KR-20260068112-A - L-glutamic acid producing bacteria and method for producing L-glutamic acid

Abstract

L-glutamic acid is produced by a method for producing L-glutamic acid and a bacterium used therein. L-glutamic acid is produced by culturing a Coryne-type bacterium capable of producing L-glutamic acid in a medium, the Coryne-type bacterium being modified to retain a variant acetyl CoA hydrolase gene encoding a variant acetyl CoA hydrolase having a substitution of a serine residue at position 383 in the amino acid sequence of wild-type acetyl CoA hydrolase with another amino acid residue, and collecting L-glutamic acid from the medium and/or the bacterium.

Inventors

• 하마노 코세키 치에
• 이노우에 코타
• 오니시 후미토

Assignees

• 아지노모토 가부시키가이샤

Dates

Publication Date

20260513

Application Date

20231228

Claims (14)

1. As a Corynebacterium bacterium capable of producing L-glutamic acid, Coryne-type bacteria modified to retain a variant acetyl CoA hydrolase gene encoding a variant acetyl CoA hydrolase having a substitution of a serine residue at position 383 in the amino acid sequence of wild-type acetyl CoA hydrolase with another amino acid residue.
2. Corynebacterium bacteria according to claim 1, wherein the other amino acid is lysine, glutamic acid, threonine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or methionine.
3. In claim 1, Coryne-type bacteria in which the other amino acid is cysteine.
4. Corynebacterium bacteria according to any one of claims 1 to 3, wherein the wild-type acetyl CoA hydrolase is a protein described in (a), (b), or (c) below. (a) A protein comprising the amino acid sequence shown in SEQ ID NO. 2; (b) an amino acid sequence comprising, wherein the amino acid sequence of SEQ ID NO. 2 comprises a substitution, deletion, insertion and/or addition of 1 to 10 amino acid residues, and also has acetyl CoA hydrolase activity; (c) A protein comprising an amino acid sequence having 90% or more identity with respect to the amino acid sequence shown in SEQ ID NO. 2, and also having acetyl CoA hydrolase activity.
5. A Corynebacterium according to any one of claims 1 to 4, wherein the Corynebacterium is a bacterium belonging to the genus Corynebacterium.
6. A Corynebacterium, wherein in any one of claims 1 to 4, the Corynebacterium is Corynebacterium glutamicum.
7. In any one of paragraphs 1 to 6, it is modified to retain the variant yggB gene, and Coryne-type bacteria, wherein the above variant yggB gene is a gene encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and/or added to the amino acid sequence of SEQ ID NO. 8.
8. In claim 7, the Coryne-type bacterium, wherein the variant yggB gene is a gene encoding a protein having the amino acid sequence of SEQ ID NO. 10.
9. As a method for producing L-glutamic acid, Culturing Coryne-type bacteria according to any one of claims 1 to 8 in a medium, and accumulating L-glutamic acid in the medium and/or within the cells of said bacteria, and Extracting L-glutamic acid from the above medium and/or the above cells A manufacturing method comprising
10. A variant acetyl CoA hydrolase having a substitution of a serine residue at position 383 in the amino acid sequence of wild-type acetyl CoA hydrolase with another amino acid residue.
11. In paragraph 10, a variant acetyl CoA hydrolase in which the other amino acid is lysine, glutamic acid, threonine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or methionine.
12. In item 10, a variant acetyl CoA hydrolase in which the other amino acid is cysteine.
13. A variant acetyl CoA hydrolase according to any one of claims 10 to 12, wherein the protein encoded by the acetyl CoA hydrolase gene is the protein described in (a), (b), or (c) below. (a) A protein comprising the amino acid sequence shown in SEQ ID NO. 2; (b) an amino acid sequence comprising, wherein the amino acid sequence of SEQ ID NO. 2 comprises a substitution, deletion, insertion and/or addition of 1 to 10 amino acid residues, and also has acetyl CoA hydrolase activity; (c) A protein comprising an amino acid sequence having 90% or more identity with respect to the amino acid sequence shown in SEQ ID NO. 2, and also having acetyl CoA hydrolase activity.
14. A variant acetyl CoA hydrolase gene encoding a variant acetyl CoA hydrolase according to any one of claims 10 to 13.

Description

L-glutamic acid producing bacteria and method for producing L-glutamic acid The present invention relates to the fermentation industry, and more specifically, to a method for producing L-glutamic acid and bacteria used therein. L-glutamic acid is industrially useful as a raw material for seasonings, etc. L-amino acids are industrially produced, for example, by fermentation using microorganisms such as bacteria capable of producing L-amino acids (Non-patent Literature 1). As such microorganisms, strains isolated from nature or mutant strains thereof are used. In addition, the L-amino acid production capacity of microorganisms can be enhanced by recombinant DNA technology. Acetyl-CoA hydrolase is an enzyme that has the activity to catalyze the reaction of hydrolyzing acetyl CoA to produce coenzyme A and acetic acid, and/or the reverse reaction thereof (Non-Patent Literature 2). It is known that by reducing the activity of said enzyme, the production capacity of L-glutamic acid, L-valine, and L-alanine is enhanced (Patent Literature 1). However, it is not known that a specific variation within the amino acid sequence of said enzyme contributes to the enhancement of L-glutamic acid production capacity. Figure 1 is a diagram showing the accumulation of L-glutamic acid in wild-type acetyl CoA hydrolase gene-introduced strains and mutant acetyl CoA hydrolase gene-introduced strains. Figure 2 is a diagram showing the yield of L-glutamic acid in wild-type acetyl CoA hydrolase gene-introduced strains and mutant acetyl CoA hydrolase gene-introduced strains. The present invention will be described in detail below. The method of the present invention is a method for producing L-glutamic acid, comprising culturing Corynebacterium bacteria capable of producing L-glutamic acid in a medium, accumulating L-glutamic acid in the medium and/or within the cells of the bacteria, and collecting L-glutamic acid from the medium and/or the cells, wherein the Corynebacterium bacteria are modified to retain a specific gene mutation. The bacteria used in this method are also referred to as "bacteria of the present invention." <1> Bacteria of the present invention The bacteria of the present invention are Coryne-type bacteria capable of producing L-glutamic acid, which are modified to retain a specific gene mutation. <1-1> Corynebacterium bacteria capable of producing L-glutamic acid In the present invention, "Coryne-type bacteria having L-glutamic acid production ability" refers to Coryne-type bacteria that, when cultured in a medium, produce L-glutamic acid and have the ability to accumulate it in the medium and/or within the cell in an amount that can be recovered. Coryne-type bacteria having L-glutamic acid production ability may be Coryne-type bacteria capable of accumulating a larger amount of L-glutamic acid in the medium and/or within the cell than non-variant strains. "Non-variant strain" refers to a control strain that has not been modified to retain a specific gene mutation. That is, wild strains or parent strains may be examples of non-variant strains. Furthermore, Coryne-type bacteria having L-glutamic acid production ability may be Coryne-type bacteria capable of accumulating the desired L-amino acid in the medium in an amount of preferably 0.5 g/L or more, and more preferably 1.0 g/L or more. The bacteria of the present invention may produce L-glutamic acid alone, or as a mixture of L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-type amino acids (also called L-amino acids). Examples of L-amino acids are not particularly limited but include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. In the present invention, the terms “glutamic acid” and “amino acid” refer to L-glutamic acid and L-amino acid, respectively, unless specifically stated otherwise. In addition, in the present invention, the terms “L-glutamic acid” and “L-amino acid” refer to L-glutamic acid, L-amino acid, salts thereof, or mixtures thereof, unless specifically stated otherwise. Salts will be described later. Examples of Corynebacterium bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium. Specifically, the following species can be cited as Corynebacterium bacteria. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Coryne