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

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Abstract
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.
Funding Information
  • Royal Society (NAF\R2\180721)
  • UK Research and Innovation (EP/X032078/1)

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