Production of Sialylated Oligosaccharides in Bacillus Cells
Disclosed are non-sporulating Bacillus cells for the production of a sialylated oligosaccharide wherein said Bacillus cell has been genetically engineered to possess a lactose permease, a CMP-NeuNAc biosynthesis pathway and a sialyltransferase, as well as a method for producing a sialylated oligosaccharide using said Bacillus cell.
1 . A non-sporulating Bacillus cell for the production of a sialylated oligosaccharide, wherein said Bacillus cell has been genetically engineered to possess a lactose permease, a CMP-NeuNAc biosynthesis pathway and a sialyltransferase.
2 . The Bacillus cell according to claim 1 , wherein the sporulation capability of the Bacillus cell has been impaired by deletion or functional inactivation of one or more of the genes encoding Spo0A, sigma E and sigma F.
3 . The Bacillus cell according to claim 1 or 2 , wherein the lactose permease is E. coli LacY or a functional variant thereof.
4 . The Bacillus cell according to any one of claims 1 to 3 , wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P or GlcNAc-6P as intermediate.
5 . The Bacillus cell according to any one of claims 1 to 4 , wherein the CMP-NeuNAc biosynthesis pathway uses a sialic acid salvage pathway.
6 . The Bacillus cell according to any one of claims 1 to 5 , wherein the sialyltransferase is a lactose-accepting sialyltransferase, preferably a sialyltransferase selected from the group consisting of α-2,3-sialyltransferases, α-2,6-sialyltransferases and α-2,8-sialyltransferases.
7 . The Bacillus cell according to any one of claims 1 to 6 , wherein said Bacillus cell lacks any β-galactosidase activity or possesses reduced β-galactosidase activity as compared to a wild-type progenitor Bacillus cell of the same species.
8 . The Bacillus cell according to claim 7 , wherein the Bacillus cell has been genetically engineered by deletion or functional inactivation of at least one of the genes selected from the group consisting of yesZ and ganA.
9 . The Bacillus cell according to any one of claims 1 to 8 , wherein said Bacillus cell is a Bacillus subtilis cell.
10 . The Bacillus cell according to any one of claims 1 to 9 , for use in the production of 3′-sialyllactose, wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P as intermediate, and wherein the sialyltransferase is a α-2,3-sialyltransferase.
11 . The Bacillus cell according to any one of claims 1 to 9 , for use in the production of 6′-sialyllactose, wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P as intermediate, and wherein the sialyltransferase is a α-2,6-sialyltransferase.
12 . Use of a Bacillus cell according to any one of claims 1 to 11 for the production of a sialylated oligosaccharide.
13 . A method for the production of a sialylated oligosaccharide, the method comprising
providing a non-sporulating Bacillus cell as defined in any one of claims 1 to 11 ;
cultivating said Bacillus cell in a fermentation broth and under conditions that are permissive for the production of a sialylated oligosaccharide; and optionally
retrieving the sialylated oligosaccharide from the medium and/or the Bacillus cell.
14 . The method according to claim 13 , wherein the fermentation broth contains lactose.
15 . A sialylated oligosaccharide produced by a method according to claim 13 or 14 .
16 . Use of a sialylated oligosaccharide according to claim 15 for the manufacture of a nutritional composition, preferably of an infant formula.
17 . A nutritional composition containing a sialylated oligosaccharide according to claim 15 .