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 10(7)-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.

Automated summary

This study engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. These mutants showed high sensitivity and specificity in detecting different alcohols, paving the way for in situ detection in biofuel and wine industries.