Riboswitches with desired properties, such as sensitivity, threshold, dynamic range, is important for its application. However, the property change of a natural riboswitch is difficult due to the lack of the understanding of aptamer ligand binding properties and a proper screening method for both rational and irrational design. In this study, an effective method to change the threshold of riboswitch was established in vivo based on growth coupled screening by combining both positive and negative selections. The feasibility of the method was verified by the model library. Using this method, an N-acetylneuraminic acid (NeuAc) riboswitch was evolved and modified riboswitches with high threshold and large dynamic range were obtained. Then, using a new NeuAc riboswitch, both ribosome binding sites and key gene in NeuAc biosynthesis pathway were optimized. The highest NeuAc production of 14.32 g/l that has been reported using glucose as sole carbon source was obtained.

具有所需特性（例如灵敏度，阈值，动态范围）的核糖开关对其应用至关重要。然而，由于缺乏对适体配体结合特性的理解以及对于合理和不合理设计的适当筛选方法，天然核糖开关的特性改变是困难的。在这项研究中，成立改变核糖开关的阈值的有效方法在体内通过结合正选择和负选择进行生长耦合筛选。模型库验证了该方法的可行性。利用该方法，开发了N-乙酰神经氨酸（NeuAc）核糖开关，获得了阈值高，动态范围大的修饰核糖开关。然后，使用新的NeuAc核糖开关，优化了NeuAc生物合成途径中的核糖体结合位点和关键基因。使用葡萄糖作为唯一碳源，据报道获得最高的NeuAc产量14.32 g / l。