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Title: Evolution-guided engineering of small-molecule biosensors

Abstract

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [2]; ORCiD logo [1]; ORCiD logo [1];  [3]
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  1. Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability
  2. Joint BioEnergy Institute, Emeryville, CA, USA
  3. Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Shenzhen Inst. of Advanced Technologies, Shenzhen (China). Inst. for Synthetic Biology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1605268
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 48; Journal Issue: 1; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Synthetic Biology and Assembly Cloning

Citation Formats

Snoek, Tim, Chaberski, Evan K., Ambri, Francesca, Kol, Stefan, Bjørn, Sara P., Pang, Bo, Barajas, Jesus F., Welner, Ditte H., Jensen, Michael K., and Keasling, Jay D. Evolution-guided engineering of small-molecule biosensors. United States: N. p., 2019. Web. doi:10.1093/nar/gkz954.
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Snoek, Tim, Chaberski, Evan K., Ambri, Francesca, Kol, Stefan, Bjørn, Sara P., Pang, Bo, Barajas, Jesus F., Welner, Ditte H., Jensen, Michael K., & Keasling, Jay D. Evolution-guided engineering of small-molecule biosensors. United States. https://doi.org/10.1093/nar/gkz954
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Snoek, Tim, Chaberski, Evan K., Ambri, Francesca, Kol, Stefan, Bjørn, Sara P., Pang, Bo, Barajas, Jesus F., Welner, Ditte H., Jensen, Michael K., and Keasling, Jay D. Thu . "Evolution-guided engineering of small-molecule biosensors". United States. https://doi.org/10.1093/nar/gkz954. https://www.osti.gov/servlets/purl/1605268.
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@article{osti_1605268,
title = {Evolution-guided engineering of small-molecule biosensors},
author = {Snoek, Tim and Chaberski, Evan K. and Ambri, Francesca and Kol, Stefan and Bjørn, Sara P. and Pang, Bo and Barajas, Jesus F. and Welner, Ditte H. and Jensen, Michael K. and Keasling, Jay D.},
abstractNote = {Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.},
doi = {10.1093/nar/gkz954},
journal = {Nucleic Acids Research},
number = 1,
volume = 48,
place = {United States},
year = {2019},
month = {11}
}
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https://doi.org/10.1093/nar/gkz954
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Works referenced in this record:

Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study.
journal, February 1988

Identification and nucleotide sequence of the class E tet regulatory elements and operator and inducer binding of the encoded purified Tet repressor
journal, December 1988

  • Tovar, K.; Ernst, A.; Hillen, W.
  • Molecular and General Genetics MGG, Vol. 215, Issue 1
  • DOI: 10.1007/BF00331306

Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors
journal, November 2018

  • Meyer, Adam J.; Segall-Shapiro, Thomas H.; Glassey, Emerson
  • Nature Chemical Biology, Vol. 15, Issue 2
  • DOI: 10.1038/s41589-018-0168-3

Structural characterization and corepressor binding of the Escherichia coli purine repressor.
journal, January 1992

Genomic mining of prokaryotic repressors for orthogonal logic gates
journal, December 2013

  • Stanton, Brynne C.; Nielsen, Alec A. K.; Tamsir, Alvin
  • Nature Chemical Biology, Vol. 10, Issue 2
  • DOI: 10.1038/nchembio.1411

Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae: Ordered plasmid assembly by gap repair
journal, July 2012

  • Eckert-Boulet, Nadine; Pedersen, Mette Louise; Krogh, Berit Olsen
  • Yeast, Vol. 29, Issue 8
  • DOI: 10.1002/yea.2912

Synergistic transcriptional activation by one regulatory protein in response to two metabolites
journal, May 2002

  • Bundy, B. M.; Collier, L. S.; Hoover, T. R.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 11
  • DOI: 10.1073/pnas.102605799

Tradeoffs and Optimality in the Evolution of Gene Regulation
journal, August 2011

Programmable bacteria detect and record an environmental signal in the mammalian gut
journal, March 2014

  • Kotula, Jonathan W.; Kerns, S. Jordan; Shaket, Lev A.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 13
  • DOI: 10.1073/pnas.1321321111

Engineering alternate cooperative-communications in the lactose repressor protein scaffold
journal, April 2013

  • Meyer, S.; Ramot, R.; Kishore Inampudi, K.
  • Protein Engineering Design and Selection, Vol. 26, Issue 6
  • DOI: 10.1093/protein/gzt013

Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions
journal, September 2016

Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor
journal, March 2017

Directed evolution of a synthetic phylogeny of programmable Trp repressors
journal, February 2018

  • Ellefson, Jared W.; Ledbetter, Michael P.; Ellington, Andrew D.
  • Nature Chemical Biology, Vol. 14, Issue 4
  • DOI: 10.1038/s41589-018-0006-7

A Biosensor Strategy for E. coli Based on Ligand-Dependent Stabilization
journal, July 2018

  • Brandsen, Benjamin M.; Mattheisen, Jordan M.; Noel, Teia
  • ACS Synthetic Biology, Vol. 7, Issue 9
  • DOI: 10.1021/acssynbio.8b00052

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
journal, June 1992

  • Gossen, M.; Bujard, H.
  • Proceedings of the National Academy of Sciences, Vol. 89, Issue 12
  • DOI: 10.1073/pnas.89.12.5547

Electrochemical Conversion of Muconic Acid to Biobased Diacid Monomers
journal, May 2016

  • Matthiesen, John E.; Carraher, Jack M.; Vasiliu, Monica
  • ACS Sustainable Chemistry & Engineering, Vol. 4, Issue 6
  • DOI: 10.1021/acssuschemeng.6b00679

Full-Length Structures of BenM and Two Variants Reveal Different Oligomerization Schemes for LysR-Type Transcriptional Regulators
journal, December 2010

  • Ruangprasert, Ajchareeya; Craven, Sarah H.; Neidle, Ellen L.
  • Journal of Molecular Biology, Vol. 404, Issue 4
  • DOI: 10.1016/j.jmb.2010.09.053

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
  • DOI: 10.1073/pnas.1409523111

Dynamically Reshaping Signaling Networks to Program Cell Fate via Genetic Controllers
journal, August 2013

SensiPath: computer-aided design of sensing-enabling metabolic pathways
journal, April 2016

  • Delépine, Baudoin; Libis, Vincent; Carbonell, Pablo
  • Nucleic Acids Research, Vol. 44, Issue W1
  • DOI: 10.1093/nar/gkw305

Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras
journal, July 2014

  • Shis, David L.; Hussain, Faiza; Meinhardt, Sarah
  • ACS Synthetic Biology, Vol. 3, Issue 9
  • DOI: 10.1021/sb500262f

Regulation of Benzoate Degradation inAcinetobacter sp. Strain ADP1 by BenM, a LysR-Type Transcriptional Activator
journal, January 1998

Inducer responses of BenM, a LysR-type transcriptional regulator from Acinetobacter baylyi ADP1
journal, May 2009

Inference of Macromolecular Assemblies from Crystalline State
journal, September 2007

Chimeric LysR-Type Transcriptional Biosensors for Customizing Ligand Specificity Profiles toward Flavonoids
journal, December 2018

The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors
journal, April 1997

  • Marvin, J. S.; Corcoran, E. E.; Hattangadi, N. A.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 9
  • DOI: 10.1073/pnas.94.9.4366

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
  • DOI: 10.1038/nmeth.3696

Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method
journal, January 2007

Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels
journal, June 2016

  • Roney, Ian J.; Rudner, Adam D.; Couture, Jean-François
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep27697

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum
journal, November 2015

Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins
journal, December 2008

Perturbation from a Distance:  Mutations that Alter LacI Function through Long-Range Effects †
journal, December 2003

  • Swint-Kruse, Liskin; Zhan, Hongli; Fairbanks, Bonnie M.
  • Biochemistry, Vol. 42, Issue 47
  • DOI: 10.1021/bi035116x

A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules
journal, September 2006

InterPro in 2017—beyond protein family and domain annotations
journal, November 2016

  • Finn, Robert D.; Attwood, Teresa K.; Babbitt, Patricia C.
  • Nucleic Acids Research, Vol. 45, Issue D1
  • DOI: 10.1093/nar/gkw1107

Biotechnological production of muconic acid: current status and future prospects
journal, May 2014

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

  • Suckow, Jörg; Markiewicz, Peter; Kleina, Lynn G.
  • Journal of Molecular Biology, Vol. 261, Issue 4
  • DOI: 10.1006/jmbi.1996.0479

EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae
journal, March 2014

  • Jensen, Niels B.; Strucko, Tomas; Kildegaard, Kanchana R.
  • FEMS Yeast Research, Vol. 14, Issue 2
  • DOI: 10.1111/1567-1364.12118

Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression
journal, September 2012

  • Meinhardt, Sarah; Manley, Michael W.; Becker, Nicole A.
  • Nucleic Acids Research, Vol. 40, Issue 21
  • DOI: 10.1093/nar/gks806

Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications
journal, December 2017

Transiting from Adipic Acid to Bioadipic Acid. Part II. Biosynthetic Pathways
journal, January 2015

  • Bart, Jan C. J.; Cavallaro, Stefano
  • Industrial & Engineering Chemistry Research, Vol. 54, Issue 2
  • DOI: 10.1021/ie502074d

Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR
journal, May 2006

  • Collins, Cynthia H.; Leadbetter, Jared R.; Arnold, Frances H.
  • Nature Biotechnology, Vol. 24, Issue 6
  • DOI: 10.1038/nbt1209

Automated design of specificity in molecular recognition
journal, December 2002

  • Havranek, James J.; Harbury, Pehr B.
  • Nature Structural Biology, Vol. 10, Issue 1
  • DOI: 10.1038/nsb877

Emergent genetic oscillations in a synthetic microbial consortium
journal, August 2015

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
  • DOI: 10.1021/ja7109053

Isolation of the lac Repressor
journal, December 1966

  • Gilbert, W.; Muller-Hill, B.
  • Proceedings of the National Academy of Sciences, Vol. 56, Issue 6
  • DOI: 10.1073/pnas.56.6.1891

Combinatorial Synthesis of Genetic Networks
journal, May 2002

Dissecting protein structure and function using directed evolution
journal, December 2007

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast
journal, September 2016

  • Skjoedt, Mette L.; Snoek, Tim; Kildegaard, Kanchana R.
  • Nature Chemical Biology, Vol. 12, Issue 11
  • DOI: 10.1038/nchembio.2177

Modular organisation of inducer recognition and allostery in the tetracycline repressor
journal, April 2016

  • Werten, Sebastiaan; Schneider, Julia; Palm, Gottfried Julius
  • The FEBS Journal, Vol. 283, Issue 11
  • DOI: 10.1111/febs.13723

Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes
journal, April 1996

The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor
journal, December 2009

  • Reichheld, Sean E.; Yu, Zhou; Davidson, Alan R.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 52
  • DOI: 10.1073/pnas.0911566106

Distinct Effector-binding Sites Enable Synergistic Transcriptional Activation by BenM, a LysR-type Regulator
journal, March 2007

  • Ezezika, Obidimma C.; Haddad, Sandra; Clark, Todd J.
  • Journal of Molecular Biology, Vol. 367, Issue 3
  • DOI: 10.1016/j.jmb.2006.09.090

A general strategy to construct small molecule biosensors in eukaryotes
journal, December 2015

  • Feng, Justin; Jester, Benjamin W.; Tinberg, Christine E.
  • eLife, Vol. 4
  • DOI: 10.7554/eLife.10606

Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family
journal, April 2007

  • Ezezika, Obidimma C.; Haddad, Sandra; Neidle, Ellen L.
  • Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 63, Issue 5
  • DOI: 10.1107/S1744309107019185

Activity reversal of Tet repressor caused by single amino acid exchanges: Reverse TetR
journal, June 2004

Promoter Nucleosome Organization Shapes the Evolution of Gene Expression
journal, March 2012

Adipic acid tolerance screening for potential adipic acid production hosts
journal, February 2017

  • Karlsson, Emma; Mapelli, Valeria; Olsson, Lisbeth
  • Microbial Cell Factories, Vol. 16, Issue 1
  • DOI: 10.1186/s12934-017-0636-6

Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators
journal, August 2018

  • Juárez, Javier F.; Lecube-Azpeitia, Begoña; Brown, Stuart L.
  • Nature Communications, Vol. 9, Issue 1
  • DOI: 10.1038/s41467-018-05525-6

Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems
journal, July 2015

Sensing new chemicals with bacterial transcription factors
journal, October 2016

  • Libis, Vincent; Delépine, Baudoin; Faulon, Jean-Loup
  • Current Opinion in Microbiology, Vol. 33
  • DOI: 10.1016/j.mib.2016.07.006

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae : Engineering Gene Circuits in
journal, July 2015

  • Wang, Meng; Li, Sijin; Zhao, Huimin
  • Biotechnology and Bioengineering, Vol. 113, Issue 1
  • DOI: 10.1002/bit.25676

T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension
journal, May 2011

  • Di Tommaso, P.; Moretti, S.; Xenarios, I.
  • Nucleic Acids Research, Vol. 39, Issue suppl
  • DOI: 10.1093/nar/gkr245
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