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.

~The effects of nitrogen starvation on the anaerobic physiology of Saccharomyces cerevisiae were studied in cells cultivated in a bioreactor. The composition of the mineral medium was designed such that the nitrogen source became depleted while there was still ample glucose left in the medium. The culture was characterized by acoustic gas analysis, flow injection analysis and HPLC analysis of extracellular substrates and metabolites. During the cultivation, the macromolecular composition of the cells was analysed with respect to the cellular content of RNA, protein, trehalose and glycogen. During exponential growth under anaerobic conditions, the maximum specific growth rate (p,J of 5. cerevisiae CBS 8066 (0.46 h-l) was identical to the p, , , determined under aerobic conditions. Depletion of ammonium in the medium led to an abrupt decrease in the flux through glycolysis. Subsequently, a continuous decrease in the carbon dioxide evolution rate, caused by catabolite inactivation of the hexose-transport system, was observed. The apparent halflife of the transport system under nitrogen starvation was 13 h. During the exponential growth phase, the cellular content of RNA and protein was 15y0 (w/w) and 60% (w/w), respectively. A t the end of the cultivation where the cells had been starved of nitrogen for 18 h, the cellular content of RNA and protein had decreased to 4% (w/w) and 22% (w/w), respectively. The intracellular carbohydrate content increased dramatically as trehalose and glycogen accumulated to final concentrations of 7 O/ O (w/w) and 25 O/ O (w/w), respectively. Glycerol formation during nitrogen starvation was higher than that accounted for by the formation of organic acids, suggesting a protein turnover of approximately 6% h-l. The growth energetics of 5. cerevisiae CBS 8066 also changed as a result of nitrogen starvation, and V, , was observed to increase from 80 mmol g-l during the exponential growth phase to more than 130 mmol g-1 towards the end of the cultivation. The presented results illustrate the effect of nitrogen starvation on glycerol formation, protein turnover, catabolite inactivation of the sugar-transport system, the cellular composition, the cell cycle and growth energetics.

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