Panke S, Sánchez-Romero J M, de Lorenzo V
Centro Nacional de Biotecnología-CSIC, Madrid, Spain.
Appl Environ Microbiol. 1998 Feb;64(2):748-51. doi: 10.1128/AEM.64.2.748-751.1998.
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.
为构建一种能将甲苯以及几种烷基、氯代和硝基取代衍生物生物转化为相应苯甲酸盐的细菌催化剂，恶臭假单胞菌质粒pWW0的上部TOL操纵子与其同源调控基因xylR一起被完全重新组装成一个单一基因盒。然后，通过一种基因技术将相应的DNA片段靶向导入恶臭假单胞菌菌株的染色体，该技术可删除先前克隆步骤中继承的所有重组标签，使天然菌株仅携带编码所需表型的DNA片段。所得菌株通过两步过程以甲苯作为唯一碳源生长：甲苯由上部TOL酶介导转化为苯甲酸盐，苯甲酸盐通过儿茶酚中间体的常规邻位环裂解途径进一步代谢。
