(593ad) Riboswitch-sRNA for Dual Transcript Control by a Ligand

Butanol is an alternative to gasoline and an excellent biofuel, which can be used to run cars without modifying their engines. Butanol derived from fermentation of polysaccharides by Clostridium bacteria is an established technology (ABE fermentation), similar to ethanol fermentation by yeast. However, “side reactions” from bacterial enzymes produce contaminant metabolites such as acetate and butyrate (butyric acid), increasing the economic costs of butanol recovery from scaled fermentations. One successful metabolic engineering approach has been to delete the clostridial genes encoding enzymes that produce acetate. However, deletions in genes encoding butyrate-synthesis enzymes also starve the bacteria of energy required for growth, and are lethal to the bacteria. 

To solve this gene expression problem and to make industrially fermented butanol more economically viable, we are re-engineering native bacterial RNA sequences. Specifically, existing RNA genetic control elements within bacteria, called sRNAs, are repurposed here to specifically target and decrease the messenger RNA (mRNA) produced from the butyrate synthesis genes. This hybrid sRNA is essentially a metabolite-controlled molecular device, a “dimmer switch” riboswitch that which will be used to diminish, not turn off, specific butyrate gene expression, ultimately as a dynamic feedback of specific metabolite levels. This system presents an advantage over gene deletions because, when not artificially diminished, butyrate gene expression will default to its natural state, improving the health of clostridial cultures during fermentation. Initially the system is being designed to work in E. coli with well-characterized sRNAs, riboswitch aptamer sequences and reporter genes. The long-term goal is to evolve a metabolite-binding riboswitch by directed evolution, for incorporation into a retargeted sRNA as a metabolic feedback in the gram-positive Clostridia. By reducing butyrate contaminants this project will make butanol and other locally produced biofuels a more economically feasible alternative to gasoline. 

Here we report our progress in repurposing a model sRNA regulator, the E. coli riboregulator DsrA, to build (1) a hybrid riboswitch-DsrA and (2) a retargeted DsrA that binds to the desired target mRNAs by antisense base pairing. Our initial approach has been to set up an mRNA reporter gene system that verifies and quantitatively reports the sRNA-mRNA interactions with a known mRNA target, prior to sRNA-riboswitch integration studies. We designed an arabinose-inducible hns-lacZ reporter fusion to be expressed from a plasmid, such that DsrA expressed from its native promoter on a second plasmid binds the hns RNA sequences naturally regulated by DsrA and diminishes LacZ production, as indicated by a colorimetric assay. A mutant DsrA control that is produced stably (Lease et al 2004, J Bact 186:6179) but which cannot base pair and thus cannot regulate hns RNA, does not interfere in the production of LacZ from our reporter gene, which confirms the specificity of the sRNA-mRNA interaction seen previously (Lease et al. 1998, PNAS USA 95:12456) in this reporter gene context. We will further demonstrate two simultaneous reporter gene activities separately controlled by DsrA.

For improved control of DsrA levels and to facilitate both riboswitch integration into DsrA and retargeting of DsrA, we are using an inducible DsrA which is produced from a lac promoter on a plasmid (Mandin & Gottesman, 2010 EMBO J). We have designed DsrA variants using natural restriction sites within the dsrA gene as well as engineered sites both upstream and downstream. These plasmid constructs permit ready insertion or replacement of short patches of DsrA sequence, such as insertion of riboswitch sequences, and retargeting of the sRNA by altering the nucleotide sequences that base-pair with specific mRNA targets. We will present the results of integrating a riboswitch into different positions within DsrA on its activity. Also, we will present the initial results in the reprogramming of DsrA to bind directly to reporter gene sequences that lack the natively regulated (e.g. hns RNA) sequences. These results are the initial steps towards achieving specifiable, dual RNA transcript control with small metabolite ligand-based metabolic feedback, in order to optimize fermentation culture health for butanol biofuel production at scale.