Alpha (1,2) fucosyltransferases suitable for use in the production of fucosylated oligosaccharides
View Patent ↗The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.
1. An isolated Escherichia coli ( E. coli ) bacterium comprising a genetic modification to reduce β-galactosidase activity, a defective colanic acid synthesis pathway, and an isolated nucleic acid encoding a lactose-accepting α (1,2) fucosyltransferase enzyme, wherein the sequence of said α (1,2) fucosyltransferase enzyme comprises a Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein (FutN).
2. The isolated E. coli bacterium of claim 1 , wherein an endogenous lacZ gene and an endogenous lacI gene of said E. coli bacterium are deleted or functionally inactivated.
3. The isolated E. coli bacterium of claim 2 , wherein said E. coli bacterium comprises a lacIq gene promoter upstream of a lacY gene.
4. The isolated E. coli bacterium of claim 1 , wherein an endogenous wcaJ gene of said E. coli bacterium is deleted or functionally inactivated.
5. The isolated E. coli bacterium of claim 1 , wherein said bacterium further comprises a mutation in an adenosine-5′-triphosphate (ATP)-dependent intracellular protease.
6. The isolated E. coli bacterium of claim 1 , wherein said bacterium accumulates intracellular lactose in the presence of exogenous lactose.
7. The isolated E. coli bacterium of claim 1 , wherein said bacterium accumulates intracellular guanosine diphosphate (GDP)-fucose.
8. The isolated E. coli bacterium of claim 1 , wherein the amino acid sequence of said FutN comprises SEQ ID NO:8.
9. The isolated E. coli bacterium of claim 1 , wherein said E. coli bacterium comprises the genotype ΔampC::P trp B cI, Δ(lacI-lacZ)::FRT, P lacIq lacY + , ΔwcaJ::FRT, thyA::Tn10, Δlon:npt3, (lacZ + ).
10. The isolated E. coli bacterium of claim 5 , wherein said mutation in said ATP-dependent intracellular protease is a mutation in a lon gene.
11. The isolated E. coli bacterium of claim 1 , wherein said bacterium further comprises a mutation in a lacA gene.
12. The isolated E. coli bacterium of claim 1 , wherein said bacterium further comprises a mutation in the thyA gene.