Characterization and Engineering of a Clostridium Glycine Riboswitch and Its Use To Control a Novel Metabolic Pathway for 5‑Aminolevulinic Acid Production in Escherichia coli

A riboswitch, a regulatory RNA that controls gene expression by specifically binding a ligand, is an attractive genetic element for the control of conditional gene expression and metabolic pathways. In this study, we identified a glycine riboswitch located in the 5′-untranslated regions of a glycine:proton symporter gene in Clostridium pasteurianum. The glycine riboswitch is shown to contain two tandem aptamers and to function as an activator of expression of genes fused to its expression platform. Results of singlet aptamer experiments indicated that aptamer-2 has a much higher impact on regulating gene expression than aptamer-1. Further, we successfully obtained synthetic glycine-OFF riboswitches using a dual selection approach, and one of them repressed gene expression up to 10.2-fold with an improved dynamic range. The specific glycine-OFF riboswitch can function as an independent repressor in the presence of glycine, and its repression mechanism is inferred from predicted secondary structure. The selected glycine-OFF riboswitch was used to dynamically control the biosynthesis of 5-aminolevulinic acid (5-ALA) in Escherichia coli with an unnatural 5-ALA synthetic pathway, in which glycine plays a key role. It is demonstrated that the use of a synthetic Clostridium glycine-OFF riboswitch can lead to a significant increase (11%) of 5-ALA in E. coli harboring an unnatural biosynthetic pathway.