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

Zhiqing Wang, Aarti Doshi, Ratul Chowdhury, Yixi Wang, Costas D. Maranas, Patrick C. Cirino

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
JournalProtein Engineering, Design and Selection
Volume33
DOIs
StatePublished - Sep 14 2020

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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