Engineering of a TrpR-Based Biosensor for Altered Dynamic Range and Ligand Preference.

Transcriptional factors play a crucial role in regulating cellular functions. Understanding and altering the dynamic behavior of the transcriptional factor-based biosensors will expand our knowledge in investigating biomolecular interactions and facilitating biosynthetic applications. In this study, we characterized and engineered a TrpR-based tryptophan repressor system in Escherichia coli. We found that the reconstructed TrpR1-PtrpO1 biosensor system exhibited low basal expression and narrow dynamic range in the presence of tryptophan or its analogue 5-hydroxytryptophan (5-HTP). Given the application potential of the biosensor, we introduced engineering approaches in multiple levels to optimize its dynamic behavior. First, the I57 and V58 residues in the ligand-binding pocket were rationally mutated in search of variants with altered ligand specificity. Two TrpR1 variants, V58E and V58K, successfully acquired ligand preference toward tryptophan and 5-HTP, respectively. The biosensor-induced expression levels were increased up to 10-fold with those variants. Furthermore, to pursue broader operational range, we tuned the regulator-operator binding affinity by mutating the binding box of TrpR1. Collectively, we demonstrated that the biosynthesis-significant biosensor TrpR1-PtrpO1 can be engineered to acquire extended dynamic ranges and improved ligand preference. The engineered biosensor variants with remarkable dynamic behavior can serve as key genetic elements in high-throughput screening and dynamic regulation in biosynthetic scenarios.