WO2008033001A1

WO2008033001A1 - A corynebacteria having enhanced l-lysine productivity and a method of producing l-lysine using the same

Info

Publication number: WO2008033001A1
Application number: PCT/KR2007/004478
Authority: WO
Inventors: Young Hoon Park; Sang Jo Lim; Jun Ok Moon; So Yeon Rah; Hee Jong Lee; Jae Woo Jang; Do Hyun Kwon; Hyo Jin Kim; Jin Suck Sung; Hyung Joon Kim
Original assignee: Cj Cheiljedang Corporation
Priority date: 2006-09-15
Filing date: 2007-09-17
Publication date: 2008-03-20
Also published as: JP2017221202A; JP5756259B2; CN101573438A; US9109242B2; BRPI0716980A2; JP6695308B2; US20100143984A1; JP2015171366A; KR100924065B1; AU2007295159B2; EP2066782A1; KR20080025355A; EP2066782A4; CN101573438B; JP2010503395A; AU2007295159A1; EP3170889A1

Abstract

Disclosed are a variant of Corynebacterium which shows activity greater than the endogenous activity of aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase and diaminopimelate dicarboxylase and additionally pyruvate carboxylase, and a method of producing L-lysine using the same.

Description

A CORYNEBACTERIA HAVING ENHANCED L-LYSINE PRODUCTIVITY AND A METHOD OF PRODUCING L-LYSINE

USING THE SAME

Technical Field

[1] The present invention relates to a variant of Corynebacterium which shows activity greater than the endogenous activity of aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrop- icolinate reductase and diaminopimelate dicarboxylase and additionally pyruvate carboxylase, and a method of producing L- lysine using the same. Background Art

[2] Corynebacterium, especially, Corynebacterium glutamicum, is a Gram-positive microorganism which is widely used in the production of L-amino acids. Of L-amino acids, L-lysine is applicable in a variety of industries, including animal feedstuff, pharmaceutical and cosmetic industries. For use in these industries, typically, L-lysine is produced by fermentation using Corynebacterium strains.

[3] Corynebacterium strains anchoring enhanced genes involved in lysine biosynthesis and methods of producing L-lysine are well known in the art. For example, U.S. Pat. No. 6,746,855 discloses corynebacteria strains with an enhanced lysE gene (lysine export carrier gene), to which genes selected from the group comprising a dap A gene, a lysC gene, a pyc gene and a dapB gene are additionally introduced, and a method for the production of L-lysine by cultivating the strains. U.S. Pat. No. 6,221,636 discloses a coryneform bacterium carrying a recombinant DNA comprising a DNA sequence coding for aspartokinase, in which the feedback inhibitory activity of L-lysine and L- threonine is substantially desensitized, and a DNA sequence coding for a diaminopimelate decarboxylase.

[4] Nowhere are Corynebacterium spp. which show higher activity of the six enzymes involved in the biosynthesis pathway of lysine, that is, aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase, and diaminopimelate dicarboxylase, than the endogenous activity thereof, mentioned in any document published prior to the present invention. Furthermore, Corynebacterium spp. that show more than the endogenous activity of pyruvate carboxylase in addition to the six enzymes are not found in any documents published prior to the present invention. Disclosure of Invention Technical Problem

[5] It is an object of the present invention to provide strains of Corynebacteria that have higher than endogenous activity of the six enzymes involved in the pathway of lysine biosynthesis, that is, aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase and diaminopimelate dicarboxylase.

[6] It is another object of the present invention to provide strains of Corynebacteria that have higher than endogenous activity of pyruvate carboxylase, in addition to the six enzymes.

[7] It is another object of the present invention to provide a method of producing L- lysine using the microorganisms.

Technical Solution

[8] In order to accomplish the above objects, the present invention provides a strain of

Corynebacteria anchoring aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase and diaminopimelate dicarboxylase which show higher than their respective endogenous activities.

[9] In accordance with another aspect thereof, there is provided a strain of

Corynebacteria in which the activity of pyruvate carboxylase is higher than the endogenous activity thereof, in addition to the six enzymes.

[10] In accordance with a further aspect, there is provided a method of producing L- lysine using the microorganisms.

[H]

[12] The Corynebacteria strains of the present invention show activities of the seven enzymes at higher levels than their endogenous levels. The elevated enzyme activity in accordance with the present invention is based on various factors including an increase in the number of gene copies, the replacement of native promoters with more potent promoters, and artificial mutation directed to activity enhancement. In greater detail, the number of gene copies can be increased by the introduction of an exogenous allele and/or by the amplification of the endogenous gene. As for the substitution of the gene promoter, examples thereof include the introduction of an exogenous promoter having potent activity to express the structural genes downstream thereof and replacement with an endogenous gene promoter. Gene amplification may be readily achieved using a method well known in the art, for example, by culturing under suitable conditions.

[13] In accordance with an aspect of the present invention, the Coryneform bacteria of the present invention features the presence of at least one copy of aspB (a gene encoding aspartate aminotransferase), lysC (a gene encoding aspartate kinase), asd (a gene encoding aspartate semialdehyde dehydrogenase), dap A (a gene encoding dihy- drodipicolinate synthase), dapB (a gene encoding dihydrodipicolinate reductase) and lysA (a gene encoding diaminopimelate dicarboxylate) in addition to the endogenous genes aspB, lysC, asd, dap A, dapB and lysA in the nuclear DNA thereof. In a modification of this aspect, potent exogenous promoters may be located upstream of the initiation codon of the respective structural genes.

[14] In accordance with another aspect of the present invention, the coryneform bacteria of the present invention feature the presence of at least one copy of a pyc (pyruvate carboxylase) gene in addition to the endogenous pyc gene in the nuclear DNA thereof, with at least one copy of aspB, lysC, asd, dap A, dapB and lysA located therein. In a modification of this aspect, a potent exogenous promoter is located upstream of the initiation codon of the pyc gene while potent exogenous promoters replace endogenous promoters of the six respective genes.

[15]

[16] As long as it belongs to Corynebacterium, any coryneform bacteria can be used as a mother strain into which the genes are introduced. Examples of Corynebacterium microorganisms useful in the present invention include Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum, Corynebacterium thermoaminogenes FERM BP- 1539, Brevibacterium flavumATCC 14067, Brevibacterium lactofermentum ATCC 13869, and L- amino acid-producing mutants or strains derived therefrom, such as Corynebacterium glutamicum KFCC1O881 and Corynebacterium glutamicum KFCCI lOOl, with preference for Corynebacterium glutamicum KFCC 10881.

[17] In the Corynebacteria microorganisms of the present invention, aspB, lysC, asd, dapA, dapB, lysA and pyc have nucleotide sequences of SEQ ID NOS.: 25, 26, 27, 28, 29, 30 and 37, respectively, each comprising a native promoter and a termination codon.

[18] The Corynebacteria microorganisms of the present invention may be coryneform bacteria into which vectors pDZ-2aspB, pDZ-21ysC/asd, pDZ-2dapA/dapB, pDZ- 21ysA and pDZ-2pyc, respectively having the cleavage maps of FIGS. 2 to 6, are transformed. These vectors may be introduced in a predetermined order or simultaneously. As mentioned above, respective exogenous promoters may be located upstream of the initiation codon of the genes. Preferably, the microorganism anchors at least one additional respective copy of aspB, lysC, asd, dap A, dapB, lysA and pycin the genomic DNA thereof. In an alternative or a more preferable embodiment, a potent exogenous promoter is inserted between an initiation codon and an endogenous promoter for each gene. The insertion of an exogenous gene into genomic DNA can be achieved using a method well known in the art, for example, homologous recombination. [19] Particularly useful in the present invention is Corynebacterium glutamicum KFCC-

1008-CJ5, with accession number KCCM10770P. This strain can be derived from Corynebacterium glutamicum KFCC- 10881 by introducing pDZ-2aspB, pDZ-2 lysC/ asd, pDZ-2dapA/dapB and pDZ-21ysA, respectively having the cleavagemaps of FIGS. 2 to 5, and culturing the transformant in a selection medium to allow the exogenous genes aspB, lysC, asd, dap A, dapB and lysA to undergo homologous recombination with respective endogenous alleles, thereby anchoring two copies of aspB, lysC, asd, dapA, dapB and lysA in the genomic DNA thereof. This strain can produce L-lysine at higher efficiency than the mother strain. The L-lysine production efficiency can be further increased by the additional introduction of the vector pDZ-2pyc, having the cleavage map of FIG. 6, into the transformed strain so as to anchor two copies of pyc in the genomic DNA.

[20] In accordance with another aspect thereof, the present invention providesa method of producing L-lysine at a high yield, comprising the insertion of an exogenous promoter between a native promoter and an initiation codon for individual genes.

[21] In an embodiment of the present invention, the promoters of the lysine biosynthesis genes are replaced with respective exogenous potent CJ7 promoters. The CJ7 promoter useful in the present invention is a potent promoter, having the nucleotide sequence of SEQ ID NO. 44, derived from Corynebacterium ammoniagenes, which was previously developed by the present applicant (Korean Pat. No. KR-0620092). Upon the expression of lysC of Corynebacterium glutamicum KFCC- 10881, the CJ7 promoter was found to increase the activity of aspartate kinase twice as much as the endogenous promoter. Although only examples with the CJ7 promoter are given herein, it should be understood that the CJl to CJ6 promoters, disclosed in Korean Pat. No. KR- 0620092, respectively having nucleotide sequences of SEQ ID NOS.: 45 to 50 derived from Corynebacterium ammoniagenes, can be applied to the preparation of lysine- producing strains in the same manner as that used for the CJ7 promoter. Accordingly, the CJl to CJ6 promoters, as well as the CJ7 promoter, fall within the range of the exogenous promoter useful for increasing the expression level of genes of interest in accordance with the present invention.

[22] In accordance with a further aspect thereof, the present invention provides a method for producing L-lysine, comprising:

[23] culturing the microorganism of the present invention to express L-lysine within the cells or release L-lysine into a medium; and

[24] recovering L-lysine from the cells or the medium.

[25] The culturing step is further explained below.

[26] The microorganism in the L-lysine production method according to the present invention is as described hereinbefore. [27] For the culturing step, one of various processes well known in the art can be adopted. The Corynebacteria strain can be cultured in a batch process or a continuous process, such as a fed batch process or a repeated fed batch process.

[28] For use in culturing, a medium must meet the requirements of the strain to be cultured. Culture media for Corynebacteria spp. are well known (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D. C, USA, 1981). Examples of carbon sources useful for culturing include saccharides and carbohydrates, such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose and the like, oils and lipids, such as soybean oil, sunflower oil, castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid and the like, alcohols such as glycerols, ethanol and the like, and organic acids such as acetic acid. These materials may be used alone or in combination. The nitrogen source useful in the culture medium for the bacteria of the present invention may be represented by peptone, yeast extract, broth, malt extract, a corn steep solid, soybean flour, urea, and inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used alone or in combination. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or sodium salts thereof can be used as a nitrogen source in the culture medium. Also, the culture medium requires a metal salt, such as magnesium sulfate or iron sulfate, as an essential ingredient for the growth of the microorganism. In addition, other essential ingredients, such as amino acids and vitamins, are contained in the culture medium. Instead of the ingredients themselves, their precursors may be used. The ingredients may be added to a culture of the microorganism in a batch manner or a continuous manner.

[29] The pH of the culture may be adjusted with a basic compound such as sodium hydroxide, potassium hydroxide or ammonia, or an acidic compound such as phosphoric acid or sulfuric acid. A defoaming agent such as fatty acid poly glycol ester may be added to prevent the formation of bubbles. An aerobic state may be maintained by injecting oxygen or oxygen-containing gas (e.g., air) into the culture. While the organism is cultured, the culture medium is maintained within the range from 20 to 45⁰C and preferably within the range from 25 to 4O⁰C. The culturing is continued until the production of L- amino acid reaches the maximum. In this regard, it takes 10 to 160 hours to attain the maximal amount of L- lysine. This amino acid may be released into the culture medium or may remain within the cells.

[30] The recovering step is carried out as follows. The recovery of L-lysine from cells or culture media is well known in the art. Examples of L-lysine recovery methods include, but are not limited to, filtration, anion exchange chromatography, crystallization and HPLC. Advantageous Effects

[31] Having higher enzyme activity than the endogenous activity of aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihy- drodipicolinate synthase, dihydropicolinate reductase and diaminopimelate di- carboxylase, and additionally pyruvate carboxylase, the Corynebacteria microorganisms of the present invention can produce L- lysine at higher yield.

[32] Accordingly, the method featuring the use of the Corynebacteria microorganisms can be applied for the high-yield production of L-lysine. Brief Description of the Drawings

[33] FIG. 1 is a schematic view showing a vector pDZ for a nuclear-targeted gene into

Corynebacterium.

[34] FIG. 2 is a schematic view showing a vector pDZ-2aspB for a nuclear-targeted gene into Corynebacterium.

[35] FIG. 3 is a schematic view showing a vector pDZ-21ysC/asd for a nuclear-targeted gene into Corynebacterium.

[36] FIG. 4 is a schematic view showing a vector pDZ-2dapA/dapB for a nuclear- targeted gene into Corynebacterium.

[37] FIG. 5is a schematic view showing a vector pDZ-21ysA for a nuclear-targeted gene into Corynebacterium.

[38] FIG. 6is a schematic view showing a vector pDZ-2pyc for a nuclear-targeted gene into Corynebacterium.

[39] FIG. 7 is a schematic view showing a vector pDZ-PCJ7.

[40] FIG. 8 is a schematic view showing a vector pDZ-PCJ7/aspB.

[41] FIG. 9 is a schematic view showing a vector pDZ-PCJ7/lysC.

[42] FIG. 10 is a schematic view showing a vector pDZ-PCJ7/dapA.

[43] FIG. 11 is a schematic view showing a vector pDZ-PCJ7/dapB.

[44] FIG. 12 is a schematic view showing a vector pDZ-PCJ7/lysA.

[45] FIG. 13 is a schematic view showing a vector pDZ-PCJ7/pyc.

Best Mode for Carrying Out the Invention

[46] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[47]

[48] EXAMPLE 1: Construction of Vector pDZ for Nuclear-Targeted Gene and

Insertion of Gene Using the Same

[49] On the basis of the E. coli cloning vector pACYC177 (New England Biolab,

GenBank accession # X06402), a recombinant vector pDZ for carrying a Corynebacterium chromosome was constructed as follows.

[50] A digest of a pACYClW vector resulting from treatment with Acul and Banl restriction enzymes was blunt-ended using a Klenow enzyme. For use as a selection marker, a lacZ gene from E. coli was prepared by gene amplification from the nuclear DNA of E. coli K12 W3110 through PCR, designed to contain the promoter thereof, and then by 5' end phosphorylation and blunt end generation with T4 DNA polymerase and polynucleotide kinase. These two DNA fragments were ligated to each other to give a circular DNA molecule, followed by inserting an artificially synthesized adaptor sequence containing a plurality of restriction sites into the restriction site BamHI of the circular DNA molecule to construct the vector pDZ for nuclear-targeted gene into Corynebacterium. FIG. 1 is a schematic view showing the map of the vector pDZ for nuclear-targeted gene into Corynebacterium.

[51] Afterwards, a gene of interest was inserted into the nuclear DNA of

Corynebacterium. To this end, Corynebacterium glutamicum KFCC1O881, an L- lysine-producing strain, was transformed with the pDZ vector carrying two copies of a gene of interest in tandem by electroporation (using an electrical pulse method according to Appl. Microbiol. Biotechnol. (1999) 52:541-545) and screened for transformants,in which the gene of interest was inserted into the chromosome due to homology on a selection medium containing 25 mg/L of kanamycin. The appearance of a blue color on the solid medium containing X-gal

(5-bromo-4-chloro-3-indolyl-β-D-galactoside) indicated successful gene insertion into nuclear DNA using the vector. The primary nuclear-inserted strains were cultured in a nutrient broth (3O⁰C, 8 hrs) with shaking and were then serially diluted from 10-4 to 10-10 before being spread on solid media containing X-gal. Most of the colonies that were grown were stained a blue color. White colonies, which were a minority of the grown colinies, were selected. These secondarily selected strains did not carry the inserted vector sequence in the genome thereof because it was removed from the genome by crossover. These strains were finally tested for susceptibility to kanamycin and analyzed for gene structure through PCR before final selection.

[52]

[53] EXAMPLE 2: Preparation of Lysine-Producing Corynebacterium

≠utamicum fKFCC-10881) bv Mutation

[54] A strain into which the genes responsible for the pathway of lysine biosynthesis were to be inserted was based on Corynebacterium glutamicum (KFCC- 10881), which is resistant to S-(2-aminoethyl) cysteine (hereinafter referred to as "AEC") and is homoserine-leaky.

[55] The mutant strain KFCC- 10881 was prepared from the wild-type Corynebacterium glutamicum (ATCC 13032). A medium containing the mother strain at a concentration of 10 - 10 cells/ml was treated with 500 μg/ml of the mutagen N- methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as "NTG") at 3O⁰C for 30 min, followed by the selection of colonies grown on a complex plate containing 5 g/1 of AEC. After the primary mutant strain was analyzed for ACE resistance and lysine productivity, it was led to secondary mutation with NTG. A plurality of the colonies thus formed were tooth-picked into minimal media, which were or were not supplemented with homoserine so as to separate homoserine auxotrophs (secondary mutants), which cannot grow in a minimal medium lacking homoserine. The homoserine auxotroph was allowed to undergo tertiary mutation so as to create a homoserine-leaky strain which was identified by incubation in a minimal medium containing 10 mg/L of homoserine. The strain grown in the medium was examined for lysine productivity (Table 1). The resulting lysine-producing strain, which is AEC resistant and homoserine-leaky, was deposited with the Korean Federation of Culture Collection under the accession number of KFCC- 10881. The minimal medium and the production medium used in this example are as follows.

[56] Table 1

[57] [58] Minimal Medium TpH 7.0) [59] Glucose 100 g, (NH ) SO 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH₂PO₄ 1 g, MgSO₄ 7H₂O 0.5 g, Biotin 100 D, Thiamine chloride 1000 D, Calcium pantothenate 2000 D, nicotinamide 3000 D, CaCGO g (in 1 liter of distilled water)

[60] Production Medium TpH 7.0) [61] Glucose 100 g, (NH ) SO 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH₂PO₄ 1 g, MgSO₄ 7H₂O 0.5 g, Biotin 100 D, Thiamine chloride 1000 D, CaCGO g (in 1 liter of distilled water)

[62] [63] EXAMPLE 3: Cloning of Lvsine-Producing Corvnebacterium øutamicum KFCC-10881-Derived aspB Gene. Construction of Recombinant Vector (pDZ-2aspB) and Preparation of Strain Anchoring aspB

[64] PCR was performed on the aspB gene involved in the pathway of lysine biosynthesis, with the nuclear DNA of the lysine-producing PCR Corynebαcterium glutαmicum KFCC1O881 prepared in Example 2 serving as a template. Information on the base sequence (NCBI Registration No. NC_003450, NcglO237) of the aspB gene was acquired from the data of the NIH GenBank and was used to design two pairs of primers to amplify an aspB gene ranging from the promoter region to the terminator codon (Table 2).

[65] Table 2

[66] [67] PCR was performed using the nuclear DNA of Corynebacterium glutamicum KFCC 10881 as a template and a set of oligonucleotide SEQ ID NOS. 1 and 2 or oligonucleotide SEQ ID NOS. 3 and 4 as primers in the presence of PfuUltra High- Fidelity DNA Polymerase (Stratagene), with 30 cycles of denaturing at 96⁰C for 30 sec, annealing at 53⁰C for 30 sec, and extending at 72⁰C for 2min. The PCR products thus obtained were found to be two kinds of aspB genes (aspB-A and aspB-B), each containing a l,493bp-long promoter region, which were produced respectively with a set of SEQ ID NOS. 1 and 2 and a set of SEQ ID NOS. 3 and 4. The PCR products were cloned into the E. coli vector pCR2.1 with the aid of a TOPO Cloning Kit (Invitrogen) to give recombinant vectors pCR-aspB-A and pCR-aspB-B. These pCR vectors were treated respectively with the restriction enzymes which are specific for opposite ends of aspB-A and aspB-B (aspB-A: Smal+Hindlll, aspB-B: Hindlll+Nhel) to separate the aspB genes from the pCR vectors. These fragments were cloned through 3 -piece ligation into the EcoRV- Nhel sites of a pDZ vector to produce a recombinant vector pDZ-2aspB, in which two copies of aspB were cloned in tandem. FIG. 2 is a map of a pDZ-2aspB vector for a nuclear-targeted gene into Corynebacterium.

[68] The recombinant vector pDZ-2aspB was transformed into the lysine -producing Corynebacterium glutamicum KFCC- 10881 prepared in Example 2, followed by the insertion of an additional copy of the aspB gene just next to the aspB gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring two copies of an aspB gene, which was named Corynebacterium glutamicum KFCC 10881 -CJl. The adjacent positioning of two copies of the aspB gene was demonstrated by PCR using a set of primers, SEQ ID NOS. 17 and 18 (Table 3), designed to amplify the contact region between the two copies of the aspB gene. [69] Table 3

[70] [71] EXAMPLE 4: Cloning of Lysine-Producing Corvnebacterium glutamicum KFCC-10881-Derived lvsC/asd Gene. Construction of Recombinant Vector (pDZ-21ysC/asd) and Preparation of Strain Anchoring lysC/asd

[72] Information on the base sequence (NCBI Registration No. NC_003450, Ncgl0247~0248) of the lysC/asd gene was acquired from data of the NIH GenBank and was used to design two pairs of primers to amplify an aspB gene ranging from the promoter region to the terminator codon (Table 4) in a manner similar to that of Example 3.

[73] Table 4

[74] [75] PCR was performed using the nuclear DNA of Corynebαcterium glutamicum KFCC 10881 as a template and aset of oligonucleotide SEQ ID NOS. 1 and 2 or oligonucleotide SEQ ID NOS. 3 and 4 as primers in the presence of PfuUltra High- Fidelity DNA Polymerase (Stratagene), with 30 cycles of denaturing at 96⁰C for 30 sec, annealing at 52⁰C for 30 sec, and extending at 72⁰C for 3 min.

[76] The PCR products thus obtained were found to be two kinds of lysC/asd genes, lysC/asd-A and lysC/asd-B, each containing a 2,805bp-long promoter region, which were produced respectively with a set of SEQ ID NOS. 5 and 6 and a set of SEQ ID NOS. 7 and 8. The PCR products were cloned into the E. coli vector pCR2.1 with the aid of a TOPO Cloning Kit (Invitrogen) to give recombinant vectors pCR-lysC/asd-A and pCR-lysC/asd-B.

[77] These pCR vectors were treated respectively with restriction enzymes that are specific for opposite ends of lysC/asd-A and lysC/asd-B (lysC/asd-A: BamHI+Smal, lysC/asd-B: Smal+Pvul) to separate the lysC/asd genes from the pCR vectors. These fragments were cloned through 3 -piece ligation into the BamHI-PvuI sites of a pDZ vector to construct a recombinant vector pDZ-21ysC/asd in which 2 copies of lysC/asd were cloned in tandem. FIG. 3 is a map of the pDZ-21ysC/asd vector for a nuclear- targeted gene into Corynebacterium.

[78] The recombinant vector pDZ-21ysC/asd was transformed into the lysine-producing Corynebacterium glutamicum KFCC1O881-CJ1 prepared in Example 3, followed by the insertion of an additional copy of lysC/asd gene just near the lysC/asd gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring two copies of lysC/asd gene, which was named Corynebacterium glutamicum KFCC 10881 -C J2. The adjacent positioning of two copies of aspB gene was identified through PCR using a set of primers (Table 5) designed to amplify the contact region between the two copies of aspB gene.

[79] Table 5

[80] [81] EXAMPLE 5: Cloning of Lvsine-Producing Corvnebacterium øutamicum KFCC-10881-Derived dapA/dapB Gene. Construction of Recombinant Vector (pDZ-2dapA/dapB) and Preparation of Strain Anchoring dapA/dapB

[82] Information on the base sequence (NCBI Registration No. NC_003450, Ncgll 896- 1898) of dapA/dapB gene was acquired from data of the NIH GenBank. The dapA gene was determinedto comprise a dapB gene and an operon with a function-unknown ORF (Ncgll987) located therebetween. The information was used to design two pairs of primers to amplify the overall dapB-ORF (Ncgll 897)-dapA gene ranging from the dapB promoter to the terminator codon (Table 6).

[83] Table 6

[84] [85] PCR was performed using the nuclear DNA of Corynebacterium glutamicum KFCC 10881 as a template and a set of oligonucleotide SEQ ID NOS. 9 and 10 or oligonucleotide SEQ ID NOS. 11 and 12 as primers in the presence of PfuUltra High-Fidelity DNA Polymerase (Stratagene), with 30 cycles of denaturing at 96⁰C for 30 sec, annealing at 52⁰C for 30 sec and extending at 72⁰C for 3 min.

[86] The PCR products thus obtained were found to be two kindsof dapA/dapB genes, dapA/dapB-A, dapA/dapB-B, each containing a 3,210bp-long promoter region, which were produced respectively with a set of SEQ ID NOS. 9 and 10 and a set of SEQ ID NOS. 11 and 12. The PCR products were cloned into the E. coli vector pCR2.1 with the aid of the TOPO Cloning Kit (Invitrogen) to give recombinant vectors pCR- dapA/dapB-A and pCR-dapA/dapB-B.

[87] These pCR vectors were treated with corresponding restriction enzymes, which are specific for opposite ends of dapA/dapB-A and dapA/dapB-B (dapA/dapB-A: EcoRI+SacI, dapA/dapB-B: Sacl+Xhol) to separate the dapA/dapB genes from the pCR vectors. These fragments were cloned through 3-piece ligation into the EcoRI- Xhol sites of a pDZ vector to construct a recombinant vector pDZ-2dapA/dapB in which 2 copies of dapA/dapB were cloned in tandem. FIG. 4 is a map of the pDZ- 2dapA/dapB vector.

[88] The recombinant vector pDZ-2dapA/dapB was transformed into the lysine- producing Corynebacterium glutamicum KFCC10881-CJ2 prepared in Example 4, followed by the insertion of an additional copy of dapA/dapB gene just next to the dapA/dapB gene in the nuclear DNA through crossover so as to produce a lysine- producing strain anchoring two copies of the dapA/dapB gene, which was named Corynebacterium glutamicum KFCC10881-CJ3. The adjacent positioning of two copies of dapA/dapB gene was identified by PCR using a set of primers (Table 7) designed to amplify a contact region between the two copies of dapA/dapB gene.

[89] Table 7

[90]

[91] EXAMPLE 6: Cloning of Lysine-Producing Corynebacterium glutamicum

KFCC-10881-Derived lysA Gene. Construction of Recombinant Vector

(pDZ-21ysA) and Preparation of Strain Anchoring lysA

[92] Information on the base sequence (NCBI Registration No. NC_003450,

Ncgll 132-1133) of lysA gene was acquired from data of the NIH GenBank. The lysA gene was analyzed to comprise an argS gene (arginyl-tRNA synthetase) and an operon. The information was used to design two pairs of primers to amplify the overall argS- lysA gene ranging from the argS promoter to the terminator codon (Table 8).

[93] Table 8

[94] [95] PCR was performed using the nuclear DNA of Corynebacterium glutamicum KFCC 10881 as a template and a set of oligonucleotide SEQ ID NOS. 13 and 14 or SEQ ID NOS. 15 and 16 as primers in the presence of PfuUltra™ High-Fidelity DNA Polymerase (Stratagene), with 30 cycles of denaturing at 96⁰C for 30 sec, annealing at 52⁰C for 30 sec and extending at 72⁰C for 4 min.

[96] The PCR products thus obtained were found to be two kinds of argS/lysA genes, argS/lysA-A and argS/lysA-B, each containing a 3,359bp-long promoter region, which were produced respectively with a set of SEQ ID NOS. 13 and 14 and a set of SEQ ID NOS. 15 and 16. The PCR products were cloned into the E. coli vector pCR2.1 with the aid of a TOPO Cloning Kit (Invitrogen) to give recombinant vectors pCR- argS/lysA-A and pCR-argS/lysA-B.

[97] These pCR vectors were treated with corresponding restriction enzymes which are specific for opposite ends of argS/lysA-A and argS/lysA-B (argS/lysA-A: Hindlll+Notl, argS/lysA-B: Notl+Xbal) to separate the argS/lysA genes from the pCR vectors. These fragments were cloned in a 3-piece ligation into the Hindlll-Xbal sites of a pDZ vector to construct a recombinant vector pDZ-21ysA, in which 2 copies of argS/lysA were cloned in tandem. FIG. 5 is a map of the pDZ-21ysA vector.

[98] The recombinant vector pDZ-21ysA was transformed into the lysine-producing Corynebacterium glutamicum KFCC10881-CJ3 prepared in Example 5, followed by the insertion of an additional copy of lysA gene just next to the lysA gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring two copies of lysA gene, which was named Corynebacterium glutamicum KFCC 10881 -C J4. The adjacent positioning of two copies of lysA gene was determined through PCR using a set of primers (Table 9) designed to amplify a contact region between the two copies of lysA gene.

[99] Table 9

[100] [101] The lysine-producing strain Corynebacterium glutamicum KFCC10881-CJ4, which anchors the six genes responsible for lysine biosynthesis in vivo, were deposited with the Korean Culture Center of Microorganisms on Aug. 21, 2006 under the accession number KCCM 10770P.

[102] [103] EXAMPLE 7: Cloning of Lysine-Producing Corynebacterium glutamicum KFCC-10881-Derived pycGene. Construction of Recombinant Vector (pDZ-2pyc) and Preparation of Strain Anchoring pvc

[104] Informationon the base sequence (NCBI Registration No. NC_003450, NcglO659) of pyc gene was acquired from data of the NIH GenBank and used to design two pairs of primers to amplify a pyc gene ranging from the promoter region to the terminator codon (Table 10) in a manner similar to that of Example 3.

[105] Table 10

[106] [107] PCR was performed using the nuclear DNA of Corynebacterium glutamicum KFCC 10881 as a template and a set of oligonucleotide SEQ ID NOS. 31 and 32 or SEQ ID NOS. 33 and 34 as primers in the presence of PfuUltra™ High-Fidelity DNA Polymerase (Stratagene), with 30 cycles of denaturing at 96⁰C for 30 sec, annealing at 52⁰C for 30 sec, and extending at 72⁰C for 4 min. The PCR products thus obtained were found to be two kinds of pyc genes, pyc- A and pyc-B, each containing a 3,925bp-long promoter region, which were produced respectively with a set of SEQ ID NOS. 31 and 32 and a set of SEQ ID NOS. 33 and 34. The PCR products were cloned into the E. coli vector pCR2.1 with the aid of a TOPO Cloning Kit (Invitrogen) to give recombinant vectors pCR-pyc-A and pCR-pyc-B. [108] These pCR vectors were treated with corresponding restriction enzymes, which are specific for opposite ends of pyc-A and pyc-B (pyc-A: Xbal and EcoRV, pyc-B: EcoRV and Hindlll) to separate the pyc genes from the pCR vectors. These fragments were cloned in a 3-piece ligation into the Xbal-Hindlll sites of a pDZ vector to construct a recombinant vector pDZ-2pyc, in which 2 copies of pyc were cloned in tandem. FIG. 6 is a map of the pDZ-2pyc vector.

[109] The recombinant vector pDZ-2pyc was transformed into the lysine-producing

Corynebacterium glutamicum KFCC10881-CJ4, prepared in Example 6, followed by the insertion of an additional copy of pycA gene just next to the pycA gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring two copies of pyc gene, which was named Corynebacterium glutamicum KFCC 10881 -C J5. The adjacent positioning of two copies of pyc gene was determined through PCR using a set of primers (Table 11) designed to amplify the contact region between the two copies of pyc gene.

[110] Table 11

[111]

[112] As a result, the lysine-producing strain Corynebacterium glutamicum

KFCC 10881 -C J5 anchors the seven genes involved in lysine biosynthesis. [113] [114] EXAMPLE 8: Production of Lysine from Strains Anchoring Genes Re- sponsible for Lysine-Biosynthesis [115] The L-lysine-producing strains Corynebacterium glutamicum KFCC-10881-CJ4 and KFCC- 10881-CJ5, which were prepared respectively in Examples 6 and 7, were cultured in order to produce L-lysine as follows. [116] Corynebacterium glutamicum KFCC- 10881, KFCC- 10881 -C J4 and KFCC-

10881-CJ5 were inoculated into 240 ml-corner baffle flasks containing 25 ml of the following seed medium and cultured at 3O⁰C for 20 hour with shaking at 200 rpm.

Thereafter, 1 ml of each of the cultures was inoculated into a 250 ml-corner baffle flask containing 24 ml of the following production medium and cultured at 3O⁰C for

120 hours with shaking at 200 rpm. The seed medium and the production medium comprise the following compositions. [117] [118] Seed medium TPH 7.0) [119] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH PO 4 g, K HPO 8 g, MgSO 7H O 0.5 g, biotin 100 D, thiamine HCl 1000 D, calcium pantothenate 2000 D,

4 2 nicotinamide 2000 D (in 1 liter of distilled water)

[120] Production medium φH 7.0) [121] glucose 100 g, (NH ) SO 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g, KH PO 1 g, MgSO 7H O 0.5 g, biotin 100 D, thiamine chloride 1000 D, calcium pantothenate 2000 D, nicotinamide 3000 D, and CaCGO g (in 1 liter of distilled water).

[122] [123] After the completion of culture, HPLC analysis was performed to determine the amounts of the L-lysine produced by the strains. The concentrations of L-lysine in the cultures of Corynebacterium glutamicum KFCC- 10881, KFCC 10881 -C J4 and KFCC 10881 -C J5 are summarized in Table 12, below.

[124] Table 12

[125] [126] As seen in Table 12, Corynebacterium glutamicum KFCC- 10881 -C J4, in which the six genes involved in lysine biosynthesis are anchored, was found to increase in lysine productivity by about 7%, compared with the mother strain KFCC- 10881. Also, it was measured that an increase of about 8% in lysine production was obtained with Corynebacterium glutamicum KFCC- 10881 -C J5, which anchors a pyc gene in addition to the six lysine biosynthesis genes, in comparison to the mother strain KFCC- 10881.

[127] [128] EXAMPLE 9: Preparation of Lvsine-Producing Corvnebacteήum øutamicum KFCC-10881-Derived Strain Anchoring Three Copies of lvsC/asd Gene

[129] The recombinant vector pDZ-21ysC/asd, prepared in Example 4, was transformed into the lysine-producing Corynebacterium glutamicum KFCC 10881 -C J4 prepared in Example 6, followed by the insertion of an additional copy of the lysC/asd gene just next tothe two adjacent copies of the lysC/asd gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring three copies of the lysC/asd gene, which was named Corynebacterium glutamicum KFCClOδδl-CJό. The alignment of three copies of the lysC/asd gene in tandem was determined through PCR using a set of primers (Table 13) designed to amplify contact regions among the three copies of the lysC/asd gene.

[130] Table 13

[131] [132] The lysine-producing strain Corynebacterium glutamicum KFCC10881-CJ6 was found to have aspartate kinase activity 2.1 times as high as that of KFCC10881-CJ4, as measured by the method using aspartyl hydroxamate according to Pechere J. F. and Capony J. P. (Anal Biochem 22: 536-539, 1968) (Table 14).

[133] Table 14

[134] [135] EXAMPLE 10: Preparation of Lvsine-Producing Corvnebacterium glutamicum KFCC-10881-Derived Strain Anchoring Three Copies of dapA/dapB Gene

[136] The recombinant vector pDZ-2dapA/dapB, prepared in Example 5, was transformed into the lysine-producing Corynebacterium glutamicum KFCC 10881 -C J6 prepared in Example 9, followed by the insertion of an additional copy of dapA/dapB gene just next tothe two adjacent copies of dapA/dapB gene in the nuclear DNA through crossover so as to produce a lysine-producing strain anchoring three copies of dapA/ dapB gene, which was named Corynebacterium glutamicum KFCC 10881 -C J7. The alignment of three copies of dapA/dapB gene in tandem was determined through PCR using a set of primers (Table 15) designed to amplify contact regions among the three copies of dapA/dapB gene.

[137] Table 15

[138] [139] EXAMPLE 11: Lysine Production from the Strain Anchoring Complex Genes Responsible for Lysine Biosynthesis

[140] The L-lysine-producing strain Corynebacterium glutamicum KFCC- 10881 -C J7, prepared in Example 10, was cultured to produce L-lysine as follows. [141] Corynebacterium glutamicum KFCC- 10881 -C J4 and KFCC-10881-CJ7 were inoculated into 250 ml-corner baffle flasks,containing 25 ml of the following seed medium, and were cultured at 3O⁰C for 20 hour with shaking at 200 rpm. Thereafter, 1 ml of each of the cultures was inoculated into a 250 ml-corner baffle flask, containing 24 ml of the following production medium, and was cultured at 3O⁰C for 120 hours with shaking at 200 rpm. The seed medium and the production medium comprise the following compositions.

[142] [143] Seed medium TpH 7.0) [144] glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH PO 4 g, K HPO 8 g, MgSO 7H O 0.5 g, biotin 100 D, thiamine HCl 1000 D, calcium pantothenate 2000 D, nicotinamide 2000 D (in 1 liter of distilled water)

[145] Production medium TpH 7.0) [146] glucose 100 g, (NH 4 ) 2 SO 4 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g,

KH PO 1 g, MgSO 7H O 0.5 g, biotin 100 D, thiamine chloride 1000 D, calcium pantothenate 2000 D, nicotinamide 3000 D, CaCGO g (in 1 liter of distilled water).

[147] [148] After the completion of culture, HPLC analysis was performed to determine the amounts of the L-lysine produced by the strains. Theconcentrations of L-lysine in the cultures of Corynebacterium glutamicum KFCC-10881-CJ4 and KFCC- 10881 -C J7 are summarized in Table 16, below.

[149] Table 16

[150] [151] As seen in Table 16, Corynebacterium glutamicum KFCC- 10881 -C J7, in which the three genes involved in lysine biosynthesis are anchored in triplicate, was found to increase in lysine productivity by about 11%, compared with the mother strain KFCC- 10881-CJ4.

[152] [153] EXAMPLE 12: Construction of Vector for Promoter Replacement and Preparation of Strain Replaced Exogenous Promoter for Lysine Biosynthesis Gene

[154] An exogenous potent CJ7 promoter was substituted for the native promoter for the lysine biosynthesis genes on the basis of pDZ. The CJ7 promoter is a strong, Corynebacterium ammoniagenes-deήved promoter having a base sequence represented by SEQ ID NOS. 44 to 50. It is disclosed in Korean Pat. No. 0620092, issued to the present applicant. When the nuclear lysC of Corynebacterium glutamicum KFCC- 10881 was expressed in the presence of the CJ7 promoter, aspartate kinase activity was found to increase by a multiple as high as 2 when using the inherent promoter.

[155] A vector for introducing the CJ7 promoter into the nuclear DNA of the lysine- producing strains was prepared as follows.

[156] First, a pDZ vector was treated with restriction enzymes Xbal and Ndel. PCR was performed in the presence of SEQ ID NOS. 42 and 43 (Table 17) as primers designed to insert Xbal and Ndel sites respectively at the 5'- and the 3 '-end of a PCR product amplified from the CJ7 promoter of the nuclear DNA of Corynebacterium am- moniagenes. After treatment with Xbal and Ndel, the CJ7 promoter PCR product was ligated to the truncated pDZ vector to give a pDZ-PCJ7 vector, which served as a primary plasmid for introducing a CJ7 promoter into the nuclear DNA of the strain (FIG. 7)

[157] Table 17

[158]

[159] PCR was performed, with the nuclear DNA of the lysine-producing strain

Corynebacterium glutamicum KFCC1O881 prepared in Example 2 serving as a template, to obtain two kinds of DNA fragments necessary for the substitution of the CJ7 promoter for respective native promotersof the genes introduced to the nuclear DNA, that is, a partial native promoter fragment about 300bp long and a partial ORF about 300 bp long stretching from base +2 in the downstream direction. The primers used forthe PCR were designed such that an Xbal site was inserted at opposite ends of the PCR product of the partial native promoter fragment and an Ndel site was inserted at opposite ends of the PCR product of the partial ORF. These PCR products were treated with respective restriction enzymes. pDZ-PCJ7 was digested with Xbal for ligation to the truncated PCR product of the partial native promoter fragment and then with Ndel for ligation to the truncated PCR production of the partial ORF to construct recombinant plasmids, respectively named pDZ-PCJ7-aspB, pDZ-PCJ7-lysCasd, pDZ- PCJ7-dapA, pDZ-PCJ7-dapB, pDZ-PCJ7-argSlysA and pDZ-PCJ7-pyc, each comprising a CJ7 promoter located between the two PCR DNA fragments (FIGS. 8 to 13).

[160] The plasmids were used for the recombination of target genes as follows. First, the recombinant vector pDZ-PCJ7-aspB was transformed into the lysine-producing Corynebacterium glutamicum KFCC- 10881, which then underwent crossover to the CJ7 promoter at the promoter region of the aspB gene inthe nuclear DNA. In the same way, the other recombinant plasmids were transformed in order to create a novel strain KFCC-10881-PCJ7-5 in which a CJ7 promoter is located at the promoter regions of aspB, lysCasd, dapA, dapB and argSlysA genes. In addition to the promoters, the promoter of the pyc gene was substituted with a CJ7 promoter to create KFCC- 10881-PCJ7-6.

[161]

[162] EXAMPLE 13: Production of Lysine in Strain Having Exogenous Promoter for Lysine Biosynthesis Gene

[163] The L-lysine-producing strains Corynebacterium glutamicum KFCC-10881-PCJ7-5 and KFCC- 10881-PCJ7-6 were cultured to produce L-lysine as follows.

[164] Corynebacterium glutamicum KFCC- 10881, KFCC- 10881-PCJ7-5 and KFCC-

10881-PCJ7-6 were inoculated into 250 ml-corner baffle flasks containing 25 ml of the following seed medium, and were cultured at 3O⁰C for 20 hours with shaking at 200 rpm. Thereafter, 1 ml of each of the cultures was inoculated into a 250 ml-corner baffle flask containing 24 ml of the following production medium and cultured at 3O⁰C for 120 hours with shaking at 200 rpm. The seed medium and the production medium comprise the following compositions.

[165]

[166] Seed medium TPH 7.0)

[167] glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH PO 4 g, K HPO 8 g,

MgSO 7H O 0.5 g, biotin 100 D, thiamine HCl 1000 D, calcium pantothenate 2000 D,

4 2 nicotinamide 2000 D (in 1 liter of distilled water) [168] Production medium TpH 7.0)

[169] glucose 100 g, (NH ) SO4 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g,

KH PO4 1 g, MgSO 7H O 0.5 g, biotin 100 D, thiamine chloride 1000 D, calcium pantothenate 2000 D, nicotinamide 3000 D, CaCGO g (in 1 liter of distilled water).

[170]

[171] After the completion of culture, HPLC analysis was performed to determine the amounts of the L-lysine produced by the strains. The concentrations of L-lysine in the cultures of Corynebacterium glutamicum KFCC- 10881, KFCC-10881-PCJ7-5 and KFCC-10881-PCJ7-6 are summarized in Table 18, below.

[172] Table 18

[173] [174] As seen in Table 18, Corynebacterium glutamicum KFCC- 10881-PCJ7-5, in which the six genes involved in lysine biosynthesis were anchored along with respective exogenous CJ7 promoters therefor and Corynebacterium glutamicum KFCC- 10881-PCJ7-6, in which a pyc gene and a CJ7 promoter therefor were anchored in addition to the six genes and promoters, were found to increase in lysine productivity by about 17.4% and 18.6% respectively, compared with the mother strain KFCC- 10881.

[175] [176] EXAMPLE 14: Activity of Lysine Biosynthesis Enzymes in Strain Anchoring Additional Gene Responsible for Lysine Biosynthesis and Having Replaced Exogenous Promoter therefor

[177] The L-lysine-producing strains Corynebacterium glutamicum KFCC10881-CJ4, KFCC10881-CJ7 and KFCC-10881-PCJ7-5, prepared respectively in Examples 6, 10 and 12, were assayed for the activity of aspartate kinase and diaminopimelate di- carboxylase as representatives of lysine biosynthesis enzymes because their activity is relatively easy to measure. The modified lysine-producing strains were found to increase in enzyme activity compared to the initial mother strain KFCC 10881 (Table 19) as measured by the method according to Pechere J. F. and Capony J. P. (Anal Biochem 22: 536-539, 1968) for aspartate kinase activity and the method according to J. Cremer et al. for diaminopimelate dicarboxylase activity.

[178] Table 19

Claims

[I] A Corynebacteria microorganism, showing increased enzymatic activity compared to respective endogenous activities of aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase and diaminopimelate dicarboxylase.

[2] The Corynebacteria microorganism as set forth in claim 1, further showing increased enzymatic activity compared to the endogenous activity of pyruvate carboxylase.

[3] The Corynebacteria microorganism as set forth in claim 1, wherein the increased enzyme activity of aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydropicolinate reductase and diaminopimelate dicarboxylaseis attributed to an increase in the expression of an aspB gene, a lysC gene, an asd gene, a dapA gene, a dapBgene and a lysA gene of a coryneform bacteria.

[4] The Corynebacteria microorganism as set forth in claim 2, wherein the increased enzyme activity of pyruvate carboxylase is attributed to an increase in the expression of a pyc gene of a coryneform bacteria.

[5] The Corynebacteria microorganism as set forth in claim 3 or 4, wherein the expression increase results from the introduction of one or more copies of exogenous alleles corresponding to the endogenous genes of the microorganism.

[6] The Corynebacteria microorganism as set forth in claim 5, wherein the microorganism is a coryneform bacteria having accession number KFCC 10881.

[7] The Corynebacteria microorganism as set forth in claim 5, wherein the aspB gene, the lysC gene, the asd gene, the dapA gene, the dapB gene, the lysA gene and the pyc gene have nucleotide sequences of SEQ ID NOS. 25, 26, 27, 28, 29, 30 and 37, respectively.

[8] The Corynebacteria microorganism as set forth in claim 5, wherein the introduction is performed by transforming vectors respectively having cleavage maps of FIGS. 2 to 6 into a coryneform bacteria.

[9] The Corynebacteria microorganism as set forth in claim 5, wherein the exogenous allele genes are anchored in the nuclear DNA of the microorganism.

[10] The Corynebacteria microorganism as set forth in claim 5, wherein the microorganism is Corynebacterium glutamicum KFCC10881-CJ4 having accession number KCCM 10770P.

[I I] The Corynebacteria microorganism as set forth in any one of claims 3 to 10, wherein the expression increase results from introduction of an exogenous promoter for each of the genes. [12] The Corynebacteria microorganism as set forth in claim 11, wherein the exogenous promoter has a nucleotide sequence represented by one of SEQ ID

NOS. 44 to 50. [13] The Corynebacteria microorganism as set forth in claim 12, wherein the exogenous promoter is a nucleotide sequence of SEQ ID NO. 44. [14] The Corynebacteria microorganism as set forth in claim 11, wherein the exogenous promoter is introduced by introducing at least one of the vectors of

FIGS. 8 to 13 into a coryneform bacteria. [15] A method of producing L- lysine, comprising: culturing the microorganism of any one of claims 1 to 14; and recovering lysine from the cells of the microorganism or the cell culture.