TREX: A Universal Tool for the Transfer and Expression of Biosynthetic Pathways in Bacteria

Abstract

Secondary metabolites represent a virtually inexhaustible source of natural molecules exhibiting a high potential as pharmaceuticals or chemical building blocks. To gain broad access to these compounds, sophisticated expression systems are needed that facilitate the transfer and expression of large chromosomal regions, whose genes encode complex metabolic pathways. Here, we report on the development of the novel system for the transfer and expression of biosynthetic pathways (TREX), which comprises all functional elements necessary for the delivery and concerted expression of clustered pathway genes in different bacteria. TREX employs (i) conjugation for DNA transfer, (ii) randomized transposition for its chromosomal insertion, and (iii) T7 RNA polymerase for unimpeded bidirectional gene expression. The applicability of the TREX system was demonstrated by establishing the biosynthetic pathways of two pigmented secondary metabolites, zeaxanthin and prodigiosin, in bacteria with different metabolic capacities. Thus, TREX represents a valuable tool for accessing natural products by allowing comparative expression studies with clustered genes.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

“…The H. chejuensis hap cluster (see above and Kim et al 2007Kim et al , 2008 has been expressed in a recombinant E. coli strain (Kwon et al 2010). Drepper and coworkers have successfully expressed the S. marcescens pig cluster (in total 15 genes) heterologously in Pseudomonas putida using the TREX technology (Loeschcke et al 2013). A red pigmentproducing marine bacterium, Zooshikella rubidus, was shown to be capable of producing prodigiosin and also cycloprodigiosin (Lee et al 2011); however, genetic information has yet to be published.…”

Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

“…However, there are some challenges in streamlining the transition from the discovery stage of an enzyme through its metagenomic analysis, and ultimately towards its end‐user applications (Jemli et al ., ). The major technological bottlenecks include (i) a low proportion of coding metagenomic DNA accessible for expression (Guazzaroni et al ., ), (ii) a low proportion of enzymes selected from screens perform well in industrial settings (Martínez‐Martínez et al ., ), (iii) a lack of relevant substrates for screening (Fernández‐Arrojo et al ., ), (iv) insufficient screening methods for rare enzymatic activities (Singh, ), (v) a poor performance of enzymes under non‐natural conditions (Fernández‐Arrojo et al ., ), (vi) the existence of enzymes that are inactive after expression in the widely used host Escherichia coli (Loeschcke et al ., ), (vii) the lack of reliable bioinformatics pipelines for analysis of next‐generation sequencing data generated from positive hits or direct sequencing (Nyyssönen et al ., ), and (viii) the lack of reliable functional prediction of hypothetical proteins (Mende et al ., ; Anton et al ., ; Bastard et al ., ; Chistoserdova, ). In addition, the minimization of amplification of annotation mistakes (sequence/activity incoherence) in databases (Fernández‐Arrojo et al ., ) is among the more challenging issues to be solved.…”

Emerging Strategies and Integrated Systems Microbiology Technologies for Biodiscovery of Marine Bioactive Compounds

“…Another novel approach that allows the transfer of a complete biosynthetic pathway into a different bacterium has been reported recently by Loeschcke and coworkers [210]. The TRansfer and EXpression system (TREX) involves the transfer an entire biosynthetic pathway to a suitable host.…”

“…The target gene cluster (which can be located on BAC, plasmid or cosmid) is labeled by the two TREX cassettes. The transfer of metabolic pathways can be achieved by restriction endonuclease-based cloning or more advanced techniques such as restriction enzyme-independent cloning, and recombineering techniques based on λ phage or yeast recombinases that enable a better handling of large DNA fragments [210]. In the second step, the TREX-labeled gene cluster is transferred to a bacterial host.…”

“…This module—provided on plasmid pIC20H-RL, GenBank Accession Number: JX668229—includes both TREX cassettes where the two T7 promoters point outwards. This system has been applied to the transfer and expression in bacteria of two biosynthetic pathways responsible for the production of two secondary metabolites, prodigiosin and zeaxanthin [210]. In this way, TREX can help to identify and synthesize novel bioactive compounds.…”