中性核心母乳寡糖生物合成的研究进展和发展趋势

doi:10.12211/2096-8280.2025-083

摘要/Abstract

摘要：

母乳寡糖（human milk oligosaccharide，HMO）是母乳中重要的功能和营养成分。其中，中性核心母乳寡糖（neutral core human milk oligosaccharide，ncHMO）主要包括乳糖-N-三糖（LNT Ⅱ）、乳糖-N-新四糖（LNnT）和乳糖-N-四糖（LNT），在婴幼儿健康发育过程中发挥着不可替代的生理作用。近年来，中性核心母乳寡糖的生物合成技术快速发展，其工业化生产从可能走向现实。本文综述了中性核心母乳寡糖的酶法和微生物细胞法合成研究现状，介绍了糖基转移酶和糖苷酶在酶法合成中性核心母乳寡糖的应用，讨论了糖基转移酶的催化特性、底盘细胞的种类及改造等关键因素对微生物细胞法合成中性核心母乳寡糖产量的影响，进一步对比了两种方法生物合成中性核心母乳寡糖的优缺点。目前，中性核心母乳寡糖的生物合成存在酶催化效率低、底盘细胞选择与适配性差、副产物多及内毒素污染等问题，需通过理性设计酶元件、优化安全底盘、动态调控代谢网络及强化发酵纯化工艺等策略协同攻关，有望实现母乳寡糖的低成本、高效绿色生产，为开发更具营养价值的婴幼儿健康食品提供核心支撑。

关键词: 母乳寡糖, 乳糖-N-三糖, 乳糖-N-新四糖, 乳糖-N-四糖, 生物合成

Abstract:

Human milk oligosaccharides (HMOs) are essentially functional and nutritional components found in human milk. They can be primarily classified into fucosylated, neutral core, and sialylated HMOs. Lacto-N-triose Ⅱ (LNT Ⅱ), lacto-N-neotetraose (LNnT), and lacto-N-tetraose (LNT) are common neutral core human milk oligosaccharides (ncHMOs), which can be extended to form longer-chain HMOs and play important roles in intestinal health. In recent years, the biosynthesis of ncHMOs has developed rapidly, and industrial-scale production is from theoretical possibility to practical reality. The synthesis approaches for ncHMOs include chemical synthesis, enzymatic synthesis, and microbial cell synthesis. As the rapid development in biotechnology, enzymatic and microbial cell synthesis have emerged as prominent methods in ncHMOs biosynthesis. Enzymatic synthesis is highly efficient, regioselective, and stereoselective. Currently, glycosyltransferases and glycoside hydrolases represent the two major types of enzymes used for biosynthesizing ncHMOs. Glycosidase-based enzymatic synthesis has demonstrated high conversion rates for LNT Ⅱ and LNnT production. However, the enzymatic synthesis of LNT is less efficient and requires further improvement. Notably, the production of LNnT and LNT typically relies on LNT Ⅱ as a key precursor, requiring a multi-step synthetic strategy. Microbial cell synthesis employs metabolic engineering to construct continuously synthetic pathways in microbial cells such as Escherichia coli and Bacillus subtilis. Knocking out genes in competitive pathway, optimizing genes expression, regenerating cofactors have significantly enhanced the yields of ncHMOs. The biosynthesis of ncHMOs faces several critical challenges, including the low activity and poor substrate specificity of key glycosyltransferases, such as β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase. Additionally, the transporters of LNT Ⅱ and LNnT are not clear in microbial cell. Furthermore, the yields of LNT Ⅱ should be substantially improved for industrial-scale production. Thus, it is important to overcome the interconnected limitations in enzyme engineering (particularly glycosyltransferase specificity and activity), microbial cell modification (focusing on metabolic compatibility and pathway design), and bioprocess optimization (through rational pathway redesign) via an integrated synthetic biology and fermentation engineering approach in the future. These strategies are essential for achieving efficient, cost-effective biosynthesis of ncHMO at industrial scale.

Key words: human milk oligosaccharides, lacto-N-triose Ⅱ (LNT Ⅱ), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), biosynthesis

中图分类号:

Q815

刘丹, 王建宇, 江正强. 中性核心母乳寡糖生物合成的研究进展和发展趋势[J]. 合成生物学, 2025, 6(5): 1126-1144.

LIU Dan, WANG Jianyu, JIANG Zhengqiang. Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides[J]. Synthetic Biology Journal, 2025, 6(5): 1126-1144.

图/表 9

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classification	HMOs	concentrations in colostrum/(g/L)	concentrations in mature breast milk/(g/L)
neutral HMOs	2′-FL	3.03 ± 1.79	1.64 ± 1.54
	3-FL	0.41 ± 0.43	1.35 ± 1.00
	LNFP Ⅰ	1.90 ± 0.96	0.70 ± 0.67
	LNFP Ⅱ	0.28 ± 0.37	0.54 ± 0.52
	LDFT	0.65 ± 0.71	0.19 ± 0.21
	LNDFH Ⅰ	0.47 ± 0.26	0.25 ± 0.25
	LNDFH Ⅱ	0.08 ± 0.10	0.12 ± 0.15
	DFLNH	0.05 ± 0.11	0.28 ± 0.30
neutral core HMOs	LNT Ⅱ	0.16 ± 0.08	0.03 ± 0.01
	LNT	0.59 ± 0.39	0.59 ± 0.27
	LNnT	0.40 ± 0.12	0.34 ± 0.14
sialylated HMOs	3′-SL	0.21 ± 0.06	0.10 ± 0.02
	6′-SL	0.33 ± 0.11	0.22 ± 0.09
	DSLNT	0.36 ± 0.14	0.19 ± 0.07
	LSTc	1.37 ± 0.56	0.23 ± 0.18

classification	HMOs	concentrations in colostrum/(g/L)	concentrations in mature breast milk/(g/L)
neutral HMOs	2′-FL	3.03 ± 1.79	1.64 ± 1.54
	3-FL	0.41 ± 0.43	1.35 ± 1.00
	LNFP Ⅰ	1.90 ± 0.96	0.70 ± 0.67
	LNFP Ⅱ	0.28 ± 0.37	0.54 ± 0.52
	LDFT	0.65 ± 0.71	0.19 ± 0.21
	LNDFH Ⅰ	0.47 ± 0.26	0.25 ± 0.25
	LNDFH Ⅱ	0.08 ± 0.10	0.12 ± 0.15
	DFLNH	0.05 ± 0.11	0.28 ± 0.30
neutral core HMOs	LNT Ⅱ	0.16 ± 0.08	0.03 ± 0.01
	LNT	0.59 ± 0.39	0.59 ± 0.27
	LNnT	0.40 ± 0.12	0.34 ± 0.14
sialylated HMOs	3′-SL	0.21 ± 0.06	0.10 ± 0.02
	6′-SL	0.33 ± 0.11	0.22 ± 0.09
	DSLNT	0.36 ± 0.14	0.19 ± 0.07
	LSTc	1.37 ± 0.56	0.23 ± 0.18

来源	酶	受体	供体	转化率/%	参考文献
米曲霉（Aspergillus oryzae）	NagA	乳糖	GlcNAc	0.21	[41]
两歧双歧杆菌（Bifidobacterium bifidum）	BbhI-WT	乳糖	β-D-GlcpNAcOpNP	16	[43]
两歧双歧杆菌（B. bifidum）	BbhI-D714T	乳糖	pNP-GlcNAc	84.7	[44]
两歧双歧杆菌（B. bifidum）	BbhI-D746E	乳糖	Glc-oxa	86.0	[45]
两歧双歧杆菌（B. bifidum）	BbhI-D746T	乳糖	pNP-GlcNAc	85.0	[44]
光冈链状杆菌（Catenibacterium mitsuokai）	CmHex187	乳糖	pNP-GlcNAc	44.3	[46]
居海藻黄杆菌（Flavobacterium algicola）	FlaNag2353	乳糖	pNP-GlcNAc	4.15	[47]
Haloferula sp.	HaHex74	乳糖	(GlcNAc)₂	14.4	[48]
Haloferula sp.	mHaHex74	乳糖	(GlcNAc)₂	28.2	[49]
土壤宏基因组	HEX1	乳糖	(GlcNAc)₂	16.1	[50]
紫色链霉菌（Streptomyces violascens）	Hex(Sv)-2557(D297K)	乳清粉	(GlcNAc)₂	14.85	[51]
丁氏泰泽菌（Tyzzerella nexilis）	TnHex189	乳糖	pNP-GlcNAc	57.2	[52]

来源	酶	受体	供体	转化率/%	参考文献
米曲霉（Aspergillus oryzae）	NagA	乳糖	GlcNAc	0.21	[41]
两歧双歧杆菌（Bifidobacterium bifidum）	BbhI-WT	乳糖	β-D-GlcpNAcOpNP	16	[43]
两歧双歧杆菌（B. bifidum）	BbhI-D714T	乳糖	pNP-GlcNAc	84.7	[44]
两歧双歧杆菌（B. bifidum）	BbhI-D746E	乳糖	Glc-oxa	86.0	[45]
两歧双歧杆菌（B. bifidum）	BbhI-D746T	乳糖	pNP-GlcNAc	85.0	[44]
光冈链状杆菌（Catenibacterium mitsuokai）	CmHex187	乳糖	pNP-GlcNAc	44.3	[46]
居海藻黄杆菌（Flavobacterium algicola）	FlaNag2353	乳糖	pNP-GlcNAc	4.15	[47]
Haloferula sp.	HaHex74	乳糖	(GlcNAc)₂	14.4	[48]
Haloferula sp.	mHaHex74	乳糖	(GlcNAc)₂	28.2	[49]
土壤宏基因组	HEX1	乳糖	(GlcNAc)₂	16.1	[50]
紫色链霉菌（Streptomyces violascens）	Hex(Sv)-2557(D297K)	乳清粉	(GlcNAc)₂	14.85	[51]
丁氏泰泽菌（Tyzzerella nexilis）	TnHex189	乳糖	pNP-GlcNAc	57.2	[52]

来源	酶	受体	供体	转化率/%	参考文献
大肠杆菌（Escherichia coli）O55:H7	WbgO	LNT Ⅱ	UDP-Gal	87	[66]
紫色色杆菌（Chromobacterium violaceum）	Cvβ3GalT	LNT Ⅱ	UDP-Gal	99	[67]
金杆菌（Aureobacterium sp.）L-101	—	乳糖	pNP-LNB	0.6	[57]
两歧双歧杆菌（B. bifidum）JCM 1254	LnbB-D320E	乳糖	LNB-oxa	30	[68]
两歧双歧杆菌（B. bifidum）JCM 1254	LnbB-W394F	乳糖	pNP-LNB	32	[69]
两歧双歧杆菌（B. bifidum）JCM 1254	LnbB-W394H	乳糖	LNB-oxa	72	[70]
环状芽孢杆菌（Bacillus circulans）ATCC 31382	β-gal-3	LNT Ⅱ	oNPG	20，22	[57,71]
玉米乳酪杆菌（Lacticaseibacillus zeae）	LzBgal35A	LNT Ⅱ	oNPG	45.4	[72]