母乳低聚糖生物合成研究进展

doi:10.12211/2096-8280.2025-047

摘要/Abstract

摘要：

母乳低聚糖（Human milk oligosaccharides，HMOs）是母乳中的核心营养成分，对于婴幼儿的生长发育具有无可替代的生理功效，是新一代婴幼儿配发奶粉的核心原料。目前针对HMOs的研究主要集中在生理功能、临床应用以及生物合成技术开发等方面。鉴于HMOs广阔的市场需求，高效生物合成HMOs逐渐成为研究热点。合成生物学技术的突破使微生物发酵法大规模生产HMOs成为可能，显著提升了产业化进程的经济可行性。本文系统综述近年来HMOs的研究进展并展望了HMOs的发展趋势及挑战，包括：（1）HMOs相关生理功能研究进展；（2）HMOs生物合成关键糖基转移酶研究进展；（3）基于微生物细胞工厂的HMOs生物合成路径设计；（4）用于改造HMOs合成底盘细胞的合成生物学策略。通过解析HMOs生物合成关键酶元件筛选、代谢途径通量平衡及底盘细胞代谢网络调控等核心科学问题，为HMOs的高效生物制造提供理论依据与技术参考。

关键词: 母乳低聚糖, 糖基转移酶, 合成生物学, 代谢网络调控

Abstract:

Human milk oligosaccharides (HMOs), recognized as key bioactive constituents of human milk, play indispensable roles in infant growth and development through immunomodulation, gut microbiota regulation, and pathogen defense mechanisms. These multifaceted compounds have emerged as pivotal ingredients in next-generation infant formula formulations. Current investigations primarily concentrate on three domains: elucidation of biological functions, exploration of therapeutic applications, and optimization of biosynthesis platforms. The growing commercial demand for HMOs has driven significant advancements in enzymatic synthesis and metabolic engineering approaches to achieve cost-effective production at industrial scales. The rapid development of synthetic biology has revolutionized the production of HMOs, making large-scale microbial fermentation a viable and economically feasible strategy for industrial applications. This paradigm shift has significantly accelerated the commercialization of HMOs, which are increasingly recognized for their critical roles in infant nutrition, gut microbiota modulation, and immune system development. In this comprehensive review, we systematically evaluate the latest research progress in HMOs, with particular emphasis on their biosynthesis, functional mechanisms, and biotechnological production, focusing on four key dimensions: (1) Physiological functions and mechanistic insights of HMOs-recent studies have elucidated the prebiotic, antimicrobial, and immunomodulatory properties of HMOs, highlighting their structural diversity and structure-function relationships. Advances in glycomics and microbiome research have deepened our understanding of how HMOs influence host-microbe interactions and metabolic pathways. (2) Discovery and engineering of glycosyltransferases for HMOs biosynthesis-glycosyltransferases (GTs) play a pivotal role in determining the structural diversity of HMOs. We review the latest strategies for enzyme mining, characterization, and protein engineering, including directed evolution and computational design, to enhance catalytic efficiency and substrate specificity. (3) Metabolic pathway design and optimization in microbial cell factories–the construction of efficient microbial chassis for HMOs production requires systematic pathway engineering. Key considerations include precursor supply, cofactor balancing, and the elimination of metabolic bottlenecks. We discuss recent advances in dynamic pathway regulation and modular assembly techniques to improve yield and purity. (4) Synthetic biology strategies for chassis cell development–the optimization of host strains involves genome-scale metabolic modeling, CRISPR-based genome editing, and adaptive laboratory evolution. Additionally, novel approaches such as cell-free biosynthesis and consortia-based fermentation are emerging as promising alternatives for complex HMOs synthesis. Furthermore, we discuss emerging challenges and future directions in this field.

Key words: human milk oligosaccharides, glycosyltransferases, synthetic biology, metabolic network regulation

万李, 杨龙浩, 罗国聪, 朱莺莺, 沐万孟. 母乳低聚糖生物合成研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-047.

WAN Li, YANG Longhao, LUO Guocong, ZHU Yingying, MU Wanmeng. Research progress on biosynthesis of human milk oligosaccharides[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-047.

图/表 5

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宿主菌株	基因修饰	关键基因（来源）	产物及产量（g/L）	参考文献
E. coli BL21(DE3)	删除基因lacZ、wcaJ和pgi；整合三拷贝lacY基因；质粒过表达基因setA、rcsA、rcsB、NsfutC、manC、manB、gmd和wcaG	NsfutC（Neisseria sp.）	2′-FL，26.17 g/L（摇瓶）；141.27 g/L（5 L发酵罐）	[37]
E. coli BL21 Star (DE3)	删除基因fucI、fucK、lacZ、wcaJ、lon和lacA；整合基因manC、manB、gmd、wcaG、futM2、dtpA和glpF	futM2（Bacteroides gallinaceum）	3-FL，11.26 g/L（摇瓶）；60.24 g/L（5 L发酵罐）	[38]
E. coli BL21(DE3)	删除基因lacZ和wcaJ；manA、manC-manB和gmd-wcaG的启动子强化；整合基因SAMT（两拷贝）、fut3Bc和RcsAB；质粒过表达基因SAMT和fut3Bc	SAMT（Azospirillum lipoferum）；fut3Bc（Neobacillus cucumis）	DFL，8.34 g/L（摇瓶）；53.15 g/L（5 L发酵罐）	[39]
E. coli BL21 Star (DE3)	删除基因lacZ、nanAKET、nagB、pfkB和pfkA；整合基因neuBCA（两拷贝）、nST、glmM、glmS、cmk、lacY和scrY*	nST（Neisseria gonorrheae）	3′-SL，8.12 g/L（摇瓶）；56.8 g/L（5 L发酵罐）	[40]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因lgtA、Pf、galE、udK和pyrF	lgtA（N. meningitidis）；Pf（Pseudogulbenkiania ferrooxidans）	LNT，6.16 g/L（摇瓶）；57.5 g/L（5 L发酵罐）	[41]
E. coli BL21(DE3)	删除基因lacZ、nanA、pfkA、nanK和manA；质粒过表达基因neuBCA、glnA、plst6∗、pyrG、ndk和cmk	plst6∗（Photobacterium leiognathi JT-SHIZ-119）	6′-SL，3.85 g/L（摇瓶）；25.31 g/L（3 L发酵罐）	[42]
E. coli BL21 Star (DE3)	删除基因lacZ、ugd、ushA、agp、wcaJ、otsA、wcaC、lacA和nagB；整合基因galETKM、lgtA-galE、hpgalT（四拷贝）、lgtA、galE和CmSET；基因galU和glmM的启动子强化	lgtA（N. meningitidis）；hpgalT（H. pylori）	LNnT，112.47 g/L（5 L发酵罐）；107.4 g/L（1000 L发酵罐）	[34]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE、lgtA（四拷贝）和wbgO（三拷贝）；质粒过表达基因 manC、manB、gmd、wcaG、wbgO和thspR2FT	lgtA（N. meningitidis）；wbgO（E. coli O55:H7）；thspR2FT（Thermoanaerobacterium sp. RBIITD）	LNFP I，4.42 g/L（摇瓶）；35.1 g/L（5 L发酵罐）	[43]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因 manC、manB、gmd、wcaG、wbgO和Bf13FT(K128D)	lgtA（N. meningitidis）；wbgO（E. coli O55:H7）；Bf13FT（Bacteroides fragilis NCTC 9343）	LNFP V，2.44 g/L（摇瓶）；25.68 g/L（5 L发酵罐）	[44]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因 manC、manB、gmd、wcaG、wbgO、fucT14和futM1	lgtA（N. meningitidis）；wbgO（Escherichia coli O55:H7）；fucT14（Helicobacter pylori DMS 6709）； futM1（a Bacteroidaceae bacterium from gut metagenome）	LNDFH Ⅱ，3.01 g/L（摇瓶）；18.06 g/L（5 L发酵罐）	[45]
E. coli K-12 W310	删除基因lacZY和wcaJM；质粒过表达基因HpgalT、 NplgtA、PgsfucT、rcsA和lacY	NplgtA（Neisseria polysaccharea ATCC 43768）； HpgalT（H. pylori NCTC 11637）；PgsfucT（Parabacteroides goldsteinii JCM 13446）	LNFP Ⅲ，1.05 g/L（摇瓶）；3.84 g/L（3 L发酵罐）	[46]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB、ugD、nanA、nanK和nanT；整合基因galE和lgtA（四拷贝）；质粒过表达基因neuB、neuC、neuA、wbgO和nst	lgtA（N. meningitidis）；wbgO（Escherichia coli O55:H7）；nst（Neisseria meningitidis）	LST a，1.24 g/L（摇瓶）；4.85 g/L（5 L发酵罐）	[47]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB、ugD、nanA、nanK和nanT；整合基因galE和lgtA（四拷贝）；质粒过表达基因neuB、neuC、neuA、hpgalT和ed6st	lgtA（N. meningitidis）；hpgalT（H. pylori J99）；ed6st（Vespertiliibacter pulmonis）	LST c，1.72 g/L（摇瓶）；9.74 g/L（5 L发酵罐）	[36]

宿主菌株	基因修饰	关键基因（来源）	产物及产量（g/L）	参考文献
E. coli BL21(DE3)	删除基因lacZ、wcaJ和pgi；整合三拷贝lacY基因；质粒过表达基因setA、rcsA、rcsB、NsfutC、manC、manB、gmd和wcaG	NsfutC（Neisseria sp.）	2′-FL，26.17 g/L（摇瓶）；141.27 g/L（5 L发酵罐）	[37]
E. coli BL21 Star (DE3)	删除基因fucI、fucK、lacZ、wcaJ、lon和lacA；整合基因manC、manB、gmd、wcaG、futM2、dtpA和glpF	futM2（Bacteroides gallinaceum）	3-FL，11.26 g/L（摇瓶）；60.24 g/L（5 L发酵罐）	[38]
E. coli BL21(DE3)	删除基因lacZ和wcaJ；manA、manC-manB和gmd-wcaG的启动子强化；整合基因SAMT（两拷贝）、fut3Bc和RcsAB；质粒过表达基因SAMT和fut3Bc	SAMT（Azospirillum lipoferum）；fut3Bc（Neobacillus cucumis）	DFL，8.34 g/L（摇瓶）；53.15 g/L（5 L发酵罐）	[39]
E. coli BL21 Star (DE3)	删除基因lacZ、nanAKET、nagB、pfkB和pfkA；整合基因neuBCA（两拷贝）、nST、glmM、glmS、cmk、lacY和scrY*	nST（Neisseria gonorrheae）	3′-SL，8.12 g/L（摇瓶）；56.8 g/L（5 L发酵罐）	[40]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因lgtA、Pf、galE、udK和pyrF	lgtA（N. meningitidis）；Pf（Pseudogulbenkiania ferrooxidans）	LNT，6.16 g/L（摇瓶）；57.5 g/L（5 L发酵罐）	[41]
E. coli BL21(DE3)	删除基因lacZ、nanA、pfkA、nanK和manA；质粒过表达基因neuBCA、glnA、plst6∗、pyrG、ndk和cmk	plst6∗（Photobacterium leiognathi JT-SHIZ-119）	6′-SL，3.85 g/L（摇瓶）；25.31 g/L（3 L发酵罐）	[42]
E. coli BL21 Star (DE3)	删除基因lacZ、ugd、ushA、agp、wcaJ、otsA、wcaC、lacA和nagB；整合基因galETKM、lgtA-galE、hpgalT（四拷贝）、lgtA、galE和CmSET；基因galU和glmM的启动子强化	lgtA（N. meningitidis）；hpgalT（H. pylori）	LNnT，112.47 g/L（5 L发酵罐）；107.4 g/L（1000 L发酵罐）	[34]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE、lgtA（四拷贝）和wbgO（三拷贝）；质粒过表达基因 manC、manB、gmd、wcaG、wbgO和thspR2FT	lgtA（N. meningitidis）；wbgO（E. coli O55:H7）；thspR2FT（Thermoanaerobacterium sp. RBIITD）	LNFP I，4.42 g/L（摇瓶）；35.1 g/L（5 L发酵罐）	[43]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因 manC、manB、gmd、wcaG、wbgO和Bf13FT(K128D)	lgtA（N. meningitidis）；wbgO（E. coli O55:H7）；Bf13FT（Bacteroides fragilis NCTC 9343）	LNFP V，2.44 g/L（摇瓶）；25.68 g/L（5 L发酵罐）	[44]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB和ugD；整合基因galE；质粒过表达基因 manC、manB、gmd、wcaG、wbgO、fucT14和futM1	lgtA（N. meningitidis）；wbgO（Escherichia coli O55:H7）；fucT14（Helicobacter pylori DMS 6709）； futM1（a Bacteroidaceae bacterium from gut metagenome）	LNDFH Ⅱ，3.01 g/L（摇瓶）；18.06 g/L（5 L发酵罐）	[45]
E. coli K-12 W310	删除基因lacZY和wcaJM；质粒过表达基因HpgalT、 NplgtA、PgsfucT、rcsA和lacY	NplgtA（Neisseria polysaccharea ATCC 43768）； HpgalT（H. pylori NCTC 11637）；PgsfucT（Parabacteroides goldsteinii JCM 13446）	LNFP Ⅲ，1.05 g/L（摇瓶）；3.84 g/L（3 L发酵罐）	[46]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB、ugD、nanA、nanK和nanT；整合基因galE和lgtA（四拷贝）；质粒过表达基因neuB、neuC、neuA、wbgO和nst	lgtA（N. meningitidis）；wbgO（Escherichia coli O55:H7）；nst（Neisseria meningitidis）	LST a，1.24 g/L（摇瓶）；4.85 g/L（5 L发酵罐）	[47]
E. coli BL21(DE3)	删除基因lacZ、wecB、nagB、ugD、nanA、nanK和nanT；整合基因galE和lgtA（四拷贝）；质粒过表达基因neuB、neuC、neuA、hpgalT和ed6st	lgtA（N. meningitidis）；hpgalT（H. pylori J99）；ed6st（Vespertiliibacter pulmonis）	LST c，1.72 g/L（摇瓶）；9.74 g/L（5 L发酵罐）	[36]