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

Abstract

Summary 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 ortho ‐monooxygenase from Burkholderia cepacia G4 (TOM‐Green), a newly discovered glutathione S ‐transferase (GST) from Rhodococcus AD 45 (IsoILR1), found to have activity towards epoxypropane and cis ‐DCE epoxide, and an overexpressed E. coli mutant γ‐glutamylcysteine synthetase (GSHI*). Along with IsoILR1, another new Rhodococcus AD 45 GST, IsoILR2, was cloned that lacks activity towards cis ‐DCE epoxide and differs from IsoILR1 by nine amino acids. The recombinant strain in which TOM‐Green and IsoILR1 were co‐expressed on separate plasmids degraded 1.9‐fold more cis ‐DCE compared with a strain that lacked IsoILR1. In the presence of IsoILR1 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 IsoILR1 and GSHI* expressed compared with V max = 10 nmol min −1 mg −1 protein for the GST – GSHI* – strain. In addition, cells expressing IsoILR1 and GSHI* grew 78% faster in rich medium than a strain lacking these two heterologous genes.

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