Metagenomics harvested genus-specific single-stranded DNA annealing proteins improve and expand recombineering in Pseudomonas species

The widespread Pseudomonas genus embraces a series of relevant species with remarkable abilities to degrade and produce a whole range of compounds. While their unusual charac- teristics of tolerance and stress resistance already position them as valuable hosts for in- dustrial and environmental biotechnology, easier and more efficient DNA manipulation tools would propel them to the front ranks of the metabolic engineering platforms. To improve their genome editing capabilities, we generated a genus-specific library of potential single- stranded DNA annealing proteins, also known as recombinases, that were later screened in a high-throughput manner using a serial enriching workflow followed by Oxford Nanopore NGS analysis. Among different active candidates with variable levels of allelic replacement fre- quency, efficient recombinases were found and characterized for mediating recombineering in four different species. New variants yielded higher allelic replacement frequencies than previous standards in P. putida and P. aeruginosa, and set a precedent for efficient recom- bineering in P. taiwanensis and P. fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and boosting their potential for synthetic biology applications.