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

Autor:	Wang Z; Department of Chemical and Biomolecular Engineering, University of Houston, 4726 Calhoun Rd, Houston, TX 77204-4004 Houston, TX, USA., Doshi A; Department of Biology and Biochemistry, University of Houston, 3507 Cullen Blvd, Houston, TX 77204-5008 Houston, TX, USA., Chowdhury R; Department of Chemical and Biomedical Engineering, Penn State University, University Park, PA 16802-4400 PA, USA., Wang Y; Department of Chemical and Biomolecular Engineering, University of Houston, 4726 Calhoun Rd, Houston, TX 77204-4004 Houston, TX, USA., Maranas CD; Department of Chemical and Biomedical Engineering, Penn State University, University Park, PA 16802-4400 PA, USA., Cirino PC; 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.
Jazyk:	angličtina
Zdroj:	Protein engineering, design & selection : PEDS [Protein Eng Des Sel] 2020 Sep 14; Vol. 33.
DOI:	10.1093/protein/gzaa027
Abstrakt:	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. (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
Databáze:	MEDLINE
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