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Title: Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

Abstract

Abstract We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Authors:
 [1];  [2];  [3];  [1];  [3];  [4]
  1. Department of Chemical and Biomolecular Engineering, University of Houston, 4726 Calhoun Rd, Houston, TX 77204-4004 Houston, TX, USA
  2. Department of Biology and Biochemistry, University of Houston, 3507 Cullen Blvd, Houston, TX 77204-5008 Houston, TX, USA
  3. Department of Chemical and Biomedical Engineering, Penn State University, University Park, PA 16802-4400 PA, USA
  4. Department of Chemical and Biomolecular Engineering, University of Houston, 4726 Calhoun Rd, Houston, TX 77204-4004 Houston, TX, USA, Department of Biology and Biochemistry, University of Houston, 3507 Cullen Blvd, Houston, TX 77204-5008 Houston, TX, USA
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1731008
Grant/Contract Number:  
AC05-000R22725
Resource Type:
Published Article
Journal Name:
Protein Engineering, Design and Selection
Additional Journal Information:
Journal Name: Protein Engineering, Design and Selection Journal Volume: 33; Journal ID: ISSN 1741-0126
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Wang, Zhiqing, Doshi, Aarti, Chowdhury, Ratul, Wang, Yixi, Maranas, Costas D., and Cirino, Patrick C.. Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid. United Kingdom: N. p., 2020. Web. https://doi.org/10.1093/protein/gzaa027.
Wang, Zhiqing, Doshi, Aarti, Chowdhury, Ratul, Wang, Yixi, Maranas, Costas D., & Cirino, Patrick C.. Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid. United Kingdom. https://doi.org/10.1093/protein/gzaa027
Wang, Zhiqing, Doshi, Aarti, Chowdhury, Ratul, Wang, Yixi, Maranas, Costas D., and Cirino, Patrick C.. Fri . "Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid". United Kingdom. https://doi.org/10.1093/protein/gzaa027.
@article{osti_1731008,
title = {Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid},
author = {Wang, Zhiqing and Doshi, Aarti and Chowdhury, Ratul and Wang, Yixi and Maranas, Costas D. and Cirino, Patrick C.},
abstractNote = {Abstract We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.},
doi = {10.1093/protein/gzaa027},
journal = {Protein Engineering, Design and Selection},
number = ,
volume = 33,
place = {United Kingdom},
year = {2020},
month = {11}
}

Journal Article:
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https://doi.org/10.1093/protein/gzaa027

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