Switching the Ligand Specificity of the Biosensor XylS from meta to para-Toluic Acid through Directed Evolution Exploiting a Dual Selection System

The Pseudomonas putida transcriptional activator XylS induces transcription from the Pm promoter in the presence of several benzoic acid effectors, with m-toluic acid being the most effective and p-toluic acid being much less effective. To alter the effector specificity of XylS, we developed a dual selection system in Escherichia coli, which consists of (i) an artificial operon of an ampicillin resistance gene and tetR under Pm promoter control and (ii) a chloramphenicol resistance gene under tetR promoter control. This system enabled both positive selection to concentrate XylS mutants recognizing a desired ligand and negative selection to exclude undesired XylS mutants such as those recognizing undesired ligands and those that are active without effectors. Application of a random mutagenesis library of xylS to directed evolution that exploited this selection system yielded two XylS mutants that recognize p-toluic acid more effectively. Analysis of each missense mutation indicated three amino acid residues (N7, T74, and I205) important for p-toluic acid recognition. Then, a codon-randomized xylS library at these three residues was similarly screened, resulting in three XylS mutants with increased p-toluic acid-recognition specificity. Analysis of each amino acid substitution revealed that T74P attributes to both m-toluic acid sensitivity loss and subtle p-toluic acid sensitivity acquisition, and that N7R increases the overall ligand-sensitivity. Finally, the combination of these two mutations generated a desirable XylS mutant, which has a high p-toluic acid sensitivity and scarcely responds to m-toluic acid. These results demonstrate the effectiveness of the dual selection system in the directed evolution of biosensors.