Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of a P. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.