Premium
Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source
Author(s) -
Walker Andy W,
Keasling Jay D
Publication year - 2002
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10251
Subject(s) - pseudomonas putida , parathion , organophosphate , operon , plasmid , chemistry , energy source , transformation (genetics) , parathion methyl , biochemistry , hydrolysis , enzyme , dna , gene , mutant , biology , pesticide , organic chemistry , coal , agronomy
Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p ‐nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH ( opd ), with and without the secretory leader sequence, was cloned into broad‐host‐range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid‐harboring operons encoding enzymes for p ‐nitrophenol transformation to β‐ketoadipate was transformed into P. putida allowing the organism to use 0.5 m M PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 m M parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non‐aqueous phase liquid phase but was still bioavailable. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 715–721, 2002.