代謝工学的手法によるピルビン酸の発酵生産法の開発 Development of Fermentative production of Pyruvate by Metabolic Control

Fermentative production of pyruvate by metabolic control was developed. A new approach to the screening of novel pyruvate-producing yeasts and variations in thier vitamin-auxotrophy with regard to the productivity of pyruvate are described. Among the isolates, Torulopsis glabrata IFO 0005 proved to be a superior strain for the fermentative production of pyruvate. T.glabrata IFO 0005 has an absolute requirement for four vitamins: nicotinic acid(NA), thiamine(B1), pyridoxine(Pdx), and biotin(Bio) for growth. Consequently, the auxotrophy of multiple vitamins involved in the metabolism of pyruvate proved to be highly effective for the fermentative production of pyruvate from glucose. It has been postulated that the auxotrophy of the four vitamins noted above plays an essential role in maintaining the metabolic balance for pyruvate accumulation. The optimal practical fermentative conditions for T.glabrata IFO 0005 in a 3-l jar fermentor and the determination of several by-products are described. The system for the regeneration of NAD^+ from NADH in this fermentation of T.glabrata IFO 0005 is also discussed. In addition, three efficient methods for the isolation of pyruvate-producing mutants of T.glabrata IFO 0005 were established. After mutagenesis of the parental strain, mutants that demonstrated higher fermentative production of pyruvic acid from glucose than the parent were dirived. These were of the following three kinds: (a)mutants requiring acetate for full growth(acetate-leaky auxotrophic mutants); (b)salt-tolerant mutants; (c)L-arginine auxotrophic mutants, and L-isoleucine and L-valine double auxotrophic mutants. Finally, the establishment of a praxtical isolation and purification method using a reverse osmotic membrane is described.