Genome engineering using a synthetic gene circuit in Bacillus subtilis

Area of Science:

• Microbiology
• Synthetic Biology
• Molecular Biology

Background:

• Efficient genome engineering in Bacillus subtilis is crucial for developing industrial applications.
• Existing methods often leave foreign DNA, complicating strain development.
• A need exists for seamless genome editing techniques without leaving marker DNA.

Purpose of the Study:

• To develop and validate a novel synthetic gene circuit for counter-selectable marker-based genome engineering in Bacillus subtilis.
• To achieve precise genomic modifications, including insertions, deletions, and point mutations, without foreign DNA integration.
• To demonstrate the system's efficacy in deleting large genomic regions.

Main Methods:

• A synthetic gene circuit comprising repressible promoters (Pxyl, Pspac) and repressor genes (lacI, xylR) was designed.
• The Pxyl-lacI cassette was integrated into the B. subtilis genome, with a helper plasmid carrying xylR and Pspac-cat.
• A counter-selection mechanism was established using xylose induction, leading to chloramphenicol sensitivity unless the marker is deleted via recombination.

Main Results:

• Successful base insertion, deletion, and point mutation of the Bacillus subtilis genome were demonstrated.
• The system achieved seamless genome engineering, leaving no foreign DNA behind.
• Large genomic regions, including a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE), were successfully deleted.

Conclusions:

• The developed synthetic gene circuit provides an efficient and scarless method for Bacillus subtilis genome engineering.
• This technology is highly valuable for constructing designer Bacillus strains for diverse industrial applications.
• The system's ability to delete large DNA fragments broadens its utility in metabolic engineering and strain development.