In Sacchuromyces cerevisiue, the structural genes PDCI, PDCS and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and TnSble. A pyruvate-decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a threefold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc-strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc-and wild-type S. cerevisiae were identical. However, Pdc-S. cerevisiue was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0.10 h-') were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild‐type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate‐decarboxylase‐negative (Pdc−) mutant lacking all three PDC genes exhibited a three‐fold lower growth rate in complex medium with glucose than the isogenic wild‐type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc− strains. Furthermore, in ethanol‐limited chemostat cultures, the biomass yield of Pdc− and wild‐type S. cerevisiae were identical. However, Pdc− S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol‐limited chemostat cultures (D = 0·10 h−1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed‐substrate cultures were switched to a feed containing glucose as the sole carbon source, wash‐out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl‐CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose‐limited chemostat cultures.

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN.PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of thePDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h−1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction 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-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first 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 overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h−1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.