Protocol to obtain genetically engineered Acetobacterium woodii and Eubacterium callanderi strains

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Acetogens like Acetobacterium woodii and Eubacterium callanderi are crucial for converting CO2 and H2 into organic acids.
• Genetic tools for these acetogens are limited, hindering their biotechnological applications.
• Developing methods for genetic manipulation is essential for improving their industrial potential.

Purpose of the Study:

• To establish a reliable protocol for generating electrocompetent Acetobacterium woodii and Eubacterium callanderi cells.
• To facilitate genetic engineering of these acetogens for enhanced C1 substrate conversion.
• To provide a foundation for applying a genetic toolbox to these and similar anaerobic bacteria.

Main Methods:

• Detailed description of the electroporation procedure for Acetobacterium woodii and Eubacterium callanderi.
• Implementation of verification steps to confirm the successful generation of recombinant strains.
• Adaptation of the protocol for potential use with other anaerobic bacterial species.

Main Results:

• Successful production of electrocompetent Acetobacterium woodii DSM 1030 and Eubacterium callanderi DSM 3468 cells.
• Demonstration of the electroporation technique's efficacy in these specific acetogenic species.
• Establishment of verification methods for recombinant strain identification.

Conclusions:

• The presented protocol effectively generates electrocompetent acetogenic cells, Acetobacterium woodii and Eubacterium callanderi.
• This method opens avenues for genetic engineering and metabolic pathway optimization in these bacteria.
• The protocol is adaptable for use with a broader range of anaerobic microorganisms, expanding their synthetic biology applications.