Enhanced acetate ester production of Chinese liquor yeast by overexpressing ATF1 through precise and seamless insertion of PGK1 promoter.

As the most important group in the flavor profiles of Chinese liquor, ester aroma chemicals are responsible for the highly desired fruity odors. Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis in Saccharomyces cerevisiae. In this study, we overexpressed ATF1 in Chinese liquor yeast through precise and seamless insertion of PGK1 promoter (PGK1p) via a novel fusion PCR-mediated strategy. After two-step integration, PGK1p was embedded in the 5'-terminal of ATF1 exactly without introduction of any extraneous DNA sequence. In the liquid fermentation of corn hydrolysate, both mRNA level and AATase activity of ATF1 in mutant were pronounced higher than the parental strain. Meanwhile, productivity of ethyl acetate increased from 25.04 to 78.76 mg/l. The self-cloning strain without any heterologous sequences residual in its genome would contribute to further commercialization of favorable organoleptic characteristics in Chinese liquor.