Biosynthesis of human milk oligosaccharides in engineered bacteria
AssigneeGlycosyn LLC
InventorsMassimo Merighi, John M. McCoy, Matthew Ian Heidtman
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Abstract
The invention provides compositions and methods for engineering bacteria to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.
Claims38
A method for producing a fucosylated oligosaccharide in a bacterium, comprising
- providing a bacterium, said bacterium comprising a functional β-galactosidase gene, an exogenous fucosyltransferase gene, a GDP-fucose synthesis pathway, a functional lactose permease gene;
- culturing said bacterium in the presence of lactose;
- and
- retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
A method for producing a fucosylated oligosaccharide in a bacterium, comprising
- providing an enteric bacterium, said bacterium comprising a functional β-galactosidase gene, an exogenous fucosyltransferase gene, a mutation in a colanic acid synthesis gene, and a functional lactose permease gene;
- culturing said bacterium in the presence of lactose;
- and
- retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
A method for producing a 3′-sialyl-3-fucosyllactose (3′-S3FL) in a bacterium,
- said bacterium comprising a functional β-galactosidase gene, an exogenous sialyl-transferase gene, an exogenous fucosyltransferase gene, a GDP-fucose synthesis pathway, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, and a functional lactose permease gene;
- culturing said bacterium in the presence of lactose;
- and
- retrieving said 3′-S3FL from said bacterium or from a culture supernatant of said bacterium.
A method for producing a 3′-sialyl-3-fucosyllactose (3′-S3FL) in an enteric bacterium,
- said enteric bacterium comprising a functional lacZ gene, an exogenous fucosyltransferase gene, an exogenous sialyltransferase gene, a mutation in an endogenous colanic acid synthesis gene, a functional lactose permease gene, a deficient sialic acid catabolic pathway, and sialic acid synthetic capability;
- culturing said bacterium in the presence of lactose;
- and
- retrieving said 3′-S3FL from said bacterium or from a culture supernatant of said bacterium.
A method for phenotypic marking of a gene locus in a host cell, whose native β-galactosidase gene is deleted or inactivated, by utilizing an inserted recombinant β-galactosidase gene engineered to produce a low but detectable level of β-galactosidase activity.
A method for depleting a bacterial culture of residual lactose in a β-galactosidase negative host cell, whose native β-galactosidase gene is deleted or inactivated, by utilizing an inserted recombinant β-galactosidase gene engineered to produce a low but detectable level of β-galactosidase activity.
A method for detecting bacterial cell lysis in a culture of a β-galactosidase negative host cell, whose native β-galactosidase gene is deleted or inactivated, by utilizing an inserted recombinant β-galactosidase gene engineered to produce a low but detectable level of β-galactosidase activity.
An isolated E. coli bacterium comprising a defective colanic acid synthesis pathway, reduced level of β-galactosidase activity, and an exogenous fucosyl transferase gene.
A nucleic acid construct comprising an exogenous fucosyltransferase gene transformed into a bacterial host strain comprising a deleted endogenous β-galactosidase gene, a replacement functional β-galactosidase gene of low activity, a GDP-fucose synthesis pathway, a functional lactose permease gene, and a deleted lactose acetyltransferase gene.
Dependencies are inferred from the claim text.