Redirection of the respiro-fermentative flux distribution in Saccharomyces cerevisiae by overexpression of the transcription factor Hap4p

Abstract

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.

FIG. 1

Expression of several mRNAs in a Hap4p overexpression strain. DL1 and DL1HAP were grown to mid-logarithmic phase in medium containing 2% glucose (D) or 2% ethanol–2% glycerol (EG); 20 μg of total RNA was hybridized with probes specific for HAP4, ACT (actin), QCR8, or SUC2 mRNA.

FIG. 2

Growth characteristics and glucose consumption and ethanol production profile in fermentor batch cultivations. Shown are dry yeast biomass formation (□, ■) and glucose (◊, ⧫) and ethanol (▵, ▴) concentrations present in the culture supernatant of the wild-type strain DL1 (A) and the Hap4p overproducer DL1HAP (B) and glucose consumption (C) and ethanol production (D) relative to the amount of dry yeast biomass formed in wild-type DL1 (closed symbols) and DL1HAP (open symbols) strains.