DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

Journal Article · · Nucleic Acids Research
DOI: https://doi.org/10.1093/nar/gkw687 · OSTI ID:1396115
 [1];  [1];  [2];  [2];  [2];  [3];  [1]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
  3. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)

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.

Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOD; USDOE
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1396115
Report Number(s):
LA-UR-16-20060
Journal Information:
Nucleic Acids Research, Vol. 44, Issue 17; ISSN 0305-1048
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 22 works
Citation information provided by
Web of Science

References (66)

Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits journal December 2013
Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits journal December 2013
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
Genetically encoded sensors enable real-time observation of metabolite production journal February 2016
Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. journal January 1994
PHENIX: a comprehensive Python-based system for macromolecular structure solution. text January 2010
The crystal structure of a putative transcriptional regulator RHA06195 from Rhodococcus sp. RHA1 posted_content October 2006
PHENIX: a comprehensive Python-based system for macromolecular structure solution. text January 2010
Evolution of enzyme catalysts caged in biomimetic gel-shell beads journal July 2014
RosettaLigand Docking with Full Ligand and Receptor Flexibility journal January 2009
The oligomeric structures of plant cryptochromes journal May 2020
RosettaLigand Docking with Full Ligand and Receptor Flexibility journal January 2009
Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry journal May 2014
Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective journal June 2010
Novel screening methods—biosensors journal December 2015
Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen journal August 2020
Current issues in organophosphate toxicology journal April 2006
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
Crystal Structure of Thermotoga maritima 0065, a Member of the IclR Transcriptional Factor Family journal March 2002
Engineering an allosteric transcription factor to respond to new ligands journal December 2015
PROCHECK: a program to check the stereochemical quality of protein structures journal April 1993
Evolution of enzyme catalysts caged in biomimetic gel-shell beads text January 2014
Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance journal April 1994
Genetically encoded sensors enable real-time observation of metabolite production journal February 2016
Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. journal January 1994
Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors journal March 2006
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides journal October 1995
Evolution-guided optimization of biosynthetic pathways journal December 2014
Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry journal May 2014
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution journal February 2010
Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor: A Comparative Approach for Protein Design journal May 2015
Engineering an allosteric transcription factor to respond to new ligands journal December 2015
Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance journal April 1994
Emergence of novel functions in transcriptional regulators by regression to stem protein types journal August 2007
Novel screening methods—biosensors journal December 2015
Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase journal October 1997
Design and Application of a Mevalonate-Responsive Regulatory Protein journal December 2010
Structure prediction for CASP8 with all-atom refinement using Rosetta journal January 2009
Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor journal August 2015
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation journal February 2021
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes journal July 2006
Emergence of novel functions in transcriptional regulators by regression to stem protein types journal August 2007
MolProbity : all-atom structure validation for macromolecular crystallography journal December 2009
Coot model-building tools for molecular graphics journal November 2004
AraC Regulatory Protein Mutants with Altered Effector Specificity journal April 2008
Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis journal July 2015
A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site journal June 2002
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Teaching TetR to Recognize a New Inducer journal May 2003
Engineering allostery journal December 2014
Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase journal October 1997
A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site journal June 2002
AraC Regulatory Protein Mutants with Altered Effector Specificity journal April 2008
Design and Application of a Mevalonate-Responsive Regulatory Protein journal December 2010
Automation of protein purification for structural genomics journal March 2004
Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor journal August 2015
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution journal February 2010
Engineering allostery journal December 2014
Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors journal March 2006
Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis journal July 2015
Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides journal January 2006
Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides journal October 1995
Screening for Enhanced Triacetic Acid Lactone Production by Recombinant Escherichia coli Expressing a Designed Triacetic Acid Lactone Reporter
  • Tang, Shuang-Yan; Qian, Shuai; Akinterinwa, Olubolaji
  • Journal of the American Chemical Society, Vol. 135, Issue 27, p. 10099-10103 https://doi.org/10.1021/ja402654z
journal June 2013
Current issues in organophosphate toxicology journal April 2006
Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective journal June 2010
Purification and Properties of the Phosphotriesterase from Pseudomonas diminuta journal November 1989

Cited By (4)

Current status of water environment and their microbial biosensor techniques – Part II: Recent trends in microbial biosensor development journal May 2018
Tailor-made transcriptional biosensors for optimizing microbial cell factories journal November 2016
Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes journal November 2019
Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review journal February 2020