Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-w eight byproducts (ethanol, acetate) in biomass-directed applications of Sac charomyces cerevisiae. To investigate whether decreased expression levels o f pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, whi ch encodes a positive regulator of pyruvate-decarboxylase synthesis, was in activated in the prototrophic strain S. cerevisiae CEN.PK113-7D. This cause d a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limite d, 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 wer e the major byproducts formed by the wild type, In the pdc2 Delta strain, f ormation. of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2 Delta strain produced substantial amounts of pyruvate a fter a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2 Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic fl ux 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 metabol ism was respiratory, At dilution rates up to 0.20 h(-1), growth of the pdc2 Delta strain was respiratory and biomass yields were similar to those of w ild-type cultures. Above this dilution rate, washout occurred. The low mu(m ax) of the pdc2 Delta strain in glucose-limited chemostat cultures indicate s that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyr uvate. Furthermore, it implies that inactivation of PDC2 is not a viable op tion for reducing byproduct formation in industrial fermentations. (C) 1999 John Wiley & Sons, Inc.