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

Abstract

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.

Natural 5-Aminolevulinic Acid: Sources, Biosynthesis, Detection and Applications

“…Promoter engineering, 5′-UTR engineering and RBS library can be used to optimize the expression of the target genes, and thereby increase the biosynthesis of 5-ALA. In riboswitch engineering, a glycine-OFF riboswitch was utilized to dynamically downregulate ALAD expression in the presence of glycine, while small amounts of glycine allow hemB to be expressed normally (Zhou et al, 2019). Using CRISPRi technology, a heme-responsive regulatory system was developed to control the concentration of heme dynamically and precisely.…”

“…Recently, the highest titer of biosynthetic 5-ALA reached 18.5 g/L in C. glutamicum, which was achieved via evaluation of 5-ALA synthetases from different sources, regulating intracellular activities of 5-ALA synthetase and phosphoenolpyruvate carboxylase, and optimization of the fermentation medium (Chen et al, 2020). In the process of microbial 5-ALA production, the optimization strategies of biosynthetic pathways or metabolic networks based on genome-scale metabolic network models also play an important role in the biosynthesis of 5-ALA by solving the problems of precursor supply (Ren et al, 2018;Hara et al, 2019), proper expression of 5-ALA synthase (Yu et al, 2019;Chen et al, 2020), and regulating the expression levels of key enzymes in downstream pathways (Zhou et al, 2019;Sakurai et al, 2021). Additionally, the detection technologies applied for different samples have also made great progress (Hara et al, 2019;Ren et al, 2018;Tan Z. J. et al, 2019).…”

Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus

“…Riboswitches have multiple potential uses in drug development as targets for antibacterial compounds, and in biotechnology as a tool for strain engineering. Popular engineering strategies involve the use of riboswitches to regulate the expression of the genes in the biosynthetic pathways competitive to the one of the chosen product [ 11 , 12 ], to deactivate riboswitch-controlled feedback inhibition in selected biosynthetic pathways [ 13 ] or to achieve dynamic control of the parts of biosynthetic pathways that can constitute metabolic burden for the cells, for example membrane proteins such as product transporters [ 14 ]. The desired riboswitches can either be detected through screening of sequences selected based on transcriptomic and functional studies [ 12 , 15 ], or designed using screening [ 2 , 11 , 16 , 17 ] or based on the pre-existing data [ 13 ].…”

Improving succinate production by engineering oxygen-dependent dynamic pathway regulation in Escherichia coli

“…stress response [10], growth phase [11], metabolites [2], etc.). For post-transcriptional regulation, metabolic switches could be constructed by controlling mRNA processing and stability using riboswitch [12], endoribonuclease [13], and small regulatory RNAs [14]. For protein level regulation, native [15] or heterogenous protein degradation systems [16] could be introduced to tune protein stability and abundance.…”