文献类型：期刊文献

英文题名：Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach

作者：Liang, Mindong[1,2];Liu, Leshi[1,2];Xu, Fei[3,4];Zeng, Xiaoqian[1,2];Wang, Runjun[5];Yang, Jinling[3,4];Wang, Weishan[6];Karthik, Loganathan[1,2];Liu, Jiakun[7];Yang, Zhiheng[1,2];Zhu, Guoliang[1,2];Wang, Shuliu[1,2];Bai, Linquan[8,9];Tong, Yaojun[8,9];Liu, Xueting[1,2];Wu, Min[10];Zhang, Li-Xin[1,2];Tan, Gao-Yi[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Sch Biotechnol, Shanghai 200237, Peoples R China;[3]Zhejiang Univ, Inst Pharmaceut Biotechnol, Sch Med, Affiliated Hosp 2, Hangzhou 310058, Peoples R China;[4]Zhejiang Univ, Dept Gastroenterol, Sch Med, Affiliated Hosp 2, Hangzhou 310058, Peoples R China;[5]Huzhou Univ, Coll Life Sci, Key Lab Vector Biol & Pathogen Control Zhejiang P, Huzhou 313000, Peoples R China;[6]Chinese Acad Sci, Inst Microbiol, State Key Lab Microbial Resources, Beijing 100101, Peoples R China;[7]Chinese Acad Sci, Inst Synthet Biol, Shenzhen Inst Adv Technol, Shenzhen 518055, Peoples R China;[8]Shanghai Jiao Tong Univ, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China;[9]Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China;[10]Zhejiang Univ, Coll Life Sci, Hangzhou 310058, Peoples R China

年份：2022

卷号：50

期号：6

起止页码：3581

外文期刊名：NUCLEIC ACIDS RESEARCH

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000779485400043)】；

基金：National Key Research and Development Program of China [2020YFA0907800, 2021YFC2100600 and 2020YFA0907304]; National Natural Science Foundation of China [31720103901, 31870040 and 21877038]; National 111 Project of China [B18022]; Fundamental Research Funds for the Central Universities [22221818014]; Shanghai Science and Technology Committee [18JC1411900]; State Key Laboratory of Bioreactor Engineering. Funding for open access charge: National Key Research and Development Program of China and National Natural Science Foundation of China. Conflict of interest statement. None declared.

语种：英文

摘要：Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.