| To investigate the mechanism of EHT1 and EEB1 ecoding alcohol-Oacyltransferase on volatile components of wine during fermentation,the single and double gene deletion strain of EHT1 and EEB1 were constructed by homologous recombination technology using industrial Saccharomyces cerevisiae EC1118 as the starting strain,and the expression level of the two genes was also detected to evaluate the knock out efficiency.After detecting the growth and fermentation performance of the missing strain,the simulated grape juice and Zexiang grape juice were fermented,to study the roles of two genes in ethyl ester synthesis and discuss the indirect relationship between two genes and higher alcohols,acetic ester.Investigate the changes of aromas in the wine with the mutant fermentation,and the effects of the two genes on the overall flavor of the wine.1.Homologous knockout fragments of two target genes EHT1 and EEB1 were constructed,respectively.Then the knockout fragments were transformed into S.cerevisiae EC1118,to achieve the deletion of two target genes.Three mutants of △eht1,△eeb1 and △eht1△eeb1 were successfully constructed,and the expression levels of two genes were detected in three mutants.The expression levels of EHT1 decreased by 63.57% in △eht1,and EEB1 decreased by 43.20% in △eeb1,and EEB1 decreased by 51.7% in △eht1△eeb1,the expression level of EHT1 gene was not affected significantly.Meanwhile,from the perspective of food safety,the karnana resistance gene which was introduced during the knockout process was successfully removed.The growth and fermentation characteristics of the three mutants were detected.Results showed that low expression of these genes did not significantly affect the characteristics of the growth and fermentation of all strains,which indicated that EHT1 and EEB1 were not the key genes controlling the growth and fermentation in EC1118 strain.2.In order to detect the effects of EHT1 and EEB1 genes on volatile metabolites the three mutants were used to fermented simulated grape juice respectively.Results showed that total content of ethyl esters reduced by 35.24 %,29.13 %,15.54 %,respectively,especially decreased the content of ethyl octanate and ethyl decanoate.△eht1 had the largest effect on ethyl octanate reduced it by 32.02 %,△eeb1 had the largest effect on ethyl decanoate reduced it by 48.78 %,△eht1△eeb1 did not further reduce the total amount of ethyl ester at the base of △eht1,△eeb1.At the same time,the content of higher alcohols increased by 2.62 %,18.07 %,16.88 % respectively,which indicated that △eeb1 and △eht1△eeb1 had the most significant effect on higher alcohols but there was no significant difference between them.In addition,three mutants also increased the total content of acetate by 17.38 %,24.00 %,30.54 %,especially the phenylethyl acetate.△eht1△eeb1 showed a significant increase in phenylethyl acetate by 11.37 %.3.In order to detect the influence of EHT1 and EEB1 on the overall volatile compounds of wine,three mutants were used to ferment the grape juice of Zexiang,respectively.The effects of three mutants on esters and higher alcohols were analyzed.The results showed that total content of ethyl esters decreased by 30.55 %,19.13 %,24.36 %,especially the ethyl octanate decreased by 37.35 %,36.73 %,51.51 % respectively.In addition,three mutants increased the content of ethyl decanoate,especially the △eeb1 and △eht1△eeb1 increased by 40.67 %,51.43 %.At the same time,three mutants reduced the total content of higher alcohols by 17.08 %,12.79 % and 24.12 % respectively,especially the isoamyl alcohol.The △eht1△eeb1 had the most significant effect on higher alcohols decreased by 25.17 %,followed by △eeb1 decreased by 21.90 %.In addition,three mutants reduced the total content of acetic esters by 19.41 %,32.76 %,32.41 %,especially the isoamyl acetate decreased by 17.58 %,26.43 %,37.96 % respectively.△eeb1,△eht1△eeb1 had the largest impact on isoamyl acetate,and there was no significant difference between the two treatments.Three mutants increased the total content of acids when fermented Zexiang wine,which had the greatest influence on n-decanoic acid and caprylic acid.Among them,the highest increase rate of n-decanic acid was 15.92 %,9.36 % for △eht1△eeb1 and △eht1.And the maximum increase in the octanoic acid was 24.04 % and 21.56 % for △eht1,△eht1△eeb1.Finally,gene deletion significantly affected the grape aroma and reduced the content of terpenes by 5.82 %,19.54 %,30.35 %.In conclusion,EHT1 and EEB1 genes play important roles in the synthesis of ethyl esters and also indirectly controlling the synthesis of higher alcohols and acetic esters.The changes in expression level of these two genes significant affect the metabolic aroma in wine.EHT1 and EEB1 gene knockout changed the content of esters and higher alcohols and also changed the overall flavor of wine. |
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