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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Oliveira Monteiro, Lummy Maria [1] ; Arruda, Leticia Magalhaes [1] ; Sanches-Medeiros, Ananda [1] ; Martins-Santana, Leonardo [1] ; de Fatima Alves, Luana [2] ; Defelipe, Lucas [3, 4] ; Gustavo Turjanski, Adrian [3, 4] ; Guazzaroni, Maria-Eugenia [2] ; de Lorenzo, Victor [5] ; Silva-Rocha, Rafael [1]
Total Authors: 10
Affiliation:
[1] Univ Sao Paulo, FMRP, Cell & Mol Biol Dept, BR-14049900 Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo, FFCLRP, Biol Dept, BR-14040901 Ribeirao Preto, SP - Brazil
[3] Univ Buenos Aires, Fac Ciencias Exactas & Nat, Dept Quim Biol, RA-1428 Buenos Aires, DF - Argentina
[4] UBA, Fac Ciencias Exactas & Nat, CONICET, IQUIBICEN, RA-1428 Buenos Aires, DF - Argentina
[5] CSIC, Natl Ctr Biotechnol, Syst Biol Program, E-28049 Madrid - Spain
Total Affiliations: 5
Document type: Journal article
Source: ACS SYNTHETIC BIOLOGY; v. 8, n. 8, p. 1890-1900, AUG 2019.
Web of Science Citations: 1
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. (AU)

FAPESP's process: 12/22921-8 - Synthetic biology approaches for deciphering the logic of signal integration in complex bacterial promoters
Grantee:Rafael Silva Rocha
Support type: Research Grants - Young Investigators Grants
FAPESP's process: 18/04810-0 - Deconstructing complexity in the regulatory network for biofilm formation in gram-negative bacteria
Grantee:Ananda Sanches Medeiros
Support type: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 16/19179-9 - Deciphering the architecture/function relationship in complex bacterial promoters through synthetic biology approaches
Grantee:Lummy Maria Oliveira Monteiro
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 16/06323-4 - New tools for improving metagenomic cellulase screenings
Grantee:Luana de Fátima Alves
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 17/17924-1 - Construction of synthetic promoters for the expression of cellulases using novel cis-regulatory elements in Trichoderma reesei
Grantee:Leonardo Martins Santana
Support type: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries
Grantee:María Eugenia Guazzaroni
Support type: Research Grants - Young Investigators Grants