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

The metabolism of a vitamin-auxotrophic pyruvate-producing microorganism, T orulopsis glabrata IFO 0005, was investigated by metabolic flux analysis. P articular attention was focused on the effect of culture conditions, such a s dissolved oxygen (DO) concentration and thiamine concentration, on specif ic pathway activities. The results of metabolic flux analysis indicate that the thiamine concentration significantly affected pyruvate dehydrogenase a nd pyruvate decarboxylase activities, and plays an important role in cell g rowth and pyruvate production. Metabolic flux analysis was also utilized to clarify the metabolism of this strain during pyruvate fermentation under d ifferent 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 flues, we conducted a fed-batch experiment where the initial thiamine concentration was reduced to 30 mu g/l and thiamine was added at 10 mu g/l during fermentation when the cell growth rate decreased to 0.2 h(-1). With separate addition of thia mine, the overall pyruvate yield could be improved by 15% due to the decrea se of ethanol production.