Biosynthesis of human milk oligosaccharides in engineered bacteria
View Patent ↗The invention provides compositions and methods for engineering bacteria 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 bacterium, comprising
providing an E. coli bacterium, said bacterium comprising a deletion or functional inactivation of the endogenous β-galactosidase gene; a functional promoter-less β-galactosidase gene inserted into an endogenous gene such that the resultant bacterium comprises a low level of β-galactosidase activity, wherein said β-galactosidase activity comprises between 0.05 and 200 units; an exogenous lactose-accepting fucosyltransferase gene comprising an α(1,2) fucosyltransferase gene, an α(1,3) fucosyltransferase gene, or an α(1,4) fucosyltransferase gene; an inactivating mutation in a colanic acid synthesis gene; and a functional lactose permease gene, wherein said lactose permease gene comprises E. coli lacY;
culturing said bacterium in the presence of lactose; and
retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
2. The method of claim 1 , wherein the functional promoter-less β-galactosidase gene inserted into an endogenous gene comprises an exogenous wild type E. coli lacZ gene.
3. The method of claim 1 , wherein said colanic acid synthesis gene comprises a wcaJ, wzxC, wcaD, wza, wzb, or wzc gene.
4. The method of claim 1 , wherein said bacterium further comprises an inactivating mutation in a lon gene.
5. The method of claim 1 , wherein said bacterium comprises a functional promoter-less wild-type E. coli lacZ + gene inserted into an endogenous lon gene.
6. The method of claim 1 , wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
7. The method of claim 1 , wherein said bacterium further comprises an inactivating mutation in a lacA gene.
8. The method of claim 1 , wherein said bacterium accumulates an increased intracellular lactose pool, wherein the increased intracellular lactose pool is at least 10% more than the levels in a wild type bacterium.
9. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a Bacteroides fragilis wcfW gene.
10. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a Helicobacter pylori 26695 futA gene.
11. The method of claim 1 , wherein said bacterium comprises both an exogenous fucosyltransferase gene encoding α(1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.
12. The method of claim 1 , wherein said lactose permease gene is an endogenous lactose permease gene.
13. The method of claim 1 , wherein said colanic acid synthesis gene is an endogenous colanic acid synthesis gene.
14. The method of claim 1 , wherein said endogenous β-galactosidase gene is deleted or functionally inactivated and the downstream endogenous lacY remains intact.
15. The method of claim 1 , wherein said lactose permease gene is an exogenous lactose permease gene.
16. The method of claim 1 , wherein said low level of β-galactosidase activity comprises between 0.05 and 5 units.
17. The method of claim 1 , wherein said low level of β-galactosidase activity comprises between 0.05 and 4 units.
18. The method of claim 1 , wherein said low level of β-galactosidase activity comprises between 0.05 and 3 units.
19. The method of claim 1 , wherein said low level of β-galactosidase activity comprises between 0.05 and 2 units.
20. The method of claim 1 , wherein said fucosylated oligosaccharide is 2′-fucosyllactose, 3-fucosyllactose, or lactodifucotetraose.
21. A method for producing a fucosylated oligosaccharide in a bacterium, comprising
providing an E. coli bacterium, said bacterium comprising a deletion or functional inactivation of the endogenous β-galactosidase gene; a functional exogenous wild type β-galactosidase gene inserted into an endogenous gene such that the resultant bacterium comprises a low level of β-galactosidase activity, wherein said β-galactosidase activity comprises between 0.05 and 200 units; an exogenous lactose-accepting fucosyltransferase gene comprising an α(1,2) fucosyltransferase gene, an α(1,3) fucosyltransferase gene, or an α(1,4) fucosyltransferase gene; an inactivating mutation in an endogenous colanic acid synthesis gene, wherein said colanic acid synthesis gene comprises an E. coli wcaJ, wzxC, wcaD, wza, wzb, or wzc gene; and a functional lactose permease gene, wherein said lactose permease gene comprises E. coli lacY;
culturing said bacterium in the presence of lactose; and
retrieving a fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
22. The method of claim 21 , wherein said β-galactosidase gene inserted into an endogenous gene comprises an E. coli lacZ gene.
23. The method of claim 21 , wherein said bacterium further comprises an inactivating mutation in a lon gene.
24. The method of claim 21 , wherein said bacterium comprises a functional, wild-type, promoter-less E. coli lacZ + gene inserted into an endogenous lon gene.
25. The method of claim 21 , wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
26. The method of claim 21 , wherein said bacterium further comprises an inactivating mutation in a lacA gene.
27. The method of claim 21 , wherein said bacterium accumulates an increased intracellular lactose pool, wherein said increased intracellular lactose pool is at least 10% more than the levels in a wild type bacterium.
28. The method of claim 21 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a Bacteroides fragilis wcfW gene.
29. The method of claim 21 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a Helicobacter pylori 26695 futA gene.
30. The method of claim 21 , wherein said bacterium comprises both an exogenous fucosyltransferase gene encoding α(1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.
31. The method of claim 21 , wherein said lactose permease gene is an endogenous lactose permease gene.
32. The method of claim 21 , wherein said lactose permease gene is an exogenous lactose permease gene.
33. The method of claim 21 , wherein said endogenous β-galactosidase gene is deleted and the endogenous lacY remains intact.
34. The method of claim 21 , wherein said fucosylated oligosaccharide is 2′-fucosyllactose, 3-fucosyllactose, or lactodifucotetraose.
35. The method of claim 21 , wherein said low level of β-galactosidase activity comprises between 0.05 and 5 units.
36. The method of claim 21 , wherein said low level of β-galactosidase activity comprises between 0.05 and 4 units.
37. The method of claim 21 , wherein said low level of β-galactosidase activity comprises between 0.05 and 3 units.
38. The method of claim 21 , wherein said low level of β-galactosidase activity comprises between 0.05 and 2 units.
39. The method of claim 11 or 21 , wherein said bacteria comprises the genotype of ampC::(P trpB λcI + ), P lacI q(ΔlacI-lacZ) 158 lacY + , ΔwcaJ, thyA 748 ::Tn10, Δlon::(kan, lacZ + ).
40. The method of claim 11 or 21 , wherein said bacteria comprises the genotype of ampC::(P trpB λcI + ), P lacI q(ΔlacI-lacZ) 158 lacY +, ΔwcaJ, thyA 748 ::Tn10, Δlon::(kan, lacZ + ), ΔlacA.