面向mRNA制造的酶模块工程化策略进展: 催化调控与过程集成

doi:10.1016/S1872-2067(25)64860-1

催化学报 ›› 2026, Vol. 80: 92-112.DOI: 10.1016/S1872-2067(25)64860-1

面向mRNA制造的酶模块工程化策略进展: 催化调控与过程集成

车诗怡^a^,^b, 李正军^b, 苏志国^b, 李之考^a, 余艾冰^a, 刘闽苏^a^,^c^,^*(), 张松平^b^,^*()

^a蒙纳士大学化学与生物工程系, ARC智能过程设计与控制研究中心, 克莱顿, 维多利亚, 澳大利亚
^b中国科学院过程工程研究所,生物药制备与递送全国重点实验室, 北京 100190, 中国
^c蒙纳士大学材料科学与工程系, 克莱顿, 维多利, 澳大利亚

收稿日期:2025-06-09 接受日期:2025-09-06 出版日期:2026-01-18 发布日期:2026-01-05
通讯作者: 刘闽苏,张松平
基金资助:
国家自然科学基金(22478401);中国科学院战略先导研究项目(XDB1250000);澳大利亚研究委员会(ARC)(IH230100010)

Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration

Shiyi Che^a^,^b, Zhengjun Li^b, Zhiguo Su^b, Zhikao Li^a, Aibing Yu^a, Minsu Liu^a^,^c^,^*(), Songping Zhang^b^,^*()

^aARC Research Hub for Smart Process Design and Control, Department of Chemical and Biological Engineering, Monash University, Clayton, Victoria 3800, Australia
^bState Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
^cDepartment of Materials Science and Engineering, Monash University, Clayton, Victoria 3800, Australia

Received:2025-06-09 Accepted:2025-09-06 Online:2026-01-18 Published:2026-01-05
Contact: Minsu Liu, Songping Zhang
About author:Minsu Liu (Monash University) is a senior research fellow at Monash Suzhou Research Institute and a senior lecturer at Department of Materials Science and Engineering, Monash University. He has obtained his B.E. (Hon. 1) and Ph.D. from the University of New South Wales (Australia) and Monash University (Australia), respectively. Before joining Monash, Dr. Liu worked as postdoctoral fellow at Monash University (2017‒2018) and Tsinghua University (2018‒2020). Dr. Minsu Liu is expertise in the research of nanofabrication, 2D materials, self-assembly, and thermal management. He has published more than 50 peer-reviewed journal articles with more than 2000 citations, and applied/granted more than 50 patents.
Songping Zhang (Institute of Process Engineering, Chinese Academy of Sciences) received her B.S. in 1998 and Ph.D degree in 2002 from Tianjin University, China. From 2002‒2004, she did postdoctoral research at Lund University, Sweden. Afterward, she joined Institute of Process Engineering in Jan. 2005. Prof. Zhang’ research direction focusses on biomedicine engineering, specializing in the synthesis, assembly, purification, and formulation of biomacromolecules including mRNA, proteins, recombinant virus like particles vaccine antigens, et al. She has published over 150 research papers and holds more than 40 invention patents, with multiple achievements succeed in industrial applications. She serves as an editorial board member for J. Chromatogr. A and Biochem. Engin. J.
Supported by:
National Natural Science Foundation of China(22478401);Strategic Priority Research Program of the Chinese Academy of Sciences(XDB1250000);Australian Research Council (ARC)(IH230100010)

摘要/Abstract

摘要：

信使RNA(mRNA)作为新一代治疗性药物与预防性疫苗的平台, 在肿瘤治疗、疾病预防、基因修复等领域展现出巨大潜力. 然而, mRNA类药物的临床疗效高度依赖其分子结构的完整性, 而这一点又直接取决于其生产工艺的稳定性与可控性. 与传统小分子药物合成不同, mRNA制造依赖复杂的多酶催化级联反应, 这些反应涉及构象复杂且动态的生物大分子, 包括作为模板的质粒DNA、中间产物、以及生物催化剂. mRNA的制造包括如下四个由不同的酶及辅因子催化, 但互相高度依赖的核心酶模块: DNA模板制备(模块1)、mRNA的体外转录合成(模块2)与加帽修饰(模块3)、以及多种核酸酶参与的酶解去除杂质(模块4). 上述各酶催化模块之间缺乏单元操作的兼容性且效率较低, 传统的多酶固定化或区室化策略往往难以兼顾高通量与兼容性, 导致mRNA连续规模化工艺开发存在显著瓶颈. 因此, 系统梳理这些酶模块的工程化设计策略, 对于提升mRNA生产效率、推动产业化应用具有重要意义.

本文系统综述了mRNA生产过程中上述四大核心酶模块的研究进展, 重点分析了各模块的核心酶催化机制、催化调控策略及其在工艺集成中的应用. 首先总结了当前mRNA制造工艺的流程, 并分析其面临的两大挑战为多酶模块调控的复杂性和不兼容性. 然后, 将mRNA制造工艺分为质粒线性化制备DNA模板、体外转录合成mRNA、mRNA加帽修饰以及核酸酶参与的酶解去除杂质四个酶模块, 分别介绍了这四个酶模块的研究进展和发展趋势. 其中重点围绕体外转录和加帽修饰两个酶模块展开, 详细地阐述了酶的工程性改造(温度适应性酶、底物选择性酶、构象转化型酶)、酶和DNA模板共固定化、添加辅助酶协同反应、核苷酸底物修饰、双链RNA和未加帽mRNA等副产物抑制、提高反应动力学等策略在催化调控中的应用. 其后, 基于文献中报道的mRNA工艺集成策略, 概括为三个方向: 质粒线性化和体外转录集成、转录与加帽集成(共转录加帽、双功能酶、连续级联反应设计、连续过程控制)、反应分离耦合(固定化策略与反应相结合), 进一步探讨比较了这些策略的可行性和优缺点. 最后, 简要总结了文中的酶催化和集成策略所面临的挑战和未来的研究方向: (1) 在体外转录模块, 如何在产率和副产物控制之间取得平衡仍是关键难点, 可通过反应机理建模与人工智能辅助优化; (2) 在工艺集成方面, 不同模块对于镁离子的需求差异较大, 如何实现跨模块的统一调控对连续化生产尤为关键; (3) 探索新型反应介质和固定化策略, 有望同时实现酶回收和产物分离, 以进一步提高反应效率和经济可行性; (4) 应大力发展在线监控和过程自动化, 以满足连续化大规模工业应用的需要.

综上, 本综述系统地总结了mRNA制造中的酶模块在催化调控和工艺集成中的最新进展, 不仅梳理了当前关键酶模块的工程化策略, 也为未来实现高效、可扩展的mRNA连续制造平台提供了思路与启示, 对推动mRNA的大规模产业化发展具有重要参考价值.

关键词: mRNA制造, 酶模块, 催化调控, 工艺集成, 多酶反应

Abstract:

The clinical efficacy of mRNA-based therapeutics is critically dependent on the structural integrity of the mRNA molecule, which in turn is governed by the efficiency and robustness of its manufacturing process. Unlike conventional small-molecule synthesis, mRNA manufacturing relies on complex enzymatic cascades involving biomacromolecules with dynamic conformations as templates, intermediates, and catalysts. Key enzymatic modules, including plasmid linearization for DNA template preparation (Module 1), in vitro transcription (IVT) synthesis (Module 2), capping modification (Module 3) of mRNA, and different nucleases-aided removal of impurities (Module 4), are highly interdependent, each with specific catalytic enzymes and auxiliary cofactors. These modules present major engineering challenges of low efficiency and lack of modular compatibility across the multi-step enzymatic processes. Moreover, traditional approaches such as multienzyme immobilization or compartmentalization often fail to meet the demands of high-throughput, continuous and scalable manufacturing. This review systematically summarizes recent advances in the engineering of enzymatic modules for mRNA manufacturing, emphasizing challenges in catalytic regulation, module integration and process intensification. The potential strategies for improving reaction compatibility and enabling process integration and intensification are discussed, providing insights into future directions for engineering mRNA synthesis at scale.

Key words: mRNA manufacturing, Enzymatic modules, Catalytic regulation, Process integration, Multienzyme reaction

车诗怡, 李正军, 苏志国, 李之考, 余艾冰, 刘闽苏, 张松平. 面向mRNA制造的酶模块工程化策略进展: 催化调控与过程集成[J]. 催化学报, 2026, 80: 92-112.

Shiyi Che, Zhengjun Li, Zhiguo Su, Zhikao Li, Aibing Yu, Minsu Liu, Songping Zhang. Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration[J]. Chinese Journal of Catalysis, 2026, 80: 92-112.

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图/表 13

Fig. 1. Flow chart for manufacturing of capped mRNA from plasmid preparation to enzymatic capping, which includes four typical catalytic reaction modules. Module 1: Circular plasmid DNA is linearized by the restriction endonuclease (REase). Module 2: The linearized plasmid DNA serves as the template for IVT synthesis of mRNA catalyzed by T7 RNAP along with PPase. Module 3: Enzymatic capping of mRNA by vaccinia capping enzyme (VCE) and 2’-O-methyltransferase (2’-O-MTase) to generate the cap-1 mRNA. Module 4: A panel of nucleases mainly including RNase A, DNase I, RNase III, and RNase R, which are employed to degrade the residual RNA in the plasmid, residual plasmid DNA and dsRNA in the IVT mRNA, linear precursor RNA impurities in the circular RNA, respectively. Between each module, chromatographic processes are integrated to ensure intermediate and final product purity.

Fig. 2. REases for circular plasmid linearization. Left: Different topological forms of plasmid. Right: Two main types of Type II REase cleavages.

Fig. 3. IVT reaction catalyzed by T7 RNAP and PPase. (A) Mechanism of IVT reaction for mRNA synthesis: T7 RNAP catalysis, Mg2PPi byproduct formation and PPase catalysis. Conformational changes of initiation phase (PDB: 1QLN) (B) and elongation phase (PDB: 1H38) (C). (D) NTP insertion. Reprinted with permission from Ref. [37]. Copyright 2009, Elsevier. (E) RNA byproducts. (E-1) Incomplete RNA transcripts. (E-2) Intramolecular (cis) and intermolecular (trans) 3’extension. (E-3) Antisense RNA by promoter-less DNA. (E-4) Non-covalent mRNA aggregates.

Fig. 4. Enzyme engineering and co-immobilization of RNAP. (A) Thermophilic RNAP (G788A). (B) mRNA synthesis by psychrophilic Syn5 RNAP and VSW-3 RNAP. Reprinted with permission from Ref. [54]. Copyright 2022, Taylor & Francis. (C) RNAP for conformation transition (G47A and an extra C-terminal 884G). Reprinted with permission from Ref. [58]. Copyright 2023, Springer Nature. (D) Immobilized strategy for simultaneous tethering T7 RNAP and DNA template. (D-1) Tethering Strep-tag T7 RNAP and 5’-biotin nontemplate strand of DNA to Strep-TactinXT-coated magnetic beads. Reprinted with permission from Ref. [60]. Copyright 2021, Elsevier. (D-2) Tethering 5’-biotin template strand of DNA to streptavidin magnetic beads with promoter on the nontemplate strand linked HaloTag-T7 RNAP. Reprinted with permission from Ref. [61]. Copyright 2024, Oxford University Press.

Table 1 In-process regulation strategies in the IVT module.

Regulation		Impact	Condition	Ref.
Enzyme engineering	thermophilic RNAP	reduced 3’extension dsRNA	high-temperature	[50]
	psychrophilic RNAP	reduced 3’extension dsRNA	low-temperature	[53,54]
	P266L mutant	reduced abortive RNA	—	[56]
	S43Y mutant G47A + 884G mutant	reduced 3’extension dsRNA	—	[57,58]
	G47W mutant	reduced antisense dsRNA	—	[59]
Co-immobilization	DNA was tethered with T7 RNAP to beads	reduced 3’extension dsRNA/Higher yield	high-salt	[60,61]
Using modified NTP	Ψ, m¹Ψ, m⁵C, 5moU	reduced antisense dsRNA	—	[44,69,70]
Using modified NTP	increased ARCA/ m¹Ψ	reduced immunogenicity	—	[75]
Optimizing RNA sequence	-4 gap on the non-template strand of the DNA promoter	reduced 3’extension dsRNA/Higher yield	high-salt	[76]
Optimizing RNA sequence	additional AT-rich sequences at T7 promoter downstream	reduced abortive RNA and dsRNA/Higher yield	high-temperature	[77]
Reaction process regulation	fed-batch strategy	higher yield	—	[78,79]
	low ratio of Mg²⁺/NTP	reduced antisense dsRNA	—	[80]
	low UTP concentrations	reduced 3’extension dsRNA	—	[42,74]
	addition of urea or formamide	reduced 3’extension dsRNA	—	[81,82]

Fig. 5. Reaction process regulation and model. (A-1) The fed-batch strategy with pH-controlled addition of KOH, NTP and Mg(OAc)2. Reprinted with permission from Ref. [78]. Copyright 1999, American Institute of Chemical Engineers. (A-2) The fed-batch strategy with at-line HPLC-monitored addition of NTPs and Mg2+. Reprinted with permission from Ref. [79]. Copyright 2023, Wiley. (B) Bayesian optimization approach with a robotic liquid handler to enable high-throughput algorithmic optimization. Reprinted with permission from Ref. [94]. Copyright 2024, American Chemical Society. (C) A new IVT reaction model reported by Stover et al. Reprinted with permission from Ref. [95]. Copyright 2024, Wiley.

Fig. 6. Conformation of (A) vaccinia capping enzyme (PDB: 4CKB) and (B) 2’-O-methyltransferase (PDB: 1AV6). (A-1) Domains and mRNA capping pathways in VCE [104]. (A-2) FigureTPase with a closed hydrophilic tunnel formed by eight β-barrels. (A-3) NTase domain with GTP-binding site and OB domain in GTase. (A-4) N7-MTase domain with SAM-binding site. Engineering in-process regulation strategies of immobilization of VCE and 2’-O-MTase (C), characterization of novel FCE and its comparison to VCE at different temperatures (Reprinted with permission from Ref. [107]. Copyright 2023, Cold Spring Harbor Laboratory Press) (D), and VCE catalysis with different GTP analogs: Chemical structure and capping efficiency (E). Reprinted with permission from Ref. [113]. Copyright 2023, Oxford University Press.

Table 2 Summary of nucleases in mRNA manufacturing.

Enzyme	Source	Type	Metal ions	Application
RNase A	Bovine pancreas	Endo-	independent	RNA removal in circular plasmid preparation
DNase I	Bovine pancreas	Endo-	Mg²⁺, Ca²⁺	DNA removal after the IVT reaction
RNase III	E. coli	Endo-	Mg²⁺	dsRNA removal after the IVT reaction
RNase R	E. coli	Exo-	Mg²⁺	linear RNA precursor removal in circRNA purification

Fig. 7. Nucleases conformation and engineering in mRNA manufacturing. (A-1) Interactions between RNase A (PDB: 3DXG) and RNase inhibitor (PDB: 4PEQ). (A-2) Immobilization of RNase A by macroporous monolithic support. (B) Adsorption of DNase I (PDB: 1DNK) by AuNP with a capping agent surface. (C) The homodimeric structure of E. coli RNase Ⅲ (PDB: 7R97). (D) Structure of E. coli RNase R. Reprinted with permission from Ref. [133]. Copyright 2017, Oxford University Press.

Fig. 8. Three strategies for process integration in mRNA manufacturing and their corresponding main characteristics and challenges. Briefly, the first strategy is integration between circular plasmid linearization and IVT modules (Modules 1 and 2), the second type is integration between IVT and capping modules (Modules 2 and 3), and the third type is reaction-separation coupling, which usually requires solid-phase-immobilized mRNA. These three reaction modules involved different reaction types of site cleavage, de novo synthesis and site modification, respectively.

Fig. 9. Integration between IVT and capping modules. (A) Co-transcription with cap analogs. (B) Fusion of T7 RNAP and FCE reported by Robb et al. [124]. (C) Continuous cascade reaction by integrated buffer reported by Nwokeoji et al. Reprinted with permission from Ref. [145]. Copyright 2023, American Chemical Society. (D) Continuous process control by digital twin reported by Helgers et al. Reprinted with permission from Ref. [146]. Copyright 2021, MDPI.

Table 3 General conditions of the enzymatic reactions catalyzed by T7 RNAP, VCE and 2’-O-MTase.

Enzyme	Condition
Enzyme	Temperature	pH	Buffer	Mg²⁺	spermidine	DTT	Na⁺/K⁺/Cl^-
T7 RNAP	37 °C	7.5-8.0	Tris-HCl	10-50 mmol/L	1-2 mmol/L, promote DNA condensation	1-10 mmol/L	inhibitory at concentrations > 0.1 mol/L
VCE	37-45 °C			1 mmol/L	—	1 mmol/L
2’-O-MTase	45 °C			1 mmol/L	—	1 mmol/L

Fig. 10. Reaction-separation coupling for mRNA manufacturing. (A) The combined IVT-solid-phase reaction-separation approach published by Liu et al. Reprinted with permission from Ref. [151]. Copyright 2015, Springer Nature. (B) The chemoenzymatic method published by Thillier et al. Reprinted with permission from Ref. [152]. Copyright 2012, Cold Spring Harbor Laboratory Press. (C) The on‐column capping of poly dT-tethered mRNA proposed by Che et al. Reprinted with permission from Ref. [153]. Copyright 2024, Wiley.

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面向mRNA制造的酶模块工程化策略进展: 催化调控与过程集成

Engineering of enzymatic modules for mRNA manufacturing: Advances in catalytic regulation and process integration

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