Systematic optimization of L‐tryptophan riboswitches for efficient monitoring of the metabolite in Escherichia coli

Abstract

Riboswitches form a class of genetically encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before the riboswitch is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example. First, the constitutive expression level was adjusted to control the dynamic range of an L-tryptophan riboswitch. The dynamic range increased as the constitutive expression level increased. Then, the function of a riboswitch-encoded protein was utilized to connect the regulatory response of the riboswitch to another outcome for amplifying the dynamic range. Riboswitch-mediated control of the host cell growth enabled the amplification of the riboswitch response. Finally, L-tryptophan aptamers with different dissociation constants were employed to alter the operational range of the riboswitch.The dose-response curve was shifted towards higher L-tryptophan concentrations when an aptamer with higher dissociation constant was employed. All strategies were effective in modifying the distinct functional parameters of the L-tryptophan riboswitch, and they could be easily applied to optimization of other riboswitches owing to their simplicity. K E Y W O R D Sbiosensor, dose-response curve, L-tryptophan, riboswitch tuning, synthetic biology

Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR)

“…This was characterized by the higher levels of GFP expression recorded for the Evo1 sequence, even though its measured in vitro cleavage rates ( Supplementary Figure S8 ) were similar or lower compared to the PC. As has previously been demonstrated however ( 42 ), further work can be implemented to alter gene expression levels by varying factors such as plasmid copy numbers, promoter strengths or perhaps inducer concentrations.…”

Contemporary Tools for Regulating Gene Expression in Bacteria

“…These include tight regulation of basal expression, large dynamic range, and high signal:noise ratio. Riboswitches have also been used as biosensors to improve the production of amino acids, such as tryptophan [102], lysine [103], and vitamin B 12 [104], and for the sensing and production of the flavonoid naringenin [105,106]. Given that riboswitches sense and respond to their cognate ligand without the need for any additional proteins, these regulatory devices yield a faster response time compared with protein-based systems, and should also place reduced burden upon the cellular machinery [10].…”

Genetic tool development and systemic regulation in biosynthetic technology

“…Moreover, an artificial l -tryptophan riboswitch was used to activate gene expression. When adding 1 g/L l -tryptophan, the gene was up-regulated by 1.58-fold compared with no l -tryptophan was added [ 36 ].…”