title: Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool.
abstract: The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/NADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts. The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme, and expressed it under the control of a S. cerevisiae promoter in a strain derived from the industrial model strain S. cerevisiae CBS8066. Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD(+) and NADPH. This was further supported by measurements of the intracellular nucleotide concentrations. Thus, the (NADPH/NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass and ethanol yields were significantly lowered by the transhydrogenase.
authors: Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
authors: T L
authors: TL
authors: Nissen
authors: M
authors: M
authors: Anderlund
authors: J
authors: J
authors: Nielsen
authors: J
authors: J
authors: Villadsen
authors: M C
authors: MC
authors: Kielland-Brandt
mesh_terms: D000595:Amino Acid Sequence; D000693:Anaerobiosis; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics; D018533:Biomass; D019149:Bioreactors; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D000431:Ethanol / Q000378:metabolism; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D007656:Ketoglutaric Acids; D009243:NAD; D000431:Ethanol; D009249:NADP; D009250:NADP Transhydrogenases; D005947:Glucose; D005990:Glycerol