(647d) Dynamic Assembly of Cas6-Mediated RNA Scaffold for Colocalization of Enzymes in Saccharomyces Cerevisiae.

Slow diffusion of intracellular molecules has been a critical challenge to maintaining efficient cellular functions. Cells have naturally evolved to compartmentalize many of their metabolic pathways within intracellular organelles or to cluster their enzymes into functional metabolons for production of specific natural products. To overcome this diffusion-limited bottleneck in the biosynthesis of heterologous products in S. cerevisiae, we have devised a novel strategy to dynamically control the formation of synthetic metabolon complexes by utilizing a Cas6-mediated RNA scaffold and strand displacement mechanism. Using split luciferase as a reporter system, we have demonstrated the feasibility of utilizing these scaffolds to dynamically colocalize two enzymes in close proximity; luminescence in the strains containing functional scaffold constructs was four times higher than in the control strains. We have also adapted this novel synthetic biology tool for the dynamic assembly of a metabolon for the production of the polyketide triacetic acid lactone (TAL) in S. cerevisiae. The approach and benefits of enzyme colocalization will be presented.