In modern biotechnology, platform organisms such as E. coli and S. cerevisae are used produce small chemicals of interest through non-native enzymatic pathways. Traditional approaches for optimizing nonnative pathway productivity involve directed evolution of the key enzymes of the pathway and manipulation of the cell’s native metabolism through deletions or overexpression of enzymes todrive product formation. These approaches require multiple iterations and do not always yield the desired results. An alternative approach involves clustering nonnative enzymes together in the cell in order to maximize metabolic flux through the nonnative pathway. If the clustering is not dynamic though, metabolite imbalances can occur which can prove detrimental to maximizing product titer. To address this issue a dynamic high affinity scaffold to colocalize enzymes dynamically 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. By designing the remaining RNA sequences appropriately, assembly of the scaffold through base pair hybridization has been demonstrated. Furthermore, the scaffold can be disassembled through the 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 quickly disassociates via toehold mediated strand displacement (TMSD). In previous work, successful scaffold assembly and disassembly via trigger strand expression has been demonstrated using the split luciferase reporter system by correlating increased and decreased luminescence to scaffold assembly and disassembly. Scaffold assembly has been demonstrated to occur only when all correct scaffold components are expressed. So farr scaffold disassembly was only demonstrated using a synthetic trigger. Recently scaffold disassembly using native sRNA sequences as the trigger (RyhB)has been succesfully demonstrated. 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.

Expanding the capabilities of the Cas6 based dynamic high affinity scaffold for spatial and temporal co-localization of proteins

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.