Tavares Diogo, van der Meer Jan Roelof
Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
Front Bioeng Biotechnol. 2021 Jul 23;9:705534. doi: 10.3389/fbioe.2021.705534. eCollection 2021.
Bioreporters consist of genetically modified living organisms that respond to the presence of target chemical compounds by production of an easily measurable signal. The central element in a bioreporter is a sensory protein or aptamer, which, upon ligand binding, modifies expression of the reporter signal protein. A variety of naturally occurring or modified versions of sensory elements has been exploited, but it has proven to be challenging to generate elements that recognize non-natural ligands. Bacterial periplasmic binding proteins have been proposed as a general scaffold to design receptor proteins for non-natural ligands, but despite various efforts, with only limited success. Here, we show how combinations of randomized mutagenesis and reporter screening improved the performance of a set of mutants in the ribose binding protein (RbsB) of , which had been designed based on computational simulations to bind the non-natural ligand 1,3-cyclohexanediol (13CHD). Randomized mutant libraries were constructed that used the initially designed mutants as scaffolds, which were cloned in an appropriate bioreporter system and screened for improved induction of the GFPmut2 reporter fluorescence in presence of 1,3-cyclohexanediol. Multiple rounds of library screening, sorting, renewed mutagenesis and screening resulted in 4.5-fold improvement of the response to 1,3-cyclohexanediol and a lower detection limit of 0.25 mM. All observed mutations except one were located outside the direct ligand-binding pocket, suggesting they were compensatory and helping protein folding or functional behavior other than interaction with the ligand. Our results thus demonstrate that combinations of ligand-binding-pocket redesign and randomized mutagenesis can indeed lead to the selection and recovery of periplasmic-binding protein mutants with non-natural compound recognition. However, current lack of understanding of the intermolecular movement and ligand-binding in periplasmic binding proteins such as RbsB are limiting the rational production of further and better sensory mutants.
生物报告分子由经过基因改造的活生物体组成，这些生物体通过产生易于测量的信号来响应目标化合物的存在。生物报告分子的核心元件是一种传感蛋白或适配体，它在与配体结合后会改变报告信号蛋白的表达。人们已经利用了多种天然存在的或经过修饰的传感元件版本，但事实证明，生成识别非天然配体的元件具有挑战性。细菌周质结合蛋白已被提议作为设计非天然配体受体蛋白的通用支架，但尽管做出了各种努力，成功有限。在这里，我们展示了随机诱变和报告基因筛选的组合如何提高了一组基于计算模拟设计的、用于结合非天然配体1,3 - 环己二醇（13CHD）的核糖结合蛋白（RbsB）突变体的性能。构建了以最初设计的突变体为支架的随机突变文库，将其克隆到合适的生物报告系统中，并在1,3 - 环己二醇存在的情况下筛选增强的绿色荧光蛋白突变体2（GFPmut2）报告荧光诱导。多轮文库筛选、分选、重新诱变和筛选使对1,3 - 环己二醇的响应提高了4.5倍，检测下限降低到0.25 mM。除一个突变外，所有观察到的突变都位于直接配体结合口袋之外，这表明它们具有补偿作用，有助于蛋白质折叠或除与配体相互作用之外的功能行为。因此，我们的结果表明，配体结合口袋重新设计和随机诱变的组合确实可以导致选择和回收具有非天然化合物识别能力的周质结合蛋白突变体。然而，目前对周质结合蛋白（如RbsB）中分子间运动和配体结合的理解不足，限制了进一步和更好的传感突变体的合理产生。
