(513a) Synthetic Organelles Engineered from Phase-Separating Proteins

Engineering organelles is an emerging field within synthetic biology for bestowing cells with new biochemical functionalities. One approach is to engineer endogenous, membrane-bound organelles. However, such an approach has several obstacles: Modifying endogenous organelles may disrupt native cell physiology, and it is challenging to engineer transport through membranes for controlling organelle contents. As an alternative, we have developed a platform for generating synthetic, membraneless organelles from phase-separating proteins. Many intrinsically disordered proteins (IDPs) undergo liquid-liquid phase separation due to weak, multivalent interactions. We manipulated one such IDP, an arginine/glycine-rich RGG domain, so that it robustly forms membraneless organelles in living cells with programmable phase behavior and composition. First, we demonstrated that our RGG-based construct forms synthetic organelles that can be genetically encoded in multiple cell lines. These synthetic membraneless organelles are located in the cytoplasm and exhibit dynamic, liquid-like material properties, which are desirable features for future applications in biochemical engineering. Second, to control the presence or absence of the organelles, we modulated protein phase behavior in living cells by enzymatically altering the valency of phase-separating domains. Third, we devised strategies to recruit specific soluble, folded proteins (cargo) into the synthetic organelles in living cells. We achieved this targeted cargo recruitment by genetically fusing the cargo with modular tags that enhance cargo partitioning into the synthetic organelles. Our phase-separating material represents a modular platform to construct and control the behavior of synthetic, genetically encoded, membraneless organelles that selectively colocalize cargo proteins. This system provides a framework to compartmentalize metabolic pathways for increasing the productivity of engineered organisms.