Systematic Evaluation of Genetic and Environmental Factors Affecting Performance of Translational Riboswitches

Since
their discovery, riboswitches have been attractive tools
for the user-controlled regulation of gene expression in bacterial
systems. Riboswitches facilitate small molecule mediated fine-tuning
of protein expression, making these tools of great use to the synthetic
biology community. However, the use of riboswitches is often restricted
due to context dependent performance and limited dynamic range. Here,
we report the drastic improvement of a previously developed orthogonal
riboswitch achieved through in vivo functional selection
and optimization of flanking coding and noncoding sequences. The behavior
of the derived riboswitches was mapped under a wide array of growth
and induction conditions, using a structured Design of Experiments
approach. This approach successfully improved the maximal protein
expression levels 8.2-fold relative to the original riboswitches,
and the dynamic range was improved to afford riboswitch dependent
control of 80-fold. The optimized orthogonal riboswitch was then integrated
downstream of four endogenous stress promoters, responsive to phosphate
starvation, hyperosmotic stress, redox stress, and carbon starvation.
These responsive stress promoter–riboswitch devices were demonstrated
to allow for tuning of protein expression up to ∼650-fold in
response to both environmental and cellular stress responses and riboswitch
dependent attenuation. We envisage that these riboswitch stress responsive
devices will be useful tools for the construction of advanced genetic
circuits, bioprocessing, and protein expression.