Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli.
Lummy Maria Oliveira MonteiroLetı Cia Magalhães ArrudaAnanda Sanches-MedeirosLeonardo Martins-SantanaLuana de Fátima AlvesLucas A DefelipeAdrian Gustavo TurjanskiMarı A-Eugenia GuazzaroniVictor de LorenzoRafael Silva-RochaPublished in: ACS synthetic biology (2019)
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.
Keyphrases
- transcription factor
- molecular docking
- gene expression
- escherichia coli
- amino acid
- low dose
- dna binding
- cardiovascular events
- cancer therapy
- dna methylation
- binding protein
- antiplatelet therapy
- molecular dynamics simulations
- endothelial cells
- poor prognosis
- type diabetes
- heat shock
- biofilm formation
- cell therapy
- mesenchymal stem cells
- acute coronary syndrome
- staphylococcus aureus
- percutaneous coronary intervention
- atrial fibrillation
- cystic fibrosis