Streamlining of a Pseudomonas putida Genome Using a Combinatorial Deletion Method Based on Minitransposon Insertion and the Flp-FRT Recombination System

Abstract

Here, we document a technique to reduce the size of the genome of Pseudomonas putida by using a combinatorial mini-Tn5-targeted Flp-FRT recombination system. This method combines random insertions with the site-specific Flp-FRT recombination system to generate successive random deletions in a single strain in which parts of the genome are excised via the action of the cognate flippase. For this purpose, we have generated two mini-Tn5 transposon mutant libraries with single and double integrations of either mini-Tn5 KpF alone or mini-Tn5 KpF in parallel with mini-Tn5 TF, respectively. These mini-Tn5 transposons carry different selectable markers and each has an FRT (Flippase Recognition Target) site. Mapping of the position of both mini-Tn5 transposons in the chromosome of P. putida was conducted by Arbitrary Primed-PCR (AP-PCR). Subsequent sequencing of the PCR fragments led to the identification of the coordinates of the transposons and the orientation of both FRT sites. Under specific laboratory conditions, both FRT sites were recognized by the flippase, and the deletion of a nonessential intervening genomic segment along with the transposon backbones occurred without inheritance of any marker genes.

The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis

“…It is known that strains kept in laboratories tend to evolve (Barrick et al ., ). In order to substantiate the validity of our re‐sequencing of the genome, we compared the regions of variation with the sequence of a streamlined mutant (Leprince et al ., ): 95% of the variations were present in both sequences, showing that they were present at a very early stage and did not arise while handled in our laboratory. A significant part of the events (54) were found to affect 20 CoDing Sequences (CDSs; see Supporting Information Table S1).…”

“…The genome sequences of P. putida KT2440 DSM 615 and of a mutant strain TEC1 401‐Δ1, were obtained using Illumina sequencing technology. The wild‐type strain is the one sequenced in 2002, coming from the same original glycerol stock deposited in the DSMZ collection, and the mutant strain was generated by experimental genome reduction over strain KT2440 by the group of Vitor Martins dos Santos in Wageningen University [Microme WP3, (Leprince et al ., )]. Paired‐ends libraries were prepared with fragment size comprised between 300 and 600 bp and sequenced on HiSeq2000 (100 nt length).…”

“…In this work we set out to revisit the metabolic and physiological setup of this organism by re‐analyzing the content of its genome using several approaches. We first re‐sequenced the P. putida KT2440 wild‐type strain, in parallel with that of a streamlined derivative as a control for possible evolution in laboratory settings (Leprince et al ., ) and compared it to the original published sequence (Nelson et al ., ). Combined with transcriptomic data analysis (Frank et al ., ; Kim et al ., ), a complete structural re‐annotation of the KT2440 genome sequence led us to eliminate original erroneously predicted protein‐coding genes, to correct disrupted genes and to identify potential new genes, some of which encode enzymatic activities.…”

Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida

“…2B). The detailed method is described elsewhere (Leprince et al ., 2012). Briefly, two pairs of primers were used in two separated rounds of amplification with different sequences depending on the targeted mini‐Tn 5 transposons (Table S7).…”

Protocols for RecET‐based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

“…Particularly, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain (Federal Register, ) and exploited for the production of chemicals and products that human directly consumes, especially natural products including pharmaceuticals, nutraceuticals, and cosmetic ingredients (Loeschcke and Thies, ). Various genetic engineering elements – including counterselection markers (Galvao and de Lorenzo, ; Gross et al ., ; Graf and Altenbuchner, ; Johnson et al ., ), site‐specific recombinases (Leprince et al ., ; Ibrahim et al ., ), homing endonuclease I‐SceI (Martinez‐Garcia and de Lorenzo, ; Martinez‐Garcia et al ., ; Chen et al ., ), bacteriophage‐derived recombinases homologous to Bet protein from λ Red system (Aparicio et al ., ; Luo et al ., ), and clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR‐associated (Cas) systems (Aparicio et al ., ; Cook et al ., ; Sun et al ., ) – have been incorporated to facilitate gene knockout in P. putida . Although reduction of the genome size can be beneficial in some cases for better strain performance (Lieder et al ., ), only few cases of deleting large genomic fragment have been reported (Martinez‐Garcia et al ., ; Aparicio et al ., ).…”