Multitargeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multitargeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes. Accordingly, we began with the well-characterized E. coli native DsrA sRNA. The merits of using DsrA include its well-characterized separation of function into two independently folded stem-loop domains, wherein alterations at one stem do not necessarily abolish activity at the other stem. Expression of the sRNA and each reporter mRNA was independently controlled by small inducer molecules, allowing precise quantification of the regulatory effects of each sRNA:mRNA interaction in vivo with a microtiter plate assay. Using this system, we semirationally designed DsrA variants screened in E. coli for their ability to regulate key mRNA leader sequences from the Clostridium acetobutylicum n-butanol synthesis pathway. To coordinate intervention at two points in a metabolic pathway, we created bifunctional sRNA prototypes by combining sequences from two singly retargeted DsrA variants. This approach constitutes a platform for designing sRNAs to specifically target arbitrary mRNA transcript sequences, and thus provides a generalizable tool for retargeting and characterizing multitarget sRNAs for metabolic engineering.
Keywords: biofuels; gene regulation; metabolic engineering; noncoding sRNA; synthetic biology.