Efficient production of oligosaccharides using 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 method of producing an oligosaccharide comprising Neu5Acα-3Galβ-4Glc, the method comprising:
(a) obtaining a Lac Y+ and NanT+ Escherichia coli microorganism; and
(b) culturing the microorganism in a culture medium comprising precursors consisting of lactose and sialylic acid, wherein the microorganism comprises a heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase polypeptide which catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to the lactose to form Neu5Acα-3Galβ-4Glc, and wherein the Lac Y+ and NanT+ microorganism allows active transport of lactose and sialylic acid.
2. The method of claim 1 , further comprising culturing the microorganism such that the bifunctional Campylobacter jejuni sialyltransferase polypeptide catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to the Neu5Acα-3Galβ-4Glc to form Neu5Acα-8Neu5Acα-3Galβ-4Glc.
3. The method of claim 2 , further comprising culturing the microorganism such that the bifunctional Campylobacter jejuni sialyltransferase polypeptide catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to the Neu5Acα-8Neu5Acα-3Galβ-4Glc to form Neu5Acα-8Neu5Acα-8Neu5Acα-3Galβ-4Glc.
4. The method of claim 1 , wherein the microorganism further comprises a heterologous gene encoding a CMP-Neu5Ac synthase.
5. The method of claim 4 , wherein the gene encoding CMP-Neu5Ac synthase is from Neisseria meningitidis.
6. The method of claim 1 , wherein the bifunctional Campylobacter jejuni sialyltransferase is obtained from a Campylobacter jejuni isolate deposited under ATCC Accession No. 43438.
7. The method of claim 1 , wherein the microorganism lacks Neu5Ac aldolase activity.
8. The method of claim 1 , wherein the microorganism lacks β galactosidase activity.
9. The method of claim 1 , wherein the Neu5Ac is present in the culture medium at a concentration of about 6 mM.
10. The method of claim 1 , wherein the lactose is present in the culture medium at a concentration of about 3 mM.
11. The method of claim 1 , which is carried out on a commercial scale.