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 a sialylated oligosaccharide in an Escherichia coli ( E. coli ) bacterium, wherein said sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL) or 6′-sialyllactose (6′-SL), said method comprising:
(i) providing an E. coli bacterium, said bacterium comprising
an exogenous sialyl-transferase comprising an α(2,3) sialyl-transferase, an α(2,6) sialyl-transferase, or an α(2,8) sialyltransferase,
a mutation in an endogenous N-acetylneuraminate lyase gene (nanA), wherein said bacterium comprises an endogenous N-acetylmannosamine kinase gene (nanK) that is not mutated,
an increased UDP-GlcNAc production capability comprising overexpression of nagC, such that the bacterium produces at least 10% more UDP-GlcNAc than a native E. coli bacterium,
a sialic acid synthetic capability, 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 a null mutation in any one of the genes selected from endogenous N-acetylneuraminate lyase gene (nanA), endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE), and endogenous N-acetylneuraminic acid transporter gene (nanT), or any combination thereof.
3. The method of claim 1 , wherein said bacterium comprises a null mutation in an endogenous N-acetylneuraminate lyase gene (nanA).
4. The method of claim 1 , wherein said bacterium comprises an endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE) that is not mutated, and (i) a null mutation in the endogenous N-acetylneuraminate lyase gene (nanA), (ii) a null mutation in an endogenous N-acetylneuraminic acid transporter gene (nanT), or (iii) a null mutation in the endogenous N-acetylneuraminate lyase gene (nanA) and a null mutation in the endogenous N-acetylneuraminic acid transporter gene (nanT).
5. The method of claim 1 , wherein said bacterium comprises a null mutation in endogenous N-acetylneuraminate lyase gene (nanA), and a null mutation in endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE).
6. The method of claim 1 , wherein said sialic acid synthetic capability comprises an exogenous CMP-Neu5Ac synthetase gene (neuA), an exogenous sialic acid synthase gene (neuB), and an exogenous UDP-GlcNac 2-epimerase (neuC).
7. The method of claim 1 , wherein said α(2,3) sialyl-transferase, α(2,6) sialyl-transferase, or α(2,8) sialyltransferase, comprises a sequence of a Photobacterium sp. sialyl-transferase, Campylobacter jejuni sialyl-transferase, Neisseria meningitides sialyl-transferase, or Neisseria gonorrhoeae sialyl-transferase.
8. The method claim 1 , wherein said sialylated oligosaccharide comprises 6′ sialyllactose (6′-SL).
9. The method of claim 1 , wherein said bacterium comprises a deleted or inactivated endogenous β-galactosidase gene.
10. The method of claim 9 , wherein said deleted or inactivated β-galactosidase gene comprises an E. coli lacZ gene.
11. The method of claim 1 , wherein said bacterium comprises a recombinant β-galactosidase gene providing a level of β-galactosidase activity between 0.05 and 200 units.
12. The method of claim 1 , wherein said bacterium further comprises a deleted, inactivated, or mutated lacA gene.
13. The method of claim 1 , wherein said E. coli bacterium comprises an increased UDP-GlcNAc production capability, such that it produces at least 20% more UDP-GlcNAc than a native E. coli bacterium.
14. The method of claim 1 , wherein said increased UDP-GlcNAc production capability further comprises overexpression of a glmS gene, a glmY gene, a glmZ gene or any combination thereof.
15. The method of claim 1 , wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmS.
16. The method of claim 1 , wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmY.
17. The method of claim 1 , wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmZ.
18. A method of purifying a sialylated oligosaccharide produced by the method of claim 1 , comprising binding said sialylated oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said sialylated oligosaccharide from said column.
19. A purified sialylated oligosaccharide produced by the method of claim 1 .
20. The method of claim 1 , further comprising retrieving said sialylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
21. The method of claim 1 , wherein said bacterium comprises a mutation in an endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE).
22. The method of claim 1 , wherein said bacterium comprises an endogenous N-acetylneuraminic acid transporter gene (nanT) gene that is not mutated.
23. The method of claim 1 , wherein said sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL).
24. The method of claim 1 , wherein said mutation is within the coding region of nanA.
25. The method of claim 24 , wherein the mutation comprises an amino acid deletion or insertion.
26. The method of claim 25 , wherein the mutation causes a loss of function of a nanA gene product or loss of production of a nanA gene product.