Reverse engineering of an aspirin-responsive transcriptional regulator in Escherichia coli.
Reverse engineering of an aspirin-responsive transcriptional regulator in Escherichia coli.
复制标题
大肠杆菌中阿司匹林响应转录调节因子的逆向工程。
DOI：
10.1021/acssynbio.9b00191
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发表时间：
2019
影响因子：
4.7
通讯作者：
Rafael Silva
中科院分区：
文献类型：
--
作者：
L. Monteiro;L. M. Arruda;Ananda Sanches;Leonardo Martins;L. Alves;L. Defelipe;A. Turjanski;M. Guazzaroni;V. de Lorenzo;Rafael Silva
细菌转录因子（TF）是许多生物技术应用中复杂电路工程的关键器件。然而，很少有充分表征的诱导物响应性TF可以用于动物或人类宿主。在这项工作中，我们已经破译了诱导识别机制的两个AraC/XylS调节器从恶臭假单胞菌（BenR和XylS）创建一个新的表达系统响应乙酰水杨酸（即阿司匹林）。使用蛋白质同源性建模和与同源诱导剂苯甲酸盐和一套化学类似物的分子对接，我们鉴定了BenR和XylS的保守结合口袋。通过定点突变，我们确定了一个单一的氨基酸位置所需的有效诱导识别和转录激活。虽然BenR中的这种修饰消除了蛋白质活性，但在XylS中，它增加了对几种诱导剂的反应，包括乙酰水杨酸，使其水平接近于典型诱导剂所达到的水平。此外，通过构建嵌合蛋白与交换的N-末端结构域，我们创造了新的监管机构与混合启动子和诱导剂识别配置文件。结果，产生了一系列工程化TF，其对苯甲酸酯、3-甲基苯甲酸酯、2-甲基苯甲酸酯、4-甲基苯甲酸酯、水杨酸、阿司匹林和乙酰水杨酸（阿萨）分子的响应增强，用于在E.杆菌
Bacterial transcriptional factors (TFs) are key devices for 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. 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 modelling 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. While 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 enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin and acetylsalicylic acid (ASA) molecules for eliciting gene expression in E. coli.
影响因子：
5.5
作者：
Maier JA;Martinez C;Kasavajhala K;Wickstrom L;Hauser KE;Simmerling C
通讯作者：
Simmerling C