(307e) Engineering Protein and Biopolymer Complexation for Synthetic Organelle Assembly (Industry Candidate)

Compartmentalization allows for the spatial organization of intracellular macromolecules and has been co-opted to improve existing biological functions. One strategy for compartmentalization that has recently been acknowledged as a fundamental mechanism for organizing cellular components in eukaryotes is phase separation by protein de-mixing. Phase separated membraneless organelles create distinct environments that are essential to cellular processes ranging from cell signaling to gene expression. These membraneless compartments offer several advantages that make them highly suitable for applications in the production of pharmaceuticals and biocatalysis, including dynamic restructuring of internal constituents and free diffusion of cellular components into/out of compartments. However, the ability to independently engineer the formation and dissolution of membraneless organelles in vivo remains a challenge. Many membraneless organelles appear to have the same physical properties as complex coacervates in that they are liquid-liquid phase separated mixtures of oppositely charged polyelectrolytes. Toward this end, we are interested in utilizing the physical phenomenon of complex coacervation and principles underlying the formation of liquid-like biological condensates to create synthetic membraneless organelles. We have investigated the complex coacervation of engineered proteins with biological polyelectrolytes to determine predictive design rules for protein phase separation. We employ these design rules to create synthetic organelles by promoting phase separation of engineered proteins in E. coli. We find that coacervation is dependent on protein charge and concentration, and demonstrate the in vivo assembly of phase separated compartments that are protein-dense and dynamic.