Enzyme symphony in bio-inspired multi-enzyme cascades for enhanced biosynthesis.

Artificial multi-enzyme cascades utilize enzymatic catalysis to achieve continuous complex biosynthesis, thus, standing out as a promising approach. The remarkable efficiency of cascade reactions arises from the precise coordination among multiple enzymes, which facilitates efficient intermediate transfer and maximizing pathway flux. This coordination closely parallels the precise and synchronized collaboration of performers in a symphony orchestra. This review summarizes the value-added biosynthetic capabilities of multi-enzyme cascades, focusing on their ability to convert inexpensive substrates into high-value complex products. Various working forms of multi-enzyme systems are presented and primarily classified into four approaches: free enzymes, assembled complexes, fusion enzymes, and bio-based immobilized enzymes, with emphasis on the unique value of confined microenvironments as crucial platforms for achieving highly efficient cascade reactions. Furthermore, we emphasize the spatial architecture and dynamic regulation of multi-enzyme complexes, while exploring strategies for the rational design of artificial multi-enzyme assemblies tailored to cascade reaction requirements. Finally, emerging trends in AI-assisted design of cascade reactions and multi-enzyme complexes are highlighted to guide future developments.