Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX  

作　　者：Andreas Domrose Robin Weihmann Stephan Thies Karl-Erich Jaeger Thomas Drepper Anita Loeschcke 

机构地区：[1]Institute of Molecular Enzyme Technology,Heinrich Heine University Düsseldorf,Forschungszentrum Jülich,Jülich,Germany [2]Bioeconomy Science Center(BioSC),Forschungszentrum Jülich,Jülich,Germany [3]Institute of Bio-and Geosciences(IBG-1),Forschungszentrum Jülich,Jülich,Germany

出　　处：《Synthetic and Systems Biotechnology》2017年第4期310-319,共10页合成和系统生物技术（英文）

基　　金：The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation,Science and Research of the German federal state of North Rhine-Westphalia MIWF within the framework of the NRW Strategieprojekt BioSC(No.313/323-400-00213).

摘　　要：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.

关 键 词：Synthetic biology Yeast recombinational cloning Tn5 transposition Heterologous gene cluster expression Secondary metabolite production Pseudomonas putida 

分 类 号：F42[经济管理—产业经济]