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

Abstract

SummaryBacterial 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.

High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

“…All clones analysed (n=18; 100%) showed the correct changes, demonstrating that the observed phenotypes corresponded to mutations mediated by the HEMSE procedure. Single allelic replacements in HEMSE experiments were not confirmed by PCR and sequencing since previous works showed that virtually 100 % of Sm R , Nal R and pyrF - mutants obtained by recombineering with oligos SR, NR and LM (almost identical to RR oligo used in this work) harbored the expected changes in the target genes rpsL , gyrA and pyrF (Ricaurte et al, 2018) (Aparicio et al, 2019a). Preliminary studies in this work showed, on the other hand, that single Rif R mutants also displayed 100 % accuracy in the expected mutations of the target gene rpoB (data not shown).…”

“…S1). Given that these background levels are higher with thermal induction than with chemical induction (Ricaurte et al, 2018), it is plausible that heat shock intrinsically improves recombineering regardless of the action of heterologous recombinases. As a matter of fact, purely endogenous ssDNA recombineering at significant frequencies has been reported in a variety of Gram-negative bacteria, including E. coli and Pseudomonas syringae (Swingle et al, 2010b), a fact that play in our favour for establishing the methodology in P. putida .…”

“…For example, the RecET recombinase/exonuclease pair of P. syringae has been instrumental for executing a suite of manipulations in this species (Swingle et al, 2010a; Bao et al, 2012). Furthermore, bioinformatic mining of Pseudomona s-borne recombinases from known protein families (i.e., Redβ, ERF, GP2.5, SAK, and SAK4; Lopes et al, 2010) followed by experimental validation of the most promising in a standardized recombineering test exposed two new enzymes (Ssr and Rec2: Aparicio et al, 2016; Ricaurte et al, 2018; Aparicio et al, 2020). These recombinases delivered a comparatively high level of activity in the reference strains P. putida KT2440 and its genome-reduced derivative P. putida EM42.…”

Development of a RecE/T‐Assisted CRISPR–Cas9 Toolbox for Lactobacillus

“…Although RecT homologs have demonstrated more cross‐species activity, RecE appears to show more limited activity in an evolutionarily distant species . The Rpsl and RpoB mutagenesis‐based evaluation workflow facilitates the activity assessment of various RecTs, which will contribute to RecT‐assisted ssDNA recombineering‐related genetic engineering and, additionally, enrich the library of molecular genetic elements for Lactobacillus genome editing. However, a genome‐wide survey of RecE/T association will still be challenging for some species without an endogenous phage.…”

Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

“…where is the calculated molecular mechanics energy for the force field applied, and is the solvation energy in the generalized Born approximation. Different variations of this method are widely used in large scale drug design studies [81][82][83] as it accurately predicts mutational effects in drug-protein interaction analyses with a manageable computational demand (see e.g. references 80,81 ).…”