A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor
Abstract
A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when pNP was added as an inducer, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.
- Authors:
-
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)
- Publication Date:
- Research Org.:
- Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
- Sponsoring Org.:
- USDOD; USDOE
- OSTI Identifier:
- 1396115
- Report Number(s):
- LA-UR-16-20060
Journal ID: ISSN 0305-1048
- Grant/Contract Number:
- AC52-06NA25396
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 44; Journal Issue: 17; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Biological Science; whole-cell biosensor, transcription factor, protein design, high throughput screening, paraoxon, nerve agent, flow cytometry
Citation Formats
Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E. Â M. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States: N. p., 2016.
Web. doi:10.1093/nar/gkw687.
Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, & Strauss, Charlie E. Â M. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States. https://doi.org/10.1093/nar/gkw687
Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E. Â M. Tue .
"A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor". United States. https://doi.org/10.1093/nar/gkw687. https://www.osti.gov/servlets/purl/1396115.
@article{osti_1396115,
title = {A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor},
author = {Jha, Ramesh K. and Kern, Theresa L. and Kim, Youngchang and Tesar, Christine and Jedrzejczak, Robert and Joachimiak, Andrzej and Strauss, Charlie E. Â M.},
abstractNote = {A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when pNP was added as an inducer, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.},
doi = {10.1093/nar/gkw687},
journal = {Nucleic Acids Research},
number = 17,
volume = 44,
place = {United States},
year = {Tue Aug 30 00:00:00 EDT 2016},
month = {Tue Aug 30 00:00:00 EDT 2016}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 22 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits
journal, December 2013
- Choi, Su-Lim; Rha, Eugene; Lee, Sang Jun
- ACS Synthetic Biology, Vol. 3, Issue 3
Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits
journal, December 2013
- Choi, Su-Lim; Rha, Eugene; Lee, Sang Jun
- ACS Synthetic Biology, Vol. 3, Issue 3
Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators
journal, November 2010
- Lu, D.; Fillet, S.; Meng, C.
- Genes & Development, Vol. 24, Issue 22
Genetically encoded sensors enable real-time observation of metabolite production
journal, February 2016
- Rogers, Jameson K.; Church, George M.
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 9
Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus.
journal, January 1994
- DiMarco, A. A.; Ornston, L. N.
- Journal of Bacteriology, Vol. 176, Issue 14
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
text, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Apollo - University of Cambridge Repository
The crystal structure of a putative transcriptional regulator RHA06195 from Rhodococcus sp. RHA1
posted_content, October 2006
- Cymborowski, M. T.; Chruszcz, M.; Skarina, T.
- Protein Data Bank
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
text, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Apollo - University of Cambridge Repository
Evolution of enzyme catalysts caged in biomimetic gel-shell beads
journal, July 2014
- Fischlechner, Martin; Schaerli, Yolanda; Mohamed, Mark F.
- Nature Chemistry, Vol. 6, Issue 9
RosettaLigand Docking with Full Ligand and Receptor Flexibility
journal, January 2009
- Davis, Ian W.; Baker, David
- Journal of Molecular Biology, Vol. 385, Issue 2
The oligomeric structures of plant cryptochromes
journal, May 2020
- Shao, Kai; Zhang, Xue; Li, Xu
- Nature Structural & Molecular Biology, Vol. 27, Issue 5
RosettaLigand Docking with Full Ligand and Receptor Flexibility
journal, January 2009
- Davis, Ian W.; Baker, David
- Journal of Molecular Biology, Vol. 385, Issue 2
Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry
journal, May 2014
- Jha, Ramesh K.; Kern, Theresa L.; Fox, David T.
- Nucleic Acids Research, Vol. 42, Issue 12
Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective
journal, June 2010
- Khersonsky, Olga; Tawfik, Dan S.
- Annual Review of Biochemistry, Vol. 79, Issue 1, p. 471-505
Novel screening methods—biosensors
journal, December 2015
- Eggeling, Lothar; Bott, Michael; Marienhagen, Jan
- Current Opinion in Biotechnology, Vol. 35
Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen
journal, August 2020
- Zacarias, Sónia; Temporão, Adriana; Carpentier, Philippe
- JBIC Journal of Biological Inorganic Chemistry, Vol. 25, Issue 6
Current issues in organophosphate toxicology
journal, April 2006
- Costa, Lucio G.
- Clinica Chimica Acta, Vol. 366, Issue 1-2
Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators
journal, November 2010
- Lu, D.; Fillet, S.; Meng, C.
- Genes & Development, Vol. 24, Issue 22
Crystal Structure of Thermotoga maritima 0065, a Member of the IclR Transcriptional Factor Family
journal, March 2002
- Zhang, Rong-guang; Kim, Youngchang; Skarina, Tatiana
- Journal of Biological Chemistry, Vol. 277, Issue 21
Engineering an allosteric transcription factor to respond to new ligands
journal, December 2015
- Taylor, Noah D.; Garruss, Alexander S.; Moretti, Rocco
- Nature Methods, Vol. 13, Issue 2
PROCHECK: a program to check the stereochemical quality of protein structures
journal, April 1993
- Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.
- Journal of Applied Crystallography, Vol. 26, Issue 2
Evolution of enzyme catalysts caged in biomimetic gel-shell beads
text, January 2014
- Fischlechner, Martin; Schaerli, Yolanda; Mohamed, Mark F.
- Nature Publishing Group
Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance
journal, April 1994
- Hinrichs, W.; Kisker, C.; Duvel, M.
- Science, Vol. 264, Issue 5157
Genetically encoded sensors enable real-time observation of metabolite production
journal, February 2016
- Rogers, Jameson K.; Church, George M.
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 9
Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus.
journal, January 1994
- DiMarco, A. A.; Ornston, L. N.
- Journal of Bacteriology, Vol. 176, Issue 14
Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors
journal, March 2006
- Molina-Henares, Antonio J.; Krell, Tino; Eugenia Guazzaroni, Maria
- FEMS Microbiology Reviews, Vol. 30, Issue 2
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
journal, October 1995
- Stemmer, Willem P. C.; Crameri, Andreas; Ha, Kim D.
- Gene, Vol. 164, Issue 1
Evolution-guided optimization of biosynthetic pathways
journal, December 2014
- Raman, Srivatsan; Rogers, Jameson K.; Taylor, Noah D.
- Proceedings of the National Academy of Sciences, Vol. 111, Issue 50, p. 17803-17808
Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry
journal, May 2014
- Jha, Ramesh K.; Kern, Theresa L.; Fox, David T.
- Nucleic Acids Research, Vol. 42, Issue 12
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution
journal, February 2010
- Agresti, J. J.; Antipov, E.; Abate, A. R.
- Proceedings of the National Academy of Sciences, Vol. 107, Issue 9, p. 4004-4009
Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor: A Comparative Approach for Protein Design
journal, May 2015
- Jha, Ramesh K.; Chakraborti, Subhendu; Kern, Theresa L.
- Proteins: Structure, Function, and Bioinformatics, Vol. 83, Issue 7
Engineering an allosteric transcription factor to respond to new ligands
journal, December 2015
- Taylor, Noah D.; Garruss, Alexander S.; Moretti, Rocco
- Nature Methods, Vol. 13, Issue 2
Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance
journal, April 1994
- Hinrichs, W.; Kisker, C.; Duvel, M.
- Science, Vol. 264, Issue 5157
Emergence of novel functions in transcriptional regulators by regression to stem protein types
journal, August 2007
- Galvão, Teca Calcagno; Mencía, Mario; de Lorenzo, Víctor
- Molecular Microbiology, Vol. 65, Issue 4
Novel screening methods—biosensors
journal, December 2015
- Eggeling, Lothar; Bott, Michael; Marienhagen, Jan
- Current Opinion in Biotechnology, Vol. 35
Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase
journal, October 1997
- Watkins, Linette M.; Mahoney, Heather J.; McCulloch, Jennifer K.
- Journal of Biological Chemistry, Vol. 272, Issue 41
Design and Application of a Mevalonate-Responsive Regulatory Protein
journal, December 2010
- Tang, Shuang-Yan; Cirino, Patrick C.
- Angewandte Chemie International Edition, Vol. 50, Issue 5
Structure prediction for CASP8 with all-atom refinement using Rosetta
journal, January 2009
- Raman, Srivatsan; Vernon, Robert; Thompson, James
- Proteins: Structure, Function, and Bioinformatics, Vol. 77, Issue S9
Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor
journal, August 2015
- de los Santos, Emmanuel L. C.; Meyerowitz, Joseph T.; Mayo, Stephen L.
- ACS Synthetic Biology, Vol. 5, Issue 4
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation
journal, February 2021
- Tillu, Vikas A.; Rae, James; Gao, Ya
- Nature Communications, Vol. 12, Issue 1
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006
- Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
- Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
Emergence of novel functions in transcriptional regulators by regression to stem protein types
journal, August 2007
- Galvão, Teca Calcagno; Mencía, Mario; de Lorenzo, Víctor
- Molecular Microbiology, Vol. 65, Issue 4
MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009
- Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
Coot model-building tools for molecular graphics
journal, November 2004
- Emsley, Paul; Cowtan, Kevin
- Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
AraC Regulatory Protein Mutants with Altered Effector Specificity
journal, April 2008
- Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C.
- Journal of the American Chemical Society, Vol. 130, Issue 15
Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis
journal, July 2015
- Chen, Wei; Zhang, Shan; Jiang, Peixia
- Metabolic Engineering, Vol. 30
A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site
journal, June 2002
- Stols, Lucy; Gu, Minyi; Dieckman, Lynda
- Protein Expression and Purification, Vol. 25, Issue 1
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
Teaching TetR to Recognize a New Inducer
journal, May 2003
- Scholz, Oliver; Köstner, Martin; Reich, Marco
- Journal of Molecular Biology, Vol. 329, Issue 2
Engineering allostery
journal, December 2014
- Raman, Srivatsan; Taylor, Noah; Genuth, Naomi
- Trends in Genetics, Vol. 30, Issue 12
Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase
journal, October 1997
- Watkins, Linette M.; Mahoney, Heather J.; McCulloch, Jennifer K.
- Journal of Biological Chemistry, Vol. 272, Issue 41
A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site
journal, June 2002
- Stols, Lucy; Gu, Minyi; Dieckman, Lynda
- Protein Expression and Purification, Vol. 25, Issue 1
AraC Regulatory Protein Mutants with Altered Effector Specificity
journal, April 2008
- Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C.
- Journal of the American Chemical Society, Vol. 130, Issue 15
Design and Application of a Mevalonate-Responsive Regulatory Protein
journal, December 2010
- Tang, Shuang-Yan; Cirino, Patrick C.
- Angewandte Chemie, Vol. 123, Issue 5
Automation of protein purification for structural genomics
journal, March 2004
- Kim, Youngchang; Dementieva, Irina; Zhou, Min
- Journal of Structural and Functional Genomics, Vol. 5, Issue 1/2
Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor
journal, August 2015
- de los Santos, Emmanuel L. C.; Meyerowitz, Joseph T.; Mayo, Stephen L.
- ACS Synthetic Biology, Vol. 5, Issue 4
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution
journal, February 2010
- Agresti, J. J.; Antipov, E.; Abate, A. R.
- Proceedings of the National Academy of Sciences, Vol. 107, Issue 9, p. 4004-4009
Engineering allostery
journal, December 2014
- Raman, Srivatsan; Taylor, Noah; Genuth, Naomi
- Trends in Genetics, Vol. 30, Issue 12
Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors
journal, March 2006
- Molina-Henares, Antonio J.; Krell, Tino; Eugenia Guazzaroni, Maria
- FEMS Microbiology Reviews, Vol. 30, Issue 2
Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis
journal, July 2015
- Chen, Wei; Zhang, Shan; Jiang, Peixia
- Metabolic Engineering, Vol. 30
Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides
journal, January 2006
- Mee-Hie Cho, Catherine; Mulchandani, Ashok; Chen, Wilfred
- Protein Engineering, Design and Selection, Vol. 19, Issue 3
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
journal, October 1995
- Stemmer, Willem P. C.; Crameri, Andreas; Ha, Kim D.
- Gene, Vol. 164, Issue 1
Screening for Enhanced Triacetic Acid Lactone Production by Recombinant Escherichia coli Expressing a Designed Triacetic Acid Lactone Reporter
journal, June 2013
- Tang, Shuang-Yan; Qian, Shuai; Akinterinwa, Olubolaji
- Journal of the American Chemical Society, Vol. 135, Issue 27, p. 10099-10103
Current issues in organophosphate toxicology
journal, April 2006
- Costa, Lucio G.
- Clinica Chimica Acta, Vol. 366, Issue 1-2
Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective
journal, June 2010
- Khersonsky, Olga; Tawfik, Dan S.
- Annual Review of Biochemistry, Vol. 79, Issue 1, p. 471-505
Purification and Properties of the Phosphotriesterase from Pseudomonas diminuta
journal, November 1989
- Dumas, D. P.; Caldwell, S. R.; Wild, J. R.
- Journal of Biological Chemistry, Vol. 264, Issue 33
Works referencing / citing this record:
Current status of water environment and their microbial biosensor techniques – Part II: Recent trends in microbial biosensor development
journal, May 2018
- Nakamura, Hideaki
- Analytical and Bioanalytical Chemistry, Vol. 410, Issue 17
Tailor-made transcriptional biosensors for optimizing microbial cell factories
journal, November 2016
- De Paepe, Brecht; Peters, Gert; Coussement, Pieter
- Journal of Industrial Microbiology & Biotechnology, Vol. 44, Issue 4-5
Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes
journal, November 2019
- F. M. Machado, Leopoldo; Currin, Andrew; Dixon, Neil
- Journal of Biological Engineering, Vol. 13, Issue 1
Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review
journal, February 2020
- Li, Jia-Wei; Zhang, Xiao-Yan; Wu, Hui
- Frontiers in Bioengineering and Biotechnology, Vol. 8