A modular two-component protein cage for spatially organized enzymatic assembly and enhanced metabolic flux

Organizing enzymes on self-assembled protein cages offers a promising strategy to replicate nature's catalytic efficiency. However, most existing studies have focused on the functionalization of one-component protein cages. Here, we present a two-component protein cage scaffold, I5232_STNT, engineered with orthogonal SpyTag/SpyCatcher (ST/SC) and SnoopTag/SnoopCatcher (NT/NC) systems for flexible enzyme recruitment. This addressable scaffold enables programmable co-localization of distinct proteins containing cognate bioconjugation domains, both <i>in vitro</i> and in living cells, as demonstrated using fluorescent protein pairs. Moreover, we show that cargo proteins can be loaded onto the protein cages both before and after cage formation. By integrating a membrane-targeting peptide, we redirected the cytosolic enzyme Idi (isopentenyl diphosphate isomerase) to the <i>Escherichia coli</i> membrane, positioning it proximally to the downstream lycopene pathway enzyme CrtE. This spatial organization resulted in a 4.0-fold increase in lycopene production, demonstrating the scaffold's capacity to enhance metabolic flux through substrate channeling. Our modular platform provides a versatile tool for constructing spatially organized multi-enzyme assemblies, with broad applicability in synthetic biology and metabolic engineering.