Microorganisms and methods for producing sialylated and N-acetylglucosamine-containing oligosaccharides
The invention provides compositions and methods for engineering bacteria to produce sialylated and N-acetylglucosamine-containing oligosaccharides, and the use thereof in the prevention or treatment of infection.
1. A method for producing an N-acetylglucosamine-containing oligosaccharide in a bacterium,
wherein said N-acetylglucosamine-containing oligosaccharide comprises Lacto-N-triose 2 (LNT2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lacto-N-fucopentaose V (LNF V), Lacto-N-difucohexaose I (LDFH I), Lacto-N-difucohexaose II (LDFH II), or Lacto-N-neodifucohexaose II (LFNnDFH II), the method comprising:
(i) providing a bacterium, said bacterium comprising
β-galactosidase activity,
an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene and
a functional lactose permease gene; and
(ii) culturing said bacterium in the presence of lactose.
2. The method of claim 1 , wherein said bacterium comprises an increased UDP-GlcNAc production capability by overexpressing a positive endogenous regulator of UDP-GlcNac synthesis.
3. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of an E. coli gene selected from a nagC gene, a glmS gene, a glmY gene, a glmZ gene or any combination thereof.
4. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli nagC gene.
5. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli nagC and E. coli glmS.
6. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli nagC and E. coli glmY.
7. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli nagC and E. coli glmZ.
8. The method of claim 1 , wherein said bacterium is E. coli.
9. A method of purifying an N-acetylglucosamine-containing oligosaccharide produced the method of claim 1 , comprising binding said oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said oligosaccharide from said column.
10. The method of claim 1 , further comprising retrieving said N-acetylglucosamine-containing oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
11. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli glmS.
12. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli glmY.
13. The method of claim 2 , wherein said increased UDP-GlcNAc production capability comprises overexpression of E. coli glmZ.
14. The method of claim 1 , wherein said bacterium depletes residual lactose after producing the N-acetylglucosamine-containing oligosaccharide.