Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effecton product formation during anaerobic glucose fermentation

We studied the physiological effect of the interconversion between the NAD( H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae exp ressing the membrane-bound transhydrogenase from Escherichia coli. Our obje ctive was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD(+) in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recom binant strains exhibited reduction of 3-acetylpyridine-NAD(+) by NADPH and by NADH in the presence of NADP(+), which demonstrated that an active enzym e was present. Unlike the situation in E. coli, however, most of the transh ydrogenase activity was not present in the yeast plasma membrane; rather, t he enzyme appeared to remain localized in the membrane of the endoplasmic r eticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expr essing a high level of transhydrogenase, which indicated that increased NAD PH consumption and NADH production occurred. The intracellular concentratio ns of NADH, NAD(+) NADPH, and NADP(+) were measured in cells expressing tra nshydrogenase. The reduction of the NADPH pool indicated that the transhydr ogenase transferred reducing equivalents from NADPH to NAD(+).