Production of globosides oligosaccharieds using metabolically engineered microorganisms

A method for producing an oligosaccharide comprising the galabiose motif (Galα-4Gal), referred as globosides, the method comprising culturing a first microorganism which is LacY+ (β-galactoside permease), LacZ− (β galactosidase), and MelA− (α-galactosidase) in a culture medium comprising lactose, wherein said first microorganism comprises a heterologous lgtC gene encoding α-1,4-Gal transferase which transfers a galactose moiety from UDP-Gal to the lactose to form globotriose (Galα-4Galβ-4Glc) and wherein lactose is in excess in the culture medium.

A microorganism comprising a heterologous lgtC gene encoding α-1,4-Gal transferase and which is LacY+ (β-galactoside permease), LacZ− (β galactosidase), and MBIA− (α-galactosidase).

A living microorganism which is LacY+ (β-galactoside permease), LacZ-− (β galactosidase), melA−(α-galactosidase) and comprises (a) a heterologous lgtD gene encoding β-3 GalNAc transferase, and (b) a wbpP gene encoding for UDP-GlcNAc-C4 epimerase, or a gne gene encoding for a UDP-glucose 4-epimerase, wherein said microorganism is capable of producing globotriose and/or globotetraose in vivo.

A method for catalyzing the transfer of a galactose moiety from UDP-Gal to globotetraose to form globopentaose (β-3 Gal transferase activity) comprising catalyzing the transfer with a lgtD gene encoding β-3 GalNAc transferase, in particular the lgtD gene from Haemophilus influenzae of SEQ ID No 3.

A culture medium comprising globotriose at a concentration of 1 to 10 g/L.

A commercial scale composition comprising one or more globosides selected from the group consisting of globotriose, globotetraose, globopentaose, and galactosyl-globosides including globo-H hexasaccharide, and sialosyl galactosyl globoside (SGG) hexasaccharide.

A method for producing an oligosaccharide comprising a galabiose motif (Galα-4Gal), the method comprising culturing a first microorganism in a culture medium comprising lactose, wherein said microorganism comprises a heterologous gene encoding α-1,4-Gal transferase which transfers a galactose moiety from UDP-Gal to the lactose to form globotriose (Galα-4Galβ-4Glc) and wherein said lactose is in excess in the culture medium.

A method of catalyzing the transfer of a galactose moiety from UDP-Gal to an acceptor bearing the terminal non reducing structure GalNAcβ-3-R to form the Galβ-3GalNAcβ-3-R structure, comprising catalyzing the transfer with the glycosyltransferase encoded by the lgtD gene from Haemophilus influenzae of SEQ ID NO:3 or a sequence having at least 80% identity thereof, as a β1,3 galactosyl transferase

• wherein R is selected from the group consisting of galactose, β galactosides such as allyl-β-galactoside or propargyl-β-galactoside, α galactosides, globotriose, β globotrioside such as allyl-β-globotrioside or propargyl-β-globotrioside, a globotrioside, and galactose-X;
• and
• wherein X is a reactive group allowing the covalent coupling with an other molecule, including amino, azide and nitrophenyl groups.

A method of producing an oligosaccharide selected from Galβ-3GalNAcβ-3Gal, Galβ-3GalNAcβ-3Galα-X, Galβ-3GalNAcβ-3Galβ-X, Galβ-3GalNAcβ-3Galβ-allyl, Galβ-3GalNAcβ-3Galβ-propragyl, Galβ-3GalNAcβ-3Galα-4Galβ-4Gal (Globopentaose), Galβ-3GalNAcβ-3Galα-4Galβ-4Galα-X, Galβ-3GalNAcβ-3Galα-4Galβ-4Galβ-X, Galβ-3GalNAcβ-3Galα-4Galβ-4Galβ-allyl, and Galβ-3GalNacβ-3Galα-4Galβ-4Galβ-propargyl, wherein said oligosaccharide is produced by a microorganism comprising an heterologous lgtD gene from Haemophilus influenzae of SEQ ID NO:3 or a sequence having at least 80% identity thereof.

A method of transferring a GalNAc residue to galactose to form GalNAcβ-3Gal and to produce oligosacharrides comprising GalNAcβ-3Gal, comprising a lgtD gene encoding a GalNAc transferase, in particular the lgtD from H. influenzae (SEQ ID NO:3).

A method for producing an oligosaccharide comprising the motif GalNAcβ-3Gal, comprising culturing a microorganism which is galP (galactose permease), LacZ− (β galactosidase), MelA− (α-galactosidase) and wbpP encoding for UDP-GlcNAc-C4 epimerase, such as the Pseudomonas aeruginosa wbpP gene;

• or a gne gene encoding for a UDP-glucose 4-epimerase, such as the gne gene of C. jejuni strain NCTC 11168 of SEQ ID No 9, in a culture medium comprising galactose, wherein said microorganism comprises a heterologous lgtD gene encoding α-1,4-Gal transferase which transfers a GalNAc residue to galactose to form the an oligosaccharide comprising GalNAcβ-3Gal.