Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis

Abstract

Background: One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative 13C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions.

Results: The metabolic shift from oxidative to fermentative growth was accompanied by complex changes of carbon flux throughout the whole central metabolism. This involved a flux redirection from the pentose phosphate pathway (PPP) towards glycolysis, an increased flux through pyruvate carboxylase, the fermentative pathways and malic enzyme, a flux decrease through the TCA cycle, and a partial relocation of alanine biosynthesis from the mitochondrion to the cytosol. S. cerevisiae exhibited a by-pass of pyruvate dehydrogenase in all physiological regimes. During oxidative growth this by-pass was mainly provided via pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthase and transport of acetyl-CoA into the mitochondrion. During fermentative growth this route, however, was saturated due to limited enzyme capacity. Under these conditions the cells exhibited high carbon flux through a chain of reactions involving pyruvate carboxylase, the oxaloacetate transporter and malic enzyme. During purely oxidative growth the PPP alone was sufficient to completely supply NADPH for anabolism. During fermentation, it provided only 60 % of the required NADPH.

Conclusion: We conclude that, in order to overcome the limited capacity of pyruvate dehydrogenase, S. cerevisiae possesses different metabolic by-passes to channel carbon into the mitochondrion. This involves the conversion of cytosolic pyruvate either into acetyl CoA or oxaloacetate followed by intercompartmental transport of these metabolites. During oxidative growth mainly the NAD specific isoforms of acetaldehyde dehydrogenase and isocitrate dehydrogenase catalyze the corresponding reactions in S. cerevisiae, whereas NADPH supply under fermentative conditions involves significant contribution of sources other than the PPP such as e. g. NADPH specific acetaldehyde dehydrogenase or isocitrate dehydrogenase.

Figure 1

Metabolic network of the central cytosolic and mitochondrial metabolism of S. cerevisiae investigated in the present work. The network comprises glycolysis, pentose phosphate pathway, anaplerotic carboxylation, fermentative pathways, inter-compartmental transport of acetyl-CoA, pyruvate, and oxaloacetate, respectively, TCA cycle, malic enzyme and anabolic reactions from intermediary metabolites into anabolism.

Figure 2

Cultivation profile of S. cerevisiae in chemostat under aerobic, glucose-limited conditions at different growth rates: Concentration of cell dry mass (CDM), ethanol, acetate, and glucose.

Figure 3

Intracellular carbon flux distribution of S. cerevisiae cultivated in chemostat on [1-¹³C] glucose under aerobic glucose-limited conditions at different growth rates. All fluxes are given as relative fluxes normalized to the specific glucose uptake rate. For each reaction the fluxes corresponding to purely oxidative (μ = 0.15 h^-1, q_glc = 1.56 mmol g^-1 h^-1), respiro-fermentative (μ = 0.30 h^-1, q_glc = 4.90 mmol g^-1 h^-1), and mainly fermentative growth (μ = 0.40 h^-1, q_glc = 8.23 mmol g^-1 h^-1), respectively, are shown from top to bottom. For reversible reactions an additional arrow indicates the direction of the net flux and the values in the squared brackets are the obtained reversibilities of the corresponding enzymes. The fluxes correspond to the optimal fit between experimentally determined steady-state ¹³C labeling patterns of amino acids of the cell protein and ¹³C labeling patterns simulated via isotopomer modelling.

Figure 4

Correlation of specific growth rate and relative carbon fluxes in the central metabolism of S. cerevisiae: glucose 6-phosphate dehydrogenase (A), citrate synthase (B), and malic enzyme (C). Data shown comprise fluxes of S. cerevisiae ATCC 31267 determined at different growth rates in glucose-limited chemostat (this work, open circle), of S. cerevisiae CEN.PK 113.7D at μ = 0.1 h^-1 in glucose-limited chemostat ([27],open triangle), at μ = 0.37 h^-1 in batch culture under glucose excess ([27], closed triangle), of S. cerevisiae CEN.PK 113.7D at μ = 0.1 h^-1 in glucose-limited chemostat ([26], open square), at μ = 0.4 h^-1 in batch culture under glucose excess ([26], closed square). Note that Fiaux et al. [26] determined flux intervals instead of exact values, whereby the values given here represent the corresponding average of each interval.

Figure 5

NADPH metabolism of S. cerevisiae cultivated in chemostat under aerobic glucose-limited conditions at different growth rates. The fluxes correspond to purely oxidative (μ = 0.15 h^-1), respiro-fermentative (μ = 0.30 h^-1), and mainly fermentative growth (μ = 0.40 h^-1).

Figure 6

Absolute carbon fluxes of S. cerevisiae cultivated in chemostat under aerobic glucose-limited conditions at different growth rates: Fluxes of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl CoA synthase (A), and of intercompartmental pyruvate transport, pyruvate dehydrogenase and citrate synthase (B). For each enzyme the flux at purely oxidative (μ = 0.15 h^-1), respiro-fermentative (μ = 0.30 h^-1), and mainly fermentative growth (μ = 0.40 h^-1) is shown from left to right. Additionally the 90 % confidence intervals for the fluxes are given.