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Applied and Environmental Microbiology, June 1999, p. 2333-2340, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Mikael Anderlund,1,2 Torben L. Nissen,1,3 Jens Nielsen,3 John Villadsen,3 Jan Rydström,4 Bärbel Hahn-Hägerdal,2 and Morten C. Kielland-Brandt1,*

Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby,1 and Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby,3 Denmark, and Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, SE-40 530 Göteborg,4 and Department of Applied Microbiology, Lund University, SE-221 00 Lund,2 Sweden

Received 7 October 1998/Accepted 18 March 1999

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+.

* Corresponding author. Mailing address: Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Phone: (45) 3327 5331. Fax: (45) 3327 4765. E-mail: mkb{at}crc.dk.

Applied and Environmental Microbiology, June 1999, p. 2333-2340, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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