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

• Published in: Microbial biotechnology 2018-01, Vol.11 (1), p.176-188
• Main Authors: Ricaurte, Deirdre E., Martínez‐García, Esteban, Nyerges, Ákos, Pál, Csaba, Lorenzo, Víctor, Aparicio, Tomás
• Format: Article
• Language: English
• Subjects: Bacteria; Bioremediation; Biosynthesis; Cloning; Deoxyribonucleic acid; DNA; E coli; Editing; Escherichia coli Proteins; Experiments; Gene Editing - methods; Gene expression; Genetics, Microbial - methods; Genome editing; Genome, Bacterial; Genomes; Homology; Metabolic engineering; Metabolism; Mutation; Oligonucleotides; Phages; Proteins; Pseudomonas putida; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; PyrF gene; Recombinase; Recombinases - isolation & purification; Recombinases - metabolism; Recombination, Genetic; Ribosomal Protein S9; RpsL gene; Soil bacteria; Soil microorganisms; Strains (organisms); Surveying; Workflow
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.12846

Summary Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β – an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus‐wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recβ functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads – and beyond. Recombineering is limited by availability of recombinases that promote invasion of the replication fork by ssDNA. An experimental workflow has been designed that allows rapid prototyping of new recombinases active in G(–) bacteria. One new recombinase named Rec2 is shown to deliver unprecedented recombineering efficiency in Pseudomonas putida.