高核酸酿酒酵母规模化选育体系的建立应用及rRNA合成精细调控机制的研究

Ribonucleic acid (RNA) and its degraded products and derivatives are the important new food raw materials, food additives and pharmaceutical intermediates. Saccharomyces cerevisiae is recognized as the ideal source of RNA. Therefore, breeding of yeast with high RNA content is the basic measure to solve its low yield. rRNA, which is the most abundant RNAs, was synthesized by the precise regulation of RNA polymerase I (PolI) and other factors. It is necessary to disturb the tight regulation homeostasis to improve the rRNA synthesis. Currently, the regulation mechanism of rRNA synthesis is complicated and has not been completely elucidated. Besides, breeding of yeast strains with high RNA content have some problems, such as no large-scale screening and genetically modified (GM) breeding and so on. In this project, we will firstly construct a new expression system in industrial yeast strains, where RNA PolI regulates the expression of the GFP report gene. Based on this, we will disturb the intracellular rRNA synthesis using the atmospheric and room temperature plasma mutagenesis technique. Then the cells with fluorescence intensity increased will be preliminarily large-scale screened using flow cytometry, and secondly screened by detection of growth and RNA content to obtain the strains with high RNA content. After the breeding process, we will eliminate the new expression system in the strains with high RNA content, avoiding the GM problem. Finally, we will analyze the regulatory mechanism of rRNA synthesis, by means of transcriptome method based on the strains with high or low rRNA content, to identify the key steps or transcription factors that affect rRNA synthesis. This study will provide a theoretical basis for deeper analyzing the global and precise regulation mechanism of rRNA synthesis, and breeding strains with higher rRNA content to promote the development of ribonucleic acids and its related substances such as foods and drugs in China.

核糖核酸(RNA)及其降解物、衍生物是重要的新食品原料、食品添加剂和医药中间体。酿酒酵母是公认最理想的RNA源，选育高RNA酵母是解决其产量低的根本措施。rRNA是主要的RNA，由RNA聚合酶I(PolI)等精细调控合成，需要扰动细胞内严谨的rRNA调控稳态才能提高其积累，其合成调控机制目前还没有研究清楚，选育高核酸酵母也存在无法规模化筛选和转基因等问题。本研究拟：①构建PolI指导报告基因GFP表达的新型表达体系，建立规模化筛选体系；②高效诱变技术扰动胞内rRNA合成，流式细胞仪规模化分选荧光提高的细胞，结合生长和RNA含量检测，选育出高核酸酵母菌株，完成育种后消除表达体系，不会产生转基因问题；③基于高、低rRNA合成量菌株，开展转录组学研究，确定影响rRNA合成的因素，为更深层次解析rRNA合成精细调控机制，选育更高rRNA合成菌株提供理论研究基础，以促进我国核酸衍生食品和药物的发展。

核糖核酸（RNA）及其降解物、衍生物是重要的新食品原料、食品添加剂和医药中间体，酿酒酵母是公认最理想的RNA源，选育高RNA酵母是解决其产量低的根本措施，但目前存在无法规模化筛选和转基因等问题，rRNA是主要的RNA，由RNA聚合酶I等精细调控合成，需要扰动细胞内严谨的rRNA调控稳态才能提高其积累。本项目①突破传统育种理念，利用合成生物学技术有针对性设计、在酵母附加体质粒上构建适于酿酒酵母实验室菌株和工业菌株的RNA聚合酶I指导报告基因GFP表达的新型表达体系，通过绿色荧光强度变化反映酵母胞内rRNA合成调控扰动和核酸含量变化，结合ARTP诱变技术和流式细胞分选技术快速高通量分选出荧光强度提高的细胞，得到核酸含量提高58%和18%的高核酸酿酒酵母实验室菌株和工业菌株，选育后利用附加体质粒在酵母中的不稳定性，通过连续传代消除了新型表达体系。②采用减少酵母Crabtree效应的高细胞密度分批培养策略，即通过控制葡萄糖流加速率和控制溶氧可使酵母细胞保持较高的活力，RNA产量提高了11%。在此基础上，向培养物中添加磷酸盐，菌株的生物量提高了6%，RNA产量提高了13%。③将选育得到的高、低核酸含量的酵母实验室菌株和工业菌株进行RNA-seq分析，共同变化的基因主要在核糖体生物发生、嘌呤代谢、淀粉蔗糖代谢、RNA聚合酶、糖酵解/糖异生、半乳糖代谢、硫代谢、精氨酸合成、TCA循环、丙酮酸代谢等代谢通路富集，进一步功能验证表明，rRNA转录过程中转录起始复合物Rrn7p的表达水平、胞内GTP/ATP浓度变化均影响rRNA合成。本项目提供的高核酸酵母选育策略大大缩短了育种周期，提高了育种效率，避免了转基因问题，为核酸衍生食品安全提供了保障，项目结果对rRNA合成调控机制解析提供了理论指导。