源于Wickerhamomyces anomalus F1503醇酰基转移酶催化合成乙酸乙酯的分子机制解析

Alcohol acyltransferase (AAT) is one kind of key esterifying enzymes that plays very important role in ester formation in baijiu. However, the effect of ATT on the ester formation in baijiu has not been widely concerned, especially the molecular mechanism of esterification. Therefore, the present project intends to explore the mechanism of ester formation affect by the ATT that from Wickerhamomyces anomalus F1503, a high ethyl acetate productive strain. Some major contents are included in our research paroral: first of all, determining the AAT and its key gene related to the synthesis of ethyl acetate, with the combination of tracking method, in vitro-reaction-system method and in vivo-induction method. Secondly, the key AAT will be obtained by recombinant technique and high density fermentation method, and then enzymatic characteristics and synthesis efficiency of the ATT will be analyzed. Thirdly, with the help of homology modeling, molecular docking and crystal analysis, the three-dimensional structure, as well as the catalytic active sites will be predicted to predict the catalytic mechanism. Some mutants contains key catalytic site will be constructed under gene mutation, and the key site / region mutation structure was analyzed by bioinformatics analysis. The role of key site and regions of ATT will be well explained by constructing the "gene-enzyme-structure" research system. Finally, the molecular mechanism of the synthesis of ethyl acetate catalyzed by ATT from Wickerhamomyces anomalus will be revealed.

醇酰基转移酶（AAT）是乙酸乙酯合成的关键酶，对白酒酯形成起着至关重要作用，但该作用尚未得到广泛关注，尤其是其催化酯合成的分子机制尚未明晰。本课题以高产乙酸乙酯的异常威克汉姆酵母（Wickerhamomyces anomalus）F1503为素材，探究并解析其AAT对白酒酯的形成机理。利用发酵跟踪法、体外反应体系法和体内诱导法，从转录水平及酶催化特性确定合成乙酸乙酯关键AAT；采用重组技术及高密度发酵手段获取关键AAT，系统测定并分析其酶学特性，简析关键ATT催化合成效率；借助同源建模、分子对接和晶体解析手段，解析AAT空间结构及其催化活性位点，预测其催化机制；采用基因突变构建AAT关键催化位点突变体，辅以生物信息学解析技术，解析关键位点/区域突变结构，通过构建“基因-酶-结构”研究体系系统阐释AAT关键位点及区域的作用。最终，揭示W. anomalus AAT催化合成乙酸乙酯的分子机制。

醇酰基转移酶（AAT）是酵母合成酯类化合物的关键酶，产香酵母在白酒酿造过程中主要通过醇酰基转移酶作用合成不同种类的酯类化合物，但该作用近几年才得到关注，其催化合成酯类化合物的分子机制仍不明晰。本项目以高产乙酸乙酯的异常威克汉姆酵母为出发点，探究其合成乙酸乙酯的形成机理。结合前期优化发酵条件，通过转录组学分析确定了合成乙酸乙酯相关的酯合成相关的关键酯酶（11个）、脂肪酶（8个）、AAT（7个）、醇脱氢酶（7个）与单加氧酶（4个），并通过qPCR对7个AATs进行了验证；采用分子生物技术克隆转录组学确定的AATs，7个AATs都成功获得克隆，其中最为关键AAT酶gene00276克隆的Eat1在大肠杆菌中得到成功表达，其具有较强的乙酸乙酯合成能力，并且具有其他酯类合成能力，通过镍亲和层析获得该酶电泳级纯酶，并对其酶学性质进行了研究，同时采用同源建模和分子对接手段，初步设计了影响其催化的活性位点，预测了其催化机制。

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