Nitrogen and Redox Metabolism in Saccharomyces cerevisiae

Doctoral thesis, 2000

Anaerobic conversion of glucose to ethanol by the yeast Saccharomyces cerevisiae has been studied. Glycerol is formed as a by-product in order to maintain the overall redox balance. The excess of NADH and as a consequence the amount of glycerol formed for growth on different nitrogen sources, was estimated theoretically and the result was found to agree well with experimental data. By properly designing the growth medium so as to reduce formation of excess NADH, it was possible to increase the ethanol and decrease the glycerol production. Furthermore, the formation of ethanol was estimated from measured heat generation and carbon dioxide evolution. These estimates showed good agreement with experimental data. Alternatively, the excess NADH can be reoxidised by oxygen in the respiratory chain. A limited supply of oxygen should be used to avoid induction of respiratory catabolism of glucose. To obtain such conditions, a respiratory quotient (RQ) controller, working at high values of RQ, was constructed. It was found that the intracellular redox level correlated with the RQ, for values between 10-30. In a 14C-labelling study it was shown that, when glutamate was used as the only nitrogen source, the carbon skeleton of glutamate is transformed to 2-oxoglutarate, succinate, 2-hydroxyglutarate and to the amino acids structurally related to glutamate (i.e. glutamine, proline, arginine and lysine). This work showed that the carbon flows from glucose and glutamate are separate, thus splitting the TCA cycle at the level of 2-oxoglutarate into an upper and a lower part. During these conditions, two proteins were found to be down-regulated: the gluconeogenic form of enolase, Eno1p, and a modified form of phosphoglycerate kinase, Pgk1p, both of which are found in the lower part of glycolysis. These isoenzymes may be involved in the regulation of glycolysis. The metabolite 2-hydroxyglutarate was found in substantial amounts when glutamate was the nitrogen source. The production of this metabolite may be catalysed by a 2-hydroxyglutarate dehydrogenase, which was shown to be present in crude extracts in yeast. It is suggested that this activity is to some extent associated with the 3-phosphoglycerate dehydrogenase, which catalyses the first step of serine and glycine biosynthesis. The putative 3-phosphoglycerate dehydrogenases present in S. cerevisiae, were shown to be encoded by the SER3 and SER33 genes. However, a dual catalytic function of these enzymes could not be verified in this study.