Metabolically engineered microorganisms
View Patent ↗The present invention relates to the enzymatic synthesis of oligosaccharides, particularly, sialylated oligosaccharides comprising the carbohydrate moeities of the gangliosides GM3, GD3, and GT3.
1. A microorganism comprising a heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase polypeptide and which lacks Neu5Ac aldolase activity and β galactosidase activity, wherein the microorganism is a LacY+ and NanT+ Escherichia coli microorganism allowing active transport of lactose and sialic acid.
2. The microorganism of claim 1 , which catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to the lactose to form Neu5Acα-3Galβ-4Glc.
3. The microorganism of claim 1 , wherein the bifunctional Campylobacter jejuni sialyltransferase is from Campylobacter jejuni isolate deposited under ATCC Accession No. 43438.
4. The microorganism of claim 3 , which catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to the lactose to form Neu5Acα-3Galβ-4Glc.
5. A cell culture medium comprising lactose and Neu5Ac and the microorganism of claim 1 .
6. The cell culture medium of claim 5 , wherein the Neu5Ac is present at or continuously supplied to maintain a concentration between 2 mM and 60 mM.
7. The cell culture medium of claim 6 , wherein the Neu5Ac is present at or continuously supplied to maintain a concentration of about 6 mM.
8. The cell culture of claim 5 , wherein the lactose is present at or continuously supplied to maintain a concentration between 1 mM and 30 mM.
9. The cell culture of claim 8 , wherein the lactose is present at or continuously supplied to maintain a concentration of about 3 mM.