Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Abstract

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. 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 BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, 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 an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.

Engineering Transcription Factor XylS for Sensing Phthalic Acid and Terephthalic Acid: An Application for Enzyme Evolution

“…The mutation A111V was previously found to change the induction specificity of XylS. 46 Thus, the influence of residue 111 was investigated here by performing site-directed saturation mutagenesis. After screening, XylS variants A111C, A111L, A111V, and A111F showed clear fluorescence response to 5 mM PA and TPA (Figure S3).…”

Unraveling the Complex Interplay of Fis and IHF Through Synthetic Promoter Engineering

“…The unpredictable behaviors observed in these studies might also depict a deeper evolutionary trend in gene regulation that has selected molecular systems/mechanisms capable of promoting both evolvability and robustness of gene expression levels through non-linear gene regulation (Steinacher et al, 2016). Thus, understanding the way the architecture of cis-regulatory elements determines gene expression behavior is pivotal not only to understand natural bacterial systems but also to provide novel conceptual frameworks for the construction of synthetic promoters for biotechnological applications (Monteiro et al, 2019b). Frequently, in genetic bioengineering applications, it is also necessary to fine-tune and balance specific gene expression due to the complexity of regulatory networks (Boyle and Silver, 2012;Scalcinati et al, 2012;Steinacher et al, 2016).…”

Engineering Sensor Proteins

“…molecular docking studies with sensors and site-directed mutagenesis, Silva-Rocha et al engineered new transcription factors with enhanced responses to certain molecules, like benzoate and salicylic acid, for eliciting gene expression in E. coli. 18 In another study, Fields and colleagues recently reported on the generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. 19 These biosensors will increase the flexibility for the construction of logic gates and other potential applications in the future.…”

“…For example, bacterial transcription factors play essential roles in the engineering of complex biological circuits. Using molecular docking studies with sensors and site-directed mutagenesis, Silva-Rocha et al engineered new transcription factors with enhanced responses to certain molecules, like benzoate and salicylic acid, for eliciting gene expression in E. coli . In another study, Fields and colleagues recently reported on the generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization .…”