Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase

Chlorinated ethenes are the most prevalent groundwater pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepexyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant co-expressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wildtype EchA at low concentrations (6.8 μM) and up to 10-fold at high concentrations (540 μM). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 μM), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 μM). For complete degradation of cis-DCE to chloride ions, the apparent V_max/K_m for the recombinant Escherichia coli strain expressing the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).