Microbial cell factories enable the sustainable production of fuels, chemicals, and pharmaceuticals, yet their performance is often constrained by inefficient metabolic flux distribution, cofactor imbalance, and pathway-associated toxicity. Spatial engineering has emerged as an effective approach to address these limitations. It does so by controlling the organization of metabolic processes. This review summarizes recent advances in spatial engineering at three levels: natural organelle engineering, artificial compartmentalization, and intercellular coordination. We discuss how these strategies enhance pathway efficiency through enzyme colocalization, metabolic insulation, and division of labor. We also underscore current challenges regarding targeting efficiency and system integration. Future directions for advancing spatial design and metabolic coordination in microbial systems are outlined.
