In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli.
We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, lacto-N-neodifucohexaose Galbeta1-4(Fucalpha1-3)Glc-NAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, and lacto-N-neodifucooctaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an alpha-1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon. To prevent fucosylation at the glucosyl residue, lactulose Galbeta1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.
Dumon
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Centre de Recherches sur les Macromolécules Végétales, 601 rue de la Chimie, BP53X, 38041 Grenoble cedex 09, France.
Priem
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B
Martin
S L
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Heyraud
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A
Bosso
C
C
Samain
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E
D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D005285:Fermentation; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D007792:Lactulose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D009844:Oligosaccharides; D005643:Fucose; D007792:Lactulose; C046434:lacto-N-hexaose; C013084:lacto-N-neotetraose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D007785:Lactose