Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Greenhouse gas accumulation from fossil fuels drives climate change.
• Microbial gas fermentation offers a sustainable route for CO2 capture and biomanufacturing.
• Acetogens, like Clostridium autoethanogenum, are promising biocatalysts using CO2 and H2.

Purpose of the Study:

• To optimize plasmid curing methods for genetically engineered acetogens.
• To accelerate genetic engineering workflows for Clostridium autoethanogenum.
• To develop efficient plasmid elimination techniques for microbial cell factories.

Main Methods:

• Developed and tested CRISPR/Cas9-based and non-targeting control plasmids for curing.
• Utilized electrocompetent cells (ECCs) and non-transformative electroporation.
• Applied methods to buffer-washed glycerol stocks for plasmid elimination.

Main Results:

• CRISPR/Cas9-based curing did not outperform non-targeting controls.
• Electroporation methods achieved 14-100% curing efficiencies across five engineered C. autoethanogenum strains.
• Non-transformative electroporation of glycerol stocks efficiently cured plasmids in C. autoethanogenum and E. coli (~97%).

Conclusions:

• Optimized plasmid curing significantly accelerates genetic engineering in Clostridium autoethanogenum.
• Developed efficient and time-saving plasmid curing methods applicable to other microbial systems.
• Enhanced workflows facilitate the use of acetogens for sustainable biomanufacturing and carbon capture.