Engineered interaction elements enable enhanced multi-enzyme assembly and cascade biocatalysis for indigo synthesis

Efficient interacting peptides or protein scaffolds can be used to achieve multi-enzymatic cascade reactions to trigger substrate channeling effect, prevent intermediate diffusion, and control the flux of metabolites. However, the limited availability of existing interactive elements hinders the broad application of the multi-enzyme assembly strategy. Here, a peptide-peptide pair (PB1C/PB2N) and a protein-peptide pair (importin/PB2C) were fused to the target protein to induce protein assembly for the first time. The newly developed interactive elements, when combined with the existing RIDD/RIAD pair, can more efficiently achieve multi-enzymatic cascade reactions. The indigo synthesis pathway was optimized through cascade biocatalysis based on these interactive elements. As a result, compared with the co-expression of multiple enzymes, the interaction element-based cascade biocatalysis increased the yield of indigo by twofold. Our results demonstrate the potential of PB1C/PB2N and importin/PB2C scaffold systems as tools for enzyme assembly to control metabolic flux and increase the efficiency of biosynthetic pathways.