In vivo evolutionary engineering of riboswitch with high-threshold for N-acetylneuraminic acid production.

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.