Intracellular Construction\nof Organelle-like Compartments\nFacilitates Metabolic Flux in Escherichia coli

The formation of well-designed synthetic compartments\nor membraneless\norganelles for applications in synthetic biology and cellular engineering\nhas aroused enormous interest. However, establishing stable and robust\nintracellular compartments in bacteria remains a challenge. Here,\nwe use the structured DIX domains derived from Wnt signaling pathway\ncomponents, more specifically, Dvl2 and Axin1, as building blocks\nto generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors\nand physical properties of the DIX-based compartments can be tailored\nby genetically embedding a specific dimeric domain into the DIX domains.\nThen, a pair of interacting motifs, consisting of the aforementioned\ndimeric domain and its corresponding binding ligand, was incorporated\nto modify the client recruitment pattern of the synthetic compartments.\nAs a proof of concept, the human milk oligosaccharide lacto-<i>N</i>-tetraose (LNT) biosynthesis pathway was selected as a\nmodel metabolic pathway. The fermentation results demonstrated that\nthe co-compartmentalization of sequential pathway enzymes into intracellular\ncompartments created by DIX domain, or by the DIX domain in conjunction\nwith interacting motifs, prominently enhanced the metabolic flux and\nincreased LNT production. These synthetic protein compartments may\nprovide a feasible and effective tool to develop versatile organelle-like\ncompartments in bacteria for applications in cellular engineering\nand synthetic biology.