Study On Regulating The Synthesis Of Acetate Ester Through Promoter Modification In Saccharomyces Cerevisiae

Acetate aroma chemicals are responsible for the highly desired odors of spirit.Content and proportion of acetate ester have a significant impact on the flavor and quality of liquor.Acetate ester is mainly synthesized by the intracellular enzyme-catalyzed in the growth phase of active cells.The higher the activity of ATF1-encode alcohol acetyltransferase is,the more acetate ester can be produced.Previous research has shown that cells in which adenylate cyclase-encoding gene CYR1 was damaged prolonged their chronological lifespan by improving the resistance to heat and oxidative stress.To regulate the synthesis of acetate,promoter modification of ATF1 and CYR1 were performed to improve the activity of acetate synthetase and prolong the chronological lifespan respectively in this study.Consequently,the content of acetate ester was raised.The result strains without any heterologous gene residual improve the safety of engineering strains with excellent properties.The main research contents were as follows:(1)A method was achieved,through which any DNA fragment can be inserted into the target loci seamlessly.The strong promoter PGK1p was precisely embedded in the 5’-terminal of ATF1 ORF(Open Reading Frame)to achieve overexpression,improving the activity of alcohol acetyltransferase and promoting the synthesis of acetate.Sequencing results indicated the precise insertion of PGK1p without any extraneous DNA residual.RT-qPCR results showed transcription in mutant was 39.4-fold of the parental strain.The enzyme activity was 5-fold of the parental strain.Corn fermentation results showed total ester in engineered strain improved slightly with stable fermentation performance and various acetate increased at different degree with ethyl acetate increased from 25.04 mg/L to 78.76 mg/L.(2)To promote the synthesis of acetate,the expression of CYR1 was weaken.60、90 and 120 bp of the 3’-terminal of CYR1 promoter was deleted respectively through the seamless gene deletion method.RT-qPCR results showed transcription levels in 60 and 90 bp deletion mutants were almost the same,which were the 72%of the parental strain.While the transcription level in 120 bp deletion mutant declined to 32%of the parental strain.Both thermotolerance and chronological lifespan results showed the 120 bp deletion mutant had the most remarkable effect.Corn fermentation results showed total ester in engineered strains improved at different degree with stable fermentation performance.GC analysis after corn fermentation showed ethyl acetate was 1.24-fold of parental strain with 20%decrease in isoamyl alcohol.