Monteiro Lummy Maria Oliveira, Arruda Letı Cia Magalhães, Sanches-Medeiros Ananda, Martins-Santana Leonardo, Alves Luana de Fátima, Defelipe Lucas, Turjanski Adrian Gustavo, Guazzaroni Marı A-Eugenia, de Lorenzo Vı Ctor, Silva-Rocha Rafael
Cell and Molecular Biology Department , FMRP - University of São Paulo , Ribeirão Preto , São Paulo 14049-900 , Brazil.
Biology Department , FFCLRP - University of São Paulo , Ribeirão Preto , São Paulo 14040-901 , Brazil.
ACS Synth Biol. 2019 Aug 16;8(8):1890-1900. doi: 10.1021/acssynbio.9b00191. Epub 2019 Aug 2.
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 (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 .
细菌转录因子（TFs）是许多生物技术应用中构建复杂电路的关键元件，然而，很少有经过充分表征的诱导物响应型TFs可用于动物或人类宿主环境。我们已经解析了来自[具体来源未明确]的两种AraC/XylS调节因子（BenR和XylS）的诱导物识别机制，以创建一种对乙酰水杨酸（即阿司匹林）有响应的新型表达系统。通过蛋白质同源建模以及与同源诱导物苯甲酸和一系列化学类似物的分子对接，我们确定了BenR和XylS的保守结合口袋。通过定点诱变，我们确定了高效诱导物识别和转录激活所需的单个氨基酸位置。虽然BenR中的这种修饰会消除蛋白质活性，但在XylS中，它会增加对几种诱导物（包括乙酰水杨酸）的响应，使其接近典型诱导物所达到的水平。此外，通过构建具有交换N端结构域的嵌合蛋白，我们创建了具有混合启动子和诱导物识别谱的新型调节因子。结果，产生了一系列工程化TFs，它们对苯甲酸、3-甲基苯甲酸、2-甲基苯甲酸、4-甲基苯甲酸、水杨酸、阿司匹林和乙酰水杨酸分子的响应增强，可用于在[具体环境未明确]中引发基因表达。
