Helicobacter pylori α-1,3 fucosyltransferase gene and protein with improved soluble protein expression and activity, and thereof application for synthesis of α-1,3 fucosyloligosaccharide

Patent metadata

Field	Value
Publication number	US-10336990-B2
Application number	US-201515322072-A
Country	US
Kind code	B2
Filing date	Jun 22, 2015
Priority date	Jun 23, 2014
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

Abstract

The present invention provides an α-1,3 fucosyltransferase mutant having an increased expression level of soluble protein and increased activity, a DNA encoding the α-1,3 fucosyltransferase mutant, a recombinant vector comprising the DNA encoding the α-1,3 fucosyltransferase mutant, a host cell transformed with the recombinant DNA vector, an extract of the host cell, a method for producing 3-fucosyloligosaccharide, a method for preparing an α-1,3 fucosyltransferase mutant, and a method for screening an α-1,3 fucosyltransferase mutant. The α-1,3 fucosyltransferase mutant of the present invention has a significantly increased soluble protein expression level and activity.

First claim

The invention claimed is:

1. An α-1,3 fucosyltransferase mutant having any one sequence selected from (a) to (l):
(a) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine;
(b) a sequence wherein the amino acid at position 129 of any one of SEQ ID NOS: 1 to 3 is substituted with glutamic acid;
(c) a sequence wherein the amino acid at position 132 of any one of SEQ ID NOS: 1 to 3 is substituted with isoleucine;
(d) a sequence wherein the amino acid at position 46 of any one of SEQ ID NOS: 1 to 3 is substituted with phenylalanine;
(e) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine and the amino acid at position 129 is substituted with glutamic acid;
(f) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine and the amino acid at position 132 is substituted with isoleucine;
(g) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine and the amino acid at position 46 is substituted with phenylalanine;
(h) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, and the amino acid at position 132 is substituted with isoleucine;
(i) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, and the amino acid at position 46 is substituted with phenylalanine;
(j) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine, the amino acid at position 132 is substituted with isoleucine, and the amino acid at position 46 is substituted with phenylalanine;
(k) a sequence wherein the amino acid at position 128 of any one of SEQ ID NOS: 1 to 3 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, the amino acid at position 132 is substituted with isoleucine, and the amino acid at position 46 is substituted with phenylalanine; and
(l) a sequence wherein the amino acid at one or more positions selected from the group consisting of positions 128, 129, 132 and 46 of any one of SEQ ID NOS: 1 to 3 is substituted with another amino acid, wherein the amino acid at position 128 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, the amino acid at position 132 is substituted with isoleucine, and the amino acid at position 46 is substituted with phenylalanine.

2. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 10 wherein the amino acid at position 128 is substituted with asparagine and the amino acid at position 129 is substituted with glutamic acid.

3. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 11 wherein the amino acid at position 128 is substituted with asparagine and the amino acid at position 132 is substituted with isoleucine.

4. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 12 wherein the amino acid at position 128 is substituted with asparagine and the amino acid at position 46 is substituted with phenylalanine.

5. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 13 wherein the amino acid at position 128 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, and the amino acid at position 132 is substituted with isoleucine.

6. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 14 wherein the amino acid at position 128 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, and the amino acid at position 46 is substituted with phenylalanine.

7. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 15 wherein the amino acid at position 128 is substituted with asparagine, the amino acid at position 132 is substituted with isoleucine, and the amino acid at position 46 is substituted with phenylalanine.

8. The α-1,3 fucosyltransferase mutant of claim 1 , having the amino acid sequence of SEQ ID NO: 16 wherein the amino acid at position 128 is substituted with asparagine, the amino acid at position 129 is substituted with glutamic acid, the amino acid at position 132 is substituted with isoleucine, and the amino acid at position 46 is substituted with phenylalanine.

9. A DNA encoding the α-1,3 fucosyltransferase set forth in claim 1 .

10. The DNA of claim 9 , which has any one sequence selected from (a) to (k):
(a) the DNA sequence of SEQ ID NO: 17;
(b) the DNA sequence of SEQ ID NO: 18;
(c) the DNA sequence of SEQ ID NO: 19;
(d) the DNA sequence of SEQ ID NO: 20;
(e) the DNA sequence of SEQ ID NO: 21;
(f) the DNA sequence of SEQ ID NO: 22;
(g) the DNA sequence of SEQ ID NO: 23;
(h) the DNA sequence of SEQ ID NO: 24;
(i) the DNA sequence of SEQ ID NO: 25;
(j) the DNA sequence of SEQ ID NO: 26; and
(k) the DNA sequence of SEQ ID NO: 27.

11. A recombinant DNA vector comprising the DNA set forth in claim 9 or 10 .

12. A method for producing 3-fucosyloligosacchride, wherein either a host cell transformed with the recombinant DNA vector set forth in claim 11 , or an extract of the host cell, is used as a biocatalyst.

13. The method for producing 3-fucosyloligosacchride according to claim 12 , wherein 0.01 mM -2 mM of an inducer is used at a temperature between 15° C. and 37° C., wherein the inducer is IPTG (isopropyl β-D-1-thiogalactopyranoside), arabinose, or indole acrylic acid.

14. The method for producing 3-fucosyloligosacchride according to claim 12 , wherein a sugar receptor substrate is used at a concentration that is at least two times higher than that of a sugar donor substrate.

15. The recombinant DNA vector of claim 11 , wherein the vector comprises a strong promoter, wherein the strong promoter is trc promoter, tac promoter, T7 promoter, T5 promoter, lac promoter or trp promoter.

16. A host cell transformed with the recombinant DNA vector of claim 11 .

17. The method for producing 3-fucosyloligosacchride of claim 12 , wherein a medium supplemented with a carbon source or a nitrogen source is used.

Assignees

• Seoul Nat Univ R&Db Foundation

Inventors

• Kim Byung Gee
• Choi Yun Hee

Classifications

• C12Q1/48

  involving transferase · CPC title

• G01N2333/91102

  with definite EC number (2.4.1.-) · CPC title

• C12P19/00

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

• C12Y204/01065

  3-Galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase (2.4.1.65), i.e. alpha-1-3 fucosyltransferase · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title