蓝细菌CRISPRa系统的开发及其代谢工程应用  

Development of CRISPRa for metabolic engineering applications in cyanobacteria

作　　者：王甜甜[1,2] 朱虹 杨琛 WANG Tiantian;ZHU Hong;YANG Chen(CAS Key Laboratory of Synthetic Biology,CAS Center for Excellence in Molecular Plant Sciences,Chinese Academy of Sciences(CAS),Shanghai 200032,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院合成生物学重点实验室,中国科学院分子植物科学卓越创新中心,上海200032 [2]中国科学院大学,北京100049

出　　处：《合成生物学》2023年第4期824-839,共16页Synthetic Biology Journal

基　　金：国家重点研发计划(2021YFA0909702);国家自然科学基金(31925001,31921006);中国科学院战略性先导科技专项(XDB27020000)。

摘　　要：蓝细菌是光合作用研究的模式生物之一,也是构建光能自养细胞工厂的良好底盘。然而目前蓝细菌的遗传操作工具仍然较为缺乏且效率较低,开发高效的蓝细菌基因调控工具对于蓝细菌系统与合成生物学研究具有重要意义。本研究在模式蓝细菌聚球藻PCC 7942中开发了CRISPR激活系统,测试了多个转录激活因子,将内源的RNA聚合酶ω-亚基RpoZ与无DNA切割活性的dCas9融合表达,利用高强度启动子表达向导RNA,进而敲除内源rpoZ基因并优化了dCas9-RpoZ的表达及靶向位点。利用建立的CRISPRa系统对重要生物燃料——异戊烯醇的生物合成途径进行了工程改造,该系统不仅能够实现单基因或多基因的高表达,还可以同时对不同基因进行转录激活和抑制,将蓝细菌中异戊烯醇的产量提高了17倍,展示了该系统有望成为构建光能自养细胞工厂的有力工具。Cyanobacteria can be used as a model for photosynthesis research and as a chassis for the production of fuels and chemicals from light energy and CO2.However,the genetic tools of cyanobacteria are still relatively limited.Development of efficient tools for programming gene expression is important for cyanobacterial systems and synthetic biology.Here,we developed a CRISPR transcriptional activation system(CRISPRa)for programming heterologous gene expression in a model cyanobacterium Synechococcus elongatus PCC 7942.Among the transcriptional activators tested,endogenous RNA polymeraseω-subunit RpoZ resulted in optimal performance and was chosen for subsequent studies.We established CRISPRa by fusing dCas9 that lost DNA cleavage activity with RpoZ,and expressed single guide RNAs(sgRNAs)under a strongpromoter.We further improved heterologous reporter gene expression by deleting the rpoZ gene in S.elongatus PCC 7942,enhancing the expression of dCas9-RpoZ fusion,and optimizing the sgRNA targeting sites.Using this optimized CRISPRa system,we engineered S.elongatus PCC 7942 for improved production of isopentenol,an ideal biofuel candidate.Furthermore,we demonstrated that this system was able to simultaneously activate multiple genes of the biosynthetic pathway and repress a gene of the competing pathway,thereby increasing the isopentenol production by 17 times.Thus,this CRISPRa system could serve as a powerful tool for the construction of photoautotrophic cell factories.

关 键 词：蓝细菌 CRISPRa 异戊烯醇 

分 类 号：Q819[生物学—生物工程]