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

Abstract

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 pdc2Δ strain, formation of ethanol and acetate was reduced by 60–70%. In contrast to the wild type, the pdc2Δ 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 pdc2Δ 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 pdc2Δ strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low μmax of the pdc2Δ 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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 42–50, 1999.