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-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.
Flikweert
M T
MT
Kluyver Institute of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Kuyper
M
M
van Maris
A J
AJ
Kötter
P
P
van Dijken
J P
JP
Pronk
J T
JT
D000085:Acetates / Q000378:metabolism; D000332:Aerobiosis; D002455:Cell Division; D003470:Culture Media / Q000378:metabolism; D004790:Enzyme Induction; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D009243:NAD / Q000378:metabolism; D010641:Phenotype; D011767:Pyruvate Decarboxylase / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D000085:Acetates; D003470:Culture Media; D005656:Fungal Proteins; C103820:PDC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D009243:NAD; D000431:Ethanol; D011767:Pyruvate Decarboxylase; D005947:Glucose