3-Methylcatechol

The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l(-1)) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 micromol 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 production process. This is an important step for obtaining designer biocatalysts.

The isofunctional enzymes of catechol 1,2-dioxygenase from species of Acinetobacter, Pseudomonas, Nocardia, Alcaligenes, and Corynebacterium oxidize 3-methylcatechol according to both the intradiol and extradiol cleavage patterns. However, the enzyme preparations from Brevibacterium and Arthrobacter have only the intradiol cleavage activity. Comparison of substrate specificity among these isofunctional dioxygenases shows striking differences in the oxidation of 3-methylcatechol, 4-methylcatechol and pyrogallol.

Background:Substituted catechols are important precursors for large-scale synthesis of pharmaceuticals and other industrial products. Most of the reported chemical synthesis methods are expensive and insufficient at industrial level. However, biological processes for production of substituted catechols could be highly selective and suitable for industrial purposes.Results:We have optimized a process for bio-catalytic production of 3-substituted catechols viz. 3-nitrocatechol (3-NC) and 3-methylcatechol (3-MC) at pilot scale. Amongst the screened strains, two strains viz. Pseudomonas putida strain (F1) and recombinant Escherichia coli expression clone (pDTG602) harboring first two genes of toluene degradation pathway were found to accumulate 3-NC and 3-MC respectively. Various parameters such as amount of nutrients, pH, temperature, substrate concentration, aeration, inoculums size, culture volume, toxicity of substrate and product, down stream extraction, single step and two-step biotransformation were optimized at laboratory scale to obtain high yields of 3-substituted catechols. Subsequently, pilot scale studies were performed in 2.5 liter bioreactor. The rate of product accumulation at pilot scale significantly increased up to approximately 90-95% with time and high yields of 3-NC (10 mM) and 3-MC (12 mM) were obtained.Conclusion:The biocatalytic production of 3-substituted catechols viz. 3-NC and 3-MC depend on some crucial parameters to obtain maximum yields of the product at pilot scale. The process optimized for production of 3-substituted catechols by using the organisms P. putida (F1) and recombinant E. coli expression clone (pDTG602) may be useful for industrial application.