Author:
Xin Qinglong,Chen Yudan,Chen Qianlin,Wang Bin,Pan Li
Abstract
Abstract
Background
Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02.
Results
In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain.
Conclusions
The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.
Funder
Research and Development Plan in Key Areas of Guangdong Province
Core Technology Project of Foshan City
National Key Research and Development Program of China
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
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