2'-岩藻糖基乳糖的酶法合成研究进展和展望

doi:10.12211/2096-8280.2020-033

摘要/Abstract

摘要：

人乳寡糖（human milk oligosaccharides，HMOs）是人乳中一类结构复杂、非消化性的碳水化合物。2'-岩藻糖基乳糖（2'-fucosyllactose，2'-FL）是人乳中含量最高的寡糖，也是最早被FDA和欧盟批准可添加到婴幼儿奶粉、膳食补充剂以及医疗食品中的HMOs之一。2'-FL具有调节肠道菌群、抵抗病原菌的黏附、免疫调节及促进神经系统发育和修复等多种功能活性。2'-FL的主要合成方法有化学合成法、全细胞合成法及酶催化合成法。全细胞合成法是当前工业上生产2'-FL的主要方法，降低L-岩藻糖的成本、调节合成途径中鸟苷二磷酸-L-岩藻糖（GDP-岩藻糖）的水平与菌体生长之间的平衡、发掘新型高活性的α-1，2-岩藻糖基转移酶是降低全细胞合成2'-FL成本的关键。2'-FL的合成途径在一些更为安全表达宿主（如无抗生素大肠杆菌、枯草芽孢杆菌和酵母菌等）中的构建也面临着挑战。本文重点综述了2'-FL酶法合成的研究现状，利用α-1，2-岩藻糖基转移酶合成2'-FL专一性好，但所需糖基供体GDP-岩藻糖成本较高；利用α-L-岩藻糖苷酶的转糖苷活性也可以合成2'-FL，α-L-岩藻糖苷酶来源广泛，易获得，稳定性好，可利用天然底物作为糖基供体。将α-L-岩藻糖苷酶应用于2'-FL合成的关键在于高效的转糖苷酶以及天然、经济的岩藻糖基供体的发掘。酶法合成将来有望成为工业上生产2'-FL的方法。

关键词: 人乳寡糖, 2'-岩藻糖基乳糖, GDP-岩藻糖, 酶法合成, α-L-岩藻糖苷酶, 酶工程

Abstract:

Human milk oligosaccharides (HMOs) constitute a unique group of endogenous indigestible carbohydrates in human breast milk. HMOs play a crucial role in infant health and growth. As the most abundant HMO, 2'-fucosyllactose (Fucα1, 2Galβ1, 4Glc, 2'-FL) has been approved for infant formulas, dietary supplements and medical foods in the United States and European Union. 2'-FL has been synthesized by chemical, enzymatic synthesis and cell factory approaches, and currently mainly produced by cell factory approach. The crucial factors for 2'-FL production are the reduction of the cost of L-fucose, the discovery of novel α1,2-fucosyltransferases, and the maintenance of the balance between intracellular GDP-L-fucose level and the growth of engineering strain. Also, the construction of antibiotic-free system (such as antibiotic-free Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) is still a challenge for the synthesis of 2'-FL. In this review, the research progress of enzymatic synthesis of 2'-FL was particularly presented. 2'-FL could be enzymatically synthesized using α-1,2-fucosyltransferases or α-L-fucosidases. α-1,2-Fucosyltransferases catalyze the transformation of a fucose from a GDP-L-fucose to a lactose. The main disadvantage for 2'-FL synthesis by fucosyltransferase is the requirement for an expensive nucleotide donor. Also, α-L-fucosidases have been studied extensively since they catalyze the synthesis of 2'-FL through a transglycosylation reaction and often possess a higher availability and activity, in comparison with fucosyltransferases. The discovery of efficient transfucosidases and the availability of appropriate, fucosylated donor substrates will promote the application of α-L-fucosidases in the synthesis of 2'-FL. In the near future, enzymatic synthesis is expected to become a method for industrial production of 2'-FL.

Key words: human milk oligosaccharides, 2'-fucosyllactose, GDP-L-fucose, enzymatic synthesis, α-L-fucosidase, enzyme engineering

中图分类号:

Q814.9

史然, 江正强. 2'-岩藻糖基乳糖的酶法合成研究进展和展望[J]. 合成生物学, 2020, 1(4): 481-494.

SHI Ran, JIANG Zhengqiang. Enzymatic synthesis of 2'-fucosyllactose： advances and perspectives[J]. Synthetic Biology Journal, 2020, 1(4): 481-494.

图/表 7

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分类	化合物	结构式	浓度范围 /g·L^-1	摩尔分数/%
neutral fucosylated HMOs	2'-FL	Fucα1,2Galβ1,4Glc	0.06~3.93	31
	3-FL	Galβ1,4(Fucα1,3)Glc	0.03~1.34	5
	DFL (2',3-FL)	Fucα1,2Galβ1,4(Fucα1,3)Glc	0.28~0.43	4
	LNFP Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.001~2.08	8
	LNFP Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc	0.02~1.79	2
	LNFP Ⅲ	Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc	0.06~0.78	2
	LNFP Ⅴ	Galβ1,3GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.06	—
	LNFP Ⅵ	Galβ1,4GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.01	—
	LNDFH Ⅰ	Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc	0.43~1.87	4
	LNDFH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.02~0.25	—
	F-LNH Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.2~2.62	—
	F-LNH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4GlcNAcβ1-6Galβ1-4Glc	0.18~1.06	—
	DF-LNH Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	0.31	—
	DF-LNH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1-6)Galβ1-4Glc	0.12~1.02	—
	TF-LNH	Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	2.60~3.10	—
neutral non-fucosylated HMOs	LNT	Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.16~1.54	6
	LNnT	Galβ1,4GlcNAcβ1,3Galβ1,4Glc	0.04~2.04	6
	LNH	Galβ1,3GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.05~0.17	—
	LNnH	Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.09~0.28	—
sialylated HMOs	3'-SL	Neu5Acα2,3Galβ1,4Glc	0.09~0.30	2
	6'-SL	Neu5Acα2,6Galβ1,4Glc	0.07~0.59	6
	LSTa	Neu5Acα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.01~0.18	—
	LSTb	Galβ1,3(Neu5Acα2,6)GlcNAcβ1,3Galβ1,4Glc	0.04~0.25	—
	LSTc	Neu5Acα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glc	0.05~1.05	—
	DSLNT	Neu5Acα2,3Galβ1,3(Neu5Acα2,6)GlcNAcβ1,3Galβ1,4Glc	0.10~0.80	2
	FS-LNnH Ⅰ	Neu5Acα2,6Galβ1,4GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	0.26~0.55	—
other HMOs				13

分类	化合物	结构式	浓度范围 /g·L^-1	摩尔分数/%
neutral fucosylated HMOs	2'-FL	Fucα1,2Galβ1,4Glc	0.06~3.93	31
	3-FL	Galβ1,4(Fucα1,3)Glc	0.03~1.34	5
	DFL (2',3-FL)	Fucα1,2Galβ1,4(Fucα1,3)Glc	0.28~0.43	4
	LNFP Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.001~2.08	8
	LNFP Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc	0.02~1.79	2
	LNFP Ⅲ	Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc	0.06~0.78	2
	LNFP Ⅴ	Galβ1,3GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.06	—
	LNFP Ⅵ	Galβ1,4GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.01	—
	LNDFH Ⅰ	Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc	0.43~1.87	4
	LNDFH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4(Fucα1,3)Glc	0.02~0.25	—
	F-LNH Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.2~2.62	—
	F-LNH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4GlcNAcβ1-6Galβ1-4Glc	0.18~1.06	—
	DF-LNH Ⅰ	Fucα1,2Galβ1,3GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	0.31	—
	DF-LNH Ⅱ	Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1-6)Galβ1-4Glc	0.12~1.02	—
	TF-LNH	Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	2.60~3.10	—
neutral non-fucosylated HMOs	LNT	Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.16~1.54	6
	LNnT	Galβ1,4GlcNAcβ1,3Galβ1,4Glc	0.04~2.04	6
	LNH	Galβ1,3GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.05~0.17	—
	LNnH	Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Galβ1,4Glc	0.09~0.28	—
sialylated HMOs	3'-SL	Neu5Acα2,3Galβ1,4Glc	0.09~0.30	2
	6'-SL	Neu5Acα2,6Galβ1,4Glc	0.07~0.59	6
	LSTa	Neu5Acα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc	0.01~0.18	—
	LSTb	Galβ1,3(Neu5Acα2,6)GlcNAcβ1,3Galβ1,4Glc	0.04~0.25	—
	LSTc	Neu5Acα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glc	0.05~1.05	—
	DSLNT	Neu5Acα2,3Galβ1,3(Neu5Acα2,6)GlcNAcβ1,3Galβ1,4Glc	0.10~0.80	2
	FS-LNnH Ⅰ	Neu5Acα2,6Galβ1,4GlcNAcβ1,3(Galβ1,4(Fucα1,3)GlcNAcβ1,6)Galβ1,4Glc	0.26~0.55	—
other HMOs				13

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