Preliminary Study On The Homologous Recombination System Mediated By SPP1 Phage Recombinase In Bacillus Subtilis

Bacillus have a high capacity to secrete proteins into the growth medium and the foundation for large-scale industrial fermentation. These bacteria have been largely employed in industry because of their abilities to produce enzymes, vitamins, amino acids, nucleosides, and antiviral drugs, etc. Recently, with the rapid development of molecular techniques, a growing number of approaches have been developed for genetic manipulation of Bacillus. However, the directed evolution and construction of genetically engineered Bacillus bacteria were hindered by cumbersome experimental procedures and introducing multiple antibiotic resistance markers. Therefore, it is important to develop more efficient genetic manipulation system in Bacillus. In the present study, the model strain, B. subtilis W168, was used to construct the chassis bacterium integrated phage recombinase genes into the chromosome. In addition, the homologous recombination system mediated by phage recombinases was verified and established by direct mutation of targeted genes to construct genetic engineering strains.Integrate vector pAX01-GP34.1-GP35-GP36 carrying recombinant genes of phage SPP1 was constructed, and was transformed into B. subtilis W168ΔmutS, a mutant that had been built from B. subtilis W168. DNA sequencing proved the successful integration of phage recombinase genes into the lacA site of B. subtilis W168ΔmutS chromosome. Proteins GP34.1, GP35 and GP36, related to phage recombinases, were expressed in Escherichia coli and used for preparation of rabbit polyclonal antibodies. Western-blot assay suggested that these three recombinase genes were successfully expressed in B. subtilis W168ΔmutS.To verify the validity of the homologous recombination system mediated by phage recombinases, oligonucleotides were subsequently designed at both ends with 20-30 bp homologous sequences and several nonsense mutations were introduced. The upp gene encoded uracil phosphoribosyltransferase of chassis bacterium was directly inactive via several rounds transformation with oligonucleotides. Taken together, these results suggest that the homologous recombination system mediated by phage recombinases in B. subtilis was validated, and the genetic manipulate strategy of oligonucleotides homologous recombination was initially established in B. subtilis.In summary, a newly homologous recombination technology was exploited to efficiently genetic manipulate of Bacillus subtilis in this study. The genetic manipulations, including knockout, knockin and direct modification of target genes, did not need additional DNA digestion, ligation and other procedures, and would not introduce resistance marker. Therefore, our study provides a new scientific evidence and approach of genetic manipulation in Bacillus for direct evolution and construction of genetically engineered bacterium.