Natural living systems leverage compartmentalization as an evolutionary blueprint to orchestrate intricate biological processes through spatiotemporal segregation (Abbas et al., 2021; Fang et al., 2024). This fundamental strategy manifests in three archetypal architectures: protein-shelled microcompartments (Kerfeld et al., 2018), phospholipid membrane-bound organelles (Heald and Cohen-Fix, 2014), and liquid-liquid phase separation (LLPS)-driven membraneless organelles (MLOs) (Peeples and Rosen, 2021), which collectively enable spatial regulation of cellular materials, signaling pathways, and metabolic processes. By creating specialized physicochemical niches, these compartments can optimize metabolism through toxic intermediate sequestration, cofactor partitioning, and enzymatic cascade colocalization (Bar-Peled and Kory, 2022). Such biological principles have catalyzed the engineering of synthetic compartments—ranging from biomacromolecular assemblies and biomimetic membrane systems to de novo designed protein condensates (Qian et al., 2022).

Metabolic engineering of heterologous pathways in cellular systems frequently encounters off-target side reactions and enzymatic inefficiencies exacerbated by cellular interference, particularly through toxic intermediate accumulation that disrupts host physiology (Fang et al., 2024). To address these challenges, synthetic biologists leverage compartmentalization strategies based on diverse materials—both intracellular and extracellular—to design simplified yet specilized functional supermolecular structures. Generally, these architectures can be categorized into two primary classes based on the presence and composition of a specific physical scaffold: (i) scaffolded systems, including origami (Huang et al., 2024), bacterial microcompartments (BMCs) (Gu et al., 2024), virus-like particles (VLPs) (Cheah et al., 2023), encapsulins (Kwon et al., 2024), vault proteins (Wang et al., 2019), vesicles (Zheng et al., 2023), and other protein cages (Kang et al., 2022); (ii) scaffold-free biomolecular condensates or membraneless organelles (MLOs), and coacervates formed via LLPS that achieve dynamic molecular partitioning (Fang et al., 2024; Wang and Douglas, 2023). Scaffolded systems create diffusion-controlled bioreactors by spatially confining enzymatic activities. In contrast, phase-separated compartments leverage multivalent interactions among diverse biomacromolecules—such as nucleic acids (Guo et al., 2022), proteins (Banani et al., 2017), and their complexes (Sanders et al., 2020)—to achieve dynamic partitioning.

Contemporary strategies for spatial multienzyme organization predominantly exploit the enthalpy- and entropy-driven self-assembly of amphiphilic building blocks and engineered systems, which allows spontaneous or stimuli-responsive segregation of homogeneous/heterogeneous components into higher-order architectures (Solomonov et al., 2024). Diverging from classical self-assembly mechanisms, DNA origami-based nanocompartments, fundamentally rooted in sequence-programmed assembly, exhibit distinct engineering-guided multiscale architectures. Such scaffold or compartment systems enable spatial control over enzymatic pathways through addressable molecular connectivity rather than stochastic self-organization. In contrast, LLPS-mediated compartments rely on weak, multivalent interactions to form dynamic reaction hubs where components can rapidly exchange with the external milieus (Yuan et al., 2023). Such biomimetic systems facilitate the isolation of incompatible reactions, maintain chemical gradients, and coordinate multi-step catalysis, bridging the gap between prebiotic organization and synthetic cellular functions.

This review systematically examines six spatial engineering approaches, covering both scaffolded compartments (such as liposomes, DNA origami, polymersomes, and BMCs) and scaffoldless compartments (including MLOs and coacervates), to examine their roles in biocatalytic process control. By interrogating their distinct mechanisms in mitigating the diffusion effect of metabolites and regulating metabolic flux, this review extends to explore their potential applications across a broader range of scenarios. The discussion particularly delves into the unique physicochemical properties, reaction microenvironments and functionalization potential of LLPS-mediated artificial organelles. While highlighting recent advances in biochemical crucible construction, we identify persistent bottlenecks in current strategies and project future trajectories for the development of comprehensive metabolon designs towards protocells.