JP2022521544A - Method for producing L-amino acid using a bacterium belonging to the family Enterobacteriaceae that overexpresses the ydiJ gene - Google Patents

Abstract

The present invention provides a method for producing an L-amino acid by fermentation using a bacterium belonging to the family Enterobacteriaceae modified to overexpress the ydiJ gene.

Description

The present invention relates widely to the microbial industry, in particular, to ferment L-amino acids from bacteria belonging to the family Enterobacteriaceae modified to overexpress the ydiJ gene and enhance L-amino acid production. Regarding the manufacturing method.

Conventionally, L-amino acids have been industrially produced by a fermentation method using a microbial strain obtained from a natural source or a mutant thereof. Typically, such microorganisms have been modified to increase the yield of L-amino acids. Many techniques for increasing the yield of L-amino acids have been reported, including transformation of microorganisms with recombinant DNA (see, eg, U.S. Patent No. 4,278,765 A), promoters, leader sequences and / or attestation. There are modifications of expression control regions known to the nuator, or other traders (see, eg, US20060216796 A1 and WO9615246 A1). Other techniques for increasing production yields include increasing the activity of enzymes involved in amino acid biosynthesis and / or releasing the feedback inhibition of the enzyme of interest by the L-amino acids produced (eg WO9516042 A1). , EP0685555 A1, or U.S. Patent Nos. 4,346,170 A, 5,661,012 A, and 6,040,160 A).

As another method for increasing the production yield of L-amino acid, one or several genes involved in the degradation of the target L-amino acid, the L-amino acid from the biosynthesis pathway of the target L-amino acid. It may include reducing the expression of genes that divert precursors to other pathways, genes involved in the redistributing of carbon, nitrogen, sulfur, and phosphate flows, and genes encoding toxins.

The E. coli-specific YdiJ protein encoded by the ydiJ gene is characterized as a putative FAD-bound oxidoreductase (EcoCyc database, https://ecocyc.org/, accession ID: G6913). YdiJ has been demonstrated to be a Fe ₄ S ₄ FAD-containing protein (Estellon J. et al., An integrative computational model for large-scale identification of metalloproteins in microbial genomes: a focus on iron-sulfur cluster proteins, Metallomics, 2014, 6 (10): 1913-1930).

However, no data have been reported so far that describe the effect of overexpression of the ydiJ gene on the production of L-amino acids by fermentation of L-amino acid-producing bacteria belonging to the family Enterobacteriaceae.

This specification describes an improved method for producing L-amino acids by fermentation of bacteria belonging to the family Enterobacteriaceae. According to the present invention, it is possible to increase the production of L-amino acids by fermentation of bacteria belonging to the family Enterobacteriaceae. Specifically, the production of L-amino acid by fermentation of a bacterium belonging to the family Enterobacteriaceae modifies the bacterium so as to overexpress the ydiJ gene, thereby producing L-amino acid by the modified bacterium. It can be improved by increasing it.

The present invention provides the following.

One aspect of the present invention is a method for producing an L-amino acid.
(I) Culturing L-amino acid-producing bacteria belonging to the family Enterobacteriaceae in a medium to produce and accumulate L-amino acids in the medium and / or cells of the bacteria, and (ii). Containing the recovery of L-amino acids from the medium and / or cells,
It is to provide a method in which the bacterium has been modified to overexpress the ydiJ gene.

Another aspect of the invention is to provide the method in which the ydiJ gene is selected from the group consisting of:
(A) Gene containing the base sequence shown in SEQ ID NO: 1 or 3;
(B) A gene encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2 or 4;
(C) A gene encoding a protein containing an amino acid sequence containing substitutions, deletions, insertions, and / or additions of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 4. A gene in which the protein has the activity of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(D) A gene encoding a protein containing an amino acid sequence having 70% or more identity with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 4, wherein the protein has the amino acid sequence shown in SEQ ID NO: 2 or 4. A gene having the activity of a protein containing: and (E) a gene containing a variant base sequence of SEQ ID NO: 1 or 3, wherein the variant base sequence is due to the degeneracy of the genetic code.

In another aspect of the present invention, the ydiJ gene is overexpressed by introducing the ydiJ gene, increasing the number of copies of the ydiJ gene, and / or modifying the expression control region of the ydiJ gene. It is to provide the method, wherein the expression of the gene is enhanced as compared to the unmodified bacterium.

Another aspect of the invention is to provide the method, wherein the bacterium belongs to the genus Escherichia or Pantoea.

Another aspect of the invention is to provide the method, wherein the bacterium is Escherichia coli or Pantoea ananatis.

Another aspect of the present invention provides the method, wherein the L-amino acid is selected from the group consisting of aromatic L-amino acids, non-aromatic L-amino acids, sulfur-containing L-amino acids, and combinations thereof. That is.

Another aspect of the present invention is to provide the method in which the aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, L-tyrosine, and combinations thereof.

In another aspect of the present invention, the non-aromatic L-amino acid is L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulin, L-cysteine, L-glutamic acid, L-glutamine. From the group consisting of glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, L-valine, and combinations thereof. To provide the method of choice.

Another aspect of the invention provides the method, wherein the sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, L-cystine, and combinations thereof. That is.

Still other objects, features, and concomitant advantages of the present invention will be apparent to those skilled in the art by reading the following detailed description of embodiments constructed accordingly.

1. 1. Bacteria The bacteria described herein are L-amino acid-producing bacteria belonging to the family Enterobacteriaceae modified to overexpress the ydiJ gene. The bacteria described herein can be used in the manner described herein. Accordingly, the following description of the bacterium can be applied mutatis mutandis to any bacterium that is substitutable or equivalently used by the methods described herein.

The bacterium that can be used in the method described herein can be a bacterium that is appropriately selected depending on the type of L-amino acid of interest produced using that method.

Any L-amino acid-producing bacterium belonging to the family Enterobacteriaceae can be used by the method described herein as long as it is modified to overexpress the ydiJ gene. For example, an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae is described herein as long as it is modified to overexpress the ydiJ gene and thus the production of L-amino acids can be enhanced as compared to unmodified bacteria. It can be used by the method described in the book.

The term "L-amino acid-producing bacterium" may be used interchangeably or equivalently with the term "bacteria capable of producing L-amino acids" or "bacteria capable of producing L-amino acids".

The term "L-amino acid-producing bacterium" means that when the bacterium is cultured in a medium, L-amino acid is produced and excreted in the medium and / or in the bacterial cell (also referred to as "bacterial cell"). It can mean a bacterium belonging to the family Enterobacteriaceae that has the ability to secrete and / or accumulate.

Also, the term "L-amino acid-producing bacterium" has the ability to produce, excrete or secrete, and / or accumulate L-amino acids in a medium in larger amounts compared to, for example, unmodified bacteria. Bacteria can also mean. The term "unmodified bacterium" may be used in an alternative or equivalent manner to the term "unmodified strain". The term "unmodified strain" can mean a control strain that has not been modified to overexpress the ydiJ gene. Unmodified bacteria include, for example, Escherichia coli (E. coli).
Wild or parent strains include K-12 strains (eg, W3110 (ATCC 27325) and MG1655 (ATCC 47076)) and Pantoea ananatis (P. ananatis) AJ13355. Further, the term "L-amino acid-producing bacterium" can be used to accumulate a target L-amino acid in a medium in an amount of, for example, 0.1 g / L or more, 0.5 g / L or more, or 1.0 g / L or more. Bacteria can also mean. Also, the term "L-amino acid-producing bacterium" has the ability to produce, excrete or secrete, and / or accumulate L-amino acids in the culture medium in larger amounts compared to, for example, unmodified bacteria. However, it can also mean a bacterium capable of accumulating L-amino acids in a medium in an amount of 0.1 g / L or more, 0.5 g / L or more, or 1.0 g / L or more.

Bacteria may be inherently capable of producing L-amino acids or may be modified to be capable of producing L-amino acids. Such modifications can be achieved, for example, by mutation methods or DNA recombination techniques. The bacterium can be obtained by overexpressing the ydiJ gene in a bacterium that originally has an L-amino acid-producing ability, or in a bacterium that has already been endowed with the L-amino acid-producing ability. Alternatively, the bacterium can be obtained by imparting L-amino acid-producing ability to a bacterium that has already been modified to overexpress the ydiJ gene. Alternatively, the bacterium may have acquired L-amino acid-producing ability by being modified to overexpress the ydiJ gene. Specifically, the bacterium described in the present specification can be obtained, for example, by modifying a bacterial strain described later.

The term "L-amino acid-producing ability" is a bacterium that produces, excretes or secretes, and / or accumulates L-amino acids in the medium and / or the cells of the bacterium when the bacterium is cultured in the medium. Can mean the ability of. The term "L-amino acid-producing ability" specifically refers to L-amino acids in the medium and / or the bacterial cells to the extent that they can be recovered from the medium and / or the bacterial cells when the bacteria are cultured in the medium. Can mean the ability of bacteria belonging to the family Enterobacteriaceae to produce, excrete or secrete and / or accumulate.

The term "cultured", which refers to bacteria that may be used in the methods described herein, is substitutable or equivalent to terms such as "cultivated" that are well known to those of skill in the art. May be used for.

Bacteria can produce L-amino acids alone or as a mixture of one or more substances different from L-amino acids and L-amino acids. For example, a bacterium may use an L-amino acid of interest alone or one or more amino acids different from the L-amino acid of interest and the L-amino acid of interest (for example, an L-form amino acid (also referred to as L-amino acid)). Etc.) can be produced as a mixture. In other words, it is permissible for bacteria to be able to produce two or more L-amino acids as a mixture. Further, for example, the bacterium can produce the L-amino acid of interest alone or as a mixture of the L-amino acid of interest and one or more other organic acids (eg, carboxylic acid, etc.).

The L-amino acid is not particularly limited, but is L-alanine, L-arginine, L-aspartin, L-aspartic acid, L-citrulin, 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. Will be.

The carboxylic acid is not particularly limited, and examples thereof include formic acid, acetic acid, citric acid, butyric acid, lactic acid, propionic acid, and derivatives thereof.

The terms "L-amino acid" and "carboxylic acid" are not limited to free forms of L-amino acids and carboxylic acids, but also their derivative forms (salts, hydrates, adducts, or combinations thereof, etc.). Can be. The adduct can be a compound formed of an L-amino acid or carboxylic acid and another organic or inorganic compound. That is, the terms "L-amino acid" and "carboxylic acid" can mean, for example, L-amino acids and carboxylic acids that are in free form, derivative form, or mixtures thereof. The terms "L-amino acid" and "carboxylic acid" can mean, for example, in particular free forms of L-amino acids and carboxylic acids, salts thereof, or mixtures thereof. The terms "L-amino acid" and "carboxylic acid" are, for example, their sodium salts, potassium salts, ammonium salts, monohydrates, dihydrates, trihydrates, monohydrochlorides, dihydrochlorides. Any of the salts such as can mean. Unless otherwise stated, the terms "L-amino acid" and "carboxylic acid" (eg, "free form of L-amino acid or carboxylic acid" or "salt of L-amino acid or carboxylic acid" without reference to hydrated state. The term) can mean non-hydrates of L-amino acids and carboxylic acids, and can also mean hydrates of L-amino acids and carboxylic acids.

L-amino acids can belong to one or more L-amino acid families. As an example, the L-amino acid is a glutamic acid family containing L-arginine, L-glutamic acid, L-glutamine, and L-proline; a serine family containing L-cysteine, glycine, and L-serine; L-asparagine, L- Aspartic acid, L-isoleucine,
Aspartic acid family including L-lysine, L-methionine, and L-threonine; pyruvate family including L-alanine, L-isoleucine, L-valine, and L-leucine; and L-phenylalanine, L-tryptophan, and It may belong to an aromatic family containing L-tyrosine. Since L-histidine has an aromatic moiety such as an imidazole ring, the term "aromatic L-amino acid" may mean L-histidine in addition to the above aromatic L-amino acid.

L-amino acids can also belong to the non-aromatic family, such as L-alanine, L-arginine, L-aspartin, L-aspartic acid, L-citrulin, L-cysteine, L-glutamic acid. , L-glutamine, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, L-valine. Since the biosynthetic pathway of aromatic amino acids such as L-phenylalanine, L-tryptophane, and L-tyrosine is different from the biosynthetic pathway of L-histidine, the term "non-aromatic L-amino acid" is used as described above for non-aromatic L. -In addition to amino acids, it can mean L-histidine.

Further, the L-amino acid can also belong to the sulfur-containing L-amino acid family, and examples thereof include L-cysteine, L-methionine, L-homocysteine, and L-cystine.

Since some L-amino acids can be intermediate amino acids in the biosynthetic pathway of a particular L-amino acid, the above amino acid family also includes other L-amino acids (eg, non-protein-producing L-amino acids). good. For example, L-citrulline and L-ornithine are amino acids in the arginine biosynthetic pathway. Therefore, the glutamic acid family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.

Bacteria belonging to the family Enterobacteriaceae include Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, and Providencia. (Providencia), Salmonella (Salmonella), Yersinia (Yersinia) and other bacteria belonging to the genus can be mentioned. Such bacteria may have the ability to produce L-amino acids. Specifically, the Department of Enterobacteriaceae is based on the taxonomy used in the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543). Bacteria classified in can be used. Bacteria belonging to the family Enterobacteriaceae include, in particular, bacteria belonging to the genera Escherichia, Enterobacter, and Pantoea.

Bacteria of the genus Escherichia are not particularly limited, and specifically, the book of Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In FC Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2nd ^ed . ASM Press, Washington, DC, 1996). Bacteria of the genus Escherichia include, in particular, Escherichia coli (E. coli). Specific examples of E. coli include E. coli K-12 strains (E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), etc.), which are prototype wild strains.

Examples of the Enterobacter bacterium include Enterobacter agglomerans and Enterobacter aerogenes. Pantoea ananatis (P. ananatis (P. ananatis)
)) And so on. In recent years, some strains of Enterobacter agglomerans have been analyzed by pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii by base sequence analysis of 16S rRNA. It has been reclassified to. Any bacterium belonging to the genus Enterobacter or Pantoea can be used as long as it is a bacterium classified in the family Enterobacteriaceae. Specific examples of P. ananatis include Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), and derivative strains thereof. These strains were identified as Enterobacter agglomerans at the time of isolation and deposited as Enterobacter agglomerans, but as mentioned above, they have recently been reclassified as Pantoea ananatis by base sequence analysis of 16S rRNA.

These strains can be obtained, for example, from the American Type Culture Collection (ATCC; Address: PO Box 1549, Manassas, VA 20108, United States of America). That is, each strain is given a corresponding registration number, and an order can be placed using this registration number (see https://www.lgcstandards-atcc.org). The registration number of each strain can be found in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depositary institution where each strain was deposited.

The addition or enhancement of L-amino acid-producing ability can be carried out by a method conventionally adopted for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacterium (Amino Acid Fermentation, Amino Acid Publishing Center, 1986). First edition published on May 30, 2014, see pages 77-100). Such methods include, for example, acquisition of an auxotrophic mutant strain, acquisition of an analog-resistant strain of L-amino acid, acquisition of a metabolic control mutant strain, and recombination in which the activity of an L-amino acid biosynthetic enzyme is enhanced. The creation of strains can be mentioned. In the breeding of L-amino acid-producing bacteria, the properties such as auxotrophy, analog resistance, and metabolic control mutation imparted may be single, or may be two or more. Further, in the breeding of L-amino acid-producing bacteria, the L-amino acid biosynthetic enzyme whose activity is enhanced may be a single enzyme or two or three or more. Furthermore, the impartation of properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with the enhancement of the activity of biosynthetic enzymes.

For auxotrophic mutants, analog-resistant strains, or metabolically controlled mutants having L-amino acid-producing ability, the parent strain or wild-type strain is subjected to normal mutation treatment, and the obtained mutant strains are auxotrophic and analog. It can be obtained by selecting one that exhibits resistance or metabolic control mutation and has L-amino acid-producing ability. Normal mutation treatment includes irradiation with X-rays or ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethane sulphonate (EMS), methyl methane sulphonate (MMS), etc. Treatment with a mutant agent can be mentioned.

Further, the addition or enhancement of the L-amino acid producing ability can also be performed by enhancing the activity of the enzyme involved in the biosynthesis of the target L-amino acid. Enzyme activity can be enhanced, for example, by modifying the bacterium so that the expression of the gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935, EP1010755A and the like. The method of overexpressing the ydiJ gene described later can be similarly applied to the enhancement of the activity of any protein or the enhancement of the expression of any gene.

Further, the addition or enhancement of the L-amino acid producing ability is also carried out by reducing the activity of the enzyme that catalyzes the reaction of branching from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can be carried out. The term "enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid" is involved in the decomposition of the target L-amino acid. Enzymes can also mean. The method of weakening the expression of a gene described later can also be applied to a decrease in the activity of any protein or a weakening of the expression of any gene.

Hereinafter, the L-amino acid-producing bacteria and the method for imparting or enhancing the L-amino acid-producing ability will be specifically exemplified. The properties of the L-amino acid-producing bacteria and the modifications for imparting or enhancing the L-amino acid-producing ability as exemplified below may be used alone or in combination as appropriate.

<L-arginine-producing bacteria>
Parent strains that can be used to induce L-arginine-producing bacteria and L-arginine-producing bacteria include, for example, strains in which the activity of one or more L-arginine biosynthetic enzymes is increased. Such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetyl-γ-glutamylphosphate reductase (argC), N-acetylornithine aminotransferase (N-acetylornithine aminotransferase). argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinic acid lyase (argH), ornithine acetyltransferase (argJ), carbamoyl phosphate synthase (carAB) Can be mentioned. An example of a gene encoding the enzyme is shown in parentheses after the enzyme name (the same nomenclature applies mutatis mutandis to the following when referring to proteins / enzymes and genes). As the N-acetylglutamate synthase (argA) gene, for example, a mutant N-acetylglutamate synthase in which the amino acid residues corresponding to the 15th to 19th positions of the wild type are substituted and the feedback inhibition by L-arginine is released is used. It is preferable to use the encoding gene (EP1170361A).

Specific examples of the parent strain that can be used to induce L-arginine-producing strains and L-arginine-producing strains include, for example, E. coli 237 strain (VKPM B-7925; US2002-058315A1), mutant N-acetylglutamic acid. The inducible strain into which the argA gene encoding synthase was introduced (Russian Patent Application No. 2001112869, EP1170361A1) and the E. coli 382 strain (VKPM B-7926; EP1170358A1) derived from 237 strains with improved acetic acid assimilation ability. ), And strains belonging to the genus Escherichia such as E. coli 382ilvA + strain, which is a strain in which the wild-type ilvA gene derived from E. coli K-12 strain is introduced into 382 strains. E. coli 237 strains of VKPM B-7925 to the Lucian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on April 10, 2000. It was deposited with a deposit number and transferred to an international deposit under the Budapest Convention on May 18, 2001. E. coli 382 strains of VKPM B-7926 to the Lucian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on April 10, 2000. It was deposited with a deposit number and transferred to an international deposit under the Budapest Convention on May 18, 2001.

Examples of the parent strain that can be used to induce L-arginine-producing bacteria and L-arginine-producing bacteria include strains having resistance to amino acid analogs and the like. Such strains include, for example, α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine, or. Examples thereof include Escherichia coli mutant strains resistant to sulfaguanidine (see JP-A-56-106598).

<L-citrulline-producing bacteria and L-ornithine-producing bacteria>
L-citrulline and L-ornithine are intermediates in the L-arginine biosynthetic pathway. Therefore, as a parent strain that can be used to induce L-citrulline or L-ornithine-producing bacteria and L-citrulline or L-ornithine-producing bacteria, for example, one or more L-arginine biosynthetic enzymes can be used. Examples include strains with increased activity. Such enzymes are not limited, but for L-citrulin, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetyl-γ-glutamylphosphate reductase (argC), N- Examples thereof include acetylornithine aminotransferase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthase (carAB). In addition, such enzymes are not limited, but for L-ornithine, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetyl-γ-glutamylphosphate reductase (argC), Examples thereof include N-acetylornithine aminotransferase (argD), acetylornithine deacetylase (argE), and ornithine acetyltransferase (argJ).

As parent strains that can be used to induce L-citrulin-producing strains and L-citrulin-producing strains, specifically, but not limited to, E. coli 237 / pMADS11 strain, 237 / 12 pMADS strains, 13 237 / pMADS strains (Russian patent No. 2215783, European patent No. 1170361 B1, and US patent No. 6,790,647 B2), Escherichia coli 333 strain (VKPM B-8084) carrying carbamoyl phosphate synthesizer resistant to feedback inhibition.
) And 374 strains (VKPM B-8086) (Russian Patent No. 2,264,459 C2), increased activity of α-ketoglutaric acid synthase and activity of ferredoxin NADP ⁺ reductase, pyruvate synthase, and / or α-ketoglutaric acid dehydrogenase. Escherichia coli strain (EP2133417 A1) and other strains belonging to the genus Escherichia, and Pantoea ananantis NA1sucAsdhA strain (US patent application No. 2009286290 A1) in which the activities of succinate dehydrogenase and α-ketoglutaric acid dehydrogenase were reduced. Can be mentioned.

In addition, L-citrulline-producing bacteria can be easily obtained from any L-arginine-producing bacteria (for example, E. coli 382 strain (VKPM B-7926), etc.) by inactivating the argininosuccinate synthase encoded by the argG gene. be able to. Methods for inactivating genes are described herein.

L-ornithine-producing bacteria can be easily obtained from any L-arginine-producing bacteria (eg, E. coli 382 strain (VKPM B-7926), etc.) by inactivating ornithine carbamoyl transferase encoded by the argF and argI genes. be able to.

<L-Cysteine Producing Bacteria>
Parent strains that can be used to induce L-cysteine-producing bacteria and L-cysteine-producing bacteria include, for example, strains in which the activity of one or more L-cysteine biosynthetic enzymes is increased. Such enzymes include, but are not limited to, serine acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA). Serine acetyltransferase activity can be enhanced, for example, by introducing into the bacterium a mutant cysE gene that encodes a mutant serine acetyltransferase resistant to feedback inhibition by cysteine. The mutant serine acetyltransferase is disclosed in, for example, JP-A-11-155571 and US2005-0112731A. As such a mutant serine acetyltransferase, specifically, the Val and Asp residues at positions 95 and 96 of the wild serine acetyltransferase encoded by the cysE5 gene are Arg and Pro residues, respectively. A variant serine acetyltransferase substituted with (US2005-0112731A). In addition, 3-phosphoglycerate dehydrogenase activity can be enhanced, for example, by introducing into the bacterium a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.

As a parent strain that can be used to induce L-cysteine-producing bacteria and L-cysteine-producing bacteria, for example, one that catalyzes a reaction that branches from the biosynthetic pathway of L-cysteine to produce a compound other than L-cysteine. Or more, strains with reduced enzyme activity can also be mentioned. Examples of such an enzyme include enzymes involved in the decomposition of L-cysteine. The enzyme involved in the decomposition of L-cysteine is not particularly limited, but is cystathionine-β-lyase (metC) (Japanese Patent Laid-Open No. 11-155571; Chandra et al., Biochemistry, 1982, 21: 3064-3069), tryptophanase. Tryptophanase (tnaA) (Japanese Patent Laid-Open No. 2003-169668; Austin N. et al., J. Biol. Chem., 1965, 240: 1211-1218), O-Acetylserine sulfhydrylase B (cysM)
) (Japanese Patent Laid-Open No. 2005-245311), malY gene product (Japanese Patent Laid-Open No. 2005-245311), Pantoea ananatis d0191 gene product (Japanese Patent Laid-Open No. 2009-232844), cysteine desulfhydrase (aecD) (Japanese Patent Laid-Open No. 2002-233384). Can be mentioned.

In addition, examples of the parent strain that can be used to induce L-cysteine-producing bacteria and L-cysteine-producing bacteria include strains in which the activity of the L-cysteine excretion system and / or the sulfate / thiosulfate transport system is increased. Be done. Examples of the L-cysteine excretion protein include a protein encoded by the ydeD gene (Japanese Patent Laid-Open No. 2002-233384), a protein encoded by the yfiK gene (Japanese Patent Laid-Open No. 2004-49237), emrAB, emrKY, yojIH, acrEF, bcr, And each protein encoded by each gene of cusA (Japanese Patent Laid-Open No. 2005-287333), and a protein encoded by the yesaS gene (Japanese Patent Laid-Open No. 2010-187552). Sulfate / thiosulfate transport proteins include proteins encoded by the cysPTWA gene cluster.

As a parent strain that can be used to induce L-cysteine-producing bacteria and L-cysteine-producing bacteria, specifically, for example, traits in various cysE alleles encoding feedback-inhibiting-resistant mutant serine acetyltransferase, for example, but not limited to. Converted Escherichia coli JM15 (US Patent No. 6,218,168 B1, Russian Patent Application No. 2279477 C2), Escherichia coli W3110 (US patent) that overexpresses a gene encoding a gene suitable for excreting toxic substances to cells. No. 5,972,663), Escherichia coli strain with decreased cysteine desulfhydrase activity (JP11155571 A2), Escherichia coli W3110 (WO0127307 A1) with increased activity of the transcriptional regulator of positive cysteine regulon encoded by the cysB gene, etc. Examples include strains belonging to the genus Escherichia, Pantoea ananatis EYPSG8 overexpressing genes involved in sulfur assimilation, and derivative strains thereof (EP2486123 B1).

<L-glutamic acid-producing bacteria>
Parental strains that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria include, but are not limited to, strains in which the activity of one or more L-glutamic acid biosynthetic enzymes is increased. Such genes include glutamate dehydrogenase (gdhA), glutamine synthase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methyl. Citrate synthase (prpC), pyruvate carboxylase (pyc), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), Enolase (eno), phosphoglyceromutase (pgmA, pgmI)
, Phosphoglycerate kinase (pgk), Glyceraldehyde-3-phosphate dehydrogenase (gapA), Triosephosphate isomerase (tpiA), Fructose bisphosphate aldolase (fbp), Phosphofluctkinase (pfkA, pfkB), Glucose phosphate Genes encoding isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase can be mentioned. Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.

Strains belonging to the family Enterobacteriaceae modified to increase expression of the citrate synthase gene, the phosphoenolpyruvate carboxylase gene, and / or the glutamate dehydrogenase gene are disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2. Can be mentioned. In addition, examples of strains belonging to the family Enterobacteriaceae modified to increase the expression of genes (edd, eda) in the Entner-Dudlov pathway include those disclosed in EP1352966B.

As a parent strain that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria, the activity of an enzyme that catalyzes the biosynthesis of compounds other than L-glutamic acid is reduced by branching from the biosynthetic pathway of L-glutamic acid. Or the disappeared strain can be mentioned. Such enzymes are not particularly limited, but are not limited to isocitrate lyase (aceA), α-ketoglutaric acid dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetoxyacid synthase (ilvG), and the like. Included are acetolactate synthase (ilvI), formic acid acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate dehydrogenase (gadAB), succinate dehydrogenase (sdhABCD), 1-pyrroline-5-carboxylic acid dehydrogenase (putA). Among these enzymes, for example, it is preferable to reduce or delete the α-ketoglutaric acid dehydrogenase activity.

Escherichia bacterium with deficient or reduced α-ketoglutaric acid dehydrogenase activity,
And their acquisition methods are described in US Pat. No. 5,378,616 and US Pat. No. 5,573,945. Specific examples of these strains include the following.
Escherichia coli W3110sucA :: Km ^R
Escherichia coli AJ12624 (FERM BP-3853)
Escherichia coli AJ12628 (FERM BP-3854)
Escherichia coli AJ12949 (FERM BP-4881)

Escherichia coli W3110sucA :: Km ^R is a strain obtained by disrupting the α-ketoglutaric acid dehydrogenase gene (hereinafter, also referred to as “sucA gene”) of Escherichia coli W3110. This strain is completely deficient in α-ketoglutaric acid dehydrogenase.

The parent strains that can be used to induce L-glutamate-producing bacteria and L-glutamate-producing bacteria include Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11091), and Pantoea ananatis SC17 (0). Bacteria of the genus Pantoea such as the strain (VKPM B-9246) can also be mentioned. The AJ13355 strain is a strain isolated from soil in Iwata City, Shizuoka Prefecture, as a strain that can grow in a medium containing L-glutamic acid and a carbon source at a low pH. The SC17 strain was selected from the AJ13355 strain as a mucous low-production mutant strain (US Pat. No. 6,596,517). SC17 shares were issued on February 4, 2009, by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (currently, National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE IPOD), zip code: 292-0818.
, Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan Room 120) has been deposited and given the accession number FERM BP-11091. AJ13355 shares were issued on February 19, 1998, at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (currently NITE IPOD, postal code: 292-0818, address: Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 2- It was deposited in Room 5-8 120) under the accession number FERM P-16644, and was transferred to the international deposit under the Budapest Treaty on January 11, 1999, and the accession number FERM BP-6614 was given. SC17 (0) shares of VKPM B-9246 to the Lucian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on September 21, 2005. Deposited with the accession number.

Examples of the parent strain that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea in which α-ketoglutaric acid dehydrogenase activity is deficient or decreased, and can be obtained as described above. .. Such strains include Pantoea ananatis AJ13356 (US Pat. No. 6,331,419), which is an E1 subunit gene (sucA) -deficient strain of α-ketoglutaric acid dehydrogenase of AJ13355 strain, and Pantoea ananatis, which is a sucA gene-deficient strain of SC17 strain. SC17sucA (US Patent No. 6,596,517) can be mentioned. Pantoea ananatis AJ13356 was issued on February 19, 1998 by the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (currently NITE IPOD, postal code: 292-0818, address: Kazusakamatari 2, Kisarazu City, Chiba Prefecture, Japan). -5-8
It was deposited in Room 120) under the accession number FERM P-16645, and was transferred to the international deposit under the Budapest Convention on January 11, 1999, and the accession number FERM BP-6615 was given. The AJ13356 strain was identified as Enterobacter agglomerans at the time of isolation and was deposited as Enterobacter agglomerans AJ13356. However, in recent years, the strain has been reclassified as Pantoea ananatis by base sequence analysis of 16S rRNA. AJ13356 has been deposited with the above depository as Enterobacter agglomerans, but is described as Pantoea ananatis for the purposes of this specification. Pantoea ananatis SC17sucA was given the private number AJ417, and on February 26, 2004, the Patent Organism Depositary Center (currently NITE IPOD, postal code: 292-0818, address: Chiba Prefecture, Japan) It has been deposited as a deposit number FERM BP-8646 in Room 120, 2-5-8 Kazusakamatari, Kisarazu City.

The parent strains that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria include Pantoea ananatis SC17sucA / RSFCPG + pSTVCB strain and Pantoea ananatis AJ13601.
Strains, Pantoea ananatis NP106 strains, Pantoea ananatis NA1 strains and other strains belonging to the genus Pantoea are also included. The SC17sucA / RSFCPG + pSTVCB strain is a combination of the SC17sucA strain with the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli.
It was obtained by introducing the plasmid RSFCPG containing the above-mentioned plasmid and the plasmid pSTVCB containing the citrate synthase gene (gltA) derived from Brevibacterium lactofermentum. The AJ13601 strain was selected from this SC17sucA / RSFCPG + pSTVCB strain as a strain exhibiting resistance to high concentrations of L-glutamic acid at low pH. The NP106 strain was obtained by dropping the plasmid RSFCPG + pSTVCB from the AJ13601 strain. AJ13601 shares were released on August 18, 1999, at the Institute of Biotechnology and Industrial Technology, Faculty of Industrial Technology, Ministry of International Trade and Industry (currently NITE IPOD, zip code: 292-0818).
, Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan Room 120) was deposited under the deposit number FERM P-17516, and was transferred to the international deposit based on the Budapest Treaty on July 6, 2000, and the deposit number. FERM
BP-7207 has been granted.

The auxotrophic mutant strain can also be mentioned as a parent strain that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria. Specific examples of the auxotrophic mutant include E. coli VL334thrC ⁺ (VKPM B-8961; EP1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine demanding strain with mutations in the thrC and ilvA genes (US Pat. No. 4,278,765). E. coli VL334thrC ⁺ is an L-isoleucine-requiring L obtained by introducing a wild-type allele of the thrC gene into VL334.
-Glutamic acid-producing bacteria. The wild-type allele of the thrC gene was introduced by a common transduction method using bacteriophage P1 grown in cells of wild-type E. coli K-12 strain (VKPM B-7).

Parent strains that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria include strains resistant to aspartic acid analogs. These strains may, for example, lack α-ketoglutaric acid dehydrogenase activity. As a strain that is resistant to aspartic acid analogs and lacks α-ketoglutaric acid dehydrogenase activity, specifically, for example, Escherichia coli AJ13199 (FERM BP-5807; US Pat. No. 5,908,768)
Escherichia coli FERM P-12379 (US Pat. No. 5,393,671) with reduced L-glutamic acid resolution, Escherichia coli AJ13138 (FERM BP-5565; US Pat. No. 6,110,714)
No.) is mentioned.

<L-histidine-producing bacteria>
Parent strains that can be used to induce L-histidine-producing bacteria and L-histidine-producing bacteria include strains in which the activity of one or more L-histidine biosynthetic enzymes is increased. Such enzymes are not particularly limited, but are limited to ATP phosphoribosyltransferase (hisG), phosphoribosyl-ATP pyrophosphatase (hisE), phosphoribosyl-AMP cyclohydrolase (hisI), and bifunctional phosphoribosyl-AMP cyclohydrolase / phosphoribosyl-ATP. Pyrophosphatase (hisIE), phosphoribosylformimimino-5-aminoimidazole carboxamideribotide isomerase (hisA), amide transferase (hisH), histidineol phosphateaminotransferase (hisC), histidineol phosphatase (hisB), histidi Noor dehydrogenase (hisD) can be mentioned.

The L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine. Thus, L-histidine production capacity can be efficiently enhanced, for example, by introducing mutations that confer resistance to feedback inhibition on ATP phosphoribosyl transferase (Russian Patent No. 2,003,677 C1 and Russian Patent No. 2). No. 2,119,536 C1).

Specific, but not limited to, Escherichia coli 24 strain (VKPM B-5945; RU2003677 C1), Escherichia coli NRRL B-12116, as parent strains that can be used to induce L-histidine-producing bacteria and L-histidine-producing bacteria. ~ B-12121 (US Patent No. 4,388,405), Escherichia coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US Patent No. 6,344,347 B1), Escherichia coli H-9341 (FERM BP) -6674; EP1085087 A2), Escherichia coli AI80 / pFM201 (US Pat. No. 6,258,554 B1), Escherichia coli FERM P-5038 and FERM P-transformed with a vector carrying DNA encoding L-histidine biosynthetic enzyme. For 5048 (JP 56-005099 A), Escherichia coli strain (EP1016710 A2) transformed with rht, a gene for excretion of amino acids, sulfaguanidine, DL-1,2,4-triazol-3-alanine, and streptomycin. Escherichia coli 80 strain (VKPM B-7270; RU2119536 C1), Escherichia coli MG1655 + hisGr hisL'_Δ ΔpurR (RU2119536 and Doroshenko V.G. et al., The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants, Strains belonging to the genus Escherichia such as Prikl. Biochim. Mikrobiol. (Russian), 2013, 49 (2): 149-154) can be mentioned.

<L-isoleucine-producing bacteria>
Parental strains that can be used to induce L-isoleucine-producing and L-isoleucine-producing bacteria include, but are not limited to, strains with increased activity of one or more L-isoleucine biosynthetic enzymes. Examples of such an enzyme include, but are not limited to, threonine deaminase and acetoxyacid synthase (Japanese Patent Laid-Open No. 2-458, EP0356739A, US Pat. No. 5,998,178).

Specific examples of the parent strain that can be used to induce L-isoleucine-producing bacteria and L-isoleucine-producing bacteria include, but are not limited to, a mutant strain (JP 5-304969 A) resistant to 6-dimethylaminopurine, Chia. Examples thereof include isoleucine, isoleucine hydroxamate and other isoleucine analog-resistant mutant strains, and Escherichia genus bacteria such as DL-ethionine and / or arginine hydroxamate-resistant mutant strain (JP 5-130882 A).

<L-leucine-producing bacteria>
Parent strains that can be used to induce L-leucine-producing bacteria and L-leucine-producing bacteria include strains in which the activity of one or more L-leucine biosynthetic enzymes is increased. Examples of such an enzyme include, but are not limited to, an enzyme encoded by the gene of the leuABCD operon. Further, for the enhancement of the enzyme activity, for example, a mutant leuA gene (US Pat. No. 6,403,342) encoding an isopropylmalate synthase in which feedback inhibition by L-leucine is released can be preferably used.

In addition, examples of parent strains that can be used to induce L-leucine-producing bacteria and L-leucine-producing bacteria include strains in which the expression of a gene encoding a protein that excretes L-amino acids from bacterial cells is increased. Such genes include the b2682 and b2683 genes (ygaZH).
Gene) (EP1239041 A2).

As a parent strain that can be used to induce L-leucine-producing bacteria and L-leucine-producing bacteria, specifically, but not limited to, a leucine-resistant Escherichia coli strain (eg, 57 strains (VKPM B-7386; US Patent No. 1)). 6,124,121))), Escherichia coli strains resistant to leucine analogs such as β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8- 70879 A), Escherichia coli strain obtained by the genetic engineering method described in WO96 / 06926, strains belonging to the genus Escherichia such as Escherichia coli H-9068 (JP 8-70879 A) can be mentioned.

<L-lysine-producing bacteria>
Parent strains that can be used to induce L-lysine-producing bacteria and L-lysine-producing bacteria include mutant strains belonging to the genus Escherichia and resistant to L-lysine analogs. L
-Lysine analog inhibits the growth of bacteria belonging to the genus Escherichia, but this inhibition is L-
When lysine coexists in the medium, it is completely or partially desensitized. Examples of the L-lysine analog include, but are not limited to, oxalysine, lysine hydroxymate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam and the like. Variant strains resistant to these lysine analogs can be obtained by subjecting bacteria belonging to the genus Escherichia to conventional artificial mutation treatment.

Parent strains that can be used to induce L-lysine-producing bacteria and L-lysine-producing bacteria include strains in which the activity of one or more L-lysine biosynthetic enzymes is increased. Such enzymes include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate deductase. Diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat. No. 6,040,160), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase. (Asd), aspartate aminotransferase (aspartate transaminase) (aspC), diaminopimelate epimerase (dapF)
, Tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (dapE), and aspartase (aspA) (EP1253195 A1). Among these enzymes are, for example, dihydrodiaminopimelate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and It is preferable to enhance the activity of one or more of succinyldiaminopimelate deacylase. In addition, in the parent strain that can be used to induce L-histidine-producing bacteria and L-histidine-producing bacteria, it encodes a gene involved in energy efficiency (cyo) (EP1170376 A1) and nicotinamide nucleotide transhydrogenase. Gene (pntAB)
(US Pat. No. 5,830,716A), ybjE gene (WO2005 / 073390), or combinations thereof may have increased expression levels. Since Aspart Kinase III (lysC) is subject to feedback inhibition by L-lysine, in order to enhance the activity of the enzyme, a mutant lysC gene encoding Aspart Kinase III in which the feedback inhibition by L-lysine is released is used. May be used (US Pat. No. 5,932,453). Aspartkinase III in which the feedback inhibition by L-lysine was released was the substitution of the methionine residue at position 318 with the isoleucine residue, the substitution of the glycine residue at position 323 with the aspartic acid residue, and threonine at position 352. Examples thereof include aspartic kinase III derived from Escherichia coli having one or more mutations such as substitution of a residue with an isoleucine residue (US Pat. No. 5,661,012, US Pat. No. 6,040,160). In addition, since dihydrodipicolinic acid synthase (dapA) is subject to feedback inhibition by L-lysine, in order to enhance the activity of the enzyme, a mutation encoding dihydrodipicolinic acid synthase in which the feedback inhibition by L-lysine is released is released. The type dapA gene may be used. As the dihydrodipicolinic acid synthase from which the feedback inhibition by L-lysine was released, the dihydrodipicolinic acid synthase derived from Escherichia coli having a mutation in which the histidine residue at position 118 is replaced with a tyrosine residue is used. (US Patent No. 6,040,160).

In parent strains that can be used to induce L-lysine-producing or L-lysine-producing bacteria, the activity of the enzyme that catalyzes the reaction that catalyzes the reaction of branching from the L-amino acid biosynthetic pathway to produce other compounds is reduced or eliminated. You may be. Further, in a parent strain that can be used to induce L-lysine-producing bacteria or L-lysine-producing bacteria, the activity of an enzyme that negatively acts on the synthesis or accumulation of L-lysine may be reduced or eliminated. Such enzymes include, but are not limited to, homoserine dehydrogenase, lysine.
Decarboxylase; cadA, ldcC), malic enzyme, and strains in which the activity of these enzymes is reduced or deleted are disclosed in WO95 / 23864, WO96 / 17930, WO2005 / 010175 and the like. Lysine decarboxylase activity can be reduced or deleted, for example, by reducing the expression of both the cadA and ldcC genes encoding lysine decarboxylase. Expression of both genes can be reduced, for example, by the method described in WO2006 / 078039.

Bacterial strains useful for L-lysine production include, for example, E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and E. coli VL611. Will be. In these strains, the feedback inhibition by L-lysine in aspart kinase is released.

Specific parent strains that can be used to induce L-lysine-producing strains or L-lysine-producing strains include Escherichia coli WC196 strain (FERM BP-5252, U.S. Patent No. 5,827,698) and Escherichia coli WC196ΔcadAΔldcC strain (FERM BP). -11027; also called "WC196LC"), Escherichia coli WC196ΔcadAΔldcC / pCABD2 strain (WO2006 / 078039).

The WC196 strain was bred by conferring AEC resistance on the W3110 strain derived from E. coli K-12 (US Pat. No. 5,827,698). The WC196 strain was named E. coli AJ13069, and on December 6, 1994, the Institute of Biotechnology and Industrial Technology, Institute of Industrial Technology (currently NITE IPOD, zip code: 292-0818, address:
Deposited as deposit number FERM P-14690 in Kazusakamatari Room 2-5-8, Kisarazu City, Chiba Prefecture, Japan, and transferred to international deposit based on the Budapest Treaty on September 29, 1995, with deposit number FERM BP- 5252 has been granted (US Pat. No. 5,827,698).

The WC196ΔcadAΔldcC strain was constructed from the WC196 strain by disrupting the cadA and ldcC genes encoding lysine decarboxylase. The WC196ΔcadAΔldcC strain was named AJ110692, and on October 7, 2008, the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (
Currently, NITE IPOD, postal code: 292-0818, address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, Room 120) was deposited internationally under the accession number FERM BP-11027.

The WC196ΔcadAΔldcC / pCABD2 strain was constructed by introducing the plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene into the WC196ΔcadAΔldcC strain. pCABD2 is a mutant dapA gene encoding dihydrodiaminopimelate synthase (DDPS) derived from Escherichia coli, which has a mutation (H118Y) in which feedback inhibition by L-lysine is released, and feedback by L-lysine. A mutant lysC gene encoding Aspartkinase III from Escherichia coli with a mutation (T352I) that is released from inhibition, and dapB encoding dihydrodiaminopimelate reductase from Escherichia coli. It contains the gene and the ddh gene encoding diaminopimelate dehydrogenase from Brevibacterium lactofermentum.

Parent strains that can be used to induce L-lysine-producing bacteria or L-lysine-producing bacteria also include Escherichia coli AJIK01 strain (NITE BP-01520). The AJIK01 strain was named Escherichia coli AJ111046, and on January 29, 2013, the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NITE NPMD; Postal Code: 292-0818, Address: Kisarazu City, Chiba Prefecture, Japan) It was deposited at Kazusakamatari Room 2-5-8 122) and transferred to an international deposit based on the Budapest Treaty on May 15, 2014, and has been given the accession number NITE BP-01520.

<L-Methionine-producing bacteria>
The parent strain that can be used to induce L-methionine-producing bacteria and L-methionine-producing bacteria is not particularly limited, but is limited to Escherichia coli AJ11539 strain (NRRL B-12399), AJ11540.
Strain (NRRL B-12400), AJ11541 strain (NRRL B-12401), AJ 11542 strain (NRRL B-12402) (patent GB2075055); Escherichia coli 218 strain resistant to the L-methionine analog norleucine (VKPM B-8125) RU2209248 C2) and 73 strains (VKPM B-8126; RU2215782 C2); and other strains belonging to the genus Escherichia. Escherichia coli 73 strains commissioned VKPM B-8126 to Lucian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on May 14, 2001. It was deposited by number and transferred to an international deposit under the Budapest Convention on February 1, 2002. In addition, methionine repressor-deficient strains and recombinant strains (JP 2000-139471 A) transformed with genes encoding proteins involved in L-methionine biosynthesis (homoserin transsuccinylase, cystathionine γ-synthase, etc.) are also L. -Can be used as a methionine-producing bacterium or a parent strain. Another example of a parent strain that can be used to induce L-methionine-producing and L-methionine-producing bacteria of the genus Escherichia is the lack of the L-methionine biosynthesis repressor (MetJ) and intracellular homoserine transxi. E. coli strain (US7611873 B1) with increased homoserine transsuccinylase (MetA) activity, cobalamin-independent methionine synthase (MetE) activity suppressed, cobalamin-dependent methionine synthase (Cobalamin-dependent methionine synthase
) (MetH) E. coli strain (EP2861726 B1) with increased activity, capable of producing L-threonine, threonine dehydratase (tdcB, ilvA) and at least O-succinyl homoserine lyase (O) -succinylhomoserine lyase (metB), cystathionine beta-lyase (metC), 5,10-methylenetetrahydrofolate reductase (metF), and serine hydroxymethyltransferase (serine) hydroxymethyltr
It can be an E. coli strain (US7790424 B2) transformed with a vector expressing ansferase (glyA), an E. coli strain (EP2633037 B1) with enhanced activity of transhydrogenase (pntAB), and the like.

The L-methionine-producing bacterium may be modified to overexpress the cysteine synthase gene.

The term "cysteine synthase gene" can mean a gene encoding cysteine synthase. The term "cysteine synthase" can mean a protein having cysteine synthase activity (EC 2.5.1.47). Examples of the cysteine synthase gene include the cysM gene and the cysK gene. The cysM gene is
Cysteine synthase B with thiosulfate as a substrate may be encoded. The cysK gene may encode cysteine synthase A with a sulfide as a substrate. Specific examples of the cysteine synthase gene include the cysM gene unique to P. ananatis. The nucleotide sequence of the cysM gene peculiar to P. ananatis is shown in SEQ ID NO: 14.

The L-methionine-producing bacterium may be modified to have a mutant metaA gene.

The metA gene encodes homoserine transsuccinylase MetA (EC 2.3.1.46). The term "mutant metaA gene" can mean a gene encoding a mutant MetA protein. The term "mutant MetA protein" has an R34C mutation, which is a mutation in which the arginine (Arg) residue at position 34 in the amino acid sequence of the wild-type MetA protein is replaced with a cysteine (Cys) residue). Can mean MetA protein. "
The term "wild-type metaA gene" can mean a gene encoding a wild-type MetA protein. The term "wild-type MetA protein" can mean a MetA protein that does not have the R34C mutation. Wild-type metaA genes include P. ananatis-specific metaA genes and variants thereof that do not have mutations that result in R34C mutations in the encoded protein. Examples of the wild-type MetA protein include a P. ananatis-specific MetA protein and a variant thereof that does not have the R34C mutation. In other words, the mutant metaA gene may be identical to any wild-type metagene, except that it has a mutation that results in an R34C mutation in the encoded protein. The mutant MetA protein may also be identical to any wild-type MetA protein except that it has the R34C mutation. The amino acid sequence of the MetA protein peculiar to P. ananatis is shown in SEQ ID NO: 36. Specifically, an example of the amino acid sequence of the mutant MetA protein can be the amino acid sequence set forth in SEQ ID NO: 38, which can be encoded by the mutant metaA gene having the base sequence set forth in SEQ ID NO: 37. That is, the mutant metaA gene may be a gene having the base sequence shown in SEQ ID NO: 37 (for example, DNA), and the mutant MetA protein may be a protein having the amino acid sequence shown in SEQ ID NO: 38. The mutant metaA gene may be a gene (eg, DNA) having a base sequence having a mutation that results in the R34C mutation of the protein encoded by the mutant base sequence of SEQ ID NO: 37. The mutant MetA protein may be a variant amino acid sequence of SEQ ID NO: 38 and a protein having an amino acid sequence having an R34C mutation. The mutant MetA protein may be a homoserine transsuccinylase resistant to feedback inhibition by L-methionine. In other words, the mutant MetA protein may be a protein having homoserine transsuccinylase activity and resistant to feedback inhibition by L-methionine. The description of the ydiJ gene and the mutant of the YdiJ protein described later can be applied mutatis mutandis to the mutant of the metaA gene and the mutant of the MetA protein. The term "position 34" does not necessarily indicate the absolute position in the amino acid sequence of the wild-type MetA protein, but rather the relative position based on the amino acid sequence shown in SEQ ID NO: 36 in the wild-type MetA protein. be.

The L-methionine-producing bacterium may be modified so that the expression of the metJ gene is weakened.

The metJ gene encodes a Met repressor, which can suppress the expression of methionine regulon and the enzymes involved in SAM synthesis. Examples of the metJ gene include those unique to host bacteria such as P. ananatis. The nucleotide sequence of the metaJ gene peculiar to P. ananatis is shown in SEQ ID NO: 25.

The parent strain that can be used to induce L-methionine-producing bacteria and L-methionine-producing bacteria of the genus Pantoea is not particularly limited, and examples thereof include P. ananatis AJ13355 strain (FERM BP-6614).

<L-Phenylalanine-producing bacteria>
Escherichia coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197), carrying the mutant pheA34 gene, is not limited as a parent strain that can be used to induce L-phenylalanine-producing bacteria and L-phenylalanine-producing bacteria. Escherichia coli HW1089 (ATCC 55371, US Patent No. 5,354,672), Escherichia coli MWEC101-b (KR8903681), Escherichia coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (US Patent No. 1) 4,407,952), Escherichia coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), Escherichia coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), Escherichia coli K-12 [ W3110 (tyrA) / pPHATerm] (FERM BP-12662), as well as Escherichia coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579, EP488424 B1) named AJ12604. Furthermore, as a parent strain that can be used to induce L-phenylalanine-producing bacteria and L-phenylalanine-producing bacteria, L-phenylalanine-producing bacteria belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or yddG gene is enhanced is also included. (US Pat. Nos. 7,259,003 and 7,666,655).

<L-proline-producing bacteria>
The parent strain that can be used to induce L-proline-producing bacteria and L-proline-producing bacteria is not limited, but Escherichia coli that lacks the ilvA gene and can produce L-proline.
Strains belonging to the genus Escherichia such as 702ilvA (VKPM B-8012, EP1172433 A1) can be mentioned. Parent strains that can be used to induce L-proline-producing and L-proline-producing strains include strains with enhanced expression of one or more genes involved in L-proline biosynthesis. An example of such a gene that can be used in L-proline-producing bacteria is the proB gene, which encodes a glutamate kinase in which the feedback inhibition by L-proline has been released (DE3127361 A1). In addition, the parent strain that can be used to induce L-proline-producing and L-proline-producing bacteria is the expression of one or more genes encoding proteins responsible for the excretion of L-amino acids from bacterial cells. There is also a strain in which is enhanced. Examples of such genes include the b2682 gene and the b2683 gene (ygaZH gene) (EP1239041 A2).

Bacteria belonging to the genus Escherichia having L-plasmid-producing ability are described in NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent No. 2207371 C2), DE3127361 A1. Examples thereof include plasmid mutant strains and Escherichia coli strains such as the plasmid mutant strain described in Bloom FR et al “The 15th ^Miami winter symposium, 1983, p.34”.

<L-threonine-producing bacteria>
Parent strains that can be used to induce L-threonine-producing bacteria and L-threonine-producing bacteria include strains in which the activity of one or more L-threonine biosynthetic enzymes is increased. Such enzymes are not particularly limited, but aspart kinase III (lysC).
, Aspartate semialdehyde dehydrogenase (asd), aspart kinase I (thrA), homoserine kinase (thrB), threonine synthase (threo)
Examples include nine synthase) (thrC) and aspartate aminotransferase (aspartate transaminase) (aspC). Among these enzymes, it is preferable to enhance the activity of one or more enzymes such as aspart kinase III, aspartate semialdehyde dehydrogenase, aspart kinase I, homoserine kinase, aspartate aminotransferase, threonine synthase and the like. .. The L-threonine biosynthesis gene may be introduced into a strain in which threonine degradation is suppressed. Examples of the strain in which threonine degradation is suppressed include E. coli TDH6 strain (Japanese Patent Laid-Open No. 2001-346578) in which threonine dehydrogenase activity is deficient.

The activity of L-threonine biosynthetic enzymes is inhibited by the end product L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is preferable to modify the L-threonine biosynthesis gene so as not to receive feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute a threonine operon, and the threonine operon forms an attenuator structure. The expression of threonine operon is inhibited by isoleucine and threonine in the culture medium and is suppressed by attenuation. Enhanced expression of the threonine operon can be achieved by removing the leader sequence or attenuator in the attenuation region (Lynn S.P. et al., J. Mol. Biol., 1987, 194: 59-69; WO 02 / 26993; WO2005 / 049808; WO2003 / 097839).

Although a unique promoter exists upstream of the threonine operon, the promoter may be replaced with an unnatural promoter (WO98 / 04715). In addition, the threonine operon may be constructed so that the gene involved in threonine biosynthesis is expressed under the control of the repressor and promoter of the lambda phase (EP0593792B). Bacteria modified to avoid feedback inhibition by L-threonine are L-threonine analog α-amino-.
It can also be obtained by selecting a strain resistant to β-hydroxyisovaleric acid (AHV).

The threonine operon thus modified so as not to be inhibited by L-threonine feedback is improved in the expression level in the host by increasing the copy number or by being linked to a strong promoter. Is preferable. Increased copy numbers can be achieved by introducing a plasmid containing the threonine operon into the host. The increase in the number of copies can also be achieved by transferring the threonine operon onto the genome of the host using a transposon, Mu phage, or the like.

Moreover, as a method of imparting or enhancing L-threonine production ability, a method of imparting L-threonine resistance to a host and a method of imparting L-homoserin resistance can also be mentioned. Tolerance can be achieved, for example, by enhancing the expression of a gene that imparts resistance to L-threonine, a gene that imparts resistance to L-homoserin. The genes that confer resistance include the rhtA gene (Livshits V.A. et al., Res. Microbiol., 2003, 154: 123-135), rhtB gene (EP0994190A), rhtC gene (EP1013765A), yfiK gene, and yeaS gene (EP1016710A). ). Examples of the method for imparting L-threonine resistance to the host include the methods described in EP0994190A and WO90 / 04636.

Escherichia coli TDH-6 / pVIC40 (VKPM B-3996, US Pat. No. 5,175,107 and No. 5, but not specifically limited, as a parent strain that can be used to induce L-threonine-producing bacteria and L-threonine-producing bacteria. 5,705,371), Escherichia coli 472T23 / pYN7 (ATCC 98081, US Patent No. 5,631,157), Escherichia coli NRRL B-21593 (US Patent No. 5,939,307), Escherichia coli FERM BP-3756 (US Patent No. 5,474,918), Escherichia coli FERM BP-3519 and FERM BP-3520 (US Pat. No. 5,376,538), Escherichia coli MG442 (Gusyatiner et al., Genetika (Russian), 1978, 14: 947-956), Escherichia coli VL643 and VL2055 (EP1149911 A2), Escherichia coli VKPM B-5318 (EP0593792 A1) and other Escher
Strains belonging to the genus ichia can be mentioned.

The TDH-6 strain is deficient in the thrC gene, is sucrose assimilating, and has a leaky mutation in its ilvA gene. This strain also has mutations that confer resistance to high concentrations of threonine or homoserine on the rhtA gene. VKPM B-3996 strain is plasmid pVIC40
This strain is obtained by introducing the plasmid pVIC40 into the TDH-6 strain. The plasmid pVIC40 was obtained by inserting a thrA * BC operon containing the mutant thrA gene into an RSF1010-derived vector. This mutant thrA gene encodes the aspart kinase homoserine dehydrogenase I, which is substantially deprived of threonine-induced feedback inhibition. The VKPM B-3996 strain was deposited with the All-Union Scientific Center of Antibiotics (Russian Federation, 117105 Moscow, Nagatinskaya Street 3-A) on November 19, 1987 under accession number RIA 1867. The VKPM B-3996 strain was also deposited on April 7, 1987 with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) under accession number B-3996.

The B-5318 strain is isoleucine non-requiring, and the control region of the threonine operon in the plasmid pVIC40 is replaced by the temperature-sensitive lambda phage C1 repressor and PR promoter. The VKPM B-5318 strain was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number VKPM B-5318.

The parent strain that can be used to induce L-threonine-producing bacteria or L-threonine-producing bacteria may be further modified to enhance the expression of one or more of the following genes:
--A mutant thrA gene encoding the aspart kinase homoserine dehydrogenase I resistant to threonine-induced feedback inhibition,
--ThrB gene encoding homoserine kinase,
--ThrC gene encoding threonine synthase;
--The rhtA gene, which encodes a presumed transmembrane protein of the threonine and homoserine excretion system,
--Aspartate-asd gene encoding β-semialdehyde dehydrogenase,
--The aspC gene that encodes aspartate aminotransferase (aspartate transaminase).

The thrA gene encoding aspart kinase I-homoserin dehydrogenase I in Escherichia coli has been identified (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b0002, GenBank accession No. NC_000913.2, nucleotide positions 337-2,799). , Gene ID 945803). The thrA gene is located between the thrL and thrB genes on the chromosome of Escherichia coli K-12.

The thrB gene encoding homoserine kinase in Escherichia coli has been identified (KEGG, entry No. b0003, GenBank accession No. NC_000913.2, nucleotide positions 2,801-3,733, Gene ID 947498). The thrB gene is located between the thrA and thrC genes on the chromosome of Escherichia coli K-12.

The thrC gene encoding threonine synthase in Escherichia coli has been identified (KEGG, entry No. b0004, GenBank accession No. NC_000913.2, nucleotide positions 3,734-5,020, Gene ID 945198). The thrC gene is located between the thrB gene and the yaaX gene on the chromosome of the Escherichia coli K-12 strain. All three of these genes function as a single threonine operon thrABC. In order to enhance the expression of the threonine operon, it is desirable to remove the attenuator region that affects transcription from the operon (WO).
2005/049808 A1, WO2003 / 097839 A1).

The mutant thrA gene encoding aspart kinase I-homoserin dehydrogenase I resistant to feedback inhibition by threonine, as well as the thrB and thrC genes, is a well-known plasmid pVIC40 present in the Escherichia coli threonine-producing strain VKPM B-3996.
Can be obtained as a single operon from. Details of the plasmid pVIC40 are described in US Pat. No. 5,705,371.

The rhtA gene encoding the threonine and homoserine efflux system (inner membrane transporter) proteins of Escherichia coli has been identified (KEGG, entry No. b0813, GenBank accession No. NC_000913.2, nucleotide positions 848,433-849,320, complementary. Chain, Gene ID 947045). The rhtA gene is located on the chromosome of the Escherichia coli K-12 strain, between the dps and ompX genes near the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is identical to the ybiF gene (KEGG, entry No. B0813).

The asd gene encoding aspartate-β-semialdehyde dehydrogenase in Escherichia coli has been identified (KEGG, entry No. b3433, GenBank accession No. NC_000913.2, nucleotide positions 3,571,798-3,572,901, complementary strand, Gene ID 947939. ). The asd gene is located on the chromosome of the Escherichia coli K-12 strain, between the glgB and gntU genes located on the same strand (the yhgN gene is on the opposite strand).

In addition, the aspC gene encoding the aspartate aminotransferase of Escherichia coli has been clarified (KEGG, entry No. b0928, GenBank accession No. NC_000913.2, nucleotide positions 983,742-984,932, complementary strand, Gene ID 945553). The aspC gene is located on the chromosome of Escherichia coli K-12, between the ycbL gene on the opposite strand and the ompF gene on the same strand.

<L-Tryptophan-producing bacteria>
The parent strains that can be used to induce L-tryptophan-producing bacteria and L-tryptophan-producing bacteria are not limited, but Escherichia coli JP4735 / pMU3028 (DSM10122) and Escherichia coli JP4735 / pMU3028 (DSM10122) lacking the tryptophanal-tRNA synthetase encoded by the mutant trpS gene. JP6015 / pMU91 (DSM10123) (US Pat. No. 5,756,345), Escherichia with serA allele encoding phosphoglycerylate dehydrogenase not subject to feedback inhibition by serine and trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan. coli SV164 (pGH5) (US Patent No. 6,180,373 B1), Escherichia coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) (US Patent No. 4,371,614) lacking the enzyme tryptophanase No.), Escherichia coli AGX17 / pGX50, pACKG4-pps (WO97 / 08333, US Patent No. 6,319,696 B1) with enhanced phosphoenolpyruvate production ability, and other strains belonging to the genus Escherichia. The parent strain that can be used to induce L-tryptophan-producing bacteria and L-tryptophan-producing bacteria is L belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or yddG gene is enhanced.
-Tryptophan-producing bacteria are also mentioned (US Patent Application Publication Nos. 2003148473 A1 and 2003157667 A1).

Parent strains that can be used to induce L-tryptophan-producing bacteria and L-tryptophan-producing bacteria include strains with enhanced activity of one or more of anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase. .. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that release the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include Escherichia coli SV164, which carries anthranilate synthase with de-feedback inhibition, and a mutant serA gene encoding a phosphoglycerate dehydrogenase with de-feedback inhibition. Escherichia plasmid pGH5 (WO94 / 08031 A1)
Examples thereof include transformants obtained by introduction into coli SV164.

As a parent strain that can be used to induce L-tryptophan-producing bacteria and L-tryptophan-producing bacteria, a strain into which a tryptophan operon containing a gene encoding uninhibited anthranilate synthase is introduced (Japanese Patent Laid-Open No. 57-71397). , Japanese Patent Application Laid-Open No. 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by enhancing the expression of the gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by the trpA and trpB genes, respectively. Furthermore, the L-tryptophan production capacity may be improved by enhancing the expression of the isocitrate lyase-malate synthase operon (WO2005 / 103275).

<L-valine-producing bacteria>
Parent strains that can be used to induce L-valine-producing and L-valine-producing strains include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). It is desirable to remove the region required for attenuation in the ilvGMEDA operon so that the L-valine produced does not attenuate the expression of the operon. Furthermore, it is desirable that the ilvA gene in the operon is disrupted and the threonine deaminase activity is reduced.

Parent strains that can be used to induce L-valine-producing bacteria and L-valine-producing bacteria also include mutant strains having a mutation in aminoacyl t-RNA synthetase (US Pat. No. 5,658,766). An example of such a strain is Escherichia coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase. Escherichia coli VL1970 was deposited on June 24, 1988 with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) under accession number VKPM B-4411.

In addition, mutant strains that require lipoic acid for growth and / or lack H ⁺ -ATPase can also be used as L-valine-producing strains or their parent strains (WO96 / 06926 A1).

Parent strains that can be used to induce L-valine-producing and L-valine-producing bacteria include Escherichia coli H81 strain (see VKPM B-8066, eg EP1942183 B1), Escherichia coli NRRL B-12287 and NRRL B-12288 (see Escherichia coli NRRL B-12287 and NRRL B-12288). US Pat. No. 4,391,907), Escherichia coli VKPM B-4411 (US Pat. No. 5,658,766), Escherichia coli VKPM B-7707 (EP1016710 A2), etc.

The gene and protein used for breeding the L-amino acid-producing bacterium may have, for example, known base sequences and amino acid sequences of the above-exemplified genes and proteins, respectively. In addition, the genes and proteins used for breeding L-amino acid-producing bacteria are variants of the above-exemplified genes and proteins (for example, as long as the original functions (for example, in the case of proteins, their respective enzyme activities) are maintained). It may be a variant of a gene and protein having such a known base sequence and amino acid sequence). For gene and protein variants, the description of the ydiJ gene and encoded protein variants described herein can be applied mutatis mutandis.

The bacteria described herein have been modified to overexpress the ydiJ gene.

The ydiJ gene unique to P. ananatis is a putative FAD-binding redox enzyme YdiJ (BioCyc database, https://biocyc.org/, accession ID: PAJ_RS05850; UniParc, accession No. UPI0002FB0FA6; KEGG entry No. PAJ_1060). To code. The ydiJ gene peculiar to P. ananatis has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence of the YdiJ protein encoded by the gene is shown in SEQ ID NO: 2.

E. coli-specific ydiJ gene is a putative FAD-binding oxidoreductase YdiJ (EcoCyc database,
https://ecocyc.org/, accession ID: G6913; UniProt accession No. P77748; KEGG entry No. b1687), which encodes the menI and ydiK genes on the same strand on the chromosome of the E. coli K-12 strain. Located between. The ydiJ gene peculiar to E. coli has the nucleotide sequence shown in SEQ ID NO: 3, and the amino acid sequence of the YdiJ protein encoded by the gene is shown in SEQ ID NO: 4.

That is, the ydiJ gene may be a gene having the base sequence shown in SEQ ID NO: 1 or 3 (for example, DNA), and the YdiJ protein may be a protein having the amino acid sequence shown in SEQ ID NO: 2 or 4. The expression "a gene or protein has a base sequence or amino acid sequence" can mean that the gene or protein contains the base sequence or the amino acid sequence in a longer sequence, unless otherwise specified. It can also be meant that the protein has only the base sequence or the amino acid sequence.

Homologs of YdiJ proteins unique to various bacteria belonging to the family Enterobacteriaceae are known, and examples are shown in Table 1.

The following description of the gene having the nucleotide sequence shown in SEQ ID NO: 1 is any gene (SEQ ID NO: 3) that can be used in place of or equivalent to the gene having the nucleotide sequence shown in SEQ ID NO: 1 in the method described herein. It can also be applied mutatis mutandis to (including genes having the base sequence shown in). Similarly, the following description of a protein having the amino acid sequence set forth in SEQ ID NO: 2 is any protein that can be used as a substitute for or equivalent to the protein having the amino acid sequence set forth in SEQ ID NO: 2 in the methods described herein. It can also be applied mutatis mutandis to (including proteins having the amino acid sequence shown in SEQ ID NO: 4).

There can be differences in DNA sequences between genera, species, or strains of bacteria belonging to the family Enterobacteriaceae. Therefore, the ydiJ gene is not limited to the gene having the base sequence shown in SEQ ID NO: 1, but has a variant base sequence for SEQ ID NO: 1 and the YdiJ protein (protein having the amino acid sequence shown in SEQ ID NO: 2 and its mutation). It may include genes encoding (including body proteins). Similarly, the YdiJ protein is not limited to the protein having the amino acid sequence shown in SEQ ID NO: 2, and may include the protein having the variant amino acid sequence of SEQ ID NO: 2.

The term "variant base sequence" refers to any synonymous amino acid codon according to the standard gene code table (see, eg, Lewin B., “Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Can be used to mean a base sequence encoding a YdiJ protein (eg, a protein having the amino acid sequence set forth in SEQ ID NO: 2). Therefore, the DNA encoding the YdiJ protein having the amino acid sequence shown in SEQ ID NO: 2 can be a gene having the variant base sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code.

The term "variant base sequence" refers to a non-modified protein whose three-dimensional structure has the amino acid sequence shown in SEQ ID NO: 2 or that the activity or function of the protein having the amino acid sequence shown in SEQ ID NO: 2 is maintained. As long as it encodes a protein that has not been significantly altered, it also means a base sequence complementary to the sequence shown in SEQ ID NO: 1 or a base sequence capable of hybridizing with a probe that can be prepared from the base sequence under stringent conditions. Can be. The term "stringent condition" is defined as the parameter "identity" when using a specific hybrid, for example the computer program blastn, with 70% or more, 75% or more, 80% or more, 85% homology. 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more hybrids are formed and non- It can mean a condition in which a specific hybrid, for example a hybrid having a lower homology than the above, is not formed. Stringent conditions include, for example, a salt concentration of 1 x SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulfate) at 60 ° C, and a salt concentration of 0.1 x SSC, 0.1% SDS. Conditions include washing at 60 ° C. or at a salt concentration of 0.1 × SSC, 0.1% SDS at least once at 65 ° C., in another example two or three times. The wash time may depend on the type of membrane used for blotting, but should generally be recommended by the manufacturer. For example, the recommended cleaning time for Amersham Hybond ^TM -N + positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. Cleaning process is 2
Or it can be done 3 times. As the probe, a part of the sequence complementary to the sequence shown in SEQ ID NO: 1 may be used. Such a probe uses an oligonucleotide prepared based on the sequence shown in SEQ ID NO: 1 as a primer and a DNA fragment containing a base sequence as a template for PCR (polymerase chain reaction; White TJ et al., The. It can be prepared by polymerase chain reaction, Trends Genet., 1989, 5: 185-189). The length of the probe is recommended to be greater than 50 bp, but can be appropriately selected depending on the bleeding conditions and is usually 100 bp to 1 kbp. For example, when using a DNA fragment with a length of about 300 bp as a probe, the post-hybridization wash conditions are, for example, 2 × SSC, 0.1% SDS at 50 ° C, 60 ° C, or 65 ° C. obtain.

The term "mutant base sequence" can also mean a base sequence encoding a mutant protein.

The term "mutant protein" can mean a protein having the variant amino acid sequence of SEQ ID NO: 2.

The term "variant protein" is specifically any of the substitutions, deletions, insertions, and / or additions of one or several amino acid residues as compared to the amino acid sequence set forth in SEQ ID NO: 2. In any case, whether the protein has one or more mutations in the sequence and the activity or function of the protein having the amino acid sequence shown in SEQ ID NO: 2 is maintained, or its three-dimensional structure is shown in SEQ ID NO: 2. It can mean a protein that has not been significantly altered relative to an unmodified protein having an amino acid sequence. The number of mutations in a variant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of changes in the mutant protein is not strictly limited, but is 1 to 300 in SEQ ID NO: 2, 1 to 250 in another example, 1 to 200 in another example, 1 to 1 in another example. 150, 1-100 in another example, 1-90 in another example, 1-80 in another example, 1-70 in another example, 1-60 in another example, 1-50 in another example, 1-40 in another example, 1-30 in another, 1-20 in another, 1-15 in another, 1-10 in another, or 1-5 in another. It's okay. This is because the amino acids can have a high degree of homology to each other, and the activity or function of the protein is not affected, or the three-dimensional structure of the protein has the amino acid sequence shown in SEQ ID NO: 2. This is possible because it does not change significantly. Therefore, the variant protein is prominent for unmodified proteins in which the activity or function of the protein having the amino acid sequence shown in SEQ ID NO: 2 is maintained or the three-dimensional structure of the protein has the amino acid sequence shown in SEQ ID NO: 2. 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% of the entire amino acid sequence of SEQ ID NO: 2 unless changed to
As the parameter "identity" when using the computer program blastp, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It may be a protein having an amino acid sequence having the defined homology. As used herein, the term "homology" may mean "identity", which is the identity between amino acid residues. Sequence identity between two sequences is calculated as the ratio of matching residues between the two sequences when the two sequences are aligned for maximum match.

Examples of substitutions, deletions, insertions, and / or additions of one or several amino acid residues include conservative mutations. Representatives of conservative mutations can be conservative substitutions. Conservative substitutions are not limited, but if the substitution site is an aromatic amino acid, between Phe, Trp, Tyr,
Substitution between Ala, Leu, Ile and Val when the substitution site is a hydrophobic amino acid, and between Glu, Asp, Gln, Asn, Ser, His and Thr when the substitution site is a hydrophilic amino acid. Between Gln and Asn when the site is a polar amino acid, between Lys, Arg and His when the substitution site is a basic amino acid, and between Asp and Glu when the substitution site is an acidic amino acid. When the substitution site is an amino acid having a hydroxyl group, it is a substitution that substitutes each other between Ser and Thr. Examples of conservative substitutions are Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln. Substitution to, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr replacement, Ile to Leu, Met, Val or Phe replacement, Leu to Ile, Met, Val or Phe replacement, Lys to Asn, Glu, Gln, His Or replacement with Arg, Met to Ile
, Leu, Val or Phe replacement, Phe to Trp, Tyr, Met, Ile or Leu replacement, Ser to Thr or Ala replacement, Thr to Ser or Ala replacement, Trp to Phe or Tyr Substitutions include Tyr to His, Phe or Trp, and Val to Met, Ile or Leu. In addition, the substitution, deletion, insertion, addition, etc. of amino acid residues as described above includes mutations naturally occurring due to individual differences in the organism from which the amino acid sequence is derived.

Examples of substitutions, deletions, insertions, and / or additions of one or several amino acid residues include non-conservative mutations, provided that the mutation is at one or more of the different positions in the amino acid sequence. The above-mentioned second mutation maintains the activity or function of the mutant protein, or does not significantly change the three-dimensional structure of the protein with respect to the unmodified protein having the amino acid sequence shown in SEQ ID NO: 2. In addition, it is compensated.

The percentage of polypeptide identity can be calculated by the blastp algorithm. More specifically, the percentage of polypeptide identity is the default setting of Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1) by the blastp algorithm provided by the National Center for Biotechnology Information (NCBI). ; Compositional Adjustments: Conditional compositional score matrix adjustment) can be used. The percentage of polynucleotide identity can be calculated by the blastn algorithm. More specifically, the percentage of polynucleotide identity can be calculated using the default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) by the blastn algorithm provided by NCBI.

The term "bacteria modified to overexpress the ydiJ gene" refers to the corresponding gene product (eg, YdiJ protein) in the modified bacterium as compared to an unmodified strain (eg, wild strain or parent strain). ) Is increased (ie, higher) or the expression level (ie, expression level) of the ydiJ gene is increased (ie, higher). It can mean that you are. As an unmodified strain that can be used as a control for the above comparison, a wild strain of a bacterium belonging to the family Enterobacteriaceae (for example, E. coli W3110)
Strains (ATCC 27325), E. coli MG1655 strains (ATCC 47076), P. ananatis AJ13355 strains (FERM BP-6614), etc.).

That is, the term "overexpressing the ydiJ gene" means that the total amount and / or total activity of the corresponding gene product (eg, YdiJ protein) is increased (ie, higher) compared to the unmodified strain. Can mean. The total amount and / or total activity of the corresponding gene product (eg, YdiJ protein) is, for example, by increasing (ie, enhancing) the expression level of the gene relative to an unmodified bacterial strain, or encoded by the gene. It can be increased by increasing the activity per molecule (also referred to as specific activity) of the protein relative to the unmodified strain (eg, wild or parent strain). An increase in the total amount or activity of a protein can be measured, for example, as an increase in the amount or activity of the protein per cell, which may be the average amount or activity of the protein per cell. Bacteria can be modified to increase the amount and / or activity of the YdiJ protein per cell to 150% or more, 200% or more, or 300% or more of the amount and / or activity in the unmodified strain.

The term "activity of a protein having the amino acid sequence shown in SEQ ID NO: 2" utilizes flavin adenine dinucleotide (abbreviated as FAD) as a coenzyme to catalyze the transfer of electrons from one molecule to another. Can mean the activity of a protein that can. The same applies to the term "activity of a protein having the amino acid sequence shown in SEQ ID NO: 4". As a method for measuring the activity of a protein that can trigger the catalytic action of electron transfer from one molecule to another by using FAD as a coenzyme, FAD and its reduced form (for example, FADH and FADH2) are used. Examples include spectrophotometric assays that can assess interconversion (see, for example, the Flavin adenine dinucleotide (FAD) assay kit (Abcam, cat. No. ab204710)).

Protein concentration can be determined by the Bradford protein assay with bovine serum albumin (BSA) as standard or the Lowry method (Bradford M.M., Anal. Biochem., 1976, 72: 248-254; Lowry O.H. et. al., J. Biol. Chem., 1951, 193: 265-275).

The term "overexpressing the ydiJ gene" can also mean that the expression level (ie, expression level) of the ydiJ gene is increased (ie, higher) as compared to the unmodified strain. Therefore, the term "ydiJ gene overexpression" replaces the term "ydiJ gene expression is enhanced or increased" or "ydiJ gene expression level is enhanced or increased". Can be used as possible or equivalent. An increase in the expression level of a gene can be measured, for example, as an increase in the expression level of the gene per cell, which may be the average expression level of the gene per cell. The terms "gene expression level" or "gene expression level" can mean, for example, the amount of gene expression product (eg, the amount of mRNA of the gene or the amount of protein encoded by the gene). Bacteria may be modified, for example, to increase the expression level of the ydiJ gene per cell to 150% or more, 200% or more, or 300% or more of the expression level in the unmodified strain.

Methods that can be used to enhance gene expression of the ydiJ gene include, but are not limited to, the number of copies of the gene, eg, the number of copies of the gene in the bacterial genome (ie, chromosome) and / or autonomous replication retained by the bacterium. Examples include methods for increasing the number of copies of a gene in a vector (eg, a plasmid). The number of copies of a gene can be increased, for example, by introducing the gene into the chromosome of the bacterium and / or by introducing an autonomously replicating plasmid containing the gene into the bacterium. Increasing the number of copies of such a gene can be carried out by genetic engineering techniques well known to those of skill in the art.

Vectors that can be used by bacteria of the family Enterobacteriaceae include, but are not limited to, conditional replication vectors (eg, vectors that replicate from an R6K (oriRγ) origin, such as the pAH162 vector), narrow host range plasmids (eg, pMW118 / 119, etc.). pBR322, pUC19, etc.), broad host range plasmids (RSF1010, RP4, etc.). The ydiJ gene can also be introduced into bacterial chromosomal DNA by, for example, homologous recombination or Mu-driven integration. Only one copy of the ydiJ gene may be introduced, or two copies or more may be introduced. For example, by performing homologous recombination targeting a base sequence in which a plurality of copies are present in a chromosomal DNA, a plurality of copies of the ydiJ gene can be introduced into the chromosomal DNA. Nucleobase sequences in which multiple copies are present in chromosomal DNA include, but are not limited to, repetitive DNA and inverted repeats present at the ends of transposable factors. Furthermore, by incorporating the gene into a transposon and transferring it, multiple copies of the gene can be introduced into the chromosomal DNA. Interchromosomal amplification methods can be used to introduce multiple copies of a gene into chromosomal DNA. Mu-driven transfer allows the introduction of more than 3 copies of genes into the chromosomal DNA of the recipient strain in one step (Akhverdyan VZ et al., Biotechnol. (Russian), 2007, 3: 3-20).
..

The genes introduced into the bacteria described herein can connect downstream of the promoter. The promoter is not particularly limited as long as a promoter that functions in the host bacterium is selected, and may be a promoter derived from the host bacterium or a promoter of a heterologous origin. The term "promoter functioning in the host bacterium" is used. , Can mean a promoter having promoter activity in a host bacterium. Specific examples of promoters that function in Enterobacteriaceae bacteria include, but are not limited to, the following potent promoters.

Examples of the method that can be used to enhance the expression of a gene such as the ydiJ gene include a method of increasing the expression level of the gene by modifying the expression control region of the gene. Modification of the gene expression control region can be adopted in combination with an increase in the number of copies of the gene. The gene expression control region can be modified, for example, by substituting the gene's native expression control region with a native and / or modified foreign expression control region. The term "expression control region" may be substituted or equivalent to the term "expression control sequence". If the ydiJ gene may be organized into an operon structure with one or more other genes, a method that can be used to enhance the expression of the gene is an operon containing the gene by modifying the expression control region of the operon. Modifications can be carried out, for example, by substituting the native expression control region of the operon with a native and / or modified foreign expression control region, including increasing the expression level of the operon. This method can simultaneously enhance the expression of two or more genes, including the ydiJ gene.

Expression control regions include promoters, enhancers, operators, attenuators and termination signals, anti-termination signals, ribosome binding sites (RBS), and other expression control elements (eg, regions to which repressors or activators bind, and regions. / Or, for example, the binding site of a transcriptional and translational control protein in a transcribed mRNA). Such control regions are described, for example, in known literature (eg Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory Manual”, 2nd ^ed ., Cold Spring Harbor Laboratory Press (1989); Pfleger. BF et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes, Nat. Biotechnol., 2006, 24: 1027-1032; Mutalik VK et al., Precise and reliable gene expression via standard transcription and translation initiation elements, Nat. Methods, 2013, 10: 354-360). Modification of the gene expression control region can be combined with an increase in the number of copies of the gene (eg, Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871; Tyo KEJ et al., Nature Biotechnol., 2009,
See 27: 760-765).

Suitable promoters for enhancing the expression of the ydiJ gene include strong promoters. The term "strong promoter" can mean a stronger promoter than the innate promoter of the ydiJ gene. Strong promoters that function in enterobacteriaceae bacteria are not limited, but are limited to lac promoter, trp promoter, trc promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter (derived from Bifidobacterium genus). , _Pnlp8 promoter ( _WO2012 / 137689), and the PR or PL promoter of lambda (λ) phage. As a strong promoter, a highly active form of a conventional promoter may be obtained by using various reporter genes. For example, the strength of the promoter can be increased by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (WO0018935 A1). Promoter strength can be defined by the frequency of initiation of RNA synthesis. Methods for assessing promoter strength and examples of potent promoters are described in Goldstein MA et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). Strong promoters can be used that give high levels of gene expression in bacteria (eg, Enterobacteriaceae). Alternatively, the effect of the promoter can be enhanced, for example, by introducing mutations into the promoter region of the ydiJ gene to obtain stronger promoter function, thus increasing the transcriptional level of the ydiJ gene located downstream of the promoter. Can be increased. In addition, the replacement of several nucleotides in the Shine-Dalgarno (SD) sequence and / or the spacer between the SD sequence and the start codon and / or the sequence immediately upstream or downstream of the start codon in the ribosome binding site is the mRNA. It is known that it greatly affects translation efficiency. Therefore, these regions can be examples of gene expression control regions. For example, 20-fold range of expression levels have been found, depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol.,,
1981, 35: 365-403; Hui A. et al., EMBO J., 1984, 3: 623-629).

The number of copies of a gene or the presence or absence of a gene can be determined, for example, by limiting treatment of chromosomal DNA and then using a probe based on the gene sequence, or fluorescent in.
It can be measured by performing situ hybridization (FISH) or the like. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods such as Northern blotting and quantitative RT-PCR. The amount of protein encoded by the gene can be measured by known methods such as SDS-PAGE and subsequent immunoblotting (Western blotting) and mass spectrometry of protein samples.

Methods for manipulating and cloning DNA recombinant molecules, such as plasmid DNA preparation, DNA cleavage, DNA binding, DNA transformation, selection of oligonucleotides as primers, introduction of mutations, etc., are available to those skilled in the art. It may be a well-known usual method. Such methods are, for example, Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory”.
Manual ”, 2nd ^ed ., Cold Spring Harbor Laboratory Press (1989) or Green MR and Sambrook JR,“ Molecular Cloning: A Laboratory Manual ”, 4th ^ed ., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick , Jack J. Pasternak and Cheryl L. Patten, “Molecular Biotechnology: principles and applications of recombinant DNA”, 4th ^ed ., Washington, DC, ASM Press (2009).

As the operation method using the recombinant DNA, any method can be used, including, for example, conventional methods such as transformation, transfection, infection, conjugation, and mobilization. Bacterial transformation, transfection, infection, conjugation, or mobilization with a protein-encoding DNA can impart the bacterium the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization include any method. For example, for efficient DNA transformation and transfection, methods of treating receiving cells with calcium chloride to increase the permeability of E. coli K-12 cells to DNA have been reported (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Biol., 1970, 53: 159-162). Specialized and / or generalized transformation methods have been described (Morse ML et al.,
Transduction in Escherichia coli K-12, Genetics, 1956, 41 (1): 142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Other methods for random and / or targeted integration of DNA into host microorganisms, such as "Mu-driven integration / amplification" (Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871), "Red / ET-driven integration" or "λRed / ET-mediated integration" (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97 (12): 6640-45; Zhang Y., et al., Nature Genet., 1998, 20: 123-128), can be applied. In addition, for multiple insertions of the desired gene, recA results in Mu-driven recombination (Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871) and amplification of the desired gene. In addition to chemically inducible chromosomal evolution based on dependent homologous recombination (Tyo KEJ et al., Nature Biotechnol., 2009, 27: 760-765), transference, site-specific and / or homologous Red / Recombination via ET,
And / or other methods utilizing various combinations of P1-mediated generalization transduction (eg, Minaeva NI et al., BMC Biotechnology, 2008, 8:63; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93 (2): 815-829).

The base sequence of the ydiJ gene unique to the bacterial species (eg, P. ananatis and E. coli and others listed in Table 1) and the amino acid sequence of the YdiJ protein encoded by the gene have already been elucidated (above). (See) So, for the same gene or mutant base sequence specific to such a bacterial species, an oligonucleotide primer prepared based on the DNA of the same bacterial species and the base sequence of the ydiJ gene unique to the same bacterial species is used. By cloning from the same bacterial species by PCR, or mutation methods in which DNA containing the ydiJ gene is treated in vitro, for example with hydroxylamine, or bacterial species carrying the ydiJ gene are usually used for UV (UV) irradiation or such treatment. It can be obtained by a mutation method treated with a mutant agent such as N-methyl-N'-nitro-nitrosoguanidine (NTG) or nitrite, or by chemical synthesis as a full-length gene structure. The ydiJ gene and its mutant base sequences unique to any other organism, including other bacterial species, can be obtained as well.

The term "native to" when referring to a protein or nucleic acid means that the protein or nucleic acid is unique to a particular organism (eg, mammal, plant, insect, bacterium, or virus, etc.). obtain. That is, a protein or nucleic acid specific to a particular organism can mean a protein or nucleic acid naturally occurring in the organism, respectively. Proteins or nucleic acids specific to a particular organism can be isolated from the organism and sequenced by methods known to those of skill in the art. In addition, the term "unique" when referring to a protein or nucleic acid is used because the amino acid or base sequence of the protein or nucleic acid isolated from the organism in which the protein or nucleic acid is present can be readily determined. Any protein or nucleic acid resulting in the amino acid sequence or base sequence is identical to the amino acid sequence or base sequence of the naturally occurring, naturally expressed and / or naturally produced protein or nucleic acid in the organism. It can also mean a protein or nucleic acid obtained by means, such as genetic engineering techniques or chemical synthesis methods, including recombinant DNA technology. The term "protein" can mean any, but not limited to, peptides, oligopeptides, polypeptides, proteins, enzymes and the like. The term "nucleic acid" can mean, but is not limited to, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and in particular, expression control sequences comprising promoters, attenuators, terminators, etc. It can mean any of a base sequence encoding a gene, an intergenic sequence, a signal peptide, a prosite of a protein, an artificial amino acid sequence, or the like. For example, a gene can be, in particular, DNA. The amino acid sequences and base sequences as well as their homologues endemic to various organisms are described herein, and examples of these are proteins having the amino acid sequence set forth in SEQ ID NO: 2 (which is a bacterium of the P. ananatis species). It can be unique to and can be encoded by a gene having the base sequence shown in SEQ ID NO: 1).

The term "non-modified" (which is "native", "natural") when referring to genes (eg, "unmodified genes") and proteins (eg, "unmodified proteins"). (Alternatively or equivalently used) to the terms "natural" and "wild-type") are naturally present in the organism, specifically in the unmodified strain of the bacterium, respectively. , Naturally expressed and / or naturally produced genes and proteins. Such organisms include any organism having the corresponding gene or protein, and specifically, for example, E. coli W3110 strain, E. coli MG1655 strain, P. ananatis.
There are 13355 shares. The unmodified gene can encode an unmodified protein.

The term "bacteria modified to weaken gene expression" can mean that the bacteria have been modified to weaken gene expression in the modified bacteria. Gene expression can be attenuated, for example, by inactivating the gene.

The term "gene is inactivated" means that the modified gene encodes a protein that is completely inactive or non-functional as compared to a gene that encodes a protein with inorganic pyrophosphatase activity. Can mean. Modified DNA regions include partial or whole gene deletions, one or more base substitutions that result in amino acid substitutions in the gene-encoded proteins (missense mutations), stop codon introductions (nonsense mutations), and gene mutations. Deletion of one or two bases leading to a leading frame shift, insertion of drug resistance genes and / or transcription termination signals, or expression control regions (promoters, enhancers, operators, attenuators and termination signals, anti-termination signals, ribosome binding) By modification of the site (RBS), and other expression control elements, etc.), the gene may not be naturally expressed. Gene inactivation can be performed, for example, by UV irradiation or mutation treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene disruption using homologous recombination, and / Or insertion-deletion mutagenesis based on "Red / ET-driven integration" or "λRed / ET-mediated integration" (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97 (11) : 5978-5983; Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645;
It can be carried out by a conventional method such as Zhang Y. et al., Nature Genet., 1998, 20: 123-128).

The term "the bacterium has been modified to weaken gene expression" refers to the region in which the modified bacterium is operably linked to the gene (which is the promoter, enhancer, operator, attenuator and termination signal, Includes sequences that control gene expression, such as ribosome binding sites (RBS) and other expression control elements), modified to weaken gene expression levels compared to unmodified strains. And other examples (see, eg, WO95 / 34672; Carrier TA and Keasling JD, Biotechnol. Prog., 1999, 15: 58-64) can also be meant. The term "operably linked" when referring to a gene means that the regulatory region is the expression of the gene (eg, enhanced).
Increased, constitutive, basal, anti-terminated, weakened, deregulated, reduced or suppressed expression) can be achieved and / or into the mRNA and / or gene of the gene. It can mean that the encoded amino acid sequence (also referred to as an expression product) is linked to the base sequence of the gene so that it can be produced as a result of the expression of the gene.

The term "bacteria modified to weaken gene expression" is used in the modified bacteria as compared to unmodified strains (eg, wild strains or parent strains) in which the expression level (ie, expression level) of the gene is. It can also mean that the bacteria have been modified to weaken. A decrease in the expression level of a gene can be measured, for example, as a decrease in the expression level of the gene per cell, which may be the average expression level of the gene per cell. The terms "gene expression level" or "gene expression level" can mean, for example, the amount of gene expression product (eg, the amount of mRNA of the gene or the amount of protein encoded by the gene). Bacteria may be modified such that the expression level of the gene per cell is reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

The term "bacteria modified to weaken gene expression" refers to the total amount and / or total activity of the corresponding gene product (ie, Met repressor) in the modified bacterium as compared to the unmodified strain. It can mean that the bacteria have been modified to be reduced. A decrease in the total amount or activity of a protein can be measured, for example, as a decrease in the amount or activity of the protein per cell, which may be the average amount or activity of the protein per cell. Bacteria can be modified such that the amount and / or activity of the Met repressor per cell is reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain. ..

Specifically, gene expression can also be attenuated by, for example, substituting a weaker gene expression control sequence, such as a promoter on chromosomal DNA. Promoter strength can be defined by the frequency of initiation of RNA synthesis. Examples of methods for assessing promoter strength are described in Goldstein MA et al. (Goldstein MA and Doi RH, Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). Also, as disclosed in WO0018935 A1, the promoter can be modified to weaken by introducing one or more base substitutions into the promoter region of the gene. In addition, the Shine-Dalgarno (SD) sequence and / or the spacer between the SD sequence and the start codon, and / or the substitution of several nucleotides in the sequence immediately upstream and / or downstream of the start codon in the ribosome binding site. It is known that it greatly affects the translation efficiency of mRNA.

Gene expression is specifically expressed, for example, by inserting a transposon or insertion sequence (IS) into the coding region of the gene (US Patent No. 5,175,107) or the region controlling gene expression, or by UV irradiation or nitrosoguanidine. It can also be weakened by conventional methods such as mutagenesis with (N-methyl-N'-nitro-N-nitrosoguanidine; NTG). In addition, site-specific mutation integration is, for example, λRed / ET-mediated recombination (Datsen).
It can be carried out by a known chromosome editing method based on ko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645).

Bacteria can have specific properties such as various nutritional requirements, drug resistance, drug sensitivity, drug dependence, etc., in addition to the above-mentioned properties, without departing from the scope of the present invention.

2. 2. Methods The methods described herein include methods of producing L-amino acids using the bacteria described herein. In the method for producing L-amino acid using the bacterium described in the present specification, the bacterium is cultivated (also referred to as culturing) in a medium and the L-amino acid is used as a medium or a bacterium of the bacterium (“bacterial bacterium”). It may include a step of producing, excreting or secreting and / or accumulating in (also referred to as "body") or both, and a step of recovering L-amino acids from the culture medium and / or bacterial cells. The method may further optionally include the step of purifying the L-amino acid from the medium and / or bacterial cells. The L-amino acid can be produced in the form as described above. L-amino acids can be produced, in particular, in free form, or salts thereof, or mixtures thereof. For example, salts such as sodium salts, potassium salts, ammonium salts and the like of L-amino acids or intramolecular salts such as zwitterions can be produced by the above method. This is possible because amino acids can react under fermentation conditions with each other or with neutralizers such as inorganic or organic acids or alkaline substances by a typical acid-base neutralization reaction to form salts. , This is a chemical characteristic of amino acids that is obvious to those skilled in the art. Specifically, L-cysteine monohydrochloride (L-cysteine × HCl) or L-cysteine monohydrate monohydrochloride (L-cysteine × H ₂ O × HCl) can be produced by the above method. ..

Bacterial culture and recovery and optionally purification of L-amino acids from media and the like can be carried out in the same manner as in conventional fermentation methods for producing L-amino acids using microorganisms. That is, the culture of bacteria and the recovery and purification of L-amino acids from a medium or the like are carried out by applying the conditions suitable for culturing bacteria and the conditions suitable for recovery and purification of L-amino acids, which are well known to those skilled in the art. It may be carried out.

The medium used is not particularly limited as long as it contains at least a carbon source and the bacteria described herein can grow to produce L-amino acids. The medium may be a synthetic medium or a natural medium, such as a typical medium containing carbon sources, nitrogen sources, sulfur sources, phosphorus sources, inorganic ions, and other organic and inorganic components as needed. Carbon sources include glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, sugars such as starch hydrolysate, alcohols such as ethanol, glycerol, mannitol and sorbitol, gluconic acid and fumaric acid. , Citric acid, malic acid, organic acids such as succinic acid, fatty acids and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolyzate, ammonia gas, and aqueous ammonia can be used. Further, peptone, yeast extract, meat extract, malt extract, corn steep liquor and the like can also be used. The medium can contain one or more of these nitrogen sources. Examples of the sulfur source include ammonium sulfate, magnesium sulfate, iron sulfate, manganese sulfate, sodium thiosulfate, ammonium thiosulfate, sodium sulfide, ammonium sulfide and the like. The medium may contain a phosphorus source in addition to a carbon source, a nitrogen source, and a sulfur source. As the phosphorus source, a phosphoric acid polymer such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or pyrophosphoric acid can be used. Vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12 and other vitamins and other necessary substances such as adenine, RNA and other nucleic acids, amino acids, peptone, cazaminoic acid, yeast extract and other organic nutrients. , Appropriate amount (may be trace amount) can be present. In addition to these, if necessary, a small amount of calcium phosphate, iron ion, manganese ion or the like may be added. As other organic and inorganic components, one kind of component may be used, or two or more kinds of components may be used in combination. In addition, when an auxotrophic strain that requires amino acids or the like for growth is used, it is preferable to supplement the required nutrients in the medium.

Culturing can be carried out under conditions suitable for culturing the bacteria used in the method for producing L-amino acids. For example, the culture can be carried out under aerobic conditions for 16-72 hours, or 16-24 hours. The culture temperature during culturing can be controlled within the range of 30 to 45 ° C, or 30 to 37 ° C. The pH can be adjusted between 5 and 8 or between 6 and 7.5. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance such as urea, calcium carbonate, an inorganic acid, an inorganic alkali, or ammonia gas.

After culturing, L-amino acids can be recovered from the medium. Specifically, L-amino acids existing outside the cells can be recovered from the medium. In addition, after culturing, L-amino acids can be recovered from bacterial cells. Specifically, the cells are crushed, solids such as cells and cell crushed suspension (also referred to as cell debris) are removed to obtain a supernatant, and L-amino acids are recovered from the supernatant. Can be done. Crushing of the cells can be carried out by a well-known method such as ultrasonic crushing using high-frequency sound waves. Removal of solids can be carried out, for example, by centrifugation or membrane filtration. Recovery of L-amino acids from media, supernatants, etc. may be, for example, concentration, crystallization, membrane treatment, ion exchange chromatography, flash chromatography, thin layer chromatography, medium pressure or high pressure liquid chromatography, or theirs. It can be carried out by conventional techniques such as combination. These methods may be used alone or in combination as appropriate.

The invention will be described more accurately with reference to the following non-limiting examples.

Example 1 Construction of P. ananatis strain with enhanced expression of ydiJ gene
The promoter region of the ydiJ gene (SEQ ID NO: 1) of the P. ananatis SC17 strain was replaced with the P _nlp8 promoter by λRed-dependent integration. For this purpose, P. ananatis SC17 (0) strain (US patent No. ^8383372 B2, VKPM B-9246) was used in LB liquid medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual (3rd ed.). ), Cold Spring Harbor Laboratory Press, 2001) overnight culture. Then, 1 mL of the culture solution was inoculated into 100 mL of an LB liquid medium containing isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM, and the cells were shaken at 32 ° C. for 3 hours. 250 rpm) cultured. The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Using the plasmid DNA pMW-Km-Pnlp8 (see WO2011043485) as a template, PCR was performed using primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6), and the recombinant sequence of the promoter region of the ydiJ gene was present at both ends. Amplified DNA fragments were obtained. PCR conditions are as follows: denaturation step at 95 ° C for 3 minutes; profile of two first cycles of 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; 20 seconds at 94 ° C. , Profile of the last 30 cycles at 54 ° C for 20 seconds, 72 ° C for 90 seconds; final step at 72 ° C for 5 minutes. The amplified DNA fragment (about 1,6 kbp size) was purified by agarose gel electrophoresis and introduced into competent cells by electroporation. LB containing 20 mg / L kanamycin after culturing the cells in SOC medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual (3rd ^ed .), Cold Spring Harbor Laboratory Press, 2001) for 2 hours. It was applied on a plate and cultured at 34 ° C. overnight until each colony was visible. The desired transformant was identified by PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). As a result, P. ananatis SC17 (0) -Pnlp8-ydiJ strain was obtained.

Example 2 Production of L-Cysteine by P. ananatis strain with enhanced expression of ydiJ gene To test the effect of enhanced expression of ydiJ gene on L-cysteine production, SC17 (0)-.
Chromosome DNA was isolated from the Pnlp8-ydiJ strain and 10 μg was used for electroporation transformation of P. ananatis EYP197 (s). The L-cysteine producing strain EYP197 (s) of P. ananatis was constructed as described in RU2458981 C2 or WO2012 / 137689. That is, the P. ananatis EYP197 (s) strain was constructed by introducing the cysE5 and yeaS genes from P. ananatis SC17 and replacing the native promoter of the cysPTWA gene cluster with the Pnlp8 promoter. Transformants were applied on LB plates containing 20 mg / L kanamycin and cultured at 34 ° C. overnight until each colony was visible. The desired transformant was identified by PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). As a result, P. ananatis EYP Pnlp8-ydiJ strain was obtained.

P. ananatis EYP197 (s) strain and EYP Pnlp8-ydiJ strain at 32 ° C using LB liquid medium, respectively.
Incubated for 18 hours. Next, 0.2 mL of the obtained culture medium was inoculated into 2 mL of the fermentation medium shown in Table 2 in a 20 × 200 mm test tube, and the temperature was 32 ° C. on a rotary shaker at 250 rpm until glucose was consumed.
Incubated for 24 hours.

After culturing, the amount of L-cysteine accumulated in the medium was used by partially modifying the method described in Gaiton de MK (Biochem J., 104 (2): 627-633 (1967)) as follows. Measured: 150 μL of each sample was mixed with 1 MH ₂ SO ₄ 150 μL, incubated at 20 ° C for 5 minutes, then 700 μL of H ₂ O.
Was added to the mixture, 150 μL of the resulting mixture was transferred to a new vial, and 800 μL of solution A (1 M Tris-HCl pH 8.0, 5 mM dithiothreitol (DTT)) was added. Incubate the resulting mixture at 20 ° C for 5 minutes, spin at 13000 rpm for 10 minutes, and then add 100 μL of the mixture to 20 × 200.
Transferred to a mm test tube. Next, add 400 μL H ₂ O, 500 μL glacial acetic acid, and 500 μL solution B (0.63 g ninhydrin, 10 mL glacial acetic acid, 10 mL 36% HCl) and bring the mixture to a boiling water bath. Incubated in for 10 minutes. Then 4.5 mL of ethanol was added and OD ₅₆₀ was measured. The concentration of cysteine was calculated using the formula: C (Cys, g / L) = 11.3 × OD ₅₆₀ .

Table 3 shows the results of fermentation in eight independent tubes (mean ± standard deviation). As can be seen from Table 3, the modified strain P.ananatis EYP Pnlp8-ydiJ is a control strain P.ananatis EYP197 (s).
It was possible to accumulate a larger amount of L-cysteine in comparison with.

Example 3: Production of L-methionine by P. ananatis strain with enhanced expression of ydiJ gene To test the effect of enhanced expression of ydiJ gene on L-methionine production, SC17 (0) -Pnlp8-ydiJ strain (implemented). The chromosomal DNA of Example 1) was isolated and 10 μg of DNA was used for transformation of P. ananatis C3568 by electroporation. The L-methionine-producing strain C3568 of P. ananatis was constructed as described in Preliminary Examples. Transformants were applied on LB plates containing 20 mg / L kanamycin and cultured at 34 ° C. overnight until each colony was visible. The desired transformant was identified by PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). As a result, P. ananatis C3568 Pnlp8-ydiJ strain was obtained.

P. ananatis C3568 strain and C3568 Pnlp8-ydiJ strain were each cultured in LB liquid medium at 32 ° C. for 18 hours. Then, 0.2 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 4 in a 20 × 200 mm test tube, and glucose is consumed at 32 ° C. for 48 hours on a rotary shaker at 250 rpm. Was cultured to.

After culturing, the amount of L-methionine accumulated in the medium was measured with an Agilent 1260 amino-acid analyzer.

The results of four independent in vitro fermentations (mean ± standard deviation) are shown in Table 5. As can be seen from Table 5, the modified strain P. ananatis C3568 Pnlp8-ydiJ was able to accumulate a larger amount of L-methionine as compared with the parent strain P. ananatis C3568.

Example 4 Construction of E. coli MG1655 strain with enhanced expression of ydiJ gene 4.1 Construction of E. coli MG1655 Ptac-ydiJ (Km) strain
E. coli MG1655 (ATCC 700926) / pKD46 strain is cultured overnight in LB liquid medium. Then, 1 mL of the culture solution is inoculated into 100 mL of LB liquid medium containing isopropylarabinose at a final concentration of 50 mM and ampicillin at 50 mg / L, and the cells are cultured at 37 ° C. for 2 hours with shaking (250 rpm). The cells are collected and washed 3 times with ice-cooled 10% glycerol to obtain competent cells. The amplified λatt L-Km ^R -λatt R-Ptac fragment (Example 4.2 described later) is purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, incubate on an L-agar plate containing 25 mg / L kanamycin at 37 ° C. for 18 to 24 hours. Purify the emerging colonies in the same medium. Next, primers P5 (SEQ ID NO: 9) and P6 (SEQ ID NO: 10) were used to confirm that the promoter region of the ydiJ gene on the chromosome was replaced with the attL-Km ^R -attR-Ptac fragment. Perform a PCR reaction. Then, the E. coli MG1655 Ptac-ydiJ (Km) strain is obtained.

4.2 Construction of λattL-Km ^R -λattR-Ptac fragment The λattL-Km ^R -λattR-Ptac fragment (SEQ ID NO: 11) is constructed as follows. Primer P7 (SEQ ID NO: 12) and P8 (SEQ ID NO: 13) and Prime Star polymerase using the chromosome of P. ananatis SC17 (0) λattL-Km ^R -λattR-Ptac-lacZ strain (US patent No. 9,051,591 B2) as a template. Perform PCR using (Takara Bio Inc.). The reaction is prepared according to the composition included in the kit and amplifies the DNA at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 cycles per minute per kbp. As a result, a λatt L-Km ^R -λatt R-Ptac gene fragment (SEQ ID NO: 11) having a recombinant sequence of the promoter region of the ydiJ gene and a region complementary to the region adjacent to the ydiJ gene at both ends is obtained.

Example 5: Production of L-arginine by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-arginine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-arginine-producing strain 382ilvA + of E. coli by P1 transduction to obtain the E. coli 382ilvA + Ptac-ydiJ (Km) strain.

The 382ilvA ⁺ strain is obtained from the L-arginine-producing strain 382 (VKPM B-7926, EP1170358 A1) by P1 transduction of the wild-type ilvA gene of the E. coli K-12 strain. The 382 shares were published on April 10, 2000 by the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny).
It was deposited with proezd., 1, Moscow 117545, Russian Federation) under accession number VKPM B-7926, and was subsequently transferred to a deposit under the Budapest Convention on May 18, 2001.

E. coli 382ilvA + strain and 382ilvA + Ptac-ydiJ (Km) strain, respectively, at 37 ° C for 18 hours, 3
Shake culture in mL nutrient medium, inoculate 0.3 mL of the resulting culture into 2 mL of fermentation medium shown in Table 6 in a 20 × 200 mm test tube, and place on a rotary shaker at 32 ° C. for 48 hours. Incubate.

After culturing, the amount of L-arginine accumulated in the medium is measured by paper chromatography using a mobile phase consisting of butane-1-ol: acetic acid: water = 4: 1: 1 (v / v). Ninhydrin solution (2%; in acetone) is used as a visualization reagent. Spots containing arginine are excised, L-arginine is eluted with 0.5% CdCl ₂ aqueous solution, and the amount of L-arginine is measured spectroscopically at 540 nm.

Example 6: Production of L-citrulin by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-citrulin production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-citrulin-producing strain 382ΔargG of E. coli by P1 transduction to obtain the E. coli 382ΔargG Ptac-ydiJ (Km) strain.

The 382ΔargG strain was first developed by Datsenko K.A. and Wanner B.L. called "λRed / ET-mediated integration" (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97 (12): It is obtained by deleting the argG gene on the chromosome of E. coli arginine-producing strain 382 (VKPM B-7926, EP1170358 A1) by 6640-6645). According to this method, homologous PCR primers are constructed in both the argG gene and the region adjacent to the gene conferring antibiotic resistance in the template plasmid. The plasmid pMW118-λattL-cat-λattR (WO05 / 010175) is used as a template for PCR.

E. coli 382ΔargG strains and 382ΔargG Ptac-ydiJ (Km) strains are shake-cultured at 37 ° C. for 18 hours in 3 mL of nutrient medium, respectively. Then, 0.3 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 7 in a 20 × 200 mm test tube, and cultured on a rotary shaker at 32 ° C. for 48 hours.

After culturing, the amount of L-citrulin accumulated in the medium is measured by paper chromatography using a mobile phase consisting of butane-1-ol: acetic acid: water = 4: 1: 1 (v / v). Ninhydrin solution (2%; in acetone) is used as a visualization reagent. Spots containing L-citrulline are excised, L-citrulline is eluted with 0.5% CdCl ₂ aqueous solution, and the amount of L-citrulline is measured spectroscopically at 540 nm.

Example 7: Production of L-glutamic acid by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-glutamic acid production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-glutamic acid-producing strain VL334thrC ⁺ (EP1172433 A1) of E. coli by P1 transduction to obtain the E. coli VL334thrC ⁺ Ptac-ydiJ (Km) strain.

The VL334thrC ⁺ strain was deposited with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on December 6, 2004 under accession number VKPM B-8961 and then December 2004. It was transferred to an international deposit under the provisions of the Budapest Treaty on the 8th of March.

E. coli VL334thrC ⁺ strain and VL334thrC ⁺ Ptac-ydiJ (Km) strain are cultured at 37 ° C. for 18-24 hours on L-agar plates, respectively. Then, 1 loopful of cells is transferred to 2 mL of the fermentation medium shown in Table 8 in a 20 × 200 mm test tube, and cultured at 30 ° C. with shaking for 3 days.

After culturing, the amount of L-glutamic acid accumulated in the medium is butane-1-ol: acetic acid: water = 4
Measured by paper chromatography using a mobile phase consisting of 1: 1 (v / v), stained with ninhydrin (1% solution in acetone), and L-glutamic acid in 50% ethanol containing 0.5% CdCl ₂ . Elute and measure the amount of L-glutamic acid at 540 nm.

Example 8: Production of L-histidine by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-histidine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-histidine-producing strain 80 of E. coli by P1 transduction to obtain the E. coli 80 Ptac-ydiJ (Km) strain.

The 80 strains are listed in Russian Patent No. 2119536 C1 on October 15, 1999.
Deposited with the National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) under accession number VKPM B-7270 and then on July 12, 2004 for international deposit under the provisions of the Budapest Treaty. It has been transferred.

E. coli 80 strains and 80 Ptac-ydiJ (Km) strains are cultured at 29 ° C. for 6 hours in 2 mL L-broth, respectively. Then, 0.1 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 9 in a 20 × 200 mm test tube, and cultured on a rotary shaker (350 rpm) at 29 ° C. for 65 hours. ..

After culturing, the amount of L-histidine accumulated in the medium is measured by thin layer chromatography (TLC). Use a 10 × 15 cm TLC plate coated with a 0.11 mm layer of Sorbfil silica gel containing a non-fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russian Federation). The Sorbfil plate develops in a mobile phase consisting of propane-2-ol: acetone: 25% ammonia water: water (6: 6: 1.5: 1 (v / v)). A ninhydrin solution (2%, w / v; in acetone) is used as a visualization reagent. After unfolding, the plates are dried and scanned using the Camag TLC Scanner 3 with winCATS software (version 1.4.2) in absorbance mode at 520 nm.

Example 9: Production of L-leucine by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-leucine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) DNA fragment from the chromosome was introduced into E. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121) by P1 translation, and E. coli 57 Ptac-ydiJ (Km). Get a stock.

57 shares were deposited on May 19, 1997 with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) under accession number VKPM B-7386.

E. coli 57 strains and 57 Ptac-ydiJ (Km) strains are cultured on L-agar plates at 37 ° C. for 18-24 hours, respectively. Each strain is grown in a rotating shaker (250 rpm) for 18 hours at 32 ° C. in 2 mL of L-broth containing 4% sucrose in a 20 × 200 mm test tube to obtain seed material. Then, inoculate the fermentation medium with 0.2 mL (10%) of seed material. Fermentation is 2 mL in a 20 x 200 mm test tube
Perform with the minimum fermentation medium of. The cells are grown at 32 ° C. for 48-72 hours by shaking at 250 rpm.

After culturing, the amount of L-leucine accumulated in the medium is measured by paper chromatography using a mobile phase consisting of butane-1-ol: acetic acid: water = 4: 1: 1 (v / v).

Example 10: Production of L-lysine by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-lysine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-lysine-producing strain WC196LC / pCABD2 of E. coli by P1 transduction to obtain the E. coli WC196LC Ptac-ydiJ (Km) / pCABD2 strain.

The L-lysine-producing strain WC196LC of E. coli is constructed as described in EP 2083083 A1. L-lysine production plasmid pCABD2 carrying the dapA, dapB, lysC, and ddh genes
WC196LC strain is transformed by a conventional method with (WO95 / 16042 and WO01 / 53459) to obtain WC196LC / pCABD2 strain.

The plasmid pCABD2 is a mutant dapA gene encoding a dihydrodipicolinate synthase derived from E. coli, which has a mutation in which feedback inhibition by L-lysine is released, and a mutation in which feedback inhibition by L-lysine is released. E. coli-derived mutant lysC encoding aspartokinase III
It contains the gene, the dapB gene encoding dihydrodipicolinate reductase from E. coli, and the ddh gene encoding diaminopimelate dehydrogenase from Brevibacterium lactofermentum (WO95 / 16042 and WO01 / 53459).

E. coli WC196LC / pCABD2 strain and WC196LC Ptac-ydiJ (Km) / pCABD2 strain, 37 respectively
Incubate in L-medium containing streptomycin (20 mg / L) at ° C.

Then, 0.3 mL of the obtained culture is inoculated into 2 mL of fermentation medium in a 20 × 200 mm test tube, and cultured on a rotary shaker (240 rpm) at 34 ° C. for 48 hours. The composition of the fermentation medium is the same as that described in Example 5, except that 30 mg / L streptomycin was added.

After culturing, the amount of L-lysine accumulated in the medium is estimated as described in Example 5.

Example 11: Production of L-ornithin by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-ornithin production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-ornithine-producing strain 382ΔargFΔargI of E. coli by P1 transduction to obtain the E. coli 382ΔargFΔargI Ptac-ydiJ (Km) strain.

The 382ΔargFΔargI strain was first developed by Datsenko KA and Wanner BL, called “λRed / ET-mediated integration” (Datsenko KA and Wanner BL, Proc.
Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) sequentially deletes the argF and argI genes on the chromosome of E. coli arginine-producing strain 382 (VKPM B-7926, EP1170358 A1). It is obtained by. According to this method, two pairs of PCR primers are constructed that are homologous to both the region adjacent to the argF or argI gene and the region adjacent to the gene conferring antibiotic resistance in the template plasmid. The plasmid pMW118-λattL-cat-λattR (WO05 / 010175) is used as a template for PCR.

E. coli 382ΔargFΔargI strains and 382ΔargFΔargI Ptac-ydiJ (Km) strains are shake-cultured at 37 ° C. for 18 hours in 3 mL of nutrient medium, respectively. Then, 0.3 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 11 in a 20 × 200 mm test tube, and cultured on a rotary shaker at 32 ° C. for 48 hours.

After culturing, the amount of L-ornithine accumulated in the medium is measured by paper chromatography using a mobile phase consisting of butane-1-ol: acetic acid: water = 4: 1: 1 (v / v). Ninhydrin solution (2%; in acetone) is used as a visualization reagent. Spots containing ornithine are excised, ornithine is eluted with 0.5% CdCl ₂ aqueous solution, and the amount of ornithine is measured spectroscopically at 540 nm.

Example 12: Production of L-phenylalanine by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-phenylalanine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) DNA fragment from the chromosome was introduced into the L-phenylalanine-producing strain AJ12739 of E. coli by P1 transduction, and E. coli AJ12739 Ptac-ydiJ.
Obtain (Km) stock.

The AJ12739 strain was deposited with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on November 6, 2001 under accession number VKPM B-8197, and then in August 2002. It was transferred to an international deposit under the provisions of the Budapest Treaty on the 23rd.

E. coli AJ12739 strain and AJ12739 Ptac-ydiJ (Km) strain are each cultured at 37 ° C. for 18 hours in nutrient medium. Then, 0.3 mL of the obtained culture is inoculated into 3 mL of the fermentation medium shown in Table 12 in a 20 × 200 mm test tube, and cultured on a rotary shaker at 37 ° C. for 48 hours.

After culturing, the amount of L-phenylalanine accumulated in the medium is measured by thin layer chromatography (TLC).
Measured by. Use a 10 × 15 cm TLC plate coated with a 0.11 mm layer of Sorbfil silica gel (Stock Company Sorbpolymer, Krasnodar, Russian Federation) containing a non-fluorescent indicator. The Sorbfil plate develops in a mobile phase consisting of propane-2-ol: ethyl acetate: 25% aqueous ammonia: water (40:40: 7:16 (v / v)). Ninhydrin solution (2%; in acetone) is used as a visualization reagent.

Example 13: Production of L-proline by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-proline production, MG1655 Ptac-ydiJ (Km) strain (Example 4). ) Is introduced into the L-proline-producing strain 702ilvA of E. coli by P1 transduction to obtain the E. coli 702ilvA Ptac-ydiJ (Km) strain.

The 702ilvA strain was deposited with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on July 18, 2000 under accession number VKPM B-8012, and then in May 2001. It was transferred to an international deposit under the provisions of the Budapest Treaty on the 18th.

E. coli 702ilvA and 702ilvA Ptac-ydiJ (Km) strains are cultured on L-agar plates at 37 ° C. for 18-24 hours, respectively. Then, each of these strains is cultured under the same conditions as in Example 7 (production of L-glutamic acid).

Example 14: Production of L-tryptophan by E. coli strain with enhanced expression of ydiJ gene In order to test the effect of enhanced expression of ydiJ gene on L-tryptophan production, MG1655
A DNA fragment from the chromosome of the Ptac-ydiJ (Km) strain (Example 4) was introduced into the L-tryptophan-producing strain SV164 (pGH5) of E. coli by P1 translation, and E. coli SV164 (pGH5) Ptac- Obtain ydiJ (Km) stock.

The SV164 (pGH5) strain is a strain obtained by introducing the plasmid pGH5 into the E. coli SV164 strain. The SV164 strain has a trpE allele encoding anthranilate synthase that is not subject to feedback inhibition by tryptophan. The SV164 strain is a strain obtained by introducing a mutation into the trpE gene of E. coli YMC9 strain (ATCC 33927). YMC9 strains are available from the American Type Culture Collection (PO Box 1549, Manassas, VA 20108, United States of America). The plasmid pGH5 contains a mutant serA gene encoding a phosphoglycerate dehydrogenase that is not subject to feedback inhibition by serine. The SV164 (pGH5) strain is described in detail in US Pat. No. 6,180,373 B1 or EP0662143 B1.

E. coli SV164 (pGH5) and SV164 (pGH5) Ptac-ydiJ (Km) strains, 18 at 37 ° C, respectively.
For hours, shake culture in 3 mL nutrient medium containing tetracycline (20 mg / L, marker for pGH5 plasmid). Then, 0.3 mL of the obtained culture was inoculated into 3 mL of fermentation medium containing tetracycline (20 mg / L) in a 20 × 200 mm test tube, respectively, and placed on a rotary shaker at 250 rpm at 37 ° C. Incubate for 48 hours.

After culturing, the amount of L-tryptophan accumulated in the medium is measured by TLC as described in Example 12 (Production of L-Phenylalanine). The fermentation medium components are shown in Table 13. Fermentation medium components are sterilized separately in each of the groups shown in the table (A, B, C, D, E, F, G, and H) to avoid unwanted interactions during sterilization.

Example 15: L-valine production by E. coli strain with enhanced ydiJ gene expression In order to test the effect of enhanced ydiJ gene expression on L-valine production, MG1655 Ptac-ydiJ (Km) strain (Example 4). A DNA fragment from the chromosome of E. coli is introduced into the L-valine production strain H81 of E. coli by P1 transduction to obtain the E. coli H81 Ptac-ydiJ (Km) strain.

The H81 strain was deposited with the Russian National Collection of Industrial Microorganisms (VKPM; 1 ^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on January 30, 2001 under accession number VKPM B-8066, February 1, 2002. It has been transferred to an international deposit under the provisions of the Budapest Treaty.

E. coli H81 strain and H81 Ptac-ydiJ (Km) strain are each cultured at 37 ° C. for 18 hours in a nutrient broth. 0.1 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 14 in a 20 × 200 mm test tube, and cultured on a rotary shaker (250 rpm) at 32 ° C. for 72 hours.

After culturing, the amount of L-valine accumulated in the medium is measured by TLC. Use a 10 × 15 cm TLC plate coated with a 0.11 mm layer of Sorbfil silica gel (Stock Company Sorbpolymer, Krasnodar, Russian Federation) containing no fluorescent indicator. Sorbfil plate is propane-2
-All: Ethyl acetate: 25% Ammonia Water: Water (40: 40: 7: 16 (v / v)). Ninhydrin solution (2%; in acetone) is used as a visualization reagent.

Example 16: Production of L-threonine by E. coli strain with enhanced expression of ydiJ gene
In order to test the effect of enhancing the expression of the ydiJ gene on L-threonine production, a DNA fragment from the chromosome of MG1655 Ptac-ydiJ (Km) strain (Example 4) was collected from E. coli L-threonine-producing strain B-3996Δtdh. E. coli B-3996Δtdh Ptac-ydiJ (Km) strain is obtained by P1 transduction.

The B-3996Δtdh strain uses the λRed / ET-mediated integration method (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci.) For the tdh gene on the chromosome of E. coli B-3996 (US Patent Nos. 5,175,107 and 5,705,371). Obtained by deletion in USA, 2000, 97 (12): 6640-6645).

E. coli B-3996Δtdh and B-3996Δtdh Ptac-ydiJ (Km) in 2 mL L-broth with 4% (w / w) glucose in a 20 × 200 mm tube, respectively, at 32 ° C. Incubate for 18 hours. Then, 0.2 mL of the obtained culture is inoculated into 2 mL of the fermentation medium shown in Table 15 in a 20 × 200 mm test tube, and cultured on a rotary shaker (250 rpm) at 32 ° C. for 65 hours.

After culturing, the amount of L-threonine accumulated in the medium can be measured by paper chromatography using the following mobile phase: butanol: acetic acid: water = 4: 1: 1 (v / v). Ninhydrin solution (2%; in acetone) is used as a visualization reagent. After unfolding, the plates are dried and scanned using the Camag TLC Scanner 3 with winCATS software (version 1.4.2) in absorbance mode at 520 nm.

Preliminary Example P. Construction of L-methionine-producing strain C3568 of ananatis 1. P. ananatis SC17 (0) λatt L-kan ^R -λatt R-Pnlp8sd22-cys Construction of M strain
A P. ananatis SC17 (0) λatt L-kan ^R -λatt R-Pnlp8sd22-cysM strain in which the promoter region of the cysM gene (SEQ ID NO: 14) was replaced with the cassette λatt L-kan ^R -λatt R-Pnlp8sd22 was constructed by λRed-dependent integration. bottom. For this purpose, P. ananatis SC17 (0) strain (US Patent)
No. 8383372 B2, VKPM B-9246) was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells.
PCR was performed using the pMW118-attL-kan-attR-pnlp8sd22 plasmid (SEQ ID NO: 17) as a template using primers P9 (SEQ ID NO: 15) and P10 (SEQ ID NO: 16), and the recombinant sequence of the promoter region of the cysM gene was obtained at both ends. Amplified DNA fragments of λatt L-kan ^R -λatt R-Pnlp8sd22 possessed by λatt L-kan R-Pnlp8sd22 were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, primers P11 (SEQ ID NO: 18) and P12 (SEQ ID NO: 19) were used to confirm that the promoter region of the cysM gene on the chromosome was replaced with the λatt L-kan ^R -λatt R-Pnlp8sd22 cassette. PCR analysis (TaKaRa Speed Star (R); 92 ° C for 10 seconds, 56 ° C for 10 seconds, 72 ° C for 60 seconds for 40 cycles) was performed. As a result, P. ananatis SC17 (0) λatt L-kan ^R -λatt R-Pnlp8sd22-cysM strain (abbreviation: C2338) was obtained.

2. 2. Construction of P. ananatis C2597 strain (SC17 (0) ΔmdeA :: λattL-kan ^R -λattR-Pnlp8sd22-cysM) Silent gene mdeA (SEQ ID NO: 20) is cassetted λattL-kan ^R -λattR-Pnlp8sd22-cysM
The P. ananatis SC17 (0) ΔmdeA :: λatt L-kan ^R -λatt R-Pnlp8sd22-cysM strain substituted with λRed-dependent integration was constructed. For this purpose, P. ananatis SC17 (0) strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. PCR was performed using the chromosomes isolated from the C2338 strain (preliminary example 1) using primers P13 (SEQ ID NO: 21) and P14 (SEQ ID NO: 22) as templates, and λattL-kan having a recombinant sequence of the mdeA gene at both ends. Amplified DNA fragments of ^R -λattR-Pnlp8sd22-cysM were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, they were applied onto an LB plate containing 35 mg / L kanamycin, and 34
Incubated at ° C for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR was performed using primers P15 (SEQ ID NO: 23) and P16 (SEQ ID NO: 24) to confirm that the mdeA gene on the chromosome was replaced with the λatt L-kan ^R -λatt R-Pnlp8sd22-cysM cassette. Analysis (TaKaRa Speed Star (R); 92 ° C for 10 seconds, 56 ° C for 10 seconds, 72 ° C for 60 seconds for 40 cycles) was performed. As a result, P. ananatis SC17 (0) ΔmdeA :: λatt L-kan ^R -λatt R-Pnlp8sd22-cysM strain (abbreviation: C2597) was obtained.

3. 3. Construction of P. ananatis C2603 strain (SC17ΔmdeA :: λattL-kan ^R -λattR-Pnlp8sd22-cysM)
Chromosome DNA was isolated from the C2597 strain (SC17 (0) ΔmdeA :: λattL-kan ^R -λattR-Pnlp8sd22-cysM) using the EdgeBio PurElute Bacterial Genomic kit as directed by the manufacturer. The obtained chromosomal DNA was used for transformation of P. ananatis SC17 strain (FERM BP-11091). For this purpose, P. ananatis SC17 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of the LB liquid medium, and the cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C2597 strain (preliminary example 2) was introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, they were applied onto an LB plate containing 35 mg / L kanamycin, and 34
Incubated at ° C for 16 hours. The colonies that emerged were purified in the same medium. Next, in order to confirm the substitution of the mdeA gene on the chromosome, PCR analysis was performed as described in Preliminary Example 2.
As a result, P. ananatis SC17 Δmde A :: λatt L-kan ^R -λatt R-Pnlp8sd22-cysM strain (abbreviation: C2603) was obtained.

4. Deletion of the kan gene from the C2603 strain (SC17ΔmdeA :: λattL-kan ^R -λattR-Pnlp8sd22-cysM)
The kanamycin resistance gene (kan) was deleted from the C2603 strain using the RSF (TcR) -int-xis plasmid (US20100297716 A1). RSF (TcR) -int-xis was introduced into the C2603 strain by the electroporation method, and the cells were coated on an LB medium containing tetracycline (15 mg / L) and cultured at 30 ° C., and then C2603 / RSF (TcR). Obtained -int-xis strain.

The obtained plasmid-carrying strain was purified with LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain a single colony. The single colony was then applied on medium containing 50 mg / L kanamycin and cultured at 37 ° C. overnight with shaking (250 rpm). Kanamycin-sensitive strains were applied on LB medium containing 10% sucrose (weight ratio) and 1 mM IPTG to remove the RSF (TcR) -int-xis plasmid from the strain and cultured overnight at 37 ° C. .. Tetracycline-sensitive colonies were selected and the corresponding strain was C2614.

5. P. ananatis SC17 (0) ΔmetJ :: λatt L-cat ^R -λatt R Strain construction
A P. ananatis SC17 (0) ΔmetJ :: λatt L-cat ^R -λattR strain lacking the metJ gene (SEQ ID NO: 25) was constructed by λRed-dependent integration. For this purpose, P. ananatis SC17 (0)
The strains were cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. PCR was performed using primers P17 (SEQ ID NO: 26) and P18 (SEQ ID NO: 27) using the pMW118-attL-cat-attR plasmid (Minaeva NI et al., BMC Biotechnol., 2008, 8:63) as a template, and metJ. Amplified DNA fragments of λatt L-cat ^R -λatt R having the recombinant sequence of the gene at both ends were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L chloramphenicol and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR analysis (TaKaRa Speed) was performed using primers P19 (SEQ ID NO: 28) and P20 (SEQ ID NO: 29) to confirm that the metJ gene on the chromosome was replaced with the λatt L-cat ^R -λatt R cassette. Star (R); 92 ° C for 10 seconds, 56 ° C for 10 seconds, 72 ° C for 60 seconds for 40 cycles) was carried out. As a result, P. ananatis SC17 (0) ΔmetJ :: λatt L-cat ^R -λattR strain (abbreviation: C2607) was obtained.

6. Construction of P. ananatis C2634 strain (C2614ΔmetJ :: λatt L-cat ^R -λattR)
Chromosome DNA was isolated from the C2607 strain (SC17 (0) ΔmetJ :: λatt L-cat ^R -λattR) using the EdgeBio PurElute Bacterial Genomic kit as instructed by the manufacturer. The obtained chromosomal DNA was used for transformation of C2614 strain (preliminary example 4). For this purpose, the C2614 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of the LB liquid medium, and the cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C2607 strain (preliminary example 5) was introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L chloramphenicol and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, in order to confirm the substitution of the metJ gene on the chromosome, PCR analysis was performed as described in Preliminary Example 5. As a result, P. ananatis C2614 Δmet J :: λatt L-cat ^R -λatt R strain (abbreviation: C2634) was obtained.

7. Construction of P. ananatis C2605 strain (SC17 (0) attL-kan ^R -attR-Ptac71 φ10-metA)
The promoter region of the metA gene (SEQ ID NO: 30) is the cassette λatt L-kan ^R -λatt R-Ptac71
A strain of P. ananatis SC17 (0) λatt L-kan ^R -λatt R-Ptac71 φ10-metA substituted with φ10 was constructed by λRed-dependent incorporation. For this purpose, P. ananatis SC17 (0) strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. PCR was performed using primers P21 (SEQ ID NO: 31) and P22 (SEQ ID NO: 32) using the pMW118-attL-kan-attR-Ptac71 φ10 plasmid (SEQ ID NO: 33) as a template, and the recombinant sequence of the promoter region of the metA gene was obtained at both ends. Amplified DNA fragments of λatt L-kan ^R -λatt R-Ptac71 φ10 possessed by λatt L-kan R-Ptac71 φ10 were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, primers P23 (SEQ ID NO: 34) and P24 (SEQ ID NO: 34) and P24 (SEQ ID NO: 34) and P24 (SEQ ID NO: 34) were used to confirm that the promoter region of the metaA gene on the chromosome of the SC17 (0) strain was replaced with the λatt L-kan ^R -λatt R-Ptac71 φ10 cassette. PCR analysis (TaKaRa Speed Star (R); 92 ° C. for 10 seconds, 56 ° C. for 10 seconds, 72 ° C. for 60 seconds for 40 cycles) was performed using No. 35). As a result, P. ananatis SC17 (0) λatt L-ka
n ^R -λatt R-Ptac71 φ10-met A strain (abbreviation: C2605) was obtained.

8. Construction of P. ananatis C2611 strain (SC17λattL-kan ^R -λattR-Ptac71 φ10-metA)
Chromosome DNA was isolated from the C2605 strain (SC17 (0) λatt L-kan ^R -λatt R-Ptac71 φ10-metA) (preliminary example 7) using the EdgeBio PurElute Bacterial Genomic kit as instructed by the manufacturer. The obtained chromosomal DNA was used for transformation of SC17 strain. For this purpose, P. ananatis
The SC17 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of the LB liquid medium, and the cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C2605 strain was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR analysis was performed as described in Preliminary Example 7 to confirm the substitution of the promoter region of the metaA gene on the chromosome. As a result, P. ananatis
SC17 λatt L-kan ^R -λatt R-Ptac71 φ10-met A strain (abbreviation: C2611) was obtained.

9. Construction of P. ananatis C2619 strain (C2611ΔmetJ :: λatt L-cat ^R -λattR)
Chromosome DNA was isolated from the C2607 strain (SC17 (0) ΔmetJ :: λatt L-cat ^R -λattR) (preliminary example 5) using the EdgeBio PurElute Bacterial Genomic kit as instructed by the manufacturer. The obtained chromosomal DNA was used for transformation of C2611 strain (preliminary example 8). For this purpose, the C2611 strain was cultured overnight in LB liquid medium. Then, inoculate 1 mL of the culture solution into 100 mL of the LB liquid medium.
The cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C2607 strain was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L chloramphenicol and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, in order to confirm the substitution of the metJ gene on the chromosome, PCR analysis was performed as described in Preliminary Example 5. As a result, P. ananatis C2611 Δmet J :: λatt L-cat ^R -λatt R strain (abbreviation: C2619) was obtained.

10. Selection of P. ananatis strains having a mutant allele of the metaA gene encoding feedback-resistant MetA
Inoculate the cells of C2619 strain (SC17 λatt L-kan ^R -λatt R-Ptac71 φ10-metA Δmet J :: λatt L-cat ^R -λatt R) into a 50 mL flask containing LB liquid medium so that OD600 becomes 0.05, and inoculate at 34 ° C. In 2
Incubated for hours, aeration (250 rpm). The log-growing cell culture of the same strain with an OD600 of 0.25 was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (final concentration 25 mg / L) for 20 minutes. The resulting culture was centrifuged, washed twice with fresh LB liquid medium and then seeded on M9 agar plates containing glucose (0.2%) and norleucine (600 g / L). The production ability of L-methionine was examined for the obtained mutant strain. The strain with the highest L-methionine-producing ability was selected, and the nucleotide sequence of the metA gene of that strain was determined. Sequence analysis revealed a mutation in the metaA gene that resulted in the substitution of the arginine (Arg) residue at position 34 in the amino acid sequence of wild-type MetA (SEQ ID NO: 36) with a cysteine residue (R34C mutation). The amino acid sequence of the mutant MetA protein having the R34C mutation is shown in SEQ ID NO: 38, and the base sequence of the mutant metaA gene encoding the mutant MetA protein is shown in SEQ ID NO: 37. In this way, the P. ananatis SC17 λatt L-kan ^R -λatt R-Ptac71 φ10-met A (R34C) Δmet J :: λatt L-cat ^R -λatt R strain (abbreviation: C2664) was constructed.

11. Construction of P. ananatis C2669 strain (C2634 λatt L-kan ^R -λatt R-Ptac71 φ10-met A (R34C))
Chromosome DNA was isolated from the C2664 strain (SC17 λatt L-kan ^R -λatt R-Ptac71 φ10-met A (R34 C) Δmet J :: λatt L-cat ^R -λatt R) using the EdgeBio PurElute Bacterial Genomic kit as instructed by the manufacturer. The obtained chromosomal DNA was used for transformation of C2634 strain (preliminary example 6). For this purpose, P. ananatis C2634 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of the LB liquid medium, and the cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm).
The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C2664 strain was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR analysis was performed as described in Preliminary Example 7 to confirm the substitution of the promoter region of the metaA gene on the chromosome. As a result, P. ananatis C2634 λatt L-kan ^R -λatt R-Ptac71 φ10-met A (R34C)
A strain (abbreviation: C2669) was obtained.

12. Deletion of kan and cat genes from C2669 strain (C2614ΔmetJ :: λattL-cat ^R -λattR λattL-kan ^R -λattR-Ptac71 φ10-metA (R34C))
The RSF (TcR) -int-xis plasmid was used to delete the kanamycin and chloramphenicol resistance genes (corresponding to kan and cat) from the C2669 strain (preliminary example 11). RSF (TcR) -int-xis was introduced into the C2669 strain (preliminary example 11) by the electroporation method, and the cells were applied to an LB medium containing tetracycline (15 mg / L) and cultured at 30 ° C. A C2669 / RSF (TcR) -int-xis strain was obtained.

The obtained plasmid-carrying strain was purified with LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain a single colony. The single colonies were then applied on medium containing 50 mg / L kanamycin and 35 mg / L chloramphenicol and cultured overnight at 37 ° C. with shaking (250 rpm). Strains sensitive to both kanamycin and chloramphenicol were applied on LB medium containing 10% sucrose (weight ratio) and 1 mM IPTG to remove the RSF (TcR) -int-xis plasmid from the strain. Then, the cells were cultured at 37 ° C. overnight. Tetracycline-sensitive colonies were selected and the corresponding strain was C2691.

13. Construction of P. ananatis C3208 strain (SC17 (0) λatt L-cat ^R -λatt R-Ptac71 φ10-C)
P. ananatis SC17 (0) λatt L-cat ^R -λatt R-Ptac71 φ10-C strain in which the promoter region of the PAJ_RS05335 gene (SEQ ID NO: 39; abbreviation: C gene) was replaced with the cassette λatt L-cat ^R -λatt R-Ptac71 φ10 Built by dependent embedding. For this purpose, P. ananatis SC17 (0) strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. PCR was performed using primers P25 (SEQ ID NO: 41) and P26 (SEQ ID NO: 42) using the pMW118-attL-cat-attR-Ptac71 φ10 plasmid (SEQ ID NO: 43) as a template, and the recombinant sequence of the promoter region of the C gene was obtained at both ends. Amplified DNA fragments of λatt L-cat ^R -λatt R-Ptac71 φ10 possessed by λatt L-cat R-Ptac71 φ10 were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L chloramphenicol and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, primers P27 (SEQ ID NO: 44) and P28 (SEQ ID NO: 44) and P28 (SEQ ID NO: 44) were used to confirm that the promoter region of the C gene on the chromosome of the SC17 (0) strain was replaced with the λatt L-cat ^R -λatt R-Ptac71 φ10 cassette. PCR analysis (TaKaRa Speed Star (R); 92 ° C. for 10 seconds, 56 ° C. for 10 seconds, 72 ° C. for 60 seconds for 40 cycles) was performed using No. 45). As a result, P. ananatis SC17 (0) λatt L-cat ^R -λatt R-Ptac71 φ10-C strain (abbreviation: C3208) was obtained.

14. Construction of C3293 strain (SC17 (0) ΔybhK :: λattL-cat ^R -λattR-Ptac71 φ10-CE2) Silent gene ybhK (SEQ ID NO: 46) is cassetted λattL-cat ^R -λattR-Ptac71 φ10-CE2
(This includes the PAJ_RS05335 gene (SEQ ID NO: 39; abbreviation: C gene) and the PAJ_RS05340 gene (SEQ ID NO: 40; abbreviation: E2 gene) under the control of the Ptac71 φ10 promoter). The ^R -λatt R-Ptac71 φ10-CE2 strain was constructed by λRed-dependent incorporation. For this purpose, P. ananatis SC17 (0) strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32 ° C. for 3 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. PCR was performed using the chromosomes isolated from the C3208 strain (preliminary example 13) using primers P29 (SEQ ID NO: 47) and P30 (SEQ ID NO: 48) as templates, and λattL-cat having a recombinant sequence of the ybhK gene at both ends. Amplified DNA fragments of ^R -λatt R-Ptac71 φ10-CE2 were obtained. The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by an electroporation method. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR was performed using primers P31 (SEQ ID NO: 49) and P32 (SEQ ID NO: 50) to confirm that the ybhK gene on the chromosome was replaced with the λatt L-cat ^R -λatt R-Ptac71 φ10-CE2 cassette. Analysis (TaKaRa Speed Star (R); 92 ° C for 10 seconds, 56 ° C for 10 seconds, 72 ° C for 60 seconds for 40 cycles) was performed. As a result, P. ananatis SC17 (0)
ΔybhK :: λatt L-cat ^R -λatt R-Ptac71 φ10-CE2 strain (abbreviation: C3293) was obtained.

15. Construction of C3568 strain (C2691ΔybhK :: λatt L-cat ^R -λatt R-Ptac71 φ10-CE2)
Chromosome DNA was isolated from the C3293 strain (SC17 (0) ΔybhK :: λatt L-cat ^R -λatt R-Ptac71 φ10-CE2) using the EdgeBio PurElute Bacterial Genomic kit as instructed by the manufacturer. The obtained chromosomal DNA was used for transformation of the C2691 strain. For this purpose, the C2691 strain (Preliminary Example 12) was cultured overnight in LB liquid medium. Then, 1 mL of the culture solution was inoculated into 100 mL of the LB liquid medium, and the cells were cultured at 32 ° C. for 2 hours with shaking (250 rpm). The cells were collected and washed 3 times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the C3293 strain (preliminary example 14) was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells were applied onto an LB plate containing 35 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The colonies that emerged were purified in the same medium. Next, PCR analysis was performed as described in Preliminary Example 14 to confirm the substitution of the ybhK gene on the chromosome. As a result, P. ananatis
C2691Δybh K :: λatt L-cat ^R -λatt R-Ptac71 φ10-CE2 strain (abbreviation: C3568) was obtained.

Although the present invention has been described in detail with reference to exemplary embodiments, it will be apparent to those skilled in the art that various modifications and equivalents can be employed without departing from the scope of the invention.

The method of the present invention is useful for producing L-amino acids by fermentation of bacteria.

Claims (9)

A method for producing L-amino acids
(I) Culturing L-amino acid-producing bacteria belonging to the family Enterobacteriaceae in a medium to produce and accumulate L-amino acids in the medium and / or cells of the bacteria, and (ii). Containing the recovery of L-amino acids from media and / or cells,
A method in which the bacterium has been modified to overexpress the ydiJ gene. The method of claim 1, wherein the ydiJ gene is selected from the group consisting of:
(A) Gene containing the base sequence shown in SEQ ID NO: 1 or 3;
(B) A gene encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2 or 4;
(C) A gene encoding a protein containing an amino acid sequence containing substitutions, deletions, insertions, and / or additions of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 4. A gene in which the protein has the activity of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(D) A gene encoding a protein containing an amino acid sequence having 70% or more identity with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 4, wherein the protein has the amino acid sequence shown in SEQ ID NO: 2 or 4. A gene having the activity of a protein containing: and (E) a gene containing a variant base sequence of SEQ ID NO: 1 or 3, wherein the variant base sequence is due to the degeneracy of the genetic code. The ydiJ gene is overexpressed by introducing the ydiJ gene, increasing the number of copies of the ydiJ gene, and / or modifying the expression control region of the ydiJ gene, whereby the expression of the gene is unmodified bacteria. The method of claim 1 or 2, which is enhanced as compared to. The method according to any one of claims 1 to 3, wherein the bacterium belongs to the genus Escherichia or the genus Pantoea. The method according to any one of claims 1 to 4, wherein the bacterium is Escherichia coli or Pantoea ananatis. The invention according to any one of claims 1 to 5, wherein the L-amino acid is selected from the group consisting of aromatic L-amino acids, non-aromatic L-amino acids, sulfur-containing L-amino acids, and combinations thereof. Method. The method of claim 6, wherein the aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, L-tyrosine, and combinations thereof. The non-aromatic L-amino acids are L-alanine, L-arginine, L-aspartin, L-aspartic acid, L-citrulin, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L- 60. The method described. The method according to claim 6, wherein the sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, L-cystine, and combinations thereof.