Engineering Artificial Protein Cages for Metabolic Pathway Regulation by Compartment Reinforcement

Microbial cell factories provide a cost-effective platform for high-value chemical production. However, the lack of intracellular compartmentalization can lead to the diffusion of pathway intermediates and their diversion into competing reactions, thereby decreasing the yield of target chemicals. To overcome this, spatial organization of pathway enzymes using synthetic protein cages can enhance the catalytic efficiency and direct intermediate transfer. Here, we constructed a substrate channel system by employing the heterologous artificial protein cage as a modular scaffold. The design was validated through the precise recruitment and colocalization of sequential enzymes via specific docking domains. The biosynthesis of Lacto-<i>N</i>-tetraose (LNT) in <i>Escherichia coli</i> was used as a model pathway, and we demonstrated that clustering the required enzymes within the subcellular compartment resulted in 34.26% improvement in LNT titer, compared to the nonscaffolded control. This study establishes a compartmentalized strategy that accelerates pathway flux through engineered enzyme colocalization.