Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas

Riboswitches are
RNA regulatory elements that bind specific ligands
to control gene expression. Because of their modular composition,
where a ligand-sensing aptamer domain is combined with an expression
platform, riboswitches offer unique tools for synthetic biology applications.
Here we took a mutational approach to determine functionally important
nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch
in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana.
These chimeric riboswitches displayed a distinct specificity and dynamic
range of responses to different ligands. Our studies demonstrate ease
of assembly as 5′UTR DNA parts, predictability of output, and
utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation
in current design platforms will facilitate the generation of genetic
circuits to advance synthetic biology and metabolic engineering of
microalgae.