A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Bacterial recombineering, crucial for genetic manipulation, traditionally uses Escherichia coli λ phage recombinase β.
• Its application is largely confined to enterobacterial strains, with limited efficiency in other bacteria.
• Developing efficient recombineering tools for diverse bacterial species like Pseudomonas putida is essential for advancing synthetic biology and metabolic engineering.

Purpose of the Study:

• To identify and characterize an efficient Recβ homologue for recombineering in the model soil bacterium Pseudomonas putida.
• To establish a robust workflow for deploying recombineering in new bacterial hosts.

Main Methods:

• Conducted a genus-wide protein survey to identify potential recombinase candidates.
• Assayed selected novel proteins for their ability to introduce specific mutations (K43T substitution in rpsL gene) in P. putida.
• Validated the efficiency of the identified recombinase by introducing various mutations into the pyrF gene.

Main Results:

• Discovered an ERF superfamily protein, named Rec2, exhibiting eightfold greater recombineering activity than previously known recombinases in P. putida.
• Demonstrated Rec2's capability to efficiently introduce a range of mutations into the pyrF gene of P. putida.
• Established a complete workflow for implementing recombineering in P. putida and potentially other bacterial strains.

Conclusions:

• Rec2 is a highly efficient functional analogue of Recβ for recombineering in Pseudomonas putida.
• The developed workflow facilitates the deployment of recombineering in diverse bacterial systems.
• This advancement holds significant implications for genome editing in P. putida for metabolic engineering and broader applications in Pseudomonads and beyond.