多位点基因组编辑改善酿酒酵母多重胁迫耐性及机理研究

During the industrial production, Saccharomyces cerevisiae often suffers from the harsh conditions, such as high temperature, high glucose concentration and high ethanol concentration, etc. These harsh conditions would produce the multiple stress to Saccharomyces cerevisiae, which would result in the arrest of cell growth and physiological activities. Thus the substrate can not be consumed and conversed into the products. Therefore, it is neceaary to improve the multiple stress tolrance and to enhance the production perfomance of Saccharomyces cerevisiae, especially in the real industrial conditions. Here, based on the premilirary results of thermotoerant, ethanol tolerant yeast construction and performance analysis, we propose to investigate the posibility to improve the the multiple stress tolerance of Saccharomyces cerevisiae by TALENs (Transcription activator-like effector (TALE) nucleases) assisted multiplex genome editing, and elucidat the underlining molecuar mechanism. First, the natural promoters of the multiple key genes related to the thermotolerance, ethanol tolerance and osmotolerance in Saccharomyces cerevisiae genome were substituted by the synthetic promoters which contain the binding region of TALENs. Then TALENs will be triggered to edit the yeast genome at multiplex points, and the mutated yeast strains with improved multiple stress tolerance were selected for evaluation and analyisis. By DNA cloing, sequencing, and real-time PCR analysis, the relationship of the mutation and combinatoral gene expression were confirmed. At last, based on the transcriptomic and quantitative proteomic analysis, the systems response mechanism and molecular basis will be ecluciated. This study will constribute to developing the the system stratigies for engineering the next generation industrial Saccharomyces cerevisiae for future industrial application.

在工业生产中酿酒酵母经常处于高温、高糖（高渗）、高乙醇等复杂环境，使其受多重胁迫而导致细胞生长或生理活动停滞，无法利用底物生产目标产品，严重影响生产能力和效率。因此如何改善酿酒酵母多重胁迫耐性，并解析其分子机理是构建高效酿酒酵母进行工业生产的前提。本项目在前期耐高温及耐乙醇酵母选育及机理研究的基础上，利用TALENs（转录激活因子样效应物核酸酶）辅助的基因组编辑，进行合成启动子替换与胁迫耐性相关的关键基因天然启动子，在约30个目标基因引入TALEN作用位点，进行基因组水平多位点编辑改造与组合表达调控，改善酿酒酵母对高温、高渗和高乙醇等多重胁迫耐性。通过荧光定量PCR和基因克隆测序研究目标基因功能表达变化和突变组成，解析影响酿酒酵母多重胁迫性状的作用规律；通过转录组和定量蛋白质解析，研究和阐明多重胁迫耐性酿酒酵母的系统分子作用机理，为构建新一代多重胁迫耐性工业酿酒酵母奠定基础。

在工业生产中酿酒酵母经常处于高温、高糖（高渗）、高乙醇等复杂环境，使其受多重胁迫而导致细胞生长或生理活动停滞，无法利用底物生产目标产品，严重影响生产能力和效率。因此如何改善酿酒酵母多重胁迫耐性，并解析其分子机理是构建高效酿酒酵母进行工业生产的前提。本项目在酿酒酵母中建立并优化了TALENs和CRISPR-Cas9辅助的多位点基因组编辑技术，用于改造并提高了酿酒酵母耐受高乙醇、高温和高渗的抗胁迫性能，同时对胁迫抗性菌株进行组学分析，进一步阐明了酿酒酵母的胁迫抗性机理。首先，我们在酿酒酵母中发展了TALENs介导的多位点基因组编辑（TAME）技术，靶向TALENs特异性识别修饰启动子区保守序列TATA框与GC框之间的关键区域，以控制98个基因的表达，进而加速底盘细胞基因组进化。结果表明，TAME技术能够实现酿酒酵母基因组的高效半理性改造，定点修饰效率可达到27.3%，成功用于提高酿酒酵母耐受乙醇、高温和高渗的胁迫性能。对突变菌株进行全基因组重测序分析，发现大量突变基因参与细胞壁和细胞膜相关的生物过程，以及线粒体DNA拷贝数的倍增。电子显微镜和流式细胞仪分析也显示突变菌株的细胞壁和细胞膜发生重构，表明细胞壁和细胞膜是保证细胞抗胁迫性能的重要因素。其次，通过分析25个转录因子单敲除菌株对长时热胁迫和短时热激条件的响应情况，确定了3个耐高温胁迫核心转录因子（SIN3、SRB2和FHL1）。利用CRISPR/Cas9技术对这3个转录因子的启动子区进行多位点编辑，提高了菌株的耐高温性能。最后，针对酿酒酵母经过基因组编辑技术编辑后，本身非同源末端引入突变效率和多样性较低的问题，通过改造DNA修复途径增加基因组编辑工具引入突变的能力，达到丰富细胞表型加速酵母育种的目的。另外，转录组和定量蛋白质组分析进一步解析了酿酒酵母耐高温的转录调控网络和分子机理。总之，本项目建立的多位点基因组编辑技术提高了酿酒酵母的多重胁迫耐性，以及酿酒酵母胁迫耐性分子机理的解析，为构建新一代多重胁迫耐性工业酿酒酵母奠定基础。