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.