Pseudomonas putidaKT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxPsite-specific recombination

Abstract

Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/PmekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.

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

“…In contrast, it fails to function in species such as P. syringae (Swingle et al ., 2010) and C. glutamicum (Binder et al ., 2013). Recβ activity in P. putida KT2440 has been documented at ~10 −7 mutants/viable cell (Luo et al ., 2016), a result equal to, if not lower than, basal recombineering rates in this organism. Notwithstanding our use of an alternative plasmid/reporter system, we take note of Recβ activity reaching ~10 −3 mutants/viable cell, a value nearly four orders of magnitude greater than the one reported above.…”

“…Due to its reported levels of activity in P. syringae (Swingle et al ., 2010), we selected RecT Psy , a homologue of the E. coli Rac prophage recombinase RecT. Finally, we included the original E. coli λ phage recombinase Recβ , which has been shown to demonstrate low levels of activity in P. putida KT2440 (Luo et al ., 2016). As a control, we added to our study the current leading candidate for recombineering in P. putida , the T1E_1405 protein of P. putida DOT‐T1E (named Ssr).…”

Engineering Pseudomonas putida KT2440 for simultaneous degradation of carbofuran and chlorpyrifos

“…P. putida KT2440 has been highlighted as an optimal chassis for implantation of various organic‐degrading genes to create a multifunctional engineered strain for simultaneous degradation of multiple contaminants (Gong et al ., ). Recently, a two‐step markerless genome modification method was developed for P. putida KT2440, which combined λRed‐mediated homologous recombination with Cre ‐recombinase catalysed site‐specific recombination (Luo et al ., ). Graf and Altenbuchner () developed a chromosomal marker‐free modification method for P. putida KT2440 by combining homologous recombination with upp counter‐selection system.…”

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

“…Yet, their applicability to species such as Pseudomonas putida has a special interest because of the value of environmental microorganisms as useful platforms for metabolic engineering (Nikel et al, 2014; Nikel et al, 2016; Martínez-García and de Lorenzo, 2019). Attempts of functional expression of the lambda Red system in various species of Pseudomonas have been reported, but recombination frequencies were low in the absence of selection (Lesic and Rahme, 2008; Liang and Liu, 2010; Luo et al, 2016; Chen et al, 2018; Yin et al, 2019). Red-like counterparts found in Pseudomonas prophages have been more successful to the same ends.…”