Enzyme Assembly Guided via Modified Signal Peptide for Enhanced Cascade Biocatalysis

Enzyme aggregation holds great promise for enhancing the efficiency of multienzyme cascade reactions. To date, the lack of a universal and tunable strategy to induce enzyme aggregation remains a key bottleneck in the field. Herein, we establish a rational design-based strategy to reprogram the native signal peptide from an α-helical conformation into a β-sheet architecture, thereby imparting it with defined aggregation-inducing properties. This engineered peptide tag enabled enzyme clustering while maintaining the solubility and augmented activity of fusion proteins. By optimizing the linker, the tag induced tunable IB formation, which in cellular systems enhanced sequential production of EGT and 6-HHA by 173% and 111%, respectively. The function of the tag was validated across multiple bacterial strains, confirming its broad compatibility and flexibility. This work represents a significant improvement over current methods by providing a simple, genetically encodable strategy for designing signal peptides to construct self-assembling biocatalysts with highly efficient capabilities.