Jolanda Blom

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 bakers 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.

Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae

The tendency of Saccharomyces cerevisiae to favor alcoholic fermentation over respiration is a complication in aerobic, biomass-directed applications of this yeast. Overproduction of Hap4p, a positive transcriptional regulator of genes involved in respiratory metabolism, has been reported to positively affect the balance between respiration and fermentation in aerobic glucose-grown batch cultures. In this study, the effects of HAP4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN.PK 113-7D under a variety of growth conditions. In aerobic glucose-limited chemostat cultures, overexpression of HAP4 increased the specific growth rate at which aerobic fermentation set in by about 10% relative to the isogenic wild-type. Upon relief of glucose-limited conditions, the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain. The effect of Hap4p overproduction was most drastic in aerobic, glucose-grown chemostat cultures in which ammonium was limiting. In such cultures, the biomass yield on glucose was double that of the wild-type.

Dissection of the promoter of the HAP4 gene in S. cerevisiae unveils a complex regulatory framework of transcriptional regulation.

In S. cerevisiae, the heteromeric Hap2/3/4/5 complex is necessary for induced transcription of a large number of genes involved in oxidative metabolism on non‐fermentable carbon sources. The Hap4p subunit is the activator subunit and at the same time also the regulatory part of the complex, since it is the only one whose level is regulated by carbon source itself. HAP4 promoter analysis shows a 265 bp activating region at position −1006/−741 bp upstream of the ATG start codon. Specific and differential protein‐binding to a 30 nt CSRE‐like sequence within this region was observed with extracts from repressing and inducing carbon sources. Carbon source‐dependent activation mediated by the 265 bp fragment, as well as protein binding to the 30 nt CSRE‐like region, is dependent on the presence of CAT8 function, unveiling a complex framework by which the expression of the HAP4 gene is coordinated. Copyright