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US 9,453,230 B2Granted
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.
Claims40
A method for producing a fucosylated oligosaccharide in a bacterium, comprising
- providing an E. coli bacterium, said bacterium comprising a deletion or functional inactivation of the endogenous β-galactosidase gene;
- a functional promoter-less β-galactosidase gene inserted into an endogenous gene such that the resultant bacterium comprises a low level of β-galactosidase activity, wherein said β-galactosidase activity comprises between 0.05 and 200 units;
- an exogenous lactose-accepting fucosyltransferase gene comprising an α(1,2) fucosyltransferase gene, an α(1,3) fucosyltransferase gene, or an α(1,4) fucosyltransferase gene;
- an inactivating mutation in a colanic acid synthesis gene;
- and a functional lactose permease gene, wherein said lactose permease gene comprises E. coli lacY;
- culturing said bacterium in the presence of lactose;
- and
- retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
5 more elements
A method for producing a fucosylated oligosaccharide in a bacterium, comprising
- providing an E. coli bacterium, said bacterium comprising a deletion or functional inactivation of the endogenous β-galactosidase gene;
- a functional exogenous wild type β-galactosidase gene inserted into an endogenous gene such that the resultant bacterium comprises a low level of β-galactosidase activity, wherein said β-galactosidase activity comprises between 0.05 and 200 units;
- an exogenous lactose-accepting fucosyltransferase gene comprising an α(1,2) fucosyltransferase gene, an α(1,3) fucosyltransferase gene, or an α(1,4) fucosyltransferase gene;
- an inactivating mutation in an endogenous colanic acid synthesis gene, wherein said colanic acid synthesis gene comprises an E. coli wcaJ, wzxC, wcaD, wza, wzb, or wzc gene;
- and a functional lactose permease gene, wherein said lactose permease gene comprises E. coli lacY;
- culturing said bacterium in the presence of lactose;
- and
- retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
5 more elements
Dependencies are inferred from the claim text.