Siu, K.-H.; Jiang, V.; Gaffney, E. M.; Walls, M. T.; Joseph, J.; Avalos, J. L.

Cells spatially organize metabolic enzymes to optimize flux through complex biochemical networks. Here, we engineered synthetic Membrane-Associated Condensates (sMACs), artificial assemblies that form on intracellular membranes through liquid-liquid phase separation, to couple metabolic compartmentalization with cofactor partitioning. Using intrinsically disordered regions (IDRs) characterized by simulations and validated by experiments, we identified an IDR sequence, Dbp1n, that selectively enrich NADH and NADPH, establishing distinct local redox microenvironments. At the endoplasmic reticulum (ER) membrane, NADPH-enriched sMACs enhanced cytochrome P450 activity by improving electron transfer and supporting in situ regeneration. At the plasma membrane, NADH-enriched sMACs provided scaffolds for a xylose uptake and assimilation pathway that exploits localized NADH/NAD recycling to improve flux. These results collectively demonstrated that condensate composition and membrane context can be engineered to enhance metabolic functions. sMACs thus provide a modular framework for creating programmable, membrane-associated microreactors, enabling new strategies for metabolic engineering and offering insights into how phase separation can be exploited in living cells.