Construction Of High Yield C6-C10 Ethyl Esters Saccharomyces Cerevisiae Strains

C6-C10 ethyl ester is an important aroma substance in Chinese strong aroma baijiu and wines,which can impart flavor to baijiu,wines,beer and so on.The synthesis of C6-C10 ethyl ester in Saccharomyces cerevisiae is a PDH(pyruvate metabolism)bypass pathway,which is mainly affected by acetyl-CoA,malonyl-CoA,and medium-chain acyl-CoA.In this study,yeasts with higher ester-producing ability were screened from the existing ester-producing yeasts in the laboratory as starting strain.Secondly,the acetyl-CoA,malonyl-CoA and medium-chain acyl-CoA anabolic pathways of the selected strains were strengthened,and the exogenous alcohol acyltransferase catalytic pathway was introduced to promote the synthesis of C6-C10 ethyl ester in Saccharomyces cerevisiae.The main contents and results were as follows:The first is to screen the starting strain.In the experiment,the existing strains CA and C01,AY 12 and AY12-α in the laboratory,and the strain CA producing higher ethyl ester of C6-C10 was screened,with ethyl caproate of 0.27 mg/L,ethyl octanate of 1.65 mg/L and ethyl decanoate of 2.92 mg/L,respectively,and was used as a starting strain for molecular modification in the later stage.The second is to enhance the synthesis of acetyl-CoA to increase the amount of C6-C10 ethyl ester.Using CA as a starting strain,the overexpressing strains C-adh2,C-ald6,C-acsl and simultaneously were obtained by using the strong promoter and the overexpressing genes ADH2,ALD6 and ACS1 are directly related to the acetyl-CoA synthesis pathway.The mutants were expressed,and the content of acetyl-CoA was characterized by detecting the C6-C10 ethyl ester content of the strain.The results showed that the over-expressed ACS1 strain C-acs1 had higher ethyl hexanoate production than C-adh2 and C-ald6,indicating that the ACS1 gene effect may be better than ADH2 and ALD6 in increasing the content of acetyl-CoA.The yields of ethyl hexanoate,ethyl octanoate and ethyl decanoate of strain C-ald6acsl were 2.54 times,6.93 times and 6.78 times,respectively,of the starting strain.Further,the overexpression of ADH2 gene showed almost no change in the content of C6-C10 ethyl ester,indicating that the combination of ACS1 and ALD6 genes was better.Therefore,C-ald6acsl was selected as the starting strain for late molecular transformation.Then,the synthesis of malonyl CoA was enhanced to increase the content of C6-C10 ethyl ester.Overexpression and free overexpression of the acetyl-CoA carboxylase gene ACC1*were integrated using a strong promoter.The fermentation experiment showed that the yields of ethyl octanoate and ethyl decanoate of C-ald6acslA*integrated with ACC1*were increased by 1.36 times and 1.49 times,respectively,compared with the starting strain C-ald6acs1;On the basis of further free expression of ACC1*,the yield of C6-C10 ethyl ester was not significantly improved considering that free expression lacked of the instability.Therefore,C-ald6acs1A*was selected as the starting strain for the next transformation.Further,experiments were performed to overexpress FAS1,FAS2 to enhance the synthesis of mid-chain acyl-CoA.On the basis of C-ald6acslA*,FAS1 and FAS2 were overexpressed separately and simultaneously,named as C-ald6acslA*F1,C-ald6acs1A*F2 and C-ald6acs1A*F1F2 respectively.The content of medium chain acyl-CoA was characterized by detecting the yield of C6-C10 ethyl ester in the mutant strain.The results showed that the yield of ethyl hexanoate,ethyl octanoate and ethyl decanoate of C-ald6acs1A*Fl was increased by 65%,19.3%and 18.5%,respectively,compared with the starting strain C-ald6acs1A*.However,the yield of C6-C10 ethyl ester in C-ald6acslA*F2 were slightly lower.The results indicated that the overexpression of the gene FAS1 encoding the β subunit in the fatty acid synthase is superior to FAS2 in enhancing the C6-C10 ethyl ester.The content of ethyl hexanoate,ethyl octanoate and ethyl citrate produced by strain C-ald6acs 1 A*F1F2 was 2.77mg/L,11.19 mg/L and 11.32mg/L,respectively,which was 54.7%,41.5%and 43.4%times higher than that of the starting strain C-ald6acs1A*,indicating that coexpression of FAS1 and FAS2 genes can further increase the content of C6-C10 ethyl ester.In the early stage of the experiment,the metabolic flow caused more flow to the fatty acyl-CoA.Therefore,it was necessary to introduce the alcohol acyltransferase gene SAAT in the strawberry to enhance the alcohol acyltransferase catalytic pathway.The amount of ethyl hexanoate produced by C-ald6acslA*F1F2S was 7.53 mg/L,which was 2.72 times that of the starting strain C-ald6acs1A*F1F2.Moreover,The content of ethyl octanoate and ethyl decanoate were 7.00 times and 8.78 times that of the strain CA,indicating that the specificity of the alcohol acyltransferase SAAT to ethyl hexanoate was better than that of ethyl octanoate and ethyl decanoate,which was consistent with the literature.