Title

Protein engineering of toluene monooxygenases for synthesizing hydroxylated aromatics

Date of Completion

January 2005

Keywords

Engineering, Chemical

Degree

Ph.D.

Abstract

Toluene monooxygenases from pseudomonads are powerful enzymes for aromatic hydroxylation. The goal of this study is to engineer toluene monooxygenases to explore different hydroxylation reactions for making industrially-important singly- or doubly-hydroxylated aromatic products such as 1-naphthol (15,000 ton/yr market). It was discovered that wild type toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO, formally toluene 3-monooxygenase) from Ralstonia pickettii PKO1, and toluene ortho-monooxygenase from Burkholderia cepacia G4 perform three successive hydroxylations of benzene. To control these successive hydroxylations, two-phase reactors have also been designed to produce phenol from benzene and 2-naphthol from naphthalene using T4MO. In addition, toluene 3-monooxygenase was discovered to hydroxylate monosubstituted benzenes predominantly at the para position, instead of the meta position as was reported previously, and was renamed as TpMO. Hence, the toluene degradation pathway was modified for R. pickettii PKO1 to explain the physiological significance of the different hydroxylation pattern. ^ T4MO was discovered here to oxidize nitrobenzene to 4-nitrocatechol, to oxidize naphthalene to 1-naphthol (52%) and 2-naphthol (48%), to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), as well as to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). T4MO and TpMO mutants capable of synthesizing hydroxylated products with high efficiency and different regiospecificity were discovered through protein engineering via directed evolution and saturation mutagenesis at various positions of the α subunits TmoA and TbuA1 (I100, G103, A107, T201, F205, A213, and E214). The combination of error-prone PCR and saturation mutagenesis created the best 4-nitrocatechol producing mutant T4MO TmoA I100A with a 16-fold higher rate of 4-nitrocatechol formation than wild-type T4MO. The regiospecific oxidation of o-methoxyphenol and o-cresol was changed for the significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone by the variants TmoA G103A/A107S, G103S, G103S/A107T, and G103S/A 107G. (Abstract shortened by UMI.) ^