Engineering of redox metabolism in yeast: New strategies for improved glycerol production

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Engineering of redox

metabolism in yeast:

New strategies for

improved glycerol production

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Engineering of redox metabolism in yeast:

New strategies for improved glycerol production

Proefschrift

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magniﬁ cus Prof. dr. ir. J.T. Fokkema, voorzitter van het College voor Promoties,

in het openbaar te verdedigen op dinsdag 5 september 2006 te 17:30 uur

door

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Dit proefschrift is goedgekeurd door de promotoren Prof. dr. J.T. Pronk

Prof. dr. J.P. van Dijken

Samenstelling promotiecommissie:

Rector Magniﬁ cus, voorzitter

Prof. dr. J.T.Pronk, Technische Universiteit Delft, promotor

Prof. dr. J.P. van Dijken, Technische Universiteit Delft, promotor

Prof. dr. ir. J.J. Heijnen Technische Universiteit Delft

Prof. dr. G. Lidén Lunds Universitet, Lund, Zweden

Prof. dr. L. Olsson Danmarks Tekniske Universitet, Lyngby, Denemarken

Dr. M.J. Teixeira de Mattos Universiteit van Amsterdam

Dr. ir. A.J.A. van Maris Technische Universiteit Delft

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Prologue

‘Yeast may prove to be the salvation of our modern civilization.

–

The efﬁ cient use of replenishable sources of energy, in which yeast fermentation will play a major role,

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Abbreviations

3-HPA 3-Hydroxypropionaldehyde

2/3PG Combined pool of 2- and 3-phosphoglycerate

6PG 6-Phosphogluconate

ADP Adenosine diphosphate

AMP Adenosine monophosphate

ATP Adenosine triphosphate

COSY Correlation spectrometry

DHAP Dihydroxyacetone phosphate

Δμ˜ H+ _{Proton motive force}

DOT Dissolved oxygen tension

ΔpH Chemical component of proton motive force (alkaline inside)

DPN Diphosphopyridine nucleotide

∆Ψ Electrical membrane potential difference

ε Extinction coefﬁ cient

FAD(H) Flavin adenine dinucleotide

F6P Fructose-6-phosphate

FBP Fructose-1,6-bisphosphate

FDA Food and Drug Administration

GAP Glyceraldehyde-3-phosphate

GC-MS Gas chromatography-mass spectrometry

GRAS Generally regarded as safe

GTBE Glycerin tertiary-butyl ether

G3P Glycerol-3-phosphate G6P Glucose-6-phosphate

k ATP requirement for cell formation

k_M Michaelis-Menten constant

LC-MS Liquid chromatography-mass spectrometry

lb. Pound avoirdupois

μ(max)_(Maximum)_{speciﬁ c growthrate}

M6P Manose-6-phosphate

m_ATP Cellular maintenance

MG Methylglyoxal

MTBE Methyl tertiary-butyl ether

NAD(H) Nicotinamide dinucleotide

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10 Abbreviations

MFA Metabolic ﬂ ux analysis

NMR Nuclear magnetic resonance

OD₍₆₆₀₎ Optical density (at 660 nm)

ORF Open reading frame

P5P Combined pool of xylulose-5-phosphate, ribose-5-phosphate and

ribulose-5-phosphate

PCR Polymerase chain reaction

PDO 1,3-Propanediol

PEP Phosphoenol pyruvate

P_i Inorganic phosphate used in the conversion of ADP to ATP

pK_a Acid dissociation constant

PPP Pentose phosphate pathway

PTT Polytrimethylene therephtalate

P/O ATP synthesized per electron pair transferred to oxygen

R_f/g Molar formate to glucose ratio

S7P Sedoheptulose-7-phosphate

SS_res Residual sum of squares

TAM3KO pdc1,5,6Δnde1,2Δgut2Δ in TAM background

TAM pdc1,5,6Δ S. cerevisiae selected for C₂ independence and rapid growth

on excess glucose UQ Ubiquinone

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Chapter 1

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General introduction

13 INDUSTRIAL BIOTECHNOLOGY

‘Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for speciﬁ c use’ (282). This deﬁ nition describes biotechnology as agreed upon in the Convention on Bio-logical Diversity, which was signed by 157 World Leaders present at the 1992 ‘Earth Summit’ in Rio de Janeiro. The summit, organized by the United Nations Environ-ment Programme, intended to guide the world community towards a more secure, equitable and sustainable future without affecting biodiversity.

Biotechnology: a colorful world

A broad deﬁ nition of biotechnology such as described above, which covers most - if not all - areas of biotechnology, required subdivision into different ﬁ elds. In an attempt to facilitate public comprehension, EuropaBio, the political voice of the biotechnology industry in Europe, took the initiative to distinguish different biotechnological disciplines. Soon after, this resulted in a generally accepted color assignment to four subdivisions (Table 1). Drs. Feike Sijbesma, Board member of DSM, coined the term ‘white’ for biotechnology of industrial processes (EuropaBio,

personal communication). Marine and aquatic applications were associated with

the color blue, whereas green was associated with agricultural processes and red with medical biotechnology.

The ‘labeling’ of the biotechnological disciplines was not limited to these four biotechnological colors. Soon after the fi rst color assignments, industrial biotech-nology was split into white and grey biotechbiotech-nology. ‘White’ would refer to bioin-dustries using genetically modifi ed organisms and ‘grey’ would capture classical fermentation and bioprocess technology. Recently, an editor of the electronic journal of biotechnology, even suggested to include fi ve more colors in the world of biotechnology: Yellow (food biotechnology and nutrition science), brown (arid zone and desert biotechnology), purple (patents, publications and inventions),

Table 1 Four (or ﬁ ve) generally accepted color codes identifying the subdivisions of biotechnology.

Color Subﬁ eld

Blue Marine and aquatic applications Green Agricultural processes Red Medical sciences White Industrial processes

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14

Chapter 1

gold (bioinformatics and nanotechnology) and even dark (bioterrorism, biowarfare, biocrimes) (56). In practice, the use of all nine proposed colors for different biotech-nological ﬁ elds probably does not serve the initial idea to facilitate public compre-hension by color-coding. However, addition of a ﬁ fth color (yellow) to identify food and nutrition sciences (Table 1), might arguably have a positive impact on public understanding, knowledge and awareness.

The remainder of this thesis will focus on industrial (white) biotechnology, which can be described as the area of biotechnology in which living cells or derivatives of living cells, such as enzymes, are used for industrial production of (commod-ity) chemicals. These products are often easily degradable, although this is not a prerequisite and sometimes even undesirable. In general, production of chemi-cals via a biochemical route rather than chemical synthesis requires fewer steps, is performed under more moderate conditions and usually requires less energy and creates less waste in comparison to traditional chemical processes. Historically, industrial biotechnology has had a hard time competing with more economical oil-based chemical industries, despite these numerous advantages. Although there are several examples of biotechnologically produced compounds (e.g. ethanol, cit-ric acid, lysine and lactic acid), organic chemicals are still predominantly produced by the petrochemical industry. As a consequence, most processes in industrial biotechnology are relatively new (less than a few decades) and as such have not had the same time of optimization of production processes as the petrochemical industry (89).

Increased awareness for the need for sustainability, increased environmental awareness, commitment to international treaties (e.g. Kyoto Protocol) and the desire for independence from oil import are increasingly turning the political will towards increased use of industrial biotechnology. As a result, present-day biopro-cesses are sometimes supported by tax-breaks and subsidies. Progress in industrial biotechnology is also accelerated by technological breakthroughs and thereby overcoming (or decreasing signiﬁ cance of) economic restraints. More recently, a signiﬁ cant increase of crude oil prices has given extra momentum to bio-based industrial applications as an alternative to classic petrochemistry.

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15 renewable fuels (41). In Europe, politicians seem reluctant to publicly and explicitly express such geopolitical concerns, although crude imports account for about 75% of total oil consumption, thus making the European Union even more dependent on imported energy than the USA (329). However, with crude oil prices increasing by roughly US$30 per barrel (increasing the EU15 oil import bill by €120 billion a year (329)) during the last few years, the need for alternatives has become an important issue on the European agenda once again. Other advantages of a long-term switch to a carbohydrate-based economy are the use of nature’s short-term renewable re-sources, accompanied by limited greenhouse gas emissions due to a closed carbon cycle (Figure 1) and the boost given to the local agricultural economy.

RENEWABLE FEEDSTOCKS AND A CARBOHYDRATE ECONOMY

In the long run when fossil fuels become a scarce commodity, energy can be de-rived from a variety of alternative sources, such as wind, sun, water, alternative fossil energy deposits, nuclear ﬁ ssion and fusion. Production of organic substances and materials however, is entirely dependent on a carbon skeleton originating from traditional organic carbon deposits or from alternative (renewable) carbon skel-etons. In the proposed new carbohydrate economy, sugars may form the central component. In the form of polysaccharides these sugars are stored in vast amounts in biomass, particularly plant biomass (129). According to U.S. Congress, the term biomass means ‘any organic matter that is available on a renewable or recurring basis (excluding old-growth timber), including dedicated energy crops and trees, agricultural food and feed crop residues, aquatic plants, wood and wood residues, animal wastes, and other waste materials’ (280). Out of the 170 billion tons of bio-mass produced annually by photosynthesis on earth, currently only 6 billion tons are used by man, mainly in food production (129).

sunlight biofuels renewable carbohydrates CO2 biomass biorefinery industrial biotechnology combustion photosynthesis = 100-102y (bio)chemicals platform biochemicals fossil hydrocarbon rserves = 106-108y sunlight fuels fossil hydrocarbon reserves CO2 biomass geo(bio)logy refinery combustion photosynthesis chemicals platform chemicals

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16

Chapter 1

In contrast to the fossil carbon cycle, the renewable carbon cycle can directly use biomass as the feedstock and as such is not dependent on millions of years of geological processes (Figure 1). Pretreatment of the raw material (biomass) to make it accessible to further processing is essential for the use of renewable feedstocks. The vast majority of plant material consists of cellulose (38%-50%), hemicellulose (23%-32%) and lignin (15%-25%), although substantial amounts of starch and sugar can be found in fruits, vegetables, certain roots and tubers as well. Analogous to refi ning crude oil, plant material will have to be refi ned into different carbohydrate fractions and further processed to suitable platform chemicals and energy carriers. The integration of (i) processing solid plant material, (ii) hydrolysis thereof to fermentable sugars and lignin and (iii) conversion by microorganisms to desired building blocks, constitutes the very heart of the new carbohydrate economy and is appropriately named biorefi nery. There are currently several biorefi nery concepts being pursued in research and development (129).

1. The whole crop bioreﬁ nery uses sugars and starch stored in raw materials such as cereals or maize.

2. The green bioreﬁ nery can process materials with relatively high water content, such as green grass, clover or immature cereals

3. The lignocellulose feedstock bioreﬁ nery uses ‘nature-dry’ raw material with high cellulose content, such as straw, wood, paper waste and corn stovers.

Plant material consists of a complex network of polymerized sugars and aromatic alcohols. Only a minor fraction of available plant biomass consists of easily degrad-able starch, of which two forms exist. (Figure 2). The unbranched form, amylose, is a glucose polymer linked with α-1,4-glycosidic bonds. Amylopectin is the branched form, which has about one α-1,6-glycoside linkage per thirty α-1,4-glycoside links and resembles the yeast storage carbohydrate glycogen, but with a lower degree of branching. Rapid hydrolysis of amylopectin and amylose to oligosaccharides takes place by amylases, a variety of enzymes naturally present in many organisms.

Due to their structural function in plant cellulose, hemicellulose and lignin are much more resistant to degradation than starch. Cellulose is an unbranched poly-mer of D-glucose units, linked together with β-1,4-glycoside bonds (Figure 2). In

this complex structure, the cellulosic ﬁ bers are surrounded by hemicellulose and/or lignin. The branched polysaccharide hemicellulose consists of a mixture of pentose and hexose sugars like D-xylose, D-fructose, L-arabinose, D-mannose, D-glucose and

D-galactose (Figure 2). Hemicellulose has a weak undifferentiated structure

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17

biomass conversion to biocommodities can be found in the current relatively poor accessibility of the sugars in woody material. Advances in pretreatment technology have made cellulosic material accessible to enzymatic hydrolysis. At the same time, progress in carbohydrate accessibility is likely to accelerate because of improved microbial production and increased activity of these hydrolyzing enzymes (163). Industrially, lignocellulosic material is ﬁ nely chopped and treated by dilute acid hydrolysis to saccharify hemicellulose. This way, the cellulosic sugar chains can be degraded to mono- and oligosaccharides by appropriate cellulases (endogluca-nase, exoglucanase and β-glucosidase). Cellulases are not as omnipresent in nature as amylases, but can be found in a variety of fungi and in several bacteria and

Actinomycetes. During World War II, Trichoderma reesei was found to produce

cellulases when the fungus broke down cotton clothing and canvas tents of the U.S. army in the South Paciﬁ c (239). Lignin cannot be degraded enzymatically, but part of the lignin fraction is hydrolyzed during the dilute acid treatment. Remaining solid lignin can be separated, dried and used for energy generation. A drawback of the acidic conditions during hydrolysis is degradation of glucose and xylose, which results in the formation of hydroxymethylfurfural and furfural. These and other in-hibiting compounds decrease the ability of microorganisms to ferment the high sugar-content hydrolysate to the desired platform chemical. Eventually, consolida-tion of enzymatic degradaconsolida-tion of woody material in conjuncconsolida-tion with conversion of a broad spectrum of soluble carbohydrates to the desired platform chemicals will result in the ultimate bioreﬁ nery. However, in order to reach that goal, biocatalysts (microorganisms and/or enzymes) are required that can convert all carbon sources in the complex hydrolysates to a range of desired products. For this the under-standing and development of (new) platform microorganisms is essential.

Figure 2 General and chemical structure of starch illustrating the α-1,4 (A) and α-1,6 bonds (B). Simpliﬁ ed structure of cellulose (C) made up of β-1,4-linked D-glucose units (gluc) and

hemicellulose (C) in which also a mixture of other sugars such as D-xylose (xyl), L-arabinose

(ara), D-galactose (gal), D-fucose (fuc) is polymerized at various linkages, with acetal (ac)

groups in between.

gluc gluc gluc gluc gluc gluc gluc gluc

A

B

gluc gluc gluc gluc gluc gluc gluc gluc gluc gluc gluc gluc gluc gluc ac gluc gluc ara xyl gal xyl xyl xyl fuc

gluc gluc gluc gluc gluc gluc

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18

Chapter 1

MICROORGANISM, METABOLIC ENGINEERING AND METABOLISM For many years, improvement of strains used in a variety of biological produc-tion processes consisted of random mutagenesis of microbial strains, followed by screening for the best-producing mutants. This classic way of strain improvement is widely accepted and has an impressive record of success. Production of the antibi-otic penicillin was increased more than thousand fold using many rounds of random mutagenesis and selection (152, 273). Nature’s fi rst principle in natural selection, survival of the fi ttest (55), can also be used to improve biological characteristics (e.g. increase substrate affi nity).

To make optimal use of nature’s ways to transform substrates into products, while deriving energy needed for the assimilation of nutrients to biomass, several requirements have to be met: (i) A suitable biological catalyst or microbial back-ground is required. (ii) Within this backback-ground, redox and energy availability should be balanced. (iii) A more economical conversion is obtained when a broad range of substrates can be used. (iv) The desired product should be made with an optimal product yield and production rate, preferably without signiﬁ cant generation of by-products.

The advent of genetic engineering in 1973 introduced a completely new way to optimize, introduce, develop, modify or eliminate pathways in microorganisms (48). Subsequent developments resulted in a completely new area of biotechnology: metabolic engineering, which was deﬁ ned as ’the improvement of cellular activities by manipulations of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology’ (17).

Metabolic engineering and functional genomics

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19 Genomics, transcriptomics, proteomics, metabolomics and ﬂ uxomics together con-stitute the heart of present day metabolic engineering design (Figure 3).

The central dogma of molecular biology, which describes the transcription pro-cess from DNA to RNA and subsequent translation of RNA to proteins, constitutes the bottom part of the metabolic engineering triangle (Figure 3). Each level is part of the chain that forms a dynamic link between the genome, growth conditions and the cellular phenotype. The regulation of gene expression is a key process for adaptation to changes in environmental conditions and thus for survival. Genom-ics, which comprehensively analyzes DNA structure, quantitatively studies genes, regulatory and non-coding sequences and the replication of DNA, constitutes the base of the metabolic engineering triangle. The subset of genes transcribed to messenger RNA in organisms is called the transcriptome. Transcriptomics studies this process in a genome-wide range relative to a reference. A powerful tool used in transcriptomics is the DNA-microarray, which allows for analysis of the cellular mRNA level of practically every gene on an organism’s genome. This level is the resultant of newly transcribed mRNA, degraded mRNA and processed mRNA. An-other important metabolic engineering approach (Figure 3) is proteomics, which involves the systematic study of proteins in order to provide a comprehensive view of the structure, function and regulation of biological systems. Proteomics not only studies protein production proﬁ ling, but also analyzes post-translational modiﬁ cations and protein-protein interactions. Directly linked to proteins are their substrates, products and co-factors. Formation of biomass and the production of

Figure 3 Metabolic engineering comprising target, design, synthesis and analysis with at the center the various ’omics’ used (193).

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20

Chapter 1

primary and secondary metabolites represent the end products of genetic expres-sion. The comprehensive analysis of metabolites has been termed metabolomics. Additional information can be obtained by determining the material ﬂ ows (ﬂ ux) through different pathways. Flux patterns can be derived using isotopic labeling of substrate (e.g. 13_{C glucose) in combination with in silico metabolic network models}

(fl uxomics) (252, 302, 328). The completion of the fi rst genome sequencing proj-ects, combined with the various ‘-omics’ in a cyclic strategy comprising design, synthesis and analysis has signifi cantly advanced metabolic engineering or path-way engineering.

Microorganisms as biocatalysts

Within industrial biotechnology, microorganisms perform an essential role as bio-logical catalysts. With the use of metabolic engineering, these organisms’ char-acteristics can be improved such that they produce the desired compound (e.g. ethanol, glycerol, proteins) from the desired resources (e.g. glucose, xylose) (Figure 4). Escherichia coli and Saccharomyces cerevisiae in particular have proven to be ideal backgrounds for metabolic engineers. Both organisms have a long history of research and as a result are well understood, genetically well accessible and these organisms are relatively easy to cultivate. With advancing sequencing techniques, improved analysis tools and integration with information technology other microor-ganisms, with intrinsic advantages, are rapidly gaining ground as well.

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21 In this thesis, research focuses on the use of S. cerevisiae as a platform for metabolic engineering. Its intrinsic advantages include its status as a GRAS organ-ism (Generally Regarded As Safe, FDA, USA) and the availability of a complete and extensively annotated genome, which has been publicly available since April 24th_,

1996 (103). The fact that S. cerevisiae grows relatively rapidly (doubling times of wild type strains are under 2 h) on simple well-deﬁ ned media, which enables com-plete control over its chemical and physical environment, has further contributed to its popularity as a model system for eukaryotic cell biology research.

Bakers’ yeast: a brief reﬂ ection on its past and present

Used by Sumerian, Assyrian, Babylonian, Chinese and Inca cultures for fermenta-tion, yeasts have built a historical relation with humans dating back for more than 8000 years (54). The English word ‘yeast’ stems from the Greek term ζεστός (zestos). In particular the genus Saccharomyces has played a key role in processes used for commercial exploitation (333). Interestingly, the fact that we now know S. cerevisiae may be considered a result of human domestication (166) as S. cerevisiae is a mi-nority organism in natural habitats such as fruits and berries of wild plants (225).

In the early days bakers’ yeast was used for fermentation of fruits to wine and brewing of beer from water and grains (Figure 5), later bakers’ yeast was used to leaven bread as well. Of course, back then, the fermentative reaction nor the link with yeasts and the nature thereof, was understood neither from a biological

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22

Chapter 1

nor chemical point of view. It was Antonie van Leeuwenhoek who ﬁ rst observed yeast cells under his self-made microscope and envisioned yeast as tiny globules in 1680 (Figure 6). The awareness that yeast is the link between sugars and alcoholic fermentation only came in 1858 as Louis Pasteur described his studies on yeast fermentation in his ‘mémoires sur la fermentation alcoolique’ (219, 220). Pasteur was the ﬁ rst to recognize yeast as a living organism that actively converts sugars into ethanol and carbon dioxide under concomitant formation of acetate, succinate and glycerol. Few years later, Kühne emphasized that enzymes were inseparably associated with living cells, which at the time were called ‘ferments’ (139).

Carbon, energy and redox metabolism

The survival and success of microorganisms is for a large part determined by the availability of nutrients in their environment. Nutrients function as building blocks for the formation of biomass (assimilation). In addition to this, nutrients are required for making available the free energy that is needed both for growth as well as for maintenance of already existing biomass. Essential macronutrients for

Saccha-romyces cerevisiae are a carbon source, a source of nitrogen, phosphorous and

sulfur, which generally contain the essential elements hydrogen and oxygen as well. Micronutrients consist of some trace elements and vitamins (312). Nutrient assimila-tion, the process of biosynthesis from these nutrients, requires substantial amounts of energy. Carbon, energy and redox metabolism are therefore closely related.

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Carbon dissimilation

Adenosine triphosphate (ATP) is the molecular ‘currency’ of intracellular free en-ergy. In heterotrophic organisms such as S. cerevisiae the ATP needed in a variety of cellular processes, such as biosynthesis and cellular maintenance, is provided by oxidation of organic molecules to carbon dioxide and water or alternatively by fermentation of sugars to ethanol and carbon dioxide. The carbon source, which in many cases is glucose, thereby acts as a the raw material for biosynthesis in addi-tion to being a source of free energy (94). S. cerevisiae is a facultative anaerobe and is able to utilize glucose under both aerobic and anaerobic conditions. In this yeast, glucose is dissimilated via the Embden-Meyerhof-Parnas pathway, commonly re-ferred to as glycolysis, to 2 molecules of pyruvate. The return on initial investment of 2 ATP per glucose in the upper part of glycolysis is 4 ATP and 2 nicotinamide dinucleotide (NADH) per glucose in the lower part of the pathway (Figure 7).

In yeasts, pyruvate is located at an important branch point between fermenta-tive and oxidafermenta-tive metabolism (234). Under strictly anaerobic growth conditions, the sole mode of ATP generation in S. cerevisiae is substrate level phosphorylation and the NADH produced in the glyceraldehyde-3-phosphate reaction has to be reoxidized, which in S. cerevisiae takes place via alcoholic fermentation. In this metabolic route, pyruvate is ﬁ rst decarboxylated to acetaldehyde, which subse-quently acts as electron acceptor for NADH reoxidation (Figure 7).

Pyruvate dissimilation can also take place via oxidative metabolism. Pyruvate is then further metabolized in the mitochondria, the cell’s molecular ‘power plants’ (151), separated from the cytosol by two surrounding membranes. After pyruvate translocation from the cytosol to the mitochondrial matrix, pyruvate is

metabo-lized to acetyl-coenzyme A under concomitant formation of CO₂ and generation

of NADH, by pyruvate dehydrogenase (235). Subsequently acetyl-CoA enters the tricarboxylic acid (TCA) cycle, where citrate synthase binds the acetyl group to oxa-loacetate to form citrate. In the TCA cycle, citrate is dissimilated in a series of reac-tions that ﬁ nally yield oxaloacetate, 2 CO₂, 1 ATP, 1 FADH and 3 NADH. The redox carrying cofactors FADH and NADH are then regenerated as will be discussed later in this paragraph.

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24

Chapter 1

Redox metabolism and compartmentalization

Nicotinamide dinucleotide coenzymes were discovered in 1906 as ‘co-zymase’ and were found to be required for glycolytic activity in cell extracts of yeast (112). Warburg and coworkers ﬁ rst puriﬁ ed and determined the chemical composition of the hydrogen transferring coenzyme (wasserstoffübertragendes Co-Ferment) and the fermentation coenzyme (Gärungs-Co-Ferment), currently known as NADPH

glucose

GAP DHAP

glycerol pyruvate acetaldehyde

ethanol

CO2

2 ATP

fructose-1,6-bisP

NADH

NADH 2 ATP NADH

pyruvate 3 CO2 1 ATP 3 NADH 1 FADH mitochondrion cytosol A B C D E F G RH₂ R NADH L RH₂ R NADH G3P Pi NADH G3P DHAP NADH UQ biosynthesis ATP H I J K H+ ADP ATP NADH Acetyl-CoA NADH CO2

Figure 7 Schematic overview of fermentative and oxidative glucose metabolism of

Saccharomyces cerevisiae. (A) upper part of glycolysis, which includes two sugar

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25 and NADH, respectively (322, 323). Later, NAD(H) - at the time referred to as

diphosphorpyridine nucleotide (DPN_OX, DPN_RED) - was suggested to serve as a

‘transport metabolite’ of reducing equivalents in a variety of reactions (39). In the discussion following Büchner’s paper the term ‘transport metabolite’ was limited to compounds functioning in a closed cycle. This is in agreement with the present day term ‘conserved moiety’, describing other carrier molecules as well (e.g. the previously mentioned adenosine phosphates ATP/ADP/AMP).

The oxidation-reduction properties of the pyridine ring give NAD(H) and NADP(H) an important role in many biological reactions. Compared to the NADH/

NAD+_{redox couple, the NADPH/NADP}+_{redox system is in a much more reduced}

state (38, 96, 249). This reﬂ ects the different functions that NAD(H) and NADP(H) have in yeast metabolism. NADPH is preferentially used in assimilatory reactions

(consuming NADPH), in which reactions are favored by a high NADPH/NAPD+

ratio. However, dissimilatory reactions (producing NADH), are favored by a low

ratio between reduced and oxidized (NADH/NAD+_{) cofactors. Presently, around}

100 enzyme reactions are known to involve oxidation or reduction of NAD(P)+_and

NAD(P)H. Among these are enzymes involved in precursors assimilation and de-toxiﬁ cation processes.

Biomass is slightly more reduced than glucose, which suggests a net consump-tion of reducing equivalents (i.e. NADPH). However, an imbalance exists between NADPH consumption and NADH production in biosynthetic reactions, which de-pends on the nitrogen source that is used (38, 204), and because of CO₂ formation associated with precursor generation a net production of reducing equivalents oc-curs (i.e. NADH) (106, 204, 288). As a result, formation of 1 g of biomass from glu-cose and ammonia is accompanied by a net generation of around 10 mmol NADH (311). In addition, excess NADH can result from excretion of oxidized components, such as pyruvate, acetate and acetaldehyde during growth on glucose (194, 205, 288).

Because of their function as conserved moieties, oxidation and reduction of these redox carriers in the cell have to be continuously balanced. This redox bal-ancing act is complicated by subcellular compartmentation, which is characteristic for eukaryotes, and therefore also for S. cerevisiae. The mitochondrial membrane consists of an inner and outer layer. During biogenesis of the mitochondria, de

novo synthesis of NAD+_{takes place in the cytosol. This newly synthesized NAD}+_is

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26

Chapter 1

impact on pyridine nucleotide metabolism, as redox carriers must be reoxidized in the compartment where they are generated to maintain a cellular redox balance.

NADPH providing reactions, such as the pentose phosphate pathway and acetaldehyde dehydrogenase are mainly located in the cytosol, whereas NADH turnover takes place both in the cytosol and in mitochondria. The NAD(H)-depen-dent enzymes of glycolysis and major enzymes of alcoholic fermentation are local-ized in the cytosol. Oxidative pyruvate dissimilation via the tricarboxylic acid cycle is associated with NADH production and is located in the mitochondrial matrix. Some NAD(H)-dependent enzymes, such as malate dehydrogenase, not only have isoenzymes in the cytosol and/or mitochondria but also in microbodies (: e.g. per-oxisomes, glyoxysomes).

In S. cerevisiae, the pentose phosphate pathway serves as a generator for NADPH and of precursors for biosynthesis (ribose and erythrose-4-phosphate). Yeast cannot reoxidize cytosolic NADPH by coupling the electrons of the reduc-tion equivalents to respiratory chain, nor does it have a transhydrogenase that can

transfer electrons from NADPH to NAD+_{(37, 59, 288). As a consequence, the}

pen-tose phosphate pathway does not play a signiﬁ cant role in glucose dissimilation in

S. cerevisiae (19).

In contrast to its inability to respire NADPH, S. cerevisiae harbors several mechanisms to reoxidize NADH via mitochondrial respiration. The most important systems for regeneration of cytosolic NADH are the external NADH

dehydroge-nases (Nde1,2p), that reoxidize NADH to NAD+_{directly upon electron transfer to}

the ubiquinone pool of the mitochondrial respiratory chain. The glycerol-3-phos-phate shuttle indirectly oxidizes cytosolic NADH (160, 207). In this shuttle, NADH is oxidized by reduction of dihydroxyacetone phosphate (DHAP) to

glycerol-3-phos-phate (G3P) by a NAD+_{-linked glycerol-3-phosphate dehydrogenase encoded by}

GPD1,2 (4, 77). Subsequently G3P is reoxidized to DHAP by a mitochondrial

FAD-dependent glycerol-3-phosphate dehydrogenase (Gut2p) (147, 246) that donates electrons to the ubiquinone pool. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembrane space (160, 260). In addition, intramitochondrial NADH can be reoxidized via an internal NADH:ubiquinone oxidoreductase (Ndi1p) (58, 165).

Symmetric and asymmetric redox shuttles

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27 while reducing NAD+_{in the other compartment. Therefore shuttles enable}

reoxida-tion by the NADH dehydrogenase in the other compartment. Prerequisites of such a redox shuttle mechanisms are symmetric metabolic pathways present in both compartments or asymmetric routes that complement one another, that result in the net consumption of NADH in one cellular compartment while producing it in the other compartment. Cross membrane transport of carrier molecules either by free diffusion or via transporters is involved, provided the overall energy balance allows the process.

With the exception of the ethanol-acetaldehyde shuttle, no direct evidence for the actual in vivo functioning of the below described mechanisms to shuttle NADH equivalents across the mitochondrial membrane has been presented to date (18, 19). However, indirect evidence, such as functional presence of all compounds has been presented.

Ethanol-acetaldehyde shuttle

A well-known shuttle is the ethanol-acetaldehyde shuttle. The alcohol dehydroge-nases involved are localized in different compartments and are completely revers-ible in action. Both acetaldehyde and ethanol freely diffuse across the mitochon-drial membranes and the action of mitochonmitochon-drial Adh3p and cytosolic Adh1p and Adh2p results in a fully symmetrical shuttle that is capable of a net translocation of redox equivalents across compartmental barriers. Under aerobic conditions, the driving force in this process is the reoxidation of NADH via the internal NADH dehydrogenase (19).

The ethanol-acetaldehyde shuttle plays an import role under anaerobic condi-tions and it was shown that an adh3Δ mutant has a 30% lower growth rate under anaerobic conditions compared to a wild type (3, 194). The reason for this is that excess NADH is generated in the mitochondrial matrix as a result of biosynthesis (e.g. pyruvate dehydrogenase and isocitrate dehydrogenase). These assimilatory reactions result in the generation of excess mitochondrial NADH. To maintain mi-tochondrial redox balance, translocation of this NADH to the cytosol is required, because reoxidation via the internal NADH dehydrogenase is not possible under anaerobic conditions (316). Once in the cytosol, the excess NADH originating from the mitochondria can be reoxidized by forming glycerol.

Malate-oxaloacetate shuttle

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28

Chapter 1

occurs via a mitochondrially located malate dehydrogenase (Mdh1p) (272) and a cytosolic isoenzyme Mdh2p (176, 262). However, neither malate nor oxaloacetate can freely diffuse across the inner mitochondrial membrane. The dicarboxylate car-rier Dic1p catalyzes the exchange of dicarboxylic acids (e.g. malate and succinate) for inorganic phosphate. Its primary function is thought to be import of malate (214). Amongst other compounds, the mitochondrial oxaloacetate transporter (Oac1p) can translocate malate and oxaloacetate though a uniport or exchange mechanism. Although the primary direction is thought to be oxaloacetate import, export may be possible in exchange for malate under energetically favorable oxa-loacetate and malate concentrations (60, 213). Under aerobic conditions, import of malate coupled to export of oxaloacetate may thus result in net translocation of cytosolic NADH to the mitochondria (19).

Malate-aspartate shuttle

If the mitochondrial membrane were impermeable to oxaloacetate, redox translo-cation might be possible in an extended version of the above-described shuttle, the malate-aspartate shuttle. Cytosolic NADH can be translocated to the mito-chondria by combined action of aminotransferase, malate dehydrogenases and antiporters (19). Cytosolic oxaloacetate, generated from aspartate by a cytosolic aspartate amino transferase (Aatp2), is reduced to malate by Mdh2p, while

cyto-solic NADH is oxidized to NAD+_{. Malate is then transported across the}

mitochon-drial membranes in exchange for α-ketoglutarate (2-oxoglutarate) by means of the oxodicarboxylate antiporter (Odc1p and Odc2p) (212). Malate then undergoes the symmetrical reaction in the reversed direction, in which the main driving force is the

imbalance of NADH and NAD+_{between the cytosol and the mitochondrial matrix.}

Mdh1p oxidizes malate to oxaloacetate, which is converted to aspartate by the action of the mitochondrial aminotransferase (Aat1p) with simultaneous conversion of glutamate to α-ketoglutarate. The glutamate-aspartate antiporter (Agc1p) then exports aspartate to the cytosol in exchange for glutamate where it completes the cycle (45). One cycle of the shuttle results in a net translocation of cytosolic NADH across the mitochondrial membrane.

Malate-pyruvate shuttle

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29 where malic enzyme (Mae1p) converts malate back to pyruvate (31, 214). Pyruvate will have to be transported back to the cytosol for a functional shuttle (117), but such a transporter has not been found (276). Bakker et al. suggested that this system may be considered an alternative means of pyruvate transport to the mitochondria (19). Moreover, a closed malate-pyruvate shuttle during aerobic growth on glucose is highly unlikely as the direction of pyruvate transport is from the cytosol to the mitochondria and not in the reverse direction.

The malate-oxaloacetate, malate-aspartate and malate-pyruvate shuttles involve the in vivo function of the cytosolic isoenzyme of malate dehydrogenase. However, since Mdh2p is rapidly phosphorylated in the presence of excess glucose (177, 178) and because the anaerobic expression of MDH2 is very low (269), it seems unlikely that in vivo translocation of NADH via these three shuttles is possible under anaerobic or aerobic glucose excess conditions.

Mitochondrial complexes

The mitochondrial matrix, which has a high protein content, limits diffusion of metabolites and it has therefore been suggested that channeling processes occur between mitochondrial enzymes involved in NADH metabolism (78, 181). To allow for such rapid channeling of substrates and products, the concept of a TCA cycle enzymes ‘metabolon’ (supramolecular complex) has been proposed (218, 261). The kinetic regulation of Gut2p as a function of Nde1,2p may reﬂ ect the presence of these proteins in a supramolecular complex involved in mitochondrial oxidation of cytosolic NADH (210). In fact, a supramolecular organization of most mitochon-drial redox-related enzymes has recently been proposed in yeast. The TCA cycle enzymes Sdh1p (succinate dehydrogenase), Fum1p (fumarate hydratase), Mdh1p (malate dehydrogenase), Cit1p (citrate synthase), Idh2p (Isocitrate dehydrogenase)

and Ald4p (NAD+_{-dependent acetaldehyde dehydrogenase) are associated with}

Ndi1p and the ﬁ ve intermembrane-space facing dehydrogenases Nde1p, Nde2p,

Gut2p, Dld1p (D-lactate-cytochrome c oxidoreductase) and Cyb2p (L

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30

Chapter 1

described above may result in additional prioritization (40). At the metabolic level, expression of NDI1 is repressed by glucose, whereas NDE1 and NDE2 are not (58). In addition, NDE2 is induced after diauxic shift, i.e. growth on ethanol (260)

Electron transfer and ATP generation

In contrast to anaerobic conditions, where glycolytic NADH and excess biosyn-thetic NADH are regenerated by alcoholic fermentation and glycerol formation respectively, aerobic NADH reoxidation is mainly linked to activity of the respiratory chain. Under these aerobic conditions, electrons captured in the reduced forms of conserved moieties, such as NADH and FADH, are passed onto the mitochondrial respiratory chain and are ﬁ nally accepted by oxygen (oxidative phosphorylation). This cascade of electron transfers across the closely associated complexes of the respiratory chain, to the ﬁ nal electron acceptor oxygen via cofactors, such as ubi-quinone and cytochrome c results in the build-up of an electrochemical proton gradient (Δμ˜H+_{) across the mitochondrial membrane (Figure 8) (34). This proton}

mo-tive force (Δμ˜H+_{) consists of an electrical membrane potential difference (∆Ψ), which}

is negative on the inside, and a chemical component (∆pH) that is inside alkaline (179). In contrast to many other eukaryotes, including several yeasts, S. cerevisiae

NADH NAD+ H+ NAD(P)H NAD(P)+ I II III IV V Ex In Cyt c UQ Suc Fum Gut2p G3P DHAP NADH NAD+ _H+ _H+ _H+ ADP+Pi ATP ½ O2 H2O inner membrane outer membrane inter membrane matrix e -e -e -e- e -e

-Figure 8 Schematic representation of oxidative phosphorylation in the electron transport chain. From left to right: A proton pumping NADH dehydrogenase (complex I) is absent in Saccharomyces cerevisiae. The internal (In) and external (Ex) dehydrogenases transfer electrons (e-_{) directly to the ubiquinone pool (UQ). FAD-dependent succinate}

dehydrogenase (Complex II) donates electrons to the ubiquinone pool. The

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31 does not have a classical complex I NADH:ubiquinone oxidoreductase. Thus, it lacks the intrinsic proton-pumping activity of this initial step of the respiratory chain (66, 125). Electrons from FADH generated by succinate dehydrogenase (Sdh1p) as well as electrons from the three NADH dehydrogenases (Nde1,2p and Ndi1p) and those from Gut2p are transferred to the ubiquinone pool (Figure 8). From the ubiquinone pool electrons enter complex III and via cytochrome c they arrive in complex IV where they are ﬁ nally accepted by oxygen. In yeast, complexes III and IV translocate protons and thus generate a proton motive force, which drives the ATP synthase (complex V), which converts ADP and P_i to ATP (Figure 8). The gener-ated ATP can be use in free energy requiring cellular processes. The respiration efﬁ ciency, which can be expressed as the number of ATP molecules synthesized per electron pair transferred to oxygen (P/O ratio) depends on the H+_/ATP

stoichi-ometry of the ATP synthase and amount of translocated protons. In S. cerevisiae the effective P/O ratio in growing cells has been estimated to be close to unity (78, 313).

Glycerol metabolism

In anaerobic cultures of Saccharomyces cerevisiae, reoxidation of surplus NADH derived from biosynthetic reactions, proceeds via glycerol formation, which func-tions as an exclusive sink for reducing equivalents under anaerobic condifunc-tions (10, 25, 120, 288). Formation of one glycerol from glucose requires the net input of one ATP. The glycolytic intermediate dihydroxyacetone phosphate (DHAP) is ﬁ rst

reduced to glycerol-3-phosphate (G3P) by NAD+_{-linked glycerol-3-phosphate}

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intracel-32

Chapter 1

lular glycerol (268) and on the other hand it allows for regulated release of glycerol under osmotic down-shift (161, 162). Fps1p also mediates glycerol uptake, albeit at a lower rate than export (200). Recently, a member of the major facilitator family (Stl1p), amongst which are sugar transporters, was found (79) to enable proton symport of glycerol into cells during an immediate response to osmotic shock. Intra/extra cellular glycerol concentration ratios of up to 600 can be achieved by S.

cerevisiae (3, 201). Intra/extra cellular ratios up to 10,000 to 1 have been reported

in some osmotolerant yeasts (289, 308).

Reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by glyc-erol-3-phosphate dehydrogenase is differentially regulated. Although the two isoenzymes encoded by the GPD1 and GPD2 genes catalyze the same reaction they have different metabolic functions. Gpd1p functions during osmotic stress induced glycerol production (4, 149) and appears to be responsible for the dehy-drogenase activity in the G3P shuttle (147). The primary role of Gpd2p is redox mediated DHAP reduction during anaerobicity or (in)directly inhibited respiratory activity (9, 10, 25, 76). Under anaerobic or respiratory deﬁ cient conditions, Gpd2p is proposed to help establish mitochondrial redox balance by driving the ethanol-acetaldehyde shuttle (see earlier) through oxidation of NADH in the mitochondrial intermembrane space (283). G3P dephosporylation to glycerol is catalyzed by two isoenzymes of glycerol-3-phosphate phosphatase, encoded by GPP1 and GPP2 (196, 277). Both Gpp1p and Gpp2p are induced upon osmotic stress, although Gpp2 is more responsive (195). Gpp1p appears to be induced by redox mediation in similar way as Gpd2p (196, 209).

Increased Gpdp activity by overexpression of the encoding genes resulted in an increased glycerol production. However, neither overexpression of Gpp1p nor Gpp2p enzyme levels (10-fold) resulted in increased glycerol formation. This, ac-cording to the authors, indicates that Gpdp and not Gppp is the rate-limiting step in glycerol production (61, 62, 175, 241).

Mixed substrate utilization and cofactor regeneration

In addition to glycolytic and assimilatory formation of redox equivalents, cytosolic NADH can be generated by addition of an auxiliarly substrate. While in batch cul-tures the simultaneous utilization of substrates may be prevented by catabolite repression and/or kinetic interactions, substrate-limited cultivation generally allows for the simultaneous utilization of different carbon and energy sources by microor-ganisms (70, 131, 184).

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33 when a mixture of glucose and n-alkane is used. In this example oxaloacetate is synthesized de novo by heterotrophic CO₂ fi xation, or replenished by the glyoxyl-ate cycle, while acetyl-CoA is generglyoxyl-ated from the n-alkane. Without the n-alkane, acetyl-CoA would have to be generated from glucose as well, resulting in a lower carbon conversion effi ciency (16). Growth of Saccharomyces cerevisiae on a mixture of ethanol and glucose as the carbon source results in a higher biomass yield than could be expected from the growth yields on the separate substrates (57). The main reason for this phenomenon is more effi cient use of the different substrates in assimilatory and dissimilatory processes (301).

Of particular interest are the single carbon (C₁) substrates methanol, formal-dehyde and formate. C₁ substrates can often serve as a carbon source, but not all microorganisms have this ability (236, 309). Instead of serving as a carbon source,

C₁ compounds can be oxidized to carbon dioxide. During this process, redox

equivalents can be generated, which can be used to generate ATP via oxidative phosphorylation (16, 287). Many yeasts are capable of metabolizing methanol and the more oxidized derivatives in the linear degradation pathway to CO₂ (e.g.

Han-senula sp., Candida sp. and Pichia sp.) (115, 250). Methanol conversion to

form-aldehyde can take place by methanol oxidase, which uses molecular oxygen as a substrate and generates hydrogen peroxide. In Gram-positive bacteria, an NADH dehydrogenase has been described that can oxidize methanol to formaldehyde

(11, 12). As such, complete microbial oxidation of methanol to CO₂ can yield 2

or 3 NADH equivalents, depending on the linear oxidation pathway (Figure 9).

However, Saccharomyces cerevisiae only harbors NAD+_{-dependent formaldehyde}

(247) and NAD+_{-dependent formate dehydrogenases (208). Therefore S. cerevisiae}

is able to oxidize formaldehyde and formate, but not methanol, to carbon dioxide. Full oxidation yields 2 or 1 NADH respectively.

Figure 9 The linear methanol oxidation pathway to CO2 generates 3 NADH equivalents using

a methanol dehydrogenase (MeOHDH), while using alcohol oxidase only yields the two NADH in the formaldehyde dehydrogenase (Sfap) and formate dehydrogenase (Fdhp) steps.

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34

Chapter 1

Formate is a popular electron donor for cofactor regeneration in enzyme ca-talysis and whole-cell biotransformation processes. Formate oxidation does not result in toxic byproducts, as CO₂ does not accumulate in solution (320). Therefore, formate has been used as an electron donor for the production of mannitol by engineered Escherichia coli strains (131). Similarly, it has been demonstrated that formate oxidation by glucose-grown anaerobic cultures of E. coli leads to a shift towards fermentation products that are more reduced than glucose (22).

In S. cerevisiae CEN.PK113-7D, two isoenzymes of NAD+_{linked formate}

dehy-drogenase (FDH, EC 1.2.1.2), encoded by the FDH1 and FDH2 genes, are induced upon cultivation in the presence of formate (208). In aerobic, glucose-limited cul-tures, co-utilization of formate replaces redox equivalents derived from glucose dissimilation to enable respiration. At optimal formate-to-glucose ratios, this results in a scenario in which glucose is solely used for carbon assimilation, whereas for-mate functions entirely as a source of energy supply. This situation represents the ultimate separation of assimilation and dissimilation (Figure 10) (106, 310).

METABOLIC ENGINEERING OF SUBSTRATE UTILIZATION AND PRODUCT FORMATION

In the new carbohydrate economy, the bioreﬁ nery concept focuses on the produc-tion of biocommodities from hydrolysate feedstocks. The efﬁ cient conversion of

glucose biomass energy glucose biomass NADH, ATP formate glucose biomass NADH, ATP formate energy

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35 hydrolysate sugars to the desired products, herein is required. To achieve this goal, improvement of current and development of new platform organisms is essential. To illustrate this, two examples will be discussed below: (i) Metabolic engineering of S. cerevisiae, known for its capacity to produce ethanol, applied to broaden the substrate range. (ii) Since not only traditional, but also new less traditional platform chemicals can be produced, metabolic engineering of product formation will be discussed using glycerol, and one of its derivatives 1,3-propanediol as an example.

Metabolic engineering of substrate utilization: xylose fermentation by

Saccharomyces cerevisiae

For efﬁ cient conversion of the sugars in lignocellulosic hydrolysates to ethanol by

Saccharomyces cerevisiae, the substrate range of this yeast needs to be

broad-ened. A fi rst target would be the utilization of xylose, which besides glucose is the most abundant sugar in such hydrolysates. A fi rst attempt at metabolic engineering of xylose utilization by S. cerevisiae, therefore introduced xylose reductase (Xyl1p) and xylitol dehydrogenase (Xyl2p) from the yeast Pichia stipitis to convert xylose into xylulose. Xylulose can subsequently be phosphorylated by naturally present xylulose kinase, which links xylose metabolism to the pentose phosphate pathway (Figure 11) (138). However, the result of the introduced Xyl1p and Xyl2p was disap-pointing; growth on xylose as the sole carbon source was slow and xylose was almost entirely oxidized, without the formation of ethanol (7, 137). Simultaneous overexpression of the gene encoding xylulose kinase (XKS1) from P. stipitis further improved ethanol production (72). An intrinsic problem of the reductase/dehydro-genase system is cofactor specifi city. Xyl1p prefers NADPH for xylose reduction and Xyl2p preferentially reduces NAD+_{upon conversion of xylitol conversion. This}

results in an impairment of the redox balance (35, 36). The solution to this redox

Figure 11 Heterologous expression (grey) of xylose reductase (XR) and xylitol

dehydrogenase (XDH) in Saccharomyces cerevisiae links xylose metabolism to endogenous (dark) xylulose kinase (XK) and the pentose phosphate pathway. As an alternative, xylose isomerase can be heterologously expressed to convert xylose to xylulose without cofactor intercession.

xylose xylitol xylulose

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36

Chapter 1

problem was found in functional expression of a xylose isomerase that directly converts D-xylose to D-xylulose without the intercession of cofactors (Figure 11).

However, functional expression of a xylose isomerase from various sources in S.

cerevisiae was unsuccessful (97, 319). In 2003, a breakthrough was realized when

the xylose isomerase gene originating from Piromyces sp. E2 was functionally ex-pressed in S. cerevisiae (141). Subsequent targeted metabolic engineering strate-gies such as overexpression of all enzymes involved in the conversion of xylulose to glycolytic intermediates and deletion of the gene encoding for the xylitol forming enzyme aldose reductase, resulted in a S. cerevisiae strain growing anaerobically on xylose (142). Evolutionary engineering, a term used to describe rationally designed natural selection as a metabolic engineering tool (253), was used to further increase applicability of the strain for future industrial ethanol production by improving xy-lose consumption in a glucose/xyxy-lose mixture (143). Further broadening of the S.

cerevisiae substrate range (e.g. L-arabinose and uronic acids (294)), will eventually

lead to even more efﬁ cient and more economical fermentation of hydrolyzed plant material. This may help to decrease the burden on natural resources and can pos-sibly guide our economy to a more sustainable future.

Metabolic engineering of product formation

The majority of organic chemicals can be produced based on routes starting from only a few basic petrochemical building blocks. As an alternative, these building blocks, commonly referred to as platform chemicals, can be made from sugars via biochemical pathways, which are naturally present in organisms or which have been introduced via genetic engineering.

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metaboli-General introduction

37 cally engineering Saccharomyces cerevisiae. Not only has glycerol production itself shown to be important throughout history, it also serves as a model for other (po-tential) products which are more reduced than the carbohydrate feedstock, such as for example 1,3-propanediol.

Glycerol formation in a historical perspective

In the beginning of the 20th_{century, glycerol was primarily used for production}

of nitroglycerin as an explosive needed in mining and ordnance industries. Some glycerol was also used in cosmetics, drugs, toothpastes and tobacco processing (1). At the time, glycerol was mainly formed as a byproduct of saponifi cation of fats and oils during soap production (232). While studying the production of wine and beer, Pasteur found glycerol was formed to the extent of 2.0-3.6% of the glu-cose fermented by weight (219, 224). Evidently, with such low yields, commercial production of glycerol through regular yeast fermentation was not feasible. At the dawn of World War I, research on the mechanism behind alcoholic fermentation resulted in the fi nding of bisulfi te steered glycerol accumulation (52). While in-creasing amounts of glycerol were needed to provide Germany with glycerol for its weaponry and explosives industry, the blockade imposed by Britain on imperial Germany decreased availability of commodities, like glycerol (202). Germany was compelled to use the sulfi te steered process, patented by Connstein and Lüdecke, as an alternative way to produce glycerol (52, 53). Glycerol production surged to approximately 1000 tons per month during World War I. The fermentation of beet sugar in some 24 factories in Germany can be considered one of the fi rst large scale applications of industrial biotechnology (150). After the war, Carl Neuberg analyzed this process and found that instead of normal alcoholic fermentation, which he named the ‘fi rst form’ of fermentation, two new forms of fermentation could be distinguished.

In Neuberg’s ‘second form’ the fermentation was steered towards glycerol by trapping acetaldehyde with bisulﬁ te, which prevented its reduction with NADH to ethanol (189, 190). Instead, NADH reoxidation then occurs by reduction of dihy-droxyacetone phosphate (DHAP), resulting in glycerol-3-phosphate (G3P) followed by subsequent dephosphorylation to glycerol. Under optimal conditions glycerol yields could be increased to 27% by weight on the basis of sugar supplied at a maximum concentration of 45 g·L-1_{(90, 224). The sulﬁ te process was later improved}

using a mixture of bisulfi te and sulfi te, which is less toxic to the fermenting yeast than bisulfi te alone (47).

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38

Chapter 1

alkaline conditions by addition of high concentrations of sodium carbonate. After improvement of the latter process by Eoff and co-workers this mode of fermenta-tion gave a 10.5-24.2% conversion of sugars into glycerol (74, 75, 188, 190).

From the late 1940s, glycerol was increasingly chemically synthesized via more economical propylene oxidation and fermentative glycerol production slowly phased out. The availability of cheap molasses, the awareness that petroleum based glycerol production is not sustainable in the long run and an increased demand for glycerol were a drive for the reintroduction of biochemical routes some de-cades later (1). Processes based on Neuberg’s bisulﬁ te and alkali steered processes received interest once again. The productivity was improved even until the late 1980s and early 1990s by addition of CO₂ and application of a vacuum for effective removal of toxic acetaldehyde (126, 128), controlled aeration (127), immobilization of S. cerevisiae cells and the use of different fermentation modes (fed-batch, batch) (24, 116, 121, 315).

Osmotolerant yeasts have also been used for the production of glycerol. Con-trary to S. cerevisiae where glycerol is the primary osmolyte, formation of a vari-ety of other polyols like D-mannitol, erythritol and D-arabitol is inevitable during

fermentation with osmotolerant yeasts. Glycerol yields using such osmotolerant yeasts, are generally higher than in the traditional Neuberg processes employing

S. cerevisiae. The overall yield of polyhydric alcohols can be increased to 40% of

glucose supplied (224). However, none of the above-mentioned improvements on the sulﬁ te process were able to signiﬁ cantly increase glycerol productivity and lead to substantially reduced production costs.

Rational reprogramming of glycerol metabolism

With the advent of ‘modern’ metabolic engineering research on glycerol produc-tion shifted towards strategies employing recombinant DNA technology for a ra-tional reprogramming of cellular metabolism (17, 193). Initially, the sulﬁ te process was mimicked by blocking ethanol fermentation by genetic engineering. In an attempt to prevent NADH reoxidation by acetaldehyde reduction to ethanol, an

adh1∆adh3∆adh4∆ and inactive Adh2p mutant was constructed (68). A glycerol

yield on glucose of 0.51 mol glycerol·(mol glucose)-1_{was obtained. Unfortunately,}

the adh0_{strain was hampered by formation of toxic acetaldehyde and undesired}

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39 type reference strain (191). Combination with a second strategy, involving overex-pression of essential enzymes in the glycerol pathway (e.g. glycerol-3-phosphate dehydrogenase), resulted in a further increase of the yield (175, 191).

Instead of blocking NADH reoxidation in the fermentative branch, a forced split of fructose-1,6-bisphophate into equimolar amounts of DHAP and glyceraldehyde-3-phosphate (GAP) can in theory result in a yield of 1.0 mol glycerol·(glucose)-1_.

Although glycerol production was signiﬁ cantly improved upon deletion of trios-ephosphate isomerase (tpi1∆) (49-51), however growth on glucose as the sole carbon source was impossible. This was probably a result of accumulation of cytotoxic compounds such as DHAP and methylglyoxal (a product of spontane-ous degradation of triosephosphates) (167, 237). In this mutant all cytosolically generated NADH is required to reduce DHAP to glycerol and cytosolic NADH regeneration by mechanisms other than glycerol-3-phosphate dehydrogenase needed to be prevented. Indeed, additional deletion of nde1∆, nde2∆ and gut2∆ effectively decreased cytosolic NADH competition and restored growth, albeit slow (0.03 h-1_{). After additional natural selection for an increased growth rate, the}

tpi1∆nde1∆nde2∆gut2∆ strain produced glycerol up to a concentration of 219 g·L-1

at a yield of 0.99 mol glycerol·(mol glucose)-1_{in aerobic batch fermentations (206).}

This glycerol producing strain requires aerobic conditions to be able to suffi ciently provide ATP needed for growth. This approach successfully demonstrated the use of rigid carbon stoichiometry at the fructose-1,6-bisphosphate (FBP) branch point along with suffi cient redox and energy availability. However, by fi xing the carbon stoichiometry the maximum theoretical glycerol yield is limited to 1 mol·(mol glu-cose)-1_{and a further yield increase in this strain is not possible.}

Decline and prospects of the glycerol market

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ex-40

Chapter 1

plosive growth of glycerol supply, while demand is currently projected to increase only by 2.2% per year (132).

The upside of the collapsed glycerol market value is renewed interest in the use of glycerol as a platform chemical. The Soap & Detergent Association even spon-sors an annual US$3000 prize for research into new applications for glycerol (170). Examples are manifold: Valuable citric acid can be produced from raw glycerol using Yarrowia lipolytica (216). Another striking example is the development of glycerin butyl ether (GTBE) as an alternative fuel additive to methyl tertiary-butyl ether (MTBE). The use of MTBE as oxygenate has been partially banned pri-marily because of its high water solubility and offensive taste and odor, but MTBE is also feared for its suspected carcinogenicity (67, 172). The alternative GTBE, was developed by researchers of the University of Dortmund (Germany). Although toxicological studies have not been performed, GTBE is less likely to be toxic than MTBE due to water insolubility of GTBE (8, 21). However, whereas water insolubility limits spreading to the environment, build-up of the GTBE in the lipid structure of organisms may result in new health concerns.

Another way to valorize glycerol is fermentation to 1,3-propanediol (PDO). Due to its bifunctional organic nature, PDO has high potential for the use in poly-condensations (122). The PDO polymer, polytrimethylene therephtalate (PTT), is

marketed under the names Sorona®_{by DuPont and Corterra}®_{by Shell. For more}

than a century PDO is known to be a product of bacterial glycerol fermentation (331) and is produced by species of Klebsiella, Clostridia, Citrobacter, Enterobacter and Lactobacillus (13, 20, 23, 122, 173, 217, 332). Conversion yields of up to 0.82 mol PDO·(mol glycerol)-1_{have been achieved in genetically modiﬁ ed E. coli}

spe-cies (259) and 0.88 mol·mol-1_{in altered Klebsiella pneumoniae species (331). A}

100% PDO recovery can be achieved by liquid/liquid extraction (20). Owing to the unique nature and properties of PDO, prices of up to US$3.50 per lb. are reason-able in comparison to similar polymer feedstocks (42). However, market values are expected to be US$0.70-2.10 per lb. at production volumes ranging from 1 to 200 million pounds per year (42). Therefore, the use of the (even) lower cost feedstock glucose instead of glycerol is beneﬁ cial from an economical point of view.

PDO production from glucose

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41 A one-stage process would not only use a cheaper feedstock, but would also sig-niﬁ cantly reduce fermentation costs (144).

In an ambitious joint metabolic engineering effort by DuPont and Genencor International, construction of a single microorganism capable of utilizing D-glucose

was made possible. An Escherichia coli K12 strain was metabolically engineered to produce PDO at a rate of 3.5 g·(L·h)-1_{, a titer of 135 g·L}-1_{and a yield of 0.51 g}

PDO·(g glucose)-1_{(186). In this strain the conversion to glycerol was facilitated by}

overexpression of Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (encoded by GPD1) and glycerol-3-phosphatase (encoded by GPP2). A challenging next step was the complex reduction of glycerol to PDO. This involves a difﬁ cult chemical rearrangement of glycerol to 3-hydroxypropionaldehyde (3-HPA) by a

coenzyme B₁₂-dependent glycerol dehydratase. In this process, coenzyme B₁₂ is

occasionally rendered inactive and therefore requires regeneration to sustain glyc-erol dehydratase activity. The multiple enzymes needed in the ﬁ rst step of glycglyc-erol conversion to PDO encoded by the pdu operon, are heterologously expressed from Klebsiella pneumoniae. NADPH-dependent reduction of 3-HPA to PDO is en-coded by yqhD, endogenous to E. coli. Some undesired reactions, such as glycerol kinase and glycerol dehydrogenase were eliminated. In addition, the yield

restrain-ing PEP-dependent D-glucose phosphorylation is replaced with ATP-dependent

phosphorylation and facilitated diffusion comprising galactose permease (galP) and glucokinase (glk) endogenous to E. coli (63, 73, 185, 186). The attributes of three different microorganisms combined in one production strain, resulted in a 500-fold productivity improvement. Corn-derived PDO production is now a commercially vi-able process and DuPont plans to construct a large-scale PDO fermentation facility by 2006

AIM, SCOPE AND OUTLINE OF THESIS

This thesis focuses on metabolic engineering of redox metabolism in

Saccharomy-ces cerevisiae for improved glycerol production. Although industrial relevance for

glycerol production per se has dwindled with the explosive growth of the bioetha-nol and biodiesel markets, glycerol production still provides an interesting model system for one-step microbial production of compounds with a higher degree of reduction than the substrate, such as 1,3-propanediol.

Engineering of redox metabolism in yeast: New strategies for improved glycerol production

Engineering of redox

metabolism in yeast:

New strategies for

improved glycerol production

Engineering of redox metabolism in yeast:

New strategies for improved glycerol production

Prologue

Contents

Abbreviations

Chapter 1