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Engineering Transcription Factor BmoR Mutants for Constructing Multifunctional Alcohol Biosensors.

Tong Wu1, Zhenya Chen1, Shuyuan Guo1

  • 1Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, 100081 Beijing, China.

ACS Synthetic Biology
|February 17, 2022
PubMed
Summary

Engineered alcohol biosensors (TFBs) can now distinguish specific alcohol isomers and detect them at low concentrations, even with high ethanol background noise, enabling new industrial applications.

Keywords:
BmoRbiosensorhigh sensitivityspecificitytranscription factorwider detection

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Chemical Engineering

Background:

  • Transcription factor-based biosensors (TFBs) offer in situ detection but suffer from ligand promiscuity, low sensitivity, and narrow detection ranges.
  • Existing TFBs cannot differentiate structurally similar alcohols, limiting their industrial use.
  • Alcohols' similar structures pose a challenge for specific detection by TFBs.

Purpose of the Study:

  • To engineer an alcohol-regulated transcription factor (BmoR) with enhanced specificity, sensitivity, and detection range.
  • To overcome the limitations of native TFBs for industrial applications.
  • To explore the ultimate detection limits of TFBs for small-molecule ligands.

Main Methods:

  • Engineering of an alcohol-regulated transcription factor, BmoR, and its mutants.
  • Testing BmoR mutants for specificity in distinguishing alcohol isomers (e.g., n-butanol and isobutanol).
  • Evaluating BmoR mutant performance in the presence of high ethanol concentrations and assessing sensitivity and detection limits.

Main Results:

  • Generated BmoR mutants capable of distinguishing between constitutional isomers like n-butanol and isobutanol.
  • Achieved insensitivity to high ethanol concentrations (up to 800 mM) while detecting specific higher alcohols.
  • Developed mutants with significantly increased sensitivity (over 10^7-fold) and an expanded upper detection limit for specific alcohols.

Conclusions:

  • Engineered BmoR variants overcome key limitations of native TFBs, enabling specific alcohol detection.
  • The developed TFBs demonstrate high sensitivity and specificity, even in complex mixtures with high ethanol background.
  • This work paves the way for advanced in situ monitoring in industries such as biofuels and wine production.