Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source

ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H+symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane.P. putidaPaW340 containingmhbRΔTDHIMcould not grow on 3-hydroxybenzoate; however, supplyingmhbTintransallowed the bacterium to grow on the substrate.K. pneumoniaeM5a1 andP. putidaPaW340 containing recombinant MhbT transported14C-labeled 3-hydroxybenzoate but not14C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake byP. putidaPaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.

Section: Methodsmentioning

confidence: 99%

MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Gao

Wang

et al. 2012

“…Isolation of the organophosphate hydrolase (opd) and phosphodiesterase (pde) genes has been described previously (31,33). Native opd was PCR amplified from pWM513 (23) [14]).…”

Section: Methodsmentioning

confidence: 99%

“…In general, these enzymes have a broad substrate range and can hydrolyze a number of organophosphate contaminants. While one of these enzymes or some variant can allow the initial detoxification of an organophosphate contaminant, the organism may not degrade the hydrolysis products, some of which are toxic and inhibit bacterial growth (13,33).…”

mentioning

confidence: 99%

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

Mattozzi

Tehara²,

Hong

et al. 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 ␤-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.Organophosphates have been widely used as nerve agents and pesticides. Examples of these compounds include the pesticides coumaphos and parathion and the nerve gas agents soman, sarin, and VX. These compounds irreversibly inhibit acetylcholine degradation in the human body and can kill quickly by causing persistent and uncontrolled muscle stimulation. Parathion, methyl parathion, and paraoxon, three insecticides, are common model organophosphates because they are much less toxic (in rats, the 50% lethal dose of paraoxon is 1.8 mg/kg, compared to 0.001 mg/kg for VX) and less volatile than many nerve agents. Synthetic in origin, many organophosphates are persistent in the environment and resist degradation by naturally extant microorganisms.Although incineration and chemical hydrolysis have been widely used to destroy these toxic compounds (20), many microorganisms can detoxify organophosphates by hydrolyzing them using organophosphate acid anhydrases. In general, these enzymes have a broad substrate range and can hydrolyze a number of organophosphate contaminants.

“…The bioremediation of sites contaminated with OPs is of concern in circumstances where there is a high risk of contact with humans. Escherichia coli, Moraxella sp., and Pseudomonas putida expressing recombinant bacterial phosphotriesterases (PTEs) have been used to detoxify solutions of phosphotriester pesticides (24,34,47) and could potentially be used for the bioremediation of contaminated soil and water. PTEs have been characterized with high activity towards particular OPs (e.g., paraoxon), but relatively low activities towards other OPs (e.g., demeton) (12,20,29).…”

mentioning

confidence: 99%

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

McLoughlin

Jackson

Liu

et al. 2004

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.Organophosphates (OPs) have been used as pesticides and chemical warfare agents during the last 60 years. OPs are considered nonpersistent compounds, breaking down in the environment over time to form relatively nontoxic compounds due to the action of light, water, and soilborne organisms. The bioremediation of sites contaminated with OPs is of concern in circumstances where there is a high risk of contact with humans. Escherichia coli, Moraxella sp., and Pseudomonas putida expressing recombinant bacterial phosphotriesterases (PTEs) have been used to detoxify solutions of phosphotriester pesticides (24, 34, 47) and could potentially be used for the bioremediation of contaminated soil and water. PTEs have been characterized with high activity towards particular OPs (e.g., paraoxon), but relatively low activities towards other OPs (e.g., demeton) (12,20,29). For the bioremediation of sites contaminated with poor PTE substrates, it is desirable to evolve PTEs with enhanced activity towards such substrates.Several groups have undertaken the directed evolution of PTEs for higher activity towards poor substrates (7,8,49). The major difficulty in generating such an enzyme is effectively screening large mutant libraries for mutants with higher activity. To this end, we are investigating the possibility of coupling PTE activity with cell growth that uses a phosphotriester as the sole phosphorus source. The enzyme expression leve...