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

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journal contribution
posted on 2019-11-21, 14:33 authored by Yuki Ogawa, Yohei Katsuyama, Kento Ueno, Yasuo Ohnishi
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.