P1 trisaccharide (Galα1,4Galβ1,4GlcNAc) synthesis by enzyme glycosylation reactions using recombinant Escherichia coli
P1 trisaccharide (Galα1,4Galβ1,4GlcNAc) synthesis by enzyme glycosylation reactions using recombinant Escherichia coli
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DOI：
10.1128/aem.69.4.2110-2115.2003
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发表时间：
2003-04-01
影响因子：
4.4
通讯作者：
Wang, PG
中科院分区：
文献类型：
--
作者：
Liu, ZY;Lu, YQ;Wang, PG
大肠杆菌感染的频率导致了人们对我们食品供应中的致病菌的担忧，以及对治疗药物的需求。肠道细胞上的糖脂是大肠杆菌产生的志贺样毒素的受体。这些糖脂的寡糖部分类似物可以与受体竞争毒素，从而起到抗菌剂的作用。本研究对低聚糖类似物P1三糖(Galalpha1，4Galbeta1，4GlcNAc)的酶促合成进行了研究。在所提出的合成途径中，UDP-葡萄糖是以蔗糖和鱼腥藻为原料合成的。蔗糖合成酶，然后用大肠杆菌UDP-葡萄糖4-差向异构酶转化为UDP-半乳糖。用幽门螺杆菌的β-1，4-半乳糖基转移酶和脑膜炎奈瑟氏菌的α-1，4-半乳糖基转移酶将两个半乳糖分子连接到N-乙酰氨基葡萄糖上，生成一个分子的P1三糖。这四种酶在单一的基因工程大肠杆菌菌株中共表达，然后被渗透并用于催化酶反应。以N-乙酰氨基葡萄糖的用量计算，P1三糖的积累量最大为50 mM(200ml反应体积为5.4g)，产率为67%。本研究为重组菌制备规模化合成P1三糖提供了一条有效途径。
The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galalpha1,4Galbeta1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori beta-1,4-galactosyltransferase and a Neisseria meningitidis alpha-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.