Engineering of the ligand specificity of transcriptional regulator XylS by deep mutational scanning

Deep mutational scanning was applied to alter the ligand specificity (m-toluic-acid to p-toluic-acid) of the transcriptional regulator XylS from Pseudomonas putida. A xylS mutant library in which the ligand-binding domain was codon-randomized was applied to an antibiotic resistance gene (ampicillin and chloramphenicol)-based dual screening system. The cells were cultured in the presence of ampicillin or chloramphenicol and m-toluic acid or p-toluic acid, and the frequency of each mutation presents in the library was examined using a next-generation sequencer before and after cultivation. From the obtained data, we estimated the amino acid substitutions important for switching the ligand specificity.