Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in Methanococcus maripaludis

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Methanococcus maripaludis is a genetically tractable methanogen with potential for bioproduction.
• Efficient genetic tools are crucial for engineering M. maripaludis for converting CO2 and H2 into valuable products.
• Existing genetic tools are insufficient for complex metabolic engineering in M. maripaludis.

Purpose of the Study:

• To design and validate a novel genome-editing toolbox for M. maripaludis.
• To enable precise gene deletions and characterization of promoter strengths for metabolic engineering.
• To expand the biotechnological applications of M. maripaludis.

Main Methods:

• Utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) with endogenous homology-directed repair.
• Demonstrated gene deletion efficiency up to 95% in hyperpolyploid M. maripaludis.
• Quantified the relative strength of 15 promoters under different growth conditions.

Main Results:

• Successfully deleted target genes with high efficiency.
• Achieved a large deletion by replacing the flagellum operon with the E. coli beta-glucuronidase gene.
• Quantified promoter activities crucial for metabolic engineering and flux balancing.

Conclusions:

• The developed CRISPR/LbCas12a toolbox is a reliable and efficient method for genome editing in methanogens.
• This tool facilitates metabolic engineering and expands the potential of M. maripaludis for bioproduction.
• The promoter characterization data aids in optimizing metabolic pathways for fuel and chemical synthesis.