Assessment of the Two Helicobacter pylori α‐1,3‐Fucosyltransferase Ortholog Genes for the Large‐Scale Synthesis of LewisX Human Milk Oligosaccharides by Metabolically Engineered Escherichia coli

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Abstract
We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto‐ N‐neotetraose Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (LNnT). The major product obtained was lacto‐ N‐neofucopentaose‐V Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]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(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]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 futBled to equal amounts of both lacto‐ N‐neofucopentaose‐V and lacto‐ N‐neofucopentaose‐II Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)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(β1–4)[Fuc(α1–3)]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.