Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid.
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| Title: | Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid. |
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| Authors: | Wang, Zhiqing1 (AUTHOR), Doshi, Aarti2 (AUTHOR), Chowdhury, Ratul3 (AUTHOR), Wang, Yixi1 (AUTHOR), Maranas, Costas D3 (AUTHOR), Cirino, Patrick C1,2 (AUTHOR) pccirino@uh.edu |
| Source: | PEDS: Protein Engineering, Design & Selection. 2020, Vol. 33, p1-9. 9p. |
| Subjects: | Biosensors, Polyketide synthases, Engineering, Biosynthesis, Protein engineering, Acids |
| 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. [ABSTRACT FROM AUTHOR] |
| Copyright of PEDS: Protein Engineering, Design & Selection is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
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| 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. [ABSTRACT FROM AUTHOR] |
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| ISSN: | 17410126 |
| DOI: | 10.1093/protein/gzaa027 |
