Environmental Biotechnology - Applications - Paraoxon Degradation

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Researchers

Matt Mattozzi, Joy Su, Scott Stevenson

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Project Description

Figure 1. Engineered paraoxon degradation pathway, constructed using enzymes from various soil bacteria.
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Figure 1. Engineered paraoxon degradation pathway, constructed using enzymes from various soil bacteria.

Organophosphate compounds, widely used as pesticides and chemical warfare agents, are potent cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We developed a strain of Pseudomonas putida str. KT2440 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. ENV2030, which encodes enzymes that transform p-nitrophenol (PNP) into β-ketoadipate, and a synthetic operon encoding an organophosphate hydrolase (opd) from Flavobacterium sp. ATCC 27551, a phosphodiesterase (pde) from Delftia acidovorans, and an alkaline phosphatase (phoAP) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. We devised new methods to analyze paraoxon and DEP using LC-MS/MS in order to determine the rates of mineralization. The engineered strain can efficiently mineralize up to 1 mM (275 mg/L) paraoxon within 48 hours, using the contaminant as its sole carbon and phosphorus source. 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.

Most recent work includes examining the accumulation of the intermediate p-nitrophenol (PNP) which appears to be accumulating in the media and restricting further growth of the cells (Figure 3).

Figure 3. In our experiments, PNP typically accumulates and then degrades as the cells use it as a carbon source.
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Figure 3. In our experiments, PNP typically accumulates and then degrades as the cells use it as a carbon source.
Figure 2. Growth of P. putida cells on MOPS-based minimal medium on 2% noble agar. 0.5 mM paraoxon is the sole carbon and phosphorus source available to the cells. The two sides of the plate represent cells expressing a native periplasmic leader sequence on opd (left) or a truncated leader (right). After 1 day, the cells begin to accumulate PNP, visible as a growing yellow streak. At 2 days, nearly all of the paraoxon has been converted. At 3 days, the medium becomes colorless again as the cells start using the accumulated PNP as a carbon source.
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Figure 2. Growth of P. putida cells on MOPS-based minimal medium on 2% noble agar. 0.5 mM paraoxon is the sole carbon and phosphorus source available to the cells. The two sides of the plate represent cells expressing a native periplasmic leader sequence on opd (left) or a truncated leader (right). After 1 day, the cells begin to accumulate PNP, visible as a growing yellow streak. At 2 days, nearly all of the paraoxon has been converted. At 3 days, the medium becomes colorless again as the cells start using the accumulated PNP as a carbon source.
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Relevant publications

  • M. D. Mattozzi, S. K. Tehara, T. Y. Hong and J. D. Keasling. 2006. “Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida.” Appl. Environ. Microbiol. 72:6699-6706.
  • S. K. Tehara and J. D. Keasling. 2003. “Gene cloning, purification, and characterization of a phosphodiesterase from Delftia acidovorans.” Appl. Environ. Microbiol. 69:504-508.
  • E. S. Gilbert, A. W. Walker and J. D. Keasling. 2003. “A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion.” Appl. Microbiol. Biotechnol. 61:77-81.
  • A. W. Walker and J. D. Keasling. 2002. “Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source.” Biotechnol. Bioeng. 78:715-721.
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Current funding

University of California Toxic Substances Research and Teaching Program

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