Construction Of Saccharomyces Cerevisiae Engineering Bacteria Based On The Cloned Expression Of Alcohol Acetyltransferase

Ethyl acetate is an ester substance with fruity aroma,which can impart rich aromas to white wine,fruit wine,flower wine,etc.,and has important significance for the formation of wine flavor.At present,a large number of low-grade fermented wines still have shortcomings such as few aroma components and insufficient fragrance.In this study,EAT（encoding alcohol acetyltransferase gene）in the metabolic pathway of alcohol acetyltransferase was used as the research object.The research uses genetic engineering methods to clone and express EAT in Saccharomyces cerevisiae from Wickerhamomyces anomalus FBKL2.00K8,and explores the effect of overexpression of the target gene on the amount of ethyl acetate produced in Saccharomyces cerevisiae,and evaluates the performance and aroma of low fermentation wines.The main research results are as follows:1.The research uses PCR method to amplify the target gene EAT from Wickerhamomyces anomalus FBKL2.00K8,connects to p MD 19-T vector and sequences to get the correct sequence.Bioinformatics analysis of the target gene revealed that the target protein sequence has a similarity of up to 99%with the protein in Wickerhamomyces anomalus NRRL Y-366-8,which is composed of 490 amino acids,and the protein molecular weight is 53.9 KDa,The theoretical isoelectric point（p I）is 6.8,and the extinction coefficient（λ=280nm）is 66740 M^-1cm^-1.This protein is a hydrophobic protein.Its secondary structure mainly exists in three forms:α-helix,β-turn and random coils,which account for 40%,2.86%and 40.41%of the total number of amino acids,respectively.It has no transmembrane structure and it is a secreted protein.The research uses lightning cloning technology to connect the target gene EAT to the shuttle vector Yep-PGK to construct the recombinant plasmid Yep-PEK,and uses the yeast lithium acetate transformation method to introduce the recombinant plasmid into the competent cells of Saccharomyces cerevisiae W303-1A to successfully construct a engineering strain W303-EAT expressing the target protein.2.Based on the metabolic pathway of alcohol acetyltransferase,the research analyzes the changes of metabolites by monitoring the content changes of acetyl-Co A,pyruvate and ethyl acetate in different fermentation periods.It proved that the target gene EAT is in the engineering strain W303-EAT and it can effectively catalyze the production of ethyl acetate from acetyl-Co A and ethanol,which has an important influence on the amount of ethyl acetate produced.The research on the enzymatic properties of the engineering strain W303-EAT found that the alcohol acetyltransferase activity of the engineering strain W303-EAT is significantly increased by 5-6 times compared with the original strain W303-1A.The optimal reaction temperature of the recombinase is 30^°C,and the enzyme activity is stable below 30^°C.The optimal p H of the recombinase is 7,and the enzyme molecule is relatively stable in the p H range of 7-8.Metal ions including Mg²⁺,K⁺and Ca²⁺can increase the enzyme activity.Fe³⁺and Mn²⁺have little effect on the enzyme activity.Zn²⁺,Cu²⁺,Hg²⁺,Ag⁺,Cd²⁺,Ba²⁺and Pb²⁺will reduce the activity of the enzyme seriously.3.The fermentation performance test of the engineering strain W303-EAT showed that:compared with the original strain W303-1A,there was no significant difference in the amount of CO₂loss,alcohol content,and reducing sugar content of the engineering strain W303-EAT（P>0.05）.The ability of the engineering strain W303-EAT to produce acetate is significantly improved,in which the content of ethyl acetate increased by 6.14 times,and the production of phenethyl acetate increased by1.5 times.The content of higher alcohols（isobutanol and isoamyl alcohol）all decreased.The research applies the engineering strain W303-EAT to low-grade fermented wines（rice wine,blueberry wine,grapefruit wine and rose wine）and explores the differences in aroma production characteristics between the original strain and commercial dry yeast.The results showed that the capacity of engineering strain W303-EAT to produce ethyl acetate was slightly higher than that of commercially available yeast,and was significantly higher than that of original strain W303-1A by 4.28 times,0.18 times,0.24 times and 1.57 times,respectively.The engineering strain W303-EAT has a good effect of improving the ester and flavoring in rice wine and rose wine.The engineering strain W303-EAT has the lowest higher alcohol content in fermented wine and has a certain effect on lowering higher alcohol.