无细胞多酶系统的模块化构建

doi:10.1016/S1872-2067(21)64002-0

摘要/Abstract

摘要：

无细胞多酶催化是绿色生物制造领域的新兴技术之一. 该技术通过设计和构建新的多酶合成路线, 使得一些从天然途径难以获得的化学品的生产成为可能, 具有广阔的应用前景. 尽管无细胞多酶系统的构建具有高度的灵活性和可调性, 但如何实现人工系统中多酶反应的兼容性和协同性仍是目前多酶体系构建的难点.

针对这一难题, 模块化构建和优化无细胞多酶体系的策略近年来受到广泛关注. 尽管该策略已被应用于实践, 但人们对于模块化的概念仍存在不同的理解. 本文通过功能性来定义多酶模块, 一个模块通常指共同作用并执行特定功能的一组酶, 且同一模块内部的酶与酶之间应具有紧密的互作关系. 另外, 多酶体系的模块化还发生在从分子到反应器等多个不同的水平. 介绍了自然界中的多酶模块, 包括途径水平和分子水平的多酶模块, 并简要介绍了这些天然模块的特点和功能. 途径水平的多酶模块主要指生物体中一些承担特定生理生化功能的多酶反应(如糖酵解、糖异生、柠檬酸循环等). 分子水平的多酶模块则主要包括多酶复合物和模块化酶, 重点介绍了丙酮酸脱氢酶复合体、纤维小体、嘌呤体和非核糖体多肽合成酶等典型的例子. 本文还梳理了近年来模块化构建人工无细胞多酶体系的重要进展, 并根据模块在反应路线中的功能, 将其划分为主反应模块和辅助模块. 主反应模块部分重点介绍了用于碳代谢、碳同化、蛋白质糖基化和非核糖体多肽合成等四类具有代表性的新型人工构建的主反应模块. 辅助模块按功能分为能量平衡、多酶体系保护和催化功能强化三类, 并介绍了相关的研究进展及其在多酶催化过程中的实际应用. 讨论了在反应器水平上多酶催化过程的模块化, 通过将多酶反应进行合理拆分或组合并分配在不同的反应器模块中, 可以进一步优化多酶催化过程, 提高反应效率.

综上, 天然多酶体系和人工多酶体系的例子均表明, 模块化是构建和调控多酶催化过程的有力手段. 从分子到反应器多个水平的模块化策略使人们能够多尺度地设计、组织、优化和调控多酶催化过程, 将在生物制造领域发挥越来越重要的作用.

关键词: 无细胞生物合成, 体外合成生物学, 多酶系统, 模块化构建, 人工模块

Abstract:

The implementation of multiple enzymes for chemical production in a cell-free scenario is an emerging field in biomanufacturing. It enables the redesign and reconstitution of new enzymatic routes for producing chemicals that may be hard to obtain from natural pathways. Although the construction of a cell-free multienzyme system is highly flexible and adaptable, it is challenging to make all enzymatic reactions act in concert. Recently, modular construction has been conceptualized as an effective way to harmonize diverse enzymatic reactions. In this review, we introduce the concept of a multienzyme module and exemplify representative modules found in Nature. We then categorize recent developments of synthetic multienzyme modules into main-reaction modules and auxiliary modules according to their roles in reaction routes. We highlight four main-reaction modules that can perform carbon metabolism, carbon assimilation, protein glycosylation and nonribosomal peptide synthesis, and exemplify auxiliary modules used for energy supply, protection and reinforcement for main reactions. The reactor-level modularization of multienzyme catalysis is also discussed.

Key words: Cell-free biosynthesis, In vitro synthetic biology, Multienzyme system, Modular construction, Synthetic module

张寅晨, 聂宁, 张一飞. 无细胞多酶系统的模块化构建[J]. 催化学报, 2022, 43(7): 1749-1760.

Yinchen Zhang, Ning Nie, Yifei Zhang. Toward modular construction of cell-free multienzyme systems[J]. Chinese Journal of Catalysis, 2022, 43(7): 1749-1760.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(21)64002-0

https://www.cjcatal.com/CN/Y2022/V43/I7/1749

图/表 6

Fig. 1. Example of natural multienzyme modules. (a) A section view of pyruvate dehydrogenase complex. Reprinted with permission from Ref. [25]. Copyright 2001, National Academy of Sciences of the United States of America. (b) Schematic representation of cellulosome. CipA, cellulosome integrating protein A; OlpB, outer layer protein B; SLH, bacterial SLH domain (for S-layer homology). (c) A fluorescence image of purinosomes and the illustration of protein-protein interactions among purinosome proteins. ATIC, a bifunctional enzyme composed of aminoimidazolecarboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase; ASL, adenylosuccinate lyase; PAICS, a bifunctional enzyme composed of carboxyaminoimidazole ribonucleotide synthase and succinoaminoimidazolecarboxamide ribonucleotide synthetase; FGAMS, formylglycinamidine ribonucleotide synthase; PPAT, phosphoribosylpyrophosphate amidotransferase; GARS, glycinamide ribonucleotide synthetase; AIRS, aminoimidazole ribonucleotide synthetase; TGART, a trifunctional enzyme composed of glycinamide ribonucleotide synthetase, GAR formyltransferase and aminoimidazole ribonucleotide synthetase; GAR Tfase, GAR transformylase. Reprinted with permission from Ref. [34]. Copyright 2013, Royal Society of Chemistry. (d) A non-ribosomal peptide synthtase for the production of surugamide A.

Fig. 2. Schematic representation of carbon metabolism pathways for hydrogen generation from biomass and the production of myo-inositol from starch. αGP, α-glucan phosphorylase; PGM, phosphoglucomutase; stav CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PPGK, polyphosphate glucokinase; CTec2, Cellic CTec2 cellulase; XK, xylulokinase; IPS, inositol 1-phosphate synthase; IMP, inositol monophosphatase; G6PDH, glucose 6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; H2ase, hydrogenase; R5PI, ribose 5-phosphate isomerase; Ru5PE, ribulose 5-phosphate epimerase; TK, transketolase; TAL, transaldolase; TIM, triose phosphate isomerase; ALD, aldolase; FBP, fructose 1,6-bisphosphate; PGI, phosphoglucose isomerase.

Fig. 3. An artificial starch anabolic pathway (ASAP) that can produce starch from CO2 and H2. The pathway consists of four modules: C1, C3, C6 and Cn modules (individually colored). aox, alcohol oxidase; fls, formolase; dak, dihydroxyacetone kinase; tpi, triosephosphate isomerase; fba, fructose-1,6-bisphosphate aldolase; fbp, fructose-bisphosphatase; pgi, phosphoglucose isomerase; pgm, phosphoglucomutase; agp, ADP-glucose pyrophosphorylase; ss, starch synthase; ppa, pyrophosphatase; ppk, polyphosphate kinase. Reprinted with permission from Ref. [53]. Copyright 2021, AAAS.

Fig. 4. A platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). GTs, glycosyltransferases; ApNGT, the N-linked glycosyltranferase from A. pleuropneumoniae. Reprinted with permission from Ref. [55]. Copyright 2019, Nature Publishing Group.

Fig. 5. Reprogramming NRPS systems with DNA scaffolds. (a) Gramicidin S is synthesized by two NRPS enzymes, designated GrsA and GrsB. (b) Assembling individual modules of GrsB on DNA templates can successfully produce the precursor of Gramicidin S, fPVOL.

Fig. 6. Reactor-level modularization of multienzyme systems. (a) The continuous flow system can produce a series of amines through different packed bed modules. Reprinted with permission from Ref. [92]. Copyright 2021, Wiley-VCH. (b) A three-enzyme cascade capillary bioreactor for digestion of genomic DNA strands. SVP, snake venom phosphodiesterase; ALPase, alkaline phosphatase. Reprinted with permission from Ref. [93]. Copyright 2018, American Chemical Society.

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