BAT2基因缺失葡萄酒酵母的构建及其对葡萄酒高级醇的影响

该研究以葡萄酒酵母BV-0为出发菌，利用融合聚合酶链式反应（PCR）构建BAT2基因敲除组件，通过电转化导入菌株BV-0进行同源重组，获得BAT2单等位基因敲除菌株BV-bk；再由菌株BV-bk产孢分离出两种不同配型的单倍体BAT2基因敲除菌进行杂交融合，获得BAT2双等位基因敲除菌株BV-BK；最后利用Cre/loxP重组系统去除菌株BV-bk、BV-BK的KanMX抗性基因得到菌株BV-b、BV-B。采用菌株BV-0、BV-b及BV-B分别发酵葡萄酒，并测定葡萄酒的基本理化指标及高级醇含量。结果表明，与出发菌株BV-0相比，菌株BV-b和BV-B发酵的葡萄酒的基本理化指标基本一致，异丁醇含量分别下降14.9%、67.0%，异戊醇含量分别下降17.87%、31.10%，且菌株BV-B发酵葡萄酒中正丙醇含量上升36.55%，说明两株BAT2基因敲除菌株可应用于葡萄酒发酵中以降低异丁醇和异戊醇含量，从而降低葡萄酒的苦味和醉度，具有潜在的工业应用价值。

Construction of BAT2 gene-deficient wine yeast and its effect on higher alcohols in wine

In this study, using wine yeast BV-0 as the starting strain, the BAT2 gene knockout component was constructed by fusion polymerase chain reaction (PCR). The BAT2 monoallelic knockout strain BV-bK was obtained by homologous recombination through electrical transformation into strain BV-0. Then two haploid BAT2 gene knockout strains with different matching types were isolated from strain BV-bK by sporulation and hybridized to obtain BAT2 biallelic knockout strain BV-BK. Finally, the strains BV-b and BV-B were obtained through removing the KanMX resistance genes of strains BV-bk and BV-BK by the Cre/loxP recombinant system. The wine was fermented by strain BV-0, BV-b and BV-B, respectively, and the basic physicochemical indexes and higher alcohol contents of wine were determined. The results showed that compared with the starting strain BV-0, the basic physicochemical indexes of the wine fermented by strain BV-b and BV-B were almost the same. The isobutyl alcohol content decreased 14.9% and 67.0%, respectively. The isoamyl alcohol content decreased 17.87% and 31.10%, respectively, and the n-propanol content increased 36.55% in wine fermented by strain BV-B. It indicated that the two BAT2 gene knockout strains could be used in wine fermentation to reduce isobutyl alcohol and isoamyl alcohol content, thereby reducing the bitterness and intoxicating degree of wine, which had potential industrial application value.