Rethinking aerobic glucose metabolism in Saccharomyces cerevisiae: is glucose completely fermented to ethanol at high concentrations?

Saccharomyces cerevisiae is a Crabtree-positive yeast capable of producing ethanol under aerobic conditions when exposed to high glucose concentrations. This behavior has traditionally been interpreted as the coexistence of fermentative and respiratory metabolism, assuming that a fraction of glucose may be directly oxidized while another fraction is fermented. However, experimental observations accumulated over several years with different S. cerevisiae strains, both wild-type and genetically modified, in aerobic batch cultures containing high glucose concentrations point to a different possibility: that virtually all glucose carbon is initially channelled into alcoholic fermentation, producing ethanol and CO₂, with no significant direct respiratory contribution from glucose. Oxygen consumption under these conditions could correspond, at least to a substantial extent, to the subsequent oxidation of ethanol generated from glucose. The present work formulates this possibility as a quantitative metabolic hypothesis and proposes an experimental design to discriminate between the two models. Aerobic batch cultures initiated with 20 g/L glucose and isotope tracing using [U-¹³C₆]glucose are proposed, together with time-course monitoring of glucose, ethanol, biomass, O₂ consumption, and CO₂ production, and analysis of ¹³C distribution between ethanol and metabolites associated with oxidative metabolism. A complementary experiment using [U-¹³C₂]ethanol would assess the contribution of ethanol to oxidative metabolism while glucose remains available. The objective is to determine what proportion of glucose carbon enters oxidative metabolism directly and what proportion reaches it only after passing through the fermentative pathway and being converted into ethanol. The hypothesis is experimentally falsifiable and can be tested using isotope-tracing and metabolic-flux analysis approaches.