基因组复制工程辅助的连续进化技术提高蓝细菌高pH耐受性的研究

Cyanobacteria cell factories would face types of harsh environmental stress in large scale cultivation processes. To achieve stable and efficient photosynthetic production of biomass and bioproducts, physiological tolerances optimization of cyanobacteria chassis strain would be necessary. Physiological tolerances of microbial cells were complex phenotypes related with multiple targets, which were usually improved by evolutionary engineering strategies. Efficient optimization of cyanobacteria physiological tolerances called for development of novel evolutionary engineering technologies. In this project, we aimed to develop the Genome Replication Engineering Assisted Continuous Evolution (GREACE) technology in cyanobacteria to enable effective and continuous generation and isolation of evolved strains. Taking synthetic biology tools and strategies, a bistable control system holding mutation rates of genome replication would be developed to achieve artificially regulated hypermutable status in cyanobacteria chassis strain. Combined with continuous selective pressure, evolved strain with improved pH-tolerance would be generated and isolated through an in vivo mutagenesis process. Efficacy of the cyanobacteria GREACE technology would be confirmed and evaluated by high-pH tolerance engineering in cyanobacteria chassis strain. Based on the isolated strain with evolved high-pH tolerance, mechanisms for survival and adaptation of cyanobacteria in high-pH environments would be disclosed by comparative genomics, transcriptomics, and proteomics. In addition, the high-pH tolerant cyanobacteria strain obtained from GREACE would be engineered for ethanol photosynthetic production and evaluated in large scale cultivation systems. This project would provide an effective evolutionary engineering tool for engineering complex physiological tolerances traits in cyanobacteria, and get through the technological routes from physiological functionality optimization to large scale cultivation of cyanobacteria cell factories.

蓝细菌光合细胞工厂在规模化培养时会面临各种严苛的环境胁迫，为了保证生物量和代谢产品稳定、高效的合成和积累，需要对蓝细菌底盘藻株的生理耐受性进行优化。微生物细胞生理耐受性机制复杂、靶点众多，需要用进化工程策略进行改造，而蓝细菌缺乏高效的进化工程工具。本研究将在蓝细菌中开发基因组复制工程辅助的连续进化（GREACE）技术，以合成生物学手段实现对底盘藻株基因组复制过程保真/突变状态的双稳态控制，同时结合持续的筛选压力快速获取进化藻株。蓝细菌GREACE技术的效能将以底盘藻株高pH耐受性的改造来验证，在此基础上通过基因组、转录组和蛋白质组层面上的系统分析和比较，揭示蓝细菌对高pH条件的适应与耐受机制；本研究还将分析GREACE技术改造获得的高pH耐受型底盘藻株应用于乙醇光合细胞工厂构建和规模化培养时的表现与潜力，从而验证并打通光合微生物平台从改造生理功能到应用于规模化培养的技术路线。

蓝细菌光合细胞工厂在规模化培养时会面临各种严苛的环境胁迫，为了保证生物量和代谢产品稳定、高效的合成和积累，需要对蓝细菌底盘藻株的生理耐受性进行优化。微生物细胞生理耐受性机制复杂、靶点众多，需要用进化工程策略进行改造，而蓝细菌缺乏高效的进化工程工具。本研究核心研究任务为蓝细菌中基因组复制工程辅助的连续进化技术的开发以及通过其应用对蓝细菌逆境胁迫耐受能力的改造和机制研究。通过系统的基因挖掘和表达调控，获得了进行可控超突变的底盘细胞，基因组复制突变率提升了两个数量级；通过针对性的环境胁迫调控，实现了超突变状态的跃升，将突变率提高至野生型的8000倍；应用可控超突变变体系，成功筛选获得对高温高光、高pH等环境胁迫的耐受能力显著提升的聚球藻突变藻株，并对其机制进行解析，对其底盘进行了改造利用，获得新型光合细胞工厂。在本项目资助下，还针对合成生物技术优化蓝细菌光合生理和工程应用属性，提升光合生物制造技术应用潜力的技术发展情况进行系统总结，提出了新的观点和认识，在生物技术领域权威期刊上发表多篇高水平综述论文。

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