Abstract:

Amidst accelerating global efforts toward carbon neutrality, sustainable biomanufacturing has emerged as a critical replacement for energy-intensive and environmentally detrimental 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, providing 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 (e.g., protein scaffolds, 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 and microenvironmental optimization (e.g., pH, ionic strength gradients) is shown to dramatically enhance biocatalytic efficiency, stability, and operational lifetime. Industrial relevance is substantiated through compelling case studies demonstrating MECR-driven synthesis of high-value compounds, including optically pure pharmaceutical intermediates, bio-based polymers, and platform chemicals, achieving superior atom economy (>90%), drastic E-factor reductions, and near-quantitative yields under mild aqueous conditions. The inherent competitive advantages of MECRs—unparalleled 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 articulate 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.

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

摘要：

在全球碳中和战略背景下，绿色生物制造正加速取代传统高污染、高能耗的化工生产方式。多酶级联反应（multi-enzyme cascade reaction， MECR）作为新一代生物催化平台技术，通过模块化酶网络实现“一锅式”高效转化，展现出卓越的原子经济性、显著降低的能耗以及突出的环境友好性。本综述明确聚焦于体外多酶级联催化系统（in vitro multi-enzyme systems），涵盖以下研究范畴：1）体外多酶级联体系的定义；2）体外多酶级联体系的分类；3）体外多酶级联体系的相关技术及应用。系统解析了体外多酶组装与生物级联催化的分子机制与技术体系：基于反应拓扑学特征，提出四类级联模型（线性/趋同/平行/循环），阐明其动力学优势；突破性技术涵盖AI驱动的酶理性设计、纳米限域空间组织及光/电辅因子再生系统。通过智能适配设计理念，本研究深入解析了跨尺度酶模块的拓扑优化与催化耦合机制，整合了计算流体力学建模、载体界面分子工程及微环境传质调控等关键技术。产业化实践表明，该技术已成功实现手性药物中间体、高值天然产物等的高效绿色合成，推动医药、材料等领域的工艺革新。展望未来，动态微环境精准适配、人工智能辅助的酶网络设计及连续流规模化制备等方向将引领技术发展，为绿色生物制造的产业化升级提供重要理论支撑与技术路径。

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