Engineering transcription factor BmoR for screening butanol overproducers.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Wild-type transcription factors have limited detection ranges, hindering their use as biosensors for industrial screening.
• Developing biosensors with expanded detection capabilities is crucial for optimizing microbial production processes.

Purpose of the Study:

• To engineer the transcription factor BmoR to expand its detection range for screening alcohol overproducers.
• To enhance the utility of transcription factors for industrial applications.

Main Methods:

• Created a BmoR mutation library with random mutations across functional domains.
• Modeled the BmoR-isobutanol complex to identify binding sites, confirmed via site-directed mutagenesis.
• Constructed combinatorial mutants combining mutations for increased detection range and enhanced output.

Main Results:

• Identified specific mutations that expand the detection range and improve output of BmoR.
• Engineered mutants F276A/E627N BmoR and D333N/E627N BmoR exhibit wider detection ranges (0-100 mM).
• These mutants show potential for screening isobutanol overproduction strains due to their performance with quantitative and intracellular isobutanol.

Conclusions:

• Demonstrated successful engineering of a wild-type transcription factor for expanded industrial utility.
• Developed BmoR variants with significantly improved detection ranges for isobutanol screening.
• This approach provides a framework for modifying transcription factors for biosensing applications.