Engineering of the Ligand Specificity of Transcriptional Regulator XylS by Deep Mutational Scanning

Deep
mutational scanning is a method for protein engineering. Here,
we applied it to alter the ligand specificity of the transcriptional
regulator XylS from Pseudomonas putida to recognize p-toluic acid instead of the native
ligand m-toluic acid. For this purpose, we used an
antibiotic resistance gene-based dual screening system, which was
constructed for the directed evolution of XylS toward the above-mentioned
ligand specificity. We constructed a xylS mutant
library in which each codon for the amino acid residue of the putative
ligand-binding domain (residues 1–213, except 7th residue)
was randomized to generate all possible single amino acid-substituted
XylS variants and introduced it into Escherichia coli harboring the selection plasmid for the screening system. The cells
were cultured in the presence of appropriate antibiotics and m-toluic acid or p-toluic acid, and the
frequency of each mutation present in the library was examined using
a next-generation sequencer before and after cultivation. Heatmaps
showing the enrichment score of each XylS variant were obtained. By
searching for a p-toluic-acid-specific heatmap pattern,
we focused on G71 and H77. Analysis of the ligand specificities of
G71- or H77-substituted XylS variants revealed that several G71-substituted
XylS variants responded specifically to p-toluic
acid. Thus, the 71st residue was found to be an unprecedented residue
that is important for switching ligand specificity. Our study demonstrated
the usefulness of deep mutational scanning in engineering the ligand
specificity of a transcriptional regulator without structural information.
We also discussed the advantages and disadvantages of deep mutational
scanning compared with directed evolution.