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.
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+.
Anderlund
M
M
Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.
Nissen
T L
TL
Nielsen
J
J
Villadsen
J
J
Rydström
J
J
Hahn-Hägerdal
B
B
Kielland-Brandt
M C
MC
D000693:Anaerobiosis; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D009243:NAD; D009250:NADP Transhydrogenases; D005947:Glucose