Spatial control of enzyme orientation at the lipid droplet interface enables regio- and chemoselective metabolic flux

Precise spatial control over multienzyme cascade reactions is increasingly recognized as a design lever in synthetic biology for enhancing catalytic performance and directing metabolic flux. By emulating principles of cellular compartmentalization, synthetic scaffolds and engineered interfaces have emerged as powerful platforms to modulate intermediate channeling and reaction efficiency in complex biocatalytic systems. However, robust and spatially defined immobilization of multienzyme assemblies within physicochemically optimal compartments remains challenging because of the dynamic intracellular environment. Here, we reprogram the amphiphilic interface of yeast lipid droplets (LDs) as a programmable scaffold system to support regio- and chemoselective localization of enzymes. Guided by molecular dynamics simulations, we designed LD-anchoring motifs to direct the enzyme orientation at the LD monolayer interface, yielding cytosol-facing or LD inner-facing configurations. Using this designed LD-anchoring motif, we spatially reorganize a trienzyme cascade for prodeoxyviolacein (PDV) biosynthesis across the cytosol-LD interface, facilitating directional conversion of hydrophilic substrates into hydrophobic products. This spatial reorganization substantially improved PDV production by 1300%, achieving 46.25 milligrams per liter. Our approach offers a modular and tunable platform for engineering spatially organized and readily deployable enzyme cascades, opening avenues for biosynthetic catalysis at organellar interfaces.