~ ~~~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 S'charomyces cerevisiae at different dilution rates (D) ranging from 0.05 to 0.315 h-l. The activity of most enzymes measured remained constant over this range except for alcohol dehydrogenase 1/11 which decreased proportionally with increasing dilution rate. A decrease in phosphoglucomutase activity occurred with increasing dilution rate but reached a minimum at D 0.2 h-l and from thereon remained constant. A decrease in pyruvate decarboxylase activity and a slight decrease in phosphoglucoisomerase activity was observed. At D 0-29/0.315 h-l , at the onset of the Crabtree effect, most glycolytic enzymes remained constant except for pyruvate decarboxylase and glucose-6-phosphate dehydrogenase which increased at D 0.315 h-l and alcohol dehydrogenase 1/11 which decreased. The ADHIIN and PDCl mRNA levels obtained at the different dilution rates were in accordance with the activity measurements.The mRNA level of HXKl decreased with increasing dilution rates, whereas the transcription of HXK2 increased.Pyruvate dehydrogenase (PDAI) and PGIl mRNA fluctuated but no significant change could be detected. These results indicate that there is no transcriptional or translational regulation of glycolytic flux between D 0.05 h-' and 0.315 h-l except at the branch point between oxidative and fermentative metabolism (pyruvate decarboxylasel pyruvate dehydrogenase) at D 0.315 h-l. Surprisingly regulation of the Crabtree effect does not seem to involve transcriptional regulation of PDAI. The concentrations of ATP and CAMP decreased slightly during the increase in dilution rate, but increased again at D 0.315 h-l. 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 D 0.29 and 0.315 h-l, whereas the fructose 1,6-diphosphate concentration increased from 0.05 h-l to 0-315 h-l. These data indicate that glycolytic flux in S. cerevisiae 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.
For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).
In the yeast Kluyveromyces marxianus two forms of inulinase were present, namely, an inulinase secreted into the culture fluid and an inulinase retained in the cell wall. Both forms were purified and analyzed by denaturing and nondenaturing polyacrylamide gel electrophoresis. With the use of endo-ID-N-acetyl-glucosaminidase H, it was established that the enzyme retained in the cell wall and the enzyme secreted into the culture fluid have similar subunits consisting of a 64-kDa polypeptide with varying amounts of carbohydrate (26 to 37% of the molecular mass). The two forms of inulinase differed in size because of their differences in subunit aggregation. The enzyme present in the culture fluid was a dimer, and the enzyme retained in the cell wall was a tetramer. The differences in oligomerization did not affect the apparent Km values towards the substrates sucrose and raffinose. These findings support the hypothesis that the retention of glycoproteins in the yeast cell wall may be caused by a permeability barrier towards larger glycoproteins. The amino-terminal end of inulinase was determined and compared with the amino terminus of the closely related invertase. The kinetic and structural evidence indicates that in yeasts two distinct ,3-fructosidases exist, namely, invertase and inulinase.
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