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 gamma-glutamylcysteine synthetase

Author:

Rui, LY  Kwon, YM  Reardon, KF  Wood, TK  

Journal:

ENVIRONMENTAL MICROBIOLOGY

Issue Date:

2004

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 AD45 (IsoILR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E. coli mutant gamma-glutamylcysteine synthetase (GSHI*). Along with IsoILR1, another new Rhodococcus AD45 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 muM), 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 muM) 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 muM). 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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