Developing Clostridium diolis as a biorefinery chassis by genetic manipulation

Area of Science:

• Microbiology
• Metabolic Engineering
• Synthetic Biology

Background:

• Clostridium diolis efficiently converts renewable resources into chemicals and fuels.
• Efficient genetic manipulation tools are lacking for C. diolis, hindering research.
• Plasmids and electro-transformation are crucial for microbial strain development.

Purpose of the Study:

• To develop an efficient electroporation protocol for Clostridium diolis.
• To identify functional replicons for plasmid replication in C. diolis.
• To demonstrate the feasibility of metabolic engineering in C. diolis.

Main Methods:

• Optimization of electroporation parameters for C. diolis.
• Identification and validation of two functional replicons.
• Heterologous gene overexpression using engineered plasmids for metabolic pathway modification.

Main Results:

• An efficient electroporation protocol for C. diolis was established, enhancing transformation efficiency by over 100-fold (5 to 692 transformants/µg DNA).
• Two replicons functional in C. diolis were identified, enabling plasmid maintenance.
• Metabolic engineering successfully improved 1,3-propanediol and butanol production and enabled de novo butyl acetate synthesis.

Conclusions:

• The developed electroporation protocol significantly advances genetic manipulation capabilities for C. diolis.
• This study establishes a foundation for molecular-level studies and strain improvement of C. diolis.
• Metabolic engineering of C. diolis opens new avenues for producing valuable chemicals from renewable feedstocks.