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

Title: Engineering an allosteric transcription factor to respond to new ligands

Journal Article · · Nature Methods
DOI: https://doi.org/10.1038/nmeth.3696 · OSTI ID:1239401
 [1];  [1];  [2];  [3];  [3];  [3];  [1];  [4]; ORCiD logo [5];  [2];  [6];  [1];  [1]
  1. Harvard Univ., Boston, MA (United States). Wyss Inst. for Biologically-Inspired Engineering; Harvard Medical School, Boston, MA (United States). Dept. of Genetics
  2. Univ. of Washington, Seattle, WA (United States). Dept. of Biochemistry. Howard Hughes Medical Inst.
  3. Univ. of California, Los Angeles, CA (United States). UCLA-USDOE Inst. for Genomics and Proteomics
  4. Yale Univ., New Haven, CT (United States). Dept. of Molecular, Cellular and Developmental Biology; Yale Univ., West Haven, CT (United States). Systems Biology Inst.
  5. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
  6. Univ. of Washington, Seattle, WA (United States). Dept. of Genome Sciences. Dept. of Medicine. Howard Hughes Medical Inst.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. In this paper, we engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). Finally, the ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Research Organization:
Harvard Univ., Boston, MA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Inst. of Health (NIH) (United States)
Contributing Organization:
Yale Univ., New Haven, CT (United States); Univ. of California, Los Angeles, CA (United States); Univ. of Washington, Seattle, WA (United States)
Grant/Contract Number:
FG02-02ER63445; FC02-02ER63421; AC02-06CH11357
OSTI ID:
1239401
Journal Information:
Nature Methods, Journal Name: Nature Methods Journal Issue: 2 Vol. 13; ISSN 1548-7091
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

References (63)

Next-generation synthetic gene networks journal December 2009
Refinement of Macromolecular Structures by the Maximum-Likelihood Method journal May 1997
Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor journal August 2015
Comparison of 15 dinoflagellate genomes reveals extensive sequence and structural divergence in family Symbiodiniaceae and genus Symbiodinium journal April 2021
Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector. journal February 1987
A family of bacterial regulators homologous to Gal and Lac repressors. journal August 1992
FLASH: fast length adjustment of short reads to improve genome assemblies journal September 2011
The TetR Family of Transcriptional Repressors journal June 2005
Features and development of Coot journal March 2010
Programming cells by multiplex genome engineering and accelerated evolution journal July 2009
BLAT---The BLAST-Like Alignment Tool journal March 2002
Use of TLS parameters to model anisotropic displacements in macromolecular refinement journal January 2001
Engineering allostery journal December 2014
Teaching TetR to Recognize a New Inducer journal May 2003
A comparison of normalization methods for high density oligonucleotide array data based on variance and bias journal January 2003
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements journal March 1997
Flexibility in the Inducer Binding Region Is Crucial for Allostery in the Escherichia coli Lactose Repressor journal June 2009
AraC Regulatory Protein Mutants with Altered Effector Specificity journal April 2008
Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips journal November 2010
Complete Genome Sequence of Clostridium botulinum CJ0611A1, a Type A(B) Isolate Associated with an International Outbreak of Botulism from Commercial Carrot Juice journal January 2021
Engineering and characterization of a superfolder green fluorescent protein journal December 2005
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. journal June 1992
De Novo Computational Design of Retro-Aldol Enzymes journal March 2008
Controlling Gene Expression in Living Cells Through Small Molecule-RNA Interactions journal October 1998
PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome journal December 2019
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products journal May 2000
Genetic Studies of the Lac Repressor XV: 4000 Single Amino Acid Substitutions and Analysis of the Resulting Phenotypes on the Basis of the Protein Structure journal August 1996
Charting a course through RNA polymerase journal September 2000
Clustal W and Clustal X version 2.0 journal September 2007
High-Throughput Metabolic Engineering: Advances in Small-Molecule Screening and Selection journal June 2010
Transcription Factor-Based Screens and Synthetic Selections for Microbial Small-Molecule Biosynthesis journal September 2012
Recombineering with tolC as a Selectable/Counter-selectable Marker: remodeling the rRNA Operons of Escherichia coli journal December 2007
Conformer Generation with OMEGA: Algorithm and Validation Using High Quality Structures from the Protein Databank and Cambridge Structural Database journal March 2010
Evolution-guided optimization of biosynthetic pathways journal December 2014
Evolutionarily conserved networks of residues mediate allosteric communication in proteins journal December 2002
Design and Application of a Mevalonate-Responsive Regulatory Protein journal December 2010
Kemp elimination catalysts by computational enzyme design journal March 2008
Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones: Directed evolution of LuxR journal December 2004
Computational design of ligand-binding proteins with high affinity and selectivity journal September 2013
Molecular Biology of the LysR Family of Transcriptional Regulators journal October 1993
Perturbation from a Distance:  Mutations that Alter LacI Function through Long-Range Effects journal December 2003
Molecular basis for control of antibiotic production by a bacterial hormone journal February 2021
Arac/XylS family of transcriptional regulators. journal January 1997
Directed evolution of protein switches and their application to the creation of ligand-binding proteins journal August 2005
Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis journal May 2006
Using Orthologous and Paralogous Proteins to Identify Specificity-determining Residues in Bacterial Transcription Factors journal August 2002
Prediction of functional specificity determinants from protein sequences using log-likelihood ratios journal November 2005
XDS journal January 2010
Genetic Studies of the lac Repressor. XIV. Analysis of 4000 Altered Escherichia coli lac Repressors Reveals Essential and Non-essential Residues, as well as "Spacers" which do not Require a Specific Sequence journal July 1994
A Directed Approach for Engineering Conditional Protein Stability Using Biologically Silent Small Molecules journal July 2007
Generation of Novel Bacterial Regulatory Proteins That Detect Priority Pollutant Phenols journal January 2000
Conformer Generation with OMEGA: Learning from the Data Set and the Analysis of Failures journal November 2012
Synthetic biosensors for precise gene control and real-time monitoring of metabolites journal July 2015
Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers journal February 1997
Plasticity of quaternary structure: Twenty-two ways to form a LacI dimer journal February 2001
A three-hybrid system for detecting small ligand-protein receptor interactions journal November 1996
Design and Application of a Mevalonate-Responsive Regulatory Protein journal December 2010
Screening and Identification of a Fungal β-Glucosidase and the Enzymatic Synthesis of Gentiooligosaccharide journal October 2010
Genome Scale Reconstruction of a Salmonella Metabolic Model: COMPARISON OF SIMILARITY AND DIFFERENCES WITH A COMMENSAL Escherichia coli STRAIN journal August 2009
Emergence of novel functions in transcriptional regulators by regression to stem protein types journal August 2007
Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression journal September 2012
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs journal September 1997
Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor: A Comparative Approach for Protein Design journal May 2015

Cited By (63)

Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol journal November 2019
Assessing glycolytic flux alterations resulting from genetic perturbations in E. coli using a biosensor journal July 2017
SensiPath: computer-aided design of sensing-enabling metabolic pathways journal April 2016
Biosensor-based enzyme engineering approach applied to psicose biosynthesis journal January 2019
Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion journal January 2019
Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol preprint February 2019
A progesterone biosensor derived from microbial screening journal March 2020
A load driver device for engineering modularity in biological networks journal November 2014
Genetically encoded biosensors for lignocellulose valorization journal October 2019
The evolving interface between synthetic biology and functional metagenomics journal July 2018
DNA-based memory devices for recording cellular events journal September 2018
A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor journal August 2016
Numerical operations in living cells by programmable RNA devices journal August 2019
De novo design of programmable inducible promoters journal September 2019
Commentary: Synthetic Addiction Extends the Productive Life Time of Engineered Escherichia coli Populations journal June 2018
Computational design of a modular protein sense-response system journal November 2019
Overcoming genetic heterogeneity in industrial fermentations journal July 2019
Evolution-guided engineering of small-molecule biosensors journal November 2019
Scaling up genetic circuit design for cellular computing: advances and prospects journal October 2018
MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect journal November 2019
Lighting up yeast cell factories by transcription factor-based biosensors journal September 2017
Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors journal November 2018
Phosphatase activity tunes two-component system sensor detection threshold journal April 2018
Directing evolution: the next revolution in drug discovery? journal September 2017
Crosstalk between transcription and metabolism: how much enzyme is enough for a cell?: Crosstalk between transcription and metabolism journal August 2017
Lighting up yeast cell factories by transcription factor-based biosensors journal September 2017
Next-generation biocontainment systems for engineered organisms journal May 2018
Dynamic metabolic control: towards precision engineering of metabolism journal January 2018
Tailor-made transcriptional biosensors for optimizing microbial cell factories journal November 2016
Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol journal November 2019
Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex journal April 2017
Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes journal December 2019
Scaling up genetic circuit design for cellular computing: advances and prospects journal October 2018
De novo design of programmable inducible promoters journal September 2019
Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression journal August 2019
Numerical operations in living cells by programmable RNA devices journal August 2019
Programmable T7-based synthetic transcription factors journal August 2018
Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography journal January 2019
A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor journal August 2016
Tools and systems for evolutionary engineering of biomolecules and microorganisms journal May 2019
Phosphatase activity tunes two-component system sensor detection threshold journal April 2018
Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol journal May 2017
A self-inducible heterologous protein expression system in Escherichia coli journal September 2016
Genetically encoded biosensors for lignocellulose valorization journal October 2019
Archimedes’ principle for characterisation of recombinant whole cell biocatalysts journal February 2018
Archimedes’ principle for characterisation of recombinant whole cell biocatalysts text January 2018
A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs journal March 2019
Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools journal November 2017
Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast journal January 2017
Archimedes’ principle for characterisation of recombinant whole cell biocatalysts text January 2018
The Role of Protein Engineering in Biomedical Applications of Mammalian Synthetic Biology text January 2020
Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein journal May 2018
Unified rational protein engineering with sequence-based deep representation learning journal October 2019
Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function journal December 2018
MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect journal November 2019
Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes journal November 2019
Programmable T7-based synthetic transcription factors journal August 2018
Synthetic addiction extends the productive life time of engineered Escherichia coli populations journal February 2018
Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion journal January 2019
The Role of Protein Engineering in Biomedical Applications of Mammalian Synthetic Biology journal October 2019
Synthetic Biology-The Synthesis of Biology journal April 2017
Structural and functional analyses of the cellulase transcription regulator CelR journal August 2018
Binary addition in a living cell based on riboregulation journal July 2018