SUMMARY: mRNA steady-state levels and activities of enzymes of intermediary carbon metabolism (hexokinase, phosphoglucoisomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase) and glucose-regulated enzymes (pyruvate decarboxylase, pyruvate dehydrogenase, invertase, alcohol dehydrogenase) were determined in glucose-limited continuous cultures of an industrial strain of at different dilution rates () ranging from 0.05 to 0.315 h. The activity of most enzymes measured remained constant over this range except for alcohol dehydrogenase I/II which decreased proportionally with increasing dilution rate. A decrease in phosphoglucomutase activity occurred with increasing dilution rate but reached a minimum at 0.2 h and from thereon remained constant. A decrease in pyruvate decarboxylase activity and a slight decrease in phosphoglucoisomerase activity was observed. At 0.29/0.315 h, at the onset of the Crabtree effect, most glycolytic enzymes remained constant except for pyruvate decarboxylase and glucose-6-phosphate dehydrogenase which increased at 0.315 h and alcohol dehydrogenase I/II which decreased. The and mRNA levels obtained at the different dilution rates were in accordance with the activity measurements. The mRNA level of decreased with increasing dilution rates, whereas the transcription of increased. Pyruvate dehydrogenase () and mRNA fluctuated but no significant change could be detected. These results indicate that there is no transcriptional or translational regulation of glycolytic flux between 0.05 h and 0.315 h except at the branch point between oxidative and fermentative metabolism (pyruvate decarboxylase/pyruvate dehydrogenase) at 0.315 h. Surprisingly regulation of the Crabtree effect does not seem to involve transcriptional regulation of . The concentrations of ATP and cAMP decreased slightly during the increase in dilution rate, but increased again at 0.315 h. The concentrations of glucose 6-phosphate and fructose 6-phosphate did not increase when the dilution rate increased as expected from the activities of hexokinase, phosphoglucoisomerase and phosphofructokinase. Instead, a decrease in the glucose 6-phosphate and fructose 6-phosphate concentrations was observed. The concentration of glucose 1-phosphate also decreased with increasing dilution rate but increased again at 0.29 and 0.315 h, whereas the fructose 1,6-diphosphate concentration increased from 0.05 h to 0.315 h. These data indicate that glycolytic flux in is regulated mainly by allosteric regulation of glycolysis when growth rate is increased. Invertase was present (mRNA and activity) at every dilution rate which indicates that glucose-specific repression of enzyme systems is not present in glucose-limited continuous cultures, not even when the yeast produces ethanol. This also indicates that the Crabtree effect is not related to glucose repression.


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