Improvement of the Bioethanol Yield in Saccharomyces cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta Constructed by the CRISPR-Cas9 Approach with the Decrease of By-Product Formation

Abstract Background: Bioethanol plays an important value in renewable liquid fuel. The inhibition of by-product formation would enhance the ethanol production of Saccharomyces cerevisiae. In fact, the excessive accumulation of glycerol and organic acids caused the decrease of ethanol content in the process of industrial ethanol production. Results: In this study, S. cerevisiae engineering strains were constructed using the CRISPR-Cas9 approach to delete GPD2, FPS1, and ADH2 to improve the yield of ethanol with the decrease of by-product contents. Engineered S. cerevisiae SCGFA by the GPD2, FPS1, and ADH2 deletion produced 23.1 g/L with 50 g/L of glucose as substrate. SCGFA strain exhibited the ethanol conversion rate of 0.462 g per g of glucose. In addition, the contents of glycerol, lactic acid, acetic acid, and succinic acid in SCGFA decreased by 22.7, 12.7, 8.1, 19.9, and 20.7% compared with the wild-type strain, respectively. Conclusions: The co-knockout of GPD2, FPS1, and ADH2 dramatically improved the ethanol yield of S. cerevisiae by the inhibition of glycerol release and the prevention of ethanol consumption. This study developed a new S. cerevisiae engineering strain SCGFA with high-level ethanol production after GPD2, FPS1, and ADH2 deletion. The engineering strain SCGFA could apply in ethanol production with less formation of by-products.