Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02

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.

Bacillus amyloliquefaciens: Harnessing Its Potential for Industrial, Medical, and Agricultural Applications—A Comprehensive Review

“…For instance, if a modification to the amyE gene successfully integrates and improves the bacterium's performance, the applied strategy was effective [22]. Previous research has confirmed the successful application of genetic engineering techniques in B. amyloliquefaciens and shown the potential for further optimization and industrial utilization [9,10,13,22,23]. For instance, studies [13,20,[23][24][25][26][27] have exhibited enhancements in the production of enzymes, such as protease and lipase, due to genetic engineering.…”

“…Xin et al (2022) recently reported the development of a CRISPR-based genetic toolkit designed for efficient gene editing, knockout, and integration in Bacillus amyloliquefaciens LB1ba02. By employing a single-plasmid CRISPR/Cas9n system, the researchers achieved an impressive 93% knockout efficiency for a single gene and simultaneous editing of three loci with 53.3% efficiency using a base editing CRISPR/Cas9n-AID system [ 9 ]. This innovative toolkit was successfully applied to four genes ( aprE , nprE , wprA , and bamHIR ), showcasing its potential as a rapid gene knockout and integration tool for B. amyloliquefaciens LB1ba02.…”

“…Additionally, numerous genes involved in amino acid metabolism, fatty acid me-tabolism, and energy metabolism were upregulated or downregulated [53]. Wang et al (2022) employed a different approach to improve the yield of fengycin and iturin using metabolically engineered B. amyloliquefaciens. They utilized B. amyloliquefaciens WH1 as a wild-type strain and knocked out several genes, including bdh to block the carbon over-flow metabolic pathway, kinA to disrupt bacterial sporulation and extend the production period, dhbF to enhance the availability of amino acids and fatty acids, and rapA to in-crease Spo0A~P levels.…”

“…These tools range from classical counter-selection marker strategies to the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)—based genetic toolkits. The diverse protein secretion systems, along with the novel artificial promoter and ribosome binding site (RBS) libraries, further facilitate the production of extracellular enzymes [ 9 , 10 , 11 , 12 , 13 ].…”

Improved Recombinant Expression of Maltogenic α-Amylase AmyM in Bacillus subtilis by Optimizing Its Secretion and NADPH Production

“…The identification of the successful role of the editing plasmid was based on the method of Qinglong Xin [17], with slight improvements. The correct knockout and knockin plasmids were transformed into B. subtilis WB800 (∆amyE) according to the electrotransformation method of Guoqiang Cao [18], then spread on an LB plate containing 50 ug/mL of kanamycin and cultured at 30 • C for about 14 h. After the transformants on LB plate were verified via colony PCR, the transformants with the correct plasmids were picked into liquid LB medium containing 50 µg/mL kanamycin and 100 µM/mL IPTG for induction at 30 • C for 24 h at 220 rpm.…”