Adaptive laboratory evolution of Saccharomyces cerevisiae CEN.PK 113-7D to enhance ethanol tolerance

Saccharomyces cerevisiae is a widely used yeast for industrial production of ethanol. However, elevated ethanol, temperature, and osmotic stress adversely affect fermentation efficiency. In this study, adaptive laboratory evolution for S. cerevisiae CEN.PK 113–7D on higher concentrations of ethanol was performed. After 144 days, the maximum specific growth rate (µmax) increased from 0.0240 h−1 to 0.1150 h−1 for the strain evolved on 9% v/v ethanol, and from 0.0002 h−1 to 0.0530 h−1 for the strain evolved on 11% v/v ethanol, and the specific glucose uptake rate increased by 30%. The 11%-evolved strain produced 94.5 g/L ethanol in a fermentation as compared to 78.5 g/L production by a non-evolved strain. By whole-genome sequencing of the evolved clones, we identified multiple coding mutations in genes involved in processes such as stress response, cell growth regulation, pentose phosphate pathway, lipid synthesis, and redox balance. The selected mutations in RKI1, CYC2, ANR2, RGA2, RGA1, LPX1, and LRE1 genes were validated by introducing them in the non-evolved yeast, showing 1.7- to 5-fold growth improvement at 9% ethanol (p < 0.05). Notably, RGA2, RGA1 and LPX 1 carried an identical missense mutation across three independent clones. The RKI1I208V mutant showed the highest ethanol tolerance, while CYC2N342A achieved the highest ethanol production.