BIOSYNTHESIS OF HUMAN MILK OLIGOSACCHARIDES IN ENGINEERED BACTERIA
The invention provides compositions and methods for engineering bacteria to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.
1 . 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.
2 . The method of claim 1 , wherein said β-galactosidase gene comprises an E. coli lacZ gene.
3 . The method of claim 1 , wherein said β-galactosidase gene is an endogenous β-galactosidase gene or an exogenous β-galactosidase gene.
4 . The method of claim 1 , wherein said bacterium accumulates an increased intracellular lactose pool, and produces a low level of β-galactosidase.
5 . The method of claim 1 , wherein said exogenous fucosyltransferase gene encodes α(1,2) fucosyltransferase or α(1,3) fucosyltransferase.
6 . The method of claim 5 , wherein said α(1,2) fucosyltransferase gene comprises a Bacteroides fragilis wcfW gene.
7 . The method of claim 5 , wherein said α(1,3) fucosyltransferase gene comprises a Helicobacter pylori 26695 futA gene.
8 . The method of claim 5 , wherein said bacterium comprises both an exogenous fucosyltransferase gene encoding α(1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.
9 . The method of claim 1 , wherein said GDP-fucose synthesis pathway comprises endogenous enzymes or exogenous enzymes.
10 . The method of claim 1 , wherein said lactose permease gene is an endogenous lactose permease gene or an exogenous lactose permease gene.
11 . 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.
12 . The method of claim 11 , wherein said β-galactosidase gene comprises an E. coli lacZ gene.
13 . The method of claim 11 , wherein said exogenous fucosyltransferase gene encodes α(1,2) fucosyltransferase or α(1,3) fucosyltransferase.
14 . The method of claim 11 , wherein said enteric bacterium comprises E. coli.
15 . The method of claim 14 , wherein said colanic acid synthesis gene comprises a wcaJ gene
16 . The method of claim 14 , wherein said bacterium further comprises a mutation in a lon gene.
17 . The method of claim 14 , wherein said bacterium comprises a functional, wild-type E. coli lacZ + gene inserted into an endogenous lon gene.
18 . The method of claim 14 , wherein an endogenous lacZ gene of said E. coli is deleted.
19 . The method of claim 14 , wherein said bacterium further comprises an exogenous rcsA or rcsB gene.
20 . The method of claim 14 , wherein said bacterium further comprises a mutation in a lacA gene.
21 . 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.
22 . The method of claim 21 , wherein said exogenous sialyl-transferase gene encodes α(2,3)sialyl-transferase.
23 . The method of claim 21 , wherein said exogenous fucosyltransferase gene encodes α(1,3) fucosyltransferase.
24 . The method of claim 21 , wherein said deficient sialic acid catabolic pathway comprises a null mutation in endogenous N-acetylneuraminate lyase or N-acetylmannosamine kinase genes.
25 . The method of claim 21 , wherein said sialic acid synthetic capability comprises an exogenous UDP-GlcNAc 2-epimerase gene, an exogenous Neu5Ac synthase gene, or an exogenous CMP-Neu5Ac synthetase gene.
26 . 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.
27 . The method of claim 26 , wherein said exogenous fucosyltransferase gene encodes α(1,3) fucosyltransferase.
28 . The method of claim 26 , wherein said exogenous sialyltransferase gene encodes an α(2,3)sialyl transferase.
29 . The method of claim 26 , wherein said deficient sialic acid catabolic pathway comprises a null mutation in endogenous N-acetylneuraminate lyase or N-acetylmannosamine kinase genes.
30 . The method of claim 26 , wherein said sialic acid synthetic capability comprises an exogenous UDP-GlcNAc 2-epimerase gene, an exogenous Neu5Ac synthase gene, or an exogenous CMP-Neu5Ac synthetase gene.
31 . 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.
32 . 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.
33 . 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.
34 . A method of purifying a fucosylated oligosaccharide produced by the method of claim 1 , comprising binding said fucosylated oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said fucosylated oligosaccharide from said column.
35 . An isolated E. coli bacterium comprising a defective colanic acid synthesis pathway, reduced level of β-galactosidase activity, and an exogenous fucosyl transferase gene.
36 . A purified fucosylated oligosaccharide produced by the method of claim 1 .
37 . 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.
38 . The nucleic acid construct of claim 37 , wherein said exogenous fucosyltransferase gene encodes α(1,2) fucosyltransferase or α(1,3) fucosyltransferase.