A NITROGEN-LIMITED, GLUCOSE-REPRESSED, CONTINUOUS-CULTURE OF SACCHAROMYCES-CEREVISIAE

Glucose-repressed growth of Saccharomyces cerevisiae was analysed in a nitrogen-limited continuous culture at different dilution rates (D). The glucose consumption of the yeast decreased from 3.4 g g(-1) h(-1) to 3.0 g g(-1) h(-1) when D was decreased from 0.3 h(-1) to 0.15 h(-1) . No transcripts of the SUC2 and HXK1 genes, encoding, respectively, i nvertase and hexokinase isoenzyme 1, could be detected. Because both g enes are regulated by glucose repression at the transcriptional level, this confirmed that the culture was glucose repressed at every D. Dur ing the decrease in D, no change in the activities or mRNA revels of k ey enzymes in carbon metabolism was observed, except for alcohol dehyd rogenases I and II and phosphoglucomutase. these enzymes increased in activity and/or mRNA level when D was decreased, which was also observ ed in glucose- and galactose-limited continuous cultures. This demonst rates that the expression levels of alcohol dehydrogenases I and II, a nd also phosphoglucomutase, are coupled to the growth rate of the orga nism. A comparison between the alcohol dehydrogenase II activity in gl ucose- and nitrogen-limited continuous cultures demonstrated that the growth rate contributes as much to repression of alcohol dehydrogenase II activity as does glucose. Both the glucose consumption and the act ivity of the glycolytic enzymes were relatively constant when D was de creased and, as a consequence, the concentrations of intracellular met abolites remained constant. A slight decrease in the glucose 6-phospha te concentration was observed, which could be caused by the slight dec rease in glucose consumption at low D values. The 2-oxoglutarate and c AMP concentrations increased twofold when D was decreased. The first p robably reflects the increased NH4+ consumption at high D values, whil e the latter is caused by the high amount of extracellular cAMP compar ed with the amount of intracellular cAMP. The decrease in growth rate raised the amount of biomass, while nitrogen was limiting in all cases . Analysis of the biomass composition at the different D values reveal ed that the amount of nitrogen per gram dry weight was constant, white the amounts of carbon, hydrogen and oxygen were higher at low D value s. In addition, higher concentrations of trehalose and glycogen were f ound at low D values. This demonstrates that the glucose which is used at high D values for the production of biomass is converted into stor age carbohydrates, e.g. trehalose and glycogen, at tow D values. A nit rogen pulse did not result in a dramatic response of S. cerevisiae exc ept for a rapid change in the free amino acid pool. Thus S. cerevisiae is not able rapidly to enhance growth upon a nitrogen pulse, which is in contrast with the response to a glucose pulse of a carbon-limited continuous culture of S. cerevisiae.