酵母YF1503中合成乙酸乙酯脂肪酶互作蛋白的筛选及功能鉴定

Ethyl acetate is the key compound that affects and determines the aroma, taste and style of baijiu. It is important to study the scientific problems of ethyl acetate in the brewing process to improve the quality and stability of baijiu. Studies have shown that lipase does play an important role in the synthesis of ethyl acetate, but the research on the function of lipase secreted by microorganisms in the process of baijiu brewing is still in the preliminary study and discussion stage. A high yield yeast strain YF1503 was obtained by isolation and purification of traditional strains and analysis of ethyl acetate by liquid chromatography. Wickerhamomyces anomalus yeast was identified by morphology and molecular biology. The ethyl acetate production conditions of strain YF1503 were optimized. The yield of ethyl acetate reached 17.35 g/L. The application of yeast YF1503 in the production of baijiu was investigated by solid-state fermentation simulation experiment. The flavor of fermented grains was improved significantly, the kinds and contents of acetates increased significantly, and the yield of ethyl acetate increased significantly. The mechanism of lipase-catalyzed synthesis of ethyl acetate in yeast YF1503 was further elucidated, and its function in the pathway of ethyl acetate synthesis was explored, and the regulatory proteins affecting the catalytic synthesis of key lipases were searched, so as to reveal the important mechanism of lipase-catalyzed synthesis of ethyl acetate in liquor brewing comprehensively and systematically. In this project, screening and functional identification of ethyl acetate lipase interaction protein from yeast YF1503 as the research target, the key to explore the function of lipase in ethyl acetate synthesis approach, using yeast two hybrid system on critical lipase catalyzed synthesis of protein interactions (regulatory proteins), and using H2Y, CRISPR technologies to research the internal relations between interaction protein and the lipase catalyzed synthesis.The resolution of this scientific problem not only provides a new perspective and thought for comprehensively and systematically revealing the important mechanism of lipase catalyzed synthesis of ethyl acetate, but also provides theoretical guidance for fundamentally improving baijiu quality and product stability.

乙酸乙酯是影响并决定白酒香气、口感和风格的关键化合物，关注并探讨乙酸乙酯在酿造过程中的科学问题，对于从根本上改善白酒品质，提高产品稳定性具有重要作用。研究表明脂肪酶对白酒中乙酸乙酯合成确实具有重要作用，但是关于白酒酿造过程中微生物分泌的脂肪酶在行使酯化功能研究尚处于初步研究和探讨阶段。本项目组通过传统菌株分离纯化，结合液相色谱进行乙酸乙酯分析，分离获得一株高产乙酸乙酯酵母 YF1503。经形态学和分子生物学鉴定为异常威克汉姆酵母。对其产乙酸乙酯条件进行优化，得到乙酸乙酯产量达到 17.35 g/L。通过白酒固态模拟发酵实验考察酵母 YF1503 在生产中的应用情况，酒醅的风味得到明显改善，乙酸酯种类增多且含量显著增加，乙酸乙酯产量显著提高。进一步解析酵母YF1503中脂肪酶催化合成乙酸乙酯的机理，探究其在乙酸乙酯合成途径中的功能，寻找影响关键脂肪酶的催化合成的调节蛋白，从而全面系统地揭示白酒酿造过程中脂肪酶催化合成乙酸乙酯的重要机制。本项目以酵母YF1503中合成乙酸乙酯脂肪酶互作蛋白的筛选及功能鉴定为研究目标，重点探究脂肪酶在乙酸乙酯合成途径中的功能，采用酵母双杂交系统筛选出影响关键脂肪酶催化合成作用的互作蛋白（调节蛋白），并运用酵母双杂、CRISPR等技术明确互作蛋白与脂肪酶催化合成的内在关系。这一科学问题的解决不仅为全面系统地揭示脂肪酶催化合成乙酸乙酯重要机制提供新的视角和思路，而且为从根本上改善白酒品质，提高产品稳定性提供理论指导。

乙酸乙酯是一种具有菠萝香和苹果香的挥发性酯类，广泛存在于各类白酒中，是影响并决定酿造白酒风味和品质的关键化合物。脂肪酶对白酒中乙酸乙酯的催化合成具有重要作用，其用量及酶活会严重影响乙酸乙酯的产量。但是在实际酿造过程中，微生物分泌的脂肪酶如何掌控量效关系并发挥酯化功能还无法解释。因此，本项目组以前期分离获得一株高产乙酸乙酯的酵母菌株（命名为异常威克汉姆酵母YF1503）为研究对象，根据其基因组测序、注释及比对结果，成功克隆26个脂肪酶基因，并在大肠杆菌表达体系表达重组酶，体外模拟乙酸乙酯合成含量，明确候选脂肪酶基因，命名为LipA。建立了异常威克汉姆酵母YF1503双杂交cDNA文库，文库容量达标（库容量>1×107cfu/mL），在制备好的cDNA文库中筛选与诱饵蛋白相互作用的蛋白，筛选出6个与YF1503脂肪酶LipA互作的蛋白，初步鉴定结果表明这6个基因可能参与到YF1503中脂肪酶LipA催化合成乙酸乙酯中。利用同源重组的方法构建突变菌株，并结合乙酸乙酯合成实验验证异常威克汉姆酵母YF1503野生菌株与突变菌株lipa-18催化乙酸乙酯合成的差异。结果表明，与异常威克汉姆酵母YF1503野生菌株相比，突变菌株lipa-18合成乙酸乙酯含量明显减少。首次尝试借助转录组测序技术寻找到直接可以调控乙酸乙酯合成的调节因子，结合异常维克汉姆酵母F1503全基因组测序结果，根据基因组功能注释信息挖掘调控因子，利用调控因子的表达情况，结合关键酶基因转录水平和酯合成动态变化分析，确定关键调控因子。项目以催化合成乙酸乙酯的脂肪酶为研究对象，通过多组学技术交叉运用，较好的解释了异常维克汉姆酵母YF1503中合成乙酸乙酯脂肪酶互作蛋白的功能及调控机制，关注并探讨在酿造过程中微生物代谢的酶类合成乙酸乙酯产生这一科学问题，对于从根本上改善白酒品质，提高产品稳定性具有重要作用。