Abstract In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis , Bacillus amyloliquefaciens , and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. Key points
• Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide.
• Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system.
• The process of immobilizing BlGGT with epoxy resin was optimized.
Weidan Chang,Weipeng Liu,Haoran Shen,Sisi Chen,Peizhen Liao,Yingju Liu
Precise detection of single-nucleotide mutations (SNMs) is extremely important in various biomedical applications, but the simultaneous detection of multiple SNMs remains a great challenge. Herein, we developed a new method based on CRISPR/Cas9 system for multiple SNMs detection. The CRISPR/Cas9 system transduces the nucleic acid into an intermediate trigger to initiate the isothermal amplification reaction and further form fluorescence signals. According to this strategy, we established nucleic acid bio-computing operations-molecule logic gate in simultaneous distinction of the genetic locus . We demonstrate that the fluorescence signals generated from different input combinations can be used to discriminate the multiple genetic locus, and the molecular logic gate has great potential in single-base mismatch detection. In addition, the successful assay of real samples indicates that the novel strategy could further adapt for the pathogenic monitoring and biomedical research.
Xin Qinglong,Chen Yudan,Chen Qianlin,Wang Bin,Pan Li
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.
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