Construction Of Wine Yeast With Appropriate Low-production Of Higher Alcohols

The higher alcohols are the main flavor compounds in alcoholic beverages, mainly originated from the secondary metabolite of Saccharomyces cerevisiae. The relatively high content of higher alcohols was present in grape wine. In this study, we cut off the production of a-ketoacid as well as increase the decomposition of a-ketoacid according to the metabolic pathway of higher alcohols in order to decrease the content of higher alcohols in grape wine, and detailed operations included: the BAT1 (encoding the mitochondrial branched-chain amino-acid aminotransferase) was overexpressed; in addition, the BAT2(encoding the cytoplasmic branched-chain amino-acid aminotransferase) was knocked out combined with overexpressing BAT1; the ILV3 (encoding the dihydroxy-acid dehydratase)was knocked out combined with overexpressing BAT1. The main researches were as follows:(1) The recombinant SY-BAT1f was constructed by overexpressing the BAT1 individually in the arginine auxotrophic adiploid wine yeast strain YZ22. The difference of growth and fermentation properity between in SY-BAT1f and YZ22 was not obvious.However, compared with YZ22, the content of isobutanol, isoamylol, ?-phenylethanol,n-propanoland total higher alcohols produced by SY-BAT1f reduced by 38.54%, 37.52%,15.17%, 34.35% and 30.07%, respectively. Additionally, the mutant SYBBK was constructed by deleting one allele of BAT2 simultaneously overexpressing the BAT1, and the mutant SYBB3 was constructed by deleting two allele of BAT2 simultaneously overexpressing the BAT1 from the strain YZ22. The mutant SYBBK was fairly equivalent to parental strain in terms of growth and fermentation characteristics, but the growth rate of SYBB3 was slightly slower than that of YZ22. Fermentation results showed that the content of isobutanol, isoamylol, n-propanol and toal higher alcohols produced by SYBBK deareased by 30.46%, 25.11%, 39.32% and 26.78% compared with that produced by YZ22,similarly, all these compounds mentationed above in SYBB3 decreased by 39.41%, 37.18%,22.34% and 37.68%, respectively.(2) The mutants SYV31BK and SYV32BK were constructed by further deleting one allele of ILV3 and two allele of ILV3 based on overexpressing the BAT1 from the strain YZ22, respectively. The SYV31BK and SYV32BK were substantially equivalent to YZ22 in terms of growth and fermentation characteristics. The results showed that the content of isobutanol, isoamylol, and toal higher alcohols reduced by 13.96%, 12.97% and 10.23%,the difference in content of n-propanol and (3-phenylethanol produced by SYV31BK and YZ22 was not obvious. SYV32BK had not any decline in content of each higher alcohol compared with YZ22.(3) The influence of different grape cultivars and fermentation temperatures on the performance of low-higher alcohols production in mutant was investigated, and the results showed that the content of total higher alcohols in wine produced by SYBB3, with cultivars of Cabernet Sauvignon, Gernischet, Syrah and Yan-73, was 386.43 mg/L, 314.66 mg/L,426.71 mg/L and 281.88 mg/L respectively and that the content of isobutanol and isoamylol produced by mutant SYBB3 both reduced by 20?30% in every grape cultivars used compared with YZ22. Additionally, in temperatures of 20?,25? and 28?, the content of total higher alcohols produced by SYBB3 was both in the range of 250?350 mg/L,SYBB3 maintained the high decline rate for isobutanol and isoamylol, the decline rate of isobutanol and isoamylol gradually rised with temperature rising.