Engineering Transcription Factor BmoR Mutants for Constructing Multifunctional Alcohol Biosensors

Native transcription factor-based biosensors (TFBs) have the potential for the in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogues. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Furthermore, we obtained two mutants with raised outputs and over 10⁷-fold higher sensitivity and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol). Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB toward its small-molecule ligands, paving the way for in situ detection in biofuel and wine industries.