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Evolution-guided engineering of small-molecule biosensors.

Tim Snoek1, Evan K Chaberski1, Francesca Ambri1

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Summary
This summary is machine-generated.

Researchers developed a high-throughput method to engineer allosteric transcription factors (aTFs) in yeast. This platform enables custom biosensor design for biotechnology by evolving ligand specificity and function.

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Area of Science:

  • Synthetic Biology
  • Biotechnology
  • Molecular Biology

Background:

  • Allosteric transcription factors (aTFs) are valuable biosensors for monitoring and regulating cellular processes.
  • Optimizing aTF specificity and transfer functions is crucial for practical applications in biotechnology.
  • Current methods for aTF engineering often require extensive optimization and are chassis-dependent.

Purpose of the Study:

  • To develop a versatile, high-throughput platform for evolving prokaryotic aTF specificity and transfer functions in a eukaryotic chassis (Saccharomyces cerevisiae).
  • To engineer aTFs with altered ligand specificities, improved dynamic ranges, and modified operational ranges.
  • To demonstrate the creation of a host-agnostic biosensor platform for user-defined small-molecule detection.

Main Methods:

  • Mutagenesis of the effector-binding domain (EBD) of the cis,cis-muconic acid-inducible transcription factor BenM.
  • Application of toggled selection regimes in baker's yeast (Saccharomyces cerevisiae) to select for desired aTF variants.
  • Characterization of evolved biosensors for DNA-binding affinity and functionality in both eukaryotic and prokaryotic systems.

Main Results:

  • Successfully evolved BenM variants with altered ligand specificities and improved dynamic output ranges.
  • Achieved shifts in the operational range and a complete inversion of function from activation to repression.
  • Evolved biosensors retained similar DNA-binding affinities to the parent aTF and were functional when transferred to a prokaryotic chassis.

Conclusions:

  • The developed platform enables robust engineering of aTF specificity and transfer functions in a eukaryote chassis.
  • This method facilitates the creation of novel, host-agnostic biosensors with user-defined small-molecule specificities.
  • The platform significantly advances the application of aTFs in biotechnology and synthetic biology by providing tailored biosensing tools.