Extracellular multi-enzyme assembly and biocatalytic cascade: advances and prospects

MA Muqing; WU Yan; QU Maohua; LU Xiafeng; CAO Min; DU Feng; JI Rongtao; DONG Leichi; LUO Zhibo

doi:10.12211/2096-8280.2025-056

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Extracellular multi-enzyme assembly and biocatalytic cascade: advances and prospects

Invited Review | 更新时间：2025-09-05

- Extracellular multi-enzyme assembly and biocatalytic cascade: advances and prospects
- Synthetic Biology Journal Vol. 6, Issue 4, Pages: 920-939(2025)
- 作者机构：
  
  杭州微远生物科技有限公司，浙江杭州 310024
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2025-056
  CLC： Q814
- Received：06 June 2025，
  
  Revised：2025-06-30，
  
  Published：31 August 2025
- 稿件说明：
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马牧青，吴彦，曲茂华，卢夏锋，曹敏，杜峰，季荣涛，董磊迟，罗志波. 体外多酶组装与生物级联催化：进展与展望［J］. 合成生物学， 2025， 6（4）： 920-939

MA Muqing， WU Yan， QU Maohua， LU Xiafeng， CAO Min， DU Feng， JI Rongtao， DONG Leichi， LUO Zhibo. Extracellular multi-enzyme assembly and biocatalytic cascade： advances and prospects［J］. Synthetic Biology Journal， 2025， 6（4）： 920-939
马牧青，吴彦，曲茂华，卢夏锋，曹敏，杜峰，季荣涛，董磊迟，罗志波. 体外多酶组装与生物级联催化：进展与展望［J］. 合成生物学， 2025， 6（4）： 920-939 DOI： 10.12211/2096-8280.2025-056.

MA Muqing， WU Yan， QU Maohua， LU Xiafeng， CAO Min， DU Feng， JI Rongtao， DONG Leichi， LUO Zhibo. Extracellular multi-enzyme assembly and biocatalytic cascade： advances and prospects［J］. Synthetic Biology Journal， 2025， 6（4）： 920-939 DOI： 10.12211/2096-8280.2025-056.

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，本文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。

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.

关键词

Keywords

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