WO2021060438A1

WO2021060438A1 - Method for producing l-amino acids by bacterial fermentation

Info

Publication number: WO2021060438A1
Application number: PCT/JP2020/036193
Authority: WO
Inventors: Mikhail Kharisovich Ziyatdinov; Andrey Olegovich Lobanov
Original assignee: Ajinomoto Co., Inc.
Priority date: 2019-09-25
Filing date: 2020-09-25
Publication date: 2021-04-01
Also published as: JP2022550084A

Abstract

The present invention provides a method for producing L-amino acids by fermentation using a bacterium belonging to the order Enterobacterales which has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity (ipdC) and/or a gene encoding a protein having threonine deaminase activity (ilvA).

Description

METHOD FOR PRODUCING L-AMINO ACIDS BY BACTERIAL FERMENTATION

Field of the Invention

The present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium belonging to the order Enterobacterales which has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity, 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, US4278765 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 US4346170 A, US5661012 A, and US6040160 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.

An ipdC gene encodes an IpdC protein which was characterized as indolepyruvate decarboxylase (Enzyme Commission (EC) number: 4.1.1.74). It was demonstrated that indolepyruvate decarboxylase (IpdC) takes part in biosynthesis of indole-3-acetic acid from tryptophan via indole-3-pyruvic acid and indole-3-acetaldehyde in various bacteria including Pantoea strains (Brandl M.T. and Lindow S.E. Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola, Appl. Environ. Microbiol., 1996, 62(11):4121-4128; Estenson K. et al., Characterization of indole-3-acetic acid biosynthesis and the effects of this phytohormone on the proteome of the plant-associated microbe Pantoea sp. YR343, J. Proteome Res., 2018, 17(4):1361-1374).

However, no data has been previously reported that describes the effect of attenuation of expression of ipdC gene on production of L-amino acids by fermentation of an L-amino acid-producing bacterium belonging to the order Enterobacterales.

Disclosure of the Invention

An improved method of producing L-amino acids by fermentation of a bacterium belonging to the order Enterobacterales is described herein. According to the presently disclosed subject matter, production of an L-amino acid by fermentation of a bacterium belonging to the order Enterobacterales can be increased. Specifically, production of an L-amino acid by fermentation of a bacterium belonging to the order Enterobacterales can be improved by attenuating expression of a gene encoding a protein having indolepyruvate decarboxylase activity, so that the production of the L-amino acid by the modified bacterium can be enhanced. The production of the L-amino acid by fermentation of the modified bacterium can be improved further when the bacterium is modified further to attenuate expression of a gene encoding a protein having threonine deaminase activity.

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 in a culture medium an L-amino acid producing bacterium belonging to the order Enterobacterales to produce and accumulate the L-amino acid in the culture medium or cells of the bacterium, or both, and
(ii) collecting the L-amino acid from the culture medium or the cells of the bacterium, or both,
wherein said bacterium has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity and/or a gene encoding a protein having threonine deaminase activity.

It is another aspect of the invention to provide a method for producing an L-amino acid comprising:
(i) cultivating in a culture medium an L-amino acid producing bacterium belonging to the order Enterobacterales to produce and accumulate the L-amino acid in the culture medium or cells of the bacterium, or both, and
(ii) collecting the L-amino acid from the culture medium or the cells of the bacterium, or both,
wherein said bacterium has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity.

It is another aspect of the invention to provide the method as described above, wherein said gene encoding a protein having indolepyruvate decarboxylase activity is an ipdC gene.

It is another aspect of the invention to provide the method as described above, wherein said protein having indolepyruvate decarboxylase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55,
(B) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55, but which includes substitution, deletion, insertion, and/or addition of 1 to 250 amino acid residues, and wherein said protein has indolepyruvate decarboxylase activity, and
(C) a protein comprising an amino acid sequence having an identity of not less than 50% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 55, and wherein said protein has indolepyruvate decarboxylase activity.

It is another aspect of the invention to provide the method as described above, wherein said gene is selected from the group consisting of:
(a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 54,
(b) a gene comprising a nucleotide sequence that is able to hybridize under stringent conditions with a nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1 or 54, and wherein the gene encodes a protein having indolepyruvate decarboxylase activity,
(c) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55, but which includes substitution, deletion, insertion and/or addition of 1 to 250 amino acid residues, and wherein said protein has indolepyruvate decarboxylase activity, and
(d) a gene comprising a variant nucleotide sequence of SEQ ID NO: 1 or 54, wherein the 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 expression of the gene encoding a protein having indolepyruvate decarboxylase activity is attenuated due to inactivation of the gene.

It is another aspect of the invention to provide the method as described above, wherein said gene encoding a protein having indolepyruvate decarboxylase activity is deleted.

It is another aspect of the invention to provide the method as described above, wherein said bacterium has been modified further to attenuate expression of a gene encoding a protein having threonine deaminase activity.

It is another aspect of the invention to provide the method as described above, wherein said gene encoding a protein having threonine deaminase activity is an ilvA gene.

It is another aspect of the invention to provide the method as described above, wherein said bacterium belongs to the family Enterobacteriaceae or Erwiniaceae.

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, and a sulfur-containing L-amino acid.

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, and L-tyrosine.

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, and L-valine.

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, and L-cystine.

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 can be an L-amino acid-producing bacterium belonging to the order Enterobacterales that has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity. 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 for 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 order Enterobacterales can be used in the method as described herein, provided that the bacterium can be modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity. It is also acceptable that any L-amino acid-producing bacterium belonging to the order Enterobacterales can be used in the method as described herein, provided that the bacterium can be modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity, 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”, the phrase “a bacterium having an ability to produce an L-amino acid”, or the phrase “a bacterium having an L-amino acid-producing ability”.

The phrase “an L-amino acid-producing bacterium” can mean a bacterium belonging to the order Enterobacterales 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 (i.e. the 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 attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Examples of the non-modified strain can include a wild-type or parental strain such as Pantoea ananatis (P. ananatis) AJ13355 and Escherichia coli (E. coli) K-12 strains such as W3110 (ATCC 27325) and MG1655 (ATCC 47076). The phrase “an L-amino acid-producing bacterium” can also mean a bacterium that is able to cause accumulation in a culture 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.

Furthermore, the bacterium belonging to the order Enterobacterales and modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity, which has an L-amino acid-producing ability, can also be used. The bacterium may inherently have the ability to produce L-amino acid or may be modified to have an L-amino acid-producing ability. Such modification can be attained by using, for example, a mutation method or DNA recombination techniques. The bacterium can be obtained by attenuating expression of a gene encoding a protein having indolepyruvate decarboxylase activity in a bacterium that inherently has L-amino acid-producing ability. Alternatively, the bacterium can be obtained by imparting L-amino acid-producing ability to a bacterium already modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Alternatively, the bacterium may be imparted with L-amino acid-producing ability by being modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity. The bacterium as described herein can be obtained, specifically, for example, by modifying a bacterial strain described hereinafter.

The phrase “an L-amino acid-producing ability” can mean the ability of a bacterium belonging to the order Enterobacterales to produce, excrete or secrete, and/or cause accumulation of L-amino acid in a culture medium and/or the bacterial cells. The phrase “an L-amino acid-producing ability” can specifically mean the ability of a bacterium belonging to the order Enterobacterales 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 is grown in a medium and used according to the method as described herein may be used interchangeably or equivalently to the phrase “cultivated”, “grown”, 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. 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, and derivatives thereof.

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 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 these. An adduct can be a compound formed by the amino acid or the 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 them. 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 them. Unless otherwise stated, the phrases “L-amino acid” and “carboxylic acid” without referring to hydration, such as the phrases “an L-amino acid in a free form”, “a carboxylic acid in a free form”, “a salt of an L-amino acid”, and “a salt of a carboxylic acid”, can refer to an L-amino acid and a carboxylic acid not in a hydrate form, a hydrate of an L-amino acid and a carboxylic acid, or a mixture of these.

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. It is known that biosynthetic pathways for sulfur-containing L-amino acids are closely interrelated in bacteria (see, for example, Ferla M.P. and Patrick W.M., Bacterial methionine biosynthesis, Microbiology, 2014, 160(Pt 8):1571-1584). In particular, in E. coli L-cysteine is derived biochemically from L-serine. Specifically, L-serine is activated by serine acetyltransferase (CysE) to obtain O-acetylserine which is reduced then by O-acetylserine(thiol)-lyase (CysM) using a reduced sulfur source such as, for example, hydrogen sulfide to produce L-cysteine. L-Cysteine can then be converted via L-cystathionine to L-homocysteine in transsulfuration pathway which is catalyzed successively by O-succinylhomoserine(thiol)-lyase/O-succinylhomoserine lyase (MetB) and cystathionine β-lyase/L-cysteine desulfhydrase (MetC). L-Methionine is synthesized from L-homocysteine using homocysteine transmethylase (MetE) and/or methionine synthase (MetH). L-cystine is normally produced in culture medium along with L-cysteine as a result of oxidation of the L-cysteine (Nakamori S. et al., Overproduction of L-cysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase, Appl. Environ. Microbiol., 1998, 64(5):1607-1611).

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.

The bacteria belonging to the family Enterobacteriaceae were recently reclassified on the basis of comprehensive comparative genomic analysis which includes phylogenetic reconstructions based on 1548 core proteins, 53 ribosomal proteins and four multilocus sequence analysis proteins (Adelou M. et al., Genome-based phylogeny and taxonomy of the ‘Enterobacteriales’: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599).

According to the reclassification, the genera previously belonging to the family Enterobacteriaceae have entered different families within the order Enterobacterales such as, for example, the families Enterobacteriaceae, Erwiniaceae, and so forth. Based on the above analysis, a bacterium that can be used in the method as described herein and belonging to the order Enterobacterales can be from the families Enterobacteriaceae, Erwiniaceae, and so forth, the genera Enterobacter, Escherichia, Klebsiella, Salmonella, Erwinia, Pantoea, Morganella, Photorhabdus, Providencia, Yersinia, and so forth, and can have the ability to produce L-amino acid. Specifically, those classified into the order Enterobacterales according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. Examples of strains belonging to the order Enterobacterales that can be modified include a bacterium of the family Enterobacteriaceae or Erwiniaceae, and, specifically, the genus Escherichia, Enterobacter, or Pantoea.

The Escherichia bacterial species are not particularly limited, and examples include species classified into the genus Escherichia according to the taxonomy known to those skilled in the field of microbiology. Examples of the Escherichia bacterium include, for example, 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.

The Enterobacter bacteria are not particularly limited, and examples include species classified into the genus Enterobacter according to classification known to a person skilled in the art of microbiology. Examples of the Enterobacter bacterium include, for example, Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Specific examples of Enterobacter agglomerans strains include, for example, the Enterobacter agglomerans ATCC 12287. Specific examples of Enterobacter aerogenes strains include, for example, the Enterobacter aerogenes ATCC 13048, NBRC 12010 (Sakai S. and Yaqishita T., Microbial production of hydrogen and ethanol from glycerol-containing wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine, Biotechnol. Bioeng., 2007, 98(2):340-348), and AJ110637 (FERM BP-10955). Examples of the Enterobacter bacterial strains also include, for example, the strains described in European Patent Application Laid-open (EP-A) No. 0952221. In addition, Enterobacter agglomerans also include some strains classified as Pantoea agglomerans.

The Pantoea bacteria are not particularly limited, and examples include species classified into the genus Pantoea according to classification known to a person skilled in the art of microbiology. Examples of the Pantoea bacterial species include, for example, Pantoea ananatis (P. ananatis), Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples of P. ananatis strains include, for example, the P. ananatis LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (FERM BP-11091), and SC17(0) (VKPM B-9246). Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like on the basis of nucleotide sequence analysis of 16S rRNA etc. (Mergaert J. et al., Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the Genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii (Smith 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov., Int. J. Syst. Evol. Microbiol., 1993, 43:162-173). The Pantoea bacteria include those reclassified into the genus Pantoea as described above.

Examples of the Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Examples of the Klebsiella bacteria include Klebsiella planticola.

These strains are available from, for example, the American Type Culture Collection (Address: P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to the respective strains, and the strains can be ordered by using these registration numbers (refer to atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.

The bacterium may be a bacterium inherently having an L-amino acid-producing ability, or may be a bacterium modified so that it has an L-amino acid-producing ability. The bacterium having an L-amino acid-producing ability can be obtained by imparting an L-amino acid-producing ability to such a bacterium as mentioned above, or by enhancing an L-amino acid-producing ability of such a bacterium as mentioned above.

To impart or enhance an L-amino acid-producing ability, methods conventionally employed in the breeding of amino acid-producing strains of Escherichia bacteria, and so forth (see “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 typical 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 typical mutagenesis treatments 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/or 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, EP 1010755 A, and so forth.

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 biosynthesis pathway of an objective L-amino acid to generate a compound other than the objective L-amino acid. The “enzyme that catalyzes a reaction branching away from the biosynthesis pathway of an objective L-amino acid to generate a compound other than the objective L-amino acid” includes an enzyme involved in decomposition of the objective amino acid. An enzyme activity can be reduced by, for example, modifying a bacterium so that the gene encoding the enzyme is inactivated. The method for reducing enzyme activity will be described later.

Hereafter, 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, but are not limited to, strains belonging to the genus Escherichia such as E. coli strain 237 (VKPM B-7925) (US2002058315 A1) and its derivative strains harboring mutant N-acetylglutamate synthase (RU2215783 C2), E. coli strain 382 (VKPM B-7926, EP1170358 A1), which is an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced (EP1170361 A1), E. coli strain 382 ilvA⁺, which is a strain obtained from the strain 382 by introducing the wild-type allele of ilvA gene native to E. coli K-12 strain thereto, and the like. Examples of mutant N-acetylglutamate synthase include, for example, 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 (EP1170361 A1).

Examples of L-arginine-producing bacteria and parental strains which can be used to derive L-arginine-producing bacteria also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetyl-γ-glutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), N-acetylornithine aminotransferase (argD), ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthetase (carAB), in addition to the gene encoding N-acetylglutamate synthase (argA).

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 E. 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-Aspartic acid-producing bacteria
Examples of L-aspartic acid-producing bacteria and parental strains which can be used to derive L-aspartic acid-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli fumaric acid-producing strain CGMCCNO: 2301 (CN101240259 A) and its derivative aspartic acid-producing strain ΔiclR (CCTCC NO: M 2018521) (CN109370971 A) in which a gene iclR encoding glyoxylate shunt regulator is deleted, and AS12 (CN105296411 B) in which aceBA genes encoding malate synthetase and isocitrate lyase are deleted and aspA gene encoding aspartate ammonia-lyase is inserted instead of them.

The E. coli CM-AS-115 (CCTCC NO: M 2016457) (CN106434510 A) is a genetically engineered strain capable of directly producing L-aspartic acid by fermentation. The starting strain is wild-type Escherichia coli W1485 (ATCC12435), which is knocked out of multiple genes (icdA (EG10489), mdh (EG10576), sfcA (EG10948), maeB (EG14193), fumAC (EG10356 and EG10358)) to obtain a recombinant strain CM-AS-100. The above strain CM-AS-100 was subjected to evolutionary metabolism and domestication to obtain a mutant strain CM-AS-105. Overexpression of two genes of mutant CM-AS-105: ppc encoding phosphoenolpyruvate carboxylase and aspA encoding aspartase resulted in E. coli CM-AS-115 strain.

Examples of L-aspartic acid-producing bacteria and parental strains which can be used to derive the L-aspartic 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, E. coli AJ13199 (FERM BP-5807, US5908768), and E. coli AJ13138 (FERM BP-5565, US6110714).

Examples of L-aspartic acid-producing bacteria and parental strains which can be used to derive L-aspartic acid-producing bacteria include bacteria which have been modified to have decreased activity of α-ketoglutarate dehydrogenase (encoding by sucA, sucB, lpdA); decreased activity of citrate synthase (gltA); increased activity of phosphoenolpyruvate carboxylase (ppc); and increased activity of glutamate dehydrogenase (gdhA) or glutamate synthase (gltBD). Bacteria can be further modified to have attenuated expression of the gene encoding aspartate ammonia-lyase (aspartase, aspA).

Examples of L-aspartic acid-producing bacteria and parental strains which can be used to derive the L-aspartic acid-producing bacteria also include Pantoea bacteria, such as the P. ananatis AJ13355 strain (FERM BP-6614), P. ananatis SC17 strain (FERM BP-11091), and P. ananatis SC17(0) strain (VKPM B-9246). P. ananatis L-aspartic acid-producing strain 5ΔP2RM (WO2010038905 A1) is a derivative of the SC17(0) strain, where such modifications as ΔaspA, ΔsucA, ΔgltA, ΔpykA, ΔpykF, Δppc, ppc^K620S, encoding feedback-resistant phosphoenolpyruvate carboxylase of E. coli, ΔmdhA were consistently introduced.

L-Citrulline-producing bacteria
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 (RU2215783 C2, EP1170361 B1, US6790647 B2), E. coli strains 333 (VKPM B-8084) and 374 (VKPM B-8086), both harboring mutant feedback-resistant carbamoyl phosphate synthetase (R2264459 C2), E. 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 (US2009286290 A1), and the like.

As L-citrulline is an intermediate of L-arginine biosynthetic pathway, examples of L-citrulline-producing bacteria and parental strains which can be used to derive L-citrulline-producing bacteria, include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme is enhanced. Examples of such genes include, but are not limited to, genes encoding N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argFI), and carbamoyl phosphate synthetase (carAB), and combinations thereof.

An L-citrulline-producing bacterium can be also easily obtained from any L-arginine-producing bacterium, 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.

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, 21 (1982) 3064-3069), tryptophanase (tnaA, Japanese Patent Laid-open (Kokai) No. 2003-169668; Austin Newton et al., J. Biol. Chem., 240 (1965) 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.

Examples of L-cysteine-producing bacteria and parental strains which can be used to derive L-cysteine-producing bacteria also include, but are not limited to, strains belonging to the genus Escherichia such as E. coli JM15 transformed with different cysE alleles encoding feedback-resistant serine acetyltransferases (US6218168 B1, RU2279477 C2), E. coli W3110 having overexpressed genes which encode proteins suitable for secreting substances toxic for cells (US5972663 A), E. coli strains having a lowered cysteine desulfhydrase activity (JP11155571 A2), E. 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 L-glutamic acid-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL334thrC⁺(EP1172433 A1). The E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in thrC and ilvA genes (US4278765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage P1 grown on the wild-type E. coli strain K-12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.

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 expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme 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 (pntAB).

Examples of strains 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 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).

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 US5378616 and US5573945. Specifically, these strains include the following:
E. coli W3110sucA::Km^R,
E. coli AJ12624 (FERM BP-3853),
E. coli AJ12628 (FERM BP-3854),
E. coli AJ12949 (FERM BP-4881).

E. coli W3110sucA::Km^R is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (sucA) of E. 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 P. ananatis AJ13355 strain (FERM BP-6614), P. ananatis SC17 strain (FERM BP-11091), and P. 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 (US6596517). 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; FGUP GosNII Genetika, Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) 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 P. ananatis AJ13356 (US6331419 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. P. 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 P. 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 P. ananatis.

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 P. ananatis SC17sucA/RSFCPG+pSTVCB strain, P. ananatis AJ13601 strain, P. ananatis NP106 strain, and P. 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 E. 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 NA1 strain was obtained from the NP106 strain by introduction of the plasmid RSFPPG (EP2336347 A1). 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, E. coli AJ13199 (FERM BP-5807, US5908768), E. coli FFRM P-12379, which additionally has a lowered L-glutamic acid-decomposing ability (US5393671), and E. coli AJ13138 (FERM BP-5565, US6110714).

L-Histidine-producing bacteria
Examples of L-histidine-producing bacteria and parental strains which can be used to derive L-histidine-producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strain 24 (VKPM B-5945, RU2003677 C1), E. coli strain 80 (VKPM B-7270, RU2119536 C1), E. coli NRRL B-12116 - B-12121 (US4388405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US6344347 B1), E. coli H-9341 (FERM BP-6674) (EP1085087 A2), E. coli AI80/pFM201 (US6258554 B1), and the like.

Examples of L-histidine-producing bacteria and parental strains which can be used to derive L-histidine-producing bacteria also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding 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), histidinol dehydrogenase (hisD), and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase (RU2003677 C1 and RU2119536 C1).

Specific examples of strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048, which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains transformed with rht, a gene for an amino acid-export (EP1016710 A2), E. coli 80 strain, which has been imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, RU2119536 C1), E. 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 L-isoleucine-producing bacteria include, but are not limited to, mutant strains having resistance to 6-dimethylaminopurine (JP5-304969 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 (JP5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, can also be used as L-isoleucine-producing bacteria or parental strains (JP2-458 A, EP0356739 A1, and US5998178).

L-Leucine-producing bacteria
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 E. coli strains resistant to leucine, for example, the strain 57 (VKPM B-7386, US6124121); E. coli strains resistant to leucine analogs includingβ-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP62-34397 B and JP8-70879 A); E. coli strains obtained by the gene engineering method described in WO9606926; E. coli H-9068 (JP8-70879 A), and the like.

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 involved in L-leucine biosynthesis is enhanced. Examples of such genes include genes of the leuABCD operon, which can be represented by a mutant leuA gene encoding α-isopropylmalate synthase freed from feedback inhibition by L-leucine (US6403342 B1). 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).

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. Specific examples of bacterial strains useful for producing L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US4346170) and E. coli VL611. In these strains, feedback inhibition of aspartokinase by L-lysine is desensitized.

Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme is enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US6040160), 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). In addition, the parental strains may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP1170376 A1), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US5830716 A), the ybjE gene (WO2005073390), or combinations thereof. Since aspartokinase III (lysC) is subjected to feedback inhibition by L-lysine, a mutant lysC gene encoding an aspartokinase III desensitized to feedback inhibition by L-lysine (US5932453) 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 selected from a mutation for replacing the methionine residue at position 318 with an isoleucine residue; a mutation for replacing the glycine residue at position 323 with an aspartic acid residue; and a mutation for 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 encoding 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 involved in L-lysine production include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, and so forth, and strains in which activities of these enzymes are decreased or deleted are disclosed in WO9523864, WO9617930, WO2005010175, and so forth.

Expression of both the cadA and ldcC genes encoding lysine decarboxylase can be decreased in order to decrease or delete the lysine decarboxylase activity. Expression of the both genes can be decreased by, for example, the method described in WO2006078039.

Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include the E. coli WC196 strain (FERM BP-5252, US5827698), the E. coli WC196ΔcadAΔldcC strain (FERM BP-11027), also named as WC196LC, and the E. coli WC196ΔcadAΔldcC/pCABD2 strain (WO2006078039).

The WC196 strain was bred from the W3110 strain, which was derived from E. coli K-12, by conferring AEC resistance to the W3110 strain (US5827698). The WC196 strain was designated E. 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 (US5827698).

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 lysine biosynthesis genes (US6040160) into the WC196ΔcadAΔldcC strain. The plasmid pCABD2 contains a mutant dapA gene derived from E. coli and encoding a dihydrodipicolinate synthase (DDPS) having a mutation for desensitization to feedback inhibition by L-lysine (H118Y), a mutant lysC gene derived from E. coli and encoding aspartokinase III having a mutation for desensitization to feedback inhibition by L-lysine (T352I), the dapB gene native to E. coli and encoding dihydrodipicolinate reductase, and the ddh gene native to Brevibacterium lactofermentum and encoding diaminopimelate dehydrogenase.

Examples of L-lysine-producing bacteria and parental strains which can be used to derive L-lysine-producing bacteria also include E. coli AJIK01 (NITE BP-01520). The AJIK01 strain was designated E. coli AJ111046, and deposited at NITE IPOD (#120, 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 E. coli strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (GB2075055); and E. 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 E. coli 218 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) on May 14, 2001 under the accession number VKPM B-8125. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on February 1, 2002. The strain E. coli 73 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) 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 (JP2000-139471 A) can also be used as L-methionine-producing bacteria or parental strains.

Other examples of L-methionine-producing bacteria of the genus Escherichia and parental strains thereof that can be used to derive L-methionine-producing bacteria can be E. coli strain that is deficient in repressor of L-methionine biosynthesis system (MetJ) and has increased activity of intracellular homoserine transsuccinylase (MetA) (US7611873 B1), 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), 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 be modified to overexpress a cysteine synthase-encoding gene.

The phrase “a cysteine synthase-encoding 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-encoding 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-encoding 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: 13.

L-Methionine-producing bacteria may 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 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: 35. Specifically, an example of the amino acid sequence of a mutant MetA protein can be the amino acid sequence shown in SEQ ID NO: 37, which can be encoded by the mutant metA gene having the nucleotide sequence shown in SEQ ID NO: 36. That is, the mutant metA gene may be a gene, such as DNA, having the nucleotide sequence of SEQ ID NO: 36, and the mutant MetA protein may be a protein having the amino acid sequence of SEQ ID NO: 37. The mutant metA gene may also be a gene, such as DNA, having a variant nucleotide sequence of SEQ ID NO: 36, 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: 37, 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. Descriptions concerning variants of a gene encoding a protein having indolepyruvate decarboxylase activity and indolepyruvate decarboxylase encoded thereby described herein 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: 35.

L-Methionine-producing bacteria may 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 S-adenosylmethionine (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: 24.

L-Methionine-producing bacteria may be modified to have a mutant thrA gene encoding a mutant aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine. Examples of the mutant thrA gene include thrA442 gene.

L-Methionine-producing bacteria may be modified to overexpress an aminomethyltransferase gene.

The phrase “an aminomethyltransferase gene” can refer to a gene encoding an aminomethyltransferase. The phrase “an aminomethyltransferase” can refer to a protein having aminomethyltransferase activity (EC 2.1.2.10). Examples of the aminomethyltransferase gene can include a gcvT gene.

An example of L-methionine-producing bacteria of the genus Pantoea and parental 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). This strain is also known as P. ananatis strain SC17 (FERM BP-11091).

L-Ornithine-producing bacteria
As L-ornithine is an intermediate of L-arginine biosynthetic pathway, examples of L-ornithine-producing bacteria and parental strains which can be used to derive L-ornithine-producing bacteria, include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme, such as those described above, is enhanced.

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

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 E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (US5354672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (US4407952), E. coli K-12 [W3110 (tyrA)/pPHAB] (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662), and E. 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 (US7259003 and US7666655).

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 E. 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 E. coli strains: NRRL B-12403 and NRRL B-12404 (GB2075056), VKPM B-8012 (RU2207371 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 include, but are not limited to, strains belonging to the genus Escherichia such as E. coli TDH-6/pVIC40 (VKPM B-3996) (US5175107 and US5705371), E. coli 472T23/pYN7 (ATCC 98081) (US5631157), E. coli NRRL-21593 (US 5939307), E. coli FERM BP-3756 (US5474918), E. coli FERM BP-3519 and FERM BP-3520 (US5376538), E. coli MG442 (Gusyatiner M.M. et al., Study of relA gene function in the expression of amino acid operons. II. Effect of the relA gene allelic state on threonine over-production by Escherichia coli K-12 mutants resistant to β-hydroxynorvaline acid, Genetika (Russian), 1978, 14(6):957-968), E. coli VL643 and VL2055 (EP1149911 A2), E. 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; Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) 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 E. coli has been elucidated (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b0002; GenBank, accession No. NC_000913.3; nucleotide positions: 337 to 2,799; Gene ID: 945803). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12.

The thrB gene which encodes homoserine kinase of E. coli has been elucidated (KEGG, entry No. b0003; GenBank, accession No. NC_000913.3; 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 E. coli K-12.

The thrC gene which encodes threonine synthase of E. coli has been elucidated (KEGG, entry No. b0004; GenBank, accession No. NC_000913.3; 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 E. 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 E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in US5705371.

The rhtA gene which encodes a protein of the threonine and homoserine efflux system (an inner membrane transporter) of E. coli has been elucidated (KEGG, entry No. b0813; GenBank, accession No. NC_000913.3; nucleotide positions: 849,210 to 850,097, complement; Gene ID: 947939). The rhtA gene is located between the dps and ompX genes on the chromosome of E. 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 E. coli has been elucidated (KEGG, entry No. b3433; GenBank, accession No. NC_000913.3; nucleotide positions: 3,573,775 to 3,574,878, 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 E. coli K-12.

Also, the aspC gene which encodes aspartate aminotransferase of E. coli has been elucidated (KEGG, entry No. b0928; GenBank, accession No. NC_000913.3; nucleotide positions: 984,519 to 985,709, complement; Gene ID: 945553). The aspC gene is located between the gloC gene on the opposite strand and the ompF gene on the same strand on the chromosome of E. 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 E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (US5756345), E. 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 (US6180373 B1), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (US4371614), E. coli AGX17/pGX50,pACKG4-pps having an enhanced phosphoenolpyruvate-producing ability (WO9708333, US6319696 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 and the yedA gene or the yddG gene (US2003148473 A1 and US2003157667 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 the enzymes selected from 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 E. coli SV164, which harbors desensitized anthranilate synthase, and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO9408031 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 (JP57-71397 A, JP62-244382 A, US4371614). 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 (WO2005103275).

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 (US5998178). 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 (US5658766). Examples of such strains include E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase. E. coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) 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 (WO9606926 A1).

Examples of L-valine-producing bacteria and parent strains for deriving L-valine-producing bacteria also include E. coli H81 strain (VKPM B-8066; see, for example, EP1942183 B1), E. coli NRRL B-12287 and NRRL B-12288 (US4391907), E. coli VKPM B-4411 (US5658766), E. 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 gene encoding a protein having indolepyruvate decarboxylase activity and indolepyruvate decarboxylase encoded thereby described herein can be similarly applied.

The bacterium as described herein belonging to the order Enterobacterales may be modified to, at least, attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity.

The phrase “a gene encoding a protein having indolepyruvate decarboxylase activity” can mean a gene encoding a protein having enzymatic activity of catalyzing the following reaction: indol-3-pyruvate + H⁺ -> indole-3-acetaldehyde + CO₂ (EC: 4.1.1.74). Methods for determining the indolepyruvate decarboxylase activity of a protein can be exemplified by those described, for example, in Koga J. et al. (Purification and characterization of indolepyruvate decarboxylase, J. Biol. Chem., 1992, 267(22):15823-15828). 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., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72:248-254; Lowry O.H. et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, 193:265-275), or a Western blot analysis (Hirano S., Western blot analysis, Methods Mol. Biol., 2012, 926:87-97).

Specific example of the gene which encodes an enzyme having indolepyruvate decarboxylase activity includes an ipdC gene which encodes indolepyruvate decarboxylase. The gene encoding an enzyme having indolepyruvate decarboxylase activity can be the ipdC gene and its homolog(s) or variant nucleotide sequence(s). The more specific description of ipdC and its homologs and variant nucleotide sequences is given hereinafter.

The ipdC gene native to P. ananatis encodes indolepyruvate decarboxylase protein IpdC (synonym: indole-3-pyruvate decarboxylase, 3-(indol-3-yl)pyruvate carboxy-lyase) (BioCyc database, biocyc.org, accession ID: PAJ_RS11190; UniProtKB/Swiss-Prot, accession No. A0A0H3L5I5; UniParc, accession No. UPI0002040446; KEGG entry No. PAJ_2029).

The ipdC gene (GenBank, accession No. NC_017531.2/AP012032.2; nucleotide positions: 2461340 to 2462992, complement; Gene ID: BAK12109.1) is located between the glk gene (BAK12108.1) on the same strand and the yjhZ gene (BAK12110.1) on the opposite strand on the chromosome of P. ananatis strain AJ13355. The ipdC gene native to P. ananatis strain AJ13355 has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence of the IpdC protein encoded by this gene native to P. ananatis strain AJ13355 is shown in SEQ ID NO: 2.

That is, the ipdC gene can be a gene, such as DNA, having the nucleotide sequence of SEQ ID NO: 1, and the IpdC protein can be a protein having the amino acid sequence of SEQ ID NO: 2. 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.

The protein homologues of IpdC native to different bacteria belonging to the order Enterobacterales are known that have indolepyruvate decarboxylase activity as described above. Examples of such homologous proteins that are native to bacteria belonging to the order Enterobacterales are described in Table 1, with accession numbers of amino acid sequences in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov/protein), taxonomy data, and indication of a homology value (as “identity”, that is the identity of amino acids). In particular, an ipdC gene native to E. coli having the nucleotide sequence shown in SEQ ID NO: 54 is known which encodes the IpdC protein having the amino acid sequence shown in SEQ ID NO: 55 (Table 1, accession No.: WP_069192931.1).

The bacterium as described herein can also be an L-amino acid-producing bacterium belonging to the order Enterobacterales that has been modified to attenuate expression of a gene encoding a protein having threonine deaminase activity. That is, the bacterium that can be used in the method as described herein belonging to the order Enterobacterales can also be modified to attenuate expression of a gene encoding a protein having threonine deaminase activity. The bacterium can also be modified in such a way so that expression of a gene encoding a protein having indolepyruvate decarboxylase activity and a gene encoding a protein having threonine deaminase activity is attenuated. The descriptions concerning the L-amino acid-producing bacterium belonging to the order Enterobacterales that has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity can also be similarly applied to the L-amino acid-producing bacterium belonging to the order Enterobacterales that has been modified to attenuate expression of a gene encoding a protein having threonine deaminase activity.

The phrase “a gene encoding a protein having threonine deaminase activity” (synonym: threonine dehydratase, threonine ammonia-lyase) can mean a gene encoding a protein having enzymatic activity of catalyzing the following reaction: L-threonine -> 2-oxobutanoate + ammonium (EC: 4.3.1.19). The threonine deaminase activity of a protein can be determined, for example, by measuring α-ketobutyrate formed as the phenylhydrazone derivative, or by using other known methods (Calhoun D.H. et al., Threonine deaminase from Escherichia coli. I. Purification and properties, J. Biol. Chem., 1973, 248(10):3511-3516; Eisenstein E., Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli, J. Biol. Chem., 1991, 266(9):5801-5807).

A specific example of the gene which encodes an enzyme having threonine deaminase activity includes an ilvA gene which encodes threonine deaminase. The gene encoding an enzyme having threonine deaminase activity can be the ilvA gene and its homolog(s) or variant nucleotide sequence(s). The more specific description of ilvA and its homologs and variant nucleotide sequences is given hereinafter.

The ilvA gene native to P. ananatis encodes pyridoxal-phosphate dependent threonine dehydratase protein IlvA (BioCyc database, biocyc.org, accession ID: PAJ_RS16870; UniParc, accession No. UPI0002323460; KEGG entry No. PAJ_3043).

The ilvA gene (GenBank, accession No. NC_017531.2/AP012032.2; nucleotide positions: 3640258 to 3641871, complement; Gene ID: BAK13123.1) is located between the ilvY gene (BAK13122.1) on the opposite strand and the ilvD gene (BAK13124.1) on the same strand on the chromosome of P. ananatis strain AJ13355. The ilvA gene native to P. ananatis strain AJ13355 has the nucleotide sequence shown in SEQ ID NO: 3, and the amino acid sequence of the IlvA protein encoded by this gene native to P. ananatis strain AJ13355 is shown in SEQ ID NO: 4.

That is, the ilvA gene may be a gene, such as DNA, having the nucleotide sequence of SEQ ID NO: 3, and the IlvA protein may be a protein having the amino acid sequence of SEQ ID NO: 4.

The protein homologues of ilvA native to different bacteria belonging to the order Enterobacterales are known that have threonine deaminase activity as described above. Examples of such homologous proteins that are native to bacteria belonging to the order Enterobacterales are described in Table 2, with accession numbers of amino acid sequences in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov/protein), taxonomy data, and indication of a homology value (as “identity”, that is the identity of amino acids).

The explanations given hereinafter to, for example, attenuation of gene expression, a protein, including a variant protein, and a gene, including a variant nucleotide sequence, can be applied with appropriate changes to any protein and gene described in this specification including, but is not limited to, IpdC and IlvA proteins, and the genes encoding them such as ipdC and ilvA genes, and homologs thereof.

There may be some differences in DNA sequences between the families, genera, species or strains belonging to the order Enterobacterales. Therefore, the ipdC and ilvA genes are not limited to the genes having the nucleotide sequences shown in SEQ ID NOs: 1 and 3, accordingly, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1 and 3, accordingly, and which encode variants of the IpdC and IlvA proteins. Similarly, the IpdC and IlvA proteins are not limited to the proteins having the amino acid sequences shown in SEQ ID NOS: 2 and 4, but may include proteins having variant amino acid sequences of or homologous to SEQ ID NO: 2 and 4.

The phrase “a variant nucleotide sequence” can mean the nucleotide sequence which encodes a protein having the wild-type amino acid sequence 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 gene encoding a protein having the wild-type amino acid sequence can be a gene having a variant nucleotide sequence due to the degeneracy of the genetic code.

The phrase “a variant nucleotide sequence” can also mean, but is not limited to, a nucleotide sequence that is able to hybridize under stringent conditions with the nucleotide sequence complementary to the wild-type nucleotide sequence or a probe that can be prepared from the nucleotide sequence provided that it encodes a protein having desired activity such as, for example, indolepyruvate decarboxylase activity or threonine deaminase activity as described above. “Stringent conditions” can include those 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 50%, of not less than 55%, of not less than 60%, of not less than 65%, 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, or in another example, 0.1 × SSC, 0.1% SDS at 60°C or 65°C. Duration of washing depends 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 wild-type nucleotide sequence 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 wild-type nucleotide sequence 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 exemplified by 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 having a variant amino acid sequence.

The phrase “a variant protein” can mean a protein which has one or more mutations in the amino acid sequence as compared with the wild-type amino acid sequence of the protein whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but still maintains an activity or function similar to that of the wild-type protein, or the three-dimensional structure of the variant protein is not significantly changed relative to the non-modified protein. The number of changes in a variant protein depends on the position of amino acid residues in the three-dimensional structure of the protein or the type of amino acid residues. 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 the wild-type amino acid sequence of the protein. This is because some amino acids have high homology to one another so that the activity or function is not affected by such a change, or the three-dimensional structure of the protein is not significantly changed relative to the wild-type or 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 50%, of not less than 55%, of not less than 60%, of not less than 65%, 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 wild-type amino acid sequence of the protein, as long as the activity or function of the protein is maintained, or the three-dimensional structure of the protein is not significantly changed relative to the wild-type or non-modified protein. 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) so that an 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 such as, for example, the wild-type protein. The representative conservative mutation is 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 such as, for example, the wild-type protein.

The calculation of a percent identity of an amino acid sequence can be carried out using the algorithm blastp. More specifically, the calculation of a percent identity of an amino acid sequence 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 nucleotide sequence can be carried out using the algorithm blastn. More specifically, the calculation of a percent identity of a nucleotide sequence 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 attenuate expression of a gene” can mean that the bacterium has been modified in such a way that in the modified bacterium expression of the gene is attenuated. Exemplary, the expression of the gene can be attenuated due to 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 a wild-type or non-modified gene. 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 adjacent region of the gene, including sequences controlling gene expression such as promoter, enhancer, attenuator, ribosome-binding site, etc. 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., An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; Datsenko K.A. and Wanner B.L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., A new logic for DNA engineering using recombination in Escherichia coli, 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 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, and other expression control elements, which is modified resulting in the decrease of the expression level of the gene; and other examples (see, for example, WO9534672 A1; Carrier T.A. and Keasling J.D., Library of synthetic 5’ secondary structures to manipulate mRNA stability in Escherichia coli, Biotechnol. Prog., 1999, 15:58-64).

The phrase “operably linked to the gene” can mean that the regulatory region(s) is/are linked to the nucleotide sequence of the nucleic acid molecule or gene in such a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, antiterminated, attenuated, deregulated, decreased, or repressed expression) of the nucleotide sequence, specifically, the expression of a gene product encoded by the nucleotide sequence.

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, that the amount of the expression product of the 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 bacterium.

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 (i.e. the encoded protein) is decreased as compared with a non-modified bacterium. 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 activity of the protein 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 bacterium.

Examples of a non-modified bacterium serving as a reference for the above comparisons can include wild-type strains of a bacterium belonging to the genus Escherichia, such as the E. coli MG1655 strain (ATCC 47076), E. coli W3110 strain (ATCC 27325), or a bacterium belonging to the genus Pantoea, such as the P. ananatis AJ13355 strain (FERM BP-6614), and so forth. Examples of a non-modified bacterium serving as a reference for the above comparisons can also include a parental strain which has not been modified to attenuate expression of the gene or a bacterium in which expression of the gene is not attenuated.

Expression of a gene can be attenuated by 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 is 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 (RBS) greatly affects the translation efficiency of mRNA.

Expression of a gene can also be attenuated by inserting a transposon or an insertion sequence (IS) into the coding region of the gene (US5175107) 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 copy number, presence or absence of the 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 persons of ordinary skill 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, for example, “Mu-driven integration/amplification” (Akhverdyan 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, for multiple insertions of desired genes in addition to Mu-driven replicative transposition (Akhverdyan et al., Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria-mini review, 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., Stabilized gene duplication enables long-term selection-free heterologous pathway expression, Nature Biotechnol., 2009, 27:760-765), other methods can be used, which 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., Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure, BMC Biotechnology, 2008, 8:63; Koma D. et al., A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome, Appl. Microbiol. Biotechnol., 2012, 93(2):815-829).

As the genes encoding wild-type proteins native to the species E. coli and P. ananatis have already been elucidated (see above), the variant nucleotide sequences encoding variant proteins of the wild-type proteins can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., The polymerase chain reaction, Trends Genet., 1989, 5(6):185-189) utilizing primers prepared based on the nucleotide sequences of the wild-type genes; or the site-directed mutagenesis method by treating a DNA containing wild-type genes in vitro, for example, with hydroxylamine, or a method for treating a microorganism, for example, a bacterium belonging to the species E. coli or P. ananatis harboring wild-type genes with ultraviolet (UV) irradiation or a mutating agent such as N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the such treatment; or chemically synthesized as full-length gene structure. Genes encoding the proteins or its variant proteins from other bacteria belonging to the order Enterobacterales can be obtained in a similar manner.

The phrase “wild-type”, which can be equivalent to the phrases “native” and “natural”, as used herein as to a protein (for example, “a wild-type protein”) and a gene (for example, “a wild-type gene”) can mean, respectively, a native protein and a native gene that exist, and/or is expressed naturally in, and/or produced by a wild-type bacterium, for example, a wild-type strain of a bacterium belonging to the order Enterobacterales such as, for example, the family Enterobacteriaceae or Erwiniaceae such as, for example, the E. coli MG1655 strain (ATCC 47076), the E. coli W3110 strain (ATCC 27325), the P. ananatis AJ13355 strain (FERM BP-6614), and so forth. As a protein is encoded by a gene, “a wild-type protein” can be encoded by “a wild-type gene” naturally occurring in genome of a wild-type bacterium.

The phrase “native to” in reference to a protein or a nucleic acid native to a particular organism such as, for example, a bacterial species can refer to a protein or a nucleic acid that is native to that organism. That is, a protein or a nucleic acid native to a particular organism can mean the protein or the nucleic acid, respectively, which exists naturally in the organism and 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, 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, accordingly, to the amino acid sequence of the protein or the nucleotide sequence of the nucleic acid that exists naturally in the organism. Examples of amino acid sequences native to particular species include, but are not limited to, peptides, oligopeptides, polypeptides, including proteins, specifically enzymes, and so forth. Examples of nucleotide sequences native to particular species include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and these are not limited to expression regulatory sequences, including promoters, attenuators, terminators, and the like, genes, intergenic sequences, and nucleotide sequences encoding signal peptides, pro-moieties of proteins, artificial amino acid sequences, and so forth. Specific examples of amino acid sequences and nucleotide sequences, and homologues thereof native to various species are described herein, and these examples include the proteins IpdC and IlvA having the amino acid sequence shown in SEQ ID NOs: 2 and 4, respectively, which are native to the bacterium of the species P. ananatis and can be encoded by the corresponding genes ipdC and ilvA having the nucleotide sequence shown in SEQ ID NOs: 1 and 3, respectively.

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.

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 a bacterium as described herein can include the steps of cultivating (also called culturing) the bacterium in a culture medium to allow an L-amino acid to be produced, excreted or secreted, and/or accumulated in the culture medium or in the 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 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 persons 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 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 hydrolyzates 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 6.0 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 having ipdC gene deleted
The P. ananatis SC17(0)ΔipdC::λattL-kan^R-λattR strain having deleted the ipdC gene (SEQ ID NO: 1) was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain (US8383372 B2, VKPM B-9246) harboring RSF-Red-TER plasmid (US8383372 B2) was cultured overnight in 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 to 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR DNA fragment having sequences homologous to the upstream and downstream of ipdC gene at both termini was obtained by PCR using the primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6), and pMW118-attL-kan^R-attR plasmid (US7919284 B2) 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 (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 35 mg/L 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 97°C for 20 seconds, 54°C for 20 seconds and 72°C for 100 seconds) was performed using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8) to confirm that the ipdC gene on the chromosome was replaced with the λattL-kan^R-λattR cassette. As a result, the P. ananatis SC17(0)ΔipdC::λattL-kan^R-λattR strain was obtained.

Example 2. Production of L-methionine using P. ananatis strain having ipdC gene deleted
To test the effect from the deletion of ipdC gene on L-methionine production, the chromosomal DNA from the strain SC17(0)ΔipdC::λattL-kan^R-λattR (Example 1) was isolated using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions, and 10 μg of the DNA was used to transform P. ananatis C2691 by electroporation. The L-methionine-producing P. ananatis strain C2691 was constructed as described in Auxiliary example 1. The resulting transformants were plated on plates with LB agar containing kanamycin (20 mg/L), and 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) to confirm the replacement of ipdC gene. As a result, the P. ananatis C2691ΔipdC::λattL-kan^R-λattR strain (abbreviated as C3793) was obtained.

The P. ananatis C2691 and C2691ΔipdC::λattL-kan^R-λattR strains 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 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.

The composition of the fermentation medium (g/L) was as follows:
Glucose 40.0
(NH₄)₂SO₄ 15.0
KH₂PO₄ 1.5
MgSO₄-7H₂O 1.0
Thiamine-HCl 0.1
CaCO₃ 25.0
LB medium 4% (v/v)

The fermentation medium was sterilized at 116°C for 30 min, except that glucose and CaCO₃ were sterilized separately as follows: glucose at 110°C for 30 min and CaCO₃ at 116°C for 30 min. The pH was adjusted to 7.0 by KOH solution.

After cultivation, the amount of L-methionine which accumulated in the medium was determined using Agilent 1260 amino-acid analyzer. The results of three 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 C2691ΔipdC::λattL-kan^R-λattR strain was able to accumulate a higher amount of L-methionine as compared with the parental P. ananatis C2691 strain.

Example 3. Production of L-cysteine using P. ananatis strain having ipdC gene deleted
To test the effect from the deletion of ipdC on L-cysteine production, the chromosomal DNA from the strain SC17(0)ΔipdC::λattL-kan^R-λattR (Example 1) is transferred to the L-cysteine-producing P. ananatis EYP197(s) strain (RU2458981 C2 or WO2012/137689) by electroporation to obtain the P. ananatis EYP ΔipdC::λattL-kan^R-λattR strain. The P. ananatis strain EYP197(s) was constructed from P. ananatis SC17 (FERM BP-11091) by introducing cysE5 and yeaS genes and replacing the native promoter of cysPTWA gene cluster with Pnlp8 promoter.

The P. ananatis strains EYP197(s) and EYP ΔipdC::λattL-kan^R-λattR are separately cultivated at 32°C for 18 hours in 3 mL of LB liquid culture medium, and 0.2 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 32°C for 24 hours on a rotary shaker.

After cultivation, the amount of L-cysteine which accumulates in the medium is determined by the method described by Gaitonde M.K. (A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids, Biochem. J., 1967, 104(2):627-633) with some modifications as follows: 150 μL of each sample is mixed with 150 μL of 1 M H₂SO₄, incubated for 5 min at 20°C, then 700 μL H₂O is added to the mixture, 150 μL of the obtained mixture is transferred into the new vial, and 800 μL of solution A (1 M Tris-HCl pH 8.0, 5 mM dithiothreitol (DTT)) is added. The obtained mixture is incubated for 5 min at 20°C, rotated for 10 min at 13000 rpm, and then 100 μL of the mixture is 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) are added, and the mixture is incubated for 10 min in a boiling water bath. Then 4.5 mL ethanol is added and the OD₅₆₀is determined. The concentration of cysteine is calculated using the formula: C (Cys, g/L) = 11.3 × OD₅₆₀.

The composition of the fermentation medium (g/L) is as follows:
Glucose 40.0
(NH₄)₂S₂O₃ 12.0
KH₂PO₄ 1.5
MgSO₄-6H₂O 0.825
Thiamine-HCl 0.1
CaCO₃ 25.0
LB medium 4% (v/v)

The fermentation medium is sterilized at 116°C for 30 min, except that glucose, (NH₄)₂S₂O₃ and CaCO₃ are sterilized separately as follows: glucose at 110°C for 30 min, (NH₄)₂S₂O₃by means of filtering through 0.2 μm-membrane, and CaCO₃ at 116°C for 30 min. The pH is adjusted to 7.0 by KOH solution.

Example 4. Production of L-glutamic acid using P. ananatis strain having ipdC gene deleted
To test the effect from the deletion of ipdC on L-glutamic acid production, the chromosomal DNA from the strain SC17(0)ΔipdC::λattL-kan^R-λattR (Example 1) is transferred to the L-glutamic acid-producing P. ananatis NA1 strain (EP2336347 A1) by electroporation to obtain the P. ananatis NA1ΔipdC::λattL-kan^R-λattR strain.

The P. ananatis strains NA1 and NA1ΔipdC::λattL-kan^R-λattR are separately cultivated at 32°C for 18 hours in 3 mL of LB liquid culture medium, and 0.2 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 32°C for 24 hours on a rotary shaker.

After 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.

The composition of the fermentation medium (g/L) is as follows:
Glucose 30.0
MgSO₄-6H₂O 0.5
(NH₄)₂SO₄ 20.0
KH₂PO₄ 2.0
Yeast extract 2.0
FeSO₄-7H₂O 0.02
MnSO₄-4H₂O 0.02
Thiamine-HCl 0.01
L-lysine hydrochloride 0.2
L-methionine 0.2
DL-α,ε-diaminopimelic acid 0.2
CaCO₃ 20.0

The fermentation medium is sterilized at 116°C for 30 min, except that glucose and CaCO₃ are sterilized separately as follows: glucose at 110°C for 30 min and CaCO₃ at 116°C for 30 min. The pH is adjusted to 7.0 by KOH solution.

Example 5. Production of L-aspartic acid using P. ananatis strain having ipdC gene deleted
To test the effect from the deletion of ipdC on L-aspartic acid production, the chromosomal DNA from the strain SC17(0)ΔipdC::λattL-kan^R-λattR (Example 1) is transferred to the L-aspartic acid-producing P. ananatis 5ΔP2RM strain (WO2010038905 A1) by electroporation to obtain the P. ananatis 5ΔP2RMΔipdC::λattL-kan^R-λattR strain.

The P. ananatis strains 5ΔP2RM and 5ΔP2RMΔipdC::λattL-kan^R-λattR are separately cultivated at 32°C for 18 hours in 3 mL of LB liquid culture medium, and 0.2 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 32°C for 72 hours on a rotary shaker. After cultivation, the amount of L-aspartic acid which accumulated in the medium is determined by paper chromatography.

The composition of the fermentation medium (g/L) is as follows:
Glucose 40.0
MgSO₄-7H₂O 1.0
(NH₄)₂SO₄ 16.0
KH₂PO₄ 0.3
KCl 1.0
MES 10.0
Calcium pantothenate 0.01
Betaine 1.0
FeSO₄-7H₂O 0.01
MnSO₄-5H₂O 0.01
Thiamine-HCl 0.01
L-lysine hydrochloride 0.1
L-methionine 0.1
DL-α,ε-diaminopimelic acid 0.1
L-Glutamic acid 3.0
CaCO₃ 30.0

The fermentation medium is sterilized at 116°C for 30 min, except that glucose and CaCO₃ are sterilized separately as follows: glucose at 110°C for 30 min and CaCO₃ at 116°C for 30 min. The pH is adjusted to 6.5 by NaOH solution.

Example 6. Construction of E. coli L-methionine-producing strain having ipdC gene deleted
An E. coli L-methionine-producing strain, the examples of which are described above, harboring pKD46 plasmid (Datsenko K.A. and Wanner B.L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) having a temperature-sensitive replication 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 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR DNA fragment having sequences homologous to the upstream and downstream of ipdC gene at both termini is obtained by PCR using pMW118-attL-kan^R-attR plasmid (US7919284 B2) as a template. The resulting DNA fragment 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 applied onto the LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Emerging colonies are refined in the same culture medium. The replacement of the ipdC gene on the chromosome with the λattL-kan^R-λattR cassette is confirmed by PCR. As a result, the E. coli L-methionine-producing strain ΔipdC::λattL-kan^R-λattR is obtained.

Example 7. Production of L-methionine using E. coli strain having ipdC gene deleted
The E. coli L-methionine-producing strain ΔipdC::λattL-kan^R-λattR and its parent strain are separately cultivated with shaking at 37°C for 18 hours in LB liquid culture medium. Then, 0.2 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker at 250 rpm. After cultivation, the amount of L-methionine which accumulated in the medium is determined using Agilent 1260 amino-acid analyzer.

The composition of the fermentation medium (g/L) is as follows:
Glucose 40.0
(NH₄)₂SO₄ 15.0
KH₂PO₄ 1.5
MgSO₄-7H₂O 1.0
Thiamine-HCl 0.1
Threonine 0.5
CaCO₃ 20.0
LB medium 4% (v/v)

Example 8. Construction of P. ananatis strain having ilvA gene deleted
The P. ananatis SC17(0)ΔilvA::λattL-kan^R-λattR strain having deleted the ilvA gene (SEQ ID NO: 3) was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER strain (US8383372 B2) was cultured overnight in the LB liquid culture medium. Then, 1 mL of the cultured medium was inoculated to 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR DNA fragment having sequences homologous to the upstream and downstream of ilvA gene at both termini was obtained by PCR using the primers P5 (SEQ ID NO: 9) and P6 (SEQ ID NO: 10), and pMW118-attL-kan^R-attR plasmid (US7919284 B2) 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 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); 25 cycles at 97°C for 20 seconds, 55°C for 20 seconds and 72°C for 100 seconds) was performed using primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12) to confirm that the ilvA gene on the chromosome was replaced with the λattL-kan^R-λattR cassette. As a result, the P. ananatis SC17(0)ΔilvA::λattL-kan^R-λattR strain was obtained.

Example 9. Production of L-methionine using P. ananatis strain having ilvA gene deleted
To test the effect from the deletion of ilvA on L-methionine production, the chromosomal DNA from the strain SC17(0)ΔilvA::λattL-kan^R-λattR (Example 8) was isolated using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions, and 10 μg of the DNA was used to transform P. ananatis C2792 by electroporation. The L-methionine-producing P. ananatis strain C2792 was constructed as described in Auxiliary example 2. The resulting transformants were plated on plates with LB agar containing kanamycin (20 mg/L), and incubated at 34°C overnight until individual colonies were visible. The desired transformants were identified by PCR analysis as described in Example 8 to confirm the replacement of ilvA gene. As a result, the P. ananatis C2792ΔilvA::λattL-kan^R-λattR strain (abbreviated as C3653) was obtained.

The P. ananatis C2792 and C2792ΔilvA::λattL-kan^R-λattR strains 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 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 three independent test-tube fermentations (as average values ± standard deviations) are shown in Table 4. As one can see from the Table 4, the modified P. ananatis C2792ΔilvA::λattL-kan^R-λattR strain was able to accumulate a higher amount of L-methionine as compared with the parental P. ananatis C2792 strain.

Example 10. Construction of E. coli MG1655 strain having ilvA gene deleted
The E. coli MG1655 (ATCC 700926) strain, which contains the plasmid pKD46 having a temperature-sensitive replication, 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 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR DNA fragment having sequences homologous to the upstream and downstream of ilvA gene at both termini is obtained by PCR using pMW118-attL-kan^R-attR plasmid (US7919284 B2) as a template. The resulting DNA fragment 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 applied onto the LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Emerging colonies are refined in the same culture medium. The replacement of the ilvA gene on the chromosome with the λattL-kan^R-λattR cassette is confirmed by PCR. As a result, the E. coli MG1655ΔilvA::λattL-kan^R-λattR strain is obtained.

Example 11. Production of L-methionine using E. coli strain having ilvA gene deleted
To test the effect from the deletion of ilvA on L-methionine production, the chromosomal DNA from the strain MG1655ΔilvA::λattL-kan^R-λattR (Example 10) is transferred to the L-methionine-producing E. coli 218 strain (VKPM B-8125, RU2209248 C2) by P1-transduction. As a result, the E. coli 218ΔilvA::λattL-kan^R-λattR strain is obtained.

E. coli strains 218 and 218ΔilvA::λattL-kan^R-λattR are separately cultivated with shaking at 37°C for 18 hours in LB liquid culture medium. Then, 0.2 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 × 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker at 250 rpm.

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

Auxiliary example 1. Construction of P. ananatis L-methionine-producing strain C2691
1.1. 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: 13) replaced with cassette λattL-kan^R-λattR-Pnlp8sd22 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR-Pnlp8sd22 DNA fragment having sequences homologous to the upstream and downstream of promoter region of cysM gene at both termini was obtained by PCR using the primers P9 (SEQ ID NO: 14) and P10 (SEQ ID NO: 15), and pMW118-attL-kan-attR-Pnlp8sd22 plasmid (SEQ ID NO: 16) 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 (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 35 mg/L 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: 17) and P12 (SEQ ID NO: 18) 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.

1.2. 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: 19) with cassette λattL-kan^R-λattR-Pnlp8sd22-cysM was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR-Pnlp8sd22-cysM DNA fragment having sequences homologous to the upstream and downstream of mdeA gene at both termini was obtained by PCR using the primers P13 (SEQ ID NO: 20) and P14 (SEQ ID NO: 21), and chromosome isolated from the strain C2338 (Auxiliary example 1.1) 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 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: 22) and P16 (SEQ ID NO: 23) 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.

1.3. Construction of P. ananatis C2603 strain (SC17ΔmdeA::λattL-kan^R-λattR-Pnlp8sd22-cysM)
Chromosome 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 chromosome 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 (v/v) to obtain competent cells. Chromosome DNA isolated from the strain C2597 (Auxiliary example 1.2) 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 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 1.2 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.

1.4. 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 tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L 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.

1.5. Construction of P. ananatis C2607 strain (SC17(0)ΔmetJ::λattL-cat^R-λattR)
The P. ananatis SC17(0)ΔmetJ::λattL-cat^R-λattR strain having deleted the metJ gene (SEQ ID NO: 24) was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER strain was cultured in the LB liquid culture medium overnight. Then, 1 mL of the cultured medium was inoculated to 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 (v/v) to obtain competent cells. An amplified λattL-cat^R-λattR DNA fragment having sequences homologous to the upstream and downstream of metJ gene at both termini was obtained by PCR using the primers P17 (SEQ ID NO: 25) and P18 (SEQ ID NO: 26), 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 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: 27) and P20 (SEQ ID NO: 28) 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.

1.6. Construction of P. ananatis C2634 strain (C2614ΔmetJ::λattL-cat^R-λattR)
Chromosome DNA was isolated from the strain C2607 (SC17(0)ΔmetJ::λattL-cat^R-λattR) (Auxiliary example 1.5) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of C2614 strain (Auxiliary example 1.4). 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 (v/v) to obtain competent cells. Chromosome DNA isolated from the strain C2607 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 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 1.5 to confirm the replacement of metJ gene. As a result, the P. ananatis C2614ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2634) was obtained.

1.7. Construction of P. ananatis C2605 strain (SC17(0) λattL-kan^R-λattR-Ptac71φ10-metA)
The P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metA strain having replaced promoter region of metA gene (SEQ ID NO: 29) with cassette λattL-kan^R-λattR-Ptac71φ10 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR-Ptac71φ10 DNA fragment having sequences homologous to the upstream and downstream of promoter region of metA gene at both termini was obtained by PCR using the primers P21 (SEQ ID NO: 30) and P22 (SEQ ID NO:31), and pMW118-attL-kan-attR-Ptac71φ10 plasmid (SEQ ID NO:32) 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 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: 33) and P24 (SEQ ID NO: 34) 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.

1.8. Construction of P. ananatis C2611 strain (SC17λattL-kan^R-λattR-Ptac71φ10-metA)
Chromosome DNA was isolated from the strain C2605 (SC17(0)λattL-kan^R-λattR-Ptac71φ10-metA) (Auxiliary example 1.7) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome 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 (v/v) to obtain competent cells. Chromosome 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 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 1.7 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.

1.9. Construction of P. ananatis C2619 strain (C2611ΔmetJ::λattL-cat^R-λattR)
Chromosome DNA was isolated from the strain C2607 (SC17(0)ΔmetJ::λattL-cat^R-λattR) (Auxiliary example 1.5) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of C2611 strain (Auxiliary example 1.8). 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 (v/v) to obtain competent cells. Chromosome 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 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 1.5 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.

1.10. 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). 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: 35). The amino acid sequence of the mutant MetA protein having the R34C mutation is shown in SEQ ID NO: 37, and the nucleotide sequence of the mutant metA gene encoding the mutant MetA protein is shown in SEQ ID NO: 36. Thus, the P. ananatis SC17λattL-kan^R-λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat^R-λattR strain (abbreviated as C2664) was constructed.

1.11. Construction of P. ananatis C2669 strain (C2634λattL-kan^R-λattR-Ptac71φ10-metA(R34C))
Chromosome DNA was isolated from the strain C2664 (SC17λattL-kan^R-λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat^R-λattR) (Auxiliary example 1.10) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of C2634 strain (Auxiliary example 1.6). 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 (v/v) to obtain competent cells. Chromosome 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 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 1.7 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.

1.12. 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 1.11) using an RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into C2669 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 C2669/RSF(TcR)-int-xis strain.

The resulting plasmid-harboring strain was refined in the LB culture medium containing 15 mg/L tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L 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.

Auxiliary example 2. Construction of P. ananatis L-methionine-producing strain C2792
2.1. Construction of P. ananatis SC17(0)ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442
The P. ananatis SC17(0)ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442 strain having replaced gene metE1 (c0742) (SEQ ID NO: 38) with cassette λattL-kan^R-λattR-Ptac71φ10-thrA442 (SEQ ID NO: 39) was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR-Ptac71φ10-thrA442 DNA fragment having sequences homologous to the upstream and downstream of metE1 gene at both termini was obtained by PCR using the primers P25 (SEQ ID NO: 40) and P26 (SEQ ID NO: 41). 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 kanamycin, and cultured at 34°C for 16 hours. Emerging colonies were refined in the same culture medium. Then, a PCR analysis was performed using primers P27 (SEQ ID NO: 42) and P28 (SEQ ID NO: 43) to confirm that the metE1 gene on the chromosome was replaced with the λattL-kan^R-λattR-Ptac71φ10-thrA442 cassette. As a result, the P. ananatis SC17(0)ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442 strain was obtained.

2.2. Construction of P. ananatis C2707 strain (C2691ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442)
Chromosome DNA was isolated from the strain SC17(0)ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442 using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of P. ananatis C2691 strain (Auxiliary example 1). For this purpose, P. ananatis C2691 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 (v/v) to obtain competent cells. Chromosome DNA isolated from the strain SC17(0)ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442 (Auxiliary example 2.1) 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 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 2.1 to confirm the replacement of metE1 gene. As a result, the P. ananatis C2691ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442 strain (abbreviated as C2707) was obtained.

2.3. Deletion of kan gene from C2707 strain (C2691ΔmetE1::λattL-kan^R-λattR-Ptac71φ10-thrA442)
The kanamycin resistant gene (kan) was deleted from C2707 strain using an RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into C2707 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 C2707/RSF(TcR)-int-xis strain.

The resulting plasmid-harboring strain was refined in the LB culture medium containing 15 mg/L tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L 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 С2743.

2.4. Construction of P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metH strain
The P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metH strain having a promoter region of metH gene (SEQ ID NO: 44) replaced with cassette λattL-kan^R-λattR-Ptac71φ10 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-kan^R-λattR-Ptac71φ10 DNA fragment having sequences homologous to the upstream and downstream of promoter region of metH gene at both termini was obtained by PCR using the primers P29 (SEQ ID NO: 45) and P30 (SEQ ID NO: 46), and pMW118-attL-kan^R-attR-Ptac71φ10 plasmid (SEQ ID NO: 32) 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 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); 25 cycles at 97°C for 20 seconds, 54°C for 20 seconds and 72°C for 100 seconds) was performed using primers P31 (SEQ ID NO: 47) and P32 (SEQ ID NO: 48) to confirm that the promoter region of metH gene on the chromosome was replaced with the λattL-kan^R-λattR-Ptac71φ10 cassette. As a result, the P. ananatis SC17(0)λattL-kan^R-λattR-Ptac71φ10-metH strain was obtained.

2.5. Construction of P. ananatis C2761 strain (С2743 λattL-kan^R-λattR-Ptac71φ10-metH)
Chromosome DNA was isolated from the strain SC17(0)λattL-kan^R-λattR-Ptac71φ10-metH (Auxiliary example 2.4) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of P. ananatis С2743 strain (Auxiliary example 2.3). For this purpose, P. ananatis С2743 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 (v/v) to obtain competent cells. Chromosome DNA isolated from the strain SC17(0) λattL-kan^R-attR-Ptac71φ10-metH 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 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 2.4 to confirm that the promoter region of metH gene on the chromosome was replaced with the λattL-kan^R-λattR-Ptac71φ10 cassette. As a result, the P. ananatis С2743λattL-kan^R-λattR-Ptac71φ10-metH strain (abbreviated as C2761) was obtained.

2.6. Construction of P. ananatis SC17(0)λattL-cat^R-λattR-Pnlp8-gcvT strain
The P. ananatis SC17(0)λattL-cat^R-λattR-Pnlp8-gcvT strain having a promoter region of gcvT gene (SEQ ID NO: 49) replaced with cassette λattL-cat^R-λattR-Pnlp8 was constructed using λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER 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 (v/v) to obtain competent cells. An amplified λattL-cat^R-λattR-Pnlp8 DNA fragment having sequences homologous to the upstream and downstream of promoter region of gcvT gene at both termini was obtained by PCR using the primers P33 (SEQ ID NO: 50) and P34 (SEQ ID NO: 51), and pMW118-attL-cat^R-attR-Pnlp8 plasmid (WO2011043485) 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 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 97°C for 20 seconds, 54°C for 20 seconds and 72°C for 120 seconds) was performed using primers P35 (SEQ ID NO: 52) and P36 (SEQ ID NO: 53) to confirm that the promoter region of gcvT gene on the chromosome was replaced with the λattL-cat^R-λattR-Pnlp8 cassette. As a result, the P. ananatis SC17(0)λattL-cat^R-λattR-Pnlp8-gcvT strain was obtained.

2.7. Construction of P. ananatis C2777 strain (C2761 λattL-cat^R-λattR-Pnlp8-gcvT)
Chromosome DNA was isolated from the strain SC17(0)λattL-cat^R-λattR-Pnlp8-gcvT (Auxiliary example 2.6) using EdgeBio PurElute Bacterial Genomic kit according to manufacturer instructions. The resulting chromosome DNA was used for transformation of P. ananatis C2761 strain (Auxiliary example 2.5). For this purpose, P. ananatis C2761 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 (v/v) to obtain competent cells. Chromosome DNA isolated from the strain SC17(0)λattL-cat^R-λattR-Pnlp8-gcvT 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 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 2.7 to confirm that the promoter region of gcvT gene on the chromosome was replaced with the λattL-cat^R-λattR-Pnlp8 cassette. As a result, the P. ananatis C2761λattL-cat^R-λattR-Pnlp8-gcvT strain (abbreviated as C2777) was obtained.

2.8. Deletion of kan and cat genes from C2777 strain (C2761λattL-cat^R-λattR-Pnlp8-gcvT)
The kanamycin and chloramphenicol resistant genes (kan and cat, correspondingly) were deleted from C2777 strain (Auxiliary example 2.7) using an RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into C2777 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 C2777/RSF(TcR)-int-xis strain.

The resulting plasmid-harboring strain was refined in the LB culture medium containing 15 mg/L tetracycline and 1 mM IPTG to obtain single colonies. Then, a single colony was applied onto the culture medium containing 50 mg/L 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 C2792.

While the invention has been described in detail with reference to preferred 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 in a culture medium an L-amino acid producing bacterium belonging to the order Enterobacterales to produce and accumulate the L-amino acid in the culture medium or cells of the bacterium, or both, and
(ii) collecting the L-amino acid from the culture medium or the cells of the bacterium, or both,
wherein said bacterium has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity.
The method according to claim 1, wherein said gene encoding a protein having indolepyruvate decarboxylase activity is an ipdC gene.
The method according to claim 1 or 2, wherein said protein having indolepyruvate decarboxylase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55,
(B) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55, but which includes substitution, deletion, insertion, and/or addition of 1 to 250 amino acid residues, and wherein said protein has indolepyruvate decarboxylase activity, and
(C) a protein comprising an amino acid sequence having an identity of not less than 50% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 55, and wherein said protein has indolepyruvate decarboxylase activity.
The method according to any one of claims 1 to 3, wherein said gene is selected from the group consisting of:
(a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 54,
(b) a gene comprising a nucleotide sequence that is able to hybridize under stringent conditions with a nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1 or 54, and wherein the gene encodes a protein having indolepyruvate decarboxylase activity,
(c) a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 55, but which includes substitution, deletion, insertion and/or addition of 1 to 250 amino acid residues, and wherein said protein has indolepyruvate decarboxylase activity, and
(d) a gene comprising a variant nucleotide sequence of SEQ ID NO: 1 or 54, wherein the variant nucleotide sequence is due to the degeneracy of the genetic code.
The method according to any one of claims 1 to 4, wherein said expression of the gene encoding a protein having indolepyruvate decarboxylase activity is attenuated due to inactivation of the gene.
The method according to claim 5, wherein said gene encoding a protein having indolepyruvate decarboxylase activity is deleted.
The method according to any one of claims 1 to 6, wherein said bacterium has been modified further to attenuate expression of a gene encoding a protein having threonine deaminase activity.
The method according to claim 7, wherein said gene encoding a protein having threonine deaminase activity is an ilvA gene.
The method according to any one of claims 1 to 8, wherein said bacterium belongs to the family Enterobacteriaceae or Erwiniaceae.
The method according to claim 9, wherein said bacterium belongs to the genus Escherichia or Pantoea.
The method according to claim 10, wherein said bacterium is Escherichia coli or Pantoea ananatis.
The method according to any one of claims 1 to 11, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid, a non-aromatic L-amino acid, and a sulfur-containing L-amino acid.
The method according to claim 12, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, and L-tyrosine.
The method according to claim 12, 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, and L-valine.
The method according to claim 12, wherein said sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, and L-cystine.