RNA is at the heart of gene expression control in cells, yet small regulatory RNAs (sRNAs) of bacteria are only in the early stages of development as synthetic biology tools. Benefits of sRNA-level genetic control include the precise sRNA sequence-specific targeting of protein-coding messenger RNA for translation control. The ubiquity of sRNAs in diverse bacteria bodes well for sRNA control in organisms with few genetic tools. Some regulatory sRNAs have been repurposed for metabolic engineering control of specialty chemicals fermentation pathways. Targeting multiple mRNAs in a metabolic pathway with multi-targeting sRNAs could coordinate control of metabolic pathway flux with a single genetic intervention. Therefore, we have retargeted a native multi-target bacterial regulatory sRNA for simultaneous intervention at two non-native mRNAs. We developed a regulatory sRNA prototyping system in Escherichia coli to assay and retarget sRNAs to non-native mRNA sequences. We fused two mRNA translation initiation regions to red and green fluorescent reporter protein genes, thus creating an assay of simultaneous translation control during cell growth in a 96-well format. We describe two main experiments, retargeting the sRNA and structural optimization of the sRNA scaffold. First, sRNA antisense base pairing sequences were altered to bind two mRNA control sequences derived from Clostridium acetobutylicum ATCC 824 expressed in E. coli. These mRNA targets were chosen to optimize n-butanol fermentation selectivity while balancing NADH redox potential for improved n-butanol yield. For retargeting we used a semi-rational modular design that exploited native E. coli DsrA sRNA stem-loop antisense structure motifs. Second, we used the genetic retargeting assay system to optimize the size and strength of sRNA stem-loop structures containing antisense sequences for mRNA targeting and genetic control. We hypothesize that the stem-loop antisense structures in DsrA can minimize off-target hybridization, a potential problem in sRNA retargeting systems that may be addressed by this natural structure motif. These findings validate our choice of scaffold and semi-rational design approach for multiple mRNA control in metabolic engineering applications.

Genetic system for regulatory sRNA retargeting and scaffold optimization: Metabolic engineering applications

253rd National Meeting (2017)

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