Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

Abstract

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinantSaccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiaeand thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

“…Indeed, the resulting strains exhibited a 38% reduction of the glycerol yield and a 10% increase of the ethanol yield on glucose in anaerobic batch cultures [81]. Recently, it has been attempted to further reduce glycerol production by the functional expression of heterologous transhydrogenases in S. cerevisiae [86,87]. The rationale for these studies is that introduction of a transhydrogenase (catalysing the reaction NADH+NADP HNAD +NADPH) might allow for a conversion of NADH into NADPH, thus reducing the net production of NADH in biosynthesis.…”

“…The rationale for these studies is that introduction of a transhydrogenase (catalysing the reaction NADH+NADP HNAD +NADPH) might allow for a conversion of NADH into NADPH, thus reducing the net production of NADH in biosynthesis. Although transhydrogenases from Escherichia coli [87] and Azotobacter vinelandii [86] were functionally expressed in S. cerevisiae, this did not result in a decreased production of glycerol. Instead of reducing NADH production, the heterologous transhydrogenases catalyzed the reverse reaction, i.e.…”

“…Instead of reducing NADH production, the heterologous transhydrogenases catalyzed the reverse reaction, i.e. the transfer of electrons from NADPH to NAD in anaerobic cultures [86,87].…”

Stoichiometry and compartmentation of NADH metabolism inSaccharomyces cerevisiae

“…Indeed, the resulting strains exhibited a 38% reduction of the glycerol yield and a 10% increase of the ethanol yield on glucose in anaerobic batch cultures [81]. Recently, it has been attempted to further reduce glycerol production by the functional expression of heterologous transhydrogenases in S. cerevisiae [86,87]. The rationale for these studies is that introduction of a transhydrogenase (catalysing the reaction NADH+NADP + ↔NAD + +NADPH) might allow for a conversion of NADH into NADPH, thus reducing the net production of NADH in biosynthesis.…”

“…The rationale for these studies is that introduction of a transhydrogenase (catalysing the reaction NADH+NADP + ↔NAD + +NADPH) might allow for a conversion of NADH into NADPH, thus reducing the net production of NADH in biosynthesis. Although transhydrogenases from Escherichia coli [87] and Azotobacter vinelandii [86] were functionally expressed in S. cerevisiae , this did not result in a decreased production of glycerol. Instead of reducing NADH production, the heterologous transhydrogenases catalyzed the reverse reaction, i.e.…”