WO2015030019A1 - A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING ATTENUATED EXPRESSION OF THE znuACB GENE CLUSTER - Google Patents

Abstract

The present invention provides a method for producing L-amino acids such as L-amino acids belonging to the glutamate family by fermentation using a bacterium of the familyEnterobacteriaceae, particularly a bacterium belonging to the genusEscherichia, which has been modified to attenuate expression of at least one of the genes from theznuACB gene cluster.

Description

DESCRIPTION

A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING ATTENUATED EXPRESSION OF

THE znuACB GENE CLUSTER

Background of the Invention

Technical Field

The present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium of the family Enterobacteriaceae that has been modified to attenuate expression of at least one of the genes from the znuACB gene cluster.

Background 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 acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Patent No. 4,278,765 A) and alteration of regulatory regions such as promoter, leader sequence, and/or attenuator, or others known to the person skilled in the art (see, for example, US20060216796 Al and W09615246 Al). 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, W09516042 Al, EP0685555 Al or U.S. Patent Nos. 4,346,170 A, 5,661,012 A, and 6,040,160 A).

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

The znuACB (zinc uptake) gene cluster encodes a ZnuABC protein complex, which is a high-affinity zinc uptake system belonging to the ATP-binding cassette (ABC) transporter superfamily, one of the largest protein families known (Wu L.F. and Mandrand-Berthelot M.A. A family of homologous substrate-binding proteins with a broad range of substrate specificity and dissimilar biological functions, Biochimie, 1995, 77(9):744-750). ABC transporters have a common distinctive architecture, which consists of two transmembrane domains (TMDs) embedded in the membrane bilayer and two nucleotide-binding domains (NBDs) located in the cytoplasm (Rees D.C. et al., ABC transporters: the power to change, Nat. Rev. Mol. Cell Biol, 2009, 10(3): 218-227). The prokaryotic ABC transporters can recruit a binding protein to translocate substrates. ATP binding to the nucleotide-binding domains and hydrolysis drive the conformational changes that result in the translocation of a substrate. Functioning as importers or exporters, ABC transporters can transport a wide variety of substrates, which include ions, toxins, antibiotics, lipids, polysaccharides, nutrients such as amino acids, peptides, sugars, and so forth.

ZnuA encodes the periplasmic zinc-binding component of the transporter, while znuB encodes the membrane component, and znuC encodes the ATPase subunit. High nucleotide sequence similarity was observed for the three components of ZnuABC from Escherichia coli (E. coli, Enterobacteriaceae) and the corresponding components of the AdcABC zinc transporter of Streptococcus pneumoniae (Streptococcaceae) as well as subunits of other ABC metal ion transporters (Patzer S.I. and Hantke K. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli, Mol. Microbiol., 1998, 28(6): 1199-1210).

Until now, no data has been reported demonstrating the effect from attenuating at least one of the genes from the znuACB gene cluster on L-amino acids production by the modified bacterial strains of the family Enterobacteriaceae.

Summary of the Invention

An aspect of the present invention is to provide a bacterium belonging to the family Enterobacteriaceae, which can belong to the genus Escherichia and, more specifically, to the species E. coli, which has been modified to attenuate expression of at least one of the genes from the znuACB gene cluster.

Another aspect of the present invention is to provide a method for producing L- amino acids such as 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 using a bacterium of the family Enterobacteriaceae as described hereinafter. These aims were achieved by the unexpected finding that attenuation of expression of at least one of the genes from the znuACB gene cluster, in particular inactivation of the znuACB genes, on the chromosome of a bacterium belonging to the family Enterobacteriaceae, which can belong to the genus Escherichia and, more specifically, to the species E. coli, confers on the microorganism higher productivity of an L-amino acid, in particular, but is not limited to the L-amino acid belonging to the glutamate family such as L-arginine. This finding has resulted in the following non- limiting aspects of the present invention.

An aspect of the present invention is to provide a method for producing an L- amino acid comprising:

(i) cultivating an L-amino acid-producing bacterium of the family

Enterobacteriaceae in a culture medium to produce said L-amino acid in the bacterium and/or the culture medium,

(ii) collecting said L-amino acid from the bacterium and/or the culture medium, wherein the bacterium has been modified to attenuate expression of at least one of the genes from t e znuACB gene cluster.

It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the genus Escherichia.

It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is Escherichia coli.

It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is Pantoea ananatis.

It is a further aspect of the present invention to provide the method as described above, wherein the expression of at least one of the genes from the znuACB gene cluster is attenuated due to inactivation ofsaid gene(s).

It is a further aspect of the present invention to provide the method as described above, wherein at least one of the genes from the znuACB gene cluster is deleted.

It is a further aspect of the present invention to provide the method as described above, wherein the znuACB gene cluster is deleted.

It is a further aspect of the present invention to provide the method as described above, wherein the genes from the znuACB gene cluster are znuA, znuB and znuC.

It is a further aspect of the present invention to provide the method as described above, wherein the 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-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- valine, and combinations thereof.

It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acid is selected from the group consisting of L-arginine, L- citrulline, L-glutamic acid, L-glutamine, L-ornithine, L-proline, and combinations thereof.

Description of the Embodiments

The present invention is described in detail below.

1. Bacterium

The phrase "an L-amino acid-producing bacterium" can mean a bacterium of the family Enterobacteriaceae which has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium or 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 is able to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain, such as E. coli K-12, and can mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5 g/L or not less than 1.0 g/L of the target L-amino acid. The bacterium can produce either one kind of amino acid solely, or a mixture of two or more kinds of amino acids.

The phrase "L-amino acid-producing ability" can mean the ability of the bacterium to produce, excrete or secrete, and/or cause accumulation of the L-amino acid in a medium or the bacterial cells to such a level that the L-amino acid can be collected from the medium or the bacterial cells, when the bacterium is cultured in the medium.

The phrase "L-amino acid" can mean 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.

The phrase "aromatic L-amino acid" includes, for example, L-phenylalanine, L- tryptophan, and L-tyrosine. As L-histidine has an aromatic moiety, specifically, an imidazole ring, the phrase "aromatic L-amino acid" can also include, besides the aforementioned aromatic L-amino acids, L-histidine. The phrase "non-aromatic L-amino acid" includes, for example, 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 include, besides the aforementioned non-aromatic L-amino acids, L-histidine.

An L-amino acid can belong to more than one L-amino acid family. As an example, L-amino acids belonging to the glutamate family include L-arginine, L-glutamic acid, L-glutamine, and L-proline; L-amino acids belonging to the serine family include L- cysteine, glycine, and L-serine; L-amino acids belonging to the aspartate family include L-asparagine, L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine; L- amino acids belonging to the pyruvate family include L-alanine, L-isoleucine, L-valine, and L-leucine; and L-amino acids belonging to the aromatic family include L- phenylalanine, L-tryptophan, and L-tyrosine. As some L-amino acids can be an intermediate in the biosynthetic pathway of another 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.

L-Arginine, L-cysteine, L-glutamic acid, L-histidine, L-isoleucine, L-lysine, L- ornithine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, and L-valine are particular examples. The glutamate family amino acids such as L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline are specific examples. L- Arginine is more specific example.

The phrase "L-amino acid" includes not only an L-amino acid in a free form, but may also include a salt or a hydrate of the L-amino acid, or an adduct formed by the L- amino acid and another organic or inorganic compound as described hereinafter. Salts of amino acids include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts.

The bacteria belonging to the family Enterobacteriaceae can be from the genera Escherichia and/or Pantoea, and so forth, and can have the ability to produce an L-amino acid. Specifically, those classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543) can be used. Examples of strains from the family Enterobacteriaceae that can be modified include a bacterium of the genus Escherichia, Enterobacter or Pantoea.

Strains of Escherichia bacterium which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used (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 E. coli is a particular example. Specific examples of E. coli include E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), and so forth, which are derived from the prototype wild- type strain, E. coli K-12 strain. These strains are available from, for example, the

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

Examples of the Enterobacter bacteria include Enterobacter agglomerans,

Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteria include Pantoea ananatis, and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc. A bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae. When a Pantoea ananatis strain is bred by genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above.

L-Amino acid-producing bacteria

The bacterium of the present invention belonging to the family

Enterobacteriaceae and modified to attenuate expression of at least one of the genes from the znuACB gene cluster, which is able to produce an L-amino acid, can be used. The bacterium may inherently have the L-amino acid-producing ability or may be modified to have an L-amino acid-producing ability by using a mutation method or DNA recombination techniques. The bacterium can be modified to attenuate expression of at least one of the genes from the znuACB gene cluster in a bacterium, which inherently has the ability to produce an L-amino acid. Alternatively, the bacterium can be obtained by imparting the ability to produce an L-amino acid to a bacterium already modified to attenuate expression of at least one of the genes from the znuACB gene cluster.

L-Arginine-producing bacteria

Examples of 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) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N-acetylglutamate synthase (RU2215783), E. coli strain 382 (VKPM B-7926, EPl 170358 Al), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 170361 Al), and the like.

Examples of 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-y-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).

L-Citrulline-producing bacteria

Examples of 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 mutant N-acetylglutamate synthase strains 237/pMADSl 1 , 237/pMADS12, and 237/pMADS13 (Russian patent No. 2215783, European patent No. 1 170361 Bl , U.S. Patent No. 6790647 B2). E. coli strains 333 (VKPM B-8084) and 374 (VKPM B-8086), both of which harbor mutant feedback- resistant carbamoyl phosphate synthetase

(Russian Patent RU2264459 C2), E. coli strains in which a-ketoglutarate synthase activity is increased and ferredoxin NADP⁺ reductase, pyruvate synthase or a-ketoglutarate dehydrogenase activities are additionally modified (EP 2133417 Al), and P. ananantis strain NAlsucAsdhA in which succinate dehydrogenase and a-ketoglutarate dehydrogenase activities are decreased (US Patent Application No 2009286290), and the like, are further examples.

As L-citrulline is an intermediate of L-arginine biosynthetic pathway, examples of parent 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 (argF/Γ), and carbamoyl phosphate synthetase (carAB), and combinations thereof.

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.

L-Cysteine-producing bacteria

Examples of parental strains which can be used to derive L-cysteine-producing bacteria include, but are not limited to strains belonging to the genus Escherichia such as E. coli JM15 which is transformed with different cysE alleles encoding feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent No. 2279477), E. coli W31 10 having overexpressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663), E. coli strains having lowered cysteine desulfohydrase activity (JP11 155571 A2), E. coli W31 10 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307 Al), E. coli JM15(ydeD) (U.S. Patent No. 6,218,168), and the like.

L-Glutamic acid-producing bacteria

Examples of 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⁺ (EP 1 172433). The E. coli VL334 (VKPM B- 1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in thrC and ilvA genes (U.S. Patent No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage PI 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 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 (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase ( ceEF, IpdA), 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 (flip), phosphofructokinase (pfkA, pflcB), and glucose phosphate isomerase (pgi).

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 EP 1078989 A2, EP955368 A2, and EP952221 A2.

Examples of parental strains which can be used to derive the L-glutamic acid- producing bacteria also include strains having 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), a-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase ( vG), acetolactate synthase (ilvl), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB). Bacteria belonging to the genus Escherichia deficient in the a-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Specifically, these strains include the following:

E. coli W31 10sucA::Km^R

E. coli AJ 12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ 12949 (FERM BP-4881)

E. coli W31 10sucA::Km^R is a strain obtained by disrupting the a-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W31 10. This strain is completely deficient in α-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) (U.S. Patent No. 5,908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671), AJ13138 (FERM BP-5565) (U.S. Patent No. 6,1 10,714), and the like.

Examples of L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea that are deficient in the -ketoglutarate dehydrogenase activity or have a decreased a-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356. (U.S. Patent No.

6,331,419). Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currentlyjncorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292- 0818, JAPAN) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 1 1, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in a-ketoglutarate dehydrogenase activity as a result of disruption of the a GDH-El subunit gene {sue A). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.

L-Histidine-producing bacteria

Examples of 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), E. coli strain 80 (VKPM B-7270, RU21 19536), E. coli NRRL B-121 16 - B12121 (U.S. Patent No. 4,388,405), E. coli H- 9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347), E. coli H-9341 (FERM BP-6674) (EP1085087), E. coli AI80/pFM201 (U,S. Patent No. 6,258,554), and the like.

Examples of 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-AMP cyclohydrolase (hisl), 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 L-histidine-producing ability can also be efficiently enhanced by introducing a mutation into ATP

phosphoribosyltransferase so that resistance to the feedback inhibition is

conferred(Russian Patent Nos. 2003677 and 2119536).

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 (JP 56-005099 A), E. coli strains transformed with rht, a gene for an amino acid-export (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, RU2119536), and E. coli MG1655+AwGr hisL'_ purR (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 parental strains which can be used to derive L-isoleucine-producing bacteria include, but are not limited to mutants having resistance to 6- dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 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 parental strains (JP 2-458 A, EP0356739 Al, and U.S. Patent No.

5,998,178).

L-Leucine-producing bacteria

Examples of 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, U.S. Patent No. 6,124,121)) or leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4- azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.

The bacterium can be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which can be represented by a mutant leuA gene encoding isopropylmalate synthase freed from feedback inhibition by L-leucine (U.S. Patent No. 6,403,342). In addition, the bacterium can be improved by enhancing the expression of one or more genes encoding proteins that excrete L-amino acid from the bacterial cell. Examples of such genes include b2682 and b2683 (ygaZH genes) (EP1239041 A2).

L-Lysine-producing bacteria

Examples of L-lysine-producing bacteria belonging to the family Escherichia include mutants 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, oc-chlorocaprolactam, and so forth. Mutants 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 AJ1 1442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and E. coli VL61 1. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

Examples of parental strains which can be used to derive L-lysine-producing bacteria also include, but are not limited to strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate

decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase (aspA) (EP1253195 Al). In addition, the parental strains may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP1 170376 Al), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof. L-Amino acid-producing bacteria may have 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, the bacteria may have reduced or no activity of an enzyme that negatively acts on L-amino acid synthesis or accumulation. Examples of such enzymes involved in L-lysine production include homoserine dehydrogenase, lysine decarboxylase (cadA, IdcC), malic enzyme, and so forth, and strains in which activities of these enzymes are decreased or deleted are disclosed in W095/23864, WO96/17930, WO2005/010175, and so forth.

Expression of both the cadA and IdcC 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

WO2006/078039.

Examples of L-lysine-producing bacteria can include the E. coli

WC196AcadAAldcC/pCABD2 strain (WO2006/078039). The strain was constructed by introducing the plasmid pCABD2 containing lysine biosynthesis genes (U.S. Patent No. 6,040,160) into the WC196 strain having disrupted cadA and IdcC genes which encode lysine decarboxylase.

The WC196 strain was bred from the W31 10 strain, which was derived from E. coli K-12 by replacing the wild-type lysC gene on the chromosome of the W31 10 strain with a mutant lysC gene encoding a mutant aspartokinase III in which threonine at position 352 was replaced with isoleucine, resulting in desensitization of the feedback inhibition by L-lysine (U.S. Patent No. 5,661,012), and conferring AEC resistance to the resulting strain (U.S. Patent No. 5,827,698). The WC196 strain was designated E. coli A J 13069, deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292- 0818, JAPAN) on December 6, 1994, and assigned an accession number of FERM P- 14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and assigned an accession number of FERM BP-5252 (U.S. Patent No. 5,827,698).

The WC 196AcadAAldcC strain itself is also an exemplary L-lysine-producing bacterium. The WC196AcadAAldcC was designated AJ1 10692 and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on October 7, 2008 as an international deposit under an accession number of FERM BP-1 1027.

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

Escherichia bacteria strains such as strains AJ1 1539 (NRRL B-12399), AJ1 1540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (patent GB2075055); strains 218 (VKPM B-8125) (patent RU2209248) and 73 (VKPM B-8126) (patent RU2215782) resistant to norleucine, the L-methionine analog, or the like. The strain E. coli 73 has been deposited in the Russian National Collection of Industrial

Microorganisms (VKPM) (Russian Federation, 1 17545 Moscow, 1^st Dorozhny Proezd, 1) on May 14, 2001 under accession number VKPM B-8126, and was converted to an international deposit under the Budapest Treaty on February 1, 2002. Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine

transsuccinylase and cystathionine γ-synthase (JP 2000-139471 A) can also be used as parent strains.

L-Ornithine-producing bacteria

L-oraithine-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 argl genes. Methods for inactivation of ornithine carbamoyltransferase are described herein.

L-Phenylalanine-producing bacteria

Examples of 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::Tnl0, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672), E. coli MWEClOl-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B- 12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, E. coli K-12 [W31 10 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W31 10 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662), and E. coli K-12 [W31 10 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used as a parental strain (EP488424 Bl). Furthermore, L-phenylalanine-producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).

L-Proline-producing bacteria

Examples of 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 (EP1 172433 Al). The bacterium can be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of 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 Al). In addition, the bacterium can be improved by enhancing the expression of one or more genes encoding proteins responsible for excreting L-amino acids from the bacterial cell. Such genes are exemplified by b2682 and b2683 (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B- 12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application No.

2000124295), plasmid mutants described in DE3127361 Al, 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 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) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (Russian), 1978, 14:947-956), E. coli VL643 and VL2055 (EPl 14991 1 A2), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as being sucrose- assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSFlOlO-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Russian Federation, 1 17105 Moscow, Nagatinskaya Street 3 -A) under the accession number RIA 1867. The strain 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 B-3996.

E. coli VKPM B-5318 (EP0593792 Al) may also be used as a parental strain for deriving L-threonine-producing bacteria. The strain B-5318 is prototrophic with regard to isoleucine; and a temperature-sensitive lambda-phage CI 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 of VKPM B-5318.

The bacterium 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-P-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 entry No. b0002; GenBank accession No.

NC_000913.2; nucleotide positions: 337 to 2,799; Gene ID: 945803). The thrA gene is located between the thrL and thrB genes on the chromosome of 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_000913.2; nucleotide positions: 2,801 to 3,733; Gene ID: 947498). The thrB gene is located between the thrA and thrC genes on the chromosome of 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_000913.2; nucleotide positions: 3,734 to 5,020; Gene ID: 945198). The thrC gene is located between the thrB zndyaaX 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 transcription is desirably removed from the operon (WO2005049808 Al, WO2003097839 Al).

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 U.S. Patent No. 5,705,371.

The rhtA gene which encodes a protein of the threonine and homoserine efflux system (an inner membrane transporter) of E. coli has been elucidated (KEGG entry No. b0813; GenBank accession No. NC 000913.2; nucleotide positions: 848,433 to 849,320, complement; Gene ID: 947045). The rhtA gene is located between the dps and ompX genes on the chromosome of 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-P-semialdehyde dehydrogenase of E. coli has been elucidated (KEGG entry No. b3433; GenBank accession No. NC 000913.2; nucleotide positions: 3,571,798 to 3,572,901, complement; Gene ID: 947939). The asd gene is located between the glgB and gntU gene on the same strand (yhgN gene on the opposite strand) on the chromosome of 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_000913.2; nucleotide positions: 983,742 to 984,932, complement; Gene ID: 945553). The aspC gene is located between the ycbL gene on the opposite strand and the ompF gene on the same strand on the chromosome of E. coli K-12.

L-Tryptophan-producing bacteria

Examples of 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 (U.S. Patent No. 5,756,345), 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 (U.S. Patent No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B- 12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371 ,614), E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L- tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).

Examples of 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, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include an E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

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

L-Valine-producing bacteria

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

Examples of parental strains for deriving L-valine-producing bacteria also include mutants having a mutation of aminoacyl-tRNA synthetase (U.S. Patent No. 5,658,766). For example, E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 1 17545 Moscow, 1^st Dorozhny Proezd, 1) on June 24, 1988 under the accession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lacking H⁺-ATPase can also be used as parental strains (WO96/06926).

Examples of L-valine-producing strain include E. coli strain H-81 (VKPM B- 8066), NRRL B-12287 and NRRL B-12288 (US patent No. 4,391,907), VKPM B-4411 (US patent No. 5,658,766), VKPM B-7707 (European patent application EP1016710A2), or the like.

The bacterium of the present invention belonging to the family

Enterobacteriaceae has been modified to attenuate expression of at least one of the genes from the znuACB gene cluster.

The phrase "a bacterium modified to attenuate expression of at least one of the genes from the znuACB gene cluster" can mean that the bacterium has been modified in such a way that in the modified bacterium, expression of at least one of the genes from the znuACB gene cluster is decreased as compared to a bacterium which contains a non- modified znuACB gene cluster, for example, a wild-type or parental strain, or at least one of the genes from the znuACB gene cluster is inactivated. In the modified bacterium, any of the genes znuA, znuC or znuB (also referred to as znuACB genes), or a combination thereof may be attenuated. For example, all three genes znuA, znuC and znuB constituting the entire znuACB gene cluster can be attenuated.

The phrase "at least one of the genes from the znuACB gene cluster is inactivated" can mean that a modified gene from the znuACB gene cluster such as znuA, znuC or znuB encodes a completely inactive or non-functional protein As the protein products of the znuACB genes constitute an ZnuABC transporter, the phrase "at least one of the genes from the znuACB gene cluster is inactivated" can also mean that a modified gene from the znuACB gene cluster such as znuA, znuC or znuB encodes a completely inactive or nonfunctional protein so that the ZnuABC transporter is inactive or non-functional, as compared to a bacterium which contains the non-modified gene. Furthermore, the phrase "at least one of the genes from the znuACB gene cluster is inactivated" can also mean that a combination of two or three genes selected from znuA, znuC and znuB may be chosen to inactivate the genes from the znuACB gene cluster so that the modified genes encode completely inactive or non-functional proteins so that the ZnuABC transporter is inactive or non-functional, as compared to a bacterium which contains the non-modified genes.

It is also possible that at least one of the modified DNA regions from the znuACB gene cluster 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(s), enhancer(s), attenuator(s), ribosome-binding site(s) (RBS), etc. Inactivation of at least one of the genes from the znuACB gene cluster can also be performed, for example, by conventional methods such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N- nitrosoguanidirie), site-directed mutagenesis, gene disruption using homologous recombination, and/or insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97(1 1):5978-5983; Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., Nature Genet., 1998, 20: 123-128) based on "Red/ET-driven integration" or '^Red/ET-mediated integration".

The phrase "at least one of the genes from the znuACB gene cluster is attenuated" can mean that an amount of the protein encoded by a modified gene from the znuACB gene cluster such as znuA, znuC or znuB in the modified bacterium, in which expression of the gene is attenuated, is reduced as compared with a non-modified bacterium, for example, a wild-type or parental strain such as E. coli K-12. As the protein products of the znuACB genes constitute an ZnuABC transporter, the phrase "at least one of the genes from the znuACB gene cluster is attenuated" can also mean that an amount of the

ZnuABC transporter, a component or subunit of which is encoded by the gene, in the modified bacterium, in which expression of the gene is attenuated, is reduced as compared with a non-modified bacterium. Furthermore, the phrase "at least one of the genes from the znuACB gene cluster is attenuated" can also mean that a combination of two or three genes selected from znuA, znuC and znuB may be chosen to attenuate the genes from the znuACB gene cluster so that an amount of the proteins encoded by modified genes from the znuACB gene cluster or an amount of the ZnuABC transporter in the modified bacterium, in which expression of the genes is attenuated, is reduced as compared with a non-modified bacterium.

The phrase "at least one of the genes from the znuACB gene cluster is attenuated" can also mean that the modified bacterium contains one or more regions operably linked to one or more genes from the znuACB gene cluster, including sequences controlling expression of genes from the znuACB gene cluster such as promoters, enhancers, attenuators and transcription termination signals, ribosome-binding sites (RBS), and other expression control elements, which are modified resulting in a decrease in the expression level of at least one of the genes from the znuACB gene cluster; and other examples (see, for example, W095/34672; Carrier T.A. and Keasling J.D., Biotechnol. Prog., 1999, 15:58-64). As the znuACB gene cluster comprises the znuACB genes, the phrase "one or more genes" can mean znuA, znuC and/or znuB in any combination. The phrase "operably linked to the gene" is a specific example of the phrase "operably linked to one or more genes" and can mean that the regulatory region(s) is/are linked to the nucleotide sequence of the nucleic acid molecule or gene in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, antiterminated, attenuated, deregulated, decreased or repressed expression) of the nucleotide sequence, specifically, expression of a gene product encoded by the nucleotide sequence.

Expression of at least one of the genes from the znuACB gene cluster 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 and strong 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 a nucleotide substitution for several nucleotides in a promoter region of a target gene and thereby modify the promoter to be weakened as disclosed in International Patent

Publication WO00/18935. 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. This modification of the RBS may be combined with decreasing transcription of at least one of the genes from the znuACB gene cluster.

Expression of at least one of the genes from the znuACB gene cluster can also be attenuated by insertion of a transposon or an insertion sequence (IS) into the coding region of the gene (U.S. Patent No. 5,175,107) or in the region controlling gene expression, or by conventional methods such as mutagenesis with ultraviolet irradiation (UV) irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Furthermore, the incorporation of a site-specific mutation can be conducted by known chromosomal editing methods based, for example, on Red/ET-mediated recombination.

The copy number, presence or absence of a gene from the znuACB gene cluster such as znuA, znuC and znuB 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.

For example, in the modified bacterium, the specific activity of ZnuABC transporter encoded by the znuACB genes per mg crude protein or an amount of cells expressed as an optical density (OD) at a specific wavelength can be determined by measuring the⁶⁵Zn²⁺-uptake using radioactive⁶⁵ZnCl₂ as the substrate and γ-counter (Patzer S.I. and Hantke K., Mol. Microbiol, 1998, 28(6):1 199-1210). The specific activity of a modified ZnuA, ZnuB or ZnuC, or their combination, can be determined by measuring activity of the entire ZnuABC protein complex, containing the modified ZnuA, ZnuB or/and ZnuC, as described above. The crude protein concentration can be determined by the Bradford protein assay (Bradford M.M., Anal. Biochem., 1976, 72:248-254) using bovine serum albumin as a standard.

Methods for manipulation with recombinant molecules of DNA and molecular cloning such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well-known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch E.F. and Maniatis T., "Molecular

Cloning: A Laboratory Manual", 2^nd ed., Cold Spring Harbor Laboratory Press (1989) or Green M.R. and Sambrook J.R., "Molecular Cloning: A Laboratory Manual", 4^th ed., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, "Molecular Biotechnology: principles and applications of recombinant DNA", 4^th ed., Washington, DC, ASM Press (2009). The znuA gene (synonyms yebL, yzzP) encodes a zinc transporter subunit, a periplasmic-binding component of ABC superfamily ZnuA (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. bl857; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P39172). The znuA (GenBank accession No. NC 000913.2; nucleotide positions: 1939675 to 1940607, complement; Gene ID: 946375) is located between the yebA gene on the same strand and the znuC gene on the opposite strand on the

chromosome of E. coli strain K-12. The nucleotide sequence of the znuA gene and the amino acid sequence of the ZnuA protein encoded by the znuA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The znuC gene (synonyms yebM) encodes a zinc transporter subunit, an ATP- binding component of ABC superfamily ZnuC (KEGG, entry No. bl 858;

UniProtKB/Swiss-Prot, accession No. P0A9X1). The znuC (GenBank accession No.

NC_000913.2; nucleotide positions: 1940686 to 1941441 ; Gene ID: 946374) is located between the znuA gene on the opposite strand and the znuB gene on the same strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the znuC gene and the amino acid sequence of the ZnuC protein encoded by the znuC gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The znuB gene (synonyms yebl encodes a zinc transporter subunit, a membrane component of ABC superfamily ZnuB (KEGG, entry No. M859; UniProtKB/Swiss-Prot, accession No. P39832). The znuB (GenBank accession No. NC 000913.2; nucleotide positions: 1941438 to 1942223, complement; Gene ID: 946372) is located between the znuC on the same strand and the ruvB gene on the opposite strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the znuB gene and the amino acid sequence of the ZnuB protein encoded by the znuB gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Since there may be some differences in DNA sequences between the genera, species or strains of the family Enter obacteriaceae, the znuA, znuC and znuB genes are not limited to the genes shown in SEQ ID NOs: 1, 3 and 5, respectively, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1, 3 and 5, and which encode variants of the ZnuA, ZnuC and ZnuB proteins, respectively.

The phrase "a variant protein" can mean a protein which has one or several changes in the sequence compared with SEQ ID NO: 2, 4 or 6, 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 ZnuA, ZnuC or ZnuB protein, respectively, or the three-dimensional structure of the ZnuA, ZnuC or ZnuB protein is not significantly changed relative to the wild-type or non-modified protein. The number of changes in the variant protein depends on the position 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 30, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2, 4 or 6. 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 ZnuA, ZnuC or ZnuB protein is not significantly changed relative to the wild-type or non-modified protein. Therefore, the protein variants encoded by the zn A, znuC and znuB genes may have a homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 80%, not less than 90%, not less than 95%, or not less than 98% with respect to the entire amino acid sequence shown in SEQ ID NO: 2, 4 or 6, respectively, as long as the activity or function of the ZnuA, ZnuC and ZnuB proteins is maintained, or the three-dimensional structure of the ZnuA, ZnuC and ZnuB is not significantly changed relative to the wild- type or non-modified proteins.

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s). The representative conservative mutation 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, He and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gin, Asn_> Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gin 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 Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for He, substitution of He, Met, Val or Phe for Leu, substitution of Asn, Glu, Gin, His or Arg for Lys, substitution of lie, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, He 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, He or Leu for Val. 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 mutations in the different position(s) of amino acids sequence so that the activity or function of the variant protein is maintained and similar to that of the ZnuA, ZnuC or ZnuB protein, or the three- dimensional structure of ZnuA, ZnuC or ZnuB is not significantly changed relative to the wild-type or non-modified protein. .

To evaluate the degree of protein or DNA homology, several calculation methods can be used, such as BLAST search, FASTA search and ClustalW method. The BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87:2264-2268; "Applications and statistics for multiple high- scoring segments in molecular sequences". Proc. Natl. Acad. Sci. USA, 1993, 90:5873- 5877). The computer program BLAST calculates three parameters: score, identity and similarity. The FASTA search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods EnzymoL, 1990, 183:63-98). The ClustalW method is described by Thompson J.D. et al. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res., 1994, 22:4673-4680).

Moreover, the znuA, znuC and znuB genes can be variant nucleotide sequences. The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes the ZnuA, ZnuC or ZnuB protein using any synonymous amino acid codons according to the standard genetic code table (see, e.g., Lewin B., "Genes VIIF, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458) , or "a variant protein" of the ZnuA, ZnuC or ZnuB protein. The znuA, znuC and znuB genes can be variant nucleotide sequences due to degeneracy of genetic code.

The phrase "a variant nucleotide sequence" can also mean, but is not limited to a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, 3 or 5, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it encodes active or functional protein. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 70%, not less than 80%, not less than 90%, 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 xSSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate), or in another example, O.l xSSC, 0.1% SDS at 60°C or 65°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham Hybond™-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 sequences complementary to the sequences shown in SEQ ID NO: 1, 3 or 5 may also be used. Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequences shown in SEQ ID NO: 1 , 3 and 5 and a DNA-fragment containing the nucleotide sequence as 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 hybridization can be exemplified by 2xSSC, 0.1% SDS at 50°C, 60°C or 65°C.

As the genes encoding the ZnuA, ZnuC and ZnuB proteins of the species E. coli have already been elucidated (see above), the variant nucleotide sequences encoding variant proteins of ZnuA, ZnuC and ZnuB proteins can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189) utilizing primers prepared based on the nucleotide sequences of the znuACB genes; or the site-directed mutagenesis method by treating DNA containing the wild-type znuA, znuC or znuB gene in vitro, for example, with hydroxylamine, or a method for treating a microorganism, for example, a bacterium belonging to the family Enterobacteriaceae harboring the wild-type znuA, znuC and znuB 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 structures. Genes encoding the ZnuA, ZnuC and ZnuB proteins or its variant proteins of other microorganisms can be obtained in a similar manner. The phrase "a wild-type protein" can mean a native protein naturally produced by a wild-type or parent bacterial strain of the family Enter obacteriaceae, for example, by the wild-type E. coli MG1655 strain. A wild-type protein can be encoded by the "wild- type gene", or the "non-modified gene" naturally occurring in genome of a wild-type bacterium.

The bacterium as described herein can be obtained by attenuating expression of at least one of the genes from the znuACB gene cluster in a bacterium inherently having an ability to produce an L-amino acid. Alternatively, the bacterium as described herein can be obtained by imparting the ability to produce an L-amino acid to a bacterium

alreadyattenuating expression of at least one of the genes from the znuACB gene cluster.

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

2. Method

A method of the present invention includes the method for producing an L-amino acid such as 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, or a mixture thereof. More specifically, a method of the present invention includes the method for producing an L-amino acid of the glutamate family such as L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-proline, and L-ornithine, or a mixture thereof. The method for producing an L-amino acid can include the steps of cultivating the bacterium in a culture medium to allow the L-amino acid to be produced, excreted, or accumulated in the culture medium, and collecting the L-amino acid from the culture medium and/or the bacterial cells. Collected amino acid can be further purified. The L-amino acid can be produced in a salt or a hydrate form thereof, or a combination thereof. For example, sodium, potassium, ammonium, and the like salts of the L-amino acid can be produced by the method. The L-amino acid can be produced in an adduct form thereof with, for example, another organic or inorganic compound. Specifically, a monochlorhydrate salt of an L-amino acid can be produced by the method such as monochlorhydrate salt of L-lysine (L-lysine-HCl) or monochlorhydrate salt of L-arginine (L-arginine-HCl).

The cultivation of the bacterium, and collection and purification of L-amino acid, or a salt or hydrate thereof, from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein L-amino acid is produced using a microorganism. The culture medium for production of the L-amino acid can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, inorganic ions, and other organic and inorganic components as required. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, sucrose, arabinose, maltose, xylose, trehalose, ribose, and

hydrolyzates of starches; alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; 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 hydrolyzates; ammonia gas; aqueous ammonia; and the like can be used. The sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like. Vitamins such as vitamin B 1 , required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, or 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 the like may be added, if necessary.

Cultivation can be performed under aerobic conditions for 16 to 72 h, or for 16 to 65 h; the culture temperature during cultivation can be controlled within 30 to 45°C, or within 30 to 37°C; and the pH can be adjusted between 5 and 8, or between 6.5 and 7.5. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.

After cultivation, solids such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid, or a salt or hydrate thereof, can be recovered from the fermentation liquor by any combination of conventional techniques such as concentration, ion-exchange

chromatography, and crystallization.

The collected target L-amino acid composition may contain microbial cells, medium components, moisture, and by-product metabolites of the microorganism in addition to the target substance. Purity of the collected target substance is 50% or higher, preferably 85% or higher, particularly preferably 95% or higher (U.S. Patent No.

5,431,933, Japanese Patent No. 1214636, U.S. Patent Nos. 4,956,471, 4,777,051,

4,946,654, 5,840,358, 6,238,714, U.S. Patent Published Application No. 2005/0025878). Examples

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

Example 1. Construction of a E. coli strain having an inactivated znuACB gene cluster.

An E. coli strain in which the znuACB gene cluster is inactivated was constructed by the method initially developed by Datsenko K.A. and Wanner B.L. called " Red/ET- mediated integration" (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). A DNA-fragment containing the kanamycin resistance marker (Km^R) was obtained by PCR using primers PI (SEQ ID NO: 7) and P2 (SEQ ID NO: 8), and the pMWl \S- attL-Km^R- attR plasmid (WO2011043485 Al) as the template. The pMWl \S- attL-Km^R- attR plasmid was constructed using the pMWl \S-attL-Tc^R-attR plasmid (WO2005/010175) and substituting the tetracycline resistance marker gene with the kanamycin resistance marker gene from pUC4K plasmid (Vieira J. and Messing J., Gene, 1982, 19(3):259-268). Primer PI contains a region complementary to the region located at the 5 '-end of the znuA gene and a region complementary to the attR region. Primer P2 contains a region complementary to the region located at the 3 '-end of the znuB gene and a region complementary to the attL region. The conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final elongation for 5 min at 72°C. The obtained PCR- product (about 1.6 kbp) was purified by electrophoresis in agarose gel and used for electroporation of the E. coli MG1655 strain containing the pKD46 plasmid having a temperature-sensitive replication origin. The pKD46 plasmid (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) includes a 2,154 nucleotides DNA-fragment of phage λ (nucleotide positions from 31088 to 33241, GenBank accession No.: J02459), and contains genes of the λRed homologous recombination system (γ, β exo genes) under the control of the arabinose-inducible ParaB promoter. The pKD46 plasmid is necessary for integration of the PCR-product into the chromosome of the E. coli MG1655 strain (ATCC 47076). The E. coli MG1655 strain containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA (Accession No. CGSC7669).

Electrocompetent cells were prepared as follows: the E. coli MG1655/pKD46 strain was grown at 30°C overnight in LB-medium (Luria-Bertani broth, also referred to as lysogenic broth; Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3^rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (150 mg/L); then the culture was diluted 100 times with 5 mL of SOB-medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3^rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (150 mg/L) and L-arabinose (1 mM). The cells were grown with aeration (250 rpm) at 30°C to OD₆₀o of -0.6.

Electrocompetent cells were made by concentrating 100-fold and washing three times with ice-cold deionized H₂0. Electroporation was performed using 70 of cells and -100 ng of the PCR-product. Electroporated cells were incubated with 1 mL of SOC- medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3^rd ed.), Cold Spring Harbor Laboratory Press, 2001) at 37°C for 2.5 hours, plated onto L- agar containing kanamycin (25 mg/L), and grown at 37°C to select Km^R-recombinants. To eliminate the pKD46 plasmid, two passages on L-agar supplemented with kanamycin (25 mg/L) at 42°C were performed, and the obtained colonies were tested for sensitivity to ampicillin. Thus the E. coli MG1655AznuACB::Km strain was obtained.

Example 2. Production of L-arginine by E. coli 382ilvA⁺AznuACB::Km

Clones of E. coli 382 ilvA⁺ were selected as good-growing colonies on minimal agar plates. The strain 382 ilvA⁺ was obtained from the arginine-producing strain 382 (VKPM B-7926, EP1 170358 Al) by PI -transduction of the wild-type ilvA gene from E. coli K-12 strain. The strain 382 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 1 17545 Moscow, 1^st Dorozhny proezd, 1) on April 10, 2000 under the accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001. To test the effect from inactivation of the znuACB gene cluster on L-arginine production, DNA-fragment from the chromosome of the obtained E. coli MG1655AznuACB::Km was transferred to the L-arginine-producing E. coli strain 382ilvA⁺ by PI -transduction (Miller J.H.

"Experiments in molecular genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor (1972)) to obtain the E. coli 382ilvA⁺AznuACB::Km strain.

Both E. coli strains 382ilvA⁺ and 382ilvA⁺AznuACB::Km were each cultivated with shaking (220 rpm) at 37°C for 18 hours in 3 mL of nutrient broth. Then 0.3 mL of the obtained cultures was inoculated into 2 mL of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker to an OD₅₄₀ of -40 until glucose consumption.

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

spectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) was as follows:

Glucose 50.0

(NH₄)₂S0₄ 35.0

KH₂P0₄ 2.0

MgS0₄-7H₂0 1.0

Thiamine-HCl 0.0002

Yeast extract .0

L-Isoleucine 0.1

CaC0₃ 5.0

Glucose and magnesium sulfate were sterilized separately. CaC0₃ was dry-heat sterilized at 180°C for 2 hours. The pH was adjusted to 7.0 with KOH solution.

The results of eight independent test tube fermentations (as average values) are shown in Table 1. As it can be seen from the Table 1, the modified E. coli

382ilvA⁺AznuACB::Km strain was able to produce a higher amount of L-arginine (Arg) as compared with the parent E. coli 382ilvA⁺ strain.

Table 1. Production of L-arginine.

Example 3. Production of L-citrulline by E. coli 382AargG,AznuACB::Km

To test the effect from inactivation of the znuACB gene cluster on L-citrulline production, the DNA-fragments from the chromosome of the above-described E. coli MG1655AznuACB::Km strain are transferred to the citrulline producing E. coli strain 382AargG by PI -transduction to obtain the strain 382AargG,AznuACB::Km. The strain 382AargG is obtained by deletion of argG gene on the chromosome of the L-arginine- producing strain 382 (VKPM B-7926, EPl 170358 Al) by the method initially developed by Datsenko K.A. and Wanner B.L. called '^Red/ET-mediated integration" (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). According to this procedure, the PCR primers homologous to both the region adjacent to the argG gene and the gene which confers antibiotic resistance in the template plasmid are constructed. The plasmid Μ U S-lattL-cat- attR (WO 05/010175) is used as the template in the PCR reaction.

E. coli strains 382AargG and 382AargG,AznuACB::Km are separately cultivated with shaking at 37°C for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 h on a rotary shaker.

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

spectrophotometrically at 540 nm.

The composition of the fermentation medium (g/L) is as follows:

Glucose 48.0

(NH₄)₂S0₄ 35.0

KH₂P0₄ 2.0

MgS0₄7H₂0 1.0

Thiamine-HCl 0.0002

Yeast extract 1.0

L-Isoleucine 0.1

L-Arginine 0.1

CaCC-3 5.0

Glucose and magnesium sulfate are sterilized separately. CaC0₃ is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.0.

Example 4. Production of L-glutamic acid by E. coli VL334thrC⁺AznuACB::Km

To test the effect of inactivation of the znuACB gene cluster on L-glutamic acid production, the DNA-fragments from the chromosome of the above-described E. coli MG1655AznuACB::Km strain are transferred to the glutamate-producing E. coli strain VL334thrC⁺ (EP1172433 Al) by P 1 -transduction to obtain the strain

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

E. coli strains VL334thrC⁺ and VL334thrC⁺AznuACB::Km are separately cultivated for 18-24 h at 37°C on L-agar plates. Then, one loop of the cells is transferred into test tubes containing 2 mL of fermentation medium. Cultivation is carried out at 30°C for 3 days with shaking.

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

The composition of the fermentation medium (g/L) is as follows:

Glucose 60.0

(NH₄)₂S0₄ 25.0

KH₂P0₄ 2.0

MgS0₄-7H₂0 1.0

Thiamine-HCl 0.1

L-Isoleucine 0.07

CaC0₃ 25.0

Glucose and CaC0₃ are sterilized separately. The pH is adjusted to 7.2.

Example 5. Production of L-ornithine by E. coli 382AargFAargI,AznuACB::Km

To test the effect from inactivation of the znuACB gene cluster on L-ornithine production, the DNA-fragments from the chromosome of the above-described E. coli MG1655AznuACB::Km strain are transferred to the L-ornithine producing E. coli strain 382AargFAargI by PI -transduction to obtain the strain 382AargFAargI,AznuACB::Km. The strain 382AargFAargI is obtained by consecutive deletion of argF and argl genes on the chromosome of the L-arginine-producing strain 382 (VKPM B-7926, EPl 170358 Al) by the method initially developed by Datsenko K.A. and Wanner B.L. called '^Red/ET- mediated integration" (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). According to this procedure, two pairs of PCR primers homologous to both the region adjacent to the argF or argl gene and the gene which confers antibiotic resistance in the template plasmid are constructed. The plasmid pMW \ l8-lattL-cat- attR (WO 05/010175) is used as the template in the PCR reaction. E. coli strains 382AargFAargI and 382AargFAargI,AznuACB::Km are separately cultivated with shaking at 37°C for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 h on a rotary shaker.

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

The composition of the fermentation medium (g/L) is as follows:

Glucose 48.0

(NH₄)₂S0₄ 35.0

KH₂P0₄ 2.0

MgS0₄ x 7H₂0 1.0

Thiamine-HCl 0.0002

Yeast extract 1.0

L-Isoleucine 0.1

L-Arginine 0.1

CaC0₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaC0₃ is dry-heat sterilized at 180°C for 2 h. The pH is adjusted to 7.0.

Example 6. Production of L-proline by E. coli 702ilvAAznuACB::Km

To test the effect from inactivation of the znuACB gene cluster on L-proline production, the DNA-fragments from the chromosome of the above-described E. coli MG1655AznuACB::Km strain are transferred to the proline-producing E. coli strain 702ilvA by PI -transduction to obtain the strain 702ilvAAznuACB::Km. The strain 702ilvA was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 1 17545 Moscow, 1^st Dorozhny proezd, 1) on July 18, 2000 under the accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

E. coli strains 702ilvA and 702ilvAAznuACB::Km are separately cultivated for 18-24 h at 37°C on L-agar plates. Then, these strains are cultivated under the same conditions as in Example 4 (Production of L-glutamic acid). While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated by reference as a part of this application.

Industrial Applicability

According to the present invention, production of L-amino acids such as those belonging to the glutamate family of amino acids by a bacterium of the

Enterobacteriaceae family can be improved.

Claims

1. A method for producing an L-amino acid comprising:

(i) cultivating an L-amino acid-producing bacterium of the family

Enterobacteriaceae in a culture medium to produce said L-amino acid in the bacterium and/or the culture medium,

(ii) collecting said L-amino acid from the bacterium and/or culture medium, wherein said bacterium has been modified to attenuate expression of at least one of the genes from the znuACB gene cluster.

2. The method according to claim 1, wherein said bacterium belongs to the genus Escherichia.

3. The method according to claim 2, wherein said bacterium is Escherichia coli.

4. The method according to claim 1, wherein said bacterium belongs to the genus Pantoea.

5. The method according to claim 4, wherein said bacterium is Pantoea ananatis.

6. The method according to any one of claims 1 to 5, wherein said expression of at least one of the genes from the znuACB gene cluster is attenuated due to inactivation of said gene(s).

7. The method according to claim 6, wherein said at least one of the genes from the znuACB gene cluster is deleted.

8. The method according to any one of claims 1 to 7, wherein said znuACB gene cluster is deleted.

9. The method according to any of claims 1 to 8, wherein said genes from the znuACB gene cluster are znuA, znuB and znuC.

10. The method according to any one of claims 1 to 9, wherein said 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-phenylalanine, L-proline, L- serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

1 1. The method according to claim 10, wherein said L-amino acid is selected from the group consisting of L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L- ornithine, L-proline, and combinations thereof.