KR-20260062987-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 glyA gene encoding a variant serine hydroxymethyltransferase having a substitution of a glycine residue at position 265 in the amino acid sequence of wild-type serine hydroxymethyltransferase 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

20260507

Application Date

20231228

Claims (14)

1. As a Corynebacterium bacterium capable of producing L-glutamic acid, Corynebacterium bacteria modified to retain a variant glyA gene encoding a variant serine hydroxymethyltransferase having a substitution of a glycine residue at position 265 in the amino acid sequence of wild-type serine hydroxymethyltransferase with another amino acid residue.
2. Corynebacterium bacteria according to claim 1, wherein the other amino acid is lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or tyrosine.
3. In paragraph 1, Corynebacterium bacteria in which the other amino acid is serine.
4. Corynebacterium bacteria according to any one of claims 1 to 3, wherein the wild-type serine hydroxymethyltransferase 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 substitution, deletion, insertion and/or addition of 1 to 10 amino acid residues, and also has serine hydroxymethyltransferase 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 serine hydroxymethyltransferase 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 serine hydroxymethyltransferase having a substitution of a glycine residue at position 265 in the amino acid sequence of wild-type serine hydroxymethyltransferase with another amino acid residue.
11. In paragraph 10, the above other amino acid is a variant serine hydroxymethyltransferase, which is lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or tyrosine.
12. In item 10, a variant serine hydroxymethyltransferase in which the other amino acid is serine.
13. A variant serine hydroxymethyltransferase according to any one of claims 10 to 12, wherein the protein encoded by the glyA 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 substitution, deletion, insertion and/or addition of 1 to 10 amino acid residues, and also has serine hydroxymethyltransferase 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 serine hydroxymethyltransferase activity.
14. A variant glyA gene encoding a variant serine hydroxymethyltransferase 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. Serine hydroxymethyltransferase is an enzyme that catalyzes the transfer of hydroxymethyl groups of serine and/or glycine (https://www.genome.jp/entry/2.1.2.1), but its relationship with L-glutamic acid production is unknown. Figure 1 is a diagram showing the accumulated amount of L-glutamic acid in wild-type glyA gene-introduced strains and mutant-type glyA gene-introduced strains. Figure 2 is a diagram showing the yield of L-glutamic acid in wild-type glyA gene-introduced strains and mutant-type glyA 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 (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum Corynebacterium glutamicum (formerly known as Brevibacterium lactofermentum) is a notable example of a Corynebacterium-type ba