Activating cryptic biosynthetic gene cluster through a CRISPR–Cas12a-mediated direct cloning approach-Reference-Cited by-同舟云学术

Activating cryptic biosynthetic gene cluster through a CRISPR–Cas12a-mediated direct cloning approach

Published:2022-03-22 Issue:6 Volume:50 Page:3581-3592
ISSN:0305-1048
Container-title:Nucleic Acids Research
language:en
Short-container-title:

Author:

Liang Mindong¹,Liu Leshi¹,Xu Fei²,Zeng Xiaoqian¹,Wang Ruijun³,Yang Jinling²,Wang Weishan⁴^ORCID,Karthik Loganathan¹,Liu Jiakun⁵,Yang Zhiheng¹,Zhu Guoliang¹,Wang Shuliu¹,Bai Linquan⁶,Tong Yaojun⁶,Liu Xueting¹,Wu Min⁷,Zhang Li-Xin¹,Tan Gao-Yi¹

Affiliation:

1. State Key Laboratory of Bioreactor Engineering and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China

2. Institute of Pharmaceutical Biotechnology and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China

3. Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life Sciences, Huzhou University, Huzhou 313000, China

4. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

5. Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

6. State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

7. College of Life Sciences, Zhejiang University, Hangzhou 310058, China

Abstract

Abstract 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.

Funder

National Key Research and Development Program of China

National Natural Science Foundation of China

National 111 Project of China

Fundamental Research Funds for the Central Universities

Shanghai Science and Technology Committee

State Key Laboratory of Bioreactor Engineering

Publisher

Oxford University Press (OUP)

Subject

Genetics

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