Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in Saccharomyces cerevisiae

Abstract

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.

Aerobic physiology of redox-engineered strains modified in the ammonium assimilation for increased NADPH availability

“…The onset of aerobic fermentation in yeast can be, at least in part, attributed to glucose repression. In the case of S. cerevisiae CEN.PK113-7D, two observations are in accordance with this hypothesis: the fact that the speci¢c oxygen uptake rate decreases above the critical dilution rate [29] and the fact that an increase in the critical dilution rate of this strain can be achieved via the manipulation of genes involved in glucose repression/derepression: deletion of MIG1 and MIG2 [30] and overexpression of HAP4 [31]. In light of this, a likely explanation for the lower critical dilution rates of the recombinant strains observed in the current work can be that, as the need for NADPH production is decreased by the genetic manipulations, and a relatively larger fraction of the glucose is £owing through the Embden^Meyerhof^Parnas (EMP) pathway, the increased £ux through this pathway leads to the onset of glucose repression at lower speci¢c glucose uptake rates.…”

“…However, lower values are found in the literature for the critical dilution rate of S. cerevisiae CEN.PK113-7D. In particular, the usage of chemostat cultivations indicates a critical dilution rate for this strain of around 0.3 h 31 [29]. These di¡erences are almost certainly due to varying ethanol detection limits for the di¡erent set-ups: the concentration of ethanol in the fermenter at the critical dilution rate can be estimated from the plots presented in the work of [29] to be 5.8 mg l 31 , in contrast to 30^60 mg l 31 in the current work.…”

“…The high ethanol detection limit could also explain why the critical dilution rate is, in some cases, very close to the maximum speci¢c growth rate. Nevertheless, critical dilution rates higher than the maximum speci¢c growth rate have been observed for S. cerevisiae [29] and Saccharomyces kluyveri [14], and explained by an adaptation to growth conditions that cannot occur in a batch fermentation due to its fastchanging environment.…”

Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae

“…Glycerol and organic acids were determined by HPLC [16]. Glucose in reservoir media and supernatants was determined enzymically using a commercial glucose oxidase kit (Merck systems 14144).…”

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess

“…However, this redistribution of £uxes only a¡ected the mode of fermentative metabolism, and not the relative contribution of respiration and fermentation to sugar dissimilation. It has recently been demonstrated that a 5^10-fold overexpression of pyruvate decarboxylase in S. cerevisiae led to a decrease of the dilution rate at which aerobic fermentation set in during aerobic, glucose-limited chemostat cultures [17], and thus a¡ected the balance between respiration and fermentation. Apparently, the high pyruvate decarboxylase levels in the overexpressing strain competed for pyruvate with the enzymes involved in mitochondrial oxidation of pyruvate.…”