Metabolic flux analysis for efficient pyruvate fermentation using vitamin-auxotrophic yeast of Torulopsis glabrata

The metabolism of a vitamin-auxotrophic pyruvate-producing microorganism, Torulopsis glabrata IFO 0005, was investigated by metabolic flux analysis. Particular attention was focused on the effect of culture conditions, such as dissolved oxygen (DO) concentration and thiamine concentration, on specific pathway activities. The results of metabolic flux analysis indicate that the thiamine concentration significantly affected pyruvate dehydrogenase and pyruvate decarboxylase activities, and plays an important role in cell growth and pyruvate production. Metabolic flux analysis was also utilized to clarify the metabolism of this strain during pyruvate fermentation under different oxygen supply conditions, and the reason for the enhanced pyruvate production under conditions of 30-40% DO concentration was clarified from the viewpoint of intracellular flux distributions. Based on the analysis of the effect of thiamine concentration on the metabolic fluxes, we conducted a fed-batch experiment where the initial thiamine concentration was reduced to 30 microg/l and thiamine was added at 10 microg/l during fermentation when the cell growth rate decreased to 0.2 h(-1). With separate addition of thiamine, the overall pyruvate yield could be improved by 15% due to the decrease of ethanol production.