(619f) Developing a High Affinity, Dynamic Scaffold Toolkit for Intracellular Spatial Organization of Proteins

It is common practice in modern biotechnology to introduce nonnative enzymatic pathways in platform organisms such as E. coli to produce biochemical molecules ranging from specialty chemicals to biofuels. Traditionally, product titer is maximized by fine-tuning the nonnative enzymes’ activity and expression levels; however, in many instances the work can be time consuming and fruitless. Enzyme clustering is an alternative approach that has demonstrably helped increase nonnative pathway titers. However, without dynamic control of when the enzyme cluster forms, unwanted metabolic imbalances within the cell can occur. To address the issues with these optimization techniques and provide an alternative way to improve nonnative pathway productivity, a high affinity, dynamic scaffold toolkit for intracellular spatial organization of proteins was designed. The toolkit building blocks are small RNAs and protein components taken from the CRISPR/Cas Type I systems. These Cas proteins bind to the small RNAs with high affinity and sequence specificity. Scaffold assembly is facilitated by adding short complementary regions in the RNA strands. Scaffold disassembly is coupled to the expression a third small trigger RNA which will occur when certain intracellular conditions are satisfied. The mechanism that drives the disassembly is toehold-mediated strand displacement (TMSD) which allows for displacing one DNA or RNA strand in favor of a new trigger strand. The displacement is facilitated by the presence of an unhybridized 6-18 nucleotide long toehold region at the end of one of the initially hybridized strands. The toehold region provides a foothold onto which the trigger strand can begin to hybridize on. Eventually, the trigger strand will completely hybridize with the toehold strand, essentially kicking out the second strand. TMSD is an excellent candidate for facilitating the dynamic disassembly of the scaffold because its kinetics occurs in the order of minutes to hours. Using the split luciferase reporter system, an increase in the luminescence of the system has been demonstrated to occur only when all the correct components of the scaffold are simultaneously expressed. The scaffold assembly (and luminescence increase) only occurs when appropriate complementary regions are added to the small RNAs. Furthermore, constitutive scaffold disassembly has been demonstrated in the presence of a trigger strand. Ultimately, the scaffold will also be applied to increase the productivity and specificity of a nonnative pathway. The implementation of the scaffold toolkit will allow for intracellular dynamic process control while also providing an alternative approach for increasing nonnative pathway productivity.