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

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.