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 -glutamylcysteine synthetase

摘要

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 RhodococcusAD45 (IsoILR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E. coli mutant g-glutamylcysteine synthetase (GSHI~*). Along with IsoILR1, another new RhodococcusAD45 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.