Abstract:Saccharomyces cerevisiae is a common microorganism in ethanol fermentation.During fermentation，S.cerevisiae is affected by high temperature，ethanol，osmotic pressure，acetic acid，and other stresses.To enhance the stress tolerance of this yeast，AY12a was used as the starting strain for intracellular homologous recombination to knockout the TPK3 gene encoding the catalytic subunit of protein kinase A（PKA），thus yielding AY12a-2.The regulated activity of PKA improved the stress tolerance of the yeast.During fermentation，glucose consumption of AY12a-2 increased，CO2weight loss increased by 34%，and ethanol production increased by 17.8%.