Development of a specific biosensor for sesquiterpene based on SELEX and directed evolution platforms

Area of Science:

• Synthetic biology
• Biosensor development
• Molecular biology

Background:

• Biosensors are crucial for detecting specific molecules in synthetic biology.
• Existing methods may lack specificity or efficiency for certain compounds.
• Amorpha-4,11-diene is a sesquiterpene relevant to metabolic engineering.

Purpose of the Study:

• To develop a novel riboswitch biosensor for the detection of amorpha-4,11-diene.
• To characterize the performance of the biosensor in response to varying concentrations of the target compound.
• To elucidate the molecular mechanism underlying the biosensor's function.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment with Single Molecule Beads (SELEX-SMB) for aptamer library generation.
• TBG-directed evolution platform for screening and identifying functional riboswitches.
• Computational methods including structural prediction, molecular docking, and molecular dynamics simulations.

Main Results:

• A high-affinity aptamer library of 81,520 sequences was generated.
• Riboswitch5 was identified, exhibiting downregulation of gene expression in response to amorpha-4,11-diene (10-100 mg/L).
• A log₂Foldchange of -0.51 was achieved at 100 mg/L, indicating significant repression.
• Structural analysis revealed ligand-induced conformational changes stabilizing the riboswitch.

Conclusions:

• The developed riboswitch biosensor is effective for detecting amorpha-4,11-diene.
• This biosensor offers a valuable tool for small molecule detection and regulation in synthetic biology.
• Potential applications include strain engineering, pathway regulation, and metabolic optimization.