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首页> 外文期刊>Biotechnology Progress >Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli
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Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli

机译：代谢工程大肠杆菌大规模合成LewisX人乳寡糖的两个幽门螺杆菌α-1,3-岩藻糖基转移酶直系同源基因的评估

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We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal( beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)-GlcNAc( beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Lex) was also produced. We report here a fermentation process for the large-scale production of Lex oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Lex activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Lex carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.
机译：我们以前描述了一种细菌发酵工艺，用于将乳糖体内转化为乳糖N-新四糖Gal（beta1-4）GlcNAc（beta1-3）Gal（beta1-4）Glc（LNnT）的岩藻糖基化衍生物。所获得的主要产物是乳糖-N-新葡聚糖-V Gal（β1-4）-GlcNAc（β1-3）Gal（β1-4）[Fuc（α1-3）] Glc，其在LNnT的葡糖残基上带有岩藻糖。也仅产生了少量在N-乙酰氨基葡萄糖残基上被岩藻糖基化的寡糖，并因此带有LewisX基团（Lex）。我们在这里报告了Lex寡糖大规模生产的发酵过程。为了优化体内岩藻糖基化，比较了幽门螺杆菌的两个岩藻糖基转移酶基因futA和futB（菌株26695）。发现futA对LNnT受体具有最佳活性，而futB在体外表达更好的Lex活性。两种基因均在工程化的大肠杆菌（E. coli）细胞中表达以产生寡糖。重组寡糖的岩藻糖基化模式与体外观察到的特异性密切相关，FutB有利于携带Lex的寡糖的形成。 Lacto-N-neodifucohexaose-II Gal（beta1-4）[Fuc（alpha1-3）] GlcNAc（beta1-3）Gal（beta1-4）[Fuc（alpha1-3）] Glc占总寡糖量的70％进行了futA的驱动发酵，浓度为1.7 g / L。 futB驱动的发酵导致等量的乳酸-N-新戊糖-V和乳酸-N-新戊糖-II Gal（beta1-4）[Fuc（alpha1-3）] GlcNAc（beta1-3）Gal（beta1-4 Glc，分别以280和260 mg / L生产。出乎意料的是，在futA上驱动的发酵过程中产生了显着比例（0.5 g / L）的人乳寡糖3-岩藻糖半乳糖Galβ1-4[Fuc（alpha1-3）] Glc，这说明岩藻糖基转移酶FutA的活性在大肠杆菌中的表达，并导致其对乳糖活性的重新评估。这两个fut基因的产物产生的所有寡糖都是人乳的天然化合物。

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