Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1

Abstract

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).

FIG. 1.

Time course of hydroxylated product formation from benzene determined by HPLC and of liquid benzene disappearance determined by GC from exponentially growing TG1(T4MO) (A), TG1(T3MO) (B), and TG1(TOM) (C). The initial liquid benzene concentration was 165 μM (400 μM benzene was added as if all were in the liquid phase). Representative figures of at least two independent results are shown.

FIG. 2.

Pathway for benzene oxidation by TG1(T4MO), TG1(T3MO), and TG1(TOM).