Driven by multiple waves of biocatalysis, rapid advancements in protein engineering and bioinformatics have enabled the precise engineering of enzyme properties (such as stability (Li et al., 2024a; Ming et al., 2023; Sun et al., 2019a; Xu et al., 2025a), catalytic activity (Birch-Price et al., 2023; Corbella et al., 2023; Hecko et al., 2023; Song et al., 2023), selectivity (Ding et al., 2022; Song et al., 2023; Wu et al., 2022), and solvent resistance/environmental compatibility (Cui et al., 2020a; Cui et al., 2020b; Kikani et al., 2023; Wang et al., 2023b) and enzyme design with specific functions (Bell et al., 2024; Cui et al., 2022a; Lauko et al., 2025; Liu and Arnold, 2021; Pinto et al., 2021; Vaissier Welborn and Head-Gordon, 2019; Zhou and Huang, 2024). This progress has significantly expanded the scope of enzymatic catalysis, allowing it to increasingly supplant traditional chemical methods in complex syntheses (Buller et al., 2023; O'Connell et al., 2024; Reetz, 2022; Winkler et al., 2021). Inspired by natural metabolic networks (Kohnhorst et al., 2017; Pareek et al., 2021; Schmitt and An, 2017), artificial multi-enzyme cascade systems constitute a powerful platform characterized by streamlined production processes (France et al., 2017; Huffman et al., 2019) for converting inexpensive feedstocks into a wide range of high-value compounds (Jung and Li, 2024; Wang et al., 2020a; Yi et al., 2024) and complex natural products (Benítez-Mateos et al., 2022; Naik et al., 2024; Siedentop and Rosenthal, 2022). These integrated systems enhance synthetic efficiency through several key advantages: driving unfavorable reactions via energetic coupling, enabling efficient redox transformations (Mutti et al., 2015), and supporting self-sufficient cofactor (e.g. NAD(P)H, ATP, and FAD) regeneration (Both et al., 2016; Liu et al., 2025; Mutti et al., 2015; Pickl et al., 2015; Schultheisz et al., 2008; Zhang et al., 2021; Zhang et al., 2019). Simultaneously, they operate under mild conditions, thereby improving sustainability. These features highlight the economic and ecological value of multi-enzyme cascades, while aligning closely with the principles of green chemistry and the goals of synthetic biology.

Despite these significant advantages, the practical application and implementation of multi-enzyme cascades remain constrained by various technical and systemic challenges (Muschiol et al., 2015). The integration of catalytic steps is often hindered by incompatibilities between heterologous enzymes, including differences in kinetics, mismatched optimal conditions such as pH and temperature, and mutual inhibition. In addition, the lack of spatial organization and ordered arrangement among enzymes within multi-enzyme cascade systems may lead to the diffusion or instability of reactive intermediates, thereby promoting undesired side reactions or the accumulation of toxic byproducts. These limitations underscore the need for rational cascade design strategies (Song et al., 2018), efficient enzyme organization platforms (Wang et al., 2017), and adaptive regulatory mechanisms to unlock the synthetic power of multi-enzyme cascades.

To meet the increasing biosynthetic demands, the spatial organization of enzymes has emerged as a central research focus. A number of comprehensive reviews have outlined the diversity of cascade types and prospects in the field (Bugada et al., 2018; France et al., 2017; Hwang and Lee, 2019; Oroz-Guinea and García-Junceda, 2013; Ricca et al., 2011; Schrittwieser et al., 2018), with particular emphasis placed on the immobilization of enzymes on non-biological materials—such as inorganic nanoparticles (e.g., silica and magnetic particles) (Holyavka et al., 2023) and porous materials including metal–organic frameworks (MOFs) and covalent organic frameworks (COFs) (Arana-Peña et al., 2021; Bilal et al., 2023; Cai et al., 2025). The high enzyme loading, stability, and reusability afforded by these materials have advanced multi-enzyme catalysis, primarily through static confinement. In contrast, bio-inspired artificial multi-enzyme cascades have been received fewer reviews (Dong et al., 2021; Quin et al., 2017). These systems offer unique advantages in biocompatibility, renewability, and dynamic regulation, and are further enhanced by design principles derived from the intricate structures of natural multi-enzyme assemblies.

Given the significant potential of bio-inspired multi-enzyme cascades and the existing lack of systematic reviews, this review provides a comprehensive analysis spanning their biosynthetic capabilities to emerging design methodologies. This review systematically illustrates the biosynthetic capabilities of multienzyme systems and their dependence on precise enzymatic coordination. It also introduces four organizational strategies (free enzymes, assembly complexes, fusion proteins, and bio-based immobilized enzymes) and confined environment for multi-enzyme cascade. Next, natural multi-enzyme assemblies' dynamic spatial control mechanisms are explored. These principles inform the design of programmable, stimulus-responsive artificial multi-enzyme complexes, enabling efficient “enzyme symphony” orchestration. Finally, we outline future directions highlighting AI-driven pathway design and rational construction of bio-inspired enzyme systems for sustainable biosynthesis applications.