Microorganisims used to synthesize oligosaccharides
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. An Escherichia coli microorganism comprising a heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase polypeptide that catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to lactose to form Neu5Acα-3Galβ-4Glc, wherein the Escherichia coli microorganism lacks Neu5Ac aldolase activity and β galactosidase activity, and wherein the heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4, and wherein the microorganism is LacY+ and NanT+ allowing active transport of lactose and sialylic acid.
2. A cell culture medium comprising lactose and Neu5Ac wherein the Neu5Ac is present at or continuously supplied to maintain a concentration between 2 mM and 60 mM, and an Escherichia coli microorganism comprising a heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase polypeptide that catalyzes the transfer of a sialyl moiety from an activated sialic acid molecule to lactose to form Neu5Acα-3Galβ-4Glc, wherein the microorganism lacks Neu5Ac aldolase activity and β galactosidase activity, and wherein the heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase has at least 95% sequence identity to a sequence selected from SEQ ID NOs: 1, 2, 3 and 4.
3. The cell culture medium of claim 2 , wherein the lactose is present at or continuously supplied to maintain a concentration between 1 mM and 30 mM.
4. The Escherichia coli microorganism of claim 1 , which further comprises a heterologous gene encoding a cytidine monophospho-(CMP)-sialic acid synthetase.
5. The Escherichia coli microorganism of claim 4 , wherein the heterologous gene encoding the CMP-sialic acid synthetase is the gene for CMP-sialic acid synthetase from Neisseria meningitis.
6. The Escherichia coli microorganism of claim 1 , wherein the heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4.
7. The Escherichia coli microorganism of claim 1 , wherein the heterologous gene encoding a bifunctional Campylobacter jejuni sialyltransferase is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and any conservative substitution of a nucleotide of SEQ ID NOs: 1, 2, 3 or 4.
8. The cell culture medium of claim 2 , wherein the Neu5Ac is present at or continuously supplied to maintain a concentration of about 6 mM.
9. The cell culture medium of claim 3 , wherein the lactose is present at or continuously supplied to maintain a concentration of about 3 mM.