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Applied and Environmental Microbiology, October 2006, p. 6699-6706, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00907-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Matthew de la Peña Mattozzi,1,4 Sundiep K. Tehara,2 Thomas Hong,4,5 and Jay D. Keasling1,2,3,4*

Graduate Group in Microbiology,1 Department of Chemical Engineering,2 Department of Bioengineering,3 and Department of Molecular and Cell Biology, University of California, Berkeley, California,5 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California4

Received 20 April 2006/ Accepted 4 August 2006

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 ß-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.

* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail code 3224, University of California, Berkeley, CA 94720-3224. Phone: (510) 495-2620. Fax: (510) 495-2630. E-mail: keasling{at}berkeley.edu.

Applied and Environmental Microbiology, October 2006, p. 6699-6706, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00907-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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