Microbial cell factories have emerged as versatile bioreactors capable of orchestrating complex metabolic networks to convert renewable feedstocks into high-value biochemicals. Nevertheless, the diffusion-mediated dispersion of metabolic intermediates often compromises biosynthesis efficiency, primarily attributable to the absence of artificial subcellular compartments for spatiotemporal organization of catalytic enzymes. Herein, we established a synthetic biology platform leveraging engineered biomolecular condensates to achieve precise flux control via a modular pathway compartmentalization. First, the fused sarcoma low complexity domain (FUSLCD) was designed to combine the GCN4 to rationally integrate with GCN4 scaffold proteins to create programmable artificial organelles. Second, the protein recruitment and assembly functions of artificial organelles were identified by a short peptide pair or directly fusing with the FUSLCD protein in a spatial organization way. Third, using the 2'-fucosyllactose (2'-FL) de novo biosynthesis pathway as a model system, we demonstrated enhanced pathway efficiency by colocalizing critical enzymes within artificial organelles in engineered E. coli, yielding a significant improvement in 2'-FL titer through flux compartmentalization. This study not only overcome diffusion-limited reactions via engineered spatial organization but also offer a versatile toolkit for optimizing compartmentalized biosynthesis pathways.
Keywords: 2′-fucosyllactose; Artificial organelle; Biomolecular condensate; Compartmentalized flux control; Spatial organization.