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,...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.sub-tilis 168,and the competence master regulator ComK using a double-stranded DNA substrate with short ho-mology 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.sub-tilis,and it will promote studies of genome function,construction of production strains,and genome mining in B.subtilis.展开更多
基金supported by the National Key R&D Program of China(2019YFA0904000)the National Natural Science Foundation of China(31570094,81502962,32270088)+6 种基金the 111 Project(B16030)the Shan-dong Provincial Natural Science Foundation of China(ZR2020MC015,ZR2018ZC2261)the Taishan Scholar Program of Shandong Provincethe Fundamental Research Funds of Shandong University(2018GN021)the Open Project Program of the State Key Laboratory of Bio-based Material and Green Papermaking(KF201825)the Science and Technology Project of Hunan Province(2021NK1040)Natural Science Foundation of Changsha(KQ2208130).
文摘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.sub-tilis 168,and the competence master regulator ComK using a double-stranded DNA substrate with short ho-mology 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.sub-tilis,and it will promote studies of genome function,construction of production strains,and genome mining in B.subtilis.
