Large‐Scale Synthesis of H‐Antigen Oligosaccharides by Expressing Helicobacter pylori α1,2‐Fucosyltransferase in Metabolically Engineered Escherichia coli Cells

Angewandte Chemie
2006.0

Abstract

The H antigen is defined by the terminal disaccharide structure Fuca-2Gal, which constitutes the basis of the human ABO histo-blood group system and serves as a receptor for pathogens such as Campylobacter jejuni and Norwalk virus. Human milk contains large quantities of H-antigen oligosaccharides that act as soluble receptors to protect infants against infections, but these molecules cannot be readily purified from alternative natural sources, and chemical and enzymatic syntheses of the Fuca-2Gal motif are not applicable to large-scale production of H-antigen structures. Oligosaccharide synthesis by metabolically engineered Escherichia coli strains overexpressing heterologous glycosyltransferase genes is a powerful technique for low-cost production of complex carbohydrates. We developed an efficient method for the production of fucosyl α1,2-linked oligosaccharides by high-cell-density culture of metabolically engineered E. coli strains overexpressing the Helicobacter pylori futC gene for α1,2-fucosyltransferase. The SD5 strain, constructed by cotransforming the Col1F'DlacZ,DE3 host with pET-21afutC and pLNTR (harboring Neisseria meningitidis lgtAB and rcsA genes), produced lacto-N-neofucopentaose-1 (LNnF-1, 2, an H-2 antigen oligosaccharide) and 2'-fucosyllactose (3) as main products. The SD6 strain, expressing only futC and rcsA, efficiently converted lactose to 3: with an initial lactose concentration of 15 g/L and 45 h of culture, the intra- and extracellular concentrations of 3 reached 3 and 11 g/L, respectively. In conclusion, we established an efficient method for the production of fucosyl α1,2-linked oligosaccharides. Trisaccharide 2'-fucosyllactose (3), with its recognized anti-infective properties, very high yields, and extracellular recovery facilitating purification, could be produced industrially on a multiton scale for nutraceutic applications. Pentasaccharide LNnF-1 (2), containing the H-2 antigen structure, could be used as a precursor for the synthesis of other antigens of the ABH histo-blood-group system.

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