GATA转录因子Dal80p对黄酒加工酿酒酵母代谢形成氨基甲酸乙酯的调控机制研究

Ethyl carbamate (EC) is an important metabolic pollutant presented in Chinese yellow rice wine affecting its quality and safty, which restricts the quality improvement and industrial upgrading of traditional Chinese yellow rice wine processing products. In Chinese yellow rice wine brewing system, low nitrogen habitat such as nitrogen catabolite repression (NCR) is a key scientific problem which leads to the formation of EC precursors by yeast metabolism. Therefore, based on the preliminary studies, this project constructs a Dal80p engineered yeast strain by means of gene knockout and overexpression; identifies substances and enzymatic differences of arginine metabolism during the regulation of Dal80p by means of metabolites quantitative and RT-PCR; explores molecular mechanism of Dal80p on arginine metabolism by means of transcriptomics and identifies its key differentially regulated genes; clarifies the interaction between Dal80p and key differentially expressed genes by means of molecular interaction; studies its inhibition of EC produced by Saccharomyces cerevisiae metabolism in the simulated system and actual processing system by means of brewing experiment. The results of this project not only provide a theoretical basis and scientific basis for the analysis of the nutritional stress environment, EC formation and its regulation during traditional Chinese yellow rice wine brewing to achieve the purpose of effectively inhibiting the formation of EC, but also provide reference for researches on regulating the formation of metabolic pollutants in other fermented foods in China.

黄酒中存在的氨基甲酸乙酯是影响其品质与安全的重要难题，也是制约传统黄酒酿造加工产业升级与品质提升的关键。在黄酒酿造体系中低氮生境如氮代谢抑制效应（NCR）是导致黄酒酿造酵母细胞代谢生成EC前体物的核心科学难题。鉴于此，本申请项目在原有研究基础上，借助基因敲除、过表达手段构建Dal80p工程菌株；借助代谢产物定量和RT-PCR方法鉴定Dal80p对精氨酸代谢调控的物质与酶学差异；采用转录组学探索Dal80p调控精氨酸代谢的分子机制，鉴定出其调控的关键差异基因；借助分子互作手段阐明DAL80P与关键差异基因的互作机理；借助酿造学评价方法，研究其模拟体系和实际加工体系中对酿酒酵母细胞代谢生成EC的抑制作用。本项目研究结果不仅为解析我国传统黄酒酿造营养胁迫环境与EC 形成及其调控提供理论基础和科学依据，达到有效抑制EC 的形成目的，也将为调控我国其他酿造食品中代谢性污染物形成的研究提供借鉴依据。

氨基甲酸乙酯是发酵食品特别是发酵饮品中分布最广的2A级致癌物质之一，已有研究表明其形成的主要原因是酿酒酵母氮代谢阻遏效应引起的氮源选择性和不充分利用。因此，深入理解酿酒酵母细胞的氮代谢调控机制，对发酵菌株进行代谢工程靶向改造是解决发酵饮品中氨基甲酸乙酯形成最有效的途径。课题组前期研究表明，Dal80是酿酒酵母细胞氮代谢阻遏效应负向转录调控因子之一，但其在氮代谢过程中功能作用还不甚了解，Dal80p的功能和可能的作用机理目前一直尚不清晰。本项目以黄酒发酵所处的氮营养分解代谢胁迫(NCR)环境为背景，在原有研究基础上，借助基因敲除、过表达手段构建Dal80p工程菌株，完成了3株酵母菌的代谢产物定量和RT-PCR方法鉴定Dal80p对精氨酸代谢调控的物质与酶学差异，随后采用转录组学探索Dal80p调控精氨酸代谢的分子机制，通过生信分析鉴定出其调控的关键差异基因和主要调控途径，并首次借助Chip-Seq手段阐明DAL80P与关键差异基因的互作机理，挖掘出其调控精氨酸代谢的具体靶标和可能途径，最后采用酿造学研究手段，阐明DAL80改造菌株在模拟体系和实际酿造体系中对酿酒酵母细胞代谢生成EC的抑制作用及其实际应用价值，结果发现DAL80能大幅度降低胞外尿素的生成，从而实现EC含量降低到对照组的一半，但对黄酒酿造的整体产品品质没有带来负面影响。项目项目研究结果为解析我国传统黄酒酿造营养胁迫环境与EC 形成及其调控提供理论基础和科学依据，也将为调控我国其他酿造食品中代谢性污染物的靶向调控及高品质制造提供借鉴依据。