Comparison of phage-derived recombinases for genetic manipulation ofPseudomonasspecies

Abstract

AbstractSeveral strains in thePseudomonasgenus are categorized as plant growth promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, known genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in 3 PGPR strains:P. protegensPf-5,P. protegensCHA0, andP. putidaKT2440. Using point mutations in therpoBgene, we reach maximum recombineering efficiencies of 1.5 x 10-4, 3 x 10-4, and 5 x 10-5, respectively, in these strains using λ-Red Beta recombinase fromE. coli. We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in thePseudomonasgenus.ImportanceThePseudomonasgenus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol and synthetic biology. Though several strains have been identified with beneficial properties, in situ genetic manipulations to further improve these strains for commercial applications have been limited due to lack of efficient genetic tools that have been tested across this genus. Here we test the recombineering efficiencies of 5 phage-derived recombinases across 3 biotechnologically relevantPseudomonasstrains:P. putidaKT2440,P. protegensPf-5, andP. protegensCHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across thePseudomonasgenus and a strategy that may be applied to develop analogous systems in other non-model bacteria.