Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Abstract

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

“…The reaction mixtures were incubated for 18 h at 30°C. Subsequently, the mixes were filtered through 0.22-m-pore-size PTFE filters and analyzed for 2-chlorobenzoate (2-CBA) by HPLC, performed as described previously (67), using an Accucore C 18 LC column (100 mm by 2.1 mm, 2.6-m particle size, 80-Å pore size; Thermo Scientific) on an LC10-Ai LC system (Shimadzu, Duisburg, Germany), with a gradient of water/acetonitrile (solvent A is water with 0.1% formic acid, and solvent B is acetonitrile with 0.1% formic acid; the gradient was started at 5% B with a hold at 5% B for 1.5 min; a gradient from 5% B to 98% B for 5.5 min; a hold at 98% B for 2 min; a gradient from 98% B to 5% B in 0.5 min; and a hold at 5% B for 2 min to reequilibrate) at a flow rate of 1 ml/min. The retention time of 2-CBA was determined as 4.78 min using a pure standard.…”

A Rational Design of Pseudomonas putida KT2440 capable of Anaerobic Respiration

“…To enable an anaerobic lifestyle, previous designs included the introduction of between 3 and 24 genes in P. putida KT2440 genome [13,14,4,22] but our in silico methods suggests that at least three times more genes are required. Novel methods developed specifically for integration of large operons or multiple genes like yTREX [46] allow incorporation of up to 14 genes at one time in P. putida.…”

Metabolic pathway engineering

“…These libraries may be useful for a wide range of applications, such as transcriptional and translational fine-tuning and sampling of the multidimensional gene expression space to balance multi-gene metabolic pathways. The second article on tool development, contributed by Domröse et al., describes a tool they call yTREX, which harnesses the versatility of yeast homologous recombination to carry out one-step cloning of vectors for the rapid TR ansfer, chromosomal integration by random transposition, and EX pression of gene clusters in bacteria [ 9 ]. To demonstrate this tool, the authors assembled the multigene biosynthetic pathways for phenazine, violacein, and prodiginine (with as many as 14 genes), and integrated them into P. putida .…”