酿酒酵母芳香醇脱氢酶家族的功能、进化及其在杂醇合成通路中的地位

项目名称： 酿酒酵母芳香醇脱氢酶家族的功能、进化及其在杂醇合成通路中的地位

项目编号： No.31301547

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 杨冬冬

作者单位： 浙江农林大学

项目金额： 25万元

中文摘要： 酿酒酵母通过Ehrlich Patway将苯丙氨酸等五种芳香族和脂肪族氨基酸代谢为2-苯乙醇等相应的杂醇，这些杂醇在酵母发酵食品/饮品的风味品质构成中起决定性作用。研究表明，酿酒酵母的七个预测芳香醇脱氢酶（AAD）是催化Ehrlich通路最后一步反应的关键候选基因；然而由于该家族基因全部位于高度可塑和快速进化的染色体近端粒区，其相关的生物学功能至今缺乏系统的研究。本研究从刻画七个AAD的编码蛋白的生物化学特征入手，分析该家族及相关脱氢酶同工酶家族在Ehrlich代谢物及醛酮类代谢物挑战下的转录、翻译及酶动力学特征，研究Aad组合敲除/过表达株系在Ehrlich代谢及毒性醛酮代谢物挑战下的适应性，通过实验及生物信息分析方法收集现有条件下可获得的大量亚生态株系，建立这些株系间"基因突变-酶底物亲和性动力学特征改变-环境适应性"的相关性，全面探讨该家族在杂醇合成及小生态快速适应性中的生物学意义。

中文关键词： 酿酒酵母；芳香醇脱氢酶；生化特征；定点突变；进化

英文摘要： In silico analysis reveals the existence of seven putative aryl-alcohol dehydrogenase (AAD) genes in the budding yeast Saccharomyces cerevisiae. This gene family, as well as other several candidate genes, has been implicated to catalyze the final step of the Ehrlich Pathway. Through the Ehrlich Pathway, the aromatic amino acids are converted to fusel alcohols that play key roles in determining the aromatic/flavour profile of fermented foods and beverages. To clarify details of the Ehrlich Pathway, and to thoroughly explore the biological relevance of this subtelomeric gene family, we decide to: (1) purify and characterize the biochemical properties of Aad proteins; (2) analyze the proteomic and transcriptomic behavior of the budding yeast that is challenged with overdosed aromatic amino acids and other toxic keto metabolites; (3) explore phenotype of yeast trains in which Aads are deleted or overexpressed; (4) explore the phylogenetic relation of laboratory, commercial and other sub-ecological strains, and to build relationship between "mutation of gene", "changes of kinetics properties" and "adaptations to eco-niche". These combined biochemical, genetic, proteomic, transcriptomic and in silico analysis would provide knowledge to regulate fusel alcohol formation, as well as to decipher the mystery of this subtel

英文关键词： Saccharomyces cerevisiae；Aryl-Alcohol Dehydrogenase；Biochemical Characterization；Site-direct Mutagenesis；Evolution