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

Abstract

Background “ATP wasting” has been observed in 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. Results 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. Conclusion 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.

Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS− to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio

“…Overexpression of genes encoding the ATP-hydrolyzing component of the ATPase encoded with the atpAGD operon in several strains of E. coli glucose uptake increase the carbon flux through the glycolytic pathway and compensate for the loss of ATP [ 9 , 60 ]. ATP wasting has been used as a suitable strategy for overproducing valuable compounds in E. coli and other microorganisms like Saccharomyces cerevisiae and succinic acid, recombinant protein, lactate, acetoin, ethanol, and 2,3-butanediol [ 8 , 61 , 62 ].…”

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway

“…To further improve the metabolically engineered S. cerevisiae strains producing 2,3-BDO, activation of glycolysis by RIM15 deletion is expected to be useful. For example, 2,3-BDO production yield improves when glycerol coproduction is suppressed [7] and ATP wasting is elevated [35]. The δ integration method was employed to activate the artificial pathway from pyruvate to 2,3-BDO [9].…”