Biosynthesis of oligosaccharides

Patent metadata

Field	Value
Publication number	US-9944965-B2
Application number	US-201314136466-A
Country	US
Kind code	B2
Filing date	Dec 20, 2013
Priority date	Dec 20, 2012
Publication date	Apr 17, 2018
Grant date	Apr 17, 2018

Abstract

The present invention relates to methods for production of 2′Fucosyllactose by a microbial system comprising a α-1,2 fucosyltransferase (FucT2) polynucleotide and a Guanosine 5′-diphospho-β-L-fucose (GDP-L-fucose) synthesis pathway using lactose as a substrate. Furthermore, the present invention relates to compositions comprising the microbial system.

First claim

We claim:

1. A composition comprising a recombinant microorganism wherein the microorganism expresses a phosphomannomutase (Man B) polynucleotide, a mannose 1-phosphate guanylytransferase (Man C) polynucleotide, a GDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide and a GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG) polynucleotide; and comprises a heterologous α-1,2 fucosyltransferase polynucleotide, wherein the α-1,2 fucosyltransferase polynucleotide is a Helicobacter pylori polynucleotide, wherein the recombinant microorganism overexpresses α-1,2 fucosyltransferase, and wherein 2′-fucosyllactose can be produced at an amount of 2.5 or more g/L.

2. The composition of claim 1 wherein the microorganism is a bacteria or yeast.

3. The composition of claim 2 wherein the bacteria is Escherichia coli and the yeast is Saccharomyces cerevisiae.

4. The composition of claim 3 wherein the Escherichia coli is DH5α strain or JM strain.

5. The composition of claim 4 wherein the Escherichia coli is JM strain.

6. The composition of claim 1 , wherein the microorganism has weak β-galactosidase activity.

7. The composition of claim 1 , wherein at least one of the phosphomannomutase (Man B) polynucleotide, mannose 1-phosphate guanylytransferase (Man C) polynucleotide, GDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide and GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG) polynucleotide is a heterologous polynucleotide.

8. The composition of claim 1 , wherein the recombinant microorganism is Escherichia coli.

9. The composition of claim 1 , wherein the recombinant microorganism is a bacterium.

10. The composition of claim 9 , wherein the recombinant microorganism overexpresses an Escherichia coli phosphomannomutase (Man B) polynucleotide, an Escherichia coli mannose 1-phosphate guanylytransferase (Man C) polynucleotide, an Escherichia coli GDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide, an Escherichia coli GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG) polynucleotide, or combinations thereof, and wherein the α-1,2 fucosyltransferase polynucleotide is a Helicobacter pylori polynucleotide.

11. A method for producing 2′-fucosyllactose (2-FL) in a microorganism, comprising i) providing the microorganism of claim 1 ; ii) fermenting the microorganism in the presence of lactose; and iii) collecting 2-FL from the microorganism or from a culture broth of the microorganism.

12. The method of claim 11 wherein the microorganism is a bacteria or yeast.

13. The method of claim 12 wherein the bacteria is Escherichia coli and the yeast is Saccharomyces cerevisiae.

14. The method of claim 13 wherein the Escherichia coli is DH5a strain or JM strain.

15. The method of claim 14 wherein the Escherichia coli is JM strain.

16. The method of claim 15 wherein the JM strain Escherichia coli overexpresses at least one of a phosphomannomutase (Man B) polynucleotide, a mannose 1-phosphate guanylytransferase (Man C) polynucleotide, a GDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide and a GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG) polynucleotide.

17. The method of claim 11 wherein the presence of lactose is at a concentration of more than 0.5 g/l.

18. The method of claim 11 wherein the presence of lactose is at a concentration of between 0.5 g/l to 15 g/l.

19. The method of claim 11 wherein the method of claim 11 further comprises purifying the 2-FL collected from the microorganism or from the culture broth of the microorganism by filtering through a purification column.

20. The method of claim 19 wherein the purification column is an activated charcoal and celite column.

21. The method of claim 11 , wherein the lactose is converted into the 2′-fucosyllactose (2-FL) at a rate of greater than about 0.1 g of 2-FL per gram of lactose.

22. The method of claim 11 , wherein the microorganism has weak β-galatosidase activity.

Assignees

• Univ Illinois
• Seoul National Univ Of R&Db Foundation
• Seoul Nat Univ R&Db Foundation

Inventors

• Lee Won-Heong
• Pathanibul Panchalee
• Quarterman Josh
• Miller Michael J
• Jin Yong-Su
• Seo Jin-Ho

Classifications

• C12P19/00

  Preparation of compounds containing saccharide radicals (ketoaldonic acids C12P7/58) · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C12Y204/01069

  Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69) · CPC title

• C12P19/18 Primary

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title