Breeding Of Saccharomyces Cerevisiae Strains With High Production Of C6-C10 Fatty Acid Ethyl Esters

C6-C10 fatty acid ethyl ester(ethyl hexanoate,ethyl octanoic,ethyl decanoate)is an important aroma substance in liquor.Among them,ethyl hexanoate has the aroma of pineapple,ethyl caprylate has the aroma of pineapple,apple and brandy,and ethyl caproate has the aroma of coconut.Although these esters are present in very small amounts in alcoholic beverages,they contribute to a fuller flavor and a richer mouthfeel.In order to improve the ability of synthesizing C6-C10 fatty acid ethyl esters in Saccharomyces cerevisiae,ARTP mutagenesis and means of genetic engineering were used in this study to increase the yield of C6-C10 fatty acid ethyl esters in Saccharomyces cerevisiae during fermentation.(1)The ARTP-cerulenin-sensor-maize thick mash fermentation verification screening system was established.Using this screening model,33 strains of Saccharomyces cerevisiae with sensor induction ratio higher than 6 were screened from the mutant library of 2000 strains,and 15 strains of C6-C10 fatty acid ethyl ester were further screened by corn mash fermentation combined with GC-MS detection.Saccharomyces cerevisiae strains with improved yields,of which strain 472 has the strongest ability of producing esters,and the yields of ethyl hexanoate,ethyl octanoic,and ethyl decanoate reached 2.80,4.18 and 9.78 mg/L,respectively.It is 2.8 times,3.05 times and 4.95 times of the starting bacteria.(2)A random mutation-sensor-maize mash fermentation verification screening system of KS and MPT domains was established.According to the existing research,it showed that the forward mutation sites for improving the synthesis of C6-C10 fatty acid ethyl esters were mainly concentrated in the MPT domain of the FAS1 gene and the KS domain of the FAS2 gene,and the domains were randomly mutated.The mutant strains were obtained,and 10 strains with high production of C6-C10 fatty acid ethyl ester were obtained through sensor detection and fermentation verification screening.Among them,M72 had the strongest ester-producing ability,and the highest yield of ethyl hexanoate was 3.23 mg/L,which was 2.13 times of the starting strain.The yield of ethyl octanoic was 4.65 mg/L,which was 3.52 times that of the starting strain;the yield of ethyl decanoate was 6.47 mg/L,which was 3.44 times that of the starting strain.(3)Determination of FAS mutation point and point mutation verification.Four unreported non-synonymous mutation points were obtained by sequencing the FAS gene of15 ARTP high-yielding mutant strains,namely E171 D,E172D,V1393 W and A1735 S.Combined with the literature,nine FAS point mutant strains were constructed.The results of corn mash fermentation experiments showed that the mutations of three amino acid sites E171 D,E172D and A1735 S were beneficial to the synthesis of C6-C10 fatty acid ethyl esters.Among the point mutant strains: A1735 S had the strongest ester-producing ability.The yields of the three esters reached 1.9,3.65 and 7.71 mg/L,respectively.Sequencing analysis of ten MPT domain random mutant strains with the strongest ester production ability revealed five unreported non-synonymous mutations S318 P,N390I,I407V,A424T and K441T.It is preliminarily speculated that the non-synonymous mutation point can promote the synthesis of C6-C10 fatty acid ethyl ester: S318P>A424T>K441T.