Method for producing steviol and steviol glycoside using AOBGL1 homolog

Patent metadata

Field	Value
Publication number	US-11268118-B2
Application number	US-201615771191-A
Country	US
Kind code	B2
Filing date	Oct 28, 2016
Priority date	Oct 30, 2015
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The present invention provides a method for producing a steviol glycoside and/or steviol, said method including a step in which a steviol glycoside having at least one unbranched β1,2-glycosidic bond is reacted with the glycosidase AOBGL1 and/or AOBGL3, or a variant thereof, so as to cleave the β1,2-glycosidic bond.

First claim

The invention claimed is:

1. A method for producing a steviol glycoside having no unbranched β-1,2-glucoside bond, the method comprising:
reacting a protein selected from the group consisting of proteins (a) to (c) shown below
with
a steviol glycoside having at least one unbranched β-1,2-glucoside bond, thereby cleaving said unbranched β-1,2-glucoside bond:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4;
(b) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and
(c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside.

2. The method of claim 1 , wherein the protein selected from the group consisting of the proteins (a) to (c) further comprises a secretory signal peptide.

3. The method of claim 2 , wherein the secretory signal peptide is a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 28, 30, 32, 34, and 36.

4. The method of claim 3 , wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond is selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

5. The method of claim 4 , wherein the steviol glycoside having no unbranched β-1,2-glucoside bond is selected from rubusoside, rebaudioside A, and steviolmonoside.

6. A method for producing at least one of rebaudioside B and steviol comprising reacting together:
a protein selected from the group consisting of proteins (a) to (c) shown below,
a protein selected from the group consisting of proteins (d) to (f) shown below, and
a steviol glycoside having at least one unbranched β-1,2-glucoside bond:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4;
(b) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;
(c) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside;
(d) a protein comprising the amino acid sequence of SEQ ID NO: 7 or 8;
(e) a protein comprising an amino acid sequence wherein 1 to 77 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside; and
(f) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside.

7. The method of claim 6 , wherein the steviol glycoside having at least one unbranched β-1,2-glucoside bond comprises one or more steviol glycosides selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

Assignees

• Suntory Holdings Ltd

Inventors

• Ochiai Misa
• Ono Eiichiro

Classifications

• C12N9/2434 Primary

  acting on beta-1,4-glucosidic bonds · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title