EFFECTS OF PYRUVATE DECARBOXYLASE OVERPRODUCTION ON FLUX DISTRIBUTIONAT THE PYRUVATE BRANCH POINT IN SACCHAROMYCES-CEREVISIAE

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarbox ylase; Pdc) was introduced in Saccharomyces cerevisiae CEN.PK113-5D. T he physiology of the resulting prototrophic strain was compared with t hat of the isogenic prototrophic strain CEN.PK113-7D and an empty-vect or reference strain. In glucose-grown shake-flask cultures, the introd uction of the PDC1 plasmid caused a threefold increase in the Pdc leve l, In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h(-1), Pdc levels in the overproducing strain were 14-fo ld higher than those in the reference strains. Levels of glycolytic en zymes decreased by ca, 15%, probably due to dilution by the overproduc ed Pdc protein. In chemostat cultures, the extent of Pdc overproductio n decreased with increasing dilution rate. The high degree of overprod uction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h(-1) in the reference strains to 0.23 h(-1) in the Pdc-overprodu cing strain. In the latter strain, the specific respiration rate reach ed a maximum above the dilution rate at which aerobic fermentation fir st occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc- overproducing strain. Rather, the results indicate that Pdc overproduc tion affected flux distribution at the pyruvate branch point by influe ncing competition for pyruvate between Pdc and the mitochondrial pyruv ate dehydrogenase complex. In respiratory cultures (dilution rate, <0. 23 h(-1)), Pdc overproduction did not affect the maximum glycolytic ca pacity, as determined in anaerobic glucose-pulse experiments.