High-throughput screening system based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes.
Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher k(cat)/K(m) than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology-KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.
Young-Su
YS
Jeong
Su-Lim
SL
Choi
Hyun-Ho
HH
Kyeong
Jin-Hyun
JH
Kim
Eui-Joong
EJ
Kim
Jae-Gu
JG
Pan
Eugene
E
Rha
Jae Jun
JJ
Song
Seung-Goo
SG
Lee
Hak-Sung
HS
Kim
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002619:Chemical Warfare Agents / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010755:Organophosphates / Q000378:metabolism*; D010575:Pesticides / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002619:Chemical Warfare Agents; C080133:DmpR protein, Pseudomonas; D010755:Organophosphates; D010575:Pesticides; D010636:Phenols; D015534:Trans-Activators; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases