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

Biosensors are essential in synthetic biology, particularly for detecting compounds without distinct visual markers. In this study, a riboswitch biosensor specifically responsive to a sesquiterpene — amorpha-4,11-diene was developed for the first time. Through SELEX-SMB, a high-affinity aptamer library comprising 81,520 sequences was generated. Subsequent screening via the TBG-directed evolution platform identified riboswitch5, which downregulates downstream gene expression in response to amorpha-4,11-diene within a concentration range of 10–100 mg/L, achieving a log₂Foldchange of −0.51 at 100 mg/L. Structural predictions, combined with molecular docking and molecular dynamics simulations, revealed that ligand binding induced conformational changes that stabilize the riboswitch and enhance its repressive effect. This biosensor represents a powerful tool for the detection and regulation of small molecules, with broad applications in strain engineering, pathway regulation, and metabolic optimization in synthetic biology. Specific Biosensor for Sesquiterpene Based on SELEX and Directed Evolution Platforms. [Display omitted] •SMB-SELEX procedure was established to preserve molecular integrity.•A riboswitch responsive to sesquiterpene was developed for the first time.•TBG platform was firstly introduced to promote riboswitch screening in vivo.•A Riboswitch5 downregulated gene expression in response to sesquiterpene.