LUCIFERASE-DEPENDENT, CYTOCHROME P-450-CATALYZED DEHALOGENATION IN GENETICALLY-ENGINEERED PSEUDOMONAS

To investigate the possibility of luciferase-dependent photoreduction of cytochrome P-450's in vivo, Vibrio harveyi luciferase was coexpress ed with the bacterial cytochrome P-450cam in Pseudomonas putida. Lucif erase expression was under the control of the Pm promoter from the met a-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). T his strain does not harbor the cam plasmid, which encodes the enzymes responsible for degradation of the terpene camphor. The metabolic acti vity of the resulting strain (PpW-lux-cam) toward model halogenated co mpounds was studied. In the absence of the natural cytochrome P-450cam , electron transfer partners putidaredoxin (PR) and putidaredoxin redu ctase (Fp), and at low oxygen tension, the bacterial cells efficiently dehalogenate model chlorinated hydrocarbons in a light-independent re action. Hexachloroethane and pentachloroethane were metabolized to tet rachloroethylene and trichloroethylene, respectively, at rates compara ble to that of strain PpG786, which carries the cam plasmid. Reductive dehalogenation required the expression of both luciferase and cytochr ome P-450 in the same cells. These results indicate that alternative e lectron transfer partners may be exploited for cytochrome P-450-depend ent bioremediation strategies.