Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol

Abstract

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.

Impact of Thermodynamic Principles in Systems Biology

“…It was observed that, for the above inducible enzymes, clear predictions were made on their need as function of increasing ethanol fraction. This approach predicted: l The ethanol/glucose feed ratio where a particular enzyme started to be induced l The enzyme amount then increased linear with increasing ethanol fraction These predictions were qualitatively, but more surprising also quantitatively, validated using the wild type yeast [13,14]. Later additional validation was performed with null-mutants in the above enzymes, leading to a predicted maximal ethanol uptake rate of each mutant.…”

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

“…The biggest increase in production was observed when ethanol was added to the feed, initially as a glucose/ethanol mixed feed following the glucose batch phase of growth. The rationale for adding ethanol to the feed was that direct supply of ethanol may increase the supply of cytosolic acetyl-CoA to the mevalonate pathway (30). An alternative explanation is that use of ethanol eliminated glucose repression, but this scenario is considered unlikely as glucose was still present in the feed, and the concentration of glucose in the feed stage of the fed-batch fermentations was very low, and mostly undetectable.…”

Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae causes a partial loss of glycolytic capacity

“…The reaction was started with 2 mM fructose 6-phosphate. Phosphofructokinase (PFK; EC 2.7.1.11) was assayed according to de Jong-Gubbels et al (1995), with minor modifications. The assay mixture contained: imidazole/ HCl (pH 7?0) 50 mM, MgCl 2 5 mM, NADH 0?15 mM, fructose 2,6-diphosphate 0?10 mM, fructose-1,6-diphosphate aldolase (FBA; Roche) 0?45 U ml 21 , glycerol-3-phosphate dehydrogenase (Roche) 0?6 U ml 21 , triosephosphate isomerase (TPI) 1?8 U ml 21 (Roche) and cell extract.…”

“…Enolase (ENO; EC 4.2.1.11) was assayed according to van Hoek (2000). Pyruvate kinase (PYK; EC 2.7.1.40) was assayed according to de Jong-Gubbels et al (1995), with minor modifications. The assay mixture contained the following: cacodylic acid/KOH (pH 6?2) 100 mM, KCl 100 mM, ADP 10 mM, fructose 1,6-diphosphate 1 mM, MgCl 2 25 mM, NADH 0?15 mM, lactate dehydrogenase (Roche) 11?25 U ml 21 and cell extract.…”