Bacterial Genome Editing via a Designed Toxin-Antitoxin Cassette

Area of Science:

• Microbiology
• Synthetic Genomics
• Molecular Biology

Background:

• Efficient bacterial genome manipulation is crucial for biological and biotechnological advancements.
• Existing genetic modification methods can be limited in scope and efficiency.

Purpose of the Study:

• To develop a versatile and efficient genome engineering technology for Gram-positive bacteria.
• To establish a novel counter-selectable cassette system for precise genetic modifications.

Main Methods:

• Reprogramming bacterial Type II Toxin-Antitoxin (TA) systems into a toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS).
• Utilizing the RelBE complex and its antitoxin RelB switch for conditional regulation of cell growth and death.
• Employing a single vector system for various genetic modifications in *B. subtilis* and *C. glutamicum*.

Main Results:

• Successfully performed in-frame deletion, knock-in, gene replacement, point mutation, large-scale insertion, and deletion of large DNA fragments (up to 194.9 kb in *B. subtilis* and 179.8 kb in *C. glutamicum*).
• Achieved first-time biosynthesis of lycopene in *B. subtilis* by integrating a 5.4-kb gene cluster using TCCRAS.
• Demonstrated increased mutation efficiencies (0.8-1.0-fold) compared to the SacB method in *C. glutamicum*.

Conclusions:

• TCCRAS is an effective and versatile technology for genome-scale engineering in Gram-positive bacteria.
• The system shows significant promise for applications in metabolic engineering and synthetic genomics.
• TCCRAS offers improved efficiency and broader applicability for bacterial genetic manipulation.