Process for the production of fucosylated oligosaccharides

1. A method for the production of fucosylated oligosaccharides using a genetically modified prokaryotic host cell, the method comprising:

providing a prokaryotic host cell which has been genetically modified to have:

(i) reduced or abolished activity of a fructose-6-phosphate-converting enzyme as compared to the activity in an unmodified prokaryotic host cell, wherein the fructose-6-phosphate converting enzyme is selected from the group consisting of phosphofructokinase, glucose-6-phosphate isomerase, fructose-6-phosphate aldolase, a transketolase, and a transaldolase;

(ii) overexpression of at least one gene encoding an enzyme necessary for the de novo synthesis of GDP-fucose, wherein said at least one gene encoding an enzyme necessary for the de novo synthesis of GDP-fucose is a phosphomannomutase encoding gene, a mannose-1-phosphate guanosyltransferase encoding gene, a GDP-mannose-4,6-dehydratase encoding gene, or a GDP-L-fucose synthase encoding gene; and

(iii) expression of an exogenous gene encoding an alpha-1,2-fucosyltransferase and/or an alpha-1,3-fucosyltransferase; and

cultivating said genetically modified prokaryotic host cell in a cultivation medium comprising at least one carbon and/or energy source selected from one or more of the group consisting of glycerol, succinate, malate, pyruvate, lactate, ethanol, and citrate; and

adding lactose to the cultivation medium;

wherein the fucosylated oligosaccharide is obtained from the medium in which the host cell is cultivated; and

wherein said genetically modified prokaryotic host cell has an intracellular pool of fructose-6-phosphate that is increased by increasing the activity of a fructose-1,6-bisphosphate phosphatase.

2. The method of claim 1 , wherein the fucosylated oligosaccharide is selected from the group consisting of 2′-fucosyllactose, 3-fucosyllactose or difucosyllactose.

3. The method of claim 1 , wherein the prokaryotic host cell is selected from the group consisting of bacterial cells from an Escherichia coli strain, a Lactobacillus species or a Corynebacterium glutamicum strain.

4. The method of claim 1 , wherein the phosphomannomutase encoding gene is manB, the mannose-1-phosphate guanosyltransferase encoding gene is manC, the GDP-mannose-4,6-dehydratase encoding gene is gmd, and/or the GDP-L-fucose synthase encoding gene is wcaG.

5. The method of claim 1 , wherein the gene encoding the alpha-1,2-fucosyltransferase is wbgL from E. coli O126 or fucT2 from Helicobacter pylori.

6. The method of claim 1 , wherein the gene encoding the alpha-1,3-fucosyltransferase is from the species Akkermansia muciniphila, Bacteroides fragilis, Helicobacter pylori , or Helicobacter hepaticus.

7. The method of claim 1 , wherein the host cell is further genetically modified to express a gene encoding a protein which enables or facilitates the export of a fucosylated oligosaccharide into the culture medium.

8. The method of claim 1 , wherein the host cell is further genetically modified to overexpress an endogenous or exogenous permease for the import of lactose.

9. The method of claim 1 , wherein at least one of the expressed or overexpressed genes in (ii) or (iii) is expressed or overexpressed in a constitutive manner.

10. The method of claim 7 , wherein the gene encoding a protein which enables or facilitates the export of the desired fucosylated oligosaccharide is a sugar efflux transporter selected from the group consisting of yberc0001_9420 and setA.

11. The method of claim 1 , wherein the fructose-1,6-bisphosphate phosphatase is encoded by a gene which is a functional active variant of the fructose-1,6-bisphosphate phosphatase (fbpase) from Pisum sativum.

12. The method of claim 8 , wherein the lactose permease is E. coli LacY.

13. The method of claim 1 , wherein the lactose is added from the beginning of the cultivating in a concentration of at least 5 mM, optionally in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, optionally in a concentration >300 mM.

14. The method of claim 1 , wherein providing of lactose is accomplished by adding lactose to the cultivation medium in a concentration, such that throughout the production phase of the cultivation a lactose concentration of at least 5 mM.

15. The method of claim 1 , wherein the host cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

16. The method of claim 1 , wherein the modified genes are integrated into the genome of the host strain.

17. The method of claim 14 , wherein the lactose concentration is at least 10 mM.

18. The method of claim 14 , wherein the lactose concentration is at least 30 mM.