Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
Biotechnol Bioeng. 2018 Jan;115(1):266-271. doi: 10.1002/bit.26448. Epub 2017 Oct 6.
Riboswitches form a class of genetically encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before the riboswitch is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example. First, the constitutive expression level was adjusted to control the dynamic range of an L-tryptophan riboswitch. The dynamic range increased as the constitutive expression level increased. Then, the function of a riboswitch-encoded protein was utilized to connect the regulatory response of the riboswitch to another outcome for amplifying the dynamic range. Riboswitch-mediated control of the host cell growth enabled the amplification of the riboswitch response. Finally, L-tryptophan aptamers with different dissociation constants were employed to alter the operational range of the riboswitch. The dose-response curve was shifted towards higher L-tryptophan concentrations when an aptamer with higher dissociation constant was employed. All strategies were effective in modifying the distinct functional parameters of the L-tryptophan riboswitch, and they could be easily applied to optimization of other riboswitches owing to their simplicity.
核糖开关形成了一类遗传编码的传感器 - 调节剂，被认为是监测各种代谢物的有前途的工具。在将核糖开关应用于特定的分子监测应用中之前，应该优化其功能参数，如动态范围或工作范围。然而，由于其复杂的序列 - 功能关系，核糖开关的优化并不简单，需要进行详细的研究。在这里，我们以人工 L-色氨酸核糖开关为例，提出了三种调节和优化核糖开关功能参数的方法。首先，通过调整组成型表达水平来控制 L-色氨酸核糖开关的动态范围。随着组成型表达水平的增加，动态范围增加。然后，利用核糖开关编码蛋白的功能将核糖开关的调控反应与另一个结果连接起来，以放大动态范围。通过核糖开关介导的宿主细胞生长控制，实现了核糖开关反应的放大。最后，使用具有不同解离常数的 L-色氨酸适体来改变核糖开关的工作范围。当使用具有更高解离常数的适体时，剂量 - 反应曲线向更高的 L-色氨酸浓度移动。所有策略都有效地改变了 L-色氨酸核糖开关的不同功能参数，并且由于其简单性，它们可以轻松应用于其他核糖开关的优化。
