(87f) Rational Modular Engineering of a Global RNA Regulator for Tunable Metabolic Control

Global regulators enable organisms to respond to diverse and rapidly changing environmental stimuli by affecting vast networks of targets at, frequently, multiple biological levels. For this reason, there is ample interest in tuning their regulation as a strategy for strain engineering. Using the Carbon storage regulator (Csr) as a model system, we present a novel and modular redesign approach that focuses on the rational recombination and shuffling of substructures within a regulatory molecule of interest to engineer new schemes of metabolic control. We specifically feature this approach in the context of the non-coding RNA global regulator csrB (~400nt) to tune expression of a number of mRNAs that encode for key enzymes in many essential metabolic pathways. The csrB sRNA contains repeating stem loop structures that frequently present a known consensus CsrA binding motif, GGA. In this way, csrBcan bind a CsrA molecule, the primary Csr protein regulator, at each of these structured binding sites, blocking CsrA-mRNA interactions and thus CsrA-mediated regulation of targets.

Given that csrB acts as a sponge to titrate away the amount of CsrA that directly interacts to (up or down-) regulate an mRNA, a major premise of this work is that various engineered interactions between the csrB RNA and its cognate protein CsrA lead to a regulation gradient of all mRNAs affected in this pathway. Moreover, given the modular composition of the csrB regulator (18 GGA stem loops that are not identical and exist in different contexts within the csrB molecule), this system was ideal to test the notion of combinatorial shuffling of structural motifs to engineer many regulatory variants. In this work, we determine the regional accessibility of csrB in Escherichia coli as a measure of its stem loop-specific binding preferences for CsrA by means of an in vivo RNA structural sensing system (previously developed by the group). We then engineer 25 variants of the molecule based on rational recombination of crsB stem loop structures and demonstrate the effect of this approach in engineering regulation on a number of mRNA targets. Lastly, we demonstrate process-level effects of this regulation gradient on yields of industrially-relevant metabolites as proof of concept of tunable phenotype engineering.