METHOD FOR PRODUCING THEANINE

• US 20200131546A1
• Filed: 04/12/2018
• Published: 04/30/2020
• Est. Priority Date: 04/13/2017
• Status: Active Grant

• Current Assignee: Kyowa Hakko Bio Co Ltd (Kirin Holdings Company Limited)
• Original Assignee: Kyowa Hakko Bio Co Ltd (Kirin Holdings Company Limited)
• Inventors: TABATA, Kazuhiko, OHNO, Shoto
• Granted Patent: US 11,155,845 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 9/80 acting on amide bonds in linear amides (3.5.1)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12R 2001/19 Escherichia coli
• CPC Class Codes: C12Y 305/01002 Glutaminase (3.5.1.2)
• CPC Class Codes: C12Y 603/04012 Glutamate--methylamine ligase (6.3.4.12)

Abstract

According to the present invention, theanine can efficiently be produced without exogenously adding ethylamine and without accumulation or leftover of ethylamine as a byproduct, by using a microorganism having enhanced activity to produce ethylamine with acetaldehyde and alanine as substrates and having enhanced activity of γ-glutamylmethylamide synthetase or glutaminase.

Claims

1. A microorganism producing acetaldehyde, alanine, glutamic acid, and ATP from a carbon source and having enhanced activity of a protein of any one of the following [1] to [3] and enhanced γ

-glutamylmethylamide synthetase activity compared to those of a parent strain;

[1] a protein comprising an amino acid sequence set forth in SEQ ID NO;

2, 4, 6, or 8;

[2] a mutant protein comprising an amino acid sequence modified from an amino acid sequence set forth in SEQ ID NO;

2, 4, 6, or 8 by deletion, substitution, insertion, or addition of 1 to 20 amino acids, and having activity to produce ethylamine with acetaldehyde and alanine as substrates (hereinafter, referred to as ethylamine-producing activity); and

[3] a homologous protein comprising an amino acid sequence having 95% or more identity with an amino acid sequence set forth in SEQ ID NO;

2, 4, 6, or 8, and having ethylamine-producing activity.

2. A microorganism producing acetaldehyde, alanine, and glutamine from a carbon source, and having enhanced activity of a protein of any one of [1] to [3] in claim 1 and enhanced glutaminase activity compared to those of a parent strain.

3. A method for producing theanine, comprising:

providing a protein of any one of [1] to [3] in claim 1 and γ

-glutamylmethylamide synthetase together in an aqueous medium comprising acetaldehyde, alanine, glutamic acid, and ATP to produce and accumulate theanine in the aqueous medium; and

collecting theanine from the aqueous medium.

4. A method for producing theanine, comprising:

providing a protein of any one of [1] to [3] in claim 1 and glutaminase together in an aqueous medium comprising acetaldehyde, alanine, and glutamine to produce and accumulate theanine in the aqueous medium; and

collecting theanine from the aqueous medium.

5. A method for producing theanine, comprising:

culturing the microorganism according to claim 1 in a culture medium to produce and accumulate theanine in a culture; and

collecting theanine from the culture.

6. A method for producing theanine, comprising:

providing a culture of the microorganism according to claim 1 or a processed product of the culture, acetaldehyde, alanine, glutamic acid, and ATP together in an aqueous medium to produce and accumulate theanine in the aqueous medium; and

collecting theanine from the aqueous medium.

7. A method for producing theanine, comprising:

providing a culture of the microorganism according to claim 2 or a processed product of the culture, acetaldehyde, alanine, and glutamine together in an aqueous medium to produce and accumulate theanine in the aqueous medium; and

collecting theanine from the aqueous medium.

8. The microorganism according to claim 1, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

9. The method for producing theanine according to claim 5, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

10. The microorganism according to claim 1, wherein the carbon source is sugar.

11. A method for producing theanine, comprising:

culturing the microorganism according to claim 2 in a culture medium to produce and accumulate theanine in a culture; and

collecting theanine from the culture.

12. The microorganism according to claim 2, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

13. The method for producing theanine according to claim 11, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

14. The method for producing theanine according to claim 6, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

15. The method for producing theanine according to claim 7, wherein the microorganism is a microorganism belonging to the genus Escherichia or the genus Corynebacterium.

16. The microorganism according to claim 2, wherein the carbon source is sugar.