WO2020171227A1

WO2020171227A1 - METHOD FOR PRODUCING L-AMINO ACIDS USING A BACTERIUM BELONGING TO THE FAMILY Enterobacteriaceae HAVING OVEREXPRESSED ydiJ GENE

Info

Publication number: WO2020171227A1
Application number: PCT/JP2020/007203
Authority: WO
Inventors: Viktor Vasilievich Samsonov; Mikhail Yurievich Kiryukhin; Mikhail Kharisovich Ziyatdinov; Svetlana Alekseevna SAMSONOVA; Yulia Georgievna Rostova
Original assignee: Ajinomoto Co., Inc.
Priority date: 2019-02-22
Filing date: 2020-02-21
Publication date: 2020-08-27
Also published as: JP2022521544A; BR112021014194A2

Abstract

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

Description

METHOD FOR PRODUCING L-AMINO ACIDS USING A BACTERIUM BELONGING TO THE FAMILY Enterobacteriaceae HAVING OVEREXPRESSED ydiJ GENE

Field of the Invention

The present invention relates generally to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium belonging to the family Enterobacteriaceae which has been modified to overexpress ydiJ gene, so that production of L-amino acids is enhanced.

Description of the Related Art

Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acids production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Patent No. 4,278,765 A) and alteration of expression regulatory regions such as promoters, leader sequences, and/or attenuators, or others known to persons skilled in the art (see, for example, US20060216796 A1 and WO9615246 A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042 A1, EP0685555 A1 or U.S. Patent Nos. 4,346,170 A, 5,661,012 A, and 6,040,160 A).

Another method for enhancing L-amino acids production yields is to attenuate expression of a gene or several genes which are involved in degradation of the objective L-amino acid, genes which divert the precursors of the objective L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, sulfur, and phosphate fluxes, and genes encoding toxins, etc.

A YdiJ protein native to E. coli, which is encoded by a ydiJ gene, is characterized as a putative FAD-linked oxidoreductase (EcoCyc database, https://ecocyc.org/, accession ID: G6913). It was demonstrated that YdiJ is an Fe₄S₄ 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 has been previously reported that describes the effect of overexpression of ydiJ gene on production of L-amino acids by fermentation of an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae.

Disclosure of the Invention

An improved method for producing L-amino acids by fermentation of a bacterium belonging to the family Enterobacteriaceae is described herein. According to the presently disclosed subject matter, production of an L-amino acid by fermentation of a bacterium belonging to the family Enterobacteriaceae can be increased. Specifically, production of an L-amino acid by fermentation of a bacterium belonging to the family Enterobacteriaceae can be improved when the bacterium is modified to overexpress a ydiJ gene, so that the production of the L-amino acid by the modified bacterium can be enhanced.

The present invention thus provides the following.

It is an aspect of the invention to provide a method for producing an L-amino acid comprising:
(i) cultivating an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae in a culture medium to produce and accumulate the L-amino acid in the culture medium, cells of the bacterium, or both; and
(ii) collecting said L-amino acid from the culture medium, the cells, or both,
wherein said bacterium has been modified to overexpress a ydiJ gene.

It is another aspect of the invention to provide the method as described above, wherein said ydiJ gene is selected from the group consisting of:
(A) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3,
(B) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(C) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, but wherein said amino acid sequence includes substitution, deletion, insertion, and/or addition of one or several amino acid residues, and wherein said protein has an activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) a gene encoding a protein comprising an amino acid sequence having an identity of not less than 70% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 4 and having the activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, and
(E) a gene comprising a variant nucleotide sequence of SEQ ID NO: 1 or 3, wherein variant nucleotide sequence is due to the degeneracy of the genetic code.

It is another aspect of the invention to provide the method as described above, wherein said ydiJ gene is overexpressed by introducing the ydiJ gene, by increasing the copy number of the ydiJ gene, and/or by modifying an expression regulatory region of the ydiJ gene, so that the expression of said gene is enhanced as compared with a non-modified bacterium.

It is another aspect of the invention to provide the method as described above, wherein said bacterium belongs to the genus Escherichia or Pantoea.

It is another aspect of the invention to provide the method as described above, wherein said bacterium is Escherichia coli or Pantoea ananatis.

It is another aspect of the invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid, a non-aromatic L-amino acid, a sulfur-containing L-amino acid, and combinations thereof.

It is another aspect of the invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, L-tyrosine, and combinations thereof.

It is another aspect of the invention to provide the method as described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, L-valine, and combinations thereof.

It is another aspect of the invention to provide the method as described above, wherein said sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, L-cystine, and combinations thereof.

Still other objects, features, and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed description of embodiments constructed in accordance therewith.

Detailed Description of the Invention

1. Bacterium
The bacterium as described herein is an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae that has been modified to overexpress a ydiJ gene. The bacterium as described herein can be used in the method as described herein. Hence, the explanations given hereinafter to the bacterium can be similarly applied to any bacterium that can be used interchangeably or equivalently in the method as described herein.

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

Any L-amino acid-producing bacterium belonging to the family Enterobacteriaceae can be used in the method as described herein, provided that the bacterium can be modified to overexpress a ydiJ gene. For example, an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae can be used in the method as described herein, provided that the bacterium can be modified to overexpress a ydiJ gene, so that the production of an L-amino acid by the bacterium can be enhanced as compared with a non-modified bacterium.

The phrase “an L-amino acid-producing bacterium” may be used interchangeably or equivalently to the phrase “a bacterium that is able to produce an L-amino acid” or the phrase “a bacterium having an ability to produce an L-amino acid”.

The phrase “an L-amino acid-producing bacterium” can mean a bacterium belonging to the family Enterobacteriaceae which has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium and/or cells of the bacterium (also referred to as bacterial cells) when the bacterium is cultured in the medium.

The phrase “an L-amino acid-producing bacterium” can also mean a bacterium which has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium in an amount larger than a non-modified bacterium. The phrase “a non-modified bacterium” may be used interchangeably or equivalently to the phrase “a non-modified strain”. The phrase “a non-modified strain” can mean a control strain that has not been modified to overexpress a ydiJ gene. Examples of the non-modified strain can include a wild-type or parental strain such as, for example, Escherichia coli (E. coli) K-12 strains such as W3110 (ATCC 27325) and MG1655 (ATCC 47076), and Pantoea ananatis (P. ananatis) AJ13355. The phrase “an L-amino acid-producing bacterium” can also mean a bacterium that is able to cause accumulation in the medium of an amount, for example, not less than 0.1 g/L, not less than 0.5 g/L, or not less than 1.0 g/L of the objective L-amino acid. The phrase “an L-amino acid-producing bacterium” can also mean a bacterium which has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium in an amount larger than a non-modified bacterium, and is able to cause accumulation in the medium of an amount, for example, not less than 0.1 g/L, not less than 0.5 g/L, or not less than 1.0 g/L of the L-amino acid.

The bacterium may inherently have the ability to produce an L-amino acid or may be modified to have an ability to produce an L-amino acid. Such modification can be attained by using, for example, a mutation method or DNA recombination techniques. The bacterium can be obtained by overexpressing a ydiJ gene in a bacterium that inherently has the ability to produce an L-amino acid, or in a bacterium that has been already imparted with the ability to produce an L-amino acid. Alternatively, the bacterium can be obtained by imparting the ability to produce L-amino acid to a bacterium already modified to overexpress the ydiJ gene. Alternatively, the bacterium may be imparted with the ability to produce an L-amino acid by being modified to overexpress the ydiJ gene. The bacterium as described herein can be obtained, specifically, for example, by modifying a bacterial strain described hereinafter.

The phrase “an ability to produce an L-amino acid” can mean the ability of a bacterium to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium and/or cells of the bacterium when the bacterium is cultured in the medium. The phrase “an ability to produce an L-amino acid” can specifically mean the ability of a bacterium belonging to the family Enterobacteriaceae to produce, excrete or secrete, and/or cause accumulation of L-amino acid in a culture medium and/or the bacterial cells to such a level that the L-amino acid can be collected from the culture medium and/or the bacterial cells when the bacterium is cultured in the medium.

The phrase “cultured” with reference to a bacterium which can be used in the method as described herein may be used interchangeably or equivalently to the phrase “cultivated”, or the like, that are well-known to persons skilled in the art.

The bacterium can produce an L-amino acid either alone or as a mixture of the L-amino acid and one or more kinds of substances that are different from the L-amino acid. For example, the bacterium can produce an objective L-amino acid either alone or as a mixture of the objective L-amino acid and one or more kinds of amino acids that are different from the objective L-amino acid such as, for example, amino acids in L-form (also referred to as L-amino acids). In other words, it is acceptable that the bacterium can produce two or more L-amino acids as a mixture of them. Furthermore, the bacterium can produce an objective L-amino acid either alone or as a mixture of the objective L-amino acid and one or more kinds of other organic acids such as, for example, carboxylic acids.

Examples of L-amino acids include, but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

Examples of carboxylic acids include, but are not limited to, formic acid, acetic acid, citric acid, butyric acid, lactic acid, and propionic acid, and derivatives thereof.

The phrases “L-amino acid” and “carboxylic acid” can refer not only to an L-amino acid and a carboxylic acid in a free form, but can also refer to a derivative form thereof, such as a salt, a hydrate, an adduct, or a combination of them. An adduct can be a compound formed by an L-amino acid or a carboxylic acid and another organic or inorganic compound. Hence, the phrases “L-amino acid” and “carboxylic acid” can mean, for example, an L-amino acid and a carboxylic acid in a free form, a derivative form, or a mixture of these. The phrases “L-amino acid” and “carboxylic acid” can particularly mean, for example, an L-amino acid and a carboxylic acid in a free form, a salt thereof, or a mixture of these. The phrases “L-amino acid” and “carboxylic acid” can mean, for example, any of sodium, potassium, ammonium, mono-, di- and trihydrate, mono- and dichlorhydrate, and so forth salts of these. Unless otherwise stated, the phrases “L-amino acid” and “carboxylic acid” without referring to hydration state, such as the phrases “an L-amino acid or a carboxylic acid in a free form” and “a salt of an L-amino acid or a carboxylic acid”, can refer to an L-amino acid and a carboxylic acid not in a hydrate form, or can refer to a hydrate of an L-amino acid and a carboxylic acid.

An L-amino acid can belong to one or more L-amino acid families. As an example, the L-amino acid can belong to the glutamate family including L-arginine, L-glutamic acid, L-glutamine, and L-proline; the serine family including L-cysteine, glycine, and L-serine; the aspartate family including L-asparagine, L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine; the pyruvate family including L-alanine, L-isoleucine, L-valine, and L-leucine; and the aromatic family including L-phenylalanine, L-tryptophan, and L-tyrosine. As L-histidine has an aromatic moiety such as imidazole ring, the phrase “aromatic L-amino acid” can also refer to, besides the aforementioned aromatic L-amino acids, L-histidine.

An L-amino acid can also belong to the non-aromatic family, examples of which include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine. As the biosynthetic pathway of aromatic amino acids such as L-phenylalanine, L-tryptophan, and L-tyrosine is different from the biosynthetic pathway of L-histidine, the phrase “non-aromatic L-amino acid” can also refer to, besides the aforementioned non-aromatic L-amino acids, L-histidine.

Moreover, an L-amino acid can also belong to a sulfur-containing L-amino acid family, the examples of which include L-cysteine, L-methionine, L-homocysteine, and L-cystine.

As some L-amino acids can be the intermediate amino acids in a biosynthetic pathway of a particular L-amino acid, the aforementioned families of amino acids may also include other L-amino acids, for example, non-proteinogenic L-amino acids. For example, L-citrulline and L-ornithine are amino acids from the arginine biosynthetic pathway. Therefore, the glutamate family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.

Examples of the bacteria belonging to the family Enterobacteriaceae can include bacteria belonging to the genera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, and so forth. Such bacteria can have the ability to produce an L-amino acid. Specifically, bacteria classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543) can be used. Particular examples of the bacteria belonging to the family Enterobacteriaceae include bacteria belonging to the genera Escherichia, Enterobacter, and Pantoea.

Escherichia bacteria are not particularly limited, and examples thereof include those described in the work of Neidhardt et al. (Bachmann, B.J., Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In F.C. Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2^nd ed. ASM Press, Washington, D.C., 1996). The species Escherichia coli (E. coli) is a particular example of Escherichia bacteria. Specific examples of E. coli include E. coli K-12 strain, which is a prototype wild-type strain, such as E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), and so forth.

Examples of the Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteria include Pantoea ananatis (P. ananatis), and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc. A bacterium belonging to either genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into 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 derivatives thereof. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above.

These strains are available from, for example, the American Type Culture Collection (ATCC; Address: P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are assigned to the respective strains, and the strains can be ordered by using these registration numbers (refer to https://www.lgcstandards-atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection. These strains can also be obtained from, for example, the depositories at which the respective strains were deposited.

To impart or enhance an L-amino acid-producing ability, methods conventionally employed in the breeding of amino acid-producing strains of coryneform bacteria, Escherichia bacteria, and so forth (refer to “Amino Acid Fermentation”, Gakkai Shuppan Center (Ltd.), 1st Edition, published May 30, 1986, pp.77-100) can be used. Examples of such methods include, for example, acquiring an auxotrophic mutant strain, acquiring an L-amino acid analogue-resistant strain, acquiring a metabolic regulation mutant strain, and constructing a recombinant strain in which the activity of an L-amino acid biosynthetic enzyme is enhanced. In the breeding of L-amino acid-producing bacteria, one of the above-described properties such as auxotrophy, analogue resistance, and metabolic regulation mutation may be imparted alone, or two or three or more of such properties may be imparted in combination. Also, in the breeding of L-amino acid-producing bacteria, the activity of one of L-amino acid biosynthetic enzymes may be enhanced alone, or the activities of two or three or more of such enzymes may be enhanced in combination. Furthermore, imparting property(s) such as auxotrophy, analogue resistance, and metabolic regulation mutation can be combined with enhancing the activity(s) of biosynthetic enzyme(s).

An auxotrophic mutant strain, analogue-resistant strain, or metabolic regulation mutant strain having an L-amino acid-producing ability can be obtained by subjecting a parental strain or wild-type strain to a usual mutagenesis treatment, and then selecting a strain exhibiting auxotrophy, analogue resistance, or a metabolic regulation mutation, and having an L-amino acid-producing ability from the obtained mutant strains. Examples of the usual mutagenesis treatment include irradiation of X-ray or ultraviolet and a treatment with a mutation agent such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

An L-amino acid-producing ability can also be imparted or enhanced by enhancing the activity of an enzyme involved in biosynthesis of an objective L-amino acid. An enzyme activity can be enhanced by, for example, modifying a bacterium so that the expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00/18935, EP1010755A, and so forth. Methods for overexpressing a ydiJ gene described below can be similarly applied to enhancing the activity of any protein or enhancing the expression of any gene.

Furthermore, an L-amino acid-producing ability can also be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction branching away from the biosynthetic pathway of an objective L-amino acid to generate a compound other than the objective L-amino acid. The phrase “enzyme that catalyzes a reaction branching away from the biosynthetic pathway of an objective L-amino acid to generate a compound other than the objective L-amino acid” referred to herein can mean an enzyme involved in decomposition of the objective L-amino acid. Methods for attenuating expression of a gene described below can be applied to reducing the activity of any protein or attenuating the expression of any gene.

Hereinafter, L-amino acid-producing bacteria and methods for imparting or enhancing an L-amino acid-producing ability will be specifically exemplified. All of the properties of the L-amino acid-producing bacteria and modifications for imparting or enhancing an L-amino acid-producing ability may be used independently or in any appropriate combination.

<L-Arginine-producing bacteria>
Examples of L-arginine-producing bacteria and parental strains which can be used to derive L-arginine-producing bacteria include, for example, strains in which the activity or activities of one or more of the L-arginine biosynthetic enzymes are enhanced. Examples of 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 (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthetase (carAB). Shown in the parentheses after the names of the enzymes are examples of genes encoding the enzymes (the same nomenclature shall similarly apply when reciting proteins/enzymes and genes hereinafter). As the N-acetylglutamate synthase gene (argA), for example, a gene encoding a mutant N-acetylglutamate synthase desensitized to feedback inhibition by L-arginine by substitution for the amino acid residues corresponding to the positions 15 to 19 of the wild type enzyme (EP1170361A) can preferably be used.

Specific examples of L-arginine-producing bacteria and parental strains which can be used to derive L-arginine-producing bacteria include, for example, strains belonging to the genus Escherichia such as the E. coli 237 strain (VKPM B-7925, US2002-058315A1), derivative strains thereof into which the argA gene encoding a mutant N-acetyl glutamate synthase has been introduced (Russian Patent Application No. 2001112869, EP1170361A1), E. coli 382 strain derived from the 237 strain and having an improved acetic acid-assimilating ability (VKPM B-7926, EP1170358A1), and E. coli 382ilvA+ strain, which is a strain obtained from the 382 strain by introducing the wild-type ilvA gene from E. coli K-12 strain thereto. The E. coli strain 237 was deposited at the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on April 10, 2000 under an accession number of VKPM B-7925, and the deposit was converted to an international deposit under the provisions of the Budapest Treaty on May 18, 2001. The E. coli 382 strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on April 10, 2000 under an accession number of VKPM B-7926, and the deposit was converted to an international deposit under the provisions of the Budapest Treaty on May 18, 2001.

Examples of L-arginine-producing bacteria and parental strains which can be used to derive L-arginine-producing bacteria also include strains having resistance to amino acid analogues, and so forth. Examples of such strains include Escherichia coli mutant strains having resistance to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamate, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thienylalanine, or sulfaguanidine (refer to Japanese Patent Laid-open (Kokai) No. 56-106598).

<L-Citrulline-producing bacteria and L-ornithine-producing bacteria>
L-citrulline and L-ornithine are intermediates in the biosynthetic pathway of L-arginine. Hence, examples of L-citrulline- or L-ornithine-producing bacteria and parental strains which can be used to derive L-citrulline- or L-ornithine-producing bacteria include, for example, strains in which the activity or activities of one or more of the L-arginine biosynthetic enzymes are enhanced. Examples of 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 (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthetase (carAB), for L-citrulline. Furthermore, examples of 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 (argD), acetylornithine deacetylase (argE), and ornithine acetyltransferase (argJ), for L-ornithine.

Specific examples of L-citrulline-producing bacteria and parental strains which can be used to derive L-citrulline-producing bacteria include, but are not limited to strains belonging to the genus Escherichia such as E. coli strains 237/pMADS11, 237/pMADS12, and 237/pMADS13, which have a mutant N-acetylglutamate synthase (Russian Patent No. 2215783, European Patent No. 1170361 B1, U.S. Patent No. 6790647 B2), Escherichia coli strains 333 (VKPM B-8084) and 374 (VKPM B-8086), both harboring mutant feedback-resistant carbamoyl phosphate synthetase (Russian Patent No. 2264459 C2), Escherichia coli strains in which α-ketoglutarate synthase activity is increased, and ferredoxin NADP⁺ reductase, pyruvate synthase, and/or α-ketoglutarate dehydrogenase activities are additionally modified (EP2133417 A1), and strain Pantoea ananantis NA1sucAsdhA, in which succinate dehydrogenase and α-ketoglutarate dehydrogenase activities are decreased (U.S. Patent Application No. 2009286290 A1), and the like.

An L-citrulline-producing bacterium can be easily obtained from any L-arginine-producing bacterium such as, for example, E. coli 382 stain (VKPM B-7926), by inactivation of argininosuccinate synthase encoded by argG gene. Methods for inactivation of genes are described herein.

An L-ornithine-producing bacterium can be easily obtained from any L-arginine-producing bacterium such as, for example, E. coli 382 stain (VKPM B-7926), by inactivation of ornithine carbamoyltransferase encoded by argF and argI genes.

<L-Cysteine-producing bacteria>
Examples of L-cysteine-producing bacteria and parental strains which can be used to derive L-cysteine-producing bacteria include, for example, strains in which the activity or activities of one or more of the L-cysteine biosynthetic enzymes are enhanced. Examples of such enzymes include, but are not particularly limited to, serine acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA). The serine acetyltransferase activity can be enhanced by, for example, introducing a mutant cysE gene encoding a mutant serine acetyltransferase resistant to feedback inhibition by cysteine into a bacterium. Such a mutant serine acetyltransferase is disclosed in, for example, Japanese Patent Laid-open (Kokai) No. 11-155571 and US2005-0112731A. Specific examples of such a mutant serine acetyltransferase include the mutant serine acetyltransferase encoded by cysE5 gene, in which the Val residue and the Asp residue at positions 95 and 96 of a wild-type serine acetyltransferase are replaced with Arg residue and Pro residue, respectively (US2005-0112731A). Furthermore, the 3-phosphoglycerate dehydrogenase activity can be enhanced by, for example, introducing a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into a bacterium. Such a mutant 3-phosphoglycerate dehydrogenase is disclosed in, for example, U.S. Patent No. 6,180,373.

Furthermore, examples of L-cysteine-producing bacteria and parental strains which can be used to derive L-cysteine-producing bacteria include, for example, strains in which the activity or activities of one or more of enzymes that catalyze a reaction branching away from the biosynthesis pathway of L-cysteine to generate a compound other than L-cysteine are reduced. Examples of such enzymes include, for example, enzymes involved in decomposition of L-cysteine. Examples of the enzymes involved in decomposition of L-cysteine include, but are not particularly limited to, cystathionine-β-lyase (metC) (Japanese Patent Laid-open (Kokai) No. 11-155571; Chandra et al., Biochemistry, 1982, 21:3064-3069), tryptophanase (tnaA) (Japanese Patent Laid-open (Kokai) No. 2003-169668; Austin N. et al., J. Biol. Chem., 1965, 240:1211-1218), O-acetylserine sulfhydrylase B (cysM) (Japanese Patent Laid-open (Kokai) No. 2005-245311), the malY gene product (Japanese Patent Laid-open (Kokai) No. 2005-245311), the d0191 gene product of Pantoea ananatis (Japanese Patent Laid-open (Kokai) No. 2009-232844), and cysteine desulfhydrase (aecD) (Japanese Patent Laid-open (Kokai) No. 2002-233384).

Furthermore, examples of L-cysteine-producing bacteria and parental strains which can be used to derive L-cysteine-producing bacteria include, for example, strains in which the activity or activities of the L-cysteine excretory system and/or the sulfate/thiosulfate transport system are enhanced. Examples of proteins of the L-cysteine excretory system include the protein encoded by the ydeD gene (Japanese Patent Laid-open (Kokai) No. 2002-233384), the protein encoded by the yfiK gene (Japanese Patent Laid-open (Kokai) No. 2004-49237), the proteins encoded by the emrAB, emrKY, yojIH, acrEF, bcr, and cusA genes (Japanese Patent Laid-open (Kokai) No. 2005-287333), and the protein encoded by the yeaS gene (Japanese Patent Laid-open (Kokai) No. 2010-187552). Examples of the proteins of the sulfate/thiosulfate transport system include the proteins encoded by the cysPTWA gene cluster.

Specific examples of L-cysteine-producing bacteria and parental strains which can be used to derive L-cysteine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli JM15 transformed with different cysE alleles encoding feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168 B1, Russian Patent No. 2279477 C2), Escherichia coli W3110 having overexpressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663 A), Escherichia coli strains having a lowered cysteine desulfhydrase activity (JP11155571 A2), Escherichia coli W3110 having an increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307 A1), Pantoea ananatis EYPSG8 and derivatives thereof having overexpressed the genes involved in sulphur assimilation (EP2486123 B1), and the like.

<L-Glutamic acid-producing bacteria>
Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria include, but are not limited to, strains in which the activity or activities of one or more of the L-glutamic acid biosynthetic enzymes are enhanced. Examples of such genes include genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase. It is preferable to enhance the activity or activities of one or more kinds of enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenol pyruvate carboxylase, and methylcitrate synthase, among these enzymes.

Examples of strains belonging to the family Enterobacteriaceae and modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2. Furthermore, examples of strains belonging to the family Enterobacteriaceae and modified so that the expression of a gene of the Entner-Doudoroff pathway (edd, eda) is increased include those disclosed in EP1352966B.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include strains having a decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), succinate dehydrogenase (sdhABCD), and 1-pyroline-5-carboxylate dehydrogenase (putA). It is preferable to reduce or delete, for example, the α-ketoglutarate dehydrogenase activity, among these enzymes.

Bacteria belonging to the genus Escherichia deficient in the α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Specifically, 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-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucA gene”) of Escherichia coli W3110. This strain is completely deficient in the α-ketoglutarate dehydrogenase.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include Pantoea bacteria, such as the Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11091), and Pantoea ananatis SC17(0) strain (VKPM B-9246). The AJ13355 strain is a strain isolated from soil in Iwata-shi, Shizuoka-ken, Japan as a strain that can proliferate in a low pH medium containing L-glutamic acid and a carbon source. The SC17 strain is a strain selected as a low phlegm-producing mutant strain from the AJ13355 strain (U.S. Patent No. 6,596,517). The SC17 strain was deposited at the independent administrative agency, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (currently, independent administrative agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on February 4, 2009, and assigned an accession number of FERM BP-11091. The AJ13355 strain was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, NITE IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on February 19, 1998 and assigned an accession number of FERM P-16644. Then, the deposit was converted to an international deposit under the provisions of the Budapest Treaty on January 11, 1999, and assigned an accession number of FERM BP-6614. The strain SC17(0) was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on September 21, 2005 under the accession number VKPM B-9246.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include mutant strains belonging to the genus Pantoea that are deficient in the α-ketoglutarate dehydrogenase activity or have a decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356 (U.S. Patent No. 6,331,419 B1), which is an α-ketoglutarate dehydrogenase E1 subunit (sucA) gene-deficient strain of the AJ13355 strain, and Pantoea ananatis SC17sucA (U.S. Patent No. 6,596,517), which is a sucA gene-deficient strain of the SC17 strain. Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, NITE IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on February 19, 1998 under the accession number FERM P-16645. It was then converted to an international deposit under the provisions of the Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. The AJ13356 strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis. Pantoea ananatis SC17sucA was assigned a private number of AJ417, and deposited at the independent administrative agency, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (currently, NITE IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on February 26, 2004, under an accession number of FERM BP-8646.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include strains belonging to the genus Pantoea such as the Pantoea ananatis SC17sucA/RSFCPG+pSTVCB strain, Pantoea ananatis AJ13601 strain, Pantoea ananatis NP106 strain, and Pantoea ananatis NA1 strain. The SC17sucA/RSFCPG+pSTVCB strain was obtained by introducing the plasmid RSFCPG containing the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) native to Escherichia coli, and the plasmid pSTVCB containing the citrate synthase gene (gltA) native to Brevibacterium lactofermentum, into the SC17sucA strain. The AJ13601 strain is a strain selected from the SC17sucA/RSFCPG+pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at a low pH. The NP106 strain was obtained from the AJ13601 strain by curing the RSFCPG and pSTVCB plasmids. The AJ13601 strain was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, NITE IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on August 18, 1999, and assigned an accession number FERM P-17516. Then, the deposit was converted to an international deposit under the provisions of the Budapest Treaty on July 6, 2000, and assigned an accession number FERM BP-7207.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include auxotrophic mutant strains. Specific examples of auxotrophic mutant strains include, for example, E. coli VL334thrC⁺(VKPM B-8961, EP1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC and ilvA genes (U.S. Patent No. 4,278,765). E. coli VL334thrC⁺ is an L-isoleucine-auxotrophic L-glutamic acid-producing bacterium obtained by introducing a wild-type allele of the thrC gene into the VL334 strain. The wild-type allele of the thrC gene was introduced by the method of general transduction using a bacteriophage P1 grown on the wild-type E. coli K-12 strain (VKPM B-7) cells.

Examples of L-glutamic acid-producing bacteria and parental strains which can be used to derive the L-glutamic acid-producing bacteria also include strains having resistance to an aspartic acid analogue. Such strains can also be deficient in the α-ketoglutarate dehydrogenase activity. Specific examples of strains having resistance to an aspartic acid analogue and deficient in the α-ketoglutarate dehydrogenase activity include, for example, Escherichia coli AJ13199 (FERM BP-5807, U.S. Patent No. 5,908,768), Escherichia coli FFRM P-12379, which additionally has a lowered L-glutamic acid-decomposing ability (U.S. Patent No. 5,393,671), and Escherichia coli AJ13138 (FERM BP-5565, U.S. Patent No. 6,110,714).

<L-Histidine-producing bacteria>
Examples of L-histidine-producing bacteria and parental strains which can be used to derive L-histidine-producing bacteria also include strains in which the activity or activities of one or more kinds of L-histidine biosynthetic enzymes are enhanced. Examples of such enzymes include, but are not limited to, ATP phosphoribosyltransferase (hisG), phosphoribosyl-ATP pyrophosphatase (hisE), phosphoribosyl-AMP cyclohydrolase (hisI), bifunctional phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphatase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase (hisD).

It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine. Therefore, an L-histidine-producing ability can also be efficiently enhanced by, for example, introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase (Russian Patent Nos. 2,003,677 C1 and 2,119,536 C1).

Specific examples of L-histidine-producing bacteria and parental strains which can be used to derive L-histidine-producing bacteria also include, for example, strains belonging to the genus Escherichia such as Escherichia coli strain 24 (VKPM B-5945, RU2003677 C1), Escherichia coli NRRL B-12116 to B-12121 (U.S. Patent No. 4,388,405), Escherichia coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347 B1), Escherichia coli H-9341 (FERM BP-6674) (EP1085087 A2), Escherichia coli AI80/pFM201 (U.S. Patent No. 6,258,554 B1), Escherichia coli FERM-P 5038 and 5048, which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), Escherichia coli strains transformed with rht, a gene for an amino acid-export (EP1016710 A2), Escherichia coli 80 strain, which has been imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (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, Prikl. Biochim. Mikrobiol. (Russian), 2013, 49(2):149-154), and so forth.

<L-Isoleucine-producing bacteria>
Examples of L-isoleucine-producing bacteria and parental strains which can be used to derive the L-isoleucine-producing bacteria include, but are not limited to, strains in which the activity or activities of one or more of the L-isoleucine biosynthetic enzymes are enhanced. Examples of such enzymes include, but not particularly limited to, threonine deaminase and acetohydroxy acid synthase (Japanese Patent Laid-open (Kokai) No. 2-458, EP0356739A, U.S. Patent No. 5,998,178).

Specific examples of L-isoleucine-producing bacteria and parental strains which can be used to derive L-isoleucine-producing bacteria include, but are not limited to, Escherichia bacteria such as mutant strains having resistance to 6-dimethylaminopurine (JP 5-304969 A), mutant strains having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutant strains additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A).

<L-Leucine-producing bacteria>
Examples of L-leucine-producing bacteria and parental strains which can be used to derive L-leucine-producing bacteria also include strains in which the activity or activities of one or more of the L-leucine biosynthesis enzymes are enhanced. Examples of such enzymes include, but not particularly limited to, the enzymes encoded by the genes of the leuABCD operon. Furthermore, for enhancing the activity of such an enzyme, for example, the mutant leuA gene encoding an isopropyl maleate synthase desensitized to feedback inhibition by L-leucine (U.S. Patent No. 6,403,342) can be preferably used.

In addition, examples of L-leucine-producing bacteria and parental strains which can be used to derive L-leucine-producing bacteria also include strains in which the expression of one or more genes encoding proteins which excrete L-amino acid from the bacterial cell is enhanced. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Specific examples of L-leucine-producing bacteria and parental strains which can be used to derive L-leucine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)); Escherichia coli strains resistant to leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); Escherichia coli strains obtained by the gene engineering method described in WO96/06926; Escherichia coli H-9068 (JP 8-70879 A), and the like.

<L-Lysine-producing bacteria>
Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria include mutant strains belonging to the genus Escherichia and having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in the medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and so forth. Mutant strains having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment.

Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include strains in which the activity or activities of one or more kinds of L-lysine biosynthetic enzymes are enhanced. Examples of such enzymes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent 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). It is preferable to enhance the activity or activities of one or more of, for example, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and succinyl diaminopimelate deacylase, among these enzymes. In addition, L-histidine-producing bacteria and parental strains which can be used to derive L-histidine-producing bacteria may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP1170376 A1), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716 A), the ybjE gene (WO2005/073390), or combinations thereof. Since aspartokinase III (lysC) is subjected to feedback inhibition by L-lysine, a mutant lysC gene coding for an aspartokinase III desensitized to feedback inhibition by L-lysine (U.S. Patent No. 5,932,453) may be used for enhancing the activity of this enzyme. Examples of the aspartokinase III desensitized to feedback inhibition by L-lysine include aspartokinase III derived from Escherichia coli and having one or more mutations such as replacing the methionine residue at position 318 with an isoleucine residue; replacing the glycine residue at position 323 with an aspartic acid residue; and replacing the threonine residue at position 352 with an isoleucine residue (U.S. Patent Nos. 5,661,012 and 6,040,160). Furthermore, since dihydrodipicolinate synthase (dapA) is subjected to feedback inhibition by L-lysine, a mutant dapA gene coding for a dihydrodipicolinate synthase desensitized to feedback inhibition by L-lysine may be used for enhancing the activity of this enzyme. Examples of the dihydrodipicolinate synthase desensitized to feedback inhibition by L-lysine include dihydrodipicolinate synthase derived from Escherichia coli and having a mutation for replacing the histidine residue at position 118 with a tyrosine residue (U.S. Patent No. 6,040,160).

L-Lysine-producing bacteria or parental strains which can be used to derive L-lysine-producing bacteria may have a reduced or no activity of an enzyme that catalyzes a reaction which causes a branching off from the L-amino acid biosynthesis pathway and results in the production of another compound. Also, L-lysine-producing bacteria or parental strains which can be used to derive L-lysine-producing bacteria may have a reduced or no activity of an enzyme that negatively acts on L-lysine synthesis or accumulation. Examples of such enzymes include, but are not limited to, homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), and malic enzyme, and strains in which the activities of these enzymes are decreased or deleted are disclosed in WO95/23864, WO96/17930, WO2005/010175, and so forth. The lysine decarboxylase activity can be decreased or deleted by, for example, decreasing expression of both the cadA and ldcC genes encoding lysine decarboxylase. Expression of the both genes can be decreased by, for example, the method described in WO2006/078039.

Specific examples of bacterial strains useful for producing L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and E. coli VL611. In these strains, feedback inhibition of aspartokinase by L-lysine is desensitized.

Specific examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include the Escherichia coli WC196 strain (FERM BP-5252, U.S. Patent No. 5,827,698), the Escherichia coli WC196ΔcadAΔldcC strain (FERM BP-11027), also named as WC196LC, and the Escherichia coli WC196ΔcadAΔldcC/pCABD2 strain (WO2006/078039).

The WC196 strain was bred from the W3110 strain, which was derived from Escherichia coli K-12, by conferring AEC resistance to the W3110 strain (U.S. Patent No. 5,827,698). The WC196 strain was designated Escherichia coli AJ13069, deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, NITE IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on December 6, 1994, and assigned an accession number of FERM P-14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and assigned an accession number of FERM BP-5252 (U.S. Patent No. 5,827,698).

The WC196ΔcadAΔldcC strain was constructed from the WC196 strain by disrupting the cadA and ldcC genes which encode lysine decarboxylase. The WC196ΔcadAΔldcC was designated AJ110692 and deposited at the independent administrative agency, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (currently, NITE IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on October 7, 2008 as an international deposit under the accession number FERM BP-11027.

The WC196ΔcadAΔldcC/pCABD2 strain was constructed by introducing the plasmid pCABD2 containing the lysine biosynthesis genes (U.S. Patent No. 6,040,160) into the WC196ΔcadAΔldcC strain. The plasmid pCABD2 contains a mutant dapA gene derived from Escherichia coli and coding for a dihydrodipicolinate synthase (DDPS) having a mutation for desensitization to feedback inhibition by L-lysine (H118Y), a mutant lysC gene derived from Escherichia coli and coding for aspartokinase III having a mutation for desensitization to feedback inhibition by L-lysine (T352I), the dapB gene native to Escherichia coli and coding for dihydrodipicolinate reductase, and the ddh gene native to Brevibacterium lactofermentum and coding for diaminopimelate dehydrogenase.

Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include Escherichia coli AJIK01 (NITE BP-01520). The AJIK01 strain was designated Escherichia coli AJ111046, and deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NITE NPMD, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) on January 29, 2013. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on May 15, 2014, and assigned an accession number of NITE BP-01520.

<L-Methionine-producing bacteria>
Examples of L-methionine-producing bacteria and parent strains which can be used to derive L-methionine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (Patent GB2075055); and Escherichia coli strains 218 (VKPM B-8125) (RU2209248 C2) and 73 (VKPM B-8126) (RU2215782 C2) resistant to norleucine, the L-methionine analog, or the like. The strain Escherichia coli 73 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on May 14, 2001 under the accession number VKPM B-8126. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on February 1, 2002. Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine transsuccinylase and cystathionine γ-synthase (JP 2000-139471 A) can also be used as L-methionine-producing bacteria or parent strains. Another example of L-methionine-producing bacteria of the genus Escherichia and parent strains thereof that can be used to derive L-methionine-producing bacteria can be an E. coli strain that is deficient in a repressor of L-methionine biosynthesis system (MetJ) and has increased activity of intracellular homoserine transsuccinylase (MetA) (US7611873 B1), an E. coli strain in which activity of cobalamin-independent methionine synthase (MetE) is suppressed and activity of cobalamin-dependent methionine synthase (MetH) is increased (EP2861726 B1), an E. coli strain that has an ability to produce L-threonine and is transformed with vector(s) expressing threonine dehydratase (tdcB, ilvA) and, at least, O-succinylhomoserine lyase (metB), cystathionine β-lyase (metC), 5,10-methylenetetrahydrofolate reductase (metF) and serine hydroxymethyltransferase (glyA) (US7790424 B2), E. coli strain in which activity of transhydrogenase (pntAB) is enhanced (EP2633037 B1), and so forth.

L-methionine-producing bacteria may also be modified to overexpress a cysteine synthase gene.

The phrase “a cysteine synthase gene” can refer to a gene encoding a cysteine synthase. The phrase “a cysteine synthase” can refer to a protein having cysteine synthase activity (EC 2.5.1.47). Examples of the cysteine synthase gene can include a cysM gene and a cysK gene. The cysM gene may encode a cysteine synthase B that can use thiosulfate as a substrate. The cysK gene may encode a cysteine synthase A that can use sulfide as a substrate. Specific examples of the cysteine synthase gene can include the cysM gene native to P. ananatis. The nucleotide sequence of the cysM gene native to P. ananatis is shown in SEQ ID NO: 14.

L-methionine-producing bacteria may also be modified to have a mutant metA gene.

The metA gene encodes a homoserine transsuccinylase (EC 2.3.1.46). The phrase “a mutant metA gene” can refer to a gene encoding a mutant MetA protein. The phrase “a mutant MetA protein” can refer to a MetA protein having the R34C mutation, which is a mutation wherein the arginine (Arg) residue at position 34 is replaced with a cysteine (Cys) residue in the amino acid sequence of a wild-type MetA protein. The phrase “a wild-type metA gene” can refer to a gene encoding a wild-type MetA protein. The phrase “a wild-type MetA protein” can refer to a MetA protein not having the R34C mutation. Examples of the wild-type metA gene can include the metA gene native to P. ananatis and variants thereof provided that the variants do not have a mutation resulting in the R34C mutation of the encoded protein. Examples of the wild-type MetA protein can include the MetA protein native to P. ananatis and variants thereof provided that the variants do not have the R34C mutation. In other words, the mutant metA gene may be identical to any wild-type metA gene, except that the mutant metA gene has a mutation resulting in the R34C mutation of the encoded protein. Also, the mutant MetA protein may be identical to any wild-type MetA protein, except that the mutant MetA protein has the R34C mutation. The amino acid sequence of the MetA protein native to P. ananatis is shown in SEQ ID NO: 36. Specifically, an example of the amino acid sequence of a mutant MetA protein can be the amino acid sequence shown in SEQ ID NO: 38, which can be encoded by the mutant metA gene having the nucleotide sequence shown in SEQ ID NO: 37. That is, the mutant metA gene may be a gene, such as DNA, having the nucleotide sequence of SEQ ID NO: 37, and the mutant MetA protein may be a protein having the amino acid sequence of SEQ ID NO: 38. The mutant metA gene may also be a gene, such as DNA, having a variant nucleotide sequence of SEQ ID NO: 37, provided that the variant nucleotide sequence has a mutation resulting in the R34C mutation of the encoded protein. The mutant MetA protein may also be a protein having a variant amino acid sequence of SEQ ID NO: 38, provided that the variant amino acid sequence has the 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 below mentioned descriptions concerning variants of the ydiJ gene and the YdiJ protein can be applied similarly to variants of the metA gene and the MetA protein. The phrase “position 34” does not necessarily indicate an absolute position in the amino acid sequence of a wild-type MetA protein, but indicates a relative position in the wild-type MetA protein based on the amino acid sequence shown as SEQ ID NO: 36.

L-methionine-producing bacteria may also be modified to attenuate expression of a metJ gene.

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

An example of L-methionine-producing bacteria of the genus Pantoea and parent strains thereof that can be used to derive L-methionine-producing bacteria includes, but is not limited to, P. ananatis strain AJ13355 (FERM BP-6614).

<L-Phenylalanine-producing bacteria>
Examples of L-phenylalanine-producing bacteria and parental strains which can be used to derive L-phenylalanine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197), Escherichia coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. 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 (U.S. Patent No. 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), and Escherichia coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ12604 (FERM BP-3579) (EP488424 B1). Furthermore, L-phenylalanine-producing bacteria and parental strains which can be used to derive L-phenylalanine-producing bacteria also include strains belonging to the genus Escherichia and having an enhanced activity of the protein encoded by the yedA gene or the yddG gene (U.S. Patent Nos. 7,259,003 and 7,666,655).

<L-Proline-producing bacteria>
Examples of L-proline-producing bacteria and parental strains which can be used to derive L-proline-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli 702ilvA (VKPM B-8012), which is deficient in the ilvA gene and is able to produce L-proline (EP1172433 A1). Examples of L-proline-producing bacteria and parental strains which can be used to derive L-proline-producing bacteria also include strains in which the expression of one or more genes involved in L-proline biosynthesis is enhanced. Examples of such genes which can be used in L-proline-producing bacteria include the proB gene encoding glutamate kinase with desensitized feedback inhibition by L-proline (DE3127361 A1). In addition, examples of L-proline-producing bacteria and parental strains which can be used to derive L-proline-producing bacteria also include strains in which the expression of one or more genes encoding proteins responsible for excreting L-amino acid from the bacterial cell is enhanced. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia that have an ability to produce L-proline include the following Escherichia coli strains: NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russian Patent No. 2207371 C2), plasmid mutants described in DE3127361 A1, plasmid mutants described by Bloom F.R. et al. in “The 15^th Miami winter symposium”, 1983, p.34, and the like.

<L-Threonine-producing bacteria>
Examples of L-threonine-producing bacteria and parental strains which can be used to derive L-threonine-producing bacteria also include strains in which the activity or activities of one or more kinds of the L-threonine biosynthetic enzymes are enhanced. Examples of such enzymes include, but are not particularly limited to, aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase (thrC), and aspartate aminotransferase (aspartate transaminase) (aspC). Among these enzymes, it is preferable to enhance activity or activities of one or more kinds of enzymes such as aspartokinase III, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase. Any of the genes encoding the L-threonine biosynthesis enzymes can be introduced into a bacterium having a reduced ability to decompose threonine. Examples of such a strain in which threonine decomposition is suppressed include, for example, the E. coli TDH6 strain, which is deficient in the threonine dehydrogenase activity (Japanese Patent Laid-open (Kokai) No. 2001-346578).

The activities of the L-threonine biosynthesis enzymes are inhibited by the endproduct, L-threonine. Therefore, for constructing L-threonine-producing strains, it is preferred that the genes of the L-threonine biosynthesis enzymes are modified so that the enzymes are desensitized to feedback inhibition by L-threonine. The aforementioned thrA, thrB, and thrC genes constitute the threonine operon, which forms an attenuator structure. The expression of the threonine operon is inhibited by isoleucine and threonine in the culture broth and also suppressed by attenuation. Therefore, expression of the threonine operon can be enhanced by removing the leader sequence or the attenuator in the attenuation region (Lynn S.P. et al., J. Mol. Biol., 1987, 194:59-69; WO02/26993, WO2005/049808, and WO2003/097839).

The native promoter of the threonine operon is present upstream of the threonine operon, and can be replaced with a non-native promoter (WO98/04715). Also, the threonine operon may be constructed so that the threonine biosynthesis genes are expressed under control of the repressor and promoter of λ-phage (EP0593792B). Furthermore, a bacterium modified so that it is desensitized to feedback inhibition by L-threonine can also be obtained by selecting a strain resistant to α-amino-β-hydroxyisovaleric acid (AHV), which is an L-threonine analogue.

It is preferred that the expression amount of the threonine operon that is modified so as to be desensitized to feedback inhibition by L-threonine as described above is increased in a host by increasing the copy number thereof or by ligating it to a potent promoter. The copy number can be increased by introducing a plasmid containing the threonine operon into a host. The copy number can also be increased by transferring the threonine operon to the genome of a host using a transposon, Mu-phage, or the like.

Examples of methods for imparting or enhancing L-threonine-producing ability also include, for example, a method of imparting L-threonine resistance to a host, and a method of imparting L-homoserine resistance to a host. Such resistance can be imparted by, for example, enhancing the expression of a gene that imparts L-threonine resistance or a gene that imparts L-homoserine resistance. Examples of the genes that impart the above-mentioned 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 methods for imparting L-threonine resistance to a host include those described in EP0994190A and WO90/04636.

Specific examples of L-threonine-producing bacteria and parental strains which can be used to derive L-threonine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent Nos. 5,175,107 and 5,705,371), Escherichia coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No. 5,631,157), Escherichia coli NRRL-21593 (U.S. Patent No. 5,939,307), Escherichia coli FERM BP-3756 (U.S. Patent No. 5,474,918), Escherichia coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), Escherichia coli MG442 (Gusyatiner M.M. et al., Genetika (Russian), 1978, 14:947-956), Escherichia coli VL643 and VL2055 (EP1149911 A2), Escherichia coli VKPM B-5318 (EP0593792 A1), and the like.

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

The strain B-5318 is prototrophic with regard to isoleucine; and a temperature-sensitive lambda-phage C1 repressor and PR promoter replace the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under the accession number VKPM B-5318.

L-Threonine-producing bacteria or parental strains which can be used to derive L-threonine-producing bacteria can be additionally modified to enhance expression of one or more of the following genes:
- the mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine,
- the thrB gene which encodes homoserine kinase,
- the thrC gene which encodes threonine synthase,
- the rhtA gene which encodes a putative transmembrane protein of the threonine and homoserine efflux system,
- the asd gene which encodes aspartate-β-semialdehyde dehydrogenase, and
- the aspC gene which encodes aspartate aminotransferase (aspartate transaminase).

The thrA gene which encodes aspartokinase I and homoserine dehydrogenase I of Escherichia coli has been elucidated (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b0002; GenBank, accession No. NC_000913.2; nucleotide positions: 337 to 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 which encodes homoserine kinase of Escherichia coli has been elucidated (KEGG, entry No. b0003; GenBank, accession No. NC_000913.2; nucleotide positions: 2,801 to 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 which encodes threonine synthase of Escherichia coli has been elucidated (KEGG, entry No. b0004; GenBank, accession No. NC_000913.2; nucleotide positions: 3,734 to 5,020; Gene ID: 945198). The thrC gene is located between the thrB and yaaX genes on the chromosome of Escherichia coli K-12. All three genes function as a single threonine operon thrABC. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005049808 A1, WO2003097839 A1).

The mutant thrA gene which encodes aspartokinase I and homoserine dehydrogenase I resistant to feedback inhibition by L-threonine, as well as, the thrB and thrC genes can be obtained as one operon from the well-known plasmid pVIC40 which is present in the L-threonine-producing Escherichia coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.

The rhtA gene which encodes a protein of the threonine and homoserine efflux system (an inner membrane transporter) of Escherichia coli has been elucidated (KEGG, entry No. b0813; GenBank, accession No. NC_000913.2; nucleotide positions: 848,433 to 849,320, complement; Gene ID: 947045). The rhtA gene is located between the dps and ompX genes on the chromosome of Escherichia coli K-12 close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to the ybiF gene (KEGG, entry No. b0813).

The asd gene which encodes aspartate-β-semialdehyde dehydrogenase of Escherichia coli has been elucidated (KEGG, entry No. b3433; GenBank, accession No. NC_000913.2; nucleotide positions: 3,571,798 to 3,572,901, complement; Gene ID: 947939). The asd gene is located between the glgB and gntU gene on the same strand (yhgN gene on the opposite strand) on the chromosome of Escherichia coli K-12.

Also, the aspC gene which encodes aspartate aminotransferase of Escherichia coli has been elucidated (KEGG, entry No. b0928; GenBank, accession No. NC_000913.2; nucleotide positions: 983,742 to 984,932, complement; Gene ID: 945553). The aspC gene is located between the ycbL gene on the opposite strand and the ompF gene on the same strand on the chromosome of Escherichia coli K-12.

<L-Tryptophan-producing bacteria>
Examples of L-tryptophan-producing bacteria and parental strains which can be used to derive the L-tryptophan-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as Escherichia coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345), Escherichia coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373 B1), Escherichia coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614), Escherichia coli AGX17/pGX50,pACKG4-pps having an enhanced phosphoenolpyruvate-producing ability (WO97/08333, U.S. Patent No. 6,319,696 B1), and the like. Examples of L-tryptophan-producing bacteria and parental strains which can be used to derive the L-tryptophan-producing bacteria also include strains belonging to the genus Escherichia and having an enhanced activity of the protein encoded by the yedA gene or the yddG gene (U.S. Patent Application Nos. 2003148473 A1 and 2003157667 A1).

Examples of L-tryptophan-producing bacteria and parental strains which can be used to derive the L-tryptophan-producing bacteria also include strains in which one or more activities of anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, and hence, a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include Escherichia coli SV164, which harbors desensitized anthranilate synthase, and a transformant strain obtained by introducing into the Escherichia coli SV164 the plasmid pGH5 (WO94/08031 A1), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of L-tryptophan-producing bacteria and parental strains which can be used to derive the L-tryptophan-producing bacteria also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of α and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).

<L-Valine-producing bacteria>
Examples of L-valine-producing bacteria and parental strains which can be used to derive L-valine-producing bacteria include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.

Examples of L-valine-producing bacteria and parental strains for deriving L-valine-producing bacteria also include mutant strains having a mutation in aminoacyl-tRNA synthetase (U.S. Patent No. 5,658,766). Examples of such strains include Escherichia coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase. Escherichia coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on June 24, 1988 under the accession number VKPM B-4411.

Furthermore, mutant strains requiring lipoic acid for growth and/or lacking H⁺-ATPase can also be used as L-valine-producing bacteria or parental strains (WO96/06926 A1).

Examples of L-valine-producing bacteria and parent strains for deriving L-valine-producing bacteria also include Escherichia coli H81 strain (VKPM B-8066; see, for example, EP1942183 B1), Escherichia coli NRRL B-12287 and NRRL B-12288 (U.S. Patent No. 4,391,907), Escherichia coli VKPM B-4411 (U.S. Patent No. 5,658,766), Escherichia coli VKPM B-7707 (EP1016710 A2), or the like.

The genes and proteins used for breeding L-amino acid-producing bacteria may have, for example, known nucleotide sequences and amino acid sequences of the genes and proteins exemplified above, respectively. Also, the genes and proteins used for breeding L-amino acid-producing bacteria may be variants of the genes and proteins exemplified above, such as variants of genes and proteins having known nucleotide sequences and amino acid sequences, respectively, so long as the original function thereof, such as respective enzymatic activities in cases of proteins, is maintained. As for variants of genes and proteins, the descriptions concerning variants of a ydiJ gene and the encoded protein described herein can be similarly applied.

The bacterium as described herein has been modified to overexpress a ydiJ gene.

The ydiJ gene native to P. ananatis encodes a putative FAD-linked oxidoreductase YdiJ (BioCyc database, https://biocyc.org/, accession ID: PAJ_RS05850; UniParc, accession No. UPI0002FB0FA6; KEGG entry No. PAJ_1060). The ydiJ gene native 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.

The ydiJ gene native to E. coli encodes a putative FAD-linked oxidoreductase YdiJ (EcoCyc database, https://ecocyc.org/, accession ID: G6913; UniProt accession No. P77748; KEGG entry No. b1687) is located between the menI gene and the ydiK gene on the same strand on the chromosome of E. coli strain K-12. The ydiJ gene native 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, such as DNA, having the nucleotide sequence of SEQ ID NO: 1 or 3, and the YdiJ protein may be a protein having the amino acid sequence of SEQ ID NO: 2 or 4. The phrase “a gene or protein has a nucleotide or amino acid sequence” can mean that a gene or protein includes the nucleotide or amino acid sequence among a larger sequence unless otherwise stated, and can also mean that a gene or protein has only the nucleotide or amino acid sequence.

Homologues of the YdiJ protein native to different bacterial species belonging to the family Enterobacteriaceae are known, examples of which are described in Table 1.

The explanations given hereinafter to a gene having the nucleotide sequence shown in SEQ ID NO: 1 can be similarly applied to any gene that can be used interchangeably or equivalently with the gene having the nucleotide sequence shown in SEQ ID NO: 1 in the method as described herein, including a gene having the nucleotide sequence shown in SEQ ID NO: 3. Similarly, the explanations given hereinafter to a protein having the amino acid sequence shown in SEQ ID NO: 2 can be similarly applied to any protein that can be used interchangeably or equivalently with the protein having the amino acid sequence shown in SEQ ID NO: 2 in the method as described herein, including a protein having the amino acid sequence shown in SEQ ID NO: 4.

There may be some differences in DNA sequences between the genera, species or strains of the bacteria belonging to the family Enterobacteriaceae. Therefore, a ydiJ gene is not limited to the gene having the nucleotide sequence shown in SEQ ID NO: 1, but may include genes which have variant nucleotide sequences relative to SEQ ID NO: 1, and encode a YdiJ protein including the protein having the amino acid sequence shown in SEQ ID NO: 2 and variant proteins thereof. Similarly, a YdiJ protein is not limited to the protein having the amino acid sequence shown in SEQ ID NO: 2, but may include proteins having variant amino acid sequences of SEQ ID NO: 2.

The phrase “a variant nucleotide sequence” can mean a nucleotide sequence which encodes a YdiJ protein, such as a protein having the amino acid sequence shown in SEQ ID NO: 2, using any synonymous amino acid codons according to the standard genetic code table (see, for example, Lewin B., “Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Therefore, a DNA encoding a YdiJ protein having the amino acid sequence shown in SEQ ID NO: 2 can be a gene having a variant nucleotide sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code.

The phrase “a variant nucleotide sequence” can also mean a nucleotide sequence that is able to hybridize under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1 or a probe that can be prepared from the nucleotide sequence provided that it encodes a protein that maintains activity or function of the protein having the amino acid sequence shown in SEQ ID NO: 2, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein having the amino acid sequence shown in SEQ ID NO: 2. The phrase “stringent conditions” can refer to conditions under which a specific hybrid, for example, a hybrid having homology, defined as the parameter “identity” when using the computer program blastn, of not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 91%, not less than 92%, not less than 93%, not less than 94%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of 1 × SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate) at 60°C, 0.1 × SSC, 0.1% SDS at 60°C, or 0.1 × SSC, 0.1% SDS at 65°C. Duration of washing can depend on the type of membrane used for the blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham Hybond^TM-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. As the probe, a part of the sequence complementary to the sequence shown in SEQ ID NO: 1 may also be used. Such a probe can be produced by PCR (polymerase chain reaction; refer to White T.J. et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189) using oligonucleotides as primers prepared on the basis of the sequence shown in SEQ ID NO: 1 and a DNA fragment containing the nucleotide sequence as a template. The length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp. For example, when a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions after the hybridization can be, for example, 2 × SSC, 0.1% SDS at 50°C, 60°C or 65°C.

The phrase “a variant nucleotide sequence” can also mean a nucleotide sequence that encodes a variant protein.

The phrase “a variant protein” can mean a protein which has a variant amino acid sequence of SEQ ID NO: 2.

The phrase “a variant protein” can specifically mean a protein which has one or more mutations in the sequence as compared with the amino acid sequence shown in SEQ ID NO: 2, whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but which still maintains the activity or function of the protein having the amino acid sequence shown in SEQ ID NO: 2, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein having the amino acid sequence shown in SEQ ID NO: 2. The number of changes in the variant protein depends on the position of amino acid residue(s) in the three-dimensional structure of the protein or the type of amino acid residue(s). It can be, but is not strictly limited to, 1 to 300, in another example 1 to 250, in another example 1 to 200, in another example 1 to 150, in another example 1 to 100, in another example 1 to 90, in another example 1 to 80, in another example 1 to 70, in another example 1 to 60, in another example 1 to 50, in another example 1 to 40, in another example 1 to 30, in another example 1 to 20, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2. This is possible because amino acids can have high homology to one another, so that the activity or function of a protein is not affected by a change between such amino acids, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein. Therefore, the variant protein may be a protein having an amino acid sequence having a homology, defined as the parameter “identity” when using the computer program blastp, of not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 91%, not less than 92%, not less than 93%, not less than 94%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 as long as 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 is not significantly changed relative to the non-modified protein having the amino acid sequence shown in SEQ ID NO: 2. In this specification, “homology” may mean “identity”, that is the identity of amino acid residues. The sequence identity between two sequences is calculated as the ratio of residues matching in the two sequences when aligning the two sequences so as to achieve a maximum alignment with each other.

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s). The representative conservative mutation can be a conservative substitution. The conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gln and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, Ile or Leu for Val. In addition, such substitution, deletion, insertion, addition or the like of amino acid residues as mentioned above includes a naturally occurring mutation due to an individual difference of an organism to which the amino acid sequence is native.

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/are compensated by one or more secondary mutation(s) in the different position(s) of amino acids sequence so that activity or function of the variant protein is maintained, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein having the amino acid sequence shown in SEQ ID NO: 2.

The calculation of percent identity of a polypeptide can be carried out using the algorithm blastp. More specifically, the calculation of a percent identity of a polypeptide can be carried out using the algorithm blastp in the default settings of Scoring Parameters (Matrix: BLOSUM62; Gap costs: Existence = 11, Extension = 1; Compositional adjustments: Conditional compositional score matrix adjustment) provided by National Center for Biotechnology Information (NCBI). The calculation of a percent identity of a polynucleotide can be carried out using the algorithm blastn. More specifically, the calculation of a percent identity of a polynucleotide can be carried out using the algorithm blastn in the default settings of Scoring parameters (Match/Mismatch scores = 1,-2; Gap costs = Linear) provided by NCBI.

The phrase “a bacterium has been modified to overexpress a ydiJ gene” can mean that the bacterium has been modified in such a way that in the modified bacterium the total amount and/or the total activity of the corresponding gene product such as a YdiJ protein is increased as compared with (i.e. higher than), or the expression level (i.e. expression amount) of a ydiJ gene is increased as compared with (i.e. higher than), that observed for a non-modified strain, for example, a wild-type or parental strain. Examples of a non-modified strain that can serve as a reference for the above comparison can include a wild-type strain of a bacterium belonging to the family Enterobacteriaceae such as, for example, the E. coli W3110 strain (ATCC 27325), the E. coli MG1655 strain (ATCC 47076), the P. ananatis AJ13355 strain (FERM BP-6614), and so forth.

That is, the phrase “a ydiJ gene is overexpressed” can mean that the total amount and/or the total activity of the corresponding gene protein product such as a YdiJ protein is increased as compared with (i.e. higher than) that observed for a non-modified strain. The total amount and/or the total activity of the corresponding gene protein product such as a YdiJ protein can be increased by, for example, increasing (i.e. enhancing) the expression level of said gene, or increasing the activity per molecule (may be referred to as a specific activity) of the protein encoded by said gene, as compared with a non-modified strain, for example, a wild-type or parental strain. An increase in the total amount or the total activity of a protein can be measured as, for example, an increase in the amount or activity of the protein per cell, which may be an average amount or activity of the protein per cell. The bacterium can be modified so that the amount and/or the activity of the YdiJ protein per cell is increased to 150% or more, 200% or more, 300% or more, of the amount and/or the activity in a non-modified bacterial strain.

The phrase “an activity of the protein having the amino acid sequence shown in SEQ ID NO: 2” can mean the activity of a protein that can cause the catalysis of the transfer of electrons from one molecule to another molecule by utilizing flavin adenine dinucleotide (abbreviated as FAD) as a cofactor. The same shall apply to the phrase “an activity of the protein having the amino acid sequence shown in SEQ ID NO: 4”. Methods for determining the activity of a protein that can cause the catalysis of the transfer of electrons from one molecule to another by utilizing FAD as a cofactor can be exemplified by a spectrophotometric assay in which interconversion of FAD and its reduced forms such as FADH and FADH2 can be evaluated (see, for example, Flavin Adenine Dinucleotide (FAD) Assay Kit (Abcam, cat. No. ab204710).

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

The phrase “a ydiJ gene is overexpressed” can also mean that the expression level (i.e. expression amount) of a ydiJ gene is increased as compared with (i.e. higher than) that observed for a non-modified strain. Therefore, the phrase “a ydiJ gene is overexpressed” can be used interchangeably or equivalently to the phrase “expression of a ydiJ gene is enhanced or increased” or the phrase “the expression level of a ydiJ gene is enhanced or increased”. An increase in the expression level of a gene can be measured as, for example, an increase in the expression level of the gene per cell, which may be an average expression level of the gene per cell. The phrase “the expression level of a gene” or “the expression amount of a gene” can mean, for example, the amount of an expression product of a gene, such as the amount of mRNA of the gene or the amount of the protein encoded by the gene. The bacterium may be modified so that the expression level of the ydiJ gene per cell is increased to, for example, 150% or more, 200% or more, or 300% or more, of the expression level of in a non-modified strain.

Examples of methods which can be used to enhance expression of a gene such as a ydiJ gene include, but are not limited to, methods of increasing the copy number of the gene, such as the copy number of the gene in the bacterial genome (i.e. in the chromosome) and/or in the autonomously replicating vector, such as a plasmid, harbored by the bacterium. The copy number of a gene can be increased by, for example, introducing the gene into the chromosome of the bacterium and/or introducing an autonomously replicating vector containing the gene into the bacterium. Such increasing of the copy number of a gene can be carried out according to genetic engineering methods known to the person of ordinary skill in the art.

Examples of the vectors that can be used for a bacterium belonging to the family Enterobacteriaceae can include, but are not limited to, conditionally-replicated vectors such as, for example, vectors having R6K (oriRγ) origin replication such as, for example, the pAH162 vector and the like, narrow-host-range plasmids such as pMW118/119, pBR322, pUC19 and the like, or broad-host-range plasmids such as RSF1010, RP4 and the like. The ydiJ gene can also be introduced into the chromosomal DNA of a bacterium by, for example, homologous recombination, Mu-driven integration, or the like. One copy, or two or more copies of the ydiJ gene may be introduced. For example, homologous recombination can be carried out using a nucleotide sequence the multiple copies of which exist in the chromosomal DNA as a target to introduce multiple copies of the ydiJ gene into the chromosomal DNA. Examples of a nucleotide sequences multiple copies of which exist in the chromosomal DNA can include, but are not limited to, repetitive DNA, and inverted repeats present at the end of a transposable element. In addition, it is possible to incorporate a gene into a transposon and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA. A method for intrachromosomal amplification can be used to introduce multiple copies of a gene into the chromosomal DNA. By using Mu-driven transposition, more than 3 copies of the gene can be introduced into the chromosomal DNA of recipient strain in one step (Akhverdyan V.Z. et al., Biotechnol. (Russian), 2007, 3:3-20).

A gene to be introduced into the bacterium as described herein can be ligated downstream from a promoter. The promoter is not particularly limited so long as the promoter that can function in the host bacterium is chosen, and it may be a promoter native to the host bacterium, or it may be a heterologous promoter. The phrase “a promoter that can function in a host bacterium” can refer to a promoter that possesses promoter activity in a host bacterium. Specific examples of a promoter that can function in a bacterium belonging to the family Enterobacteriaceae include, but are not limited to, potent promoters exemplified below.

Examples of methods which can be used to enhance expression of a gene such as a ydiJ gene also include methods of increasing the expression level of the gene by modification of an expression regulatory region of that gene. Modification of an expression regulatory region of a gene can be employed in combination with an increase in the copy number of the gene. An expression regulatory region of a gene can be modified by, for example, replacing the native expression regulatory region of the gene with a native and/or modified foreign regulatory region(s). The phrase “an expression regulatory region” can be used interchangeably or equivalently to the phrase “an expression regulatory sequence”. When the ydiJ gene may be organized in the operon structure in combination with one or more other gene(s), the method which can be used to enhance expression of the gene also includes increasing the expression level of the operon having that gene by modification of an expression regulatory region of the operon, wherein the modification can be carried out by, for example, replacing the native expression regulatory region of the operon with a native and/or modified foreign regulatory region(s). In this method, the expression of two or more genes, including the ydiJ gene, can be enhanced at the same time.

Expression regulatory regions can be exemplified by promoters, enhancers, operators, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements (e.g., regions to which repressors or activators bind and/or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA). Such regulatory regions are described, for example, in known documents (e.g., Sambrook J., Fritsch E.F. and Maniatis T., “Molecular Cloning: A Laboratory Manual”, 2^nd ed., Cold Spring Harbor Laboratory Press (1989); Pfleger B.F. et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes, Nat. Biotechnol., 2006, 24:1027-1032; Mutalik V.K. et al., Precise and reliable gene expression via standard transcription and translation initiation elements, Nat. Methods, 2013, 10:354-360). Modifications of an expression regulatory region of a gene can be combined with increasing the copy number of the gene (see, for example, Akhverdyan V.Z. et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871; Tyo K.E.J. et al., Nature Biotechnol., 2009, 27:760-765).

The exemplary promoters suitable for enhancing expression of a ydiJ gene can be potent promoters. The phrase “a potent promoter” can refer to a promoter that is stronger than the native promoter of the ydiJ gene. Examples of potent promoters that can function in a bacterium belonging to the family Enterobacteriaceae can include, but are not limited to, the lac promoter, the trp promoter, the trc promoter, the tac promoter, the tet promoter, the araBAD promoter, the rpoH promoter, the msrA promoter, the Pm1 promoter (derived from the genus Bifidobacterium), Pnlp8 promoter (WO2012/137689), and the P_R or the P_L promoters of lambda (λ) phage. As a potent promoter, a highly active variant of an existing promoter may also be obtained by using various reporter genes. For example, by making the -35 and -10 regions in a promoter region closer to a consensus sequence, the strength of the promoter can be enhanced (WO0018935 A1). The strength of a promoter can be defined by the frequency of initiation acts of RNA synthesis. Examples of the method for evaluating the strength of a promoter and examples of strong promoters are described in the paper of Goldstein M.A. et al. (Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1995, 1:105-128) and so forth. Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of a ydiJ gene to obtain a stronger promoter function, thus resulting in the increased transcription level of the ydiJ gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site greatly affects the translation efficiency of mRNA. Hence, these portions can be examples of expression regulatory regions of a gene. For example, a 20-fold range in the expression levels was 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 copy number of a gene or the presence or absence of a gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and 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, including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), or mass spectrometry analysis of the protein samples, and the like.

Methods for manipulation with recombinant molecules of DNA and molecular cloning such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well-known to the persons skilled in the art. These methods are described, for example, in Sambrook J., Fritsch E.F. and Maniatis T., “Molecular Cloning: A Laboratory Manual”, 2^nd ed., Cold Spring Harbor Laboratory Press (1989) or Green M.R. and Sambrook J.R., “Molecular Cloning: A Laboratory Manual”, 4^th 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”, 4^th ed., Washington, DC, ASM Press (2009).

Any methods for manipulation with recombinant DNA can be used including conventional methods such as, for example, transformation, transfection, infection, conjugation, and mobilization. Transformation, transfection, infection, conjugation or mobilization of a bacterium with the DNA encoding a protein can impart to the bacterium the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization include any known methods. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells of E. coli K-12 to DNA has been reported for efficient DNA transformation and transfection (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Biol., 1970, 53:159-162). Methods of specialized and/or generalized transduction were described (Morse M.L. et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41(1):142-156; Miller J.H., Experiments in Molecular Genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor La. Press, 1972). Other methods for random and/or targeted integration of DNA into the host microorganism can be applied such as, for example, “Mu-driven integration/amplification” (Akhverdyan V.Z. et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871), “Red/ET-driven integration,” or “λRed/ET-mediated integration” (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y. et al., Nature Genet., 1998, 20:123-128). Moreover, multiple insertions of desired genes, in addition to Mu-driven replicative transposition (Akhverdyan V.Z. et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871) and chemically inducible chromosomal evolution based on recA-dependent homologous recombination, resulted in an amplification of desired genes (Tyo K.E.J. et al., Nature Biotechnol., 2009, 27:760-765), and other methods can be used that utilize different combinations of transposition, site-specific and/or homologous Red/ET-mediated recombinations, and/or P1-mediated generalized transduction (see, for example, Minaeva N. et al., BMC Biotechnology, 2008, 8:63; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93(2):815-829).

Since the nucleotide sequence of the ydiJ gene native to bacterial species such as P. ananatis and E. coli and others listed in Table 1 and the amino acid sequence of the YdiJ protein encoded by that gene have already been elucidated (see above), the gene native to such a bacterial species or a variant nucleotide sequence thereof can be obtained by cloning from the bacterial species by PCR utilizing DNA of the bacterial species and oligonucleotide primers prepared based on the nucleotide sequence of the ydiJ gene native to the bacterial species; or a mutagenesis method of treating a DNA containing the ydiJ gene, in vitro, for example, with hydroxylamine, or a mutagenesis method of treating the bacterial species harboring the ydiJ gene with ultraviolet (UV) irradiation or a mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for such treatment; or chemical synthesis as a full-length gene structure. The ydiJ genes native to any other organisms, including other bacterial species, or a variant nucleotide sequence thereof can be obtained in a similar manner.

The phrase “native to” in reference to a protein or a nucleic acid can mean that the protein or the nucleic acid is native to a particular organism such as, for example, mammals, plants, insects, bacteria, and viruses. That is, a protein or a nucleic acid native to a particular organism can mean the protein or the nucleic acid, respectively, that exists naturally in that organism. A protein or a nucleic acid native to a particular organism can be isolated from that organism and sequenced using means known to the one of ordinary skill in the art. Moreover, as the amino acid sequence or the nucleotide sequence of a protein or nucleic acid, respectively, isolated from an organism in which the protein or nucleic acid exists, can easy be determined, the phrase “native to” in reference to a protein or a nucleic acid can also refer to a protein or a nucleic acid that can be obtained using any means, for example, a genetic engineering technique, including recombinant DNA technology, or a chemical synthesis method, or the like, so long as the amino acid sequence of the protein or the nucleotide sequence of the nucleic acid thus obtained is identical to the amino acid sequence of the protein or the nucleotide sequence of the nucleic acid that exists naturally in, is expressed naturally in, and/or is produced naturally by the organism. The phrase “a protein” can refer to, but is not limited to, any of peptides, oligopeptides, polypeptides, proteins, enzymes, and so forth. The phrase “a nucleic acid” can refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and can specifically refer to, but is not limited to, any of expression regulatory sequences, including promoters, attenuators, terminators, and the like, genes, intergenic sequences, nucleotide sequences encoding signal peptides, pro-moieties of proteins, artificial amino acid sequences, and so forth. For example, a gene can particularly be DNA. Specific examples of amino acid sequences and nucleotide sequences, and homologues thereof native to various species are described herein, and these examples include a protein having the amino acid sequences shown in SEQ ID NO: 2, which is native to the bacterium of the species P. ananatis, which can be encoded by the gene having the nucleotide sequence shown in SEQ ID NO: 1.

The phrase “non-modified”, which can be used interchangeably or equivalently with the phrases “native”, “natural”, and “wild-type”, in reference to a gene (for example, “a non-modified gene”) and a protein (for example, “a non-modified protein”), can mean, respectively, a native gene and a native protein that exist naturally in, are expressed naturally in, and/or are produced naturally by an organism, specifically a non-modified strain of a bacterium. Examples of such an organism can include any organisms having the corresponding gene or protein, and specific examples thereof can include, for example, the E. coli W3110 strain, E. coli MG1655 strain, P. ananatis 13355 strain. A non-modified gene can encode a non-modified protein.

The phrase “a bacterium has been modified to attenuate expression of a gene” can mean that the bacterium has been modified in such a way that in the modified bacterium, expression of a gene is attenuated. The expression of a gene can be attenuated due to, for example, inactivation of the gene.

The phrase “a gene is inactivated” can mean that the modified gene encodes a completely inactive or non-functional protein as compared with the gene encoding a protein that has inorganic pyrophosphatase activity. It is also acceptable that the modified DNA region is unable to naturally express the gene due to deletion of a part of the gene or deletion of the entire gene, replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a reading frame shift of the gene, insertion of a drug-resistance gene and/or transcription termination signal, or modification of an expression regulatory region such as promoters, enhancers, operators, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements. Inactivation of the gene can also be performed, for example, by conventional methods such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, and/or insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., Nature Genet., 1998, 20:123-128) based on “Red/ET-driven integration” or “λRed/ET-mediated integration”.

The phrase “a bacterium has been modified to attenuate expression of a gene” can also mean that the modified bacterium contains a region operably linked to the gene, including sequences controlling gene expression such as promoters, enhancers, attenuators and transcription termination signals, ribosome-binding sites (RBSs), and other expression control elements, which is modified so that the expression level of the gene is decreased as compared with a non-modified strain; and other examples (see, for example, WO95/34672; Carrier T.A. and Keasling J.D., Biotechnol. Prog., 1999, 15:58-64). The phrase “operably linked” in reference to a gene can mean that the regulatory region(s) is/are linked to the nucleotide sequence of the gene in such a manner so that the expression of the gene can be attained (for example, enhanced, increased, constitutive, basal, antiterminated, attenuated, deregulated, decreased, or repressed expression), and/or mRNA of the gene and/or an amino acid sequence encoded by the gene (so-called expression product) can be produced as a result of expression of the gene.

The phrase “a bacterium has been modified to attenuate expression of a gene” can also mean that the bacterium has been modified in such a way that in the modified bacterium, the expression level (that is, expression amount) of a gene is attenuated as compared with a non-modified strain, for example, a wild-type or parental strain. A decrease in the expression level of a gene can be measured as, for example, a decrease in the expression level of the gene per cell, which may be an average expression level of the gene per cell. The phrase “the expression level of a gene” or “the expression amount of a gene” can mean, for example, the amount of an expression product of a gene, such as the amount of mRNA of the gene or the amount of the protein encoded by the gene. The bacterium may be modified so 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 that in a non-modified strain.

The phrase “a bacterium has been modified to attenuate expression of a gene” can also mean that the bacterium has been modified in such a way that in the modified bacterium, the total amount and/or the total activity of the corresponding gene product, that is, a Met repressor, is decreased as compared with a non-modified strain. A decrease in the total amount and/or the total activity of a protein can be measured as, for example, a decrease in the amount or activity of the protein per cell, which may be an average amount or activity of the protein per cell. The bacterium can be modified so that the amount or activity of a Met repressor per cell is decreased to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that in a non-modified strain.

Expression of a gene can also be attenuated by, specifically, for example, replacing an expression control sequence of the gene, such as a promoter on the chromosomal DNA, with a weaker one. The strength of a promoter can be defined by the frequency of initiation acts of RNA synthesis. Examples of methods for evaluating the strength of promoters are described in Goldstein M.A. et al. (Goldstein M.A. and Doi R.H., Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1995, 1:105-128), and so forth. Furthermore, it is also possible to introduce one or more nucleotide substitutions in a promoter region of the gene and thereby modify the promoter to be weakened as disclosed in WO0018935 A1. Furthermore, it is known that substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site greatly affects the translation efficiency of mRNA.

Expression of a gene can also be attenuated by, specifically, for example, inserting a transposon or an insertion sequence (IS) into the coding region of the gene (U.S. Patent No. 5,175,107) or in the region controlling gene expression, or by conventional methods such as mutagenesis with ultraviolet (UV) irradiation or nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine, NTG). Furthermore, the incorporation of a site-specific mutation can be conducted by known chromosomal editing methods based, for example, on λRed/ET-mediated recombination (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).

The bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence, without departing from the scope of the present invention.

2. Method
The method as described herein includes a method for producing an L-amino acid using the bacterium as described herein. The method for producing an L-amino acid using the bacterium as described herein can include the steps of cultivating (also called culturing) the bacterium in a culture medium to allow the L-amino acid to be produced, excreted or secreted, and/or accumulated in the culture medium, cells of the bacterium (also called bacterial cells), or both, and collecting the L-amino acid from the culture medium and/or the bacterial cells. The method may further include, optionally, the step of purifying the L-amino acid from the culture medium and/or the bacterial cells. The L-amino acid can be produced in such a form as described above. The L-amino acid can be produced particularly in a free form or as a salt thereof, or as a mixture of them. For example, sodium, potassium, ammonium, and the like salts or an inner salt such as zwitterion of the L-amino acid can be produced by the method. This is possible as amino acids can react under fermentation conditions with each other or a neutralizing agent such as an inorganic or organic acidic or alkaline substance in a typical acid-base neutralization reaction to form a salt that is the chemical feature of amino acids which is apparent to the person skilled in the art. Specifically, a monochlorhydrate salt of L-cysteine (L-cysteine × HCl) or a monochlorhydrate salt of L-cysteine monohydrate (L-cysteine × H₂O × HCl) can be produced by the method.

The cultivation of the bacterium, and collection and, optionally, purification of the L-amino acid from the medium and the like may be performed in a manner similar to the conventional fermentation methods wherein an L-amino acid is produced using a microorganism. That is, the cultivation of the bacterium, and collection and purification of the L-amino acid from the medium and the like may be performed by applying the conditions that are suitable for the cultivation of the bacterium, and appropriate for the collection and purification of an L-amino acid, which conditions are well-known to the persons of ordinary skill in the art.

The culture medium to be used is not particularly limited so long as the medium contains, at least, a carbon source, and the bacterium as described herein can proliferate in it and produce L-amino acid. The culture medium can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, a phosphorus source, inorganic ions, and other organic and inorganic components as required. As the carbon source, saccharides such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolysates of starches; alcohols such as ethanol, glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and 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 of soy bean hydrolysate; ammonia gas; aqueous ammonia; and the like can be used. Furthermore, peptone, yeast extract, meat extract, malt extract, corn steep liquor, and so forth can also be utilized. The medium may contain one or more types of these nitrogen sources. The sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, sodium thiosulphate, ammonium thiosulphate, sodium sulfide, ammonium sulfide, and the like. The medium can contain a phosphorus source in addition to the carbon source, the nitrogen source and the sulphur source. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphate polymers such as pyrophosphoric acid and so forth can be utilized. Vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, amino acids, peptone, casamino acid, yeast extract, and the like may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, iron ions, manganese ions, and so forth may be added, if necessary. As the other various organic and inorganic components, one kind of component may be used, or two or more kinds of components may be used in combination. Furthermore, when an auxotrophic mutant strain that requires an amino acid or the like for growth thereof is used, it is preferable to supplement a required nutrient to the medium.

Cultivation can be performed under the conditions suitable for cultivating a bacterium chosen for the use in the method for producing the L-amino acid. For example, the cultivation can be performed under aerobic conditions for from 16 to 72 hours or for from 16 to 24 hours, the culture temperature during cultivation can be controlled within from 30 to 45°C or within from 30 to 37°C, and the pH can be adjusted between 5 and 8 or between 6 and 7.5. The pH can be adjusted using an inorganic or organic acidic or alkaline substance such as, for example, urea, calcium carbonate, an inorganic acid, an inorganic alkali or ammonia gas.

After cultivation, the L-amino acid can be collected from the culture medium. Specifically, the L-amino acid present outside of cells can be collected from the culture medium. Also, after cultivation, the L-amino acid can be collected from cells of the bacterium. Specifically, the cells can be disrupted, a supernatant can be obtained by removing solids such as the cells and the cell-disrupted suspension (so-called cell debris), and then the L-amino acid can be collected from the supernatant. Disruption of the cells can be performed using, for example, methods that are well-known in the art, for example, ultrasonic lysis using high frequency sound waves, or the like. Removal of solids can be performed by, for example, centrifugation or membrane filtration. Collection of the L-amino acid from the culture medium or the supernatant etc. can be performed using, for example, conventional techniques such as concentration, crystallization, membrane treatment, ion-exchange chromatography, flash chromatography, thin-layer chromatography, medium or high pressure liquid chromatography, or a combination of these. These methods may be independently used, or may be used in an appropriate combination.

Examples

The present invention will be more precisely explained below with reference to the following non-limiting Examples.

Example 1. Construction of P. ananatis strain with enhanced expression of the ydiJ gene
The promoter region of the ydiJ gene (SEQ ID NO: 1) in the P. ananatis strain SC17 was substituted with the P_nlp8 promoter using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain (U.S. patent No. 8383372 B2, VKPM B-9246) was cultured overnight in an LB liquid culture medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3^rd ed.), Cold Spring Harbor Laboratory Press, 2001). Then, 1 mL of the cultured medium was inoculated into 100 mL of the LB liquid culture medium containing isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified DNA fragment having a recombinant sequence of promoter region of ydiJ gene at both termini was obtained by PCR using the plasmid DNA pMW-Km-Pnlp8 (see WO2011043485) as a template and primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6). Conditions for PCR were as follows: denaturation step for 3 min at 95°C; profile for two first cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the last 30 cycles: 20 sec at 94°C, 20 sec at 54°C, 90 sec at 72°C; final step: 5 min at 72°C. The amplified DNA fragment, about 1,6 kbp in size, was purified by agarose gel electrophoresis and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3^rd ed.), Cold Spring Harbor Laboratory Press, 2001) for 2 hours, then applied onto the LB plate containing 20 mg/L of kanamycin, and cultured at 34°C overnight until individual colonies were visible. The desired transformants were identified by PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). As a result, the P. ananatis SC17(0)-Pnlp8-ydiJ strain was obtained.

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

The P. ananatis strains EYP197(s) and EYP Pnlp8-ydiJ were each cultivated at 32°C for 18 hours in LB liquid culture medium. Then, 0.2 mL of the obtained cultures were inoculated into 2 mL of a fermentation medium shown in Table 2 in 20 × 200-mm test tubes and cultivated at 32°C for 24 hours on a rotary shaker at 250 rpm until glucose was consumed.

After cultivation, the amount of L-cysteine which accumulated in the medium was determined by the method described by Gaitonde M.K. (Biochem J., 104(2):627-633 (1967)) with some modifications as follows: 150 μL of each sample was mixed with 150 μL of 1 M H₂SO₄, incubated for 5 min at 20°C, then 700 μL H₂O was added to the mixture, 150 μL of the obtained mixture was transferred into the new vial, and 800 μL of solution A (1 M Tris-HCl pH 8.0, 5 mM dithiothreitol (DTT)) was added. The obtained mixture was incubated for 5 min at 20°C, rotated for 10 min at 13000 rpm, and then 100 μL of the mixture was transferred into a 20 × 200-mm test tube. Then, 400 μL H₂O, 500 μL ice acetic acid, and 500 μL of solution B (0.63 g ninhydrin, 10 mL ice acetic acid, 10 mL 36% HCl) were added, and the mixture was incubated for 10 min in a boiling water bath. Then 4.5 mL ethanol was added and the OD₅₆₀was determined. The concentration of cysteine was calculated using the formula: C (Cys, g/L) = 11.3 × OD₅₆₀.

The results of eight independent test-tube fermentations (as average values ± standard deviations) are shown in Table 3. As one can see from the Table 3, the modified P. ananatis EYP Pnlp8-ydiJ strain was able to accumulate a higher amount of L-cysteine as compared with the control P. ananatis EYP197(s) strain.

Example 3. Production of L-methionine by P. ananatis strain with enhanced expression of the ydiJ gene
To test the effect of the enhanced expression of ydiJ on L-methionine production, the chromosomal DNA from the strain SC17(0)-Pnlp8-ydiJ (Example 1) was isolated, and 10 μg of the DNA was used to transform P. ananatis C3568 by electroporation. The L-methionine-producing P. ananatis strain C3568 was constructed as described in Auxiliary example. The resulting transformants were plated on plates with LB agar containing kanamycin (20 mg/L), and the plates were incubated at 34°C overnight until individual colonies were visible. The desired transformants were identified by PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). As a result, the P. ananatis C3568 Pnlp8-ydiJ strain was obtained.

The P. ananatis strains C3568 and C3568 Pnlp8-ydiJ were each cultivated at 32°C for 18 hours in LB liquid culture medium. Then, 0.2 mL of the obtained cultures were inoculated into 2 mL of a fermentation medium shown in Table 4 in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker at 250 rpm until glucose was consumed.

After cultivation, the amount of L-methionine which accumulated in the medium was determined using Agilent 1260 amino-acid analyzer.

The results of four independent test-tube fermentations (as average values ± standard deviations) are shown in Table 5. As one can see from the Table 5, the modified P. ananatis C3568 Pnlp8-ydiJ strain was able to accumulate a higher amount of L-methionine as compared with the parental P. ananatis C3568 strain.

Example 4. Construction of the E. coli MG1655 strain with enhanced expression of the ydiJ gene
4.1. Construction of E. coli MG1655 Ptac-ydiJ (Km) strain
The E. coli MG1655 (ATCC 700926) /pKD46 strain is cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium is inoculated into 100 mL of an LB liquid culture medium containing isopropyl arabinose and ampicillin at final concentrations of 50 mM and 50 mg/L, respectively, and the cells are cultured at 37°C for 2 hours with shaking (250 rpm). The microbial cells are collected and washed three times with ice cold 10% glycerol to obtain competent cells. An amplified λattL-Km^R-λattR-Ptac fragment (Example 4.2 below) is purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells are cultured in the SOC culture medium for 2 hours, then cultivated for 18-24 hours at 37°C on L-agar plates containing 25 mg/L of kanamycin. Emerging colonies are refined in the same culture medium. Then, PCR reaction is carried out using the primers P5 (SEQ ID NO: 9) and P6 (SEQ ID NO: 10) to confirm that the promoter region of ydiJ gene is substituted by the fragment attL-Km^R-attR-Ptac on the chromosome. Thus, E. coli MG1655 Ptac-ydiJ (Km) strain is obtained.

4.2 Construction of fragment λattL-Km^R-λattR-Ptac
The λattL-Km^R-λattR-Ptac fragment (SEQ ID NO: 11) is constructed as follow. A PCR is carried out using the chromosome of P. ananatis SC17(0) λattL-Km^R-λattR-Ptac-lacZ strain (U.S. patent No. 9,051,591 B2) as a template and the primers P7 (SEQ ID NO: 12) and P8 (SEQ ID NO: 13), and Prime Star polymerase (Takara Bio Inc.). A reaction solution is prepared according to the composition attached to the kit, and DNA is amplified through 30 cycles at 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 1 minute per kbp. As a result, a gene fragment λattL-Km^R-λattR-Ptac (SEQ ID NO: 11) having a recombinant sequence of promoter region of ydiJ gene and regions complementary to the region adjacent to the ydiJ gene at both termini is obtained.

Example 5. Production of L-arginine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-arginine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-arginine-producing E. coli strain 382ilvA+ by P1-transduction to obtain the E. coli 382ilvA+ Ptac-ydiJ (Km) strain.

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

E. coli strains 382ilvA+ and 382ilvA+ Ptac-ydiJ (Km) are separately cultivated with shaking at 37°C for 18 hours in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium shown in Table 6 in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-arginine which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent. A spot containing arginine is cut out, L-arginine is eluted with 0.5% water solution of CdCl2, and the amount of L-arginine is estimated spectrophotometrically at 540 nm.

Example 6. Production of L-citrulline by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-citrulline production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-citrulline-producing E. coli strain 382ΔargG by P1-transduction to obtain the E. coli 382ΔargG Ptac-ydiJ (Km) strain.

The strain 382ΔargG is obtained by deletion of argG gene on the chromosome of the arginine-producing E. coli strain 382 (VKPM B-7926, EP1170358 A1) by the method initially 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):6640-6645). According to this procedure, the PCR primers homologous to both the region adjacent to the argG gene and the gene which confers antibiotic resistance in the template plasmid are constructed. The plasmid pMW118-λattL-cat-λattR (WO05/010175) is used as the template in the PCR.

E. coli strains 382ΔargG and 382ΔargG Ptac-ydiJ (Km) are separately cultivated with shaking at 37°C for 18 hours in 3 mL of nutrient broth. Then, 0.3 mL of the obtained cultures are each inoculated into 2 mL of fermentation medium shown in Table 7 in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-citrulline which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent. A spot containing citrulline is cut out, L-citrulline is eluted with 0.5% water solution of CdCl₂, and the amount of L-citrulline is estimated spectrophotometrically at 540 nm.

Example 7. Production of L-glutamic acid by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-glutamic acid production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-glutamic acid-producing E. coli strain VL334thrC⁺ (EP1172433 A1) by P1-transduction to obtain the E. coli VL334thrC⁺ Ptac-ydiJ (Km) strain.

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

E. coli strains VL334thrC⁺ and VL334thrC⁺ Ptac-ydiJ (Km) are separately cultivated for 18-24 hours at 37°C on L-agar plates. Then, one loop of the cells is transferred into 20 × 200-mm test tubes containing 2 mL of fermentation medium shown in Table 8. Cultivation is carried out at 30°C for 3 days with shaking.

After the cultivation, the amount of L-glutamic acid which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol : acetic acid : water = 4 : 1 : 1 (v/v) with subsequent staining by ninhydrin (1% solution in acetone), elution of L-glutamic acid in 50% ethanol with 0.5% CdCl₂ and further estimation of the amount of L-glutamic acid at 540 nm.

Example 8. Production of L-histidine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-histidine production, a DNA fragment from the chromosome of the obtained MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-histidine-producing E. coli strain 80 by P1-transduction to obtain the E. coli 80 Ptac-ydiJ (Km) strain.

The strain 80 was described in Russian Patent No. 2119536 C1 and deposited in the Russian National Collection of Industrial Microorganisms (VKPM; 1^st Dorozhny proezd., 1, Moscow 117545, Russian Federation) on October 15, 1999 under the accession number VKPM B-7270 and then converted to an international deposit under the provisions of the Budapest Treaty on July 12, 2004.

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

After the cultivation, the amount of L-histidine which accumulates in the medium is determined by thin layer chromatography (TLC). The 10 × 15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing non-fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russian Federation) are used. The Sorbfil plates are developed with a mobile phase consisting of propan-2-ol : acetone : 25% aqueous ammonia : water = 6 : 6 : 1.5 : 1 (v/v). A solution of ninhydrin (2%, w/v) in acetone is used as a visualizing reagent. After development, plates are dried and scanned with the Camag TLC Scanner 3 in absorbance mode with detection at 520 nm using winCATS software (version 1.4.2).

Example 9. Production of L-leucine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-leucine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-leucine-producing E. coli strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121) by P1-transduction to obtain the E. coli 57 Ptac-ydiJ (Km) strain.

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

E. coli strains 57 and 57 Ptac-ydiJ (Km) are separately cultivated for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains are each grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20 × 200-mm test tubes containing 2 mL of L-broth supplemented with sucrose (4%). Then, the fermentation medium shown in Table 10 is inoculated with 0.2 mL of seed material (10%). The fermentation is performed in 2 mL of a minimal fermentation medium in 20 × 200-mm test tubes. Cells are grown for 48-72 hours at 32°C with shaking at 250 rpm.

After the cultivation, the amount of L-leucine which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol : acetic acid : water = 4 : 1 : 1 (v/v).

Example 10. Production of L-lysine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-lysine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-lysine-producing E. coli strain WC196LC/pCABD2 by P1-transduction to obtain the E. coli WC196LC Ptac-ydiJ (Km)/pCABD2 strain.

The E. coli WC196LC lysine-producing strain is constructed as it is described in EP 2083083 A1. The WC196LC strain is transformed with a pCABD2 plasmid for lysine production (International Patent Publications WO95/16042 and WO01/53459), which carries dapA, dapB, lysC and ddh genes, by a conventional method, to thereby yield WC196LC/pCABD2 strain.

The plasmid pCABD2 contains a mutant dapA gene derived from E. coli and encoding dihydrodipicolinate synthase that has a mutation for desensitization to the feedback inhibition by L-lysine, a mutant lysC gene derived from E. coli and encoding aspartokinase III that has a mutation for desensitization to the feedback inhibition by L-lysine, the dapB gene native to E. coli and encoding dihydrodipicolinate reductase, and the ddh gene native to Brevibacterium lactofermentum and encoding diaminopimelate dehydrogenase (International Patent Publications WO95/16042 and WO01/53459).

E. coli strains WC196LC/pCABD2 and WC196LC Ptac-ydiJ (Km)/pCABD2 are separately cultivated in L-medium containing streptomycin (20 mg/L) at 37°C.

Then, 0.3 mL of each of the obtained cultures are each inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 34°C for 48 hours on a rotary shaker (240 rpm). The composition of the fermentation medium is the same as described in Example 5 with the addition of 30 mg/L streptomycin.

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

Example 11. Production of L-ornithine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-ornithine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-ornithine-producing E. coli strain 382ΔargFΔargI by P1-transduction to obtain the E. coli 382ΔargFΔargI Ptac-ydiJ (Km) strain.

The strain 382ΔargFΔargI is obtained by consecutive deletion of argF and argI genes on the chromosome of the arginine-producing E. coli strain 382 (VKPM B-7926, EP1170358 A1) by the method initially 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):6640-6645). According to this procedure, two pairs of PCR primers homologous to both the region adjacent to the argF or argI gene and the gene which confers antibiotic resistance in the template plasmid are constructed. The plasmid pMW118-λattL-cat-λattR (WO05/010175) is used as the template in the PCR.

E. coli strains 382ΔargFΔargI and 382ΔargFΔargI Ptac-ydiJ (Km) are separately cultivated with shaking at 37°C for 18 hours in 3 mL of nutrient broth. Then, 0.3 mL of the obtained cultures are each inoculated into 2 mL of fermentation medium shown in Table 11 in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-ornithine which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent. A spot containing ornithine is cut out, ornithine is eluted with 0.5% water solution of CdCl₂, and the amount of ornithine is estimated spectrophotometrically at 540 nm.

Example 12. Production of L-phenylalanine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-phenylalanine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-phenylalanine-producing E. coli strain AJ12739 by P1-transduction to obtain the E. coli AJ12739 Ptac-ydiJ (Km) strain.

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

E. coli strains AJ12739 and AJ12739 Ptac-ydiJ (Km) are separately cultivated at 37°C for 18 hours in a nutrient broth. Then, 0.3 mL of the obtained cultures are each inoculated into 3 mL of fermentation medium shown in Table 12 in 20 × 200-mm test tubes and cultivated at 37°C for 48 hours with shaking on a rotary shaker.

After the cultivation, the amount of L-phenylalanine which accumulates in the medium is determined by thin layer chromatography (TLC). The 10 × 15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing non-fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russian Federation) are used. The Sorbfil plates are developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent.

Example 13. Production of L-proline by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-proline production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-proline-producing E. coli strain 702ilvA by P1-transduction to obtain the E. coli 702ilvA Ptac-ydiJ (Km) strain.

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

E. coli strains 702ilvA and 702ilvA Ptac-ydiJ (Km) are separately cultivated for 18-24 hours at 37°C on L-agar plates. Then, these strains are each cultivated 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 the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-tryptophan production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L- tryptophan-producing E. coli strain SV164(pGH5) by P1-transduction to obtain the E. coli SV164(pGH5) Ptac-ydiJ (Km) strain.

The strain SV164(pGH5) is a strain obtained by introducing the plasmid pGH5 into the E. coli strain SV164. The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The strain SV164 is a strain obtained by introducing a mutation into the trpE gene in the E. coli strain YMC9 (ATCC 33927). The strain YMC9 is available from the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20108, United States of America).The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SV164(pGH5) was described in detail in U.S. Patent No. 6,180,373 B1 or EP0662143 B1.

E. coli strains SV164(pGH5) and SV164(pGH5) Ptac-ydiJ (Km) are separately cultivated with shaking at 37°C for 18 hours in 3 mL of nutrient broth supplemented with tetracycline (20 mg/L, marker of pGH5 plasmid). Then, 0.3 mL of the obtained cultures are each inoculated into 3 mL of a fermentation medium containing tetracycline (20 mg/L) in 20 × 200-mm test tubes, and cultivated at 37°C for 48 hours on a rotary shaker at 250 rpm.

After the cultivation, the amount of L-tryptophan which accumulates in the medium is determined by TLC as described in Example 12 (production of L-phenylalanine). The fermentation medium components are listed in Table 13, but should be sterilized in separate groups (A, B, C, D, E, F, G, and H), as shown, to avoid adverse interactions during sterilization.

Example 15. Production of L-valine by E. coli strain with enhanced expression of the ydiJ gene
To test the effect from enhanced expression of the ydiJ gene on L-valine production, a DNA fragment from the chromosome of the strain MG1655 Ptac-ydiJ (Km) (Example 4) is transferred to the L-valine-producing E. coli strain H81 by P1-transduction to obtain the E. coli H81 Ptac-ydiJ (Km) strain.

The strain H81 was deposited in 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, and transferred from the original deposit to international deposit based on Budapest Treaty on February 1, 2002.

E. coli strains H81 and H81 Ptac-ydiJ (Km) are separately cultivated at 37°C for 18 hours in a nutrient broth. The obtained cultures (0.1 mL each) are inoculated into 2 mL of a fermentation medium shown in Table 14 in a 20 × 200-mm test tubes, and cultivated at 32°C for 72 hours with a rotary shaker at 250 rpm.

After the cultivation, the amount of L-valine which accumulates in the medium is measured by TLC. The 10 × 15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing non-fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russian Federation) are used. The Sorbfil plates are developed with a mobile phase consisting of propan-2-ol: ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent.

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

The strain B-3996Δtdh is obtained by deleting the tdh gene on the chromosome of E. coli B-3996 (U.S. Patent Nos. 5,175,107 and 5,705,371) using the method of λRed/ET-mediated integration (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).

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

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

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

2. Construction of P. ananatis C2597 strain (SC17(0)ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM)
The P. ananatis SC17(0)ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM strain having replaced silent gene mdeA (SEQ ID NO: 20) with cassette λattL-kan^R-λattR-Pnlp8sd22-cysM was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium containing IPTG at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified λattL-kan^R-λattR-Pnlp8sd22-cysM DNA fragment having a recombinant sequence of mdeA gene at both termini was obtained by PCR using the primers P13 (SEQ ID NO: 21) and P14 (SEQ ID NO: 22), and chromosome isolated from the strain C2338 (Auxiliary example, 1) as a template. The resulting DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis (TaKaRa Speed Star(R); 40 cycles at 92°C for 10 seconds, 56°C for 10 seconds and 72°C for 60 seconds) 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 λattL-kan^R-λattR-Pnlp8sd22-cysM cassette. As a result, the P. ananatis SC17(0)ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM strain (abbreviated as C2597) was obtained.

3. Construction of P. ananatis C2603 strain (SC17ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM)
Chromosomal DNA was isolated from the strain C2597 (SC17(0) ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of P. ananatis SC17 strain (FERM BP-11091). For this purpose, P. ananatis SC17 strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C2597 (Auxiliary example, 2) was introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 2 to confirm the replacement of mdeA gene. As a result, the P. ananatis SC17ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM strain (abbreviated as C2603) was obtained.

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

The resulting plasmid-harboring strain was refined in the LB culture medium containing 15 mg/L of tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L of kanamycin and cultured at 37°C overnight with shaking (250 rpm). The kanamycin-sensitive strain was applied onto the LB culture medium containing 10% sucrose (by weight) and 1 mM IPTG and cultured at 37°C overnight in order to delete the RSF(TcR)-int-xis plasmid from the strain. A colony that was sensitive to tetracycline was selected, and the corresponding strain was designated as C2614.

5. Construction of P. ananatis SC17(0)ΔmetJ::λattL-cat^R-λattR strain
The P. ananatis SC17(0)ΔmetJ::λattL-cat^R-λattR strain having the metJ gene deleted (SEQ ID NO: 25) was constructed using λRed-dependent integration. For this purpose P. ananatis SC17(0) strain was cultured in the LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated into 100 mL of the LB liquid culture medium containing IPTG at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified λattL-cat^R-λattR DNA fragment having a recombinant sequence of metJ gene at both termini was obtained by PCR using the primers P17 (SEQ ID NO: 26) and P18 (SEQ ID NO: 27), and pMW118-attL-cat-attR plasmid (Minaeva N.I. et al., BMC Biotechnol., 2008, 8:63) as a template. The resulting DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of chloramphenicol, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis (TaKaRa Speed Star(R); 40 cycles at 92°C for 10 seconds, 56°C for 10 seconds and 72°C for 60 seconds) 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 λattL-cat^R-λattR cassette. As a result, the P. ananatis SC17(0)ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2607) was obtained.

6. Construction of P. ananatis C2634 strain (C2614ΔmetJ::λattL-cat^R-λattR)
Chromosomal DNA was isolated from the strain C2607 (SC17(0)ΔmetJ::λattL-cat^R-λattR) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of C2614 strain (Auxiliary example, 4). For this purpose, P. ananatis C2614 strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C2607 (Auxiliary example, 5) was introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of chloramphenicol, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 5 to confirm the replacement of metJ gene. As a result, the P. ananatis C2614ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2634) was obtained.

7. Construction of P. ananatis C2605 strain (SC17(0)attL-kan^R-attR-Ptac71φ10-metA)
The P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metA strain having the promoter region of metA gene (SEQ ID NO: 30) replaced with cassette λattL-kan^R-λattR-Ptac71φ10 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium containing IPTG at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified λattL-kan^R-λattR-Ptac71φ10 DNA fragment having a recombinant sequence of promoter region of metA gene at both termini was obtained by PCR using the primers P21 (SEQ ID NO: 31) and P22 (SEQ ID NO: 32), and pMW118-attL-kan-attR-Ptac71φ10 plasmid (SEQ ID NO: 33) as a template. The resulting DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis (TaKaRa Speed Star(R); 40 cycles at 92°C for 10 seconds, 56°C for 10 seconds and 72°C for 60 seconds) was performed using primers P23 (SEQ ID NO: 34) and P24 (SEQ ID NO: 35) to confirm that the promoter region of metA gene on the chromosome of the strain SC17(0) was replaced with the λattL-kan^R-λattR-Ptac71φ10 cassette. As a result, the P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metA strain (abbreviated as C2605) was obtained.

8. Construction of P. ananatis C2611 strain (SC17λattL-kan^R-λattR-Ptac71φ10-metA)
Chromosomal DNA was isolated from the strain C2605 (SC17(0)λattL-kan^R-λattR-Ptac71φ10-metA) (Auxiliary example, 7) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of SC17 strain. For this purpose, P. ananatis SC17 strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C2605 introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 7 to confirm the replacement of the promoter region of metA gene. As a result, the P. ananatis SC17λattL-kan^R-λattR-Ptac71φ10-metA strain (abbreviated as C2611) was obtained.

9. Construction of P. ananatis C2619 strain (C2611ΔmetJ::λattL-cat^R-λattR)
Chromosomal DNA was isolated from the strain C2607 (SC17(0)ΔmetJ::λattL-cat^R-λattR) (Auxiliary example, 5) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of C2611 strain (Auxiliary example, 8). For this purpose, P. ananatis C2611 strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C2607 introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of chloramphenicol, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 5 to confirm that the replacement of metJ gene. As a result, the P. ananatis C2611ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2619) was obtained.

10. Selection of P. ananatis strain having the mutant allele of metA gene encoding feedback resistant MetA
The cells of C2619 strain (SC17λattL-kan^R-λattR-Ptac71φ10-metA ΔmetJ::λattL-cat^R-λattR) were inoculated into 50 mL-flask containing an LB liquid culture medium up to OD₆₀₀ of 0.05 and cultured with aeration (250 rpm) at 34°C for 2 hours. The exponentially growing cell culture of the strain at OD₆₀₀ of 0.25 was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (final concentration 25 mg/L) for 20 minutes. The obtained culture was centrifuged, washed two times with fresh LB liquid culture medium and spread onto M9-agarized plate containing glucose (0.2%) and norleucine (600 g/L). The obtained mutant strains were tested for the ability to produce L-methionine. The strain having the highest ability to produce L-methionine was selected, and the nucleotide sequence of metA gene in that strain was determined. The sequence analysis revealed the mutation in the metA gene resulting in the replacement of the arginine (Arg) residue at position 34 with cysteine residue (R34C mutation) in the amino acid sequence of the wild-type MetA (SEQ ID NO:36). The amino acid sequence of the mutant MetA protein having the R34C mutation is shown in SEQ ID NO: 38, and the nucleotide sequence of the mutant metA gene encoding the mutant MetA protein is shown in SEQ ID NO: 37. Thus, the P. ananatis SC17λattL-kan^R-λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2664) was constructed.

11. Construction of P. ananatis C2669 strain (C2634λattL-kan^R-λattR-Ptac71φ10-metA(R34C))
Chromosomal DNA was isolated from the strain C2664 (SC17λattL-kan^R-λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat^R-λattR) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of C2634 strain (Auxiliary example, 6). For this purpose, P. ananatis C2634 strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C2664 introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 7 to confirm the replacement of the promoter region of metA gene. As a result, the P. ananatis C2634λattL-kan^R-λattR-Ptac71φ10-metA(R34C) strain (abbreviated as C2669) was obtained.

12. Deletion of kan and cat genes from C2669 strain (C2614ΔmetJ::λattL-cat^R-λattR λattL-kan^R-λattR-Ptac71φ10-metA(R34C))
The kanamycin and chloramphenicol resistant genes (kan and cat, correspondingly) were deleted from C2669 strain (Auxiliary example, 11) using an RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into C2669 strain (Auxiliary example, 11) by an electroporation method, and the cells were applied onto LB culture medium containing tetracycline (15 mg/L) and cultured at 30°C to obtain C2669/RSF(TcR)-int-xis strain.

The resulting plasmid-harboring strain was refined in the LB culture medium containing 15 mg/L of tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L of kanamycin and 35 mg/L chloramphenicol and cultured at 37°C overnight with shaking (250 rpm). Strains sensitive to both kanamycin and chloramphenicol were applied onto the LB culture medium containing 10% sucrose (by weight) and 1 mM IPTG and cultured at 37°C overnight in order to delete the RSF(TcR)-int-xis plasmid from the strain. A colony that was sensitive to tetracycline was selected, and the corresponding strain was designated as C2691.

13. Construction of P. ananatis C3208 strain (SC17(0)λattL-cat^R-λattR-Ptac71φ10-C)
The P. ananatis SC17(0)λattL-cat^R-λattR-Ptac71φ10-C strain having a promoter region of PAJ_RS05335 gene (SEQ ID NO: 39; abbreviated as C gene) replaced with cassette λattL-cat^R-λattR-Ptac71φ10 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium containing IPTG at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified λattL-cat^R-λattR-Ptac71φ10 DNA fragment having a recombinant sequence of promoter region of C gene at both termini was obtained by PCR using the primers P25 (SEQ ID NO: 41) and P26 (SEQ ID NO: 42), and pMW118-attL-cat-attR-Ptac71φ10 (SEQ ID NO: 43) plasmid as a template. The resulting DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of chloramphenicol, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis (TaKaRa Speed Star(R); 40 cycles at 92°C for 10 seconds, 56°C for 10 seconds and 72°C for 60 seconds) was performed using primers P27 (SEQ ID NO: 44) and P28 (SEQ ID NO: 45) to confirm that the promoter region of C gene on the chromosome of the strain SC17(0) was replaced with the λattL-cat^R-λattR-Ptac71φ10 cassette. As a result, the P. ananatis SC17(0)λattL-cat^R-λattR-Ptac71φ10-C strain (abbreviated as C3208) was obtained.

14. Construction of C3293 strain (SC17(0)ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2)
The P. ananatis ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2 strain having a silent gene ybhK (SEQ ID NO: 46) replaced with cassette λattL-cat^R-λattR-Ptac71φ10-CE2 which contains the genes PAJ_RS05335 (SEQ ID NO: 39; abbreviated as C gene) and PAJ_RS05340 (SEQ ID NO: 40; abbreviated as E2 gene) under the control of a Ptac71φ10 promoter was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium containing IPTG at final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. An amplified λattL-cat^R-λattR-Ptac71φ10-CE2 DNA fragment having a recombinant sequence of ybhK gene at both termini was obtained by PCR using the primers P29 (SEQ ID NO: 47) and P30 (SEQ ID NO: 48), and chromosome isolated from the strain C3208 (Auxiliary example, 13) as a template. The resulting DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis (TaKaRa Speed Star(R); 40 cycles at 92°C for 10 seconds, 56°C for 10 seconds and 72°C for 60 seconds) 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 λattL-cat^R-λattR-Ptac71φ10-CE2 cassette. As a result, the P. ananatis SC17(0)ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2 strain (abbreviated as C3293) was obtained.

15. Construction of C3568 strain (C2691ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2)
Chromosomal DNA was isolated from the strain C3293 (SC17(0)ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosomal DNA was used for transformation of C2691 strain. For this purpose P. ananatis C2691 strain (Auxiliary example, 12) was cultured in an LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 100 mL of an LB liquid culture medium and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The microbial cells were collected and washed three times with 10% glycerol to obtain competent cells. Chromosomal DNA isolated from the strain C3293 (Auxiliary example, 14) introduced into the competent cells using an electroporation method. The cells were cultured in the SOC culture medium for 2 hours, then applied onto the LB plate containing 35 mg/L of kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed as described in Auxiliary example, 14 to confirm the replacement of ybhK gene. As a result, the P. ananatis C2691ΔybhK::λattL-cat^R-λattR-Ptac71φ10-CE2 strain (abbreviated as C3568) was obtained.

While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to the one of ordinary skill in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention.

The method of the present invention is useful for the production of L-amino acids by fermentation of a bacterium.

Claims

A method for producing an L-amino acid comprising:
(i) cultivating an L-amino acid-producing bacterium belonging to the family Enterobacteriaceae in a culture medium to produce and accumulate the L-amino acid in the culture medium, cells of the bacterium, or both; and
(ii) collecting said L-amino acid from the culture medium, the cells, or both,
wherein said bacterium has been modified to overexpress a ydiJ gene.
The method according to claim 1, wherein said ydiJ gene is selected from the group consisting of:
(A) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3,
(B) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(C) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, but wherein said amino acid sequence includes substitution, deletion, insertion, and/or addition of one or several amino acid residues, and wherein said protein has an activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4,
(D) a gene encoding a protein comprising an amino acid sequence having an identity of not less than 70% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 4 and having the activity of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, and
(E) a gene comprising a variant nucleotide sequence of SEQ ID NO: 1 or 3, wherein the variant nucleotide sequence is due to the degeneracy of the genetic code.
The method according to claim 1 or 2, wherein said ydiJ gene is overexpressed by introducing the ydiJ gene, by increasing the copy number of the ydiJ gene, and/or by modifying an expression regulatory region of the ydiJ gene, so that the expression of said gene is enhanced as compared with a non-modified bacterium.
The method according to any one of claims 1 to 3, wherein said bacterium belongs to the genus Escherichia or Pantoea.
The method according to any one of claims 1 to 4, wherein said bacterium is Escherichia coli or Pantoea ananatis.
The method according to any one of claims 1 to 5, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid, a non-aromatic L-amino acid, a sulfur-containing L-amino acid, and combinations thereof.
The method according to claim 6, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, L-tyrosine, and combinations thereof.
The method according to claim 6, wherein said non-aromatic L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, L-valine, and combinations thereof.
The method according to claim 6, wherein said sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, L-cystine, and combinations thereof.