Biosynthesis of human milk oligosaccharides in engineered bacteria
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 an E.coli bacterium, comprising
(a) providing an E.coli bacterium comprising a β-galactosidase activity between 0.05 and 5 units and a mutation in a lacA gene, wherein said E.coli bacterium further comprises an exogenous lactose-accepting fucosyltransferase gene;
(b) culturing said E.coli bacterium in the presence of lactose; and
(c) retrieving a fucosylated oligosaccharide from said E.coli bacterium or from a culture supernatant of said E.coli bacterium,
wherein a functional β-galactosidase gene is inserted into an endogenous gene of said E. coli bacterium.
2. The method of claim 1 , wherein said mutation prevents the formation of intracellular acetyl-lactose.
3. The method of claim 1 , wherein said E.coli bacterium further comprises a defective colanic acid synthesis pathway.
4. The method of claim 1 , wherein said defective colanic acid synthesis pathway comprises a mutation in a colanic acid synthesis gene.
5. The method of claim 4 , wherein said colanic acid synthesis gene comprises a wcaJ, wzxC, wcaD, wza, wzb, or wzc gene.
6. The method of claim 1 , further comprising an inactivating mutation in a Ion gene.
7. The method of claim 6 , wherein said bacterium comprises a functional promoter-less wild-type E. coli lacZ + gene inserted into said Ion gene.
8. The method of claim 1 , wherein said bacterium further comprises an exogenous E. coli rcsA or E. coli rcsB gene.
9. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a Bacteroides fragilis wcfW gene or a Helicobacter pylori 26695 futA gene.
10. 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.
11. The method of claim 1 , wherein said bacterium comprises an endogenous lacY gene.
12. The method of claim 1 , wherein said bacterium comprises a lactose permease gene.
13. The method of claim 12 , wherein said lactose permease gene comprises an exogenous lactose permease gene.
14. The method of claim 1 , wherein said E.coli bacterium comprises a deletion of an endogenous β-galactosidase gene.
15. The method of claim 1 , wherein said bacterium accumulates an increased cytoplasmic lactose level, wherein the increased intracellular lactose level is at least 10% more than the level in a corresponding wild type bacterium.
16. The method of claim 1 , wherein said fucosylated oligosaccharide comprises 2′-fucosyllactose, 3-fucosyllactose, or lactodifucotetraose.
17. The method of claim 1 , wherein culturing said bacterium in step (b) does not comprise an antibiotic.
18. The method of claim 1 , wherein said bacterium comprises a plasmid expression vector.
19. The method of claim 1 , wherein said bacterium comprises an exogenous fucosyltransferase gene encoding α(1,2) fucosyltransferase or an exogenous fucosyltransferase gene encoding α(1,3) fucosyltransferase.
20. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises an α(1,3) fucosyltransferase gene from Helicobacter pylori , H. hepaticus, H. bilis, Campylobacter jejuni , or a species of Bacteroides.
21. The method of claim 1 , wherein said exogenous lactose-accepting fucosyltransferase gene comprises a E. coli strain O128:B12 wbsJ gene, a Helicobacter pylori 26695 futC gene, a H. hepaticus Hh0072 gene, a H. pylori 11639 FucTa gene, or a H. pylori UA948 FucTa gene.