OVERFLOW METABOLISM IN PRE-DIAUXIC YEASTS

Abstract

Overflow metabolites, once regarded as waste products, have recently been shown to be utilized as a fuel in mammals. This context motivated exploring the metabolic role of ethanol, a yeasts' overflow metabolite, in pre-diauxic Saccharomyces cerevisiae. By employing 2H isotope tracing followed by liquid chromatography-mass spectrometry analysis, it was demonstrated that ethanol is a crucial source of reductive energy in fermenting yeasts. Strikingly, the ethanol oxidation pathway is essential under oxidative stress, supplying nearly all the cellular NADH and over half of NADPH. 13C tracing showed that, like lactate in mammals, ethanol also allows decoupling of glycolysis and the TCA cycle. Surprisingly, not pyruvate but rather ethanol is a significant source of mitochondrial acetyl-CoA. Compartmentation analysis suggested that ethanol is oxidized to acetate in the cytosol, which then freely diffuses via a mitochondrial membrane. In mitochondria, where acetyl-CoA synthetase is lacking, it was proposed that acetate can be converted into acetyl-CoA in the CoA-transferase reaction with succinyl-CoA. Thus, ethanol is not a waste product but rather an important hydride and carbon source in the model yeast. This work deepens the fundamental understanding of S. cerevisiae overflow metabolism and informs the development of new genome-scale models for metabolic engineering.