Transcription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal. Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.

中文翻译：

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PcaV变构转录因子的定向进化以产生芳香醛的生物传感器

基于转录因子的生物传感器是检测代谢物和具有工业价值的分子的有用工具，并在生物技术和生物医学中呈现出许多潜在的应用。然而，开发生物传感器的最常见方法依赖于使用一组有限的天然变构转录因子 (aTF)。因此，改变 aTF 的配体特异性以检测新的效应子是一个重要目标。在这里，MarR aTF 家族的成员 PcaV 阻遏物被用于开发一种生物传感器，用于检测羟基取代的苯甲酸，包括原儿茶酸 (PCA)。PCA 生物传感器进一步进行定向进化以改变其对香草醛和其他密切相关的芳香醛的配体特异性，从而生成 Van2 生物传感器。使用一系列生化和生物物理分析在体外探索了 Van2 的配体识别，并进行了广泛的体内遗传表型分析，以确定每种氨基酸变化对生物传感器性能的作用。这是第一项报告 MarR aTF 家族成员定向进化的研究，并通过改变其配体特异性来生成芳香醛生物传感器来证明 PCA 生物传感器的可塑性。