Metabolic pathway engineering to enhance aerobic degradation of chlorinated ethenes and to reduce their toxicity by cloning a novel glutathione S-transferase, an evolved toluene o-monooxygenase, and γ-glutamylcysteine synthetase

Aerobic, co-metabolic bioremediation of trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE) and other chlorinated ethenes with monooxygenase-expressing microorganisms is limited by the toxic epoxides produced as intermediates. A recombinant Escherichia coli strain less sensitive to the toxic effects of cis-DCE, TCE and trans-1,2-dichloroethylene (trans-DCE) degradation has been created by engineering a novel pathway consisting of eight genes including a DNA-shuffled toluene orthomonooxygenase from Burkholderia cepacia G4 (TOM-Green), a newly discovered glutathione S-transferase (GST) from Rhodococcus AD45 (IsolLR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E. coli mutant γ-glutamylcysteine synthetase (GSHI*). Along with IsolLR1, another new Rhodococcus AD45 GST, IsolLR2, was cloned that lacks activity towards cis-DCE epoxide and differs from IsolLR1 by nine amino acids. The recombinant strain in which TOM-Green and IsolLR1 were co-expressed on separate plasmids degraded 1.9-fold more cis-DCE compared with a strain that lacked IsolLR1. In the presence of IsolLR1 and TOM-Green, the addition of GSH1* resulted in a sevenfold increase in the intracellular GSH concentration and a 3.5-fold improvement in the cis-DCE degradation rate based on chloride released (2.1 ± 0.1 versus 0.6 ± 0.1 nmol min^-1 mg^-1 protein at 540 μM), a 1.8-fold improvement in the trans-DCE degradation rate (1.29 ± 0.03 versus 0.71 ± 0.04 nmol min^-1 mg^-1 protein at 345 μM) and a 1.7-fold improvement in the TCE degradation rate (6.8 ± 0.24 versus 4.1 ± 0.16 nmol min^-1 mg^-1 protein at 339 μM). For cis-DCE degradation with TOM-Green (based on substrate depletion), V_max was 27 nmol min^-1 mg^-1 protein with both IsolLR1 and GSHI* expressed compared with V_max = 10 nmol min^-1 mg^-1 protein for the GST^- GSHI*^- strain. In addition, cells expressing IsolLR1 and GSHI* grew 78% faster in rich medium than a strain lacking these two heterologous genes.