A Hybrid Riboswitch–Small RNA Molecule for Metabolic Engineering in an n-Butanol Pathway

A hybrid riboswitchâ??small RNA molecule for metabolic engineering in an n-butanol pathway

Ashwin Lahirya, Samuel Stimpleb, David W. Woodb and Richard A. Leaseb

(a) Department of Microbiology, (b) Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.

Biologically produced n-butanol is a realistic fuel alternative to gasoline1 that can be utilized in existing infrastructure and vehicles. In fermentation using Clostridium spp., balancing cell health with fermentation yield and n-butanol selectivity is a significant metabolic engineering problem. Gene knockouts to ack or ptb genes increase butanol yield by decreasing acetate or butyrate â??contaminants,â? but compromise cell health1,2. To address these challenges we are engineering bacterial small RNAs (sRNAs), flexible stress response regulators that rapidly fineâ??tune gene expression at low energetic cost to the cell3. These sRNAs specify their target mRNAs by complementary base pairing to decrease translation, thus decreasing target protein concentration and activity. In our work, we are utilizing the well-characterized E. coli sRNA DsrA as a platform for specific, targeted mRNA control, initially in E. coli but ultimately in C. tyrobutyricum. To assay DsrA sRNA function, we have created a plasmid-based E. coli system for inducible and scalable production of DsrA sRNA which natively regulates two mRNA targets (rpoS and hns) via distinct sRNA stem-loop structural domains.4 The rpoS and hns mRNA leader sequences are fused to green and cyan fluorescent reporter proteins, respectively, and are also under independent, inducible, orthogonal and scalable promoter control. This system enables us to control and optimize desired sRNA levels to produce specific expression levels of two mRNAs, measured in real time in vivo, using a 96â??well microtiterâ??plate reader. We describe a quantitative responseâ??surface model for our system that demonstrates tunability of gene expression using a small RNA, and that can be used to predict desired sRNA regulatory output based on concentrations of individual gene-inducer ligands (IPTG, arabinose, and anhydrotetracycline). Further, we have used this system to analyze two types of hybrid riboswitch-sRNA variants which incorporate specific aptamer domains that bind ligands (theophylline or thiamine) based on their ability to activate or repress DsrA sRNA activity. Recent discovery of two natural RSâ??sRNAs 5,6 as well as previous work done in E. coli with engineered ncRNAs fused to RNA aptamers7 confirm the utility of using a ligand/metabolite responsive sRNA in metabolic engineering applications. An advantage of using a RSâ??sRNA hybrid is that the pathway flux ultimately can be coupled to a ligand within the metabolic pathway that binds a riboswitch aptamer, for rapid metabolic-to-genetic feedback control.

References:

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2. Yu M et al. (2011). Metabolic engineering of Clostridium tyrobutyricum for n-Butanol production. Metabolic engineering 13, 372-382.

3. Storz, G., Vogel, J. and Wassarman, K. (2011) Regulation by Small RNAs in Bacteria: Expanding Frontiers. Mol Cell 43:880-891.

4. Lease, R.A. and Belfort, M. (2000). A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures. Proc Natl Acad Sci U S A 97, 9919-24.

5. Sruti DebRoy et al. (2014) A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator. Science 345:6199, 937-939

6. Mellin, J.R et al. (2014) Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA. . Science 345:6199, 939-941

7. Lei Qi et al. (2012) Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals. Nucleic Acids Research, 1-12.