Biologically produced n-butanol is an important and sustainable fossil fuel alternative, with other industrial applications as a chemical feedstock, solvent, lubricant and diluent. Economically viable production of n-butanol from fermentation of Clostridium spp. is limited by low butanol yield (titer and selectivity) and high costs of feed-stock and recovery. To improve butanol yield we are using metabolic engineering with synthetic biology tools for RNA-level regulation of metabolic enzymes. Our method is to re-target a native E. coli small RNA (DsrA) to decrease expression of mRNAs in the n-butanol fermentative pathway of C. acetobutylicum. To retarget sRNA (DsrA) we altered two of its sRNA anti-sense sequences. Two distinct stem-loop structures in the sRNA were altered to base pair directly to buk and hydA mRNA leaders. These engineered sRNA variants were analyzed in Escherichia coli using target mRNA leader sequences fused to fluorescent reporters. Previous efforts to knock out the essential hydrogenase gene (hydA) in C. acetobutylicum have been unsuccessful. We will use our engineered sRNA to tune down hydA gene expression and optimize the balance between hydrogen and butanol production. Targeting the HydA enzyme will alter the NADH / NAD+ redox balance and drive the carbon flux towards butanol production. Further, strains with gene-knockouts of the butyryl kinase gene (buk) have improved butanol yield and reduced butyric acid. We also targeted buk mRNA to subtly tune the carbon flux from the butyryl co-A branching point towards butanol. Fluorescence from two mRNA reporters (GFPuv and mCherry) was measured in vivo using a 96–well microtiter–plate reader or flow cytometry. By controlling native DsrA transcription we demonstrate the tuning of two native DsrA target mRNA reporter gene translation levels (RpoS-GFPuv and H-NS-mCherry) as well as for mutant variants of DsrA containing sequences antisense to buk mRNA leader (fused to GFPuv) and to hydA mRNA leader (fused to mCherry). The anti-sense sequences with the best repression at each target were combined together into two stem-loops of the same sRNA mutant and validated. This dual-acting sRNA is anticipated to simultaneously regulate the expression of both target genes in the C. acetobutylicum metabolic pathway. Our work establishes an E. coli genetic test-bed system for development of sRNAs as tools that can be engineered for different metabolic engineering applications in diverse bacteria.

Metabolic engineering with a dual-acting small RNA molecule for improved biofuels fermentations

251st National Meeting (2016)

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