分枝杆菌LY-1内源性表达元件的筛选及其在降低Cas9蛋白毒性中的应用  

Endogenous expression elements of Mycobacterium sp.LY-1:screening and application in reducing Cas9 toxicity

作　　者：薛苗苗 李会 曹雪岑 张晓梅 王淑丽 陈立营 史劲松 许正宏[4] XUE Miaomiao;LI Hui;CAO Xuecen;ZHANG Xiaomei;WANG Shuli;CHEN Liying;SHI Jinsong;XU Zhenghong(School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,Jiangsu,China;Tianjin Tianyao Pharmaceutical Limited Company,Tianjin 300301,China;Tianjin Pharmaceutical Research Institute Limited Company,Tianjin 300301,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Bioengineering,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生命科学与健康工程学院,江苏无锡214122 [2]天津天药药业股份有限公司,天津300301 [3]天津药业研究院股份有限公司,天津300301 [4]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学通报》2022年第8期3267-3278,共12页Microbiology China

基　　金：国家重点研发计划(2019YFA0905300);江苏省青蓝工程;中央高校基本科研业务费专项资金(JUSRP221025)。

摘　　要：【背景】分枝杆菌LY-1因能够将天然植物甾醇代谢转化为重要甾体药物中间体,目前已成为工业上的优势生产菌株。高效的CRISPR/Cas9基因编辑技术是工业菌株代谢工程改造进行产量性状提升的关键。然而由于Cas9蛋白的高表达毒性问题且分枝杆菌中已公开报道的可用表达元件较少,极大地限制了Cas9蛋白在该菌株中的适度表达。【目的】筛选内源性表达元件,利用合适的表达元件启动Cas9蛋白的表达,降低其对菌株的毒性。【方法】依据文献和前期研究获得的分枝杆菌基因转录组水平数据,并结合启动子在线预测网站BDGP(https://www.fruitfly.org/seq_tools/promoter.html),筛选内源性表达元件。以增强型绿色荧光蛋白作为报告基因对表达元件的强度进行评估,并采用不同强度的表达元件启动Cas9蛋白的表达。【结果】获得了23个不同表达强度的表达元件,采用中等强度的表达元件及弱表达元件都降低了Cas9蛋白对分枝杆菌LY-1的毒性,实现了Cas9蛋白在该菌株中的适度表达。【结论】建立了分枝杆菌LY-1内源性表达元件库,为后续菌株中高效CRISPR/Cas9基因编辑技术的构建及关键酶基因调控奠定了良好的基础。[Background]Mycobacterium sp.LY-1 has become a dominant strain in industrial production because of its ability to metabolize natural phytosterols into important steroid drug intermediates.CRISPR/Cas9 as an efficient gene editing technology is the key to improving the yield and traits of industrial strains through metabolic engineering.However,due to the toxicity resulted from the high expression of Cas9 and the few available expression elements that have been reported in Mycobacterium,the moderate expression of Cas9 protein in Mycobacterium is greatly limited.[Objective]The endogenous expression elements were selected to activate the expression and reduce the toxicity of Cas9.[Methods]We used the online Berkeley Drosophila Genome Project(BDGP)to predict the endogenous expression elements from the transcriptome data of Mycobacterium genes in literature and available studies.The intensity of each expression element was assessed with enhanced green fluorescent protein as the reporter,and the expression of Cas9 protein was initiated with the expression elements of different intensities.[Results]Twenty-three expression elements with different expression intensities were obtained.The medium and weak expression elements reduced the toxicity of Cas9 to Mycobacterium sp.LY-1 and realized the moderate expression of Cas9 in the strain.[Conclusion]The endogenous expression element library of Mycobacterium sp.LY-1 was established,which laid a good foundation for the subsequent construction of CRISPR/Cas9 tools and the expression regulation of key enzymes in Mycobacterium.

关 键 词：分枝杆菌 内源性表达元件 CRISPR/Cas9 增强型绿色荧光蛋白 基因表达与调控 

分 类 号：Q78[生物学—分子生物学]