关键词: 多酶级联反应, 多酶组装, 生物级联催化, 绿色生物制造, 拓扑优化, 酶工程

Abstract:

Amidst the accelerating global efforts toward carbon neutrality, sustainable biomanufacturing has emerged as a crucial alternative for energy-intensive and environmentally harmful chemical synthesis. Within this transformative landscape, Multi-Enzyme Cascade Reactions (MECRs) represent a paradigm-shifting biocatalytic platform, harnessing the orchestrated activity of spatially organized enzyme modules to enable efficient, one-pot transformations with inherent cofactor recycling capabilities. This comprehensive review synthesizes cutting-edge advances in the design, optimization, and deployment of MECRs, offering a unified analysis across molecular, technological, and application dimensions. We critically dissect the mechanistic foundations of MECRs, including enzyme-enzyme synergy, substrate channeling phenomena, and allosteric regulation governing reaction flux. A systematic classification framework delineates cascade topologies—linear, convergent, parallel, and cyclic systems—elucidating their distinct kinetic advantages and thermodynamic constraints. Transformative technological innovations are highlighted, encompassing AI-driven de novo enzyme design, advanced co-immobilization strategies (such as protein scaffolds and biomimetic mineralization), nano-confined spatial organization for enhanced mass transfer, and novel cofactor regeneration systems utilizing light or electrical energy. The integration of computational fluid dynamics (CFD) modeling with microenvironmental optimization (such as pH and ionic strength gradients) has been shown to significantly enhance biocatalytic efficiency, stability, and operational lifetime. The relevance of industrial application is substantiated by compelling case studies that demonstrate the synthesis of high-value compounds driven by MECR, including optically pure pharmaceutical intermediates, bio-based polymers, and platform chemicals. These processes achieve superior atom economy (>90%), significant reductions in the E-factor, and near-quantitative yields under mild aqueous conditions. The inherent competitive advantages of MECRs—unmatched stereoselectivity, ambient operational parameters, and intrinsic sustainability—are rigorously contrasted against conventional chemocatalytic processes. Persistent challenges in enzyme inactivation, cofactor economics, and reactor scalability are objectively evaluated, alongside emerging mitigation strategies such as continuous-flow membrane bioreactors, artificial metabolon engineering, and machine learning-guided network optimization. Forward-looking perspectives outline a roadmap for next-generation MECRs, prioritizing dynamic spatiotemporal control of microenvironments, AI-accelerated evolution of hyperstable enzymes, and modular continuous manufacturing platforms. The strategic convergence of synthetic biology, computational enzyme engineering, and intelligent process control is poised to unlock programmable biocatalytic systems for complex, multi-step syntheses. This review establishes a foundational framework for advancing MECRs from bench-scale curiosities to industrially robust, environmentally transformative technologies, ultimately positioning enzyme cascades as a cornerstone of carbon-negative manufacturing and a pivotal enabler of global net-zero ambitions. {L-End}

Key words: multi-enzyme cascade reaction, multi-enzyme assembly, bio-catalytic cascade, green biomanufacturing, topology optimization, enzyme engineering