title: 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+.
authors.affiliation: Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.
authors.forename: M
authors.initials: M
authors.lastname: Anderlund
authors.forename: T L
authors.initials: TL
authors.lastname: Nissen
authors.forename: J
authors.initials: J
authors.lastname: Nielsen
authors.forename: J
authors.initials: J
authors.lastname: Villadsen
authors.forename: J
authors.initials: J
authors.lastname: Rydström
authors.forename: B
authors.initials: B
authors.lastname: Hahn-Hägerdal
authors.forename: M C
authors.initials: MC
authors.lastname: Kielland-Brandt
mesh_terms: 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
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D009250:NADP Transhydrogenases; D005947:Glucose