Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source.

Abstract

Valine aminotransferase, a key enzyme in both biosynthesis and breakdown of branched-chain amino acids, showed consistently higher activity in Candida utilis grown in continuous culture than in Saccharomyces cerevisiae, while pyruvate decarboxylase and alcohol dehydrogenase, the other two enzymes of the Ehrlich pathway of branched-chain alcohol formation, were lower in activity. By spheroplast lysis, it was shown that valine aminotransferase followed the distribution of pyruvate decarboxylase in being located in the cytosol. Replacement of ammonium as nitrogen source by valine during conditions of carbon or nitrogen limitation caused increased specific activities of these three enzymes in S. cerevisiae, but (with one exception) decreased those of C. utilis. Of the metabolites accumulating in the culture medium, little or no ethanol or branched-chain alcohols were present during carbon-limited growth of either organism, but the change to nitrogen limitation resulted in increases in concentration of 20- to 100-fold in pyruvate, acetate and non-pyruvate keto acids as well as the accumulation of branched-chain alcohols in both organisms, and of ethanol, ethyl acetate and glycerol in S. cerevisiae. When valine was the limiting nitrogen source, there was an increase in non-pyruvate keto acids and a 10- to 16-fold increase in 2-methylpropanol. Total branched-chain alcohols formed under nitrogen limitation were 2-fold higher in S. cerevisiae than in C. utilis, irrespective of nitrogen source. Accumulation of branched-chain alcohols, ethanol, acetate and glycerol was also observed during carbon-limited growth of S. cerevisiae with valine as nitrogen source at dilution rates above the critical rate for transition to respirofermentative growth. Less than 70% of the valine carbon metabolized during growth of S. cerevisiae and only 15% of that used during growth of C. utilis was recovered in identified metabolic products. Even allowing for losses by volatilization during aeration, this suggests that a significant amount of the valine is being metabolized by a route or routes other than the Ehrlich pathway, possibly via the action of branched-chain 2-keto acid dehydrogenase. The molar growth yield for the nitrogen source under either carbon or nitrogen limitation was significantly lower for growth on valine than for growth on ammonium, suggesting that breakdown of valine requires more energy. It is evident that not all the enzymes involved in branched-chain amino acid metabolism in yeasts have yet been identified, nor are their interactions properly understood.