用于多酶组装的可编程蛋白质支架助力稀有糖的生物合成

doi:10.1016/S1872-2067(25)64675-4

催化学报 ›› 2025, Vol. 72: 95-105.DOI: 10.1016/S1872-2067(25)64675-4

用于多酶组装的可编程蛋白质支架助力稀有糖的生物合成

高鑫^a, 唐广耀^a, 闫佳俊^a, 房森彪^b, 田康明^a, 路福平^a, 秦慧民^a^,^*()

^a天津科技大学生物工程学院, 工业发酵微生物教育部重点实验室, 天津市工业微生物重点实验室, 工业酶国家工程实验室, 天津 300457
^b天津医科大学肿瘤研究所分子药理研究室, 天津 300457

收稿日期:2024-12-29 接受日期:2025-03-13 出版日期:2025-05-18 发布日期:2025-05-20
通讯作者: *电子信箱: huiminqin@tust.edu.cn (秦慧民).
基金资助:
国家重点研发计划(2022YFC2104901);国家自然科学基金(32372279);天津科技大学优秀博士学位论文创新项目(YB2023005)

Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars

Xin Gao^a, Guangyao Tang^a, Jiajun Yan^a, Senbiao Fang^b, Kangming Tian^a, Fuping Lu^a, Hui-Min Qin^a^,^*()

^aKey Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, China
^bDepartment of Molecular pharmacology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300457, China

Received:2024-12-29 Accepted:2025-03-13 Online:2025-05-18 Published:2025-05-20
Contact: *E-mail: huiminqin@tust.edu.cn (H.-M. Qin).
Supported by:
National Key Research and Development Program of China(2022YFC2104901);National Natural Science Foundation of China(32372279);Innovation Project of Excellent Doctoral Dissertation of Tianjin University of Science and Technology(YB2023005)

摘要/Abstract

摘要：

稀有糖D-阿洛酮糖是自然界中存量较少的新型功能糖, 不仅可作为低能量甜味剂的理想替代品, 还具有降低血糖浓度、减少脂肪堆积、治疗慢性糖尿病等众多生理功能, 在食品医疗健康领域具有广阔的应用前景. 生物催化法因其具有反应条件温和、无毒副产物的生成等优势, 被认为是合成稀有糖的主要方式. 目前, D-阿洛酮糖的生物制备方法主要通过酮糖3-差向异构酶催化的异构化反应、微生物转化以及多酶级联反应实现.

然而, 异构化反应中存在热力学平衡瓶颈, 多酶催化的级联反应中涉及多酶的调控与适配以及中间体通量的问题, 阻碍了D-阿洛酮糖的高效合成. 因此, 利用多酶级联反应体系规避不利的热力学平衡, 并调控复合体中多酶的适配性是亟需解决的关键问题.

本文针对稀少糖生物合成路径中多酶适配及关键限速步骤等瓶颈问题, 通过基因编辑设计构建可选择性地结合目标酶分子的蛋白支架, 实现多酶在蛋白支架上的顺序排列与可控募集. 并以D-阿洛酮糖3-差向异构酶(DAE)、核糖醇脱氢酶(RDH)和甲酸脱氢酶(FDH)为目标酶构建体外多酶复合体, 用于稀有糖的生物合成. 结果表明, 与未组装的多酶催化体系相比, 基于蛋白支架组装的多酶复合体的催化性能提高了10.4倍, 并实现了95%以上的蒜糖醇产量. 组装体系与游离体系的分子动力学模拟分析表明, 组装体系中蛋白支架的限制作用缩短了相邻酶之间的级联反应距离, 促进了反应中间体在酶活性位点之间的连续转移, 从而增强了目标酶分子对底物的亲和力, 减少了底物分子历经多酶催化位点的反应时间, 进而提高了整体级联反应的催化活性和催化效率. 进一步地, 基于仿生矿化的固定化策略与蛋白支架的可寻址性, 制备了纳米晶体共固定化多酶催化复合体. 傅里叶变换红外光谱、X-射线粉末衍射、共聚焦激光显微镜及场发射扫描电子显微镜结果表明, 多酶复合体已均匀分布于纳米晶体材料的表面. 固定化的多酶复合体相比于其未固定态展示出更强的pH以及温度耐受性, 在操作稳定性方面也较为可观. 此外, 为了进一步验证该人工蛋白支架在胞内的可编程性和可寻址性, 构建胞内多酶自组装催化体系, 并通过支架蛋白的可编程性合理解决该路径中关键酶的限速问题, 实现胞内多酶自组装体的高效催化性能. 进一步将固定化多酶催化复合体与胞内多酶自组装催化体系构建双模块催化系统, 实现以D-果糖为出发底物的D-阿洛酮糖的高效生物合成, 进而规避其合成反应中存在的热力学平衡. 结果表明, 双模块催化系实现了以300 g L^-1 D-果糖为出发底物的D-阿洛酮糖的生物合成, D-阿洛酮糖的产率达到了90.6%, 时空生产率达到了13.6 g L^-1 h^-1.

综上所述, 本研究不仅可拓宽基于固定化技术制备有效稳定的工业酶制剂的方法学基础, 完善共固定化多酶的新型固定化策略, 为D-阿洛酮糖的工业高效绿色生物制备提供理论依据, 还将加快我国功能糖产业的技术革新, 提升国际竞争力.

关键词: 多酶级联反应, 蛋白质支架, 多酶复合物, 纳米反应器, 分子动力学模拟

Abstract:

Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot, offering considerable time and cost savings compared to a series of separate batch reactions. However, challenges related to coordination and regulatory interplay among multiple enzymes reduce the catalytic efficiency of such cascades. Herein, we genetically programmed a scaffold framework that selectively and orthogonally recruits enzymes as designed. The system was then used to generate multienzyme complexes of D-allulose 3-epimerase (DAE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) for rare sugar production. This scaffolded multienzymatic assembly achieves a 10.4-fold enhancement in the catalytic performance compared to its unassembled counterparts, obtaining allitol yield of more than 95%. Molecular dynamics simulations revealed that shorter distances between neighboring enzymes in scaffold-mounted complexes facilitated the transfer of reaction intermediates. A dual-module catalytic system incorporating (1) scaffold-bound complexes of DAE, RDH, and FDH and (2) scaffold-bound complexes of alcohol dehydrogenase and NADH oxidase expressed intracellularly in E. coli was used to synthesize D-allulose from D-fructose. This system synthesized 90.6% D-allulose from 300 g L⁻¹ D-fructose, with a space-time yield of 13.6 g L⁻¹ h⁻¹. Our work demonstrates the programmability and versatility of scaffold-based strategies for the advancement of multienzyme cascades.

Key words: Multienzymatic cascade reaction, Protein scaffold, Multienzymatic complexes, Nanoreactors, Molecular dynamics simulation

高鑫, 唐广耀, 闫佳俊, 房森彪, 田康明, 路福平, 秦慧民. 用于多酶组装的可编程蛋白质支架助力稀有糖的生物合成[J]. 催化学报, 2025, 72: 95-105.

Xin Gao, Guangyao Tang, Jiajun Yan, Senbiao Fang, Kangming Tian, Fuping Lu, Hui-Min Qin. Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars[J]. Chinese Journal of Catalysis, 2025, 72: 95-105.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(25)64675-4

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Fig. 1. Scaffolded strategy to assemble a tri-enzymatic cascade system. (a) Schematic representation of the three enzymes involved in the production of allitol. (b) Design and construction of multienzymatic assemblies on the protein scaffold. MALDI-TOF MS analysis (c) and dynamic light scattering analysis (d) of the purified SpyTag-DAE, SnoopTag-RDH, PDZlig-FDH, protein scaffold, and the assemblies. The molecular weights of the SnoopTag-RDH, SpyTag-DAE, PDZlig-FDH, protein scaffolds, and the assemblies were approximately 28.46, 32.90, 41.40, 55.70, and 159.34 kDa, respectively.

Fig. 2. Catalytic performance of the tri-enzymatic cascade reaction. (a) Allitol yield at the molar ratios were 1:1:2 and 1:1:3 of DAE, RDH, and FDH for the assembled system and the unassembled free system. (b) RMSD values of the assembled and unassembled models in 800-ns MD simulations. Changes in the spatial positions of three catalytic units both in the unassembled free systems (c) and assembled systems (d) from 800-ns MD simulations. Changes in distance triggered by the movement of spatial phases in both assembled and unassembled free systems were analyzed. The transfer of intermediates in this biosynthetic pathway was analyzed in both an unassembled free system (e) and assembled system (f) using six-phase-node steered MD simulations.

Fig. 3. Design and characterization of immobilized tri-enzyme assemblies. (a) Schematic of the preparation of co-immobilized DAE/RDH/FDH-scaffold on nanocrystals (DRF-S@nanocrystals) and investigation of the bioconversion process. Physical and chemical characterization of DRF-S@nanocrystals using FT-IR spectroscopy (b), and XRD analysis (c). (d) Confocal fluorescence microscopy images of DRF-S@nanocrystals. To identify DAE, RDH, and FDH, they were labeled with rhodamine B isothiocyanate (RhBITC), fluorescein isothiocyanate (FITC), and coumarin, respectively.

Fig. 4. Catalytic performance of the multienzyme system. (a) Specific activity and allitol yield of the immobilized forms (DRF-S@nanocrystals) and scaffolded forms (DRF-S) with D-fructose concentrations of 10-300 g/L. (b) Relative activities of DRF-S@nanocrystals and DRF-S were assessed after incubation at various temperatures and pH values. c) Relative activity and allitol yield of DRF-S@nanocrystals during 10 consecutive reaction cycles. d) Storage stability of DRF-S and DRF-S@nanocrystals.

Fig. 5. Biosynthesis of D-allulose using an organized bi-enzyme system. (a) Schematic diagram of the parallel reactions that generate D-allulose. (b) Cartoon images illustrating how ADH and NOX are clustered differently in the two strains. (c) TEM images of the Strains 1 and 2. Scale bar: 100 nm. (d) Scaffold-mediated protein clustering in E. coli cells in vivo was investigated using confocal laser scanning microscopy (CLSM). Representative images of mCherry- labeled SpyTag-NOX and GFP-labeled PDZlig-ADH, as well as a schematic diagram of the design and construction of Strains 1-1 and 2-1.

Fig. 6. (a) Schematic illustration of the dual-modular catalytic strategy utilized in this work for the production of D-allulose from D-fructose. (b) D-allulose production in Strains 1 and 2 in the time period from 30 to 240 min. (c) Specific activity profiles of Strains 1 and 2 in the allitol concentration range of 3 to 50 g L-1. (d) Production of D-allulose from D-fructose using the dual-modular catalytic strategy. Error bars indicate the standard deviations (n = 3).

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用于多酶组装的可编程蛋白质支架助力稀有糖的生物合成

Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars

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