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 isolated E. coli bacterium comprising,
(i) a deletion or functional inactivation of an endogenous β-galactosidase gene;
(ii) an exogenous functional β-galactosidase gene comprising a detectable level of β-galactosidase activity that is reduced compared to that of a wild-type E. coli bacterium, wherein the level of β-galactosidase activity comprises between 0.05 and 200 units;
(iii) an inactivating mutation in a colanic acid synthesis gene; and
(iv) an exogenous lactose-accepting fucosyltransferase gene;
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 said colanic acid synthesis gene comprises an E. coli wcaJ, wzxC, wcaD, wza, wzb, or wzc gene.
3. The method of claim 2 , wherein said colanic acid synthesis gene comprises a wcaJ gene.
4. The method of claim 1 , wherein the bacterium comprises an increased intracellular guanosine diphosphate (GDP)-fucose level, wherein the increased intracellular GDP-fucose level is at least 10% more than the level of GDP-fucose in a wild-type bacterium.
5. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene encodes α(1,2) fucosyltransferase and/or α(1,3) fucosyltransferase.
6. The method of claim 5 , wherein said α(1,2) fucosyltransferase gene comprises a Bacteroides fragilis wcfW gene.
7. The method of claim 5 , wherein said α(1,3) fucosyltransferase gene comprises a Helicobacter pylori 26695 futA gene.
8. The method of claim 1 , wherein said exogenous functional β-galactosidase gene comprises an E. coli lacZ gene.
9. The method of claim 8 , wherein the lacZ gene is inserted into an endogenous lon gene.
10. The method of claim 1 , wherein said bacterium further comprises a functional lactose permease gene.
11. The method of claim 10 , wherein said lactose permease gene is an endogenous lactose permease gene.
12. The method of claim 10 , wherein said lactose permease gene comprises an E. coli lacY gene.
13. The method of claim 1 , wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
14. The method of claim 1 , wherein said bacterium further comprises an inactivating mutation in a lacA gene.
15. The method of claim 1 , wherein said bacterium further comprises an exogenous sialyltransferase gene.
16. The method of claim 15 , wherein said exogenous sialyltransferase gene encodes an α(2,3)sialyl transferase.
17. The method of claim 1 , wherein said bacterium further comprises a deficient sialic acid catabolic pathway comprising a null mutation in an endogenous N-acetylneuraminate lyase gene or a null mutation in an endogenous N-acetylmannosamine kinase gene.
18. The method of claim 1 , wherein the level of β-galactosidase activity comprises between 0.05 and 5 units.
19. The method of claim 1 , wherein said bacterium further comprises an inactivating mutation in a lon gene.
20. The method of claim 1 , wherein said bacterium comprises an increased intracellular lactose level, wherein the increased intracellular lactose level is at least 10% more than the level in a wild-type bacterium.
21. The method of claim 1 , wherein said exogenous functional β-galactosidase gene is an E. coli lacZ gene lacking an operably linked promoter, and said colanic acid synthesis gene comprises an E. coli wcaJ, wzxC, wcaD, wza, wzb, or wzc gene.
22. The method of claim 1 , wherein said bacterium comprises the genotype of
(a) ampC::(P trpB λcI + ), P lacI q (ΔlacI-lacZ)lacY + , ΔwcaJ, thyA::Tn10, Δlon::(kan, lacZ + ); or
(b) ampC::(P trpB λcI + ), P lacI q (ΔlacI-lacZ)lacY + , ΔwcaJ, thyA::Tn10, Δlon::(kan, lacZ + ), ΔlacA.
23. The method of claim 1 , wherein said exogenous functional β-galactosidase gene is inserted into an endogenous gene.
24. The method of claim 1 , wherein said exogenous functional β-galactosidase gene comprises a recombinant β-galactosidase gene engineered to produce a detectable level of β-galactosidase activity that is reduced compared to the level of β-galactosidase activity in a wild-type E. coli bacterium.
25. The method of claim 24 , wherein the level of β-galactosidase activity comprises between 0.05 and 5 units.
26. The method of claim 1 , wherein the level of β-galactosidase activity comprises between 0.05 and 4 units.
27. The method of claim 1 , wherein the level of β-galactosidase activity comprises between 0.05 and 3 units.
28. The method of claim 1 , wherein the level of β-galactosidase activity comprises between 0.05 and 2 units.