ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F.sub.1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation. In contrast to genomic integration, we found that episomal expression of the [alpha][beta][gamma] subunits of the F.sub.1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the [beta] subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli's F.sub.1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain. Our study shows that expression of genes of the F.sub.1-portion of E. coli's ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.