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

Programmable
intercellular signaling using components of naturally
occurring quorum sensing can allow for coordinated functions to be
engineered in microbial consortia. LuxR-type transcriptional regulators
are widely used for this purpose and are activated by homoserine lactone
(HSL) signals. However, they often suffer from imperfect molecular
discrimination of structurally similar HSLs, causing misregulation
within engineered consortia containing multiple HSL signals. Here,
we studied one such example, the regulator LasR from Pseudomonas aeruginosa. We elucidated its sequence–function
relationship for ligand specificity using targeted protein engineering
and multiplexed high-throughput biosensor screening. A pooled combinatorial
saturation mutagenesis library (9,486 LasR DNA sequences) was created
by mutating six residues in LasR’s β5 sheet with single,
double, or triple amino acid substitutions. Sort-seq assays were performed
in parallel using cognate and noncognate HSLs to quantify each corresponding
sensor’s response to each HSL signal, which identified hundreds
of highly specific variants. Sensor variants identified were individually
assayed and exhibited up to 60.6-fold (p = 0.0013)
improved relative activation by the cognate signal compared to the
wildtype. Interestingly, we uncovered prevalent mutational epistasis
and previously unidentified residues contributing to signal specificity.
The resulting sensors with negligible signal crosstalk could be broadly
applied to engineer bacteria consortia.