Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

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(+) an d NADPH/ADP(+) and has a significant influence on product formation in micr oorganisms. The enzyme pyridine nucleotide transhydrogenase, which can cata lyse transfer of reducing equivalents between the two nucleotide systems, o ccurs in several organisms, but not in yeasts, The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevis iae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase fr om Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme an d expressed it under the control of a S, cerevisiae promoter in a strain de rived from the industrial model strain S, cerevisiae CBS8066, Anaerobic bat ch cultivations in high-performance bioreactors were carried out in order t o 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 pro tein was measured in the extracts from the strain expressing the sth gene f rom 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 formati on increases when excess NADH is formed, this suggested that transhydrogena se 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 trans hydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass an d ethanol yields were significantly lowered by the transhydrogenase. Copyri ght (C) 2000 John Whey & Sons, Ltd.