INTRODUCTION

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Under most growth conditions, alcoholic fermentation is the predominant mode of sugar dissimilation in Saccharomyces cerevisiae (baker's yeast). A completely respiratory sugar metabolism is only possible in aerobic cultures grown under sugar limitation and at relatively low specific growth rates (31). This principle is applied in industrial baker's yeast production, where aerobic fermentation must be avoided because it results in a reduced biomass yield (9).

At high specific growth rates, even aerobic glucose-limited cultures exhibit mixed respirofermentative metabolism (21, 31, 33). This phenomenon is sometimes attributed to a limited respiratory capacity (36, 42). Indeed, models assuming a bottleneck in respiratory glucose dissimilation adequately describe the behavior of sugar-limited chemostat cultures (1, 2). Being purely empirical, such models cannot offer a mechanistic explanation for the onset of respirofermentative metabolism at high specific growth rates. Rate-limiting reactions in respiratory sugar metabolism might reside either in intermediary carbon metabolism (e.g., in the tricarboxylic acid cycle), in mitochondrial electron transport, or in a combination of the two (45).

An alternative explanation for the occurrence of aerobic alcoholic fermentation at high specific growth rates is the competition of respiration and fermentation for pyruvate (18, 36, 46), a metabolite located at the branch point between respiration and fermentation (Fig. 1) (35). To be respired, pyruvate must be oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA); this reaction is catalyzed by the mitochondrial pyruvate dehydrogenase complex. Alcoholic fermentation requires decarboxylation of pyruvate to acetaldehyde by the cytosolic enzyme pyruvate decarboxylase (Pdc). Isolated mitochondria and purified pyruvate dehydrogenase exhibit a much lower K_m for pyruvate than does Pdc (19, 25, 33, 46). At low rates of glucose dissimilation during respiratory glucose-limited growth, the intracellular pyruvate concentration is below the K_m of pyruvate dehydrogenase (33). Since, in addition to a high K_m, Pdc exhibits cooperativity with respect to pyruvate (10, 11, 20), pyruvate will be preferentially metabolized via pyruvate dehydrogenase at low intracellular pyruvate concentrations. In contrast, at high intracellular pyruvate concentrations, pyruvate will be predominantly metabolized via Pdc; in wild-type cells, the later reaction has a much higher V_max than does mitochondrial pyruvate oxidation (31, 35, 46).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Central role of Pdc in respiratory and respirofermentative glucose metabolism in S. cerevisiae. Numbered reactions are catalyzed by the following enzymes: 1, pyruvate dehydrogenase complex; 2, Pdc; 3, acetaldehyde dehydrogenase; 4, acetyl-CoA; 5, alcohol dehydrogenase.

Decarboxylation of pyruvate to acetaldehyde by Pdc does not necessarily lead to alcoholic fermentation. Acetaldehyde may be oxidized to acetyl-CoA by the enzymes of the pyruvate dehydrogenase bypass (Fig. 1) (19, 35). In pyruvate dehydrogenase-negative mutants of S. cerevisiae grown at low specific growth rates in glucose-limited cultures, this bypass enables respiratory glucose dissimilation. This process leads to a reduced biomass yield on glucose due to the consumption of ATP in the acetyl-CoA synthetase reaction (34).

If competition between Pdc and pyruvate dehydrogenase for pyruvate is a physiologically relevant process, it should be possible to manipulate flux distribution at the pyruvate branch point by changing the V_max and/or the K_m of either of these enzymes. In practice, it is difficult to manipulate the kinetic parameters of pyruvate dehydrogenase: this large multienzyme complex is located in the mitochondrial matrix, and genes encoding mitochondrial pyruvate transporters have as yet not been identified (35). In contrast, the structural genes encoding Pdc have been well characterized (17), facilitating genetic modification of Pdc levels.

In this study, the Pdc content of S. cerevisiae was increased by increasing the copy number of the PDC1 structural gene (23). The physiological effects of Pdc overproduction were studied with aerobic glucose-limited chemostat cultures. In addition to the effect on flux distribution at the pyruvate branch point, the effect of Pdc overproduction on the glycolytic capacity of cells pregrown under sugar limitation was investigated.

	MATERIALS AND METHODS

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Strains and plasmids. Haploid, prototrophic S. cerevisiae CEN.PK113-7D (MATa MAL2-8^c SUC2) and its auxotrophic derivative CEN.PK113-5D (MATa MAL2-8^c SUC2 ura3-52) were kindly provided by P. Kötter, Frankfurt, Germany. Precultures of yeast strains were grown to the stationary phase on mineral medium (52) adjusted to pH 6.0 and containing 2% (wt/vol) glucose in shake flasks. After glycerol (30% [vol/vol]) was added, 2-ml aliquots were stored in sterile vials at 70°C. These frozen stock cultures were used to inoculate precultures for chemostat cultivation. Escherichia coli XL1Blue (13) [recA1 endA1 gyrA96 thi-1 hsdR17 supE44 recA1 lac/F' proAB lacI^qZM15 Tn10(Tet^r)] was used for plasmid amplification. The multicopy plasmid YEplac195 (16), carrying the URA3 marker gene, was a gift from R. D. Gietz (University of Manitoba, Winnipeg, Canada). pRUL321 is a 4.3-kb SphI fragment of the S. cerevisiae PDC1 gene in pBR322 (35a).

Nucleic acid manipulation and plasmid construction. Plasmid DNA was isolated from E. coli by the ammonium acetate method (26). Restriction endonucleases (Boehringer) and T4 DNA ligase (Pharmacia) were used according to the suppliers' recommendations. A 3,484-bp SphI-XhoI fragment from pRUL321 containing the PDC1 gene was cloned in YEplac195 digested with SphI and SalI, giving pRUL178. This construct, as well as the vector YEplac195, was used to transform S. cerevisiae CEN.PK113-5D by electroporation (6), selecting for uracil prototrophy. This procedure resulted in S. cerevisiae GG393 (containing the PDC1 construct) and GG393 (containing the empty vector).

Media. E. coli was grown in LB medium (37). Yeast cultures for genetic experiments were grown on either YPD (40) or MY (55) medium supplemented as required with uracil (20 mg · liter¹). For physiological characterization of wild-type and recombinant yeast strains, a defined mineral medium containing vitamins (52) was used. For chemostat cultivation, the glucose concentration in reservoir media was 7.5 g · liter¹ (0.25 mol of carbon per liter).

Chemostat cultivation in fermentors. Aerobic chemostat cultivation was performed at 30°C with laboratory fermentors (Applikon, Schiedam, The Netherlands) at a stirrer speed of 800 rpm. All chemostat cultivation runs were started at a dilution rate (D, equal to specific growth rate in steady-state cultures) of 0.10 h¹. After steady states had been established at higher dilution rates, the culture was brought back to a D of 0.10 h¹ to check for hysteresis effects. These were not found (data not shown). A steady state was defined as a situation in which at least five volume changes had passed after the last change in growth conditions and in which the biomass concentration, as well as the specific rates of carbon dioxide production and oxygen consumption, had remained constant (<2% variation) for at least two volume changes. The working volume of the cultures was kept at 1.0 liter by a peristaltic effluent pump coupled to an electrical level sensor. This setup ensured that under all growth conditions, biomass concentrations in samples taken directly from the culture differed by <1% from biomass concentrations in samples taken from the effluent line (30). The exact working volume was measured after each experiment. The pH was kept at 5.0 ± 0.1 by an ADI 1030 biocontroller via the automatic addition of 2 mol of KOH liter¹. The fermentors were flushed with air at a flow rate of 0.5 liter · min¹ by use of a Brooks 5876 mass flow controller. The dissolved oxygen concentration was continuously monitored with an oxygen electrode (model 34 100 3002; Ingold) and remained above 60% air saturation. Steady-state data are reported for cultures without detectable oscillations in oxygen consumption and carbon dioxide production rates. Chemostat cultures were routinely checked for purity by phase-contrast microscopy and by plating on YPD agar. The minor loss of ethanol in the exhaust gas due to evaporation at high dilution rates (49) and the dilution of cultures as a result of alkali titration were not taken into account for calculating carbon recoveries of steady-state chemostat cultures. These calculations were based on a carbon content of dry yeast biomass of 48%.

Gas analysis. The exhaust gas was cooled in a condenser (2°C) and dried with a Perma Pure dryer (type PD-625-12P). O₂ and CO₂ concentrations were determined with a Servomex type 1100A analyzer and a Beckman model 864 infrared detector, respectively. The off-gas flow rate was measured as described previously (54). Specific rates of CO₂ production (qCO₂) and O₂ consumption (qO₂) were calculated according to van Urk et al. (45).

Determination of culture dry weight. Culture samples (10 ml) were filtered over preweighed nitrocellulose filters (pore size, 0.45 µm; Gelman Sciences). After the removal of medium, the filters were washed with demineralized water, dried in a Sharp type R-4700 microwave oven for 20 min at a 360-W output, and weighed. Duplicate determinations varied by <1%.

Determination of maximum glycolytic capacity. Samples containing exactly 100 mg (dry weight) of biomass from a chemostat culture were harvested by centrifugation at 10,000 × g for 5 min, washed once, and resuspended in 5 ml of 0.9% (wt/vol) NaCl solution. The cell suspension was then immediately introduced into a thermostat-controlled (30°C) vessel containing 10 ml of fivefold-concentrated mineral medium (pH 5.6). The volume was adjusted to 40 ml with demineralized water. After 10 min of incubation, a 10-ml glucose pulse (100 g · liter¹) was applied, and samples (1 ml) were taken at appropriate times. The working volume was 50 ml, with a 10-ml headspace, which was continuously flushed with CO₂ gas at a flow rate of ca. 10 ml · min¹. Ethanol production in sample supernatants was determined according to Verduyn et al. (50) with alcohol oxidase from Hansenula polymorpha (a kind gift from Bird Engineering, Schiedam, The Netherlands). Specific rates of ethanol production are expressed as millimoles of ethanol · gram of dry yeast biomass¹ · h¹.

Metabolite analysis. Glucose in reservoir media and supernatants was determined with a glucose oxidase kit (Merck kit 14144; detection limit, ca. 5 µM). Ethanol, glycerol, and pyruvate were determined by high-pressure liquid chromatography with an HPX-87H Aminex ion-exchange column (300 by 7.8 mm; Bio-Rad) at 60°C. The column was eluted with 5 mM H₂SO₄ at a flow rate of 0.6 ml · min¹. Pyruvate was detected at 214 nm with a Waters 441 UV meter coupled to a Waters 741 data module. Ethanol and glycerol were detected with an ERMA type ERC-7515A refractive-index detector coupled to a Hewlett-Packard type 3390A integrator. Acetate was determined with Boehringer test kit 148261 (detection limit, ca. 0.1 mM).

Preparation of cell extracts. For preparation of cell extracts, culture samples were harvested by centrifugation, washed twice with 10 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA, concentrated fourfold, and stored at 20°C. Before being assayed, the samples were thawed, washed, and resuspended in 100 mM potassium phosphate buffer (pH 7.5) containing 2 mM MgCl₂ and 1 mM dithiothreitol (sonication buffer). Extracts were prepared by sonication with 0.7-mm-diameter glass beads at 0°C in an MSE sonicator (150-W output, 7-µm peak-to-peak amplitude) for 3 min at 0.5-min intervals. Unbroken cells and debris were removed by centrifugation at 4°C (20 min at 36,000 × g). The clear supernatant was used as the cell extract.

Enzyme analysis. Enzyme assays were performed by use of a Hitachi model 100-60 spectrophotometer at 30°C and 340 nm (E₃₄₀ of reduced pyridine dinucleotide cofactors, 6.3 mM¹) with freshly prepared extracts. All enzyme activities are expressed as micromoles of substrate converted · minute¹ · milligram of protein¹. When necessary, extracts were diluted in sonication buffer. All assays were performed in duplicate with two concentrations of cell extract. Specific activities in these duplicate experiments differed by <10%.

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Protein determinations. The protein content of whole cells was estimated by a modified biuret method (51). Protein concentrations in cell extracts were determined by the Lowry method. Dried bovine serum albumin (fatty acid free; Sigma) was used as a standard.

	RESULTS

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Growth in shake-flask cultures. For an initial characterization, strains CEN.PK113-7D (prototrophic wild type), GG392 (CEN.PK113-5D host transformed with an empty vector), and GG393 (CEN.PK113-5D host with a multicopy PDC1 vector) were grown on glucose in shake-flask cultures. In the two plasmid-containing strains, the uracil auxotrophy of the CEN.PK113-5D host was complemented by the URA3 gene on the YEplac195 vector. Therefore, a defined medium lacking uracil could be used for all three strains.

Overexpression of PDC1 in glucose-limited chemostat cultures (D, 0.10 h¹). For aerobic glucose-limited chemostat cultures of strain GG393 (D, 0.10 h¹), the presence of multiple copies of the PDC1 gene resulted in 14-fold-higher Pdc activity in cell extracts relative to that for the two isogenic reference strains (Table 2). Since episomal vectors carrying auxotrophic markers may be unstable in chemostat cultures (12, 28), Pdc activity in cell extracts of chemostat cultures was monitored for over 100 generations at a D of 0.10 h¹. No significant loss of Pdc activity was observed in either the reference strains or the PDC1-overexpressing strain (data not shown).

View this table: [in this window] [in a new window]   TABLE 2.   Specific activities of glycolytic enzymes in cell extracts of aerobic glucose-limited chemostat cultures (D, 0.10 h1) of various S. cerevisiae strainsa

Physiology of wild-type and PDC1-overexpressing strains in chemostat cultures. In aerobic glucose-limited chemostat cultures of the reference strains CEN.PK113-7D and GG392 grown at a D of <0.30 h¹, over 98% of the substrate carbon was recovered as biomass and CO₂. The biomass yield of 0.49 g · g of glucose¹ (Fig. 2A) was similar to that of respiratory cultures of other wild-type S. cerevisiae strains (48). Completely respiratory metabolism was further evident from the absence of ethanol and other typical fermentation products in culture supernatants. In these respiratory cultures, the specific rates of O₂ consumption and CO₂ production (Fig. 2B) increased linearly with D.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Effect of D on biomass yield (Yxs; grams of dry yeast biomass · gram of glucose1) and the specific rate of ethanol production (qethanol) (A) and on the specific rates of oxygen consumption (qO2) and carbon dioxide production (qCO2) (B) in aerobic glucose-limited chemostat cultures of prototrophic wild-type S. cerevisiae CEN.PK113-7D (closed symbols) and the empty-vector control strain (GG392; open symbols). Fluxes are expressed in millimoles · g of dry yeast biomass1 · h1.

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View larger version (13K): [in this window] [in a new window]   FIG. 3.   Effect of D on biomass yield (Yxs; grams of dry yeast biomass · gram of glucose1) and the specific rate of ethanol production (qethanol) (A) and on the specific rates of oxygen consumption (qO2) and carbon dioxide production (qCO2) (B) in aerobic glucose-limited chemostat cultures of the Pdc-overproducing strain S. cerevisiae GG393. Fluxes are expressed in millimoles · gram of dry yeast biomass1 · h1. The broken lines represent data for the wild-type and control strains as depicted in Fig. 2.

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View larger version (15K): [in this window] [in a new window]   FIG. 4.   Pdc activities in cell extracts of aerobic glucose-limited chemostat cultures of the prototrophic wild-type strain (S. cerevisiae CEN.PK113-7D; ), empty-vector reference strain (GG392; ) and Pdc-overproducing strain (GG393; ) as a function of D. Values are presented as the averages ± standard deviations for two independent enzyme assays.

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Effect of Pdc overproduction on maximum glycolytic capacity. To investigate whether Pdc overproduction affected the maximum glycolytic (fermentative) capacity of chemostat-grown cells, culture samples were washed, made anaerobic, and exposed to excess glucose. During the first 30 min after glucose addition, the increase in biomass concentration was negligible and the increase in ethanol concentration was linear with time and proportional to the amount of biomass present (data not shown).

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View larger version (14K): [in this window] [in a new window]   FIG. 5.   Effect of D on the maximum specific rate of ethanol production (determined off line under anaerobic conditions with excess glucose) in aerobic glucose-limited chemostat cultures of S. cerevisiae wild-type and empty-vector control strains (A) and Pdc-overproducing strain (B) (compared to the data presented in panel A). The broken line in panel A represents the specific rate of ethanol production (qethanol) during glucose-limited growth of the reference strains as presented in Fig. 2A. The solid line in panel B represents the qethanol of the Pdc-overproducing strain as plotted in Fig. 3A. Fluxes are expressed in millimoles · g of dry yeast biomass1 · h1. Values for the Pdc-overproducing strain are presented as the average ± standard deviation for two independent off-line fermentation assays.

	DISCUSSION

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Pdc overproduction. Our data confirm the conclusion of Schaaff et al. (38) that Pdc by itself does not control the specific rate of growth of S. cerevisiae on glucose. Instead, a 3.4-fold overproduction of Pdc in batch cultures resulted in a ca. 10% decrease in the specific growth rate (Table 1). In the interpretation of this effect, it should be considered that in wild-type S. cerevisiae, Pdc is already an abundant protein. Based on a specific activity of purified Pdc of 54 U · mg¹ (4), the Pdc activity in cell extracts of batch cultures of the two reference strains (1.85 U · mg of protein¹) would correspond to 3.4% of the soluble cell protein. In the overproducing strain, this value would increase to an estimated 11.5% of the soluble protein (Table 1).

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Flux distribution at the pyruvate branch point. In aerobic glucose-limited chemostat cultures, Pdc overproduction caused a substantial decrease in the D at which aerobic fermentation set in (the so-called critical D; Fig. 4). The most straightforward explanation for this observation is that the increased level of Pdc allows the enzyme to compete more effectively with the mitochondrial pyruvate dehydrogenase complex. As mentioned above, decarboxylation by Pdc does not necessarily commit pyruvate to alcoholic fermentation, as acetaldehyde can also be channelled into respiratory dissimilation via the pyruvate dehydrogenase bypass (Fig. 1) (19, 35). This pathway plays an essential role in S. cerevisiae, probably by providing cytosolic acetyl-CoA for biosynthesis (15, 35, 43). In wild-type, glucose-limited chemostat cultures, respiratory dissimilation of pyruvate via acetyl-CoA predominantly occurs via the pyruvate dehydrogenase complex (34). However, in pyruvate dehydrogenase-negative mutants, respiratory pyruvate dissimilation can be completely redirected via the bypass at low specific growth rates. This process results in a 15% decrease in the biomass yield on glucose due to consumption of ATP in the acetyl-CoA synthetase reaction (34).

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Maximum glycolytic (fermentative) capacity. Various authors have investigated the relationship between Pdc activity and glycolytic flux. In batch cultures of S. cerevisiae mutants with different levels of PDC1 expression, fermentation rates exhibited a linear correlation with a broad range of Pdc activities in cell extracts (39). A similar correlation was found in a comparison of fermentation rates and Pdc activities in different yeast species (47).