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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
journal contribution
posted on 2019-10-10, 03:03 authored by Libang Zhou, Jie Ren, Zhidong Li, Jinglei Nie, Chuang Wang, An-Ping ZengA 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.