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 recombinant Saccharomyces 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. cerevisiae and 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+.

FIG. 1

Physical map of the yeast YEpPGKαTDHβ high-copy-number plasmid containing the E. coli genes pntA and pntB, which encode the α and β subunits, respectively, of nicotinamide nucleotide transhydrogenase. Abbreviations: PGKp, PGK1 promoter, PGKt, PGK1 terminator; TDHp, TDH3 promoter, TDHt, TDH3 terminator. The directions of the promoter-gene-terminator fragments are the same in the pRSPGKαTDHβ low-copy plasmid.

FIG. 2

Recombinant transhydrogenase produced by S. cerevisiae, as analyzed by Western blotting by using transhydrogenase-specific polyclonal antibodies. Lane A, soluble fraction of reference strain TN3 not expressing the pnt genes; lane B, total membrane fraction of strain TN3; lane C, soluble fraction of strain TN24 expressing the pnt genes; lane D, total membrane fraction of strain TN24. The sizes of the molecular mass markers (in kilodaltons) are indicated on the left. The arrows indicate the positions of the α and β subunits.

FIG. 3

pH dependence of the rate of reduction of 3-acetylpyridine-NAD⁺ by NADPH (A) and the rate of reduction of 3-acetylpyridine-NAD⁺ by NADH in the presence of NADP⁺ (B), catalyzed by transhydrogenase produced by S. cerevisiae TN24 expressing the E. coli pnt genes.

FIG. 4

Distribution of recombinant E. coli transhydrogenase in yeast membranes fractionated in a sucrose gradient. Total membranes were isolated from yeast strain TN24 and loaded onto a linear sucrose gradient. After 14 h of centrifugation, 300-μl fractions were collected from the top of the gradient and used for enzyme assays. (A) Distribution of plasma membrane H⁺-ATPase (□), vacuolar H⁺-ATPase (◊), mitochondrial cytochrome oxidase (▵), and ER NADPH-cytochrome c oxidoreductase (○). (B) Distribution of the recombinant transhydrogenase (□) and protein concentrations (⧫). The arrow in panel A indicates the position of the recombinant transhydrogenase peak in panel B.