A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived. The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation. Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations.The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO, , ethanol, glucose, glycerol, NH;, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA). The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model. All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor. Carbon balances closed within 98%. The calculations comprised flux distributions at specific growth rates of 0.10 and 030 h-I. The fluxes through reactions located around important branch points of the metabolism were compared, i.e. the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways. Also the model was used t o show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase. Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose. The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis. It was found that the accuracy of the measurements of CO, , ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.

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Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD + . The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-D1), formation of glycerol was signi®cantly impaired, which resulted in reduction of the maximum speci®c growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum speci®c growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-D1 gpd2-D1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD + by synthesis of glycerol. Also, strain TN23 (gpd1-D1 gpd2-D1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD + pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP + to NADPH and NAD + . Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-D1) and TN5 (gpd2-D1) without serious effects on the maximum speci®c growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-D1 gpd2-D1) resulted in a dramatic reduction in the maximum speci®c growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in m max from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP + to NADH and NADP + . Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.

The intracellular redox state of a cell is to a large extent de®ned by the concentration ratios of the two pyridine nucleotide systems NADH/NAD + and NADPH/NADP + and has a signi®cant in¯uence 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 speci®c 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 signi®cant 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 twofold decrease in the maximal speci®c growth rate. Also the biomass and ethanol yields were signi®cantly lowered by the transhydrogenase.