(140e) Spatiotemporal Control of Protein Localization and Intracellular Metabolic Flux with an RNA Based, High Affinity, Dynamic Scaffold (Industry Candidate)

In biotechnology, platform organisms such as E. coli and S. cerevisiae are used produce small chemicals of interest through non-native enzymatic pathways. Non-native pathway productivity is traditionally optimized through metabolic pathway manipulation and/or directed evolution of the target enzymes, which require multiple iterations and do not always yield the desired results. Alternatively, nonnative enzymes can be clustered together in the cell to maximize metabolic flux through the nonnative pathway. If the clustering is not dynamic, metabolite imbalances can occur which are detrimental to maximizing product titer. To address this issue, a dynamic high affinity scaffold to colocalize enzymes was developed. The scaffold utilizes small RNA sequences and orthogonal Cas6 proteins taken from the CRISPR/Cas Type I systems. The Cas6 proteins bind with high affinity and sequence specificity to short RNA hairpin sequences. Extending the 5’ region of the short RNA sequences allows assembly of the scaffold via base pair hybridization. Scaffold disassembly can occur via addition of a short 6-18 nt toehold sequence on one of the hybridized strands. Upon expression of a third RNA strand, the trigger, the scaffold disassociates via toehold mediated strand displacement (TMSD). Scaffold assembly and disassembly via trigger strand expression has been demonstrated using the split luciferase reporter system. Scaffold assembly has been demonstrated to occur only when all correct scaffold components are expressed. Scaffold disassembly has been demonstrated using synthetic triggers and native sRNA sequences (RyhB). Furthermore, a turn ON system which relies on the expression of the trigger to assemble the scaffold has also been developed using the split luciferase system. Finally, the toolkit has been applied to increase the product yield of model metabolic pathways. The expanded capabilities of this scaffold toolkit increase the scope of its application and allows users more freedom to design a scaffold that directly fits their needs.