白酒是我国历史悠久的传统酿造饮品的典型代表，决定其风格与品质的重要酯类四大乙酯的形成机制却并不明晰，严重阻碍我国传统酿造产业的现代化进程。前期研究发现，异常维克汉姆酵母和吡硌伯克霍尔德氏菌对白酒四大乙酯的合成影响显著。项目以浓香型白酒为载体，异常维克汉姆酵母F1503和吡咯伯克霍尔德氏B1213为对象，基于基因组测序，基因敲除/回补结合性质表征分析确定关键基因；对关键基因编码的酶进行定点突变、三维结构预测及精细比对、分子对接和反应动力学分析，阐明酶催化酯合成的机制；深度注释挖掘调控因子，分析调控因子的上调/下调表达结合转录组测序及酯合成量动态变化，确定关键调控因子，结合原位环境探讨微生物菌株“调控因子—关键酶—酯”内在关联及量效关系，建立酯合成调控机制研究体系，探究酿造过程关键乙酯合成及调控作用规律，多层面系统阐释影响白酒品质的内在机制，对于推进我国传统酿造白酒的现代化进程具有重要意义。

Baijiu is a typical representative of Chinese traditionally fermented beverages，however, the unclear underlying formation mechanisms of the four important ethyl esters which determine the style and quality of Baijiu impede the pace of Chinese traditional brewing industry modernization. Our preliminary data found Wickerhamomyces anomalus and Burkholderia pyrrocinia significantly influenced the synthesis of the four important ethyl esters in Baijiu. The present proposal will take strong flavor Chinese Baijiu as the carrier, target W. anomalus F1503 and Burkholderia pyrrocinia B1213 as the key stains, identify the key genes using genome sequencing, gene knockout and complementary expression methods; analyze the enzymes encoded by the key genes using site-specific mutagenesis, three-dimensional structure prediction, subtle comparison, molecular docking and reaction kinetics, elucidate the catalytic mechanisms of the esters synthesis by these enzymes; intensively explore and annotate the regulatory factors, identify the key regulatory factors by combining up- and down-regulating expressions of the genes with transcriptome sequencing and dynamic changes of the esters concentrations, build the study methods for the researches of key ethyl esters synthesis mechanisms by investigating intrinsic connection and dose-effect relationship of in situ environment “microbial regulatory factor-key enzymes-esters”, explore the synthetic and regulatory functional rules of the key ethyl esters in brewing process, systematically elucidate the underlying mechanism affecting Baijiu quality in multi-levels. The results of the study have an important significance in promoting the modernization process of Chinese traditionally fermented Baijiu.