Wednesday, November 11, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
Saccharomyces cerevisiae, a workhorse yeast strain of the biotechnology and food industries, has been engineered for implementing economic processes for producing biofuels and chemicals. However, this yeast has limited capabilities for producing biomolecules which are derived from acetyl-CoA because of a rigid flux partition toward ethanol production during glucose metabolism. Even after introducing heterologous pathways for the biosynthesis of target molecules via acetyl-CoA, ethanol still remains as a major product. In this study, we aimed to investigate the effect of using glucose or xylose as a carbon source on the production of isoprenoids in engineered S. cerevisiae. To this end, we constructed efficient xylose-fermenting S. cerevisiae strains with the enhanced mevalonate pathway and compared the production of squalene from glucose and xylose. In order to construct an efficient xylose-fermenting S. cerevisiae, a xylose fermentation pathway from Scheffersomyces stipitis was introduced into S. cerevisiae and the promoter of transaldolase (TAL1) was substituted with a strong constitutive promoter in order to improve the xylose assimilating capability. The resulting strain was able to ferment xylose efficiently and rapidly. In order to increase metabolic fluxes toward the production of squalene, overexpression cassettes of a truncated HMG-CoA reductase 1 (tHMG1) and acetyl-CoA C-acetyltransferase (ERG10) were introduced into the xylose-fermenting S. cerevisiae. After cultivation on glucose and xylose, amounts of accumulated squalene in the engineered yeast were measured as an indicator of metabolic fluxes in the mevalonate pathway. Overexpression of tHMG1 resulted in drastic increase in specific squalene content of the engineered cells regardless of a carbon source, but the positive effect was more outstanding under the xylose conditions as compared to glucose conditions. While overexpression of ERG10 did not lead to any increase in squalene content regardless of carbon source, there was a synergistic effect on squalene production when both tHMG1 and ERG10 were overexpressed simultaneously. Specific squalene content in the engineered yeast was five-fold higher in the cells grown on xylose as compared to the cells grown on glucose. As a result, the tHMG1 and ERG10 co-overexpressing engineered yeast produced 472 mg/L of squalene with a productivity of 6.30 mg/L·h. These results suggest that the problem of the rigid flux partition toward ethanol production during the production of acetyl-CoA derived biofuels and chemicals can be bypassed through using xylose instead of glucose as a carbon source.