High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system

The bioconversion of toluene into 3-methylcatechol was studied as a model s ystem for the production of valuable 3-substituted catechols in general. Fo r this purpose, an improved microbial system for the production of 3-methyl catechol was obtained. Pseudomonas putida strains containing the rodC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were use d as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expressio n cassette with the todC1C2BAD genes cloned under the control of the induci ble regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida Fl, which degrad es toluene via 3-methylcatechol, or into mutant P. putida F107, which accum ulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of t he expression cassette. As a result, up to 14 mM (1.74 g l(-1)) of 3-methyl catechol was accumulated and the specific production rate reached a level o f 105 mu mol min(-1) g(-1) cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the pr oduction process. This is an important step for obtaining designer biocatal ysts.