Metabolic engineering of bacteria for the environment: The degradation of parathion

60,000 tons of organophosphate pesticides are produced annually in the U.S. Recent studies found over 50% of sampled groundwater sites were contaminated with pesticides; other studies have shown consumers are not adequately protected from pesticides. In addition, due to the Chemical Weapons Convention, the U.S. has over 30,000 metric tons of chemical warfare agents that are to be destroyed via incineration. Metabolic engineering offers the capability to engineer an organism to degrade pesticides (including potentially toxic metabolites) in the environment and to destroy chemical warfare agents in an environmentally benign way.; The organophosphate pesticide parathion and its oxygen analog paraoxon were chosen as model compounds for this work due to their structural and functional similarity to other organophosphate pesticides and chemical warfare agents. The ultimate goal of this work is to achieve parathion or paraoxon degradation (including metabolites) in a single microorganism so as to make the organism as environmentally applicable as possible.; The initial step in parathion or paraoxon degradation is hydrolysis. This results in the formation of diethyl thiophosphate (DETP, for parathion) or diethyl phosphate (DEP, for paraoxon) and p-nitrophenol (PNP). Thus, parathion degradation can be thought of as a three-piece puzzle: hydrolysis, PNP degradation, and DETP or DEP degradation. Hydrolysis was achieved by introducing one of four plasmids, pAWW01, pAWW02, pAWW03 or pAWW04 into the bacterium Pseudomonas putida. Resting cell hydrolase assays indicated that gene expression was low but tightly regulated in pAWW01 and pAWW02. Cells harboring pAWW03 or pAWW04 showed a dramatic increase in activity but at the consequence of losing tight control over gene expression.; A plasmid harboring genes encoding PNP degradation enzymes (pSB337) was transformed into P. putida KT2442 and PNP degradation was observed. Two pieces of the degradation puzzle were assembled by transforming P. putida KT2442 with both pSB337 and pAWW04. This allowed the organism to utilize parathion as a carbon and energy source.; DETP and DEP degradation, the third piece of the parathion degradation puzzle, was achieved by providing Comamonas acidovorans with DETP or DEP as the sole source of phosphorous. Degradation rates were estimated using an analog, bis-p-nitrophenol phosphate (BNP). The diesterase allowing for DETP and DEP degradation by C. acidovorans was cloned and sequenced by Sundiep Tehara. Insertion of the plasmid harboring the cloned diesterase (pSKT1) into E. coli DH10B allowed the organism to utilize DEP as a sole source of phosphorus. Insertion of pAWW01 and pSKT1 into E. coli DH10B allowed the organism to utilize paraoxon as a source of phosphorus.