Engineering an <scp>AlkS</scp> ‐ <scp>PalkB</scp> Transcription Factor‐Based Biosensor With Improved Sensitivity to Isobutanol and Other Short‐Chain Alcohols

Isobutanol, a promising biofuel with higher energy content than ethanol, presents a sustainable alternative through biosynthesis. However, enhancing yield remains challenging due to the inefficiencies in microbial synthesis. This study introduces a transcription factor-based biosensor using the AlkS-PalkB system in Escherichia coli, which correlates green fluorescence with isobutanol concentration. Employing directed evolution, we modified AlkS to detect isobutanol, significantly improving biosensor specificity. Initial modifications increased the dynamic response from non-detectable to a 2.60-fold change. Subsequent optimisations through site-directed mutagenesis and promoter engineering further enhanced this response to a 5.56-fold change, equivalent to a 114% increase. Although engineered for isobutanol detection with high sensitivity, the engineered biosensor retains responsiveness to several short-chain alcohols. This biosensor provides a foundation for high-throughput screening of isobutanol and other short-chain alcohol-producing strains, though additional improvements in selectivity and operating range may be required for efficient implementation.