(646c) Retargeted Bacterial Regulatory RNAs for Metabolic Engineering

In metabolic engineering we genetically alter cells to enzymatically produce specialty chemicals such as drugs and fuels. RNA is among the most flexible and potent molecular-genetic regulatory molecules that can be used to engineer these cells, as gene sequences can be edited to create tailored RNA circuit elements with high specificity and limited off-target effects. To optimize gene expression for specific processes we have created a synthetic genetic system for engineering protein translation control by RNA. The metabolic engineering application of this work is to improve n-butanol yield and selectivity in biofuels fermentation. In bacteria, small regulatory RNAs (sRNAs, ~50- 250 nucleotides) natively govern gene expression by binding protein-coding mRNAs and enhancing either their expression (at translation) or degradation/turnover. The sRNAs are ubiquitous in bacteria and act by specific sRNA-mRNA base-pairing interactions with single or multiple mRNA targets. Altering sRNA sequences permits targeting of desired mRNA transcripts and thus tailored protein (enzyme) expression. To systematize retargeting of our sRNA, we have created a quantitative gene control assay comprising one sRNA (E. coli DsrA) that acts on two reporter gene fusions of the native mRNA targets of DsrA (rpoS-GFPuv and hns-mCherry). Each mRNA and the sRNA are under separate transcription-repression control activated by small inducer molecules, and expression of both mRNAs can be read out in separate fluorescent channels during growth in vivo (96-well plate format). We utilized this inducible, scalable expression testbed system for three main experiments. First, we re-targeted DsrA by altering one of its two pairing sequences to bind with and regulate translation of mRNAs of interest in the n-butanol synthesis pathway, specifically acetate or butyrate kinases (to alter pathway flux and improve fermentation selectivity) or a hydrogenase (to improve n-butanol yield by NAD/NADH redox balance). Newly targeted mRNA sequences were used to make a new reporter gene fusion with one of the two fluorescent proteins (e.g. mCherry). Second, in order to determine the extent and basis of partitioning of the sRNA between mRNA targets and thus predict likely sRNA dosage and response, we de-targeted the DsrA by diminishing its interaction (K_d) with one mRNA target (hns) while retaining DsrA native pairing with the other target (rpoS). Finally, to more clearly delineate those sRNA structural features relevant to its mRNA strand targeting, we used this system to assess mutated but functional structures of DsrA stem-loop 2. These experiments fine-tune target mRNA translation to optimize metabolic pathway flux, inform the design of sRNAs to target specific mRNA transcripts, and create a generalizable toolset for characterizing and retargeting sRNAs in bacteria.