Modifying monooxygenases for biosynthesis

Variants of three different monooxygenase enzymes were used to transform aromatics. Modification of the gene encoding 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at one amino acid position, increased the enzyme's activity with o-nitrophenol 47 fold, m-nitrophenol 34 fold, and o-methoxyphenol 174 fold. The modified enzyme also expanded its substrate range to include m-methoxyphenol, o-cresol, and m-cresol. Toluene-o-xylene monooxygenase from a different bacterium, Pseudomonas stutzeri OX1, oxidized toluene to 3- and 4-methylcatechol and benzene to phenol. Gene shuffling and amino acid modification generated mutants that synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene.