Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1

doi:10.13560/j.cnki.biotech.bull.1985.2018-1080

Abstract

Abstract: The hyBl（hygB-like）gene cluster,a secondary metabolic gene cluster in endophytic Streptomyces SAT1,is 52% similar to the hygromycin B gene cluster,and may play a key role in SAT1 inhibiting Cryphonectria parasitica. In order to further study the antibacterial mechanism of the strain and the function of the hyBl gene cluster,hyBl gene cluster was used to establish the CRISPR/Cas9-mediated gene cluster-knockout system in SAT1. A series of conditions were optimized,including the screening of protospacers,the construction of knockout vectors,the conjugation and transformation of donor bacteria,the selection of Ca²⁺and Mg²⁺,and conjugation time,etc.,and the knockout system of gene cluster for SAT1 was established. Finally,a mutant SAT1△hyBl（14 kb deleted）was obtained with Escherichia coli ET12567（pUZ8002/pCRISPomyces2-CB）as a donor and conjugating on the MS（containing 10 mmol Mg²⁺）for 16-18 h. The antibacterial activity of SAT1△hyBl was 77% lower than that of wild strain,indicating the synthesized secondary metabolites guided by hyBl gene cluster indeed played an important role in inhibiting chestnut blight. This study demonstrates that the CRISPR/Cas9 system can be used for gene cluster editing in endophytic Streptomyces SAT1,and provides promising technical support in further studying the antimicrobial mechanism in SAT1 and verifying the functions of unknown gene clusters.

Key words: CRISPR/Cas9 system, pCRISPomyces-2, endophytic Streptomyces SAT1, conjugation, knockout of gene cluster

TIAN Wen-jia, DOU Gui-ming, WANG Sha, SUN Jing-ya, MA Yu-chao. Establishment of a CRISPR/Cas9-mediated Gene Cluster-knockout System in the Endophytic Streptomyces SAT1[J]. Biotechnology Bulletin, 2019, 35(6): 1-8.

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