Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Metabolic engineering

Background:

• Acetogenic bacteria, like Eubacterium limosum, are valuable for producing fuels and chemicals from C1 substrates.
• E. limosum possesses native advantages for C1 catabolism and growth but lacks sufficient genetic tools for engineering.

Purpose of the Study:

• To expand the molecular biology toolbox for E. limosum.
• To facilitate its application in synthetic biology and metabolic engineering.

Main Methods:

• Evaluated plasmid origins of replication and antibiotic concentration for gene expression control.
• Assessed promoter strength and developed well-characterized expression vectors.
• Developed a genetic reporter assay and a rapid transformation protocol leveraging oxygen tolerance.
• Created two novel antibiotic selection markers.

Main Results:

• Established methods for controlling gene expression copy number and levels.
• Characterized promoter performance and created versatile expression vectors.
• Developed an efficient, benchtop-compatible transformation protocol.
• Doubled the number of available antibiotic selection markers for E. limosum.

Conclusions:

• The developed genetic tools significantly enhance the engineering capabilities of E. limosum.
• These advancements will accelerate metabolic engineering and synthetic biology applications using this acetogen.
• E. limosum is now better equipped for engineered production of fuels and chemicals.