Single Crossover-Mediated Markerless Genome Engineering in Clostridium acetobutylicum

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Clostridium acetobutylicum is a crucial industrial microorganism.
• Efficient genome engineering tools are needed to enhance its capabilities.
• Existing methods for genetic modification can be complex and time-consuming.

Purpose of the Study:

• To develop a novel and efficient genome-engineering tool for Clostridium acetobutylicum.
• To facilitate precise genetic modifications in the C. acetobutylicum genome.
• To create a method that simplifies the engineering process.

Main Methods:

• Development of a small, non-replicable plasmid (pHKO1) for genome integration via single-crossover homologous recombination.
• Utilizing Flp recombinase for in vivo excision of the integrated plasmid backbone, leaving a single target site.
• Employing a segregationally unstable plasmid (pSHL-FLP) for transient expression of Flp recombinase, enabling rapid plasmid curing.

Main Results:

• Successful integration of the pHKO1 plasmid into the C. acetobutylicum genome.
• Efficient in vivo excision of the plasmid backbone by Flp recombinase.
• Rapid and complete curing of the Flp recombinase-carrying plasmid from the engineered strain.
• Demonstrated ease of use for engineering Clostridium acetobutylicum.

Conclusions:

• A novel and simplified genome-engineering tool for Clostridium acetobutylicum has been established.
• The developed method allows for efficient gene targeting and modification.
• This tool significantly facilitates the genetic engineering of C. acetobutylicum for various applications.