Systematic optimization of L‐tryptophan riboswitches for efficient monitoring of the metabolite in <i>Escherichia coli</i>

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.