Method of producing sialylated oligosaccharides
AssigneeCentre National de la Recherche Scientifique (CNRS)
InventorEric Samain
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Abstract
The present invention relates to a method for the large scale in vivo synthesis of sialylated oligosaccharides, culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as lactose, wherein said microorganism comprises heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-6-phosphate 2 epimerase and a sialyltransferase, and wherein the endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated. The invention also relates to these micoorganisms which are capable of producing internally activated sialic acid.
Claims16
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;
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- 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.
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.
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).
Dependencies are inferred from the claim text.