Biosynthesis of human milk oligosaccharides in engineered bacteria

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.

What is claimed is: 
     
         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 ion gene. 
     
     
         17 . The method of  claim 14 , wherein said bacterium comprises a functional, wild-type  E. coli  lacZ +  gene inserted into an endogenous ion 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.