Reverse engineering of an aspirin-responsive regulator in bacteria
Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.