Alpha (1,2) fucosyltransferase syngenes 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 lacto-N-fucopentaose I (LNF I) oligosaccharide in a bacterium comprising
providing a bacterium comprising an exogenous lactose-utilizing α(1,2) fucosyltransferase enzyme, wherein said α(1,2) fucosyltransferase enzyme has at least 90% sequence identity to amino acid sequence SEQ ID NO: 13; and
culturing said bacterium in the presence of lactose, wherein said LNF I oligosaccharide is produced,
wherein said bacterium further comprises β1,3-N-acetylglucosaminyltransferase and β1,3-galactosyltransferase.
2. The method of claim 1 , wherein said α(1,2) fucosyltransferase enzyme comprises Methanosphaerula palustries FutR.
3. The method of claim 1 , further comprising retrieving the fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
4. The method of claim 1 , wherein said bacterium further produces 2′-fucosyllactose (2′-FL), lactodifucotetraose (LDFT), or lacto-N-difucohexaose I (LDFH I).
5. The method of claim 1 , wherein the bacterium further comprises an exogenous lactose-utilizing α(1,3) fucosyltransferase enzyme and/or an exogenous lactose-utilizing α(1,4) fucosyltransferase enzyme.
6. The method of claim 5 , wherein the exogenous lactose-utilizing α(1,3) fucosyltransferase enzyme comprises a Helicobacter pylori 26695 futA gene.
7. The method of claim 5 , wherein the exogenous lactose-utilizing α(1,4) fucosyltransferase enzyme comprises a Helicobacter pylori UA948 FucTa gene or a Helicobacter pylori strain DMS6709 FucT III gene.
8. The method of claim 1 , wherein said bacterium further comprises a reduced level of β-galactosidase activity, a defective colanic acid synthesis pathway, an inactivated adenosine-5′-triphosphate (ATP)-dependent intracellular protease, or an inactivated endogenous lacA gene, or any combination thereof.
9. The method of claim 8 , wherein said method further comprises culturing said bacterium in the presence of tryptophan and in the absence of thymidine.
10. The method of claim 8 , wherein said reduced level of β-galactosidase activity comprises a deleted or inactivated endogenous lacZ gene and/or a deleted or inactivated endogenous lacI gene of said bacterium.
11. The method of claim 10 , wherein said reduced level of β-galactosidase activity further comprises an exogenous lacZ gene, wherein said exogenous lacZ gene comprises an β-galactosidase activity level less than wild-type bacterium.
12. The method of claim 8 , wherein said reduced level of β-galactosidase activity comprises an activity level less than wild-type bacterium.
13. The method of claim 12 , wherein said reduced level of β-galactosidase activity comprises less than 6,000 Miller units of β-galactosidase activity.
14. The method of claim 12 , wherein said reduced level of β-galactosidase activity comprises less than 1,000 Miller units of β-galactosidase activity.
15. The method of claim 8 , wherein said bacterium comprises a lack gene promoter immediately upstream of a lacY gene.
16. The method of claim 8 , wherein said defective colanic acid synthesis pathway comprises the inactivation of a wcaJ gene of said bacterium is deleted.
17. The method of claim 8 , wherein said inactivated ATP-dependent intracellular protease is a null mutation, inactivating mutation, or deletion of an endogenous lon gene.
18. The method of claim 17 , wherein said inactivating mutation of an endogenous lon gene comprises the insertion of a functional E. coli lacZ + gene.
19. The method of claim 8 , wherein said bacterium further comprises a functional lactose permease gene.
20. The method of claim 19 , wherein said bacterium comprises E. coli lacY.
21. The method of claim 8 , wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
22. The method of claim 8 , wherein said bacterium further comprises a mutation in a thyA gene.
23. The method of claim 8 , wherein said bacterium accumulates intracellular lactose in the presence of exogenous lactose.
24. The method of claim 8 wherein said bacterium accumulates intracellular GDP-fucose.
25. The method of claim 1 , wherein said bacterium is E. coli.
26. The method of claim 1 , wherein said production strain is a member of the Bacillus, Pantoea, Lactobacillus, Lactococcus, Streptococcus, Proprionibacterium, Enterococcus, Bifidobacterium, Sporolactobacillus, Micromomospora, Micrococcus, Rhodococcus , or Pseudomonas genus.
27. The method of claim 1 , wherein said production strain is selected from the group consisting of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus thermophiles, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus , and Bacillus circulans, Erwinia herbicola ( Pantoea agglomerans ), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, Xanthomonas campestris Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Streptococcus thermophiles, Proprionibacterium freudenreichii, Enterococcus faecium, Enterococcus thermophiles ), Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Pseudomonas fluorescens and Pseudomonas aeruginosa.
28. The method of claim 1 , wherein said bacterium comprises a nucleic acid construct comprising an isolated nucleic acid encoding said α(1,2) fucosyltransferase enzyme.
29. The method of claim 28 , wherein said nucleic acid is operably linked to one or more heterologous control sequences that direct the production of the enzyme in the bacterium.
30. The method of claim 29 , wherein said heterologous control sequence comprises a bacterial promoter and operator, a bacterial ribosome binding site, a bacterial transcriptional terminator, or a plasmid selectable marker.
31. The method of claim 1 , wherein the amino acid sequence of said enzyme comprises the amino acid sequence of FutR (SEQ ID NO: 13).