Improvement of ethanol and 2,3-butanediol production in Saccharomyces cerevisiae by ATP wasting

"ATP wasting" has been observed in .sup.13C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae, such as the sake strain Kyokai 7, consume approximately two-fold as much ATP as laboratory strains. Increased ATP consumption may be linked to the production of ethanol, which helps regenerate ATP. This study was conducted to enhance ethanol and 2,3-butanediol (2,3-BDO) production in the S. cerevisiae strains, ethanol-producing strain BY318 and 2,3-BDO-producing strain YHI030, by expressing the fructose-1,6-bisphosphatase (FBPase) and ATP synthase (ATPase) genes to induce ATP dissipation. The introduction of a futile cycle for ATP consumption in the pathway was achieved by expressing various FBPase and ATPase genes from Escherichia coli and S. cerevisiae in the yeast strains. The production of ethanol and 2,3-BDO was evaluated using high-performance liquid chromatography and gas chromatography, and fermentation tests were performed on synthetic media under aerobic conditions in batch culture. The results showed that in the BY318-opt_ecoFBPase (expressing opt_ecoFBPase) and BY318-ATPase (expressing ATPase) strains, specific glucose consumption was increased by 30% and 42%, respectively, and the ethanol production rate was increased by 24% and 45%, respectively. In contrast, the YHI030-opt_ecoFBPase (expressing opt_ecoFBPase) and YHI030-ATPase (expressing ATPase) strains showed increased 2,3-BDO yields of 26% and 18%, respectively, and the specific production rate of 2,3-BDO was increased by 36%. Metabolomic analysis confirmed the introduction of the futile cycle. ATP wasting may be an effective strategy for improving the fermentative biosynthetic capacity of S. cerevisiae, and increased ATP consumption may be a useful tool in some alcohol-producing strains.