Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetylcoenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The K m for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetylcoenzyme A synthetase, respectively. Batch and glucosepulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.When Saccharomyces cerevisiae grows on acetate or ethanol, ATP-dependent activation of acetate to acetyl-coenzyme A is catalyzed by acetyl-coenzyme A synthetase (EC 6.2.1.1). In addition to serving as the fuel for the citric acid cycle, acetylcoenzyme A is an essential building block for the synthesis of lipids and some amino acids. During growth on glucose, direct formation of acetyl-coenzyme A from pyruvate is catalyzed by the mitochondrial pyruvate dehydrogenase complex. Alternatively, conversion of pyruvate into acetyl-coenzyme A can be accomplished by the concerted action of the enzymes of the pyruvate dehydrogenase bypass: pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-coenzyme A synthetase (1, 2). Recent work has indicated that the latter pathway is essential for growth, probably for the provision of cytosolic acetylcoenzyme A required for lipid synthesis (3, 4).In S. cerevisiae, a completely respiratory sugar metabolism is only observed at relatively low growth rates in aerobic, sugarlimited cultures (e.g. chemostat cultures). Upon exposure of such cultures to high sugar concentrations, metabolism becomes respirofermentative and pyruvate metabolism occurs predominantly via pyruvate decarboxylase (5, 6). Under such conditions acetate is formed as a by-product, indicating that the in vivo activity of acetaldehyde dehydrogenase exceeds that of acetyl-coenzyme A synthetase. Acetate produ...
Growth efficiency and regulation of key enzyme activities were studied in carbon- and energy-limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell-free extracts of glucose-limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose-1,6-bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. Phospho-enol-pyruvate-carboxykinase activity was not detectable in extracts from glucose-grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon-limited growth of S. cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells' requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis.
Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.
Setting up a metabolic network model for respiratory growth of Saccharomyces cerevisiae requires the estimation of only two (energetic) stoichiometric parameters: (1) the operational PO ratio and (2) a growth-related maintenance factor k. It is shown, both theoretically and practically, how chemostat cultivations with different mixtures of two substrates allow unique values to be given to these unknowns of the proposed metabolic model. For the yeast and model considered, an effective PO ratio of 1.09 mol of ATP/mol of O (95% confidence interval 1.07-1.11) and a k factor of 0.415 mol of ATP/C-mol of biomass (0.385-0.445) were obtained from biomass substrate yield data on glucose/ethanol mixtures. Symbolic manipulation software proved very valuable in this study as it supported the proof of theoretical identifiability and significantly reduced the necessary computations for parameter estimation. In the transition from 100% glucose to 100% ethanol in the feed, four metabolic regimes occur. Switching between these regimes is determined by cessation of an irreversible reaction and initiation of an alternative reaction. Metabolic network predictions of these metabolic switches compared well with activity measurements of key enzymes. As a second validation of the network, the biomass yield of S. cerevisiae on acetate was also compared to the network prediction. An excellent agreement was found for a network in which acetate transport was modeled with a proton symport, while passive diffusion of acetate gave significantly higher yield predictions.
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