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Chapter 1General introduction

From: Flikweert M.T. (1999) Physiological roles of pyruvate decarboxylase in Saccharomycescerevisiae. PhD thesis, Delft University of Technology, Delft. ISBN 90­9013020­9

The yeast Saccharomyces cerevisiae is best known for its characteristic physiological property: therapid fermentation of sugars to ethanol and carbon dioxide. This feature has been exploited formillennia in the leavening of d ough and in brewing (Chen & Chiger, 1985). Nowadays S. cerevisiaeis used for many diverse purposes and it has become one of the economically most important micro­organism that are used in large­scale biotechnological processes. The commercial applications of S. cerevisiae can be grouped in two major classes (Table 1). One classcomprises applications that are aimed at production of low­molecular­weight metabolites (ethanol,carbon dioxide, glycerol, flavour compounds). This class involves processes such as the production ofalcoholic beverages, the production of CO2 for the leavening of bread and the production of fuelethanol. The second class is aimed at production of yeast biomass or at the production of compoundsthat are directly derived from biomass. Examples of the latter class are the production of bakers' yeastand the production of yeast extracts. Furthermore, its eukaryotic nature and GRAS (GenerallyRecognised As Save) status have made S. cerevisiae a popular host for the large­scale industrialproduction of heterologous proteins (Burden and Eveleigh, 1990; Romanos et al., 1992).

Table 1. Industrial applications of Saccharomyces cerevisiae (Partly taken from: Burden and Eveleigh, 1990).

Metabolite directed

Production of alcoholic beverages (beer, wine, sake)

Production of bio­alcohol

Dough fermentation

Production of specialty chemicals

Biomass­directed

Production of baker's yeast

Production of (heterologous) proteins

Production of yeast extracts

Production of yeast's as a nutritional supplement

The fact that S. cerevisiae is still one of the most important industrial yeasts is to a large extent due toits long history in the bakers' yeast process. Because of this early application, great effort has been putin the development of fermentation technology and strain improvement, both by classical methods aswell as by genetic engineering. This research has been aimed at improving various critical productparameters, including fermentative capacity, productivity in the biomass production phase, storagestability and minimisation of byproduct formation. Additional research has been aimed at extendingthe range of applications of this multi­purpose, flexible industrial micro­organism. Compared with the tremendous deepening of fundamental knowledge on the genetics and molecularbiology of S. cerevisiae the understanding of its physiology and metabolism has somewhat laggedbehind (Walker, 1998). This is elegantly underlined by Gancedo & Gancedo (1994); "A century afterBuchner's report (1897), regulation of glycolysis is still not completely solved". Further investigationsto extend fundamental knowledge on the regulation of sugar metabolism in S. cerevisiae is not only ofscientific interest but may also be profitable for industrial applications. Furthermore, insight in thephysiology of S. cerevisiae, a popular laboratory model for 'the' eukaryotic cell, can in many cases beextrapolated to other important eukaryotic organisms, including Homo sapiens.

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A high level of knowledge about the genetics and biochemistry of S. cerevisiae has been obtained inthe last decades, with the completion of the S. cerevisiae genome­sequencing project in 1996 as animportant milestone (Bradbury, 1996; Goffeau et al., 1996). Even more recently, this has led to thedevelopment of techniques for genome­wide gene expression studies (De Risi et al., 1997).

Many of the open reading frames in the S. cerevisiae genome encode proteins of which the functionremains unknown. The functional analysis of the entire yeast genome will require a concerted effort ofscientist from different relevant disciplines (e.g. molecular genetics, microbial physiology and processengineering). In cases where optimisation of industrial cultivation methods has reached its limits, thisintegrated approach is a prerequisite for rational application of recombinant­DNA techniques('metabolic pathway engineering') to improve the cellular properties of yeasts and in particular S.cerevisiae.

Regulation of alcoholic fermentation

S. cerevisiae belongs to the physiological group of facultatively fermenting yeasts. These yeastsexhibit alcoholic fermentation under oxygen­limited conditions (Van Dijken & Scheffers, 1986).Industrial cultivation of S. cerevisiae at high biomass densities is hampered by this yeast's strongtendency towards alcoholic fermentation. For S. cerevisiae, the maintenance of aerobic cultivationconditions is not sufficient to avoid the occurrence of alcoholic fermentation. Even under fully aerobicconditions a mixed respiro­fermentative metabolism is observed when the sugar concentrationexceeds a certain threshold value (ca. 1 mM, Verduyn et al. 1984) or when the specific growth rate ishigher than the so­called critical growth rate (usually ca. two­thirds of the maximum specific growthrate on glucose; Petrik et al. 1983; Postma et al.,1989). Formation of fermentation byproducts such as ethanol, acetic acid and glycerol not only reduces thebiomass yield but also affects the formation of biomass­related products. To prevent alcoholicfermentation, tight control of the sugar concentration is needed. In industry a low sugar concentrationis maintained by aerobic sugar­limited fed­batch cultivation at growth rates below the critical growthrate (Reed, 1982; Barford, 1987; Hensing et al., 1995). However, due to imperfect mixing in large­scale bioreactors local sugar concentrations often exceed the respiro­fermentative threshold (Sweereet al. 1988). Local high sugar concentrations trigger alcoholic fermentation within seconds, resultingin a local ethanol production and thus in a decrease of the biomass yield. This rapid response is calledthe short­term Crabtree effect (Petrik et al., 1983; Van Urk, 1989). The external environment and changes within this environment strongly influence the physiologicalbehaviour of yeast. The best­documented environmental factors are the availability of glucose andoxygen. The key intermediate pyruvate, the end product of glycolysis, is located at an importantbranch point in sugar metabolism. At this point the carbon flux is distributed among respiration andfermentation. In facultatively fermenting yeasts, which can produce ATP either by respiration or byfermentation, these processes compete for pyruvate and NADH. The resulting flux distributiondepends on the environmental conditions (i.e. absence/presence of oxygen, type or concentration ofsugar) and on the strains used (Table 2). Several regulatory phenomena reflecting the distribution ofpyruvate over respiratory and fermentative dissimilation have been described, including the 'Pasteureffect', the 'Custers effect', the 'Kluyver effect' and the 'Crabtree effect'. A short explanation of theseeffects is given below.

Table 2. Example of types of glucose metabolism in Saccharomyces cerevisiae depending on the availability of oxygenand carbon source (taken from Käppeli & Sonnleitner, 1986).

Conditions Type of metabolism

Batch culture Aerobic growth

Carbon­limited Respiro­fermentative

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Oxygen­limited Respiro­fermentative

Anaerobic growth Fermentative

Chemostat culture

Aerobic growth at lowdilution rates

Carbon­limited Respiratory

Oxygen­limited Respiro­fermentative

Aerobic growth at highdilution rates

Carbon­limited Respiro­fermentative

Oxygen­limited Respiro­fermentative

Anaerobic growth Fermentative

Pasteur effectThe definition of the Pasteur effect is the inhibition of sugar consumption by aerobiosis (Lagunas,1986).This physiological behaviour or effect was named after Pasteur because of his observations in1861 on experiments with cultivated brewers' yeast in the presence and absence of air. In her excellentreview on the Pasteur effect, Lagunas (1986) identified e this observation as an artefact due to theabsence in Pasteur's experiments of the extra nutrients (unsatured fatty acids and sterols) required forgrowth of S. cerevisiae under anaerobic conditions.In S. cerevisiae, the Pasteur effect is only observed under special experimental conditions, notably atvery low dilution rates in the chemostat and in resting­cell suspensions. In sugar­limited chemostatcultures the growth rate and the glucose uptake rate is low due to a low sugar concentration; in restingcells the glucose uptake rate is low due to an inactivation of the sugar transport system. Themechanism which underlies this phenomenon is probably the higher affinity for one of theintermediates pyruvate, acetaldehyde and NADH of the respiratory system over the fermentative route(Lagunas et al., 1982; Lagunas, 1986).

Custers effectThe Custers effect is the inhibition of alcoholic fermentation when a yeast culture is transferred fromoxygen­limited to anaerobic conditions (Bruinenberg et al., 1983; Van Dijken & Scheffers, 1986).This occurs in Brettanomyces species when they are grown on glucose (Custers, 1940), due to theirinability to form glycerol or other reduced metabolites under anaerobic conditions (Custers, 1940;Wijsman et al., 1984; van Dijken & Scheffers 1986). The addition of an electron acceptor duringanaerobic conditions abolished the inhibition of alcoholic fermentation and this led to the hypothesisthat the Custers effect is caused by a redox imbalance. Conversion of glucose into ethanol involves aclosed redox balance. The NADH formed in other processes (biosynthesis, acetate production) cannotbe reoxidized in this process. Under aerobic conditions the respiratory chain reoxidizes this NADH.

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Under anaerobic conditions glucose has to be partly converted to glycerol as a redox sink at theexpense of ATP (Scheffers 1966).

Kluyver effectThe Kluyver effect has been described as follows: '.. certain yeasts can utilize particular disaccharidesaerobically, but not anaerobically, although these yeast's can use one or more of the componenthexoses anaerobically.' (Sims & Barnett, 1978). This phenomenon is widespread under facultativelyfermentative yeasts. It has been reported in more than 100 yeast species (Sims and Barnett, 1978;Weusthuis, 1994). S. cerevisiae does not exhibit this effect. The yeast Candida utilis does show theKluyver effect: it is able to ferment glucose under oxygen­limited conditions but it cannot fermentmaltose under these conditions . C. utilis is, however, able to grow rapidly on maltose under aerobicconditions by respiring this disaccharide.. Yeasts that exhibit the Kluyver effect for a particular sugarare able to ferment glucose, suggesting that t the Kluyver effect must be caused in a differencebetween the metabolism of this sugar and that of glucose. The exact mechanism underlying thisphenomenon has not yet been resolved. However there are some plausible explanations like a loweredrate of sugar transport (i.e. a transport limitation) or control of the synthesis of the sugar transporter(Barnett and Sims, 1982; Barnett, 1992, Weusthuis et al., 1994; Kaliterna et al., 1995). Furthermoresome yeasts have a reduced activity of pyruvate decarboxylase during growth on non­glucose sugars,with may contribute to a lower flux through glycolysis (Sims & Barnett, 1991). Weusthuis et al.(1994) proposed feedback inhibition of disaccharide utilisation by ethanol as a possible cause of theKluyver effect. They have shown that, indeed, added ethanol suppresses the utilisation of maltose byC. utilis (Weusthuis et al.,1994).

Crabtree effectIn general, the Crabtree effect is described as the occurrence of alcoholic fermentation under aerobicconditions (Crabtree, 1929; de Deken, 1966; Fiechter et al., 1981; Van Dijken & Scheffers, 1986).More specifically, the long­term Crabtree effect and the short­term Crabtree effect can bedistinguished (Van Urk, 1989).

The long­term Crabtree effect is defined as aerobic alcoholic fermentation under steady­stateconditions at high specific growth rates (Fiechter et al., 1981; Postma et al., 1989). When S. cerevisiae is cultivated in a glucose­limited chemostat the long­term Crabtree effect appearswhen the dilution rate (and consequently the specific growth rate) exceeds the so­called 'criticalspecific growth rate' (Figure 1). The long­term Crabtree effect has been explained from a limitedrespiratory capacity of respiratory sugar metabolism (Fiechter et al., 1981; Petrik et al., 1983; Riegeret al., 1983; Käppeli, 1986). Postma (1989) studied the correlation between the occurrence of the long­term Crabtree effect and thelevels of key enzymes in glucose metabolism in glucose­limited chemostat cultures grown at variousdilution rates (Postma et al., 1989). An uncoupling effect of respiration (reflected by an enhancedqO2; Figure 1) coincided with the accumulation of some acetic acid in the cultures. Postma et al.proposed that the increased respiration rate might reflect uncoupling by acetate. The accumulation ofacetate was explained by these authors from an insufficient amount of acetyl­CoA synthetase at highgrowth rates (Postma et al., 1989). Similarly, it was proposed that the onset at alcoholic fermentationat even higher dilution rates might be due to a limited capacity of acetaldehyde dehydrogenase, thusnecessitating the redirection of the carbon flux via alcohol dehydrogenase (Postma, 1989). The definition of the short­term Crabtree effect is the instantaneous aerobic alcoholic fermentationupon addition of excess sugar to sugar­limited and/or non­fermenting cultures (Rieger et al., 1983;Verduyn et al., 1984; Van Urk, 1989; Figure 2). This effect was explained in terms of an 'overflow' inmetabolism. When a culture of S. cerevisiae is exposed to excess sugar the glucose consumption rateincreases until the respiratory capacity becomes limiting (Petrik et al.,1983; Käppeli and Sonnleitner,1986). Van Urk (1989) studied the short­term Crabtree effect and compared some Crabtree­positive withCrabtree­negative yeasts. Several differences were revealed between Crabtree­positive and Crabtree­negative yeasts, including different kinetics of glucose uptake, rates of glycolysis and glycogen

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formation and the levels of a number of key enzymes involved in pyruvate metabolism. Moststrikingly, ethanol production rates showed a clear positive correlation with the level of pyruvatedecarboxylase in Crabtree­positive yeasts. All Crabtree­positive yeasts contained higher levels of thisfermentative key enzyme than the Crabtree negative yeasts. Furthermore, high levels of acetaldehydedehydrogenase and acetyl­CoA synthetase were observed in the Crabtree­negative yeasts. These may'pull' the carbon flux towards respiration, despite the presence of some pyruvate decarboxylase. Recently, Pronk and co­workers (1998) increased pyruvate decarboxylase levels in the Crabtree­negative yeast Kluyveromyces lactis by more than ten­fold by introduction of multiple copies of the S.cerevisiae PDC1 gene. When an aerobic steady­state chemostat culture of this Pdc­overproducingstrain was exposed to excess sugar, aerobic alcoholic fermentation was absent and the strain retainedits Crabtree­negative phenotype. This indicates that, apparently, a high level of pyruvatedecarboxylase is not the sole reason for ethanol formation under aerobic conditions. The hypothesis that a limited activity of acetyl­CoA synthetase is the cause of acetate excretion by S.cerevisiae has recently been tested by overproducing this enzyme. When respiring cultures of suchengineered strains were exposed to glucose excess, acetate production were not reduced, despite a 3­6fold overproduction of acetyl­CoA synthetase (de Jong­Gubbels et al., 1998). This indicates that a'bottle­neck' at the level of acetyl­CoA synthetase is not the sole cause for acetate excretion.

Figure 1. Example of the long­term Crabtree effect in Saccharomyces cerevisiae. Panel A: Specific rate of oxygen uptake,carbon dioxide production and biomass yield as a function of the dilution rate (i.e. specific growth rate) in glucose­limited cultures of S. cerevisiae CBS 8066. Panel B: Steady­state concentrations of metabolites in glucose­limitedchemostat cultures of S. cerevisiae CBS 8066. (Taken from Postma et al., 1989)

Figure 2. Example of the short­term Crabtree effect in Saccharomyces cerevisiae. Production of ethanol and acetate aftera pulse of glucose (50 mM) to an aerobic glucose­limited chemostat culture (Van Urk et al., 1988).

Pyruvate metabolism

After uptake, yeasts and other micro­organisms degrade sugars via catabolic pathways to produceATP. A small number of common intermediates in these catabolic pathways is required forbiosynthesis of biological macromolecules. These biosynthetic reactions, which involve anabolicpathways, require a free­energy input (ATP).

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In S. cerevisiae, the major pathway involved in the initial steps of glucose catabolism is the Embden­Meyerhof­Parnas pathway (EMP­pathway or glycolysis). This pathway couples the conversion ofglucose into pyruvate to the formation of only 2 ATP via substrate­level phosphorylation.

Pyruvate, the end­product of glycolysis, is an important intermediate in the metabolism ofcarbohydrates by S. cerevisiae (for a review see Pronk et al., 1996). As pyruvate is located at thebranch­point between respiratory and fermentative carbon metabolism, flux distribution at the level ofpyruvate is of crucial importance for byproduct formation by S. cerevisiae. The three major pathwaysof pyruvate metabolism are depicted in Figure 3. It is important to note that byproduct formation isunlikely to be completely governed by carbon metabolism. Besides competition for pyruvate, themitochondria (respiration) and alcohol dehydrogenase (fermentation) also compete for the cytosolicNADH formed in glycolysis. This interaction between respiration and fermentation at the redox levelis the subject of the PhD project of ir. Karin Overkamp (project started in 1998). A majorcomplicating factor in this area is that S. cerevisiae possesses different systems for mitochondrialoxidation of cytosolic NADH, including an external NADH dehydrogenase and the glycerol­3­phosphate shuttle (Luttik et al.1998; Larsson et al. 1998).

Figure 3. Schematic representation of the key enzymic reactions of pyruvate metabolism in Saccharomyces cerevisiae.Pyc, pyruvate carboxylase; Pdh, pyruvate­dehydrogenase complex; Pdc, pyruvate decarboxylase; Adh, alcohol

dehydrogenase; Ald, acetaldehyde dehydrogenase; Acs, acetyl­coenzyme A synthetase.

Under anaerobic conditions, pyruvate dissimilation involves its conversion to ethanol and carbondioxide. The physiological function of this conversion is to maintain the redox balance by reoxidationof NADH formed in glycolysis which, during fermentative growth, is the only way to provide theyeast with ATP. The intermediate in this reaction is acetaldehyde. During fermentation, pyruvate isfirst converted into acetaldehyde, a reaction catalysed by the enzyme pyruvate decarboxylase (Figure3). The acetaldehyde acts as an electron acceptor for NADH reoxidation. The reaction is catalysed byalcohol dehydrogenase (Figure 4).An alternative route for the regeneration of NAD+ is the conversion of dihydroxyacetone phosphate toglycerol. If acetaldehyde is trapped by the non­toxic hydrogen sulfite, the rate of glycerol formationincreases (Neuberg, 1919). This modified fermentation is known as Neuberg's second fermentation.This redirection of metabolic fluxes to stimulate the formation of a commercially importantcompound (glycerol was used in the production of explosives) can be considered as 'metabolic

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engineering avant la lettre'.A completely respiratory sugar metabolism requires that glycolysis be coupled to the tricarboxylicacid (TCA) cycle, which takes place in the mitochondria. Respiratory dissimilation of pyruvaterequires its conversion to acetyl­CoA, the fuel of the TCA­cycle. In S. cerevisiae there are twopathways via which this can occur. The first is a direct oxidative decarboxylation by the mitochondrialpyruvate­dehydrogenase complex after pyruvate is transported, probably via a carrier, into themitochondria (Nalecz et al., 1991). Secondly, pyruvate can be converted to acetyl­CoA by theconcerted action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl­CoA synthetase(Figure 3; Holzer & Goede, 1961; Pronk et al., 1994). This indirect route is often referred to as thepyruvate­dehydrogenase bypass.

Figure 4. Reoxidation of NADH via the alcoholic fermentation pathway in Saccharomyces cerevisiae. Pdc, pyruvatedecarboxylase; Adh, alcohol dyhdrogenase.

The subcellular localisation of the enzymes of this PDH­bypass is not entirely known. However,enzyme activities have been shown to be present at least partially in the cytosol (Van Urk et al., 1989;Jacobson & Bernofski, 1974; Kispal et al., 1991). Cytosolic acetyl­CoA can be transported into themitochondrion via the carnitine­acetyl transferase shuttle. In a Pdh­negative mutant of S. cerevisiae itwas observed that this Pdh­bypass can completely meet the acetyl­CoA requirement. Operation of thisroute results in a lower biomass yield due to the increased ATP requirement for the synthesis ofacetyl­CoA (Pronk et al., 1994).Only under aerobic sugar­limited conditions at low specific growth rates S. cerevisiae exhibits a fullyrespiratory metabolism. A well­suited cultivation method to meet these requirements is the chemostat(Pirt, 1975).Under these conditions, pyruvate can be completely oxidized to carbon dioxide and watervia the TCA­cycle. Being a Crabtree­positive yeast, S. cerevisiae shows a respiro­fermentativemetabolism under aerobic conditions in the presence of excess sugar. Holzer (1961) proposed that thismay be the result of a competition for pyruvate between respiration and fermentation. This hypothesiswas based on the different kinetic properties of the PDH complex and pyruvate decarboxylase. Thepyruvate dehydrogenase complex has a tenfold higher affinity for pyruvate whilst its capacity forpyruvate is about tenfold lower compared to pyruvate decarboxylase (Figure 5). The PDH complex islocated in the mitochondria, but experiments have shown that the Km of isolated mitochondria issimilar to that of the PDH­complex (Van Urk et al., 1989). Dissimilation of pyruvate via pyruvatedecarboxylase is favoured at high pyruvate concentrations (Holzer, 1961; Rieger et al., 1983). At lowpyruvate concentrations the main flux is directed via the PDH­complex (Boiteux & Hess, 1970;Holzer & Goede, 1961; Kresze & Ronft, 1981). At high glucose concentrations or at high dilutionrates the pyruvate concentration is high. Consequently a relatively large part of glucose is fermentedrather than respired under these conditions. On a longer timescale this effect is enhanced because the

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expression of respiratory enzymes is repressed by glucose while the expression of pyruvatedecarboxylase is several fold induced by glucose.Several intermediates of the TCA­cycle are used for biosynthetic purposes. To keep the cycle fromterminating oxaloacetate is transported into the mitochondria for replenishing purposes. Thisoxaloacetate is synthesised from pyruvate via the anaplerotic (replenishing) carboxylation of pyruvateby pyruvate carboxylase (Figure 3). The two major dissimilatory enzymes (PDH and PDC) which directly compete for pyruvate play animportant role in the distribution of the glycolytic flux over fermentation and respiration and thereforethey will be discussed separately below.

Pyruvate­ dehydrogenase complex

The yeast pyruvate­dehydrogenase complex (PDH­complex) is an enzyme system that catalyses alipoic­acid­mediated oxidative decarboxylation of a­oxo­acids (Reed, 1974; Yeaman, 1989). Thislarge multi­enzyme complex has an apparent molecular weight of 8·106. It is a member of a family ofrelated dehydrogenase complexes, consisting of 2­oxoglutarate dehydrogenase, branched­chain 2­oxoacid dehydrogenase and pyruvate dehydrogenase. Each of the three complexes consists of threemajor components, E1 (substrate specific dehydrogenase; EC 1.2.4.1) which, in the case of the PDHcomplex, consists of two subunits (E1 and E1), E2 dihydrolipoamide acyltransferase (EC 2.3.1.12)and E3 (dihydrolipoamide dehydrogenase; EC 1.6.4.3), which all act in sequence (Reed and Yeaman,1987). The E1 and E2 subunits are complex specific, whilst the E3 subunit is common in all threecomplexes (Reed, 1974). A fourth component, protein X, is probably involved in the assembly of thecomplex (McCartney et al., 1997). When all five proteins (E1, E1, E2, E3 and X) of the PDHcomplex are assembled, an active complex is formed (Kresze & Ronft, 1981). The composition of thePDH complex is summarised in table 3.The E1 subunit catalyses the TPP­dependent decarboxylation of pyruvate. In mammalian cells theactivity of the PDH­complex is regulated by phosphorylation of three serine residues in the E1subunit resulting in inactivation of the PDH­complex. In S. cerevisiae only one site containing aserine residue is found which can be phosphorylated in vitro (Uhlinger et al., 1986; James et al.,1995). In the mammalian PDH­complex, phosphorylation and dephosphorylation are catalysed by aPDH­specific kinase and phosphatase, respectively. However, no PDH­specific kinase or phosphatasehave been isolated from S. cerevisiae.Kinetic studies with purified PDH complex from S. cerevisiae have revealed a Km value for pyruvateof 625 µM at a pH of 8.1 (Kresze & Ronft, 1981). The Km decreased to 200 µM when the pH wasdecreased to 6.5 (Kresze & Ronft, 1981; Keha et al., 1982).

Table 3. Structural genes, peptide size and catalysed reaction of the subunits of the pyruvate dehydrogenase complex.Abbreviations; TPP, thiamine pyrophosphate, FAD flavin adenine dinucleotide. (Partly taken from Wenzel, 1994 andPronk, 1996)

Enzyme Structuralgene

Peptidesize(kDa)

Cofactor Reaction

E1 PDA1 45 TPP Decarboxylation ofpyruvate

E1 PDB1 35

E2 LAT1 56 Lipoamide Transfer of acetyl group toCoA

E3 LPD1 54 FAD Reoxidation of lipoamide(E2)

X PDX1 50 Lipoamide Binding of E3 to E2 core

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Pyruvate decarboxylase (EC 4.1.1.1)

Pyruvate decarboxylase (Pdc) catalyses the irreversible cleavage of pyruvate to acetaldehyde andcarbon dioxide. The PDC enzyme and/or the genes encoding for PDC have been isolated fromdifferent organisms (Table 4). Pyruvate decarboxylase was first detected in yeast in 1911 by Neubergand Karczag who described the decarboxylation of pyruvate to acetaldehyde by fermenting yeast. Pdcis also known for the cleavage of other a­oxo­acids, in particular the conversion of branched chainoxo­acids to its corresponding aldehydes in the Ehrlich pathway. Although it is not sure whether ornot Pdc is involved in this route.In S. cerevisiae, several PDC­genes have been described. Beside the structural genes PDC1 (Schmittet al., 1983), PDC5 (Seeboth et al., 1990) and PDC6 (Hohmann, 1991a,b), encoding the active PDCisoenzymes, also regulatory genes have been described. These genes are PDC2 (Schmitt &Zimmerman, 1982), PDC3 (Wright et al., 1989) and PDC4 (Seehaus, 1986) which are probablyinvolved in the control of expression of the structural genes PDC1 and PDC5. Expression of thesegenes is enhanced during growth on glucose. The glucose induction of the PDC1 mRNA level wassignificantly stronger (fivefold) than that of the PDC5 mRNA level (Smith et al., 1983).Deletion of the PDC1 gene resulted in a fivefold increase of the transcription of the PDC5 genesuggesting that the PDC genes are be subject to autoregulation (Schaaf et al., 1989; Hohmann &Cederberg, 1990). Deletion mutants expressing neither PDC1 nor PDC5 lacked pyruvatedecarboxylase activity (Seeboth et al., 1990; Hohmann, 1991b). With this double mutant the existenceof a third PDC gene (PDC6) was shown and it was isolated by low­stringency hybridisation of agenomic library with a PDC1 probe (Hohmann, 1991a). The PDC6 sequence had a high similarity tothose of PDC1 and PDC5. Expression could not be demonstrated (Hohmann, 1991b). However,spontaneous revertants of the pdc1pdc5 double mutant have been found in which a recombination hadoccurred resulting in a fusion of the PDC6 open reading frame and the PDC1 promotor. This yielded afunctional Pdc enzyme (Hohmann 1991a). The role of PDC6 is unclear. It is not known either underwhich conditions it is expressed. PDC2 regulates the expression of the structural PDC genes. Deletionof the PDC2 gene resulted in a ten fold reduction of the PDC activity (Hohmann, 1993). Furthermore,PDC2 is involved in the regulation of thiamine pyrophosphate (TPP) synthesis (Hohmann &Meacock, 1998).

Table 4. Pyruvate decarboxylases of various yeast's, fungi, bacteria and plants (partly taken from Pohl, 1997).

Organism Species Subunit (kDa)

Yeast Saccharomyces cerevisiae 61.5 (PDC1)

62.0 (PDC5)

Kluyveromyces marxianus 61.9

Hanseniaspora uvarum 61.1

Kluyveromyces lactis ­

Fungi Neurospora crassa 62.3

Aspergillus parasiticus 64

Bacteria Zymomonas mobilis 60.9

Plants Zea mays (maize) 65.4

Oriza sativa (rice) 65.1

Lycopersicon esculentum(tomato) 62 (a) 64 (b)

Pisum sativum (pea) 64.0

Nicotiana tabacum (tobacco) 67.1

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The catalytically active PDC enzyme is a tetramer composed of two dimers. The enzyme functionsonly with the cofactor TPP along with the metal ion Mg2+ (Hübner et al., 1992). The four subunitsare identical and have a molecular mass of approximately 60 kDa each (Kuo et al., 1986). In thedimers the two subunits are tightly bound. The catalytic activity of the PDC tetramer is at its optimumat pH 6.2. At the alkaline pH 8.4 the tetramer is fully dissociated into the inactive dimers (König et al.,1992). Between these two pH values an equilibrium exists between the concentration of the tetramerand the dimers. This pH­dependent reversible dissociation of the tetramer state to the dimer state ofPdc is also found in pea seeds (Gounaris et al., 1971; Mücke et al., 1996). Since there is a pH of about6.8 in the cytosol, the tetramer is active in vivo. At high glycolytic fluxes, the cytosolic pyruvate concentration is high. During conditions of glucoseexcess significant concentrations of pyruvate are observed extracellularly, which can be taken as anindicator for the intracellular concentration (Postma et al., 1989). High concentrations of pyruvatecould bring down the pH, thus favouring formation of the PDC tetramer. Yeast PDC is activated by itssubstrate, pyruvate (Boiteux & Hess, 1970; Hübner et al., 1978,1988). Inorganic phosphate is acompetitive inhibitor of the enzyme (Hübner et al., 1978; Van Urk et al.JAAR, chapter IV). Howevera transient exposure to glucose will lead to an immediate reduction of the intracellular phosphateconcentration (Van Urk, 1989). These properties of PDC together with the observation that bothstructural genes encoding PDC (PDC1 and PDC5) are induced during growth on glucose (Hohmann,1991; Chapter 2), indicate that pyruvate will be metabolised preferentially via pyruvate decarboxylaseduring exposure to excess glucose.

Context and scope of this thesis

The work described in this thesis is aimed at elucidating the physiological functions of pyruvatedecarboxylase in S. cerevisiae. Furthermore, the feasibility of minimising the formation of ethanol andacetate in biomass­directed applications of this yeast by modifying pyruvate­decarboxylase isexperimentally investigated. These problems have been addressed by quantitative physiologicalstudies on wild­type and genetically engineered yeast strains. The molecular­genetic part of the workis part of collaborations inside the Research School BSDL (Biotechnological Sciences Delft­Leiden;with the group of Dr. H.Y. Steensma and Prof. P.J.J. Hooykaas at Leiden University) and outside theresearch school (with the group of Dr. P. Kötter and Prof. K.D. Entian in Frankfurt, Germany).Within BSDL, carbon and redox metabolism of industrial yeasts is investigated by a combination ofgenetic, physiological and process­engineering techniques. Distribution of metabolic fluxes at thepyruvate branchpoint is a subject which receives special attention, as is evident from three recentlypublished PhD theses. The thesis of Thibaut Wenzel (Leiden University, 1994) deals with themolecular genetics, regulation and physiological function of the pyruvate­dehydrogenase complex inS. cerevisiae. The thesis of Marco van den Berg (Delft University of Technology, 1997) focuses onthe molecular genetics and regulation of acetyl­CoA synthetase in this yeast. Finally, the thesis ofPatricia de Jong­Gubbels (Delft University, 1998) addresses the physiological role of acetyl­CoAsynthetase, pyruvate carboxylase and malic enzyme in S. cerevisiae.The research described in this thesis was part of a national project entitled "Metabolic Fluxes inYeasts and Fungi", subsidised by the Dutch Ministry of Economic Affairs via the Dutch Associationof Biotechnological Research Schools (ABON). This project, coordinated by Prof. K. van Dam(University of Amsterdam) involved the active participation of a number of academic research groups,addressing various aspects of glycolytic flux in industrially important yeasts and filamentous fungi.Simultaneous with the work described in this thesis, a second BSDL PhD project dealing withpyruvate metabolism was part of the ABON program. This project, carried out by Anne­MarieZeeman (Leiden University; PhD thesis in preparation) focuses on the genetics and physiology ofpyruvate metabolism in the Crabtree­negative yeast Kluyveromyces lactis.During the research described in this thesis, the Delft yeast group were involved in a EuropeanCommission research project entitled: "From gene to product in yeast: a quantitative approach". Theobjective of this project was to provide a quantitative description of S. cerevisiae as a cell factory forthe production of high­added­value products, particularly heterologous proteins (Osseweijer & Van

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Dijken, 1998). The choice of the pyruvate node as one of the focal points of research in this project,which involved 10 research groups from 8 European countries, offered a platform for discussion ofthe work with colleagues from outside the Netherlands.

Outline of this thesis

Chapter 2 of this thesis describes a physiological characterisation of a Saccharomyces cerevisiaestrain in which all three structural genes encoding pyruvate decarboxylase isoenzymes (PDC1, PDC5and PDC6) were disrupted. Growth of the Pdc­ mutant in batch and chemostat cultures on definedminimal media was compared with growth of the isogenic, prototrophic wild­type strain. The experiments described in Chapter 2 led to the conclusion that absence of pyruvate decarboxylasecauses a requirement for C2­compounds. In Chapter 3, the hypothesis that pyruvate decarboxylase isessential for the provision of cytosolic acetyl­CoA is investigated in more detail. Furthermore, thischapter addresses an apparent discrepancy in the growth characteristics of Pdc­ strains when grown oncomplex and media..Chapter 4 focuses on the response of steady­state, respiring cultures of Pdc­ S. cerevisiae to glucosepulses. Furthermore, this chapter addresses the question whether the S. cerevisiae PDH­complex isinactivated upon exposure of respiring cells to excess glucose. The experiments discussed in Chapter 4 demonstrated that the glycolytic flux observed after exposureof respiring cultures of Pdc­ S. cerevisiae to excess glucose was much lower than that of wild­typecultures. In Chapter 5, the hypothesis was tested that the reduced glycolytic flux in the mutant strainwas due to a reduced capacity for reoxidation of glycolytic (cytosolic) NADH. To this end, attemptswere made to restore the glycolytic flux of the Pdc­ mutant to wild­type levels by addition of anexternal electron acceptor.To investigate whether the occurrence of aerobic fermentation may be a consequence of a competitionfor pyruvate between respiration and fermentation, the effects of overproduction of pyruvatedecarboxylase on the flux distribution at the pyruvate branchpoint was studied in steady­statechemostat cultures. These experiments are discussed in Chapter 6The C2­requirement that results from complete elimination of pyruvate decarboxylase from the cells(Chapters 2 and 3) implies that this is not a viable option for reduction of byproduct formation inindustrial strains. Therefore, in Chapter 7, byproduct formation is studied in a strain with a reduced(non­zero) level of pyruvate decarboxylase. In addition to its role in pyruvate metabolism, it has beenproposed that pyruvate decarboxylase is a key enzyme in the production of fusel alcohols frombranched­chain 2­oxo acids. The role of pyruvate decarboxylase in this so­called 'Ehrlich pathway' isinvestigated in Chapter 8

References

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