Research Of Related Genes For Higher Alcohols Metabolism In Wine Yeast

Higher alcohols are important flavor components of wine,while fusel alcohols at high concentrations are harmful to human health and impart off-flavors.In this study,an excellent wine yeast strain WY-1 with low yield of higher alcohols was screened out to be used as the parental strain for genetic modification.There are two pathways generating higher alcohols,namely the amino acid catabolic pathway(the Ehrlich pathway)and the glucose biosynthetic metabolism pathway(the Harris pathway).To study the impact of gene modification on the production of higher alcohols,we knocked out or over-expressed the relevant genes of the two pathways,including ILV2(encoding the catalytic subunit of acetolactate synthase),ILV3(encoding the dihydroxyacid dehydratase),ILV5(encoding the acetohydroxyacid reductoisomerase),ILV6(encoding the regulatory subunit of acetolactate synthase),BAT2(encoding the cytoplasmic branched-chain amino-acid aminotransferase)and BAT](encoding the mitochondrial branched-chain amino-acid aminotransferase).(1)In this experiment,we analyzed the production of higher alcohols of eight wine yeast strains(WY-1,WY-2,WY-3,WY-4,WY-5,WY-6,VL1 and QA23).Duchenne fermentation tube experiment was adopted to compare these yeast strains' tolerance,the growth performance and fermentation performance of the strains was compared through wine fermentation.The results indicated that the higher alcohols production of WY-1 strain was 206.31 mg/L which is relatively low.And WY-1 strain could tolerate 16%(V/V)alcohol,pH 2.65,400 g/L glucose and 400 mg/L SO2 very well.Besides,WY-1 strain possessed faster growth rate and better fermentation performance.The content of alcohol produced by WY-1 strain could reach 11.01%(V/V)which is higher than other strains.WY-1 strain screened out was an excellent wine yeast strain with low yield of higher alcohols.(2)For the Harris pathway,with genes ILV2,ILV3,ILV5 knockouted respectively and gene ILV6 overexpressed,we obtained a series of recombinant strains WY-V21,WY-V22,WY-V23,WY-V31,WY-V32,WY-V51,WY-V52,WY-V53 and WY-V6.The growth and fermentation performance of the strains were no difference.The fermentation results showed that compared with WY-1,the n-propanol production of WY-V23 with three ILV2 alleles knockouted was increased by 38.46%,the isobutanol production decreased by 31.42%,the isoamylol production decreased by 10.24%;the n-propanol production of WY-V32 with two ILV3 alleles knockouted was increased by 15.87%,the isobutanol production decreased by 22.76%;the isobutanol production of WY-V53 with three ILV5 alleles knockouted was decreased by 21.10%;the higher alcohols production of WY-V6 with ILV6 gene overexpressed had no difference.(3)For the Ehrlich pathway,with genes BAT2 and BAT1 knockouted respectively,we obtained a series of recombinant strains WY-T21,WY-T22,WY-T11,WY-T12 and WY-T22T11.The growth and fermentation performance of the strain WY-T12 with two BAT1 alleles knockouted was significantly lower than the strain WY-1,there were no difference between other recombinant strains and the parent strain.The fermentation results showed that compared with WY-1,the isobutanol production of WY-T22 with two BAT2 alleles knockouted was decreased by 41.02%,the active-amyl alcohol production decreased by 25.54%;the n-propanol production of WY-T12 was increased by 37.57%,the isobutanol production increased by 26.24 times,the isoamylol production increased by 3.42 times,the active-amyl alcohol production increased by 2.61 times;the n-propanol production of WY-T22T11 with two BAT2 alleles and one BAT1 allele knockouted was decreased by 19.52%,the isobutanol production decreased by 35.51%,the active-amyl alcohol production decreased by 30.54%.