Identifying LasR Quorum Sensors with Improved Signal Specificity by Mapping the Sequence-Function Landscape.

Area of Science:

• Synthetic Biology
• Microbial Engineering
• Molecular Biology

Background:

• Programmable intercellular signaling is key for engineering coordinated functions in microbial consortia.
• LuxR-type regulators, activated by homoserine lactone (HSL) signals, are commonly used but often lack specificity.
• Imperfect discrimination of HSLs leads to misregulation in engineered consortia with multiple signals.

Purpose of the Study:

• To elucidate the sequence-function relationship for LasR regulator ligand specificity.
• To engineer LasR variants with improved molecular discrimination of HSL signals.
• To reduce signal crosstalk for enhanced control in engineered microbial consortia.

Main Methods:

• Targeted protein engineering of LasR regulator from *Pseudomonas aeruginosa*.
• Creation of a pooled combinatorial saturation mutagenesis library targeting six residues in LasR's β5 sheet.
• Multiplexed high-throughput biosensor screening using sort-seq assays with cognate and noncognate HSLs.

Main Results:

• Identified hundreds of LasR variants with significantly improved specificity for HSL signals.
• Engineered sensors exhibited up to 60.6-fold improved relative activation by cognate signals compared to wildtype.
• Uncovered prevalent mutational epistasis and identified novel residues critical for signal specificity.

Conclusions:

• Developed highly specific LasR variants with negligible signal crosstalk.
• These engineered sensors offer broad applicability for precise control in synthetic bacteria consortia.
• Enhanced specificity advances the engineering of complex microbial communities for various applications.