Increasing export of 2'fucosyllactose from microbial cells through the expression of a heterologous nucleic acid

Patent metadata

Field	Value
Publication number	US-2021238574-A1
Application number	US-201817049814-A
Country	US
Kind code	A1
Filing date	Apr 23, 2018
Priority date	Apr 23, 2018
Publication date	Aug 5, 2021
Grant date	—

Abstract

Microbial cells genetically engineered with a heterologous nucleic acid sequence that increases export of 2′ fucosyllactose are disclosed. Methods of increasing export of 2′ fucosyllactose from a microbial cell and for identifying a heterologous nucleic acid sequence that increases export of 2′ fucosyllactose from a microbial cell are also disclosed.

First claim

1 . A genetically engineered microbial cell, wherein:
the cell comprises:
a) at least one heterologous nucleic acid molecule encoding a transporter protein that facilitates the export of 2′ fucosyllactose from the microbial cell;
b) at least one heterologous nucleic acid molecule encoding a GDP-mannose-4,6-dehydratase (EC 4.2.1.47);
c) at least one heterologous nucleic acid molecule encoding a GDP-4-keto-6-D-deoxymannose epimerase-reductase (EC 1.1.1.271); and
d) at least one heterologous nucleic acid molecule encoding a 2-□-L-fucosyltransferase (EC 2.4.1.69); and
said microbial cell produces 2′ fucosyllactose.

2 . The genetically engineering microbial cell of claim 1 wherein the microbial cell is a yeast.

3 . The genetically engineering microbial cell of claim 2 wherein the yeast is selected from the group of genera consisting of Saccharomyces, Yarrowia, Kluyveromyces, Candida, Hansenula, Pichia, Schizosaccharomyces, Zygosaccharomyces, Debaryomyces, Brettanomyces, Pachysolen, Issatchenkia, Trichosporon , and Yamadazyma.

4 . The genetically engineering microbial cell of claim 1 wherein the microbial cell is a bacteria.

5 . The genetically engineering microbial cell of claim 4 wherein the bacteria is selected from the group of genera consisting of Escherichia, Bacillus, Methylomonas, Pseudomonas, Lactobacillus , and Corynebacterium.

6 . The genetically engineering microbial cell of claim 1 wherein the transporter protein is selected from the group consisting of the SWEET family of transporters, the SetA family of transporters, and the Sugar porter family of transporters.

7 . The genetically engineering microbial cell of claim 6 wherein the transporter is a SWEET transporter having at least 90% identity to an amino acid sequence selected from the group consisting if SEQ ID NO: 93, 94, 95, and 96.

8 . The genetically engineering microbial cell of claim 6 wherein transporter is a SetA protein having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 88, 105, 106, 107, and 108.

9 . The genetically engineering microbial cell of claim 6 wherein the transporter is a Sugar porter protein having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 65 and 66.

10 . The genetically engineering microbial cell of claim 1 wherein the at least one heterologous nucleic acid molecule of any of parts b) c) or d) are derived from a bacteria or a fungus.

11 . The genetically engineered microbial cell of claim 1 wherein the heterologous nucleic acid molecule of part a) further comprises a nucleic acid sequence which encodes an amino acid sequence which facilitates localization of the protein to the plasma membrane of the cell.

12 . The genetically engineered microbial cell of claim 1 wherein the cell further comprises at least one nucleic acid sequence encoding a lactose transporter.

13 . The genetically engineered microbial cell of claim 12 wherein the lactose transporter is a lactose permease.

14 . The genetically engineered microbial cell of claim 12 wherein the lactose transporter has an amino acid sequence having 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 and 28.

15 . A method for the production of 2′ fucosyllactose from a microbial cell comprising growing the genetically engineered microbial cell of claim 1 comprising at least one transporter protein, under suitable conditions and in suitable media wherein 2′ fucosyllactose is produced and exported to the media.

16 . The method of claim 15 wherein the genetically engineered microbial cell exports 2′ fucosyllactose in to the media at a rate at least 1.5× the rate of export of a similar genetically engineered microbial cell which lacks a transporter protein.

17 . A method of the production of 2′ fucosyllactose from a microbial cell comprising:
a) providing a genetically engineered microbial cell comprising:
i) at least one nucleic acid molecule encoding a transporter protein that facilitates the export of 2′ fucosyllactose from the microbial cell;
ii) at least one heterologous nucleic acid molecule encoding a GDP-mannose-4,6-dehydratase (EC 4.2.1.47);
iii) at least one heterologous nucleic acid molecule encoding a GDP-4-keto-6-D-deoxymannose epimerase-reductase (EC 1.1.1.271); and
iv) at least one heterologous nucleic acid molecule encoding a 2-10-L-fucosyltransferase (EC 2.4.1.69);
b) growing the microbial cell of step a) in media comprising a first carbon source, at a suitable temperature, and suitable pH to obtain a suitable cell concentration to produce a seed culture;
c) seeding the seed culture of step b) into a fermentation media comprising a second carbon source;
d) growing the seeded culture of step c) at a suitable, temperature and suitable pH until the point of exhaustion of the second carbon source wherein 2′ fucosyllactose is produced; and
e) optionally recovering the 2′ fucosyllactose.

18 . The method of claim 17 wherein the at least one nucleic acid molecule encoding a transporter protein that facilitates the export of 2′ fucosyllactose from the microbial cell is heterologous the cell.

19 . The method of claim 17 wherein the genetically engineering microbial cell is a yeast.

20 . The method of claim 18 wherein the transporter protein is selected from the group consisting of the SWEET family of transporters, the SetA family of transporters, and the Sugar porter family of transporters.

21 . The method of claim 17 wherein the microbial cell further comprises at least one nucleic acid sequence encoding a lactose transporter.

22 . The method of claim 17 wherein the at least one nucleic acid molecule encoding a 2-α-L-fucosyltransferase is under the control of an inducible promoter.

23 . The method of claim 17 wherein the first and second carbon source selected from the group consisting of glucose, sucrose, lactose and fructose.

24 . The method of claim 17 wherein the first carbon source is selected from the group consisting of glucose, sucrose and fructose and wherein the second carbon source is lactose.

25 . The method of claim 17 wherein the suitable temperature of steps b) and d) range from about 30 C to about 35 C.

26 . The method of claim 17 wherein the suitable pH of steps b) and d) range from about 5.4 to about 5.6.

27 . The method of claim 24 wherein the suitable pH of step b) ranges from about pH 5.4 to about 5.6 and wherein the suitable pH of step d) ranges from about 6.0 to about 7.0.

28 . The method of claim 27 wherein at step d) an inducer is added that induces the expression of the at least one nucleic acid molecule encoding a 2-α-L-fucosyltransferase.

29 . The method of claim 17 wherein the total 2′ fucosyllactose produced is about 10 g/l to about 50 g/l.

30 . A method for identifying a heterologous nucleic acid sequence that, when expressed in a microbial cell, increases the export of 2′ fucosyllactose from the microbial cell, the method comprising:
a) obtaining a 2′FL-producing yeast cell;
b) expressing a candidate heterologous nucleic acid sequence in the 2′FL-producing yeast cell of (a) whereby a screening cell is produced;
c) growing the screening cell of (b) in a growth medium under conditions where 2′FL is present

Assignees

• Dupont Nutrition Biosci Aps

Inventors

• Hollands Kerry
• Maggio-Hall Lori Anne
• Rothman Steven Cary

Classifications

• C12Y101/01

  with NAD+ or NADP+ as acceptor (1.1.1) · CPC title

• C12Y402/01047

  GDP-mannose 4,6-dehydratase (4.2.1.47), i.e. GMD · CPC title

• C12Y204/01069

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

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/18

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