Spatial Organization of the Metabolic Pathway for Enhancing <scp>l</scp>-Fucose Biosynthesis in Engineered <i>Escherichia coli</i>

Synthetic biology and metabolic engineering have revolutionized the microbial biosynthesis of high-value compounds, yet conventional strategies are frequently confronted with metabolic burden and flux competition. Inspired by natural systems that employ spatial organization to enhance catalytic efficiency, we have developed advanced techniques to optimize pathway performance in engineered microbes. In this study, we present a systematic implementation of spatial organization strategies─consisting of scaffold-free enzyme assembly mediated by interacting peptides and engineered protein compartments─to improve l-fucose biosynthesis in <i>Escherichia coli</i>. We first constructed a de novo l-fucose biosynthetic pathway by introducing α-l-fucosidase for 2'-fucosyllactose (2'-FL) hydrolysis. Key pathway enzymes WbgL and AfcA were subsequently organized into multienzyme complexes via RIAD-RIDD peptide interactions with both stoichiometry and spatial configuration systematically optimized. Furthermore, DIX domain-mediated protein compartments facilitated enzyme colocalization through orthogonal RIAD-RIDD tagging, which effectively channeled metabolic flux toward l-fucose production. Our findings establish that both self-assembling enzyme complexes and programmable protein compartments can serve as robust platforms for the spatial organization of the metabolic pathways. By addressing challenges such as flux imbalance, intermediate toxicity, and pathway cross-talk, these strategies significantly improve microbial biosynthesis efficiency. Overall, this work provides a generalizable framework for optimizing metabolic pathways through synthetic spatial organization, with broader applications in biobased chemical production.