糖质ivBT生物合成的核心元件及关键技术

doi:10.12211/2096-8280.2025-064

摘要/Abstract

摘要：

糖质（glycans）是构成生命的基本生物分子，在生物过程中发挥重要作用，与人类健康和疾病密切相关。由于糖质结构的复杂性，糖质所携带的密码信息大大超过核酸及蛋白质等任何其它生物大分子。为了破译糖质携带的密码信息，就需要获得一定量的、结构明确的糖质。合成仍然是获得糖质的最佳方式。体外生物转化（in vitro biotransformation， ivBT）是一个基于体外多酶催化的新型工业生物制造技术，由酶、辅酶等元件体外重构生化反应途径，实现目标产物的高效合成，正逐渐发展成为合成糖质的一种重要技术。本文综述了ivBT在糖质合成中的应用、ivBT糖质合成相关的糖核苷合成、糖基转移、NTP再生等核心元件及酶的固定化、微流控、酶促自动化合成、动力学模拟、离子液体等关键技术，展望了糖质ivBT合成相关的酶的协同性、特殊重组酶生产成本的降低、仿生辅酶的循环再生、多酶复合体的应用、硫酸化糖质的合成、AI辅助的反应条件优化等发展方向，旨在为糖质ivBT合成的未来发展提供指导。

关键词: 糖质的体外生物合成, 体外生物转化, 酶催化模块, NTP再生, 糖质体外生物合成的关键技术

Abstract:

Glycans are the fundamental biomolecules that make up life and play important roles in biological processes, including cell growth and proliferation, immune responses, angiogenesis and tumor cell metastasis, toxin interaction, protein folding and degradation, cell-cell communications, and cell-pathogen interactions, and are closely related to human health and disease such as occurrence, development, and metastasis of tumor, inflammation, viral or bacterial infections, etc. Due to their structural complexity arising from the number of chemically similar (and often isomeric) monosaccharide building blocks, the position and orientation of glycosidic linkages, branching, and non-template-driven biosynthesis, the coding information they carry far exceeds that of any other biological macromolecules such as nucleic acids and proteins, which are generally linear structures and biosynthesized based on templates. To decipher the code information carried by glycans, it is necessary to obtain some structurally defined pure glycans. Since it is not possible to separate and purify glycans with defined structures from natural environment, synthesis remains the best approach to obtaining glycans. In vitro biotransformation (ivBT), a novel industrial biomanufacturing platform based on multi-enzyme catalysis in vitro, has become an essential approach to obtain high value-added products due to its appealing advantages in rapid construction of nonnatural enzymatic pathways, higher product yields, faster reaction rates, and better tolerances to toxic compounds. ivBT is gradually being developed into an important means of glycan synthesis. This article reviews application of ivBT in glycan synthesis, the core elements such as synthesis of sugar-nucleotides, glycosyl transfer and NTP regeneration, and key technologies of ivBT-based glycan synthesis like enzyme immobilization, microfluidics, enzyme-mediated automatic synthesis, dynamic simulation, and ion liquids, aiming to provide guidance for its future development, including synergism of multi-enzyme, reduction of production costs for special recombinant enzymes, regeneration of biomimetic coenzymes, application of complexes of multi-enzyme, ivBT synthesis of sulfated glycans and AI-assisted optimization of reaction conditions of ivBT synthesis, etc.

Key words: ivBT-based glycan synthesis, In vitro biotransformation (ivBT), Enzymatic modules, NTP regeneration, Key technologies of ivBT-based glycan synthesis

中图分类号:

Q814.9

裴彩霞, 李建军, 杜昱光. 糖质ivBT生物合成的核心元件及关键技术[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-064.

PEI Caixia, LI Jianjun, DU Yuguang. Core Elements and Key Technologies of ivBT-based Glycan Synthesis[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-064.

图/表 8

表1 三种糖质合成方法的比较

Table 1 Comparison of three methods for glycan synthesis

	化学法	酶法	细胞工厂法
专一性	低	最高	高
选择性（区域、立体选择性）	低	最高	高
反应过程	繁琐（涉及到保护、脱保护以及活化单糖的过程）、反应时间长	反应过程简单、反应时间短	反应过程简单、反应时间短
反应条件	某些反应需要剧烈或特殊的条件（如高温、低温、高压等）	温和（37 ℃左右、pH 接近中性等）	温和（37 ℃左右、pH接近中性等）
环境	不友好（使用有机溶剂及重金属催化剂等）	友好（水溶液中反应、不用重金属催化剂）	友好（水溶液中反应、不用重金属催化剂
设备	某些反应需要特殊的设备（高压、低温等）	传统设备（发酵罐、反应罐）	传统设备（发酵罐）
转化率	低	高	低
产量	低	高	低
规模化生产	不适合	可以	可以

图1 中性（A）和唾液酸化（B）糖质的ivBT合成（非NTP再生条件）

Fig. 1 ivBT synthesis of neutral (A) and sialylated (B) oligosaccharides under conditions of non-NTP-regeneration

图2 NTP再生条件下中性（A）和唾液酸化（B）糖质的ivBT合成

Fig. 2 ivBT synthesis of neutral (A) and sialylated (B) oligosaccharides under conditions of NTP-regeneration

图3 UDP-Gal（A）、UDP-GlcA（B）、UDP-GlcNAcA（C）、UDP-GalNAcA（D）的ivBT合成GalK：半乳糖激酶；BLUSP：UDP-糖焦磷酸化酶；PPA：焦磷酸酶；GlcAK：葡萄糖醛酸激酶；NahK：N-乙酰己糖胺1-激酶；GlmU：N-乙酰葡萄糖胺-1磷酸尿苷酰转移酶或UDP-GlcNAc焦磷酸化酶

Fig. 3 ivBT synthesis of UDP-Gal(A)、UDP-GlcA(B)、UDP-GlcNAcA(C)、UDP-GalNAcA(D)GalK: galactokinase; BLUSP: UDP-sugar pyrophosphorylase; PPA: pyrophosphatase; GlcAK: glucuronokinase; NahK: N-acetylhexosamine 1-kinase; GlmU: UDP-GlcNAc pyrophosphorylase or N-acetylglucosamine-1-phosphateuridyltransferase

图4 UDP-Glc（A）、UDP-Xyl（B）、GDP-Man（C）、GDP-Fuc（D）、CMP-Sia（E）的ivBT合成HXK:己糖激酶; PGM:磷酸葡萄糖变位酶;UGP1:葡萄糖-1磷酸尿苷酰转移酶或UDP-葡萄糖焦磷酸化酶;UGHD:UDP-葡萄糖6-脱氢酶;UGD:UDP-葡萄糖醛酸脱羧酶;ScGalK:来源于沼泽红假单胞菌的半乳糖激酶;ScGPUT:来源于沼泽红假单胞菌的葡萄糖-1-磷酸尿苷酰转移酶或UDP-葡萄糖焦磷酸化酶;Glk:葡萄糖激酶;ManB:磷酸甘露糖变位酶;ManC:甘露糖-1-磷酸鸟嘌呤转移酶或GDP-甘露糖焦磷酸化酶;FKP:岩藻糖激酶/GDP-岩藻糖焦磷酸化酶或岩藻糖-1-(双功能酶);CSS:CMP-唾液酸合成酶

Fig. 4 ivBT synthesis of UDP-Glc(A)、UDP-Xyl(B)、GDP-Man(C)、GDP-Fuc(D) 、CMP-Sia(E)HXK: hexokinase; PGM: phosphoglucomutase; UGP1: glucose-1-phosphateuridyltransferase or UDP-glucose pyrophosphorylase; UGHD: UDP-glucose-6-dehydrogenase; UGD: UDP-glucuronate decarboxylase; ScGalK: galactokinase from Solitalea canadensis; ScGPUT: glucose-1-phosphate uridyltransferase or UDP-Glc pyrophosphorylase from Solitalea canadensis; Glk: glucokinase; ManB: phosphomannomutase; ManC: mannose-1-phosphate-guanyltransferase or GDP-Man pyrophosphorylase; FKP: fucokinase/GDP-Fuc pyrophosphorylase or fucose-1-phosphateguanyltransferase; CSS: CMP-sialic acid synthetase

图5 基于数字微流控的HMOs的ivBT合成［78］

Fig. 5 ivBT synthesis of HMOs on the DMF platform

图6 PAPS的再生［104］ASAK：双功能酶，ATP硫酸化酶 + 3′-磷酸腺苷-5′-磷酰硫酸激酶

Fig. 6 Regeneration of PAPSASAK: bifunctional enzyme, ATP sulfurylase + 3′-phosphoadenosine 5′-phosphosulfate kinase

表2 ivBT和细胞工厂的优劣势比较

Table 2 Comparison of pros and cons of ivBT and cell factory

	优势	劣势
ivBT	用几个工程菌株表达酶，每个菌株的负担轻；已知HMOs结构有不到20种糖苷键，只需要构建相应模块就可以合成所有的HMOs；能保证每步酶催化反应的转化率在90%以上；反应的副产物少，分离纯化相对简单；不需要解决产物进出细胞的问题	需要解决能量ATP再生和还原力平衡的问题；酶的成本问题
细胞工厂	生产过程相对简单；不需要解决能量ATP再生和还原力平衡的问题。	所有的基因都在一个菌株里，菌株的负担重；一个产品就要构建一个细胞工厂；不能保证每一步反应的转化率都在90%以上；细胞工厂的代谢产物复杂，给后面的分离纯化带来难度；需要解决产物进出细胞的问题；高效细胞工厂的构建复杂、繁琐。

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