title: Luciferase-dependent, cytochrome P-450-catalyzed dehalogenation in genetically engineered Pseudomonas.
abstract: To investigate the possibility of luciferase-dependent photoreduction of cytochrome P-450's in vivo, Vibrio harveyi luciferase was coexpressed with the bacterial cytochrome P-450cam in Pseudomonas putida. Luciferase expression was under the control of the Pm promoter from the meta-cleavage TOL operon, incorporated into the chromosome by a mini Tn5-mediated transposition. Cytochrome P-450cam expression was controlled by the Ptac-lac promoter on the broad host range vector pMMB206. Both proteins were expressed in Pseudomonas putida strain MTCC 102 (PpW). This strain does not harbor the cam plasmid, which encodes the enzymes responsible for degradation of the terpene camphor. The metabolic activity of the resulting strain (PpW-lux-cam) toward model halogenated compounds was studied. In the absence of the natural cytochrome P-450cam, electron transfer partners putidaredoxin (PR) and putidaredoxin reductase (Fp), and at low oxygen tension, the bacterial cells efficiently dehalogenate model chlorinated hydrocarbons in a light-independent reaction. Hexachloroethane and pentachloroethane were metabolized to tetrachloroethylene and trichloroethylene, respectively, at rates comparable to that of strain PpG786, which carries the scam plasmid. Reductive dehalogenation required the expression of both luciferase and cytochrome P-450 in the same cells. These results indicate that alternative electron transfer partners may be exploited for cytochrome P-450-dependent bioremediation strategies.
authors.affiliation: National Environmental Engineering Research Institute, Nehru Marg, Nagpur, India.
authors.forename: R
authors.initials: R
authors.lastname: Shanker
authors.forename: W M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D002164:Camphor / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005798:Genes, Bacterial; D005818:Genetic Engineering; D006219:Halogens / Q000378:metabolism*; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology / Q000235:genetics
chemical_list: D006219:Halogens; D006843:Hydrocarbons, Chlorinated; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases