et al., 1995). Another important fact which has

encouraged the use of S. cerevisiae in industrial applications is its classification as GRAS

(generally regarded as safe) organism by the U.S. Food and Drug Administration (FDA).


18

Compared to other industrially used microorganisms, S. cerevisiae is more tolerant

towards low pH, osmotic pressure, high concentration of sugars, and ethanol. Tolerance

towards low pH reduces the risk of contamination during large-scale industrial

fermentation processes conducted under non-sterile conditions. The general robustness

in large-scale bioprocesses due to the mentioned features and the ability to grow

anaerobically are definitely two notable attributes which add to the fact that S. cerevisiae

has become one of the most applied and explored microorganisms in white (industrial)

biotechnology.

It is also the ease of genetic modifications, which has propelled the popularity of

S. cerevisiae. First, effective transformation methods and tools had a very positive impact

on the genetic engineering of S. cerevisiae (Parent and Bostian 1995;Gietz and Woods

2001). Second, extraordinary capabilities of homologous recombination allow targeted

chromosomal manipulations with high efficiency (Klinner and Schäfer 2006).

S. cerevisiae’s genetic toolbox has even been greatly enhanced and updated in the past

years (Tyo et al., 2007; Nielsen and Jewett 2007), making S. cerevisiae one of the most

popular industrial microbial factory. Nowadays, a number of databases such as the

Saccharomyces Genome Database5 , Comprehensive Yeast Genome Database6 , General

Repository of Interaction Datasets7, The European S. cerevisiae Archive for Functional

Analysis8, and Japanese Yeast Genetic Resource Center9 possess a vast collection of useful

information with regard to S. cerevisiae strains and engineering tools that has been

provided to the yeast scientific community.

5 www.yeastgenome.org 6 mips.gsf.de/genre/proj/yeast 7 www.thebiogrid.org 8 www.euroscarf.de/index.php?name=Description 9 yeast.lab.nig.ac.jp/nig/index_en.html

19

Project objectives | Chapter 2

PROJECT OBJECTIVES | CHAPTER 2

20

S. cerevisiae has been one of the favorable cell factories in the field of industrial

biotechnology, i.e. for the production of valuable bulk and fine chemicals from renewable

raw materials. Although 1,2-PDO production in this important yeast has been already

investigated by several authors (Table 4), no significant success has been achieved so far.

In fact, the current 1,2-PDO yields and titers are far from those required for an

economically viable industrial production process. The goal of this study is to improve

1,2-PDO production in engineered S. cerevisiae by using CBS 6412-13A strain and glycerol

as carbon source.

As a starting point, the growth of CBS 6412-13A on glycerol will be attempted to further

improve (beyond 0.13 h-1) by expressing the heterologous glycerol facilitator encoding

genes from C. jadinii (CjFPS1), K. pastoris (KpFPS1), P. tannophilus (PtFPS2) and

Y. lipolytica (YlFPS1). The expression cassettes will be integrated into the genome of

CBS 6412-13A at YGLCτ3 as a target site (Flagfeldt et al., 2009) in chromosome VII via

GIN11 counter-selectable system (3.2.3). These strains will be analyzed in the Growth

Profiler and the best glycerol facilitator exhibiting highest maximum growth rate (μmax)

and lowest lag phase will be selected to be included into the genetic engineering strategies

aimed at 1,2-PDO production.

To equip the CBS 6412-13A with 1,2-PDO pathway, a heterologous 1,2-PDO pathway will

be implemented. This heterologous pathway will be composed of mgsA, yqhD, and gldA

genes from E. coli. The expression cassettes for E. coli mgsA and gldA will be integrated at

YGLCτ3 as a target site (Flagfeldt et al., 2009) in chromosome VII, while the E. coli yqhD

will be expressed from a low copy plasmid.

In an attempt to improve 1,2-PDO production in such an engineered strain, the competing

pathways that takes away the precursor DHAP from 1,2-PDO pathway (glycolysis or

gluconeogenesis) need to be eliminated or down-regulated. In this regard, eliminating or

reducing the activity of triose isomerase Tpi1p (encoded by TPI1) is crucial (Fig. 2). The

TPI1 gene cannot be completely deleted when solely using glycerol as a carbon source, as

the cells do not have the opportunity to bypass the Tpi1p reaction and gain ATP in another

pathway. Thus, down-regulating the expression of TPI1 was the target of this work. This

was achieved by replacing the gene’s native promoter by a weak promoter of known

strength. In more detail, the native promoter of TPI1 will be replaced by the weak

TEFmut2 replacement cassette previously generated by Nevoigt et al. (2006).

PROJECT OBJECTIVES | CHAPTER 2

21

The final part of this study will address the redox imbalance issue encountered during

1,2-PDO production from glycerol in S. cerevisiae. More precisely, the FAD+-dependent

glycerol catabolic pathway will be replaced by an NAD-dependent pathway adopting the

strategy of Klein et al., (2016 b). This strategy includes the deletion of the GUT1 gene

thereby abolishing the G3P pathway and the establishment of the DHA pathway by

expressing the glycerol dehydrogenase from O. parapolymorpha accompanied with the

overexpression of endogenous DAK1. The DHA pathway is supposed to generate NADH,

that can be utilized in the envisaged 1,2-PDO pathway.

At the end of the study, the effect of overexpressing crucial genes of the heterologous

1,2-PDO pathway will be investigated. For this purpose, a second copy of E. coli mgsA and

O. parapolymorpha gdh will be introduced, separately and in combination.

22

Materials & Methods | Chapter 3

MATERIALS AND METHODS | CHAPTER 3

23

3.1 Materials

Chemicals

All chemicals were obtained either from AppliChem (Darmstadt, Germany), Roche

Diagnostics (Mannheim, Germany), Sigma Aldrich (Hamburg, Germany), Carl Roth

(Karlsruhe, Germany) and were at least of analytical grade. Restriction enzymes used in

this study were obtained from Thermo Fisher Scientific (Schwerte, Germany) or New

England Biolabs (NEB; Frankfurt am Main, Germany) unless stated otherwise. All

enzymes and chemicals were used according to the manufacturer’s instructions.

Kits

The kits used in this study are given in the Table 5.

Table 5. Kits used in this study.

Kits Company Origin

QIAprep®

Spin Miniprep Kit

QIAquick®

Gel Extraction Kit

QIAquick®

PCR Purification kit

Qiagen

Qiagen

Qiagen

Hilden, Germany

Hilden, Germany

Hilden, Germany


24

3.1

.3P

lasm

ids

Th

e plasm

ids u

sed in

this stu

dy an

d th

eir origin

are given

in th

e Tab

le 6.

Ta

ble

6. P

lasmid

s used

in th

is stud

y.

*Co

do

n-o

ptim

ized fo

r expressio

n in

Sacch

aro

myces cerevisia

e


25


26

3.1

.4P

rime

rs

Prim

ers used

in th

is stud

y are listed in

Tab

le 7. A

ll prim

ers were syn

thesized

by E

uro

fins G

eno

mics (E

bersb

erg, Germ

any

). Th

e

nu

mb

ering o

f prim

ers is accord

ing to

the p

rimers n

um

berin

g used

in th

e labo

ratory. T

he restrictio

n sites are u

nd

erlined

and

the

sequ

ences fo

r in viv

o reco

mb

inatio

n an

d in

tegration

into

the gen

om

e, or o

verlap to

adjacen

t fragmen

ts du

ring G

ibso

n A

ssemb

ly

are given

in b

old

.

Ta

ble

7. P

rimers u

sed in

this stu

dy.


27


28


29

3.1

.5M

icroo

rga

nism

s use

d in

this stu

dy

All th

e bacterial an

d yeast strain

s used

in th

is stud

y are listed in

Tab

le 8.

Ta

ble

8. M

icroo

rganism

s used

in th

is stud

y.


30

Media

A summary of different media used in this study is presented in Table 9. For the preparation of solid media, 2 % agar was added.

Table 9. Media used in this study.

Media Composition

YG 10 g L-1 yeast extract, 60 mL L-1 (75.36 g L-1) glycerol, pH adjusted to 6 with 2.0 M H3PO4.

Verduyn (synthetic)

Synthetic media according to Verduyn et al., 1992. 5 g L-1 (NH4)2SO4, 3 g L-1 KH2PO4, 0.5 g L-1 MgSO4.7H2O, 1 mL trace elements. As carbon source, 20 g L-1 glucose, 20 g L-1 galactose or 60 mL L-1 (75.36 g L-1) anhydrous glycerol was added. The pH of the media containing glucose or galactose was adjusted to 6.5 with 2 M KOH and that of the medium containing glycerol was adjusted to 4.0 with 2 M H3PO4. To change the nitrogen source, 2.8 g L-1 of urea was added instead of (NH4)2SO4. The urea was filter-sterilized and added after autoclaving the rest of the salt solution. Also 1 mL of vitamins were added to the autoclaved solution.

YPGal 10 g L-1 yeast extract, 20 g L-1 peptone, and 20 g L-1 galactose.

YPD 10 g L-1 yeast extract, 20 g L-1 peptone, and 20 g L-1 glucose. YPG 10 g L-1 yeast extract, 20 g L-1 peptone, and 20 g L-1 glycerol. LB

5 g L-1 yeast extract, 10 g L-1 tryptone, 10 g L-1 NaCl, pH was adjusted to 7.5 with 2 M NaOH.

Yeast stock 10 g L-1 yeast extract, 20 g L-1 peptone, and 329 g L-1 glycerol


31

3.2 Methods

Culturing conditions

General culturing conditions for yeast and bacteria

S. cerevisiae and other yeast strains in this study were grown at 30°C, and for liquid

cultures 200 rpm orbital shaking frequency was maintained. S. cerevisiae transformants

having dominant markers (kanMX4, ble, nat1, or hphMX) either integrated into the

genome or carried by a plasmid, were selected on solid and liquid YPD medium containing

200 μg mL-1 geneticin, 20 μg mL-1 phleomycin, 300 μg mL-1 hygromycin B or 50 μg mL-1

nourseothricin. For preparation of glycerol stocks of S. cerevisiae strains, about 80 mg of

overnight grown cells was scrapped with 1 mL sterile pipette tip from agar plates and

were suspended in stock medium (Table 9), mixed briefly and stored at -80°C.

E. coli was grown at 37°C and a shaking frequency of 250 rpm was used for liquid cultures.

Plasmids containing were maintained in E. coli by addition of 100 mg L-1 ampicillin to the

medium. For making E. coli glycerol stocks, an equal amount of E. coli LB medium

overnight culture and 50% (w/v) glycerol solution10 were mixed and frozen at -80°C.

Growth characterization of strains in Growth Profiler 1152

Verduyn medium containing 2% glucose as a carbon source (pH 6.5) was inoculated with

a single yeast colony and cultivated at 30°C and 200 rpm overnight on a rotary shaker.

This pre-culture was used to inoculate an intermediate culture of the same medium to an

OD600 of ̴0.2. The intermediate culture was incubated at 30°C and 200 rpm on a rotary

shaker for 48 hours. An appropriate amount of cells from this intermediate culture was

washed once in Verduyn medium containing 6% (v/v) glycerol as carbon source (pH 4).

The washed cells were used to inoculate Verduyn medium containing 6% glycerol to an

OD600 of ̴0.2. White Krystal 24-well clear bottom microplates (Porvair Sciences,

Leatherhead, UK) were filled with 0.75 mL of prepared cell culture. 3 biological replicates

with 2 technical replicates each were used per strain. Microplates were incubated in the

Growth Profiler 1152 at 30°C and 200 rpm for up to 4 days. The Growth Profiler 1152 was

scheduled to scan the plates every 40 minutes. The Growth Profiler 1152 determined the

10 https://www.addgene.org/plasmid-protocols/create-glycerol-stock.


32

culture densities (expressed as green value or G-value) in each single well of the

microplate. The obtained G-values were converted into OD600nm values (referred to as

OD600nm equivalents) using a calibration curve with the equation of the best-fit line for

each yeast species differently according to the equations below.

S. cerevisiae: 6.1761 x 10-8 x G-value3.4784,

C. jadinii: 1.6667 x 10-7 x G-value3.3259,

P. tannophilus: 2.4481 x 10-7x G-value3.2911,

K. pastoris: 2.6407 x 10-7 x G-value3.1582

Y. lipolytica: 3.5056 x 10-7 x G-value3.1333.

Molecular biology techniques

Plasmid isolation, restriction digestion and plasmid dephosphorylation

Generally, the plasmid DNA was isolated from E. coli using the QIAprep Spin Miniprep Kit

(Table 5). The protocol followed was that described in the manual provided with the kit.

After linearizing the vector, the 5’-termini of the vector backbone were dephosphorylated

according to Sigma Aldrich’s-Cold Shrimp Alkaline Phosphatase (SAP) dephosphorylation

protocol. For restriction digestion analysis, respective restriction enzymes were used

according to the manufacturers’ instruction.

All constructed plasmids (that are explained in the following sections) were confirmed by

sequencing through GATC-Biotech AG, Germany.

PCR conditions

PCR conditions for amplification of DNA fragments for cloning into a plasmids or genomic

integration:

The PCR reactions were performed in 50 μL PCR mixtures containing 20-30 ng of template

plasmid or genomic DNA of S288c (for S. cerevisiae coding sequence, promoters and

terminators), 1x reaction buffer, 200 μM of each dNTPs, 200 nM of each primer and 0.5

Units of Phusion® High-Fidelity DNA Polymerase (New England Biolabs). The cycling

conditions were as follows: an initial denaturation at 98 °C for 3 minutes, 30 cycles of

denaturation at 98 °C for 30 seconds, annealing at Tm of primer minus 5°C for 30 seconds

and elongation at 72 °C for 30 seconds. A final elongation step was performed at 72 °C for

3 minutes.


33

Diagnostic PCR for verification of correct S. cerevisiae transformants:

The PCR reactions were performed in 20 μL PCR mixtures containing 2 μL of 1:20 diluted

genomic DNA extracted from the obtained transformants, 1x reaction buffer E, 200 μM of

each dNTP, 400 nM of each primer and 0.5 Units of TaqE polymerase. The cycling

conditions were as follows: an initial denaturation at 94°C for 4 minutes, 30 cycles of

denaturation at 94°C for 30 seconds, annealing at melting temperature of primer minus

5°C for 30 seconds and elongation for 1 minute/kb at 72°C. A final elongation step was

performed at 72°C for 5 minutes, and kept it at 12°C at the end of the PCR.

Diagnostic PCR for E. coli DH5α transformants:

The E. coli DH5α transformants for verification of correctly constructed plasmids, the

same colony PCR conditions as described above for S. cerevisiae were applied, except that

instead of adding the extracted genomic DNA, a small amount of cells was suspended in

20 μL of PCR mixture.

Isolation of genomic DNA

An amount of 50 mg of cells were scrapped from agar plate with a sterile 1 mL pipette tip

and suspended in 200 μL of TE buffer (10 mM TRIS, 1mM EDTA, pH 8). Approximately,

0.3 g of acid-washed glass beads (diameter 425-600 μm; Sigma-Aldrich), were added to

the tube containing cells and TE buffer. 200 μL phenol-chlorophorm-isoamylalcohol

(25:24:21) solution was added afterwards. The suspension was vortexed for 2 minutes at

maximum speed and the tubes were centrifuged for 10 minutes at 13,000 rpm. 20 μL of

the aqueous layer was mixed with 380 μL of water. 2-3 μL of this solution was used as a

source of genomic DNA for various PCRs.

Construction of plasmids via conventional restriction ligation method

Construction of p416TEF-KanMX:

The p416TEF (Table 6) was used to clone the loxP-kanMX4-loxP cassette. The p416TEF

was opened at KpnI site and was dephosphorylated with alkaline phosphatase to avoid

self-ligation. The loxP-kanMX4-loxP was amplified from pUG6 (Table 6) with primers

232/233 that also adds KpnI restriction site to the PCR product. The resultant PCR


34

product was digested with KpnI enzyme to create sticky end that are complimentary to

the digested p416TEF. The digested PCR product ligated into digested p416TEF plasmid

with the help of T4 DNA ligase resulting p416TEF-KanMX plasmid.

Construction of p416TEFmgsA(c)-KanMX:

The cloning of EcmgsA coding sequence in between PTEF1 and TCYC1 was carried out in

plasmid p416TEF-KanMX (Table 6). The p416TEF-KanMX was opened with BamHI and

EcoRI and was. The codon optimized EcmgsA was amplified by Phusion® High-Fidelity

DNA Polymerase (New England Biolabs) from pMA-T-mgs plasmid (Table 6) with primers

276/277. The PCR conditions used are given below. The PCR mixture was subjected to

DpnI digestion to degrade the template plasmid that could create false positive E. coli

DH5α clones after transformation. Afterwards, the PCR products were purified using the

QIAquick PCR Purification Kit (Table 5). The primers attach the BamHI and EcoRI

recognition sequences to the PCR product which upon double digestion with respective

enzymes creates sticky ends that ligates with the help of T4 DNA ligase in the opened

p416TEF-KanMX. The resultant plasmid p416TEFmgsA(c)-KanMX was verified by

sequencing using primer 230/231.

Heat shock transformation of E. coli DH5α

For heat shock transformation of E. coli DH5α, 200 μL of competent cells were thawed on

ice and mixed gently by swirling. 100 μL of competent cells were aliquoted into

pre-chilled sterile 1.5 mL falcon tubes. 5 μL (10-30 ng in total) of plasmid or Gibson

Assembly mixture were added into the cell suspension and the tube was directly put back

on ice. After the cells were kept on ice for 10 minutes and swirled gently in between, a

heat shock was applied at 42°C in a water-bath for 45 seconds. The cells were placed back

on ice directly afterwards and kept on ice for 2 minutes. 900 μL of LB medium was added

to the cells and the cells were incubated at 37°C and 250 rpm for 60 minutes.

Gibson assembly

Gibson assembly was carried out in a master mix that contained 5X isothermal (ISO)

reaction buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1

mM each of the 4 dNTPs i.e. dATP, dCTP, dGTP, dTTP, and 5 mM NAD+), 0.005 U/ul T5

exonuclease, 0.02 U/µl Phusion polymerase, 5.3 U/µl Taq DNA ligase and water


35

(Gibson et al., 2009). The mixture was used to assemble different expression cassettes.

The PCR primers were designed via SNAPGENE in a way that creates 15-25 bp of

homologous overhangs sequences to each other and to the linearized backbone of the

plasmid. As a general rule, the PCR product contained in the mixture of PCR reaction were

digested overnight with DpnI and the linearized backbone of the plasmid was

dephosphorylated. In general, 0.05 pmol of the linearized plasmid and 3-fold excess (each

0.15 pmol) of the inserts (promoter, coding sequence and the terminator) was added to

15 μL of the reaction mixture not exceeding a final total volume of 20 μL. The mixture was

incubated at 50 °C for 1 hour. After incubation, E. coli DH5α was transformed with 5 μL of

reaction mix for further propagation. The transformants carrying the correctly assembled

plasmid were verified via diagnostic PCR as described above and also by sequencing.

The specific details of assembly of each expression cassettes used in this study is as

follows;

Construction of plasmids via Gibson assembly

pUC18-gldA:

The EcgldA coding sequence was assembled in between PTDH3 and TIDP1 in pUC18 vector

(Table 6). The pUC18 was first linearized using BamHI site. The PTDH3 and TIDP1 were

amplified from genomic DNA of S. cerevisiae strain S288c (as described above, but

partially purified using ethanol precipitation method) with primers 323/324 and

327/328 respectively, while EcgldA was PCR amplified from pGA-gldA (Table 6) with

primers 325/326.

pUC18-mgsA:

The EcmgsA coding sequence was assembled in between PTDH3 and TIDP1 in pUC18 vector

(Table 6). The pUC18 was first linearized using BamHI site. The PTDH3 and TIDP1 were

amplified from genomic DNA of S. cerevisiae strain S288c (as described above, but

partially purified using ethanol precipitation method) with primers 419/422 and

415/328 respectively, while EcmgsA was PCR amplified from pMA-T-mgs plasmid (Table

6) with primers 420/414.


36

p41ble-gdh-yqhD:

The EcyqhD expression cassette was Gibson-assembled between PACT1 and TTPS1 in

p41bleTEF-Opgdh (Table 6). The PACT1 and TTPS1 were amplified from genomic DNA of

S. cerevisiae strain S288c with primers 531/532 and 533/534 respectively, while EcyqhD

was PCR amplified from pMA-T-yqhD (Table 6) with primers 535/536. Before assembly,

the p41bleTEF-Opgdh was linearized using KpnI site.

p41ble-gdh-E:

To construct p41ble-gdh-E, the PACT1 and TTPS1 was assembled in p41bleTEF-Opgdh at the

same locus (KpnI). For this purpose, the PACT1 and TTPS1 were amplified from genomic DNA

of S. cerevisiae strain S288c with primers 531/538 and 533/537 respectively. After

Gibson assembly the plasmid was called p41ble-gdh-E.

p41ble-E-yqhD and p41ble-E-E:

The EcyqhD assembly in between PACT1 and TTPS1 (p41ble-E-yqhD) and also empty

expression cassette i.e. only PACT1 and TTPS1 (p41ble-E-E) were made in p41bleTEF plasmid

where there was no Opgdh present. The primers and assembly site in p41bleTEF were the

same for p41ble-E-yqhD and p41ble-E-E as described in preparation for p41ble-gdh-

yqhD and p41ble-gdh-yqhD respectively.


37

Methods used for genetic engineering of S. cerevisiae

Yeast transformations

Transformation of S. cerevisiae was carried via LiAc/PEG (lithium acetate/polyethylene

glycol) method (Gietz et al., 1995) with minor alterations. A 5 mL overnight YPD (Table

9) was used as a pre-culture to inoculate a 50 mL complex medium culture to a starting

OD600nm of 0.2. This culture was incubated in the shaking incubator at 30°C (200 rpm)

until the OD600 reached to 0.8. The cells were pelleted in a sterile 50 mL centrifuge tube at

3,000 rpm for 5 minutes. After the removal of the supernatant, the cells were washed with

25 mL sterile water and were resuspended in 1 mL 100 mM LiAc and then transferred to

a 1.5 mL micro centrifuge tube. Cells were pelleted at 3,000 rpm for 3 minutes and the

LiAc was removed with a micropipette. After that the cells were resuspended in 100 mM

LiAc to a final volume of 300 µl, out of which 50 µl cell suspension was used for one

transformation reaction. 50 µl aliquots of cell suspension were transferred to a new 1.5

mL tube and pelleted by centrifugation at 3,000 rpm for 1 minutes and the LiAc was again

removed with a micropipette. The following components were added in strict order: a. X

μL DNA, b. 240 μL PEG (MW 3500, 50% w/v), c. 10 μL of ss (single stranded)-DNA (boiled

for 5 minutes in a heating block), d. sterile water (74 μL minus X μL of DNA used), e. 36

μL of 1 M LiAc. The solution was mixed thoroughly and incubated at 42°C for 40 minutes

in a water bath. In case of a dominant marker (e.g. kanMX4, ble, nat1, or hphMX),

expression of the marker was allowed before plating onto selective medium. This was

done by addition of 3 mL complex medium (without any antibiotics) and incubation at

30°C in a shaking incubator at 200 rpm for 4 to 6 hours. Finally, the tube was centrifuged

at 3,000 rpm at room temperature for 1 minutes and the cells were re-suspended in 1 mL

sterile water and aliquots of 100 μL were spread on respective agar plates with

appropriate antibiotic concentration and incubated at 30°C for 2-3 days.

Seamless integration via GIN11 counter-selectable system

The seamless integration of EcmgsA and EcgldA expression cassettes in chromosome VII

at location YGLCτ3, DAK1 overexpression at YPRCΔ15 locus in chromosome XVI, and

second copies of EcmgsA with or without Opgdh at YPRCτ3 genomic location in

chromosome XVI were achieved via GIN11 counter-selectable system. In more details,

plasmid pGG119 (Akada et al., 2002) was used as a source template for GALp-GIN11M86


38

cassette amplification with primers 155 and a second primer that has 40 bp sequence at

their 5’ terminal end that is complementary to region upstream of the target locus (primer

175 for YGLCτ3, primer 298 for YPRCΔ15, and primer 300 for YPRCτ3). For amplification

of the marker gene i.e. kanMX4 or ble, pUG6 or pUG66 plasmids (Gueldener et al., 2002),

respectively, were used with the help of primer 171 and a second primer that has 40 bp

sequence at their 3’ terminal end that is complementary to region downstream of the

target locus (primer 176 for YGLCτ3, primer 299 for YPRCΔ15, and primer 301 for

YPRCτ3). A complementary region was created with primers 155 and 171 between the

downstream sequence of the GALp-GIN11M86 and with the upstream sequence of the

marker gene. This complementary sequence was used by the homologous recombination

machinery of S. cerevisiae to combine the GALp-GIN11M86 and marker. S. cerevisiae strain

was transformed with equimolar amounts of GIN11M86 and marker PCR products via

lithium acetate method as described above.

For generating the final strain with the desired genetic modification (described in the

respective strain construction chapters of the results section, the

GALp-GIN11M86/marker cassette needed to be replaced with the desired expression

cassette. This was done by transforming the PCR product/products (i.e. expression

cassette/cassettes of interest) having the flanking sequences of 60 bp homologous to

chromosomal regions upstream and downstream of the previously integrated GALp-

GIN11M86/marker cassette. The cells were plated on Verduyn (synthetic) medium having

2% galactose as a carbon source in order to induce the growth inhibitory sequence

GIN11M86 for counter selection.


39

Figure 5. Two-step genetic modification process via GIN11 counter-selectable system: In the first step the GIN11M86 growth inhibitory sequence (under the control of the GAL10 promoter) and a marker containing one side of each cassette (GIN11 and marker) homologous to the genomic location while the other ends to each other, were integrated via homologous recombination into the target locus. This step leads to the creation of an intermediate strain bearing GIN11M86 and a marker. The selection is made by the respective marker. In the second step the intermediate strain is transformed with the cassette/gene of interest that has flanking homologous DNA region to integration site. The cells were plated on Verduyn (synthetic) medium having 2% galactose as a carbon source in order to induce the growth inhibitory sequence GIN11M86 for counter selection.

Gene disruption

GUT1 was disrupted by integrating KanMX4 that confers resistance to geneticin. Primers

111 and 112 (Table 7) were used to amplify the KanMX4 cassette from pUG6 plasmid

(Table 6). The primers 111 and 112 amplifies KanMX4 cassette that has a 60 bp sequence

flanking at both upstream and downstream ends that shares complementarity to the

genomic integration site.

Marker rescue

In this study plasmid pNatCre (Table 6) was used for rescuing the markers. In this plasmid,

the Cre is under GAL1 promoter. The S. cerevisiae strains, where the markers KanMX4 and

ble were intended to be kicked out, were transformed with the plasmid pNatCre. The

transformants were selected on solid complex medium with nourseothricin (50 µg mL-1).


40

3 mL complex medium with nourseothricin (50 µg mL-1) was inoculated with a single

colony and grown at 30°C and 200 rpm overnight. Next morning, the overnight culture

was centrifuged at 3,000 rpm for 5 minutes, washed 3 times in 5 mL of YPGal medium (9),

resuspended in 3 mL YPGal medium + nourseothricin (50 µg mL-1) and finally incubated

at 30°C and 200 rpm for 6-8 h. The incubation in YPGal medium is needed to induce the

expression of Cre which is under the GAL1 promoter. After incubation for 6-8 hours, a

sample of 10 µL was taken from the culture, diluted (10-4 to 10-6) and plated on solid YPD

media. The plated cells were incubated at 30°C for 2 days to obtain single cell colonies.

For the verification, the single cell colony was streaked on solid complex medium with

and without antibiotic the marker to be lost confers resistance to.

Culturing condition for 1,2-PDO production and determination of metabolite concentration by HPLC

A 3 mL YG medium in 10 mL glass tube was inoculated with a single cell colony. The

culture was grown for 36-48 hours at 30°C and 200 rpm. This 3 mL culture was used to

inoculate a second 10 mL medium in 100 mL Erlenmeyer flask and adjusted to an initial

OD600nm of 0.2. The second pre-culture was grown for 10-12 hours until it reached to an

OD600nm of 2. Finally, the second pre-culture was used to inoculate the main culture of 50

mL in 500 mL Erlenmeyer flask to an initial OD600nm of 0.2 and grown up to 144 hours at

30°C and 200 rpm. The samples were taken at least each after 24 hours, centrifuged at

3,000 rpm for 5 minutes, filtered (with 0.2 µm filter) and stored at -20°C until analyzed in

HPLC.

Before analyzing the samples in question for the production of 1,2-PDO, acetol, ethanol,

and glycerol, following standards with different concentration were prepared in Milli-Q

water (HPLC grade) and calibration curves were prepared.

1,2-PDO (99.5%): 0.075 gL-1, 0.15 gL-1, 0.25 gL-1, 0.5 gL-1, 1 gL-1, 5 gL-1

Acetol (100%): 0.015 gL-1, 0.075 gL-1, 0.15 gL-1, 0.25 gL-1, 0.5 gL-1, 1 gL-1, 2.5 gL-1

Ethanol (99.8%): 0.394 gL-1, 0.788 gL-1, 3.94 gL-1, 7.87 gL-1, 11.81 gL-1, 39.37 gL-1

Glycerol (99.5%): 1 gL-1, 5 gL-1, 10 gL-1, 20 gL-1, 40 gL-1, 60 gL-1, 80 gL-1 90 gL-1, 100

gL-1


41

Filtered samples were assayed via HPLC with Breeze™ 2 HPLC System (Waters

Corporation, Milford, MA, U.S.), RI detector 2414 (Waters Corporation) and HPX-87H

anion exchange column (BioRad, München, Germany). Sulfuric acid (5 mM) was used as

the mobile phase (flow rate 0.45 mL/minute, column at temperature 45°C). As the peaks

of 1,2-PDO and acetol were overlapping each other, two HPX-87H anion exchange column

were put one after another and as a result the peak separation was improved.

RESULTS | CHAPTER 4

42

Results | Chapter 4

RESULTS | CHAPTER 4

43

4.1 Expression of heterologous glycerol facilitators in S. cerevisiae (CBS 6412-13A) for improvement of glycerol utilization

Glycerol growth performance of selected non-conventional yeast species in comparison to CBS 6412-13A

As mentioned in section 1.2.2, laboratory strains of the species S. cerevisiae such as

CEN.PK113-7D, W303-1A, and S288c are not able to grow on glycerol as a sole carbon

source. Keeping in view the main goal of this study i.e. 1,2-PDO production from glycerol

in S. cerevisiae, starting with one of the aforementioned laboratory strains would not be a

feasible solution. At the time point when this work started, the strain CBS 6412 and its

haploid segregant CBS 6412-13A were isolated in our group. Both S. cerevisiae isolates

can moderately grow in synthetic glycerol medium. The latter (haploid) strain grows with

a maximum specific growth rate (μmax) of ~0.13 h-1 on glycerol and was, at that point in

time, considered to be the best choice for implementing the 1,2-PDO production pathway.

Still, there are a number of other yeast species, e.g. Komagataella pastoris, Pachysolen

tannophilus, Pichia sorbitophila, Yarrowia lipolytica, and Cyberlindnera jadinii, which

exhibit a much better growth performance on glycerol compared to S. cerevisiae and the

question arose whether glycerol facilitators from these species could be used in order to

even further improve the capability to utilize glycerol by the S. cerevisiae strain CBS 6412-

13A. There have been several indications in the literature that inefficient glycerol uptake

might be one reason for the poor growth of S. cerevisiae on glycerol and that the

expression of glycerol transporters from yeast species with a much better growth on

glycerol might be one solution for improving S. cerevisiae. For example, (Liu et al., 2013)

have tested the glycerol facilitators (FPS1 homologues) and the active transporters (STL1

homologues) from P. tannophilus (PtFPS1, PtFPS2, PtSTL1, PtSTL2) when expressed in a

S. cerevisiae stl1Δ mutant11. As a control, S. cerevisiae FPS1 and STL1 (ScFPS1, ScSTL1)

were (over)expressed in the same way. The strains expressing PtFPS2 achieved a μmax of

0.07 h-1 on glycerol. All other constructed strains did not grow at all.

11 Stl1p has been shown to be the major active transporter responsible for glycerol import in S. cerevisiae

and the deletion of the respective gene (STL1) has been shown to result in completely abolished growth on

glycerol (Lages and Lucas 1997; Ferreira et al., 2005).

RESULTS | CHAPTER 4

44

The work of Liu et al. (2013) has been the first demonstrating that a gene encoding for

facilitated transport of glycerol can fully replace the active glycerol transport in

S. cerevisiae.

The proof that the glycerol facilitator Fps2p from the yeast P. tannophilus can function as

glycerol importer in S. cerevisiae (Liu et al., 2013) led to the idea of whether this glycerol

facilitator can be used for improvement of growth on glycerol of CBS 6412-13A to achieve

a maximum growth rate that even exceeds 0.13 h-1. In addition to improving the growth

rate above wild-type level, the use of a facilitator has another advantage over active

transport of glycerol in the context of the envisaged global goal of this thesis. A glycerol

facilitator does not directly require energy in the form of ATP. This might be favorable for

the envisioned fermentative production of 1,2-PDO from glycerol.

In general, the better growth on glycerol of P. tannophilus compared to S. cerevisiae

(Liu et al., 2012) and the subsequent discovery that Fps2p can act as a glycerol importer

in S. cerevisiae (Liu et al., 2013) points towards the possibility that the phenotype of better

growth on glycerol of many non-conventional yeast species might be an indicator of the

presence of an efficient glycerol facilitator. Therefore, in an attempt to indirectly explore

a better glycerol facilitator than P. tannophilus Fps2p, it was intended to first compare the

glycerol growth performance of four representative non-conventional yeasts available in

our laboratory i.e. C. jadinii DSM 2361, K. pastoris X-33, P. tannophilus CBS 4044 and Y.

lipolytica IBT 446 in synthetic media. The reasons for selecting C. jadinii DSM 2361, K.

pastoris X-33, and Y. lipolytica IBT 446 were based on some indications that were

previously reported in the literature. For instance, C. jadinii DSM 2361 has a ~105 fold

higher glycerol uptake (membrane permeability) capability as compared to S. cerevisiae

(Gancedo et al., 1968), and also proved to be a superior grower on glycerol in our

laboratory (Ganbold 2013). K. pastoris is a proven industrial production host that can

grow on glycerol (Chiruvolu et al., 1998), while Y. lipolytica is an interesting oleaginous

yeast that is known for assimilation and dissimilation of glycerolipids (Goncalves et al.,

2014).

The aforementioned four yeast species along with CBS 6412-13A were grown in Verduyn

(synthetic) medium containing 6% (v/v) glycerol as the sole carbon source at pH 4 in the

Growth Profiler 1152 according to the procedure described in section 3.2.1.

RESULTS | CHAPTER 4

45

It is obvious from the growth patterns shown in Fig. 6 that the C. jadinii DSM 2361,

P. tannophilus CBS 4044 and Y. lipolytica IBT 446 outperformed, while K. pastoris X-33

was almost even to S. cerevisiae CBS 6412-13A with regard to both µmax and duration of

lag phase in synthetic glycerol medium. The lag phases are due to the adjustment time

needed by the cells to adapt to the synthetic glycerol medium when transferred from

synthetic glucose medium used for the intermediate cultures (as described in 3.2.1). A

summary of µmax and lag phases of all analyzed strains is given in Table 10.

Figure 6. Growth performance in synthetic glycerol medium (initial pH was set to 4) of four selected non-conventional yeast species in comparison to the S. cerevisiae strain CBS 6412-13A. Growth was recorded in the Growth Profiler 1152. The laboratory S. cerevisiae strain CEN.PK113-1A is unable to grow under the conditions used and was considered as negative control. Each curve presents one representative experiment out of three independent biological replicates.

Among the non-conventional yeast strains, K. pastoris X-33 was a slow grower with a μmax

of ~0.18 h-1, however, still better than CBS 6412-13A (~0.13 h-1). C. jadinii DSM 2361

showed the highest maximum growth rate (~0.42 h-1) among all the strains tested. This

μmax of C. jadinii DSM 2361 was even better than the one reported in the literature for a

different strain (μmax of 0.32 h-1 for C. jadinii IGC 254) as mentioned in 1.2.2. The reason

could be caused by differences in the cultivation condition applied in the two studies.

Another possibility could be that intra-species diversity causes the difference in

RESULTS | CHAPTER 4

46

the glycerol growth performance between different strains of the same species.

Y. lipolytica IBT 446 and P. tannophilus CBS 4044 showed an almost two-fold higher μmax

(~0.27 h-1 and ~0.26 h-1, respectively) as compared to CBS 6412-13A (~0.13 h-1). The lag

phase of P. tannophilus CBS 4044 was the shortest (~4.4 hours), while the longest lag

phase was observed for K. pastoris X-33. Notably, these lag phases disappeared when

synthetic medium containing 6% (v/v) glycerol was used for the intermediate cultures

instead (data not shown).

Table 10. Summary of maximum specific growth rates (µmax) and lag phases of four selected yeast species

and the S. cerevisiae strain CBS 6412-13A in synthetic medium containing 6% (v/v) glycerol at initial pH of

4. Growth was recorded in the Growth Profiler 1152.

Yeast strain µmax [h-1] Lag phase [h]

S. cerevisiae CBS 6412-13A 0.13 ± 0.01 17.8 ± 1.16

C. jadinii DSM 2361 0.42 ± 0.03 09.0 ± 0.70

K. pastoris X-33 0.18 ± 0.01 22.2 ± 0.12

P. tannophilus CBS 4044 0.26 ± 0.01 13.0 ± 2.19

Y. lipolytica IBT 446 0.27 ± 0.01 04.4 ± 1.05

Comparison of heterologous expression of Fps1p homologues from

selected non-conventional yeasts in CBS 6412-13A

Keeping in view the better glycerol growth performance of C. jadinii DSM 2361, K. pastoris

X-33, P. tannophilus CBS 4044 and Y. lipolytica IBT 446 (4.1.1), as a next step, it was

interesting to check whether the expression of Fps1p homologues from these selected

non-S. cerevisiae strains can enhance the glycerol growth performance of CBS 6412-13A.

Therefore, four expression cassettes were constructed in which the coding sequences of

the glycerol facilitators from C. jadinii DSM 2361 (CjFPS1), K. pastoris X-33 (KpFPS1),

P. tannophilus CBS 4044 (PtFPS2) and Y. lipolytica IBT 446 (YlFPS1) were brought under

the control of the S. cerevisiae TEF1 promoter and the CYC1 terminator, respectively. The

cassettes were generated as described in Klein et al. (2016 a). In order to integrate the

PTEF1-CjFPS1-TCYC1, PTEF1-KpFPS1-TCYC1, PTEF1-PtFPS2-TCYC1 and PTEF1-YlFPS-TCYC1 expression

cassettes into the genome of CBS 6412-13A, a two-step method applying the GIN11

RESULTS | CHAPTER 4

47

counter-selectable system (3.2.3) was used to obtain seamless integration without

leaving any additional DNA sequences behind. For this purpose, PTEF1-CjFPS1-TCYC1,

PTEF1-KpFPS1-TCYC1, PTEF1-PtFPS2-TCYC1 and PTEF1-YlFPS-TCYC1 were amplified by PCR from

plasmids the respective plasmids (Table 6) according to Klein et al. (2016 a), with

homologous overhangs for the integration into chromosome VII of CBS 6412-13A at the

long terminal repeat (LTR) YGLCτ3 as a target site (Flagfeldt et al., 2009) in the strain CBS

6412-13A. After verification of the resulting transformants for correct integrations via

PCR using the respective verification primers shown in Table 7, the strains were named

CBS 6412-13A CjFPS1, CBS 6412-13A KpFPS1, CBS 6412-13A PtFPS2, and CBS 6412-

13A YlFPS1. These strains were analyzed in the Growth Profiler in order to compare their

growth patterns in synthetic glycerol medium to that of CBS 6412-13A and the growth

curves are shown in Fig. 7.

Figure 7. Growth performance of CBS 6412-13A (CBS) expressing Fps1 homologues from C. jadinii DSM 2361 (CBS CjFPS1), K. pastoris X-33 (CBS KpFPS1), P. tannophilus CBS 4044 (CBS PtFPS2) and Y. lipolytica IBT 446 (CBS YlFPS1) in comparison to wild-type strain CBS 6412-13A, while CEN.PK113-1A (CEN) was used as negative control. The growth was recorded in Growth Profiler 1152 using Verduyn synthetic medium containing 6% (v/v) glycerol as the sole source of carbon. Each curve is one representative experiment out of three independent biological replicates.

RESULTS | CHAPTER 4

48

It is obvious from the growth patterns shown in Fig. 7 that the expression of all

heterologous FPS1 homologues significantly improved the glycerol growth performance

of CBS 6412-13A. The μmax was improved by 30 to 40% while the lag phase duration

decreased by approx. 50% (Fig. 8).

A

B

Figure 8. Glycerol growth performance of the strain CBS 6412-13A wild-type strain (CBS) and derivatives

of this strain expressing Fps1 homologues from C. jadinii DSM 2361 (CBS CjFPS1), K. pastoris X-33 (CBS

KpFPS1), P. tannophilus CBS 4044 (CBS PtFPS2) and Y. lipolytica IBT 446 (CBS YlFPS1) in comparison to

wild-type strain CBS 6412-13A. Maximum specific growth rates (µmax) (A) and duration of lag phases (B)

are shown. CEN.PK113-1A (CEN) was used as negative control.

RESULTS | CHAPTER 4

49

Regardless of the origin of the Fps1 homologues tested in this work, the growth

performance of all engineered CBS 6412-13A derivatives was very similar with respect to

both µmax and lag phase. For the remaining part of this study, FPS1 from C. jadinii was

selected for supporting glycerol uptake in the CBS 6412-13A derivatives engineered here

for 1,2-PDO production from glycerol.

4.2 Modular metabolic engineering of S. cerevisiae for 1,2-PDO production from glycerol as a carbon source

Description of metabolic engineering modules implemented in S. cerevisiae for 1,2-PDO production from glycerol

Engineering S. cerevisiae for enhanced 1,2-PDO production from glycerol requires several

prerequisites as described in section 1.3.2. In order to implement and optimize the ability

of S. cerevisiae to convert glycerol into 1,2-PDO, a modular metabolic engineering

approach has been adopted. As it will be explained at the end of this section, all modules

envisioned for implementation in S. cerevisiae either individually or in combination were

combined with a suitable genetic modification for improved glycerol uptake via a

heterologous glycerol facilitator as this has been demonstrated to result in a significantly

improved growth on glycerol in this strain (see 4.1).

A first set of engineering steps regarded the implementation of a functional 1,2-PDO

pathway starting from DHAP (Fig. 9). The respective genetic modifications will be

referred here to as “Module 1,2-PDO”. Three heterologous enzymes i.e. methylglyoxal

synthase, aldehyde reductase, and glycerol dehydrogenase (Fig. 9) are required for this

Module. The respective genes mgsA, yqhD, and gldA were taken from E. coli,

codon-optimized for expression in S. cerevisiae and named EcmgsA, EcyqhD, and EcgldA

throughout the study.

The key metabolic precursor for the 1,2-PDO pathway is DHAP (Fig. 9). In wild-type

S. cerevisiae, this main precursor is mainly directed towards glyceraldehyde 3-phosphate

(G3P) formation and further to glycolysis. The first reaction is catalyzed by Tpi1p (1.3.2).

In order to provide an increased precursor supply (DHAP) for the heterologous 1,2-PDO

pathway, Tpi1p activity was down-regulated in this work. For the down-regulation, the

native TPI1 promoter was replaced by the weak promoter TEFmut2 replacement cassette

previously generated by Nevoigt et al. (2006). The strategy of down-regulating Tpi1p by

RESULTS | CHAPTER 4

50

replacing the native promoter of TPI1 with the engineered promoter TEFmut2 was tagged

as “Module Tpi1pdown”.

The implemented 1,2-PDO pathway (Module 1,2-PDO) in S. cerevisiae requires cytosolic

reducing equivalents. To provide cytosolic NADH, the glycerol catabolic pathway

replacement strategy developed by Klein et al. (2016 b) and described in section 1.2.2,

has therefore been adopted in the current study. In detail, Klein et al. (2016 b) replaced

the native FAD-dependent glycerol catabolic pathway (G3P pathway) by a synthetic

NAD+-dependent DHA pathway. To achieve this glycerol catabolic pathway replacement

in the current study, the gene Opgdh was expressed together with the overexpression of

the native DAK1 gene while the endogenous GUT1 gene was deleted in order to abolish

the natural glycerol catabolic pathway via G3P (Fig. 4). This set of metabolic engineering

steps was named “Module DHA”.

Low uptake of glycerol in wild-type CBS 6412-13A without an Fps1 homologue might

prove a bottleneck for enhanced 1,2-PDO production later. In order to avoid this potential

problem, all constructed strains were equipped with an expression cassette for CjFPS1.

An overview of the relevant metabolic pathways, enzymes and genes as well as the

proposed engineering modules are summarized in Fig. 9.

RESULTS | CHAPTER 4

51

Fig

ure

9. M

od

ular m

etabo

lic engin

eering

for 1

,2-P

DO

pro

du

ction

from

glycero

l in S. cerevisia

e CB

S 6

41

2-1

3A

. FP

S; imp

rov

ed gly

cerol u

ptak

e, Mo

du

le 1,2

-P

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; hetero

logo

us 1

,2-P

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path

way

, Mo

du

le Tp

i1p

do

wn ; T

pi1

p d

ow

n-regu

lation

, Mo

du

le DH

A; gly

cerol catab

olic p

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ay rep

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iation

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; L-gly

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osp

hate, D

HA

; dih

yd

roxy

aceton

e, DH

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; dih

yd

roxy

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e ph

osp

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ceraldeh

yd

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; m

ethy

lglyo

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O; 1

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rop

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nm

od

ified.

RESULTS | CHAPTER 4

52

Genetic engineering of S. cerevisiae for implementation of modules aiming at efficient 1,2-PDO production from glycerol

The objective of this chapter is to provide a detailed overview of how the strains used to

characterize the impact of the different above-described engineering modules (separately

and in combinations) were constructed. It should be mentioned that most of the genetic

modifications were carried out within the genome/chromosomes while some of the

introduced heterologous genes were expressed on a single copy CEN/ARS plasmid. It is

also noteworthy to mention that the CRISPR/Cas9 system was applied to construct one of

the strains (PDO-FPS-DHA). As a summary, a simplified scheme of strain construction

history is given in Fig. 10.

Figure 10. Simplified flowchart how strains (CBS 6412-13A derivatives) used in this study were constructed. The final strains used to analyze 1,2-PDO production from glycerol including the isogenic control strain (FPS) are shown in red color. The numbering in blue color represents the chapters where the detailed constructions of the strains including the intermediate strains are described. The round circles represent genes that have been expressed via a low copy plasmid. All other genetic modifications were incorporated into the genome. PDOmin refers to minimal 1,2-PDO pathway that is composed of EcmgsA and EcgldA. Abbreviations: Ec; E. coli, Cj; C. jadinii, Op; O. parapolymorpha, oe; overexpression.

RESULTS | CHAPTER 4

53

4.2.2.1 Implementing Module 1,2-PDO together with CjFPS1 in CBS 6412-13A

The first step in the strain construction part was to generate a S. cerevisiae strain

(PDO-FPS in Fig. 10) that has a functional 1,2-PDO pathway composed of a methylglyoxal

synthase, an aldehyde reductase, and a glycerol dehydrogenase in combination with the

expression of the glycerol facilitator CjFPS1. To accomplish this, yeast expression

cassettes for the genes EcmgsA, EcyqhD, and EcgldA had to be constructed. As a first step,

all three protein-encoding sequences from E. coli were codon-optimized for S. cerevisiae

via GeneArt® and synthesized by Life Technologies for efficient translation in S. cerevisiae

(Elena et al., 2014). The next step was to construct expression cassettes by fusing each

coding sequence between a S. cerevisiae promoter and a terminator. In order to avoid

recombination events in the final strain, it was important to select a different promoter

and terminator for each of the three expression cassettes. The expression cassette PTEF1-

mgsA-TCYC1 was prepared via the conventional restriction-ligation method (cloning the

EcmgsA coding sequence between PTEF1 and TCYC1 of the yeast expression plasmid

p416TEF-KanMX) and the resulting plasmid was named p416TEFmgsA(c)-KanMX as

described in 3.2.5. In contrast, the expression cassettes PACT1-yqhD-TTPS1 and PTDH3-gldA-

TIDP1 were prepared via Gibson assembly in the plasmid p41ble and pUC18, respectively

(3.2.2). The resulting plasmids were named p41ble-E-yqhD and pUC18-gldA. As a next

step, the expression cassettes had to be implemented in the S. cerevisiae strain CBS 6412-

13A. The expression cassettes PTEF1-mgsA-TCYC1 and PTDH3-gldA-TIDP1 were adjacently

integrated into chromosome VII at location YGLCτ3 (Flagfeldt et al., 2009), while the

expression cassette PACT1-EcyqhD-TTPS1 was brought in via the CEN/ARS-based plasmid

p41ble-E-yqhD. For genomic integration of the PTEF1-mgsA-TCYC1 and PTDH3-gldA-TIDP1

expression cassettes, the strain CBS 6412-13A GIN11-KanMx4 was used as a starting point.

This strain contains the GIN11-KanMX4 cassette in chromosome VII at location YGLCτ3.

The latter strain was co-transformed with two PCR products, reflecting the expression

cassette for EcmgsA and EcgldA. For this co-transformation and integration by

homologous recombination, the expression cassettes PTEF1-mgsA-TCYC1 and PTDH3-gldA-TIDP1

were PCR amplified from the template plasmid p416TEFmgsA(c)-KanMX (Table 6) and

pUC18-gldA (Table 6) using the primer pair 257/302 (Table 7) and 303/297 (Table 7),

respectively. The resultant S. cerevisiae strain containing EcmgsA and EcgldA expression

RESULTS | CHAPTER 4

54

cassettes was named PDOmin (Fig. 11). The third expression cassette completing the three-

step PDO pathway (EcyqhD) was later brought in via a CEN/ARS plasmid (p41ble-E-yqhD)

but only after integrating an expression cassette for CjFPS1 which is described in the next

paragraph.

The previously constructed cassette PTEF1-CjFPS1-TCYC1 (for expressing a heterologous

glycerol facilitator) contained the same promoter and terminator 12 as the expression

cassette for EcmgsA in the strain PDOmin. Therefore, a new expression cassette was

generated for CjFPS1 via Gibson assembly in pUC18 using the PGK1 promoter and the

RPL15A terminator from S. cerevisiae (section 3.2.2). The resulting CjFPS1 expression

cassette was PCR amplified from the plasmid pUC18-PPGK1-CjFPS1 (3.2.2) with primer

pair 390/391 (Table 7) and co-transferred into strain PDOmin together with the ble

marker cassette amplified from plasmid pUG66 (Table 6) with primers 392/393 (Table

7). As both expression cassettes contained appropriate overlapping sequences to each

other and to the integration site, homologous recombination resulted in integration of the

CjFPS1 expression cassette together with the ble selectable genetic marker directly

downstream of EcmgsA/EcgldA expression cassettes at YGLCτ3 locus resulting in strain

PDOmin-FPS-ble (Fig. 11). The ble expression cassette used as a selection marker was

flanked by loxP sites and later removed via a Cre-recombinase mediated mechanism

(described in 3.2.3). As a last step, the resultant strain was transformed with the above-

mentioned low-copy plasmid (p41ble-E-yqhD) carrying EcyqhD expression cassette. The

acronym PDO-FPS was used for the resultant engineered strain.

12 If the same promoter or terminator were used, the CjFPS1 expression cassette could after transformation unintentionally replace the previously integrated EcmgsA cassette via homologous recombination.

RESULTS | CHAPTER 4

55

CBS 6412-13A (Wild type)

PDOmin

YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1

CBS 6412-13A-GIN11-KanMX4

+ PACT1-yqhD-TTPS1

YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1 PPGK1-CjFPS1-TRPL15A:loxP-ble-loxP

PDO-FPS

YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1 PPGK1-CjFPS1-TRPL15A:loxP-ble:loxP

PDOmin-FPS-ble

PDOmin-FPS

Figure 11 Construction of PDO-FPS strain. First, an intermediate strain CBS 6412-13A GIN11-KanMX4

was constructed by integrating GIN11M86 inhibitory sequence and kanMX4 expression cassettes via

homologous recombination at YGLCτ3 locus. In the second step, the PTEF1-mgsA-TCYC1and PTDH3-gldA-TIDP1

expression cassettes were integrated by homologous recombination at the same position thereby replacing

the GIN11M86 inhibitory sequence and kanMX4 expression cassette. In the third step, the PPGK1-CjFPS1-

TRPL15A expression cassette was integrated along with ble expression cassette (selectable genetic marker)

downstream of EcmgsA and EcgldA. Afterwards, the ble expression cassette was removed (shown in

strikethrough) and the resultant strain was transformed with the low-copy plasmid carrying EcyqhD

expression cassette (plasmid is represented by a circle).

RESULTS | CHAPTER 4

56

4.2.2.2 Construction of strain PDO-FPS- Tpi1pdown: Combining 1,2-PDO pathway with Tpi1p down-regulation

Tpi1p down-regulation (Module Tpi1pdown) aimed at restricting the flow of DHAP towards

glyceraldehyde 3-phosphate (G3P). By doing so, it was anticipated that the availability of

the key precursor DHAP was enhanced for entering the 1,2-PDO pathway (Fig. 9). To

construct the strain PDO-FPS-Tpi1pdown, the native TPI1 promoter in the strain PDOmin-

FPS (constructed in 4.2.1.1 also shown in Fig. 11) was replaced by a cassette composed of

the KanMX4 expression cassette as a selection marker upstream of the weak TEFmut2

promoter. The KanMX4 expression cassette along with the TEFmut2 promoter was PCR-

amplified from the plasmid p416TEF_2-yECitrine-kanMX4 (Table 6) previously

generated by Nevoigt et al. (2006). For this step, the primer pair 426/427 (Table 7) was

used generating flanking homologous sequences of ~60 bp to a chromosomal region

upstream of TPI1 native promoter and a region in the beginning of the TPI1 coding

sequence. Upon transformation, the TPI1 native promoter was replaced by the TEFmut2

promoter via homologous recombination. The KanMX4 upstream of the TEFmut2

promoter was later removed using the Cre-mediated recombination method (3.2.3). The

resulting strain was transformed with the low-copy plasmid (p41ble-E-yqhD) carrying

EcyqhD expression cassette. The constructed strain was named PDO-FPS-Tpi1pdown.

RESULTS | CHAPTER 4

57

4.2.2.3 Construction of strain PDO-FPS-DHA: combining 1,2-PDO pathway with increased NADH availability

The 1,2-PDO pathway (Module 1,2-PDO) requires cytosolic NADH (Fig. 9). This NADH can

be delivered by the catabolism of glycerol as soon as the native G3P pathway is replaced

by the DHA pathway as described in 4.2.1 and shown in Fig. 9 (Module DHA). Therefore,

the aim here was to combine the 1,2-PDO pathway (Module 1,2-PDO) with the glycerol

catabolic pathway replacement (Module DHA)13.

13 It’s worth mentioning that the Module 1,2-PDO was first combined with Module DHA in the absence of

Module Tpi1pdown in order to study the sole impact of glycerol catabolic pathway replacement (and the

assumed increased cytosolic NADH availability accompanied with the latter) on 1,2-PDO production.

PDOmin-FPS-kanMX4-PTEFmut2-TPI1

PTPI1::loxP-kanMX4:loxP-PTEFmut2

PDOmin-FPS

PDO-FPS-Tpi1pdown

PTPI1::loxP-kanMX4:loxP-PTEFmut2

+ PACT1-yqhD-TTPS1

Figure 12 Construction of PDO-FPS-Tpi1pdown strain. The KanMX4-TEFmut2 cassette was amplified

from p416TEF_2-yECitrine-kanMX4 and integrated upstream to the endogenous TPI1 coding sequence on

chromosome IV via homologous recombination. The KanMX4 was afterwards removed (shown in

strikethrough) and the strain was transformed with the low-copy plasmid carrying EcyqhD expression

cassette (plasmid is represented by a circle).

PDOmin-FPS-PTEFmut2-TPI1


YGLCτ3::PGAL-GIN11M86:kanMX4

RESULTS | CHAPTER 4

58

For achieving the combination of Module 1,2-PDO with Module DHA, first the native

glycerol catabolic pathway (via G3P) was abolished by deleting the GUT1 gene in

wild-type CBS 6412-13A. This step was followed by DAK1 overexpression to increase the

enzyme activity of the second step of the envisaged DHA pathway. Afterwards, the

expression cassettes of the genes EcmgsA, EcgldA, and CjFPS1 (for Module 1,2-PDO and

improved glycerol uptake) were integrated at YGLCτ3 locus using CRISPR-Cas9-mediated

genome editing system. The expression cassettes PACT1-EcyqhD-TTPS1 and PTEF1-Opgdh-TCYC1

required for completing the genetic modifications for Module 1,2-PDO and Module DHA

in this strain, respectively, were brought in together via the low-copy plasmid.

p41ble-gdh-yqhD.

In more detail, the GUT1 gene was deleted in wild-type CBS 6412-13A by integration of

kanMX4 expression cassette via homologous recombination. This disruption cassette was

PCR-amplified from pUG6 (Table 6) using 111/112 primers (Table 7). The PCR-amplified

deletion cassette contained 40 bp flanking homologous sequences to regions upstream of

and within the GUT1 coding sequence.

As the overexpression of DAK1 is an integral part of strain engineering for implementing

Module DHA, the integration of PACT1-DAK1-TTPS1 cassette was carried out via seamless

integration using the GIN11 system with ble as a marker (3.2.3). The GALp-GIN11M86/ble

was first integrated at YPRCΔ15 locus in chromosome XVI creating strain CBS 6412-13A

gut1∆-GIN11M86-ble. The PACT1-DAK1-TTPS1 cassette was PCR-amplified with primers

388/389 (Table 7) from plasmid pUC18-DAK1 (Table 6). The primers were designed in a

way to create flanking sequences homologous to the regions upstream and downstream

of chromosomal location where GALp-GIN11M86/ble was integrated. After

transformation of the strain CBS 6412-13A gut1∆-GIN11M86-ble, the DAK1 expression

cassette replaced the GALp-GIN11M86/ble sequence via homologous recombination

leaving no scars behind.

The gut1∆ strain carrying the DAK1 overexpression cassette was further used to

simultaneously integrate the cassettes PTEF1-mgsA-TCYC1, PTDH3-gldA-TIDP1, and

PPGK1-CjFPS1-TRPL15A at YGLCτ3 locus using the CRISPR-Cas9-mediated genome editing

system. To do so, the strain was first transformed with the plasmid p414-TEF1p-Cas9-

CYC1t-nat1 bearing the expression cassette for Streptococcus pyogenes cas9 (Table 6).

Afterwards, the expression cassettes of PTEF1-mgsA-TCYC1, PTDH3-gldA-TIDP1, and

RESULTS | CHAPTER 4

59

PTEF1-CjFPS1-TCYC1 were amplified from their respective plasmids (as discussed in 4.2.1.1)

with primer combinations 257/302, 303/552, and 550/551 (Table 6) respectively, and

were co-transformed with plasmid p426-SNR52p-gRNA.YGLCτ3-SUP4t-hphMX bearing a

chimeric guide RNA (gRNA) targeting the YGLCτ3 locus. The expressed gRNA guides the

Cas9 to the YGLCτ3 locus where the endonuclease causes a double strand break. The

expression cassettes amplified with above-mentioned primer combinations create

flanking homologous sequences of ~40 bp to each other and ~60 bp to the upstream and

downstream chromosomal location. Upon transformation these expression cassettes

repair the double strand break by homologous recombination. The plasmids p414-

TEF1p-Cas9-CYC1t-nat1 and p426-SNR52p-gRNA.YGLCτ3-SUP4t-hphMX were

afterwards removed from the strain via the serial dilution method (described 3.2.3).

At this time point, the strain contains deletion of GUT1, DAK1 overexpression cassette at

YPRCΔ15 and EcmgsA, EcgldA, and CjFPS1 expression cassettes at YGLCτ3. The strain was

afterwards transformed with the CEN/ARS plasmid carrying the EcyqhD and Opgdh

expression cassettes in order to complete Modules 1,2-PDO and DHA. The resultant strain

was named PDO-FPS-DHA (Fig. 13).

RESULTS | CHAPTER 4

60

YPRCΔ15::PACT1-DAK1-TTPS1

CBS 6412-13A gut1∆-DAKoe


YPRCΔ15::PGAL-GIN11M86:ble

CBS 6412-13A gut1∆

gut1∆::loxP-kanMX4-loxP

+ p414-TEF1p-Cas9-CYC1t-nat1

Figure 13 Construction of PDO-FPS-DHA strain. The endogenous GUT1 gene was deleted by integrating

kanMX4 expression cassette in CBS 6412-13A resulting in CBS 6412-13A gut1∆ strain. This strain was

further used to integrate the GIN11M86-ble at YPRCΔ15 which was prerequisite for seamless integration of

DAK1 expression cassette. The resultant strain after DAK1 integration was named CBS 6412-13A gut1∆-

DAK1oe and was used to integrate the expression cassettes for EcmgsA, EcgldA, and CjFPS1 via

CRISPR-Cas9-mediated genome editing system at YGLCτ3 locus. The Cas9 and guideRNA plasmids used in

the CRISPR-Cas9 system were removed from the strains and the resulting strain was transformed with the

low-copy plasmid carrying EcyqhD and Opgdh (all plasmids used are shown in circles).

CBS 6412-13A gut1∆-GIN11M86-ble

CBS 6412-13A gut1∆-DAKoe-EcmgsA-EcgldA-CjFPS1-cas9-gRNA

Cas9 and gRNA plasmid removal

YGLCτ3:: PTEF1-mgsA-TCYC1: PTDH3-gldA-TIDP1: PPGK1-CjFPS1-TRPL15A

CBS 6412-13A gut1∆-DAKoe

+ p426-SNR52p-gRNA.YGLCτ3-SUP4t-hphMX

+ PACT1-yqhD-TTPS1

PTEF1-gdh-TCYC1

CBS 6412-13A gut1∆-DAKoe-EcmgsA-EcgldA-CjFPS1

PDO-FPS-DHA

CR

ISP

R-C

as9

-med

iate

d

gen

om

e ed

itin

g sy

stem

RESULTS | CHAPTER 4

61

4.2.2.4 Construction of strain PDO-FPS-Tpi1pdown-DHA: combining 1,2-PDO production with increased DHAP supply and NADH availability

The motivation to construct the next strain was to combine the 1,2-PDO production

(Module 1,2-PDO) in the CjFPS1 expression background with both an increased precursor

supply (Module Tpi1pdown) and an increased NADH availability via DHA pathway (Module

DHA). For this purpose, the strain PDO-FPS-Tpi1pdown (i.e. the strain that contains EcmgsA,

EcgldA, CjFPS1 at YGLCτ3 locus and TPI1 promoter replaced by the TEFmut2 promoter

was used to delete the GUT1 gene and overexpress DAK1 by the same strategy as

described in the previous section (4.2.2.3). The resulting strain was then transformed

with the low-copy plasmid that carries EcyqhD and Opgdh expression cassettes required

for the completion of Module 1,2-PDO and Module DHA, respectively (Fig. 14).

RESULTS | CHAPTER 4

62

The genotypes of the final strains constructed in sections 4.2.1.1 to 4.2.1.4 and the

isogenic reference strain FPS (constructed in section 4.1.2) are presented in Table 11.

PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe

PDOmin-FPS-Tpi1pdown

YPRCΔ15::PGAL-GIN11M86:ble

gut1∆::loxP-kanMX4-loxP

YPRCΔ15::PACT1-DAK1-TTPS1

+ PACT1-yqhD-TTPS1

PTEF1-gdh-TCYC1

PDO-FPS-Tpi1pdown-DHA

PDOmin-FPS-Tpi1pdown gut1∆

PDOmin-FPS-Tpi1pdown gut1∆-GIN11M86-ble

Figure 14 Construction of PDO-FPS-Tpi1pdown-DHA strain. First, the GUT1 gene was deleted in

PDOmin-FPS-Tpi1pdown strain by integration of kanMX4 expression cassette resulting in PDOmin-FPS-

Tpi1pdown-gut1∆ strain. This strain was further used to integrate the GIN11M86-ble at YPRCΔ15 which was

aimed at integration of DAK1 expression cassette. The resultant strain after DAK1 integration was named

PDOmin-FPS-Tpi1pdown-gut1∆-DAK1oe, and was transformed with the low-copy plasmid carrying EcyqhD

and Opgdh (all expression cassettes that were introduced via a plasmid are shown inside a circle).

RESULTS | CHAPTER 4

63

Ta

ble

11

. Sum

mary

of th

e mo

st imp

orta

nt ge

netically

engin

eered C

BS

64

12

-13

A d

erivativ

es for a

naly

zing th

e effects o

f the su

ggested

metab

olic en

gineerin

g

Mo

du

les aimin

g at 1,2

-PD

O p

rod

uctio

n u

sing

glycero

l as carbo

n so

urce

. Th

e FP

S strain w

itho

ut 1

,2-P

DO

path

way

was u

sed as a referen

ce.

RESULTS | CHAPTER 4

64

4.3 Effect of the cultivation medium on 1,2-PDO production

At several steps of the strain construction procedure, strains were tested for 1,2-PDO

production in a commonly used synthetic medium according to Verduyn et al. (1992).

This medium was supplemented with 6% (v/v) glycerol as the sole carbon source and

adjusted to pH 4. The pH 4 was routinely used in our lab for glycerol growth

characterization because it allowed the best growth of the CBS 6412-13A wild-type

(baseline) strain as described in the section 4.1. However, even the advanced strains did

not produce significant amounts of 1,2-PDO in this medium (e.g. less than 0.2 g/L for

strain PDO-FPS-Tpi1pdown-DHA; data not shown). The strain was also tested in synthetic

medium with an initial pH of 6. This led to the generation of slightly increased biomass

(final OD600nm reached to ~7 compared to ~5 at pH 4), and the 1,2-PDO titer was ~0.5 g/L

(data not shown). Nevertheless, the question arose whether the strain PDO-FPS-

Tpi1pdown-DHA should be tested in a different medium. In fact, the strain were not able to

grow to cell densities beyond OD600nm ~7 in the synthetic medium containing (NH4)2SO4

as a nitrogen source due to rapid drop of pH in culture medium (Hensing et al., 1995).

To overcome the problem of pH drop, the nitrogen source in the synthetic medium (6%

(v/v) glycerol) was switched from ammonium sulfate ((NH4)2SO4) to urea (Hensing et al.,

1995) (3.1.6). The resulting medium was labelled as synthetic + urea. Although a very

high optical density (OD600nm ~24 after 144 hours) was achieved in the latter medium

with strain PDO-FPS-Tpi1pdown-DHA, the 1,2-PDO production was still very low (below

0.2 g/L). The data presented in Fig. 15 also show that glycerol was completely consumed

in this medium after 72 hours and almost ~18 g/L of ethanol was detectable at the same

time point. After 72 hours, the ethanol gradually disappeared from the medium, either by

evaporation or by its use as a carbon source. It has to be noted that no ethanol was

detectable in the medium with (NH4)2SO4 at all.

Jung et al. (2011) measured up to 0.9 g/l 1,2-PDO when analyzing engineered

S. cerevisiae in complex medium, i.e. in YPG (1% yeast extract, 2% peptone, and 1%

glycerol). Although the strain used in their study contained only EcmgsA and EcgldA and

no DHA pathway or Tpi1p down–regulation, it still produced much more 1,2-PDO

compared to the more advanced strain analyzed in the current study in synthetic medium.

Therefore, the performance of strain PDO-FPS-Tpi1pdown-DHA was next analyzed in YPG

medium. Notably, Jung et al. (2011) used 1% glycerol in YPG. In the current study, 6%

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65

glycerol was used due to the fact that this concentration was used before in chapter 4.1 in

synthetic medium. In YPG with 6% glycerol, the strain PDO-FPS-Tpi1pdown-DHA grew to

high densities (OD600nm ~18 after 144 hours) although to a lower level than in synthetic +

urea (OD600nm ~24 after 144 hours). However, 1,2-PDO production was only slightly

improved (to ~0.3 g/L) compared to in synthetic + urea (less than ∼0.2 g/L). The time

course data shows remarkably strong production of ethanol reaching concentrations

reaching up to ~17g/L which was almost comparable to the ethanol production in

synthetic + urea (∼18 g/L), that was disappeared with the passage of time and hence no

ethanol was detected after 144 hours. Notably, the glycerol consumption was comparable

to that observed in synthetic medium with urea as the nitrogen source.

The next step was to investigate the impact of the concentrations of the two ingredients

of YPG medium i.e. yeast extract and peptone on 1,2-PDO production. First, peptone was

completely excluded from YPG medium; this medium was called YG. In another

experiment, the peptone amount was doubled from 2% to 4% resulting in YP(4%)G

medium. The strain was grown in both media and 1,2-PDO production was analyzed.

Surprisingly, a remarkable improvement in 1,2-PDO production, compared to all

previously tested media, was observed in the complete absence of peptone. In fact, a titer

~1.3 g/L was obtained after 144 hours of cultivation. In contrast, the concentration of 1,2-

PDO in YP(4%)G at the same point in time was below 0.2 g/L. The results suggest that the

presence of one or more components present in peptone abolish 1,2-PDO production. The

OD600nm in YG medium reached to ~18 which was almost the same as in YPG but lower

than the one achieved in synthetic + urea (OD600nm ~24). The glycerol consumption rate

seemed to be lower than in YPG since ~16 g/L of glycerol was still left in the cultures after

144 hours. In YP(4%)G medium the OD600nm reached to ~23 and glycerol was completely

consumed after 144 hours. Interestingly, the overall ethanol formation was lowest in YG

medium compared to all the tested media. After 72 hours of cultivation ~6 g/L ethanol

was detected while ~16 g/L were measured in YP(4%)G at the same point in time.

To sum up the results obtained with the different media so far, it was proven that the

exclusion of peptone from complex YPG medium has a positive impact on 1,2-PDO

production. Highest product concentration (~1.3 g/L) in the strain under investigation.

was attained when only 1% yeast extract with 6% glycerol was used (YG medium). The

next step was to examine the effect of increased yeast extract in the best YG medium, for

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66

this reason the concentration of yeast extract was increased from 1% to 2% and was

called Y(2%)G medium. It was observed that the increased yeast extract enhanced growth

compared to YG medium, but the 1,2-PDO production stopped at around ∼0.8 g/L at 48

hours and did not increase further. Ethanol concentration reached to ∼13 g/L after 72

hours and decreased after this point in time. After 144 hours, only ∼0.8 g/L of glycerol

could still be detected in the medium.

To conclude, the YG medium proved to be the best among all tested media with regard to

1,2-PDO production but by far the worst when considering the rates of biomass

production and glycerol consumption.

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67

A B

C D

Figure 15 Impact of growth medium on 1,2-PDO production of one selected S. cerevisiae strain

engineered in the current study. Characterization of the strain PDO-FPS-TPI1down-DHA in 500 mL

Erlenmeyer flasks containing 50 mL of one of the following media: synthetic + urea ( ), YPG ( ), YP(4%)G

( ), YG ( ), and Y(2%)G ( ). Each medium contained 6% (v/v) glycerol as a carbon source at pH 6.

A; 1,2-PDO (g/L), B; OD600nm, C; glycerol concentration (g/L), D; ethanol (g/L). Each curve is the average of

three independent biological replicates.

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68

4.4 Effects of the different metabolic engineering modules on physiology and 1,2-PDO production of the strains in YG medium

Among the different cultivation media tested in chapter 4.3, YG medium (1% yeast extract,

6% (v/v) glycerol) resulted in the highest 1,2-PDO titer in the strain PDO-FPS-Tpi1pdown-

DHA. For this reason, YG medium was used in the following chapters for elucidating the

effects of the different individual or combined metabolic engineering modules on the

production of 1,2-PDO and biomass, consumption of glycerol as well as the production of

relevant fermentation by-products such as acetol and ethanol.

The implementation of the heterologous 1,2-PDO pathway in combination with the glycerol facilitator only resulted in the production of trace amounts of 1,2-PDO

The strain PDO-FPS (EcmgsA, EcyqhD, EcgldA, and CjFPS1) and the corresponding isogenic

reference strain FPS (wild type CBS 6412-13A containing CjFPS1) were grown in YG

medium and the supernatants were analyzed by HPLC as described in section 3.2.4. After

144 hours of cultivation, the strain PDO-FPS produced ~0.08 g/L of 1,2-PDO while it

consumed ~30.07 g/L of glycerol and reached an OD600nm of ~11 (in Fig. 16). A very low

amount of ethanol (~0.13 g/L) was detected. When the same time point is considered,

interestingly, the reference strain FPS consumed much less (~15.91 g/L) of glycerol,

reached a lower OD600nm of ~7 while neither 1,2-PDO nor ethanol could be detected. When

comparing the results obtained with the two strains, one can conclude that the

incorporation of the heterologous 1,2-PDO pathway obviously increased the growth of

the strain resulting in a faster consumption of glycerol.

It has to be mentioned that the 1,2-PDO concentration detected for strain

PDO-FPS is at the lower end of the standard curve and should be considered with care.

Still, the 1,2-PDO peak was clearly identifiable when compared to the chromatogram

obtained for the reference strain FPS.

Tpi1p down-regulation in the strain PDO-FPS slightly increased the 1,2-PDO production

To see the effect of the anticipated increased precursor (DHAP) supply on

1,2-PDO production, the strain PDO-FPS-Tpi1pdown (4.2.2.2) was analyzed next and the

results were compared to those obtained with the strain PDO-FPS. The latter strain was

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69

characterized in the previous chapter (4.4.1). The strain PDO-FPS-TPI1down was grown in

YG medium in shake flasks. Besides measuring OD600nm, samples of supernatant were

collected in intervals of 24 hours, prepared and analyzed as described in 3.2.4. The

1,2-PDO concentration increased up to 48 hours and stayed almost constant afterwards.

After 144 hours, ~0.17 g/L of 1,2-PDO was produced and ~38.39 g/L of glycerol

consumed. The OD600nm reached to ~12. The time-courses shown in Fig. 16 reveal that

some ethanol (~0.29 g/L) could be detected after 48 hours that disappeared later.

One can conclude from the results that the down-regulation of Tpi1p doubled the amount

of produced 1,2-PDO (∼0.17 g/L versus ∼0.08 g/L) when compared to the strain PDO-

FPS after 144 of cultivation. Interestingly, the down-regulation of Tpi1p did not result in

impairment of growth or glycerol-consumption capability compared to PDO-FPS strain.

The strain PDO-FPS-Tpi1pdown even consumed slightly more glycerol. At 48 hours, ~0.29

g/L ethanol was detected in the supernatant of PDO-FPS-Tpi1pdown, compared to ~0.09

g/L for PDO-FPS. After 144 hours, no ethanol was detected in the former strain, but there

was still ~0.13 g/L left in the supernatant with regard to the latter strain. There was only

a slight difference regarding the final OD600nm of both strains after 144 hours; the strain

with Tpi1p down-regulation showed an OD600nm of ~12 while the PDO-FPS strain reached

an OD600nm of ~11.

Replacement of the native FAD-dependent glycerol catabolic pathway (G3P) by the NAD+-dependent pathway (DHA) in PDO-FPS strain slightly enhanced 1,2-PDO production

The objective of replacing the G3P pathway with DHA pathway (Module DHA) in PDO-FPS

strain was to provide cytosolic NADH that is needed for 1,2-PDO production. It should be

noted that this strategy was first tested in strain PDO-FPS, i.e. without Tpi1p down-

regulation, in order to be later able to dissect the individual impacts of the different

modules on 1,2-PDO production. By adding all genetic modifications for Module DHA

(described in 4.2.2.3) to strain PDO-FPS, the strain PDO-FPS-DHA was generated.

Upon growing this strain in YG medium, it produced ~0.11 g/L of 1,2-PDO, when

measured after 144 hours of cultivation. Surprisingly, most of the glycerol was consumed

(leaving only ~0.52 g/L out of ~74.44 g/L of total glycerol) in the presence of the DHA

pathway and a much higher OD600nm (~22) was obtained after 144 hours. Both the values

for the consumed glycerol and the OD600nm achieved were roughly two-fold higher

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70

compared to the strains PDO-FPS and PDO-FPS-Tpi1pdown. Another remarkable effect of

the presence of the DHA pathway was a much higher production of ethanol compared to

all strains analyzed so far. As shown in Fig. 16, more than 2 g/L of ethanol were detected

between 24-96 hours of cultivation with the highest concentration of ∼4.8 g/L being

detected at 72 hours. Notably, ethanol concentration detected for the strains PDO-FPS,

PDO-FPS-Tpi1pdown and FPS did never exceed 0.3 g/L. Another exceptional characteristic

of strain PDO-FPS-DHA is its capability of consuming almost all glycerol in the medium.

In this context, it was surprising to realize that the1,2-PDO production (∼0.11 g/L) was

only slightly higher compared to strain PDO-FPS (∼0.08 g/L) and still lower than in strain

PDO-FPS-Tpi1pdown (∼0.17 g/L).

The combination of Tpi1p down-regulation and glycerol catabolic pathway replacement remarkably increased the 1,2-PDO production

In sections 4.4.2 and 4.4.3, the individual impacts of both Tpi1p down-regulation (Module

Tpi1pdown) and enhanced NADH availability (Module DHA) has been investigated on 1,2-

PDO production (Module 1,2-PDO). It is evident from the results that there was only a

very low improvement with regard to 1,2-PDO production by implementing the

respective modules individually. To find out whether there is a synergistic effect of

combining Module Tpi1pdown and Module DHA on 1,2-PDO production, strain PDO-FPS-

Tpi1pdown-DHA (4.2.2) was tested in this chapter. Fig. 16 shows that the combination

indeed resulted in a dramatic increase in 1,2-PDO production. The respective

concentration reached ~1.3 g/L after 144 hours. Moreover, the time course reveals that

1,2-PDO production continued to increase until it stopped at 120 hours and later

remained steady. The OD600nm obtained with this strain (~14) was lower than obtained

with the strain PDO-FPS-DHA and less (~57.57 g/L) glycerol was consumed after 144

hours. Interestingly, even more ethanol (~5.72 g/L) compared to the strain PDO-FPS-DHA

of was detected.

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71

A B

C D

Figure 16 Physiological characterization of S. cerevisiae strains (CBS 6412-13A derivatives) engineered for 1,2-PDO production from glycerol. The strains PDO-FPS ( ), PDO-FPS-Tpi1down ( ), PDO-FPS-DHA ( ) and PDO-FPS-Tpi1down-DHA ( ) strains were analyzed along with the reference FPS ( ) strain for A; 1,2-PDO (g/L), B; OD600nm, C; glycerol concentration (g/L), and D; ethanol (g/L) formation. Experiments were conducted in 500 mL Erlenmeyer flasks containing 50 mL of YG medium (1% yeast extract, 6%(v/v) glycerol). Cells were grown at 30°C and with 200 rpm shaking frequency. The samples were taken every 24 hours over a period of 144 hours. After OD600nm

measurement, the supernatants were analyzed by HPLC. Each curve is the average of three independent biological replicates.

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72

4.5 Effects of a second copy of the expression cassettes for EcmgsA and/or Opgdh encoding for enzymes operating in crucial steps of 1,2-PDO pathway

As mentioned above, it has been assumed that the Tpi1p down-regulation (Module

Tpi1pdown) ensures an enhanced carbon flux into the 1,2-PDO pathway, while the DHA

pathway (Module DHA) is helpful in supplying the cofactor NADH. Although the individual

impacts of these modules in enhancing the 1,2-PDO production in S. cerevisiae were

marginal, their combination made a momentous difference and a total of ~1.3 g/L of 1,2-

PDO was produced by the respective strain. Still, for commercial applications, there is a

need to explore avenues for further enhancing the 1,2-PDO titer. It was assumed that one

such possibility is to overexpress the major 1,2-PDO pathway genes which encode for

rate-controlling enzymes for converting DHAP to 1,2-PDO (Fig. 9). It was therefore

decided to introduce a second copy of the gene EcmgsA encoding the methylglyoxal

synthase and the gene Opgdh 14 into the strain PDO-FPS-Tpi1pdown-DHA alone and in

combination. The following chapters will consider the strain constructions (4.5.1) and

their physiological characterizations (4.5.2).

Genomic integration of a second copy of EcmgsA and Opgdh in strain PDO-FPS-Tpi1pdown-DHA

The PDO-FPS-Tpi1pdown-DHA strain contains the Modules 1,2-PDO, Tpi1pdown and DHA as

described in chapter 4.2.1.4. The second copies of EcmgsA and Opgdh were integrated in

the chromosome XVI of this strain at the YPRCτ3 genomic location. In total, three

derivatives of the strain PDO-FPS-Tpi1pdown-DHA were constructed. One strain contained

a second copy of PTDH3-mgsA-TIDP1 (PDO-FPS-Tpi1pdown-DHA-mgsA), another strain

contained a second copy of PTEF1-gdh-TCYC1 (PDO-FPS-Tpi1pdown-DHA-gdh), while a third

strain carried second copies of both expression cassettes (PDO-FPS-Tpi1pdown-DHA-

mgsA-gdh).

To achieve the respective seamless integrations, the GIN11 system was used. First, the

GIN11 cassette with ble as a marker (3.2.3) was integrated via homologous recombination

at YPRCτ3 locus in the strain PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe. This gave rise to the

16 Preliminary experiments indicate that Opgdh can perform the same function as EcgldA i.e. it can reduce the concentration of the metabolites that is assumed to be acetol and convert it to 1,2-PDO (data not shown).

RESULTS | CHAPTER 4

73

strain PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe with GALp-GIN11M86/ble. This strain was

used to create all the above-described three derivatives and the details are described in

the following paragraph.

In the first step, the strain with only the second copy of EcmgsA and the strain with the

combined second copies of EcmgsA and Opgdh were created. Both the strains required a

common intermediate strain where the expression cassettes PTDH3-mgsA-TIDP1 (from

pUC18-mgsA) and PTEF1-gdh-TCYC1 (p41bleTEF-Opgdh) (Table 6) were integrated by

amplifying them with primer pairs 417/422 and 421/416 (Table 7), respectively. The

common intermediate strain was named PDOmin-FPS-Tpi1pdown-DHA-EcmgsA-Opgdh.

When this strain was transformed with the low-copy plasmid that carries the EcyqhD

expression cassette, the strain PDO-FPS-Tpi1pdown-DHA-mgsA was generated and

represents a strain with two copies of EcmgsA. In parallel, the common intermediate

strain was transformed with the low-copy plasmid that carries the expression cassettes

for EcyqhD and Opgdh. That resulted in a strain with two copies of EcmgsA and Opgdh each

(PDO-FPS-Tpi1pdown-DHA-mgsA-gdh).

In order to construct a corresponding strain with only a second copy of Opgdh

(PDO-FPS-Tpi1pdown-DHA-gdh), the Opgdh expression cassette was first amplified from

p41bleTEF-Pagdh (Table 6) with primers 416/417 (Table 7) and integrated at the

YPRCτ3 locus of the strain PDOmin-FPS-Tpi1pdown-gut1∆-DAKoe already containing

GALp-GIN11M86/ble at YPRCτ3 position (the same intermediate strain that was used for

the integration of second copy of EcmgsA with and without Opgdh). Afterwards the

resultant strain was transformed with the low-copy plasmid that carries PACT1-yqhD-TTPS1

and PTEF1-gdh-TCYC1 expression cassettes that are the prerequisites for completion of

Module 1,2-PDO and Module DHA, respectively.

A summary of the strains created for elucidation of the individual and combined effects of

EcmgsA and Opgdh on 1,2-PDO production in PDO-FPS-Tpi1pdown-DHA strain, is given

in Fig. 17, and their detailed genotypes are listed in Table 12.

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Figure 17 Construction of strain PDO-FPS-Tpi1pdown-DHA with second copies of Opgdh and/or

EcmgsA. As a first step, the GIN11M86 inhibitory sequence and ble expression cassette were integrated via

homologous recombination at the YPRCτ3 locus in the strain PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe. In the

second step, the Opgdh with and without EcmgsA expression cassettes were integrated by homologous

recombination at the same position thereby replacing the GIN11M86 inhibitory sequence and ble

expression cassette. In the third step, the resultant strains were transformed with the low-copy plasmid

carrying EcyqhD and Opgdh expression cassettes. The strain where only an extra copy of EcmgsA was

needed (PDO-FPS-Tpi1pdown-DHA-mgsA), the strain was generated by transformation with the low- copy

plasmid carrying only EcyqhD. Expression cassettes that were brought in by a plasmid are framed by a circle.

YPRCτ3:: PTDH3-mgsA-TIDP1

PTEF1-gdh-TCYC1

PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe

PDOmin-FPS-Tpi1pdown gut1∆-DAK1oe-GIN11-ble

YPRCτ3::PGAL-GIN11M86:ble

YPRCτ3::PTEF1-gdh-TCYC1

PDO-FPS-Tpi1pdown-DHA-gdh

+ PACT1-yqhD-TTPS1 PTEF1-gdh-TCYC1

PDOmin-FPS-Tpi1pdown-DHAmin- -Opgdh

PDOmin-FPS-Tpi1pdown-DHA- EcmgsA-Opgdh

+ PACT1-yqhD-TTPS1 PTEF1-gdh-TCYC1

PDO-FPS-Tpi1pdown-DHA-mgsA-gdh

+

PACT1-yqhD-TTPS1

PDO-FPS-Tpi1pdown-DHA-mgsA

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Table 12. Summary of the constructed derivatives of strain PDO-FPS-Tpi1pdown-DHA for elucidation of the effects of overexpressing enzymes (encoded by EcmgsA and Opgdh) that operate in crucial steps of the 1,2-PDO pathway. Strain abbreviations: PDO-FPS-Tpi1pdown-DHA-mgsA; strain with the second copy of EcmgsA, PDO-FPS-Tpi1pdown-DHA-gdh; strain with the second copy of Opgdh, and PDO-FPS-Tpi1pdown-DHA-mgsA-gdh; strain with combined second copy of EcmgsA and Opgdh.

Characterization of PDO-FPS-Tpi1pdown-DHA derivatives with a second copy of EcmgsA and/or Opgdh

The three constructed strains that are listed in Table 12, were grown, samples were

collected every 24 hours over a total period of 144 hours, prepared and analysed in HPLC

(3.2.4). The time courses for 1,2-PDO and ethanol production, biomass formation

(OD600nm), and glycerol consumption of aforementioned strains is given in Fig. 18.

The results show that the strain containing a second copy of EcmgsA and Opgdh

(PDO-FPS-Tpi1pdown-DHA-mgsA-gdh) produced the highest 1,2-PDO titer ever achieved

from glycerol in S. cerevisiae (~3.68 g/L after 144 hours). Notably, the strain that

contained only a second copy of EcmgsA (PDO-FPS-Tpi1pdown-DHA-mgsA) was even able

to produce ~3.49 g/L of 1,2-PDO. In contrast, the second copy of Opgdh did not have such

a remarkable improving effect in 1,2-PDO production. In fact, the strain PDO-FPS-

Tpi1pdown-DHA-gdh only reached a titer of ~1.58 g/L of 1,2-PDO.

The results clearly proved that the second copy of EcmgsA was crucial for enhancing the

1,2-PDO titer. Obviously, the second copy of the EcmgsA expression cassette led to a

# Strain name Genotype Plasmid (relevant expression

cassettes in bracket)

1 PDO-FPS-Tpi1pdown-DHA-mgsA YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1:PPGK1-CjFPS1-TRPL15A; gut1::loxP-KanMX-loxP; YPRCΔ15::PACT1-DAK1-TTPS1 ; YPRCτ3::PTDH3-mgsA-TIDP1:PTEF1-gdh-TCYC1

p41ble-E-yqhD

(PACT1-yqhD-TTPS1)

2 PDO-FPS-Tpi1pdown-DHA-gdh YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1:PPGK1-CjFPS1-TRPL15A ; gut1::loxP-KanMX-loxP; YPRCΔ15::PACT1-DAK1-TTPS1 ; YPRCτ3::PTEF1-gdh-TCYC1

p41ble-gdh-yqhD

( PACT1-yqhD-TTPS1, PTEF1-gdh-TCYC1)

3 PDO-FPS-Tpi1pdown-DHA-mgsA-gdh YGLCτ3::PTEF1-mgsA-TCYC1:PTDH3-gldA-TIDP1:PPGK1-CjFPS1-TRPL15A; gut1::loxP-KanMX-loxP; YPRCΔ15::PACT1-DAK1-TTPS1 ; YPRCτ3::PTDH3-mgsA-TIDP1:PTEF1-gdh-TCYC1

p41ble-gdh-yqhD

( PACT1-yqhD-TTPS1, PTEF1-gdh-TCYC1)

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76

higher activity of methylglyoxal synthase which is the first enzyme of the 1,2-PDO

pathway at the junction of DHAP and thus to an increased carbon flux towards 1,2-PDO

formation. The second copy of Opgdh has a much lower positive impact on 1,2-PDO

production, even in combination with the second copy of EcmgsA. The ethanol production

in the strain with the two copies of EcmgsA and Opgdh was lower than in the strains where

only one of the two genes was increased in copy number. The level of consumed glycerol

after 144 hours was ~48.12 g/L, ~54.18 g/L, and ~37.99 g/L for the strains with the

second copy of EcmgsA, the second copy of Opgdh, and the second copy of EcmgsA in

combination with second copy of Opgdh, respectively. The respective OD600nm–levels for

the three strains were ∼13.74, ∼13.87, and ~17.77. It is obvious that, among the three

strains, the strain with the second copy of EcmgsA in combination with the second copy

of Opgdh grew to the highest cell density. This result indicates again that an enhanced flux

to 1,2-PDO seems to have a positive impact on growth.

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A B

C D

Figure 18 Physiological characterization of PDO-FPS-Tpi1pdown-DHA derivatives expressing a

second copy of EcmgsA and/or Opgdh, i.e. genes that encode for enzymes operating in crucial steps

of the 1,2-PDO pathway. Time courses of A; 1,2-PDO (g/L), B; OD600nm, C; glycerol concentration (g/L),

and D; ethanol (g/L) of strains PDO-FPS-TPI1down-DHA-mgsA ( ), PDO-FPS-TPI1down-DHA-gdh ( ),

and PDO-FPS-TPI1down-DHA-mgsA-gdh ( ) in 500 mL Erlenmeyer flasks containing 50 mL of YG (1%

yeast extract, 6% glycerol) at initial pH of 6 grown at 30°C and 200 rpm shaking frequency. The samples

were taken every 24 hours over a period of 144 hours. Each curve is the average of three independent

biological replicates.

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78

4.6 A so far unknown metabolite with the retention time of acetol accumulates in the engineered strains

The current study sought to base the 1,2-PDO pathway engineering strategies on the route

with acetol as the intermediate (Fig. 9). For efficient conversion of MG to acetol, a

heterologous aldehyde reductase (encoded by EcyqhD) was expressed. For the

conversion of acetol to 1,2-PDO another heterologous glycerol dehydrogenase (encoded

by EcgldA or Opgdh) was used in this study, even though it has to be noted that all these

enzymes do not have a very high substrate specificity and a participation in a reaction

other than the designated one.

In principle, the accumulation of acetol can be anticipated if the flux from acetol to 1,2-

PDO was is limited. An additional peak was detected when samples of the advanced

engineered strains were analyzed by HPLC. By using an acetol standard solution, it was

visible that the accumulating metabolite has the same retention time as acetol. The

identification of the peak’s identity still has to be conducted in the future. It has to be

mentioned that, a small peak of the same retention time as acetol was also detected in the

supernatant of FPS (reference strain) strain when grown in YG medium. The respective

peak area corresponds to ∼0.250 g/L of acetol. However, the peak area remained steady

throughout 144 hours of cultivation.

The time-courses of the concentration of the acetol-like metabolite were recorded for all

the strains engineered in this study and the ∼0.250 g/L were subtracted to compensate

for the false-positive peak that was detected in the reference strain. A summary of the

time courses of the acetol production is given in the Fig. 19. It is obvious that a significant

amount (∼0.27 g/L) of the acetol-like metabolite was observed after 144 hours with the

strain where modules 1,2-PDO, DHA and Tpi1p down-regulation were combined (PDO-

FPS-DHA-Tpi1pdown). The strain with an extra copy of EcmgsA (FPS-PDO-DHA-Tpi1pdown-

mgsA) even produced∼0.40 g/L, while the strain with second copy of EcmgsA in

combination with Opgdh (PDO-FPS-DHA-Tpi1pdown-mgsA-gdh) produced ∼0.38 g/L.

RESULTS | CHAPTER 4

79

Figure 19 Time courses of the concentration of the acetol-like metabolite in the genetically engineered S. cerevisiae strains generated for 1,2-PDO production from glycerol in this study. of the strains PDO-FPS ( ), PDO-FPS-Tpi1pdown( ), PDO-FPS-DHA( ), PDO-FPS-DHA-Tpi1pdown- ( ), PDO-FPS-Tpi1pdown-DHA-mgsA( ), PDO-FPS-Tpi1pdown-DHA-gdh( ), and PDO-FPS-Tpi1pdown-DHA-gdh-mgsA( ) were cultivated in 500 mL Erlenmeyer flasks containing 50 mL of YG (1% yeast extract, 6% glycerol) at initial pH of 6 grown at 30°C and 200 rpm shaking frequency. The strain FPS ( ) was used a s a reference strain. The samples were taken every 24 hours over a period of 144 hours. Each curve is the average of three independent biological replicates.

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RESULTS | CHAPTER 4

80

Discussion & future prospects | Chapter 5

DISCUSSION & FUTURE PROSPECTS | CHAPTER 5

81

5.1 Discussion

Possible bottlenecks that hinder the efficient glycerol utilization in S. cerevisiae

It has been shown in chapter 4.1 that the glycerol growth performance of

S. cerevisiae CBS 6412-13A has been improved from μmax ∼0.13 h-1 to μmax ∼0.18 h-1 by the

heterologous expression of Fps1 homologues either from C. jadinii DSM 2361, K. pastoris

X-33, P. tannophilus CBS 4044 or Y. lipolytica IBT 446. This clearly shows that all tested

heterologous facilitators allowed a more efficient glycerol import in this strain. The

results also imply that glycerol transport was a bottleneck for glycerol utilization in wild-

type CBS 6412-13A. The expression of the Fps1 homologue not only improved the

glycerol growth performance but was surprisingly also able to replace the function of

Stl1p in the stl1∆ deletion mutant of strain CBS 6412-13A (Klein et al., 2016 b). It would

be certainly interesting to investigate the native role of CjFPS1 in glycerol growth

performance of C. jadinii DSM 2361. This can be investigated by the deletion of CjFPS1 in

C. jadinii DSM 2361 and growing the mutant strain in synthetic medium with glycerol.

Notably, there was no substantial difference in the glycerol growth improvement of CBS

6412-13A expressing any of the four tested Fps1p homologous regardless of their origin.

For instance, it was observed that there was almost an equal improvement of growth rates

(μmax ∼0.13 h-1 to μmax ∼0.18 h-1) and reduction of lag phases (from ∼17 hours to ∼9

hours) for CBS 6412-13A that express either CjFPS1, KpFPS1, PtFPS2 or YlFPS1. It seems

that, in term of glycerol transportation across the cell membrane, this was the only

possible improvement by heterologous expression of Fps1p homologues. Moreover, it

was also even found out that the expression of a second copy of CjFPS1 in CBS 6412-13A

could not improve further its glycerol growth performance (data not shown). It seems

that the bottlenecks for further improvement of glycerol utilization capacity and, hence

growth rate lie in the internal cellular metabolism of glycerol. An example of the change

in internal glycerol metabolism and its impact on glycerol growth performance can be

seen in this study. Although this study is carried out in YG and not in synthetic medium, it

can be still noticed from the result (Fig. 16), that a significant improvement in growth on

glycerol was observed in the PDO-FPS-DHA strain as soon the native FAD-dependent

glycerol catabolic pathway (G3P) was replaced by the NAD+-dependent pathway (DHA).


82

The PDO-FPS strain contain the PDO pathway also, but it has far less additional effect on

the glycerol growth of FPS strain.

As shown in chapter 4.1, the glycerol growth performance of CBS 6412-13A in synthetic

medium was improved by the expression CjFPS1, KpFPS1, PtFPS2 or YlFPS1. The CBS

6412-13A with CjFPS1 (strain FPS) was used for implementation of 1,2-PDO pathway.

However, the 1,2-PDO pathway in FPS strain could not produce1,2-PDO in the synthetic

medium and therefore complex YG medium was adopted. The better glycerol growth

performance due to the heterologous expression of Fps1 homologue in engineered strains

for 1,2-PDO production, masked and was not clearly distinguishable due to the use of

complex medium (YG) i.e. all the strains with and without CjFPS1 have the growth rates

in the same range (0.20 h-1 to 0.22 h-1, results not shown). Although the effect of CjFPS1

expression is not visible due to use of complex medium, the heterologous expression of

this gene has never been excluded in genetic engineering strategies applied in this study

aiming at 1,2-PDO production. As the ultimate goal is to switch back to synthetic media

for 1,2-PDO production due to its cost effectiveness compared to complex medium in

biotech industry.

Potential limitations for further increasing 1,2-PDO production in the

generated S. cerevisiae strains

The individual impact of implementation of heterologous 1,2-PDO pathway or in

combination with either Tpi1p down-regulation or with DHA pathway in

CBS 6412-13A carrying CjFPS1 resulted in very low amounts of 1,2-PDO (below 0.2 g/L)

(chapter 4.6). It seems that the anticipated increased precursor (DHAP) to

1,2-PDO due to Tpi1p down-regulation or the potential NADH availability due to DHA

pathway is not sufficient enough to enhance 1,2-PDO production. The combination of all

three pathways resulted in significant concentration of 1,2-PDO (∼1.3 g/L). Several

potential bottlenecks for 1,2-PDO production beyond ∼1.3 g/L might exist in this strain.

For instance, the activity of the first crucial enzyme in the 1,2-PDO pathway,

methylglyoxal synthase that is encoded by EcmgsA, seems to be rate-controlling. This has

been already demonstrated by other authors (Lee and DaSilva, 2006), who showed that

the expression of three copies of EcmgsA gene resulted in more 1,2-PDO than achieved by

one or two copies of the same gene along with 2 or 3 copies of EcgldA. The rate-controlling


83

role of this enzyme was confirmed in the current work. In fact, the introduction of a

second copy of EcmgsA (4.5) into the strain PDO-FPS-Tpi1pdown-DHA increased 1,2-PDO

production more than two-fold (∼3.7 g/L compared to ∼1.3 g/L). It would therefore be

worth testing the expression of a third copy of this gene.

The enzymes of the 1,2-PDO pathway downstream of MG are not very well characterized

with regard to 1,2-PDO production. The oxidoreductases don’t have a high substrate

specificity and can catalyze more than one reaction (Jarboe 2011). In the current study,

the enzyme encoded by EcyqhD was used for the conversion of MG to acetol due to the

lowest Km value for methylglyoxal among different substrates tested in the respective

study (Lee et al., 2010). Still, no significant impact in terms of 1,2-PDO production was

recognized in our study even though the effect of expression of EcyqhD was tested in

several strain backgrounds (data not shown). Although the specific enzyme activity has

not been analyzed in this work, it is very likely that it is actively expressed since active

expression in S. cerevisiae has been already demonstrated by Soucaille et al. (2008). The

same coding sequence has been codon-optimized for S. cerevisiae in the current work and

Sanger sequencing confirmed the DNA sequence to be correct. The gene product of

EcyqhD has been demonstrated to have a relatively broad substrate specificity (Jarboe

2011). For example, substrates such as isobutyraldehyde are also reduced and the Km

values are even lower than the one for MG. It is therefore possible that the respective

enzyme rather reduces a substrate other than methylglyoxal in the cells. It is also more

likely that the native oxidoreductases activities that are unknown to us might be sufficient

enough to completely convert MG to either acetol or S/R-lactaldehyde, leaving behind no

MG for EcyqhD product. Another reason for the lacking positive impact of EcyqhD

expression on 1,2-PDO production might be the enzyme’s need for NADPH as a cofactor.

NADPH can, in principle, be supplied by the pentose phosphate pathway and the acetate

formation pathway in S. cerevisiae. It has been shown that these pathways are upregulated

in glucose medium when NADPH demand is increased by overexpression of a modified

2,3-butanediol dehydrogenase that catalyzes the reduction of acetoin (that is NADPH-

dependent) into 2,3-butanediol (Celton et al., 2012). The third enzyme in the 1,2-PDO

pathway encoded by EcgldA is also relatively unspecific with regard to its substrate. Other

authors have reported activity of this enzyme in converting MG to lactaldehyde (Lee et al.,

2016). It is therefore difficult to dissect the actual in vivo conversions catalyzed by this


84

enzyme and their contribution to 1,2-PDO formation. For instance, the possibility of

lactaldehyde accumulation due to the action of EcgldA encoded enzyme cannot be ruled

out due to the conversion of MG to lactaldehyde and inefficiency of the cell to convert

lactaldehyde to 1,2-PDO due to non-sufficient enzymatic activities. It has to be mentioned

that there is another enzyme 1,2-PDO-oxidoreductase (E. coli fucO) that has been used by

some authors for converting S-lactaldehyde to S-1,2-PDO (Cameron et al., 1999). It would

be therefore worth testing an expression of E. coli fucO and produce 1,2-PDO via

lactaldehyde (instead of acetol) as an intermediate (Fig. 2).

Besides the low efficiency of the 1,2-PDO pathway enzymes due to any of the above-

mentioned reasons, there is also possibility of upregulation of enzymes in ethanol

formation. The Tpi1p is downregulated in this report, however there is still a need to

further downregulate this enzyme to save DHAP for 1,2-PDO pathway. To conclude, it

might be necessary to further enhance 1,2-PDO pathway enzymes activities and/or

reduce the activities of enzymes involved in alcoholic fermentation.

Differences in the strain background and cultivation conditions might

have caused the comparably low 1,2-PDO production in the strain solely

expressing the heterologous 1,2-PDO pathway

It has previously been shown that the sole introduction of EcmgsA via a low-copy plasmid

led to the formation of 0.24 g/L of 1,2-PDO (Hoffman 1999). By the same authors, this

level was doubled (∼0.5 g/L) by the additional expression of EcgldA. These results were

obtained with glucose as carbon source in complex medium. Although, a different S.

cerevisiae strain was used, similar 1,2-PDO levels (∼0.48 g/L) were achieved in the

current work when EcmgsA and EcgldA were integrated into the genome of the strain CBS

6412-13A and the resulting strain was tested in glucose medium. However, the same

strain produced less than 0.2 g/L of 1,2-PDO when characterized in complex medium

containing glycerol as a carbon source (YG). This concentration is significantly lower than

the ∼0.9 g/L of 1,2-PDO reported by Jung et al. (2011) after testing a S. cerevisiae strain

expressing EcmgsA and EcgldA and growing the strain in YPG medium. Based on the data

obtained in the current study, one can conclude that the use of YPG did certainly not result

in the higher 1,2-PDO production in the study of Jung et al. (2011). However, the use of a

high-copy plasmid for EcmgsA and EcgldA expression is a very likely explanation for the

differences. A high-copy plasmid was also tested in the current work for expressing


85

EcmgsA, but a detrimental effect on growth was observed (synthetic medium was used,

data not shown). Still, there are two additional differences in the two studies under

consideration.

First, Jung et al. (2011) added 0.1 g/L of galactose to the cultivation medium for the

induction of GAL1/10 promoters that drove the expression of EcmgsA and EcgldA.

Although galactose is only present in very low concentrations and was only used for the

purpose of promoter induction, it can be used as a second carbon source besides glycerol

potentially contributing to 1,2-PDO production.

A last reason for the difference in 1,2-PDO production in the two studies could be caused

by the different strains used. The intra-species genetic diversity of S. cerevisiae should not

be underestimated and could also affect 1,2-PDO production of in the different strains

bearing the same genetic modifications. For example, Hoffman (1999) showed that

1,2-PDO production (from glucose as carbon source) via plasmid-based EcmgsA and

EcgldA significantly varied between different S. cerevisiae strains. The genetic background

could have affected expression levels of enzymes involved in the 1,2-PDO pathway.

Furthermore, in our study, a high enzymatic activity of EcmgsA encoded methylglyoxal

synthase was noted in S288c strain compared to CBS 6412-13A (data not shown) that

might also affect the 1,2-PDO production. These observations indicate that indeed there

is an intra-species diversity in S. cerevisiae in terms of extent of enzymes expression and

1,2-PDO production.

In chapter 4.3 different types of complex and synthetic media were evaluated for

enhanced 1,2-PDO production in the strain PDO-FPS-Tpi1down-DHA. Among them, the YG

medium (i.e. YPG without peptone) resulted in the highest 1,2-PDO production. Overall, it

seems that the cells do not reach the same high density in YG medium as compared to the

remaining media tested. It might be that the lower biomass formation correlates with

improved 1,2-PDO production. One explanation could be that there is a competition for

carbon flux between biomass formation and 1,2-PDO production. In case of low biomass

in YG medium compared to the remaining media, the carbon flux is probably directed

more towards 1,2-PDO pathway. The exact link between the restricted growth of cells and

enhanced 1,2-PDO production in YG medium is still unknown to us and needs further

investigations.


86

The replacement of the native FAD-dependent glycerol catabolic pathway with an NAD+-dependent pathway enhances ethanol formation

Glycerol is known as a respiratory carbon source in S. cerevisiae, and no ethanol formation

has been detected in synthetic medium containing ammonium as nitrogen source and

glycerol as sole carbon source (Klein et al., 2016 b). In this study, the use of synthetic

medium containing urea as nitrogen source and complex medium with glycerol as carbon

source however initiated a slight respiro-fermentative mode of glycerol metabolism, i.e. a

small part of the carbon was metabolized in a fermentative manner as indicated by

ethanol formation. In YG medium even the reference strain FPS produced roughly ∼0.3

g/L of ethanol after 48-72 hours (chapter 4.4). The same amount of ethanol (at the same

time point) was detected in the same medium by the strains PDO-FPS and PDO-FPS-

Tpi1pdown. Interestingly, there was a remarkable increase in ethanol formation as soon as

the DHA pathway was introduced in these strains (Fig. 16). In order to exclude the impact

of the heterologous 1,2-PDO pathway on ethanol production, the effect of the DHA

pathway in YG medium has been separately tested in a corresponding CBS 6412-13A

derivative that only contained the genetic modifications for Module DHA. This strain also

showed a high ethanol concentration (more than 2.5 g/L, data not shown). There could

be a few reasons that might have caused this increase in ethanol formation. For instance,

the DHA pathway generates NADH during glycerol catabolism compared to the native

(FAD-dependent) G3P pathway. We therefore speculate that ethanol formation was used

as a way to regenerate NAD+ in the cells similar to what happens during the Crabtree effect

in media where glucose concentration exceeds a concentration of 0.1 g/L (Crabtree, 1929).

The switch to ethanol fermentation results from a metabolic overflow due to limited

capacity of the TCA cycle and respiration as soon as the glycolytic flux exceeds a certain

level. Another possible explanation for the fact that ethanol production increased by the

implementation of the DHA pathway could be that the enzyme encoded by the Opgdh gene

contributed to ethanol formation.

In general, the production of ethanol is counterproductive for the production of our target

molecule 1,2-PDO. The fact that the implementation of the DHA pathway increased

ethanol instead of 1,2-PDO production indicates that the flux towards 1,2-PDO is still rate

controlling either due to insufficient enzyme activities or the respective NADH-dependent

enzyme(s) in the 1,2-PDO pathway have a Km value for NADH that is higher than the one


87

for the alcohol dehydrogenase 1 mainly responsible for ethanol production in S. cerevisiae

(Lutstorf et al., 1968) and cannot compete with the latter for the surplus NADH provided

by the DHA pathway. Avenues to overcome these limitations were discussed in section

5.1.2.

Accumulation of unknown metabolite with the retention time of acetol

In the 1,2-PDO pathway starting from DHAP, the first intermediate is MG. According to

the existing knowledge about the 1,2-PDO pathway in the prokaryotes, MG can proceed

via three intermediates, R or S-lactaldehyde and acetol as it is shown in Fig. 2. The

enzymes that catalyze the reactions via R/S-lactaldehyde, i.e. from MG to R/S-

lactaldehyde and then to 1,2-PDO are not well characterized. The non-specific nature of

these enzymes may cause an accumulation of reaction product other than the expected

one in 1,2-PDO pathway such as R/S-lactaldehyde or any other metabolite inside the cell.

The identity of the unknown peak that has the same retention time as acetol, is still

pending. However, if it is assumed that the peak is indeed acetol, then the results in Fig. 19

suggest that in the strain PDO-FPS-DHA-Tpi1pdown-gdh-mgsA, the extra copy of EcmgsA

has helped in pulling the carbon flux towards 1,2-PDO pathway and the extra copy of

Opgdh has supported the flux from acetol to 1,2-PDO. In fact, there was no accumulation

of the unknown metabolite in the strain with an extra copy of Opgdh (PDO-FPS-DHA-

Tpi1pdown-gdh). With this regard, the assumption of the peak as acetol seems to be correct

as if the respective metabolite was indeed acetol, then the accumulated acetol seemed to

be efficiently converted into 1,2-PDO by the higher glycerol dehydrogenase activity due

to the second copy of Opgdh thus leaving no acetol behind.


88

5.2 Future prospects

General recommendations

In his study, most of the enzymes in the entire engineering strategies are heterologous.

Although the implementation of these enzymes empowers S. cerevisiae to consume more

glycerol and produce 1,2-PDO, there are still unknown bottlenecks probably due to the

non-native nature or the less knowledge of the extent of activity of these enzymes. This

might cause an imbalance in the metabolism inside the cells. To check if the heterologous

enzymes are more functional in other reactions besides the desired locations, the resulted

metabolite needed to be identified. For this purpose, the metabolome analysis might be

helpful to identify those targets.

Another possibility could be the in silico flux analysis at the genome scale that can predict

the preference of carbon flow resulted due to the genetic engineering aimed at enhanced

1,2-PDO production. These model predictions mostly take into the account the existing

omics data available. It would be very beneficial if the in silico modelling is feed and

refined with the experimental results obtained in this study. For instance, the in silico

modeling in the light of experimental data might help in understanding the high level of

ethanol formation that results from the combination of DHA pathway with 1,2-PDO

pathway (Fig. 16) or even without 1,2-PDO pathway. Also the in silico simulation might

indicate some target enzymes (e.g. pyruvate decarboxylases), whose regulation control

can help in diverting the flux of carbon towards heterologous 1,2-PDO pathway.

Alleviating the allosteric inhibition of methylglyoxal synthase

Methylglyoxal synthase catalyzes the formation of methylglyoxal and inorganic

phosphate from DHAP. Hopper and Cooper (1971) reported an allosteric inhibition of

E. coli methylglyoxal synthase by inorganic phosphate released during the catalysis of

DHAP to methylglyoxal. It seems to be obvious that this product inhibition will avoid an

efficient carbon flow towards 1,2-PDO production via MG. This allosteric phenomenon

has been widely stated in the literature; however, the exact location of amino acids

residues (allosteric sites) in E. coli methylglyoxal synthase protein responsible for the

attachment of allosteric agent (phosphate) has not been elucidated yet. The enzyme

clearly discriminates between the inhibitory phosphate and the phosphoryl group of

DHAP (Saadat and Harrison 1999). While the crystallographic structure of E. coli


89

methylglyoxal synthase revealed sites where the phosphoryl group from DHAP could bind,

the distinct binding site for the inhibitory phosphate is still obscure.

Basically two pathways have been proposed for the allosteric signal transduction. In one

pathway, the salt bridge formation between Asp-20 and Arg-150 in the presence of

phosphate serves as allosteric wire which communicates the allosteric information to the

neighboring subunits and eventually to the active site (Saadat and Harrison, 1999).

Mutations in Asp-20 has significantly decreased the affinity towards phosphate, however

a ~100-fold decrease in activity was also observed (Saadat and Harrison, 1998). Very

recently, 10 amino acids containing Arg-150 from the C-terminal of E. coli methylglyoxal

synthase were removed (Mohammadi et al., 2014). Structural studies and irreversible

thermo-inactivation data showed that the mutated enzyme became more flexible and less

stable as compared to wild–type methylglyoxal synthase. It seems that the

C-terminal domain of E. coli methylglyoxal synthase plays a role in allosteric regulation

but also in the stability of the overall protein. In the second proposed model, Pro-92, Arg-

107 and Val-111 are engaged in an inter-subunit crosstalk which conveys the allosteric

signals (Saadat and Harrison, 1999).

Alleviation of phosphate inhibition might require the removal of the respective allosteric

site(s) in the methylglyoxal synthase protein. Notably, a few amino acids have already

been identified as potential binding sites for inorganic phosphate. The binding causes

conformational changes within the enzyme leading to its inhibition. For example, Ser55

was very recently found to be a good candidate for the binding of phosphate to

methylglyoxal synthase in Thermus sp. GH5 (Falahati et al., 2009). In E. coli’s

methylglyoxal synthase, Asp 20 and Asp 91 residues were noted for their higher binding

affinity towards phosphate (Saadat and Harrison 1998, Saadat and Harrison 1999). To

increase the yield and concentration of 1,2-PDO, it is important to reduce or eliminate the

allosteric inhibition of methylglyoxal synthase by enzyme engineering. Hence, the

mechanisms underlying this inhibition need further investigations.

Exploration of alternative methylglyoxal synthases

It has been assumed, that MG production is one of the rate controlling steps in 1,2-PDO

production (Hoffman 1999, Clomburg and Gonzalez 2011, Niimi et al., 2011). As

mentioned in Table 4, virtually all previous studies used E. coli mgsA for 1,2-PDO


90

production in yeast. The question arises whether the expression of alternative mgsA

genes from other sources can improve 1,2-PDO production. One example is the mgsA gene

from Thermoanerobacterium thermosaccharolyticum ATCC 91960. In fact,

T. thermosaccharolyticum ATCC 91960 is the best natural producer of 1,2-PDO (9.0 g/L,

(Sanchez-Riera et al., 1987). Noteworthy, the mgsA from T. thermosaccharolyticum ATCC

91960 has been cloned and sequenced (Altaras N.E. 2002). It would be worth exploring

and testing alternative mgsA genes from other microorganisms in order to produce an

even higher titer and yield of 1,2-PDO.

Methylglyoxal is toxic for the cells; its detoxification pathways also causes competition to 1,2-PDO pathway for carbon flux

Methylglyoxal is a reactive β-dicarbonyl metabolite that reacts with amine groups of

nucleic acids and proteins causing damage to the latter and eventually stops cell division

(Thornalley 1994). In bacteria, three different methylglyoxal detoxification routes have

been reported. The first one is the glutathione-dependent glyoxalase I-II system, which

leads to the production of D-lactate (Ferguson et al., 1998). The second one is the

glutathione-independent glyoxalase III system, where glyoxalase III has been described

to be responsible for the catalysis of methylglyoxal to D-lactate without any dependency

on glutathione formation (Misra et al., 1995). Pathways composed of methylglyoxal

reductases and dehydrogenases are the third very important route involved in the control

of methylglyoxal levels in the cell. They typically convert 2-oxoaldehydes to the

corresponding 2-hydroxyaldehydes, and aldehydes to alcohols (Inoue and Kimura 1995).

As the implementation of the latter route was an initial objective of this study, the non-

specificity of the respective methylglyoxal reductases and dehydrogenases for the desired

1,2-PDO intermediate metabolites creates a bottleneck as they can usually accept more

than one substrate. Ultimately, the efficiency of these enzymes is affected in terms of

1,2-PDO production.

The methylglyoxal detoxification pathways besides methylglyoxal reductases and

dehydrogenases also takes away the carbon flux from 1,2-PDO pathway. One solution for

achieving more 1,2-PDO is to select a set of proficient and well characterized

oxidoreductase enzymes in S. cerevisiae that can readily convert methylglyoxal to 1,2-PDO.

This would also help to avoid the accumulation of MG and on the other hand the 1,2-PO


91

production can be enhanced. Additionally, the competing pathways via glyoxalase system

either need to be deleted or downregulated depending its effect on the viability of the cell.

92

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