Abstract
Metabolic engineering increasingly depends upon RNA technology to customly rewire the metabolism to maximize production. To this end, pure riboregulators allow dynamic gene repression without the need of a potentially burdensome coexpressed protein like typical Hfq binding small RNAs and clustered regularly interspaced short palindromic repeats technology. Despite this clear advantage, no clear general design principles are available to de novo develop repressing riboregulators, limiting the availability and the reliable development of these type of riboregulators. Here, to overcome this lack of knowledge on the functionality of repressing riboregulators, translation inhibiting RNAs are developed from scratch. These de novo developed riboregulators explore features related to thermodynamical and structural factors previously attributed to translation initiation modulation. In total, 12 structural and thermodynamic features were defined of which six features were retained after removing correlations from an in silico generated riboregulator library. From this translation inhibiting RNA library, 18 riboregulators were selected using a experimental design and subsequently constructed and co-expressed with two target untranslated regions to link the translation inhibiting RNA features to functionality. The pure riboregulators in the design of experiments showed repression down to 6% of the original protein expression levels, which could only be partially explained by a ordinary least squares regression model. To allow reliable forward engineering, a partial least squares regression model was constructed and validated to link the properties of translation inhibiting RNA riboregulators to gene repression. In this model both structural and thermodynamic features were important for efficient gene repression by pure riboregulators. This approach enables a more reliable de novo forward engineering of effective pure riboregulators, which further expands the RNA toolbox for gene expression modulation.
Highlights
Over the last decade, synthetic biology and systems biology spurred major advances in metabolic engineering, resulting in several economically competitive production processes for both bulk and fine chemicals from renewable resources, revolutionizing industrial biotechnology [1, 2, 3, 4, 5]
De novo developed post-transcriptional riboregulators defined as the (FP/optical density (OD))corrected in presence of the riboregulator divided by the (FP/OD)corrected in the absence of the riboregulator
Thoroughly analyzing the performance of the constructed translation inhibiting RNAs (tiRNAs) with varying features can improve the knowledge on the structure-function relationship, which correlates to better predictability of de novo created riboregulators [26, 33, 36]
Summary
Synthetic biology and systems biology spurred major advances in metabolic engineering, resulting in several economically competitive production processes for both bulk and fine chemicals from renewable resources, revolutionizing industrial biotechnology [1, 2, 3, 4, 5]. As such, maximizing various production pathways requires tools able to reduce gene expression To this end, zinc fingers and transcription activator-like effectors were engineered to dynamically control transcription of a specific gene through DNA-binding proteins [10, 11]. Various metabolic engineering efforts in multiple organisms used this CRISPRi technology to successfully repress a series of specific genes in a dynamic way, hereby ameliorating the desired product formation [13, 14, 15]
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