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 M 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 M).

Vibrational spectroscopy of intermediates in benzene-to-pheno conversion by FeO+

“…Promising direct benzene-phenol oxidation techniques include Fe-doped ZSM-5 zeolites with N 2 O as the oxidant [2,3] and direct oxidation using O 2 and H 2 through a Pd membrane [4]. Some bacteria can directly convert benzene to phenol under mild conditions using toluene monooxygenases, which have a di-iron active site [5]. The biotoxicity of benzene in mammals is enhanced by its conversion to phenol by cytochrome P450, which has an Fe-heme active site [6,7].…”

Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme

“…TG1 cells expressing T3MO were recently shown by our group to oxidize successively benzene to phenol, phenol to catechol, and catechol to 1,2,3-trihydroxybenzene (33). While studying the oxidation of toluene by these cells using GC, we discovered that they produce 90% p-cresol and 10% m-cresol ( Fig.…”

“…To stably and constitutively express the toluene monooxygenase genes from the same promoter, the expression vectors pBS(Kan)T3MO and pBS(Kan)T4MO were constructed as described earlier by Tao et al (33). The protein content of TG1(T3MO) and TG1(T4MO) was 0.24 mg of protein/ml/OD unit (33).…”

“…Pseudomonas aeruginosa PAO1 cells containing this plasmid were reported to form m-cresol from toluene oxidation and were reported to not convert the cresol to methylcatechol (21). However, Tao et al (33) have recently described the successive hydroxylation of benzene to phenol, phenol to catechol, and catechol to 1,2,3-trihydroxybenzene by Escherichia coli TG1 cells expressing T3MO. Here, we report the regiospecificity of T3MO on various aromatic compounds, show that it is capable of forming 4-methylcatechol from toluene, and indicate that the enzyme originally named T3MO is actually a para-hydroxylating enzyme, not a meta-oxidizing enzyme.…”

Szénhidrogén bontó Rhodococcus erythropolis törzsek funkcionális genomikai analízise és biotechnológiai alkalmazása

“…Első lépésben két egymást követő monooxigenáz reakció során [60][61][62], vagy egy gyűrű hidroxiláló dioxigenáz segítségével oxidálódik az aromás gyűrű [54]. Az így kialakult katekol származék szénláncát egy gyűrű-hasító dioxigenáz felnyitja.…”