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Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP
Hae Jun Kwon1, Joungmin Lee2, Soo Jae Kwon1,3
1Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Busan, Republic of Korea.
Microbial Cell Factories
|January 3, 2024
Summary
Researchers established genetic tools for Clostridium sp. AWRP, enabling genome editing for alcohol production and carbon dioxide sequestration. This breakthrough facilitates further study of this promising acetogenic bacterium.
Area of Science:
- Microbiology
- Synthetic Biology
- Metabolic Engineering
Background:
- Clostridium sp. AWRP is a novel acetogenic bacterium with potential for alcohol production from carbon oxides.
- Genetic manipulation of AWRP is currently undeveloped, hindering physiological and metabolic engineering studies.
Purpose of the Study:
- To establish genetic tools for Clostridium sp. AWRP.
- To enable genome editing and further research into its applications.
Main Methods:
- Transformation of shuttle plasmids and characterization of clostridial promoters.
- Development of a CRISPR-Cas12a genome editing system.
- Electroporation and conjugation experiments with E. coli S17-1.
Main Results:
- Successful transformation of AWRP with specific plasmids (pCB102, pAMβ1, pIP404, pIM13), with pCB102 showing best electroporation efficiency.
- Established a functional CRISPR-Cas12a system for genome editing in AWRP, while CRISPR-Cas9 was ineffective.
- Achieved single and double gene deletions (xylB, pyrE, and prophage gene clusters).
Conclusions:
- A robust genome editing system was developed for Clostridium sp. AWRP.
- This genetic toolbox enables advanced manipulation, including deletion of large prophage groups.
- Facilitates deeper investigation of AWRP for syngas fermentation and CO2 sequestration.
