Alpha (1,2) fucosyltransferases suitable for use in the production of fucosylated oligosaccharides
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. A method for producing a fucosylated oligosaccharide in a host bacterium comprising
providing said host bacterium comprising a genetic modification to reduce level of β-galactosidase activity, a defective colanic acid synthesis pathway, a mutation in an ATP-dependent intracellular protease, a mutation in a thyA gene, and an exogenous lactose-accepting α(1,2) fucosyltransferase gene from a genus consisting of Bacteroides;
culturing said host bacterium in the presence of lactose; and
retrieving the fucosylated oligosaccharide from said host bacterium or from a culture supernatant of said host bacterium, wherein less than 1% (w/w) of the level of the fucosylated oligosaccharide produced by said host bacterium is from 3-fucosyllactose (3-FL).
2. The method of claim 1 , wherein said host bacterium comprises E. coli.
3. The method of claim 1 , wherein said fucosylated oligosaccharide comprises 2′-fucosyllactose (2′-FL), lactodifucotetraose (LDFT), Lacto-N-fucopentaose I (LNF I), or lacto-N-difucohexaose I (LDFH I).
4. The method of claim 1 , wherein said method further comprises culturing said host bacterium in the presence of tryptophan and in the absence of thymidine.
5. The method of claim 2 , wherein an endogenous lacZ gene and an endogenous lacI gene of said E. coli are deleted to reduce the level of β-galactosidase activity.
6. The method of claim 5 , wherein said E. coli bacterium further comprises a lacIq gene promoter immediately upstream of a lacY gene.
7. The method of claim 2 , wherein an endogenous wcaJ gene of said E. coli is deleted to inactivate the colanic acid synthesis pathway.
8. The method of claim 1 , wherein said mutation in said ATP-dependent intracellular protease is a null mutation in a lon gene.
9. The method of claim 1 , wherein said host bacterium accumulates intracellular lactose in the presence of exogenous lactose.
10. The method of claim 1 , wherein said host bacterium accumulates intracellular guanosine diphosphate (GDP)-fucose.
11. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes an amino acid sequence comprising SEQ ID NO: 8 (FutN).
12. The method of claim 2 , wherein said E. coli bacterium comprises the genotype characterized by ΔampC::P trp B cI, Δ(lacI-lacZ)::FRT, P lacIq lacY + , ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ + ), and ΔlacA.
13. The method of claim 1 , wherein the level of 3-FL produced by said host bacterium is less than 0.5% (w/w) of the level of the produced fucosylated oligosaccharide.
14. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene is from a commensal microbe rather than a pathogen.
15. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene is derived from Bacteroides vulgatus.
16. The method of claim 1 , wherein said exogenous lactose-accepting (1,2) fucosyltransferase gene encodes a Bacteroides vulgatus glycosyl transferase family protein.
17. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein.
18. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a polypeptide having at least 10% identity at the amino acid level and less than about 40% to Helicobacter pylori 26695 α(1,2) fucosyltransferase (FutC).
19. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a polypeptide having at least 20% identity at the amino acid level and less than about 40% to Helicobacter pylori 26695 α(1,2) fucosyltransferase (FutC).
20. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a polypeptide having at least 20% identity at the amino acid level and less than about 30% to Helicobacter pylori 26695 α(1,2) fucosyltransferase (FutC).
21. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a polypeptide having at least 20% identity at the amino acid level and less than about 25% to Helicobacter pylori 26695 α(1,2) fucosyltransferase (FutC).
22. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a Bacteroides sp. lactose-accepting α(1,2) fucosyltransferase enzyme.
23. The method of claim 22 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a Bacteroides vulgatus lactose-accepting α(1,2) fucosyltransferase enzyme.
24. The method of claim 22 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a Bacteroides fragilis lactose-accepting α(1,2) fucosyltransferase enzyme.
25. The method of claim 1 , wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene encodes a polypeptide having about 27% identity at the amino acid level to Helicobacter pylori 26695 α(1,2) fucosyltransferase (FutC).
26. A method for producing a fucosylated oligosaccharide in a host bacterium comprising
providing said host bacterium comprising a reduced level of β-galactosidase activity, a defective colanic acid synthesis pathway, a mutation in an ATP-dependent intracellular protease, a mutation in a thyA gene, and an exogenous lactose-accepting α(1,2) fucosyltransferase gene;
culturing said host bacterium in the presence of lactose; and
retrieving a fucosylated oligosaccharide from said host bacterium or from a culture supernatant of said host bacterium, wherein said exogenous lactose-accepting α(1,2) fucosyltransferase gene is from a bacterial strain consisting of Bacteroides vulgatus.