黄酒酵母基因组进化与氮代谢基因调控机制研究

During the brewing process of Chinese rice wine, the budding yeast tends to accumulate the products of nitrogen metabolism, such as β-phenylethanol and ethyl carbamate, which caused serious problems of food safety and quality. Because of adapting to traditional fermentation environment, yeast strains show the significant differences in the capability of nitrogen metabolism, but its molecular mechanisms remain unknown. This project is based on the two yeast strains, screened in our laboratory, with different nitrogen metabolism capability. Our aims are to: (1) Reveal the adaptive evolution mechanism of the yeast strains living in the rice wine fermentation environment (mainly nitrogen sources), using evolutionary genomic analysis. (2)Elucidate the global landscapes of the yeast transcriptome using RNA-seq methods, and clarify the different gene expression pattern of the yeast strains with different utilization ability of nitrogen sources. (3) The gene regulatory network is constructed using systems biology approach, and the dynamics of the network between yeast strains are studied. Moreover, systemic analysis will be performed to explore the interaction relationship between gene expression, gene network and genomic variations. The key regulatory genes or loci identified in this study can be used as targets or genetic markers for genetic breeding of S. cerevisiae rice wine strains. This will enable the development of traditional brewing technology of Chinese rice wine.

黄酒酵母在酿酒过程中易积累高级醇、氨基甲酸乙酯等有害氮代谢产物，严重影响黄酒品质与食品安全。这可能是酵母菌长期适应我国传统黄酒酿造环境（富含氨基酸）的适应性进化结果，但其基因组进化与基因调控机制尚未阐明。本课题拟在实验室筛选的2株不同氮代谢能力黄酒酵母菌株的基因组测序基础上，（1）通过对多株不同来源黄酒酵母菌株的基因组变异研究，揭示酵母菌适应黄酒发酵环境的基因组进化规律；（2）通过RNA-Seq技术研究黄酒酵母的全局转录组信息，阐述不同氮源利用能力酵母菌株的基因表达差异；（3）通过系统生物学方法研究黄酒酵母的氮代谢基因调控网络，从而在基因组序列、基因表达与基因调控网络三个水平系统阐述黄酒酵母基因组进化及其调控机制。本研究不仅有助于揭示酿酒酵母适应发酵环境的进化机制，也有助于黄酒酵母菌株的定向育种，降低黄酒中有害氮代谢产物积累，提高我国传统黄酒的酿造水平。

黄酒酵母在酿酒过程中易积累高级醇、氨基甲酸乙酯(EC)等有害氮代谢产物，是影响黄酒品质的主要因素。(1)本研究完成了多株黄酒酵母菌株(N85、YJS与突变株XZ-11)的全基因组测序，并通过比较基因组学研究揭示了黄酒酵母基因组的序列与结构变异，阐明黄酒酵母适应我国传统黄酒酿造环境的基因进化规律。(2)完成了黄酒酵母菌株在不同氮源下的转录组与蛋白组学分析，构建了基因共表达网络，鉴定了多个关键氮源代谢调控基因，用于黄酒酵母菌株的改造靶点或筛选标记。(3)通过基因敲除技术研究黄酒酵母特异性基因的功能，并建立了黄酒酵母的CRISPR/Cas9基因编辑技术，完成多个基因（ARG2、CAR2等）的工程菌株改造，实验表明酵母工程菌株大大降低了发酵黄酒中有害氮代谢产物（尿素与EC）的含量。(4)通过宏基因组技术研究了不同黄酒酵母菌株与黄酒发酵体系中其它微生物群的关系，鉴定了关键微生物为Lactobacillus属细菌。本研究系统揭示了黄酒酵母菌株的基因组进化规律与酵母的氮源代谢调控的分子机制，为黄酒发酵优良工业菌株选育与微生物组工程控制技术奠定基础，具有重要的学术意义和应用前景。