Ribo-attenuators: novel elements for reliable and modular riboswitch engineering

Abstract

Riboswitches are structural genetic regulatory elements that directly couple the sensing of small molecules to gene expression. They have considerable potential for applications throughout synthetic biology and bio-manufacturing as they are able to sense a wide range of small molecules and regulate gene expression in response. Despite over a decade of research they have yet to reach this considerable potential as they cannot yet be treated as modular components. This is due to several limitations including sensitivity to changes in genetic context, low tunability, and variability in performance. To overcome the associated difficulties with riboswitches, we have designed and introduced a novel genetic element called a ribo-attenuator in Bacteria. This genetic element allows for predictable tuning, insulation from contextual changes, and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as truly modular and tunable components, thus increasing their reliability for a wide range of applications.

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…For instance, by replacing its original ORF with a new one, the secondary or tertiary structure of an aptamer domain can be subtly disrupted and thus lose its ligand specificity (Folliard et al . ). This calls into question the functional modularity of riboswitches.…”

“…Thus, the ribosome is poised to dissociate from the upstream ORF and immediately re‐initiate translation at the downstream start codon, producing the protein of interest without any N‐terminal fusion (Folliard et al . ).…”

“…Recent studies have assessed riboswitch modularity (Folliard et al . ) in different genetic contexts. For instance, by replacing its original ORF with a new one, the secondary or tertiary structure of an aptamer domain can be subtly disrupted and thus lose its ligand specificity (Folliard et al .…”

“…Recently, Folliard et al . () developed a mechanism to mitigate this issue, termed a ‘riboattenuator’, which has allowed them to successfully enhance the modularity of riboswitches and circumvent the inclusion of a 5′ fusion, increasing protein production by ∼1000 fold. The riboattenuator has a second RBS, sequestered by a local hairpin, which is directly downstream of the stop codon of the riboswitch‐controlled ORF (Fig.…”

“…As for riboswitches, the riboattenuator approach (Folliard et al . ) might prove useful for ensuring the production of a protein of interest that lacks any unwanted N‐terminal fusions. Moreover, as the structure and melting temperature of RNATs can be affected by osmolytes, salts and divalent cations (Rinnenthal et al .…”