Abstract
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.
Highlights
Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression
Chlamydomonas is increasingly being used as a chassis for metabolic engineering and for synthetic biology because of its ease of genetic manipulation,[14] the availability of a comprehensive genome-wide mutant library,[15] a standardized modular cloning (MoClo) kit,[16] and amenability to the production of high value compounds.[17]
To examine in detail the mechanism underlying CrTHI4RSmediated gene regulation, a reporter construct was generated in which the phleomycin resistance gene ble fused to the green fluorescent protein (Ble-GFP), which confers resistance to the antibiotic zeocin,[18] was placed under the control of the CrTHI4 short transcript (CrTHI4s) 5′UTR containing the riboswitch (Figure 1b, construct pPM1−044)
Summary
Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. 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. The possibility to control expression of metabolic enzymes to avoid toxicity or pathway bottlenecks provides the means for sophisticated metabolic engineering strategies
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