Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly...

Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid-liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2'-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux.