Abstract
Bacillus subtilis plays an important role in fundamental and applied research, and it has been widely used as a cell factory for the production of enzymes, antimicrobial materials, and chemicals for agriculture, medicine, and industry. However, genetic manipulation tools for B. subtilis have low efficiency. In this work, our goal was to develop a simple recombineering system for B. subtilis. We showed that genome editing can be achieved in B. subtiliis 1A751 through co-expression of YqaJ/YqaK, a native phage recombinase pair found in B. subtilis 168, and the competence master regulator ComK using a double-stranded DNA substrate with short homology arms (100 bp) and a phosphorothioate modification at the 5'-end. Efficient gene knockouts and large DNA insertions were achieved using this new recombineering system in B. subtilis 1A751. As far as we know, this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in B. subtilis. In conclusion, this new recombineering system provides a simple and fast tool for genetic manipulation of B. subtilis, and it will promote studies of genome function, construction of production strains, and genome mining in B. subtilis.