| Organophosphates, commonly used as pesticides and nerve agents, are potent neurotoxins. Their structures mimic acetylcholine, interrupting signal transduction. Parathion is a good pesticide for the same reason VX and sarin are potent nerve agents.;Metabolic engineering offers an innovative way to treat contamination in situ: Bioaugmentation has clear advantages over other methods that would require removal of contaminated soils or incomplete combustion of nerve agents. The goal of this study was to engineer and optimize a single microorganism to completely detoxify and mineralize a xenobiotic compound. Furthermore, the engineered strain can use a model organophosphate pesticide, paraoxon, as a sole carbon, energy, and phosphorus source.;The degradation of paraoxon takes place in three steps: initial hydrolysis to p-nitrophenol and diethyl phosphate, degradation of p-nitrophenol and for use as a carbon source, and degradation of diethyl phosphate for use as a phosphorus source. The soil bacterium Pseudomonas putida was transformed with two plasmids. One plasmid, pSB337, contains DNA from Pseudomonas sp. ENV2030, which encodes enzymes that transform p-nitrophenol (PNP) into beta-ketoadipate, although the specific processes by which this event occurs were not completely elucidated. A second plasmid, pMM11, contains a synthetic operon encoding an organophosphate hydrolase from Flavobacterium sp., a phosphodiesterase from Delftia acidovorans, and an alkaline phosphatase from Pseudomonas aeruginosa under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/L) paraoxon within 48 hours, using paraoxon as its sole carbon and phosphorus source and an inoculum density of OD600 = 0.03.;This project presents a significant proof-of-concept step: We can rationally design a bacterium's metabolism to detoxify and mineralize a xenobiotic environmental contaminant. Additional studies present some of the native chemotaxis of P. putida toward paraoxon or p-nitrophenol, characterization of the pathway in strains of P. putida, attempts at further optimizing the system through periplasmic export of the enzymes, a study of the genes and promoters responsible for the degradation of p-nitrophenol and an attempt to reengineer the PNP degradation pathway. |
