Method of producing sialylated oligosaccharides

1. A method for producing a sialylated oligosaccharide, comprising:

a) culturing an Escherichia coli microorganism, comprising heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, wherein endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated, and wherein said microorganism produces internally, activated sialic acid as donor substrate for said sialyltransferase; and

b) culturing said microorganism in a culture medium comprising an exogenous precursor selected from the group consisting of lactose, galactose, β-galactoside, and α-galactoside, wherein active uptake into the microorganism of said exogenous precursor occurs and wherein said exogenous precursor is the acceptor substrate for said sialytransferase for producing the sialylated oligosaccharide.

2. The method according to claim 1 , wherein the sialyltransferase is α-2,3-sialyltransferase, α-2,3- and α-2,8-sialyltransferase (cstII), or α-2,6 sialyltransferase.

3. The method according to claim 1 , wherein the CMP-Neu5Ac synthetase is neuA, the sialic acid synthase is neuB, and the GlcNAc-6-phosphate 2 epimerase is neuC.

4. The method according to claim 1 , further comprising deletion or inactivation of nanT and nanE genes in the microorganism.

5. The method according to claim 1 , wherein said microorganism is LacY+ (β-galactoside permease), LacZ− (β galactosidase), and optionally MelA− (α-galactosidase).

6. The method according to claim 1 , wherein the α-galactoside is globotriose (Galα-4Galβ-4Glc).

7. A microorganism, wherein said microorganism is an Escherichia coli strain, comprising heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, wherein endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated, and wherein said microorganism produces internally, activated sialic acid as donor substrate for said sialytransferase.

8. A cell culture medium comprising lactose and the microorganism of claim 7 , wherein the heterologous sialyltransferase gene is α-2,3-sialyltransferase, α-2,3- and α-2,8-sialyltransferase (cstII), or α-2,6 sialyltransferase.

9. The method according to claim 1 , wherein the sialylated oligosaccharide is 3′sialyllactose or 6′sialyllactose, comprising culturing said microorganism in a culture medium at high cell density on a carbon substrate selected from the group consisting of glucose and glycerol, and fed with lactose which is internalized by the lactose permease of said microorganism and sialylated by said recombinant sialyltransferase of said microorganism using CMP-Neu5Ac endogenously generated from UDP-GlcNAc.

10. The method according to claim 9 ,

wherein the microorganism further comprises heterologous sequences encoding β-1,4GalNActransferase and β-1,3-Galactosyltransferase, and

wherein the method further comprises

β-1,4GalNActransferase transferring a UDP-GalNac residue to sialyllactose (GM3) to form GalNAcβ-4(Neu5Acα-3)Galβ-4Glc (GM2) and

β-1,3-Galactosyltransferase transferring a Galactosyl residue to GM2 to form a carbohydrate portion of ganglioside Galβ-3GalNAcβ-4(Neu5Acα-3)Galβ4Glc (GM1).

11. The method according to claim 9 , wherein the sialyltransferase is α-2,3- and α-2,8-sialyltransferase (cstII), and wherein the method produces Neu5Acα-3Galβ-3GalNAcβ-4(Neu5Acα-3)Galβ-4Glc (GD1a) and Galβ-3GalNAcβ-4(Neu5Acα-3)Galβ-3GalNAcβ-4(Neu5Acα-3)Galβ-4Glc (GA1).

12. The method according to claim 1 , wherein the sialyl transferase is a α-2,3-Sialyltransferase, and wherein the microorganism further comprises a heterologous lgtA gene encoding β-1,3-GlcNAc transferase and a heterologous lgtB gene encoding β-1,4-Galactosyltransferase.

13. The method according to claim 1 , for producing sialylgalactose (Neu5Acα-3Gal) and sialylated oligosaccharides with a terminal reducing galactose, wherein the microorganism is galK−, nanA− and nanK− and expresses the gene for sialyltransferase and the neuBCA genes and is cultured in a medium with galactose.

14. The method according to claim 9 , wherein the microorganism is growing on glycerol as carbon substrate.

15. A microorganism, wherein said microorganism is an Escherichia coli strain, comprising heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, wherein endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated, wherein said microorganism produces internally, activated sialic acid as donor substrate for said sialytransferase, and wherein said Escherichia coli strain is LacY+ (β-galactoside permease), LacZ− (β-galactosidase), and optionally MelA− (α-galactosidase).

16. The microorganism of claim 15 , wherein the sialyltransferase is α-2,3-sialyltransferase, α-2,3- and α-2,8-sialyltransferase (cstII), or α-2,6 sialyltransferase.