Mineralization of an organophosphate pesticide by rationally engineered catabolic pathways.

摘要

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.