(582bh) Hybrid Riboswitch-sRNA for Dual Transcript Control By a Ligand

Metabolic engineering can be used to alter or augment existing enzymatic pathways in microorganisms, in order to turn these cells into chemical plants that produce high-value specialty chemicals such as fuels and pharmaceuticals. Host cell enzymes that lead to undesired metabolites can be deleted or altered at the DNA level. However, outright deletion of native enzyme pathways that produce (for example) organic acid side reactions during n-butanol synthesis may compromise the health of fermentation cultures by blocking sources of cellular energy and reduction potential. Here we describe a novel synthetic biology–circuit element that scales the production of enzyme catalysts from their protein-coding mRNA sequences. Native bacterial small regulatory RNA (sRNA) can control gene expression at the level of protein (e.g., enzyme) translation from mRNAs, and can target multiple mRNAs via specific binding (antisense base–pairing) interactions. The sRNA–mRNA pairing may either stabilize or destabilize the target mRNA transcripts, increasing the rate of protein translation or mRNA degradation, respectively. A plasmid DNA is used to deliver the sRNA via an inducible promoter without direct chromosomal modification. By altering certain sRNA sequences, desired target mRNAs can be specified (orthogonal pairing). Our initial experiments describe retargeting the well-characterized E. coli DsrA sRNA to reporter gene fusions with the native mRNA targets of DsrA, then re-targeting of the altered–DsrA sRNA to the reporter genes themselves. Ultimately, desired clostridial mRNAs will be targeted, e.g., acetate kinase (ack) and phosphotransbutyrylase (ptb) that catalyze production of acetate and butyrate 'contaminant' side reactions of n-butanol fermentation, respectively. To enable real-time ligand feedback control of the sRNA and thereby tune the optimal level of side reactions, a ligand-responsive RNA sensor sequence called a riboswitch (RS) will be incorporated, to create a hybrid RS-sRNA synthetic biology regulatory element. Because the RS can be altered to bind different ligands, the RS-sRNA may be used to transduce a desired ligand-binding event into a "portable" and orthogonal RNA regulator. We will present the results of three distinct approaches to RS-sRNA integration, at the level of sRNA synthesis (using an antiterminator-leader RS), sRNA stability (using an RS integrated within the sRNA), and sRNA activity (using a RS–controlled, directed ribozyme cleavage). The ultimate goal of this work is to enable real-time metabolite-to-genetic feedback control of enzyme production for optimization of biofuel fermentation. The RS-sRNA will be portable between different bacterial species for fermentations and other applications as it does not require chromosomal modification of its host cell.