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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]
  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. https://doi.org/10.1093/nar/gkz954.
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
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.
@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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