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Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Synthetic and Systems Biotechnology 2017年第4期2卷 310-319页

作者： Andreas Domrose Robin Weihmann Stephan Thies Karl-Erich Jaeger thomas drepper Anita Loeschcke Institute of Molecular Enzyme Technology Heinrich Heine University DüsseldorfForschungszentrum JülichJülichGermany Bioeconomy Science Center(BioSC) Forschungszentrum JülichJülichGermany Institute of Bio-and Geosciences(IBG-1) Forschungszentrum JülichJülichGermany

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or *** exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous *** have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial ***,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene *** show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 *** of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia ***,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 *** producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong ***,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 *** compounds accumulated to substantial titers in the mg *** thus corroborate here the suitability of *** 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 biosy

关键词： Synthetic biology Yeast recombinational cloning Tn5 transposition Heterologous gene cluster expression Secondary metabolite production Pseudomonas putida

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