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.

Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida . Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida . We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.

SummaryBacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putida KT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels.