Metabolic engineering of bacteria for the degradation of paraoxon

Metabolic engineering has emerged as a viable option for degrading recalcitrant compounds. Organophosphorus (O-P) compounds that resist natural degradation within a reasonable time threaten the environment. These synthetic compounds are the remnants of past pesticide use and chemical weapons development. The goal of this project is to metabolically engineer a microorganism which will degrade paraoxon, the model compound chosen for this project.; An organism will degrade a compound if it can be used as a nutrient source or a co-metabolite. Paraoxon can be used as a carbon and phosphorus source for an engineered bacterium. A three step mechanism is proposed. In the first step, paraoxon is hydrolyzed by organophosphate hydrolase (OPH), encoded by the opd gene of Flavobacterium sp. ATCC 27551. The hydrolysis yields two products, p-nitrophenol (PNP) and diethyl phosphate (DEP). PNP can be used as a source of carbon by expression of the pnp operon of Pseudomonas sp. ENV2030. The DEP can be used as a source of phosphorus by Delftia acidovorans, but the phosphodiesterase responsible (PdeA) has not been previously isolated. Introduction and expression of the genes encoding these enzymes into one host organism would allow paraoxon to be mineralized.; The majority of this work focused on the purification and characterization of a novel phosphodiesterase, PdeA, from Delftia acidovorans. The gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 60°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono and diesterase activity, it was most active on the phosphodiester bis (p-nitrophenyl)phosphate with a Km of (2.9 +/- 0.1) mM and a kcat of (879 +/- 73) min-1. The enzyme showed sequence similarity to cAMP phosphodiesterase and cyclic nucleotide phosphodiesterases, and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. Expression of pdeA allowed E. coli DH10b to use DEP as a source of phosphorus for growth. The IS1071 transposon insertion sequence was found downstream of the pdeA.; When assembling the paraoxon degradation pathway, it was found that P. putida KT2440 containing the pnp operon, pSB337, was able to degrade PNP by using it as a source of carbon. To decrease the number of plasmids used in assembling the pathway, a synthetic operon was constructed with opd and pdeA in pBBR1MCS2. Expression of opd was found to be dependent on its placement in the operon. Expression of the synthetic operon with the pSB337 vector will yield the complete degradation of paraoxon.