(75e) A Dual-Acting sRNA of E. coli Is Repurposed As a Genetically Portable Metabolic Engineering Controller

Lease, R. A., The Ohio State University
Lahiry, A., The Ohio State University
Stimple, S. D., The Ohio State University
Wood, D. W., The Ohio State University
Yang, H., The Ohio State University
Our understanding of RNA genetic control in cells is continuing to gain in importance and application. We are interested in using synthetic RNA biology to optimize specialty chemical production through metabolic engineering. It is important to optimize metabolite flux in the scale-up of fermentation cultures to balance cellular health with the production of economically viable levels of specialty chemicals. In bacteria, metabolic pathway flux can be "fine-tuned" with a synthetic regulatory small RNA (sRNA) to linearly scale the protein transation level of pathway enzymes from existing mRNA transcripts, produced either from host DNA genes or non-native (cloned) pathways. The sRNA antisense sequences pair with mRNA sequences to form a complex that governs ribosome access and translation rates. Linearly scaling enzyme production using a retargeted antisense sRNA sequence predictably alters enzymatic pathway flux in the "assembly line" of metabolites towards a final chemical product. To this end we have engineered DsrA, a regulatory sRNA molecule native to Escherichia coli, into a "portable" and generalizable scaffold for directing antisense-based RNA control in industrially–relevant bacteria. We developed a genetic system for prototyping sRNAs in E. coli for retargeting two antisense-sequence structural domains in the sRNA. These sequences bind and repress two user-defined mRNA targets for specific, coordinated metabolic pathway perturbations. We describe experiments that show retargeting of our sRNA to C. acetobutylicum mRNA, genetic portability to Clostridium acetobutylicum, and improved selectivity and yield of n-butanol in the ABE fermentation process by improved redox balance. Further, we demonstrate the utility of our genetic assay system in characterizing the essential sequence and structural components of our sRNA antisense genetic element, in particular with the goal of weaning the sRNA from its dependence on either the E. coli or host RNA–binding Hfq protein. Finally we demonstrate a function of the structured antisense "fingerloop" domains of E. coli DsrA in excluding off-target mRNA interactions. This off–target filtering mechanism offers precise mRNA control in both E. coli and non-native hosts.