Metabolic Engineering to Reduce Toxicity Related to the Aerobic Degradation of Chlorinated Ethenes

Toluene o-monooxygenase (TOM), encoded by tomA012345 of the soil bacterium Burkholderia cepacia G4 was evolved for their degradation using DNA shuffling and saturation mutagenesis. TOM is a multi-component enzyme (six genes) consisting of a three-component hydroxylase with a catalytic oxygen-bridged binuclear iron center, a NADH-ferredoxin oxidoreductase, and a mediating protein involved in electron transfer between the hydroxylase and reductase. After directed evolution identified position 106 of the α-subunit of the hydroxylase as critical, saturation mutagenesis was implemented and the combined mutagenesis resulted in a 6-12 fold enhancement in 1-naphthol production (from naphthalene oxidation) as well as a three-fold enhancement in the degradation of chlorinated aliphatics, e.g., trichloroethylene, chloroform. To enhance further the degradation of chlorinated ethylenes by reducing the toxicity of the chlorinated epoxides formed in the TOM reaction, a novel glutathione S-transferase (GST) from Rhodococcus sp. strain AD45 was identified that is active with cis-DCE epoxide, and it was cloned into TOM-expressing strains. GST reduces toxicity by adding glutathione (α-glutamylcysteinylglycine) across the chlorinated ethylene epoxide bond formed by TOM. This is an abstract of a paper presented at the 19th Annual International Conference on Soils, Sediments, and Water (10/20-23/2003, Amherst, MA).