Improved Ethanol Tolerance and Production in Pyruvate Dehydrogenase Mutant ofSaccharomyces cerevisiae

Ethanol, a naturally synthesized compound by Saccharomyces cerevisiae yeast through alcoholic fermentation, has previously been studied as a renewable alternative to traditional fossil fuels. However, current challenges of engineering S. cerevisiae strains for ethanol production remain: low ethanol productivity, inefficient substrate catabolism, and a buildup of toxic products to inhibitory levels. In this study, we proposed a method of metabolic rewiring via the deletion of the pda1 gene, which leads to pyruvate dehydrogenase (PDH) deficiency. The Δ pda1 mutant strain was created by CRISPR Cas-9 knockout using the constructed pCRCT-PDA1 plasmid. Subsequently, mutant candidates were screened by PCR and Sanger sequencing, confirming a 17 bp deletion in the pda1 gene. The wild-type and mutant strains were analyzed for growth under aerobic and anaerobic conditions in glucose and glycerol, as well as ethanol production and tolerance. The Δ pda1 mutant displays a ~two-fold increase in anaerobic ethanol production and an aerobic growth defect with no observed increase in ethanol production. The mutant is also hyper-tolerant to ethanol, which allows a faster buildup of products in growth media with minimal reduction in growth. This new S. cerevisiae strain deficient in PDH may provide a solution to the efficient and abundant synthesis of biofuels such as ethanol by redirecting metabolic flux and altering stress response.