文献类型：期刊文献

英文题名：Efficient editing DNA regions with high sequence identity in actinomycetal genomes by a CRISPR-Cas9 system

作者：Mo, Jingjun[1];Wang, Shuwen[1];Zhang, Wan[1];Li, Chunyu[1];Deng, Zixin[1];Zhang, Lixin[2];Qu, Xudong[1]

机构：[1]Wuhan Univ, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery, Minist Educ, Wuhan, Hubei, Peoples R China;[2]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China

年份：2019

卷号：4

期号：2

起止页码：86

外文期刊名：SYNTHETIC AND SYSTEMS BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000469827000003)】；

基金：The authors are grateful to Prof. Wen Liu (Shanghai Institute of Organic Chemistry) for gifting the strain of Sac. erythraea HL3168 E3, Dr. Ming Jiang (Shanghai Jiao Tong University) for helping to develop the HPLC condition of erythromycin and Prof. Yuhui Sun (Wuhan University) for kindly providing the pWHU2653 and pYH7. This work was supported by the National Nature Science Foundation of China Grants (Nos. 31570057, 31430002, 31320103911 and 31770063), Taishan Scholarship and "the Fundamental Research Funds for the Central Universities 22221818014.

语种：英文

外文关键词：Biosynthesis; Polyketide synthase; CRISPR/Cas9; Antimycin; Actinobacteria

摘要：Actinobacteria able to produce varieties of bioactive natural products have been long appreciated by the field of drug discovery and development. Recently, a few of CRISPR/Cas9 systems bearing different types of replicons (pSG5 and pIJ101) were developed to efficiently edit their genomes. Despite wide application in gene editing, their utility in editing challenging DNA regions e.g. high sequence identity has not been compared. In this study, we confirmed that the widely used temperature-sensitive pSG5 replicon is indeed not suitable for editing modular polyketide synthase (PKS) genes due to causing unpredicted gene recombination. This problem can be addressed by replacing the pSG5 with the segregationally unstable pIJ101 replicon. By introducing a counter-selection marker CodA, convenient cloning sites in the single guide RNAs (sgRNAs) and homologous template scaffolds, we developed a new CRISPR-Cas9 system pMWCas9. This system was successfully used to delete/replace erythromycin PKS and other biosynthetic genes in Saccharopolyspora erythraea and Streptomyces sp. AL2110. By swapping the promoters of antB and antC with ermE and kasOp, we achieved a deacyl-antimycin hyper producer which produces a 9-fold higher yield than the original Streptomyces sp. AL2110 strain. Our results provide a robust and useful Cas9 tool for genetic studies in Actinobacteria.