title: 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.
abstract: 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 gamma-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 microM), a 1.8-fold improvement in the trans-DCE degradation rate (1.29 +/- 0.03 versus 0.71 +/- 0.04 nmol x min(-1) mg(-1) protein at 345 microM) and a 1.7-fold improvement in the TCE degradation rate (6.8 +/- 0.24 versus 4.1 +/- 0.16 nmol x min(-1) mg(-1) protein at 339 microM). For cis-DCE degradation with TOM-Green (based on substrate depletion), V(max) was 27 nmol x min(-1) mg(-1) protein with both IsoILR1 and GSHI* expressed compared with V(max) = 10 nmol x 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.
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222, USA.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Rui
authors.forename: Young Man
authors.initials: YM
authors.lastname: Kwon
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Reardon
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001673:Biodegradation, Environmental; D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D004785:Environmental Pollutants / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005030:Ethylenes / Q000737:chemistry / Q000378:metabolism*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015202:Protein Engineering; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004785:Environmental Pollutants; D005030:Ethylenes; D006843:Hydrocarbons, Chlorinated; C036216:ethylene; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase