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

The ability of interacting elements PB1C/PB2N and importin/PB2C for enzyme assembly was demonstrated through optimizing the indigo synthesis pathway in Escherichia coli . The indigo production was improved. Therefore, these interacting elements may be widely used to control metabolic flux and increase the efficiency of biosynthetic pathways . • Interacting short peptides PB1C and PB2N were firstly utilized for enzyme-assembly. • The efficiency of importin/PB2C as scaffold for cascade catalysis was demonstrated. • The indigo synthesis pathway was optimized in E. coli by the interaction motifs. • The interactive elements can broad the application of multienzyme assembly strategy. • The scaffold systems can be used to optimize metabolic flux and biosynthetic pathways. 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.