Accelerated genome engineering of Pseudomonas putida by I‐SceI―mediated recombination and CRISPR‐Cas9 counterselection

Abstract

Summary Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large‐scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user‐friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3–5 days. The protocol presented here is based on DNA recombination forced by double‐stranded DNA cuts (through the activity of the I‐SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR‐Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult‐to‐delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

“…The robust catabolic pathways of P. putida , while useful for producing valuable molecules from diverse carbon sources, can also serve as an obstacle to achieving high product titers as it can often metabolize the desired products [26]. Given recent advances in gene editing techniques [27–31] and our ability to rapidly assay for gene function with transposon site sequencing [17,18,32], engineering non-model hosts like P. putida for industrial applications has become less challenging.…”

Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

“…We successfully conjugated P. putida with level 1 vectors with OriV #2, #3 and #5. Unexpectedly, the Pseudomonas shuttle OriV #4 did not yield any conjugants in our tests, although it contains no changes in the sequence from the original SEVA oriV which has previously been introduced into P. putida (31).…”

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

“…To perform mCRISPRi, we first constructed P. putida KT·YFP·mCherry, harbouring constitutively expressed yfp and mCherry genes – encoding yellow fluorescent protein (YFP) and red fluorescent protein (mCherry), respectively – and a kanamycin resistance marker integrated into the Tn 7 locus of the chromosome (Table S1 in the Supporting Information). The design and construction of P. putida KT·YFP·mCherry was done essentially according to Wirth et al (). P. putida KT·YFP·mCherry is a derivative of wild‐type strain KT2440 carrying a P 100 → mCherry and P tet → yfp cassette integrated in the Tn 7 locus via a synthetic mini‐Tn 7 transposon.…”

“…In an effort to broaden the existing toolbox, many groups have focused on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9)-based methods for knocking-out or knocking-down target genes in P. putida. Recently, type II CRISPR/Cas systems have been utilized in combination with the k-Red system, SSR recombinases or the I-SceI meganuclease for precise gene deletion (Mart ınez-Garc ıa and de Lorenzo, 2011; Mougiakos et al, 2017;Sun et al, 2018;Aparicio et al, 2019Aparicio et al, , 2020Wirth et al, 2020). Engineered catalytically inactive variants of the Cas9 protein (dead Cas9, dCas9) have been shown to act as a transcription repressor in Pseudomonas strains, including P. putida KT2440.…”