Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis

Area of Science:

• Microbiology
• Synthetic Biology
• Bacterial Genetics

Background:

• Bacillus licheniformis is a spore-forming bacterium with significant probiotic, environmental, and industrial relevance.
• Numerous wild strains with diverse functionalities have been identified, but their study is limited by a lack of efficient genetic tools.
• Engineering B. licheniformis strains is crucial for unlocking their full potential in various applications.

Purpose of the Study:

• To establish a versatile and simple genetic manipulation toolkit for Bacillus licheniformis.
• To overcome the limitations posed by the absence of efficient and universal genetic tools for this species.
• To facilitate the study and engineering of B. licheniformis for basic and applied research.

Main Methods:

• Development of a conjugative DNA transfer system as the core component of the toolkit.
• Utilizing a temperature-sensitive plasmid (pTSMK) for DNA transfer across multiple B. licheniformis strains.
• Building tools for maker-free gene knockout and knock-in, CRISPR interference (CRISPRi), and transposon mutagenesis based on the developed DNA transfer system.

Main Results:

• The conjugative DNA transfer system successfully transferred the pTSMK plasmid into all ten tested B. licheniformis strains with efficiencies between 10^-5 and 10^-3.
• The toolkit enabled the construction of systems for maker-free knockout and knock-in, CRISPRi, and transposon mutagenesis.
• A high transposition frequency of 7.68 × 10^-3 was achieved using the developed transposon mutagenesis tool.

Conclusions:

• A comprehensive and user-friendly genetic manipulation toolkit has been successfully established for Bacillus licheniformis.
• This toolkit addresses most genetic engineering tasks required for B. licheniformis.
• The developed toolkit is expected to significantly advance both fundamental and applied research involving B. licheniformis.