Simple and Versatile Toolkit for Genetic Manipulation of <i>Bacillus licheniformis</i>

<i>Bacillus licheniformis</i> is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for <i>B. licheniformis</i>. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested <i>B. licheniformis</i> strains, with efficiencies ranging from 10^-5 to 10^-3. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10^-3 was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of <i>B. licheniformis</i>.