471426 Rational Modular Engineering of a Global RNA Regulator for Tunable Metabolic Control
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.