Glycolytic flux regulation in Saccharomyces cerevisiae during anaerobic growth and starvation

Doctoral thesis, 2005

The physiology of S. cerevisiae under anaerobic growth conditions is of interest not least during implementation and development of industrial yeast-catalysed ethanol fermentations in order to maintain a productive yeast population. During growth in industrial fermentations the concentration of lactic acid can often reach considerable concentrations as a result of contamination by lactic acid bacteria. The yeast also often faces complex nutritional conditions including nutrient limitation and/or starvation. The effects of lactic or benzoic acid on glycolytic rate and product yields were studied on cells growing under carbon or nitrogen limiting conditions in anaerobic chemostat cultures (D=0.1 h-1). It was shown that during growth under nitrogen limiting conditions compared to growth under carbon limiting conditions and/or in presence of lactic acid or benzoic acid, the flow of glucose was directed to a larger extent from glycogen and biomass formation towards ethanol production. The specific ethanol production rate (mmol/gh) was also stimulated; however, the effect of lactic acid was not as large as that of benzoic acid or as the effect of growth under nitrogen limitation compared to growth under carbon limitation. High glycolytic rates/specific ethanol production rates were obtained by the weak organic acids and/or nitrogen limitation. Based on comparisons of exhibited glycolytic rate with levels of allosteric effectors of the glycolytic enzymes during the different growth conditions it was suggested that a concerted action of decreased ATP levels and increased fructose-6-phosphate levels may account for at least a part of the increase glycolytic rate during these growth conditions. Studies on the carbon or nitrogen starvation response of anaerobically growing cells were also performed. Carbon starvation of anaerobic exponentially growing cells induced an almost complete loss of fermentative capacity and viability. Nitrogen starvation reduced fermentative capacity by 70-95% depending on strain. The levels of glycolytic enzymes were not altered neither after carbon nor nitrogen starvation, however, a depletion of ATP was seen immediately upon carbon starvation but not nitrogen starvation. Growth into stationary-phase prior to carbon starvation enabled the cells to retain most of their fermentative capacity after carbon starvation. Reduction in protein synthesis prior to starvation of anaerobic exponential-phase cells could also to some extent alleviate the effects of carbon starvation. Further studies on cells grown in chemostat (D=0.1h-1) under different growth conditions that were subsequently exposed to carbon or nitrogen starvation showed that there was a positive relationship between cellular content of endogenous glycogen before carbon starvation and fermentative capacity after carbon starvation. A positive relationship after carbon starvation between the intracellular ATP levels (0-1.5 mM) and the fermentative capacity in cells grown in anaerobic chemostat cultures was also found. From these results it is suggested that anaerobically grown cells not containing any endogenous carbohydrate reserves face a lack of energy upon rapid carbon starvation which renders necessary adjustments to the starvation conditions impossible to perform, thereby reducing viability and fermentative capacity of the cell upon carbon starvation.