Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in <i>Pseudomonas putida</i>

ABSTRACT Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p -nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd ) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde ) from Delftia acidovorans , and an alkaline phosphatase (encoded by phoA ) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway ( p -nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.