Divergent directed evolution of a TetR-type repressor towards aromatic molecules

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Allosteric transcription factors (aTFs) are crucial for reprogramming cellular behavior by linking small molecule signals to cellular responses.
• Expanding the range of inducers recognized by aTFs is vital for diverse synthetic biology applications.

Purpose of the Study:

• To engineer novel allosteric transcription factors (aTFs) with expanded ligand specificities.
• To establish a framework for rapid engineering of aTFs for new inducer molecules.

Main Methods:

• Established a resorcinol-responsive aTF biosensor in Escherichia coli using the TetR-family repressor RolR.
• Performed iterative engineering (fitness landscape walk) on RolR to alter its inducer specificity.
• Transplanted engineered aTFs into Saccharomyces cerevisiae to demonstrate cross-kingdom applicability.

Main Results:

• Engineered RolR variants recognized new small molecules including catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and homovanillic acid.
• Successfully demonstrated the function of engineered aTFs in both prokaryotic (E. coli) and eukaryotic (S. cerevisiae) systems.
• Developed a scalable framework for engineering aTF specificity within laboratory timescales.

Conclusions:

• This study presents a robust method for expanding aTF ligand specificities towards novel molecules.
• The engineered aTFs and the presented framework have broad implications for protein engineering, metabolic engineering, and point-of-care diagnostics.