KR20180077528A - Corynebacteria producing L-lysine and a method of producing L-lysine using the same - Google Patents

Abstract

The present application relates to a microorganism belonging to the genus Corynebacterium which produces L-lysine and a method for producing L-lysine using the microorganism.

Description

L-lysine and a method for producing L-lysine using the same, and a method for producing L-lysine using the same,

The present application relates to a microorganism belonging to the genus Corynebacterium which produces L-lysine and a method for producing L-lysine using the microorganism.

L-lysine is one of the essential amino acids that are not synthesized in vivo and is known to be necessary for accelerating growth of pediatric growth, involvement of calcium metabolism, formation of hormones and antibodies, promotion of gastric secretion and increase of resistance to diseases. In industry, it is used in the production of animal feed, medicine, and cosmetics. Various studies are being conducted to develop high efficiency production strains and fermentation process technology for efficient production of L-lysine. In such studies, a specific substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for biosynthesis has been mainly used (Korean Patent No. 10-0838038 number).

Further, in addition to biosynthetic pathway-related genes, Corynebacterium strains having enhanced specific gene activity and a method for producing L-lysine using the same are known. For example, Korean Patent Application No. 10-2014-0042397 discloses a microorganism belonging to the genus Corynebacterium having enhanced acetic acid kinase activity as compared with the intrinsic activity, and a method for producing L-lysine using the same. Korean Patent No. 10-1498630 discloses a microorganism of the genus Corynebacterium which is modified to overexpress the NCg10862 gene of Corynebacterium glutamicum to enhance L-lysine producing ability and a method for producing L-lysine using the same .

The present inventors randomly introduced an endogenous gene of Corynebacterium sp. Microorganism in order to search for an effective trait that can increase L-lysine production ability. When the expression level of a specific gene is increased, the productivity of L-lysine is increased .

Korean Patent No. 10-0838038 Korean Patent Application No. 10-2014-0042397 Korean Patent No. 10-1498630

One aspect of the present application is to provide a microorganism of the genus Corynebacterium which enhances the activity of the polypeptide of SEQ ID NO: 1 to produce L-lysine.

Another aspect of the present application is to provide a method for producing L-lysine using the microorganism of the genus Corynebacterium.

One aspect of the present application provides a microorganism of the genus Corynebacterium which enhances the activity of the polypeptide of SEQ ID NO: 1 to produce L-lysine.

The term "L-lysine ", as used herein, is an essential amino acid that is not synthesized in the body as one of the basic alpha -amino acids. L-lysine is biosynthesized from oxaloacetate via the lysine biosynthetic pathway, and NADPH-dependent reductase catalyzes the intermediate process for lysine biosynthesis. In the L-lysine biosynthesis process of one molecule, three molecules of NADPH are directly consumed by the sugar enzymes, and one molecule of NADPH is indirectly used.

Specifically, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is interpreted to include an amino acid sequence that exhibits substantial identity to the amino acid sequence. The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present application with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art, A sequence that exhibits at least 70% homology, specifically at least 80% homology, more specifically at least 90% homology, even more specifically at least 95% homology.

For example, an amino acid sequence having such homology and exhibiting an effect corresponding to the polypeptide of the amino acid sequence of SEQ ID NO: 1 may have an amino acid sequence that is deleted, modified, substituted or added in some sequence, Included is self-explanatory. Also included within the scope of the present application are amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule. Such techniques are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York (1979)).

As used herein, the term "substantial identity or homology" refers to the percentage of identity between two polynucleotides or polypeptide moieties. The homology between sequences from one moiety to another may be determined by known techniques. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from NCBI (National Center for Biological Information) It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLAST is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The polynucleotide encoding the polypeptide of the present application is a nucleotide sequence of a polynucleotide encoding the polypeptide of SEQ ID NO: 1, and a polynucleotide capable of being translated into the polypeptide by codon degeneracy is also disclosed in the present invention It is obvious that it can be included. It also includes sequences complementary to the sequence. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence. Substantially complementary sequences refer to sequences that can hybridize with the nucleotide sequences of the polynucleotides under stringent conditions known in the art. The term "stringent conditions" as used in this application is disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) The stringency conditions can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like, and can be determined differently depending on the sequence to be hybridized.

For example, the polynucleotide may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 and has a homology of at least 70%, specifically at least 80% homology, more specifically at least 90% homology, Specifically, it may include a sequence showing a homology of 95% or more.

As used herein, the term "nucleotide" refers to a deoxyribonucleotide or ribonucleotide present in the form of a single strand or a double strand, encompassing RNA genomic sequences, cDNA and RNA sequences transcribed therefrom, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term " enhanced activity " means that the activity of a polypeptide having the amino acid sequence of SEQ ID NO: 1 is increased relative to the activity of the corresponding intrinsic or non-variable polypeptide of the host microorganism. In the present application, the term " activity increase " can be used in combination with " activity enhancement ". Furthermore, enhancing the activity of the polypeptide of the present application can result in higher levels of L-lysine than wild-type, native or non-mutant microorganisms.

Specifically, the activity increase in the present application means that,

1) increase in the number of copies of the polynucleotide encoding the protein,

2) modification of the expression control sequence to increase the expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome so as to enhance the activity of the protein,

4) introduction of an exogenous polynucleotide exhibiting the activity of the protein or a polynucleotide with a codon-optimized mutated polynucleotide of the polynucleotide, or

5), and a method of modifying it so as to be strengthened by a combination of these methods. However, the present invention is not limited thereto.

The copy number increase of the 1) polynucleotide can be carried out in a form that is not particularly limited but is operably linked to a vector or inserted into a chromosome in a host cell. Specifically, a polynucleotide encoding the protein of the present invention may be operatively linked to a vector capable of being replicated and functioned independently of the host, and introduced into the host cell. Alternatively, the polynucleotide may be inserted into a chromosome in the host cell The polynucleotide may be operably linked to a vector capable of being introduced into the host cell to increase the number of copies of the polynucleotide in the chromosome of the host cell.

Next, 2) modification of the expression control sequence so that the expression of the polynucleotide is increased is not particularly limited. However, the nucleic acid sequence may be deleted, inserted, non-conserved or conservative substitution, or the like to enhance the activity of the expression control sequence. , Or by replacing with a nucleic acid sequence having more potent activity. The expression regulatory sequence may include, but is not limited to, promoters, operator sequences, sequences encoding ribosomal binding sites, sequences regulating the termination of transcription and translation, and the like.

A strong heterologous promoter may be connected to the polynucleotide expression unit in place of the original promoter. Examples of the strong promoter include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, lysCP1 promoter 096689) or CJ7 promoter (Korea Patent No. 0620092 and WO2006 / 065095), but the present invention is not limited thereto.

In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited. However, modification of the nucleotide sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof may be used to further enhance the activity of the polynucleotide sequence. , Or by replacing the polynucleotide sequence with an improved polynucleotide sequence so as to have stronger activity.

4) introduction of an exogenous polynucleotide sequence can be carried out by introducing into a host cell an exogenous polynucleotide encoding a protein exhibiting the same / similar activity as the protein, or a codon-optimized mutant polynucleotide thereof. The foreign polynucleotide can be used without limitation in its sequence or sequence as long as it exhibits the same / similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into the host cell by optimizing its codon so that transcription and translation are optimized in the host cell. Such introduction can be carried out by appropriately selecting a person skilled in the art by a known transformation method, and by expressing the introduced polynucleotide in a host cell, a protein can be produced and its activity can be increased.

Finally, 5) a method of modifying to enhance by the combination of 1) to 4) above comprises: increasing the number of copies of the polynucleotide encoding the protein; modifying the expression control sequence so that expression thereof increases; A modification of the sequence and an exogenous polynucleotide expressing the activity of the protein or a modification of the codon optimized mutated polynucleotide thereof.

As used herein, the term "vector" means a DNA product containing the desired gene or NDA sequence operably linked to a suitable regulatory sequence so as to express the desired gene or DNA, that is, Transfected to a large number, or expressed in a protein. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be transcribed into an appropriate host cell and then cloned or functioned independently of the host genome and integrated into the genome itself. The vector may further comprise a selection marker for confirming whether it has been introduced into the host cell or inserted into the chromosome, and the selectable marker is selected from the group consisting of a drug resistance, an auxotrophy, Various markers can be used that confer a selectable phenotype, such as the expression of surface proteins. In the selective treatment environment, only the cells expressing the selection marker survive or express different phenotypes, so that the transformed cells can be selected.

The vector of the present application is typically constructed as a vector for cloning or as a vector for expression. But is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. PWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A can be used as the phage vector or cosmid vector, and as the plasmid vector, pBR, pUC, pBluescriptII, pGEM, pTZ system, pCL system, pET system, or the like can be used. For example, when the vector of the present application is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, pR? promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of detoxification and a transcription / Alternatively, plasmids known in the art (for example, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.) can be used as the vectors used in the present invention.

As used herein, the term "operatively linked" means a functional linkage between an expression control sequence (e.g., a promoter sequence) of a polynucleotide and another polynucleotide, Thereby controlling the transcription and / or translation of other polynucleotides.

The term "transformation ", as used herein, refers to a method of introducing a polynucleotide into a host to induce the polynucleotide to be inserted into the genome as an extra genomic or capable of replicating a desired gene. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. The expression cassette can typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Also, the polynucleotide may be introduced into the host cell in its own form and operatively linked to the sequence necessary for expression in the host cell, but is not limited thereto.

The method of transferring the vector or the like into a host cell in the transformation process includes any method of introducing the nucleic acid into a cell and may be carried out by selecting a suitable standard technique as known in the art depending on the host cell . For example, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 167: 557-580 (1983)) and the electric pulse method (see, for example, Kang et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)), but are not limited thereto.

As the host cell, it is preferable to use a host having a high introduction efficiency of DNA and a high efficiency of expression of the introduced DNA, for example, a microorganism belonging to the genus Corynebacterium.

The term "L-lysine producing microorganism " as used in the present application means a microorganism strain capable of producing or secreting L-lysine for the purpose of the present application, and can be produced by a process according to the present invention in a larger amount or in a higher concentration Lt; / RTI > can be a microorganism capable of producing and accumulating L-lysine in a culture medium. The microorganism may include all the Corynebacterium sp. Microorganisms whose activity of the polypeptide having the amino acid sequence of SEQ ID NO: 1 herein is enhanced as compared with the intrinsic activity. Examples thereof include Corynebacterium glutamicum , Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes), Corynebacterium thermo amino to Ness (Corynebacterium thermoaminogenes), Brevibacterium Plastic boom (Brevibacterium flavum), or Brevibacterium lactofermentum Pere (Brevibacterium fermentum , and the like may be used, but the present invention is not limited thereto. One example, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum (Corynebacterium glutamicum ) can be used. Specifically, Corynebacterium glutamicum disclosed in the present application (Accession No. KCCM11877P) can be used. The present invention is not limited to these examples, and microorganisms of the genus Corynebacterium having L-lysine producing ability known in the art may be used.

Examples of the Corynebacterium sp. Microorganism having L-lysine production ability include Korean Patent No. 10-0397322, Korean Patent No. 10-0924065, Korean Patent No. 10-0073610, and / And Binder et al., Genome Biology 2012, 13: R40, the entire contents of which are incorporated herein by reference in their entirety.

Another aspect of the present application relates to a method for producing microorganisms which comprises culturing the microorganism belonging to the genus Corynebacterium in a medium; And recovering the L-lysine from the cultured microorganism, the culture medium or a mixture of the cultured microorganism and the culture medium.

The term "culture" as used in the present application means that the microorganism is grown under moderately controlled environmental conditions. The method of culturing the microorganism of the present application can be carried out according to a suitable culture medium and culture conditions well known in the art and can be easily adjusted by those skilled in the art depending on the selected strain. Specifically, examples of the culture method include, but are not limited to, a batch culture, a continuous culture, and a fed-batch culture. These various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

As used herein, the term "media" is used in the cultivation of bacteria, particularly the transformed Corynebacterium sp. Microorganism, wherein the microorganism is a source of carbon, nitrogen, Means a medium containing salts. The culture medium may include a culture medium prepared for culturing the mutant strain, and various substances in which the microorganism is released into the culture medium during growth. Specifically, the culture medium may contain L-lysine as a target substance.

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for microorganisms of the genus Corynebacterium are known (see, for example, Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington D.C., USA, 1981). The carbon source in the medium may be, for example, sugars and carbohydrates such as glucose, sucrose, sodium citrate, fructose, lactose or maltose, soybean oil, sunflower oil, But are not limited to, oils and fats such as castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture.

The nitrogen source in the medium may be, for example, a compound including peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen , But is not limited thereto. The inorganic salts contained in the culture medium may include, but are not limited to, compounds including magnesium, manganese, potassium, calcium, iron, zinc, cobalt and the like.

The number of people that can be used in the culture medium may include, but is not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or a corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. The compounds may be used individually or as a mixture, but are not limited thereto.

In addition to the components of carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be additionally added to the medium of the present application. The above-mentioned raw materials can be added to the medium in a batch manner or in a continuous manner by an appropriate method.

On the other hand, the pH of the medium can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) may be injected into the medium to maintain the exhalation state. The temperature of the culture may be 20 to 45 캜, but is not limited thereto. The culture may continue until the desired amount of L-lysine is produced, and may be accomplished in 10 to 160 hours, but is not limited thereto. The resulting L-lysine may be released into the culture medium or contained in the microorganism. The step of recovering the L-lysine from the medium in which the microorganism has been cultured, the microorganism or the mixture of the cultured microorganism and the culture medium may be carried out by various methods well known in the art, for example, centrifugation, filtration, anion exchange chromatography, Crystallization and HPLC may be used, but are not limited thereto. Further, the recovering step may include a purification step.

The Corynebacterium sp. Microorganism introducing the recombinant vector according to the present application has improved L-lysine producing ability.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  One: Corynebacterium Glutamicum  Production of wild-type DNA library

Corynebacterium < RTI ID = 0.0 > glutamicum ) was prepared as follows.

Specifically, genomic DNA was extracted from Corynebacterium glutamicum ATCC13032, and DNA fragments of 2 to 4 kb were selectively obtained by treating with restriction enzyme Sau3AI (Takara). The resultant was ligated with pECCG117 (Korean Patent No. 10-0057684) vector having a restriction endonuclease BamHI end and then introduced into E. coli DH5α and transformed into LB solid medium containing kanamycin (25 mg / L) . When 100 colonies were randomly selected and PCR was carried out using the primers of SEQ ID NOS: 5 and 6, it was found that the ratio of the colonies containing the target DNA fragment inserted with 2 to 4 kb DNA fragments was 90% or more Respectively. All the obtained colonies were inoculated into an LB liquid medium containing kanamycin (25 mg / L), mixed and cultured, and plasmids were extracted using a conventionally known plasmid extraction method to obtain a genomic DNA library of Corynebacterium glutamicum ATCC13032 Completed.

SEQ ID NO: 5: 5'-ACGACGGGATCAGTACCGA-3 '

SEQ ID NO: 6: 5'-AGCTATCTGTCGCAGCGCC-3 '

Example  2: genomic  By introducing a DNA library Lysine Production capacity  Branch Corynebacterium genus  Strain production

The genomic DNA library prepared in Example 1 was introduced into Corynebacterium glutamicum KCCM11016P, which is a lysine-producing strain, by using a known electric pulse method (the microorganism was disclosed as KFCC010881, and under the Budapest Treaty, And has been deposited with KCCM 11016P and deposited with the Korean Patent No. 10-0159812. Then, Corynebacterium glutamicum KCCM11016P into which a genomic DNA library was introduced was plated on a composite plate medium containing kanamycin (25 mg / L) and cultured for 30 to 24 hours to obtain about 2,000 colonies. Then, 200 쨉 l of the complex liquid medium was dispensed into each well of the 96-well cell culture plate, and the obtained colonies were inoculated respectively, followed by shaking culture at 30 째 C and 1,200 rpm for 24 hours. The culture was centrifuged to separate the cells and the supernatant, and 50 μl of the supernatant was mixed with the reaction solution containing lysine oxidase.

<Composition of composite plate culture medium: 1 L of distilled water>

Glucose _{20g, (NH 4) 2 SO} 4 50g, 10g peptone, 5g yeast extract, urea _{1.5g, KH 2 PO 4 5g,} K 2 HPO 4 10g, MgSO 4 7H 2 O 0.5g, 100㎍ biotin, 1,000 of thiamine hydrochloride Mu g, calcium-pantothenic acid 2,000 g, nicotinamide 2,000 g, agar 20 g

<Compositional liquid medium composition: based on 1 L of distilled water>

Glucose 20g, peptone 10g, 5g yeast extract, urea _{1.5g, KH 2 PO 4 4g,} K 2 HPO 4 8g, MgSO 4 7H 2 O 0.5g, 100㎍ biotin, thiamine HCl 1,000㎍, calcium pantothenate 2,000㎍, nicotine Amide 2,000 g

<Reaction solution composition: Based on 1 ml of potassium phosphate buffer>

0.02 unit of lysine oxidase (Sigma-Aldrich), 0.2 unit of peroxidase (Sigma-Aldrich), 2 mg of ABTS

After mixing the supernatant and the reaction solution, the absorbance at OD ₄₀₅ was analyzed for 30 minutes and 14 kinds of test groups showing higher absorbance than the control group (KCCM11016P / pECCG117) were selected. Each strain was inoculated into a 250 ml corn-baffle flask containing 25 ml of the seed medium and cultured under shaking conditions at 30 and 200 rpm for 20 hours to prepare a seed culture. Then, 1 ml of the seed culture was inoculated in a 250 ml corn-baffle flask containing 24 ml of the production medium, and cultured with shaking at 37 and 200 rpm for 96 hours. After completion of the culture, the concentration of L-lysine was analyzed by HPLC (Table 1).

<Composition of seed medium: 1 L of distilled water>

Glucose _{20g, (NH 4) 2 SO} 4 40g, 10g peptone, 5g yeast extract, urea _{1.5g, KH 2 PO 4 4g,} K 2 HPO 4 8g, MgSO 4 7H 2 O 0.5g, EO tin 100㎍, thiamine HCl 2,000 g of calcium-pantothenic acid, 2,000 g of nicotinamide

<Production medium (pH 7.0) Composition: 1 liter of distilled water>

Glucose _{100g, (NH 4) 2 SO} 4 40g, 2.5g of soy protein, corn steep solids (Corn Steep Solids) 5g, urea 3g, KH ₂ PO ₄ 1 g of MgSO ₄ 7H ₂ OO, 100 g of biotin, 1,000 g of thiamine hydrochloride, 2,000 g of calcium-pantothenic acid, _3,000 g of nicotinamide, 30 g of CaCO ₃

From the above results, KCCM11016P / 2-254 and KCCM11016P / 5-95 showing the effect of increasing lysine production ability compared to the control group were selected and the plasmid was extracted using a conventionally known plasmid extraction method. The plasmids derived from KCCM11016P / 2-254 were pEC-2-254, and the plasmids derived from KCCM11016P / 5-95 were pEC-5- 95 &lt; / RTI &gt;

As a result of sequencing, the plasmid pEC-2-254 contained the nucleotide sequence of SEQ ID NO: 17 and the plasmid pEC-5-95 contained the nucleotide sequence of SEQ ID NO: 18, whereby the two plasmids contained SEQ ID NO: 2, which encodes the amino acid sequence of SEQ ID NO: 3, and the nucleotide sequence of SEQ ID NO: 4, which encodes the amino acid sequence of SEQ ID NO: 3. The gene encoding the amino acid sequence of SEQ ID NO: 1 is referred to as HL1622, and the gene encoding the amino acid sequence of SEQ ID NO: 3 is referred to as HL1623, hereinafter referred to as HL1622 and HL1623.

Example  3: Vector construction for additional insertion of HL1622, HL1623 gene chromosome

In order to analyze the effect of the HL1622 and HL1623 genes confirmed in Example 2 on the L-lysine production enhancing effect, pDZTn vector (Korean Patent No. 10-1126041) was used as a basic vector, Was added to the chromosome of the leiomyum.

Example  3-1: Vector construction for additional insertion of HL1622 gene chromosome

In order to prepare a vector for further insertion of HL1622, a primer (SEQ ID NOS: 7 and 8) having a SpeI restriction enzyme site inserted at the 5 'end was synthesized based on the reported nucleotide sequence and PCR was performed using the chromosome of ATCC13032 as a template A DNA fragment of about 1,520 bp containing about 149 bp of the lower end of the termination codon was obtained at the upper 500 bp region of the HL1622 start codon.

SEQ ID NO: 7: 5'-AA ACTAGT GCGGAGTACTAGGTCGTG-3 '

SEQ ID NO: 8: 5'-AA ACTAGT TTATGAATTCGGCTCCAA-3 '

(The underlined part of the base sequence indicates the recognition site for the restriction enzyme.)

The denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 90 seconds were repeated 30 times, followed by polymerization at 72 ° C for 7 minutes. The DNA fragment amplified by PCR was treated with restriction enzyme SpeI to obtain each DNA fragment. The DNA fragment was ligated to a pDZTn vector having a restriction enzyme SpeI terminal and transformed into E. coli DH5α, and 25 mg / l of kanamycin was contained Lt; / RTI &gt; solid medium. After selecting the colonies transformed with the vector into which the desired gene was inserted by PCR, a plasmid was obtained using a conventionally known plasmid extraction method and named pDZTn-HL1622.

Example  3-2: Construction of Vector for Additional Insertion of HL1623 Gene Chromosome

In order to prepare a vector for further insertion of HL1623, primers (SEQ ID NOS: 9 and 10) in which a SpeI restriction enzyme site was inserted at the 5 'end were synthesized based on the reported nucleotide sequences and PCR To obtain a DNA fragment of about 1,304 bp in length including the lower 150 bp of the termination codon from the upper 500 bp region of the HL1623 start codon. The resultant was ligated to a pDZTn vector having a restriction enzyme SpeI terminus, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin.

SEQ ID NO: 9: 5'-AA ACTAGT GGAATCGGCGGTTTAGTG-3 '

SEQ ID NO: 10: 5'-AA ACTAGT CTGATCAATAACCTCC-3 '

(The underlined part of the base sequence indicates the recognition site for the restriction enzyme.)

After selecting the colonies transformed with the vector into which the desired gene was inserted by PCR, a plasmid was obtained using a conventionally known plasmid extraction method and named pDZTn-HL1623.

Example  3-3: Vector construction for simultaneous insertion of HL1622 and HL1623 gene chromosomes

In order to construct a vector for simultaneous insertion of HL1622 and HL1623, PCR was performed using the primers of SEQ ID NO: 7 and SEQ ID NO: 10 as a template of the ATCC13032 chromosome to generate a 500 bp upper portion of the HL1622 start codon and a 2,473 bp DNA fragments were amplified. The denaturation at 94 ° C for 5 minutes, the denaturation at 94 ° C for 30 seconds, the annealing at 56 ° C for 30 seconds, and the polymerization at 72 ° C for 120 seconds were repeated 30 times, followed by a polymerization reaction at 72 ° C for 7 minutes. The DNA fragment amplified by PCR was ligated to a pDZTn vector in the same manner as above to transform E. coli DH5α and plated on LB solid medium containing 25 mg / l of kanamycin. After selecting the colonies transformed with the vector into which the desired gene was inserted through PCR, a plasmid was obtained using a conventionally known plasmid extraction method and named pDZTn-HL1622-HL1623.

Example  4: HL1622, HL1623 and of the two gene insertion strains Lysine Production capacity  analysis

In order to analyze the lysine production ability of the three vectors pDZTn-HL1622, pDZTn-HL1623 and pDZTn-HL1622-HL1623 prepared in Example 3, Corynebacterium glutamicum KCCM11016P Respectively. Then, the strains in which the desired gene was inserted on the chromosome were selectively separated by PCR and named KCCM11016P :: HL1622 (Tn), KCCM11016P :: HL1623 (Tn) and KCCM11016P :: HL1622-HL1623 (Tn).

A 250 ml Corner-Baffle flask containing 3 strains and KCCM11016P strain as a seed medium was inoculated and cultured by shaking at 30 DEG C and 200 rpm for 20 hours. Thereafter, 1 ml of the seed culture was inoculated into 250 ml of a corn-baffle flask containing 24 ml of the following production medium, and cultured at 37 DEG C and 200 rpm for 96 hours. Then, L-lysine Were measured and compared (Table 2).

<Production medium (pH 7.0): 1 liter of distilled water>

(NH ₄ ) ₂ SO ₄ 40 g, soybean protein 2.5 g, corn steep solids 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 0.5 g, and ammonium acetate 7.1 g (with or without addition) 100 μg of biotin, 1,000 μg of thiamine hydrochloride, 2,000 μg of calcium-pantothenic acid, 3,000 μg of nicotinamide, 30 g of CaCO ₃

As a result of analysis of L-lysine concentration, it was confirmed that 6% of KCCM11016P :: HL1622 (Tn) and 5% of KCCM11016P :: HL1622-HL1623 (Tn) were increased in L-lysine production ability as compared with the control KCCM11016P strain. The KCCM11016P :: HL1623 (Tn) strain into which the HL1623 gene was further inserted was equivalent to the L-lysine producing ability in comparison with the control strain. The above results confirmed that only the HL1622 gene was effective in increasing L-lysine production ability.

Example  5: Production of HL1622 overexpressing strain by strong promoter and confirmation of effect

To confirm whether HL1622 overexpression increased L-lysine production ability, the previously reported strong promoter pcj7 promoter (WO 2009/096689) was inserted in the upper part of HL1622. A pDZ vector was used as a basic vector to construct a chromosome insertion vector.

Specifically, a primer (SEQ ID NO: 11) inserted with an XhoI restriction enzyme site at the 5 'end and a primer (SEQ ID NO: 12) inserted at the 5' end with an Nde restriction enzyme site were synthesized and Corynebacterium glutamicum pcj7 PCR was performed using the chromosomal DNA of the strain as a template to amplify a promoter region of about 297 bp in length. At this time, the PCR was performed at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 30 seconds, followed by polymerization at 72 ° C for 7 minutes.

SEQ ID NO: 11: 5'-AA CTCGAG ATAGGGAGCGTTGACCTT-3 '

SEQ ID NO: 12: 5'-GG CATATG TGTTTCCTTTCGTTGGG-3 '

(The underlined part of the base sequence indicates the recognition site for the restriction enzyme.)

Thereafter, a primer (SEQ ID NO: 13) inserted with an NdeI restriction enzyme site at the 5 'end and a primer (SEQ ID NO: 14) inserted with the SpeI restriction enzyme site at the 5' end were synthesized and the chromosome of ATCC 13032 was used as a template PCR was performed under the same conditions to amplify a DNA fragment of HL1622 of about 495 bp in length.

SEQ ID NO: 13: 5'-CA CATATG CCCAAATACATTGCCAT-3 '

SEQ ID NO: 14: 5'-AA ACTAGT ATCCAGCTTCAGGTTTCGCA-3 '

(The underlined part of the base sequence indicates the recognition site for the restriction enzyme.)

Also, a primer (SEQ ID NO: 15) inserted with an XbaI restriction site at the 5 'end and a primer (SEQ ID NO: 16) inserted with the XhoI restriction site at the 5' end were synthesized and the ATCC13032 chromosome was used as a template PCR amplification was performed to amplify the 496 bp HL1622 upstream region.

SEQ ID NO: 15: 5'-AA TCTAGA AGTACTAGGTCGTGTGCTGT-3 '

SEQ ID NO: 16: 5'-AA CTCGAG TTGGTTCTGCTTTCACTAAA-3 '

(The underlined part of the base sequence indicates the recognition site for the restriction enzyme.)

DNA fragment obtained by treating the upper DNA fragment of HL1622 amplified by PCR with XbaI and XhoI, DNA fragment obtained by treating pcj7 DNA fragment with restriction enzymes XhoI and NdeI, and HL1622 DNA fragment were treated with restriction enzymes NdeI and SpeI The resulting DNA fragment was ligated to a pDZ vector having a restriction endonuclease XbaI end, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin. After selecting the colonies transformed with the vector into which the desired gene was inserted through PCR, the plasmid was obtained using a conventionally known plasmid extraction method and named pDZ-pcj7_HL1622.

The constructed vector pDZ-pcj7_HL1622 was transformed into L-lysine producing strain, Corynebacterium glutamicum KCCM11016P by homologous recombination on the chromosome. The colonies in which the promoter of the HL1622 gene was replaced with pcj by PCR were selectively separated and named KCCM11016P :: pcj7_HL1622.

The KCCM11016P :: pcj7_HL1622 strain and the control KCCM11016P strain were cultured in the same manner as in Example 2, and the concentrations of lysine contained in each culture were measured and compared. As a result, it was confirmed that the L-lysine production capacity of the KCCM11016P :: pcj7-HL1622 strain was increased by 8% as compared with that of the control KCCM11016P strain, and it was once again confirmed that the L-lysine production capacity was increased as the HL1622 expression level was increased 3).

Accordingly, the present inventors named the KCCM11016P :: pcj7-HL1622 strain having increased L-lysine production ability as Corynebacterium glutamicum "CA01-2300", and as an international depositary institution under the Budapest Treaty on August 2, 2016 Deposited at the Microbial Conservation Center (KCCM) Accession number KCCM11877P.

Example  6: From KCCM10770P  Using HL1622 over-expressed microorganisms Lysine  production

The vector pDZ-pcj7_HL1622 prepared in Example 5 was transformed into 3 lines of L-lysine biosynthesis pathway-related genes into Corynebacterium glutamicum KCCM10770P (Korea Patent No. 10-0924065), a lysine-producing strain inserted into the chromosome . The colonies were then selectively isolated by PCR and named KCCM10770P :: pcj7- HL1622. The strain was cultured in the same manner as in Example 2, and the concentration of L-lysine in the culture broth was analyzed. As a result, as shown in Table 4, it was confirmed that the L-lysine production ability of the KCCM10770P :: pcj7-HL1622 strain was increased by 5% as compared with the control KCCM10770P strain (Table 4).

Example  7: From CJ3P  Using HL1622 over-expressed microorganisms Lysine  production

The vector pDZ-pcj7_HL1622 prepared in Example 5 was inoculated to three chromosome-producing lysine-producing strains Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13: R40 ). The colonies were then selectively isolated by PCR and named CJ3P :: pcj7-HL1622. The strain was cultured in the same manner as in Example 2, and the concentration of the recovered L-lysine was analyzed. As a result, it was confirmed that the L-lysine production capacity of the CJ3P :: pcj7-HL1622 strain was increased by 32% as compared with that of the control CJ3P strain (Table 5).

Example  8: At KFCC10750  Using HL1622 over-expressing microorganisms Lysine  production

The vector pDZ-pcj7_HL1622 prepared in Example 5 was transformed into 3 types of L-lysine productivity-improving genes, Corynebacterium glutamicum KCCM11347P, a chromosomally inserted lysine producing strain (the microorganism was disclosed as KFCC10750 It has been re-deposited with the international depositary under the Budapest Treaty and granted KCCM 11347P. Korean Patent No. 10-0073610). Then, the colonies were selectively separated by PCR and named KCCM11347P :: pcj7-HL1622, and cultured in the same manner as in Example 2, and the concentration of the recovered L-lysine was analyzed. As a result, it was confirmed that the L-lysine production capacity of the KCCM11347P :: pcj7-HL1622 strain was increased by 32% as compared with that of the control KCCM11347P strain (Table 6).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in various other forms without departing from the essential characteristics thereof. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all modified forms falling within the scope of the claims shall be construed as being included in the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11877P 20160802

<110> CJ CheilJedang Corporation <120> Corynebacteria producing L-lysine and a method of producing          L-lysine using the same <130> PN150224 <160> 18 <170> KoPatentin 3.0 <210> 1 <211> 339 <212> PRT <213> Corynebacterium glutamicum <400> 1 Met Pro Lys Tyr Ile Ala Met Gln Val Ser Glu Ser Gly Ala Pro Leu   1 5 10 15 Ala Ala Asn Leu Val Gln Pro Ala Pro Leu Lys Ser Arg Glu Val Arg              20 25 30 Val Glu Ile Ala Ala Ser Gly Val Cys His Ala Asp Ile Gly Thr Ala          35 40 45 Ala Ala Ser Gly Lys His Thr Val Phe Pro Val Thr Pro Gly His Glu      50 55 60 Ile Ala Gly Thr Ile Ala Glu Ile Gly Glu Asn Val Ser Arg Trp Thr  65 70 75 80 Val Gly Asp Arg Val Ala Ile Gly Trp Phe Gly Gly Asn Cys Gly Asp                  85 90 95 Cys Ala Phe Cys Arg Ala Gly Asp Pro Val His Cys Arg Glu Arg Lys             100 105 110 Ile Pro Gly Val Ser Tyr Ala Gly Gly Trp Ala Gln Asn Ile Val Val         115 120 125 Pro Ala Glu Ala Lea Ala Ale Pro Asp Gly Met Asp Phe Tyr Glu     130 135 140 Ala Ala Pro Met Gly Cys Ala Gly Val Thr Thr Phe Asn Ala Leu Arg 145 150 155 160 Asn Leu Lys Leu Asp Pro Gly Ala Ala Val Ala Val Phe Gly Ile Gly                 165 170 175 Gly Leu Val Arg Leu Ala Ile Gln Phe Ala Ala Lys Met Gly Tyr Arg             180 185 190 Thr Ile Thr Ile Ala Arg Gly Leu Glu Arg Glu Glu Leu Ala Arg Gln         195 200 205 Leu Gly Ala Asn His Tyr Ile Asp Ser Asn Asp Leu His Pro Gly Gln     210 215 220 Ala Leu Phe Glu Leu Gly Gly Ala Asp Leu Ile Leu Ser Thr Ala Ser 225 230 235 240 Thr Thr Glu Pro Leu Ser Glu Leu Ser Thr Gly Leu Ser Ile Gly Gly                 245 250 255 Gln Leu Thr Ile Ile Gly Val Asp Gly Asp Ile Thr Val Ser Ala             260 265 270 Ala Gln Leu Met Met Asn Arg Gln Ile Ile Thr Gly His Leu Thr Gly         275 280 285 Ser Ala Asn Asp Thr Glu Gln Thr Met Lys Phe Ala His Leu His Gly     290 295 300 Val Lys Pro Leu Ile Glu Arg Met Pro Leu Asp Gln Ala Asn Glu Ala 305 310 315 320 Ile Ala Arg Ile Ser Ala Gly Lys Pro Arg Phe Arg Ile Val Leu Glu                 325 330 335 Pro Asn Ser             <210> 2 <211> 1020 <212> DNA <213> Corynebacterium glutamicum <400> 2 cgcgaatctc 60 gtgcaacctg ctccgttgaa atcgagggaa gtccgcgtgg aaatcgctgc tagtggtgtg 120 tgccatgcag atattggcac ggcagcagca tcggggaagc acactgtttt tcctgttacc 180 cctggtcatg agattgcagg aaccatcgcg gaaattggtg aaaacgtatc tcggtggacg 240 gttggtgatc gcgttgcaat cggttggttt ggtggcaatt gcggtgactg cgctttttgt 300 cgtgcaggtg atcctgtgca ttgcagagag cggaagattc ctggcgtttc ttatgcgggt 360 ggttgggcac agaatattgt tgttccagcg gaggctcttg ctgcgattcc agatggcatg 420 gacttttacg aggccgcccc gatgggctgc gcaggtgtga caacattcaa tgcgttgcga 480 aacctgaagc tggatcccgg tgcggctgtc gcggtctttg gaatcggcgg tttagtgcgc 540 ctagctattc agtttgctgc gaaaatgggt tatcgaacca tcaccatcgc ccgcggttta 600 gagcgtgagg agctagctag gcaacttggc gccaaccact acatcgatag caatgatctg 660 caccctggcc aggcgttatt tgaacttggc ggggctgact tgatcttgtc tactgcgtcc 720 accacggagc ctctttcgga gttgtctacc ggtctttcta ttggcgggca gctaaccatt 780 atcggagttg atgggggaga tatcaccgtt tcggcagccc aattgatgat gaaccgtcag 840 atcatcacag gtcacctcac tggaagtgcg aatgacacgg aacagactat gaaatttgct 900 catctccatg gcgtgaaacc gcttattgaa cggatgcctc tcgatcaagc caacgaggct 960 attgcacgta tttcagctgg taaaccacgt ttccgtattg tcttggagcc gaattcataa 1020                                                                         1020 <210> 3 <211> 267 <212> PRT <213> Corynebacterium glutamicum <400> 3 Met Pro Thr Ala Ser Pro Ile Tyr Asp Val Val Val Gly Ala Gly   1 5 10 15 Ile Ser Gly Leu Ile Ala Thr Gln Leu Leu Asp Arg Ala Gly Leu Asn              20 25 30 Ile Lys Cys Phe Glu Ala Cys Ser Arg Val Gly Gly Arg Ala Val Ser          35 40 45 Val Gln Gln Ser Asp Leu Phe Leu Asp Leu Gly Ala Thr Trp Phe Trp      50 55 60 Leu Asn Glu Pro Leu Val Gln Gln Leu Val Asn Asn Leu Gly Leu Gly  65 70 75 80 Thr Phe Pro Gln Ale Ile Glu Gly Asp Ala Leu Phe Glu Thr Leu Val                  85 90 95 Asp Ala Pro Ser Arg Leu Arg Gly Asn Pro Ile Asp Ala Ala Ser Gly             100 105 110 Arg Phe Gln Ala Gly Ala Ser Ser Leu Ala Leu Gly Leu Ala Ala Gln         115 120 125 Leu Lys Pro Gly Val Leu Glu Leu Gly Asp Pro Val His Ser Leu Ser     130 135 140 Glu Glu Asp Gly Glu Ile Val Val Lys Ser Ser Lys Gln Ile Val Arg 145 150 155 160 Ala Lys His Val Ile Ile Ala Val Pro Ala Leu Ala Ala Glu Leu                 165 170 175 Ile Gly Phe Thr Leu Asp Leu Pro Ala Asp Val Arg Lys Ala Ala His             180 185 190 Pro Gln His Ile Ala Val Met Asn Trp Ala Lys Glu Lys Tyr Thr Leu         195 200 205 Pro Thr Gln Ala Ala Ser Ala Gly Gly Phe Gly His Glu Leu Phe Gln     210 215 220 Gln Pro Leu Gly His Gly Arg Ile His Trp Ala Ser Thr Glu Val Ala 225 230 235 240 Thr Glu Phe Gly Gly His Leu Glu Gly Ala Val Arg Ala Gly Ile Gln                 245 250 255 Ala Ala Leu Gln Thr Gly Phe Asn Leu Lys Ser             260 265 <210> 4 <211> 804 <212> DNA <213> Corynebacterium glutamicum <400> 4 atgccaacag caagcccaat ttatgatgtc gttgtcgtcg gagccggcat ttctggcctc 60 atcgccacgc aactgttgga ccgcgcaggt ctaaacatca aatgcttcga agcctgctca 120 agagttggcg gccgagcagt gtctgtccaa cagtccgatt tgttcctgga cctcggcgca 180 acatggttct ggctcaacga accacttgtg cagcaactcg tcaataatct cggcctcggc 240 acattccctc aggccatcga gggtgatgcg ctttttgaga cgcttgtcga cgccccgagc 300 cgcctgcggg gtaaccccat agacgctgct tcaggcaggt tccaagcagg ggcctcctcg 360 cttgcgctcg ggcttgcagc ccagctcaag ccaggagttt tagaactcgg ggaccccgtc 420 cattctctca gtgaggaaga tggggaaatc gttgtgaagt cttccaaaca gattgtgagg 480 gcaaagcacg tcatcattgc ggttccaccg gcactcgctg ccgagttgat tggtttcacc 540 ctagatttac cagctgacgt gcgaaaagca gcgcatccac aacatatagc tgtgatgaat 600 tgggcaaagg agaaatacac cttacccaca caagccgcat cggctggggg ttttgggcat 660 gagctgttcc aacaaccact cggacatggg cgaattcatt gggcatcaac ggaagttgcc 720 actgagtttg gtggacacct tgaaggcgca gttcgtgcag gaattcaggc tgcgcttcaa 780 acaggattta atctaaaatc ttaa 804 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 5 acgacgggat cagtaccga 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 6 agctatctgt cgcagcgcc 19 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 7 aaactagtgc ggagtactag gtcgtg 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 8 aaactagttt atgaattcgg ctccaa 26 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 9 aaactagtgg aatcggcggt ttagtg 26 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 10 aaactagtct gatcaataac ctcc 24 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 11 aactcgagat agggagcgtt gacctt 26 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 12 ggcatatgtg tttcctttcg ttggg 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 13 cacatatgcc caaatacatt gccat 25 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 14 aaactagtat ccagcttcag gtttcgca 28 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 15 aatctagaag tactaggtcg tgtgctgt 28 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 16 aactcgagtt ggttctgctt tcactaaa 28 <210> 17 <211> 2195 <212> DNA <213> Corynebacterium glutamicum <400> 17 ttccagtttg gcgatgccgt tggatagcgc aggttgggtg actccgtatt cgcgggctgc 60 tgcgctgaat gagtgagttt ctgcgacggc ctgcgcatag cggagccctt caaggctgag 120 ccgtttggtc ataactatat gttatccccc ttattcagag tgatggtcta ccggagaagt 180 acccagacca atagcatcga ccaacgatag cgcgctcaga agttctttag tgaaagcaga 240 accaaatgcc caaatacatt gccatgcagg tatccgaatc cggtgcaccg ttagccgcga 300 atctcgtgca acctgctccg ttgaaatcga gggaagtccg cgtggaaatc gctgctagtg 360 gtgtgtgcca tgcagatatt ggcacggcag cagcatcggg gaagcacact gtttttcctg 420 ttacccctgg tcatgagatt gcaggaacca tcgcggaaat tggtgaaaac gtatctcggt 480 ggacggttgg tgatcgcgtt gcaatcggtt ggtttggtgg caattgcggt gactgcgctt 540 tttgtcgtgc aggtgatcct gtgcattgca gagagcggaa gattcctggc gtttcttatg 600 cgggtggttg ggcacagaat attgttgttc cagcggaggc tcttgctgcg attccagatg 660 gcatggactt ttacgaggcc gccccgatgg gctgcgcagg tgtgacaaca ttcaatgcgt 720 tgcgaaacct gaagctggat cccggtgcgg ctgtcgcggt ctttggaatc ggcggtttag 780 tgcgcctagc tattcagttt gctgcgaaaa tgggttatcg aaccatcacc atcgcccgcg 840 gtttagagcg tgaggagcta gctaggcaac ttggcgccaa ccactacatc gatagcaatg 900 atctgcaccc tggccaggcg ttatttgaac ttggcggggc tgacttgatc ttgtctactg 960 cgtccaccac ggagcctctt tcggagttgt ctaccggtct ttctattggc gggcagctaa 1020 ccattatcgg agttgatggg ggagatatca ccgtttcggc agcccaattg atgatgaacc 1080 gtcagatcat cacaggtcac ctcactggaa gtgcgaatga cacggaacag actatgaaat 1140 ttgctcatct ccatggcgtg aaaccgctta ttgaacggat gcctctcgat caagccaacg 1200 aggctattgc acgtatttca gctggtaaac cacgtttccg tattgtcttg gagccgaatt 1260 cataatgcca acagcaagcc caatttatga tgtcgttgtc gtcggagccg gcatttctgg 1320 cctcatcgcc acgcaactgt tggaccgcgc aggtctaaac atcaaatgct tcgaagcctg 1380 ctcaagagtt ggcggccgag cagtgtctgt ccaacagtcc gatttgttcc tggacctcgg 1440 cgcaacatgg ttctggctca acgaaccact tgtgcagcaa ctcgtcaata atctcggcct 1500 cggcacattc cctcaggcca tcgagggtga tgcgcttttt gagacgcttg tcgacgcccc 1560 gagccgcctg cggggtaacc ccatagacgc tgcttcaggc aggttccaag caggggcctc 1620 ctcgcttgcg ctcgggcttg cagcccagct caagccagga gttttagaac tcggggaccc 1680 cgtccattct ctcagtgagg aagatgggga aatcgttgtg aagtcttcca aacagattgt 1740 gagggcaaag cacgtcatca ttgcggttcc accggcactc gctgccgagt tgattggttt 1800 cccctagat ttaccagctg acgtgcgaaa agcagcgcat ccacaacata tagctgtgat 1860 gaattgggca aaggagaaat acaccttacc cacacaagcc gcatcggctg ggggttttgg 1920 gcatgagctg ttccaacaac cactcggaca tgggcgaatt cattgggcat caacggaagt 1980 tgccactgag tttggtggac accttgaagg cgcagttcgt gcaggaattc aggctgcgct 2040 tcaaacagga tttaatctaa aatcttaaac ctcgtatttt ccctgatagg ctcagatgcg 2100 cctgaaatcg ggcttgttga ggggagaggt gtgtgacatg aaagagttgg aactgggcga 2160 ggcgagggac gtcgctgcaa cgttggaagc gatgc 2195 <210> 18 <211> 2792 <212> DNA <213> Corynebacterium glutamicum <400> 18 cgagtccaag tgtggcccac tgttcgagta cttggtagct ggctgcttcg ccggaatagg 60 cgtttaatgc caggtcattt gtttcaaaca gttgattggt gaaagtggtt aaaccgcatg 120 tgtcgggtac cagaatgaac ggttcgtgct gagtctcgcg aagttctatg gggtcggtgc 180 tgtcctgggt ggattcgacg ataacgactg gttcggagtc aatgatgcgg tgttcaaaat 240 ggggtagtgg tttcactgcg ggaatgagaa ttacattaag ttcacctgca agaagtcctt 300 catgtagttc tttcatgttt gcttcgcgga gtactaggtc gtgtgctgtg ggaagctcac 360 gaaccgcggt atatgttcgt gcaaccagtt gagggttgat aagtggggag attccaactc 420 gaatgctgcg tgcttctgag ttaatcaaac ggtgcgcttc cgcggtgatt gcgtcgattt 480 cagtcagcgc gcgttggatc agggggagga tgtggaggcc aaaggacgtc ggggtgacgc 540 cttgagtaga tcgatcgaag agttgttcac cgagccgatc ttccagtttg gcgatgccgt 600 tggatagcgc aggttgggtg actccgtatt cgcgggctgc tgcgctgaat gagtgagttt 660 ctgcgacggc ctgcgcatag cggagccctt caaggctgag ccgtttggtc ataactatat 720 gttatccccc ttattcagag tgatggtcta ccggagaagt acccagacca atagcatcga 780 ccaacgatag cgcgctcaga agttctttag tgaaagcaga accaaatgcc caaatacatt 840 gccatgcagg tatccgaatc cggtgcaccg ttagccgcga atctcgtgca acctgctccg 900 ttgaaatcga gggaagtccg cgtggaaatc gctgctagtg gtgtgtgcca tgcagatatt 960 ggcacggcag cagcatcggg gaagcacact gtttttcctg ttacccctgg tcatgagatt 1020 gcaggaacca tcgcggaaat tggtgaaaac gtatctcggt ggacggttgg tgatcgcgtt 1080 gcaatcggtt ggtttggtgg caattgcggt gactgcgctt tttgtcgtgc aggtgatcct 1140 gtgcattgca gagagcggaa gattcctggc gtttcttatg cgggtggttg ggcacagaat 1200 attgttgttc cagcggaggc tcttgctgcg attccagatg gcatggactt ttacgaggcc 1260 gccccgatgg gctgcgcagg tgtgacaaca ttcaatgcgt tgcgaaacct gaagctggat 1320 cccggtgcgg ctgtcgcggt ctttggaatc ggcggtttag tgcgcctagc tattcagttt 1380 gctgcgaaaa tgggttatcg aaccatcacc atcgcccgcg gtttagagcg tgaggagcta 1440 gctaggcaac ttggcgccaa ccactacatc gatagcaatg atctgcaccc tggccaggcg 1500 ttatttgaac ttggcggggc tgacttgatc ttgtctactg cgtccaccac ggagcctctt 1560 tcggagttgt ctaccggtct ttctattggc gggcagctaa ccattatcgg agttgatggg 1620 ggagatatca ccgtttcggc agcccaattg atgatgaacc gtcagatcat cacaggtcac 1680 ctcactggaa gtgcgaatga cacggaacag actatgaaat ttgctcatct ccatggcgtg 1740 aaaccgctta ttgaacggat gcctctcgat caagccaacg aggctattgc acgtatttca 1800 gctggtaaac cacgtttccg tattgtcttg gagccgaatt cataatgcca acagcaagcc 1860 caatttatga tgtcgttgtc gtcggagccg gcatttctgg cctcatcgcc acgcaactgt 1920 tggaccgcgc aggtctaaac atcaaatgct tcgaagcctg ctcaagagtt ggcggccgag 1980 cagtgtctgt ccaacagtcc gatttgttcc tggacctcgg cgcaacatgg ttctggctca 2040 acgaaccact tgtgcagcaa ctcgtcaata atctcggcct cggcacattc cctcaggcca 2100 tcgagggtga tgcgcttttt gagacgcttg tcgacgcccc gagccgcctg cggggtaacc 2160 ccatagacgc tgcttcaggc aggttccaag caggggcctc ctcgcttgcg ctcgggcttg 2220 cagcccagct caagccagga gttttagaac tcggggaccc cgtccattct ctcagtgagg 2280 aagatgggga aatcgttgtg aagtcttcca aacagattgt gagggcaaag cacgtcatca 2340 ttgcggttcc accggcactc gctgccgagt tgattggttt caccctagat ttaccagctg 2400 acgtgcgaaa agcagcgcat ccacaacata tagctgtgat gaattgggca aaggagaaat 2460 acaccttacc cacacaagcc gcatcggctg ggggttttgg gcatgagctg ttccaacaac 2520 cactcggaca tgggcgaatt cattgggcat caacggaagt tgccactgag tttggtggac 2580 accttgaagg cgcagttcgt gcaggaattc aggctgcgct tcaaacagga tttaatctaa 2640 aatcttaaac ctcgtatttt ccctgatagg ctcagatgcg cctgaaatcg ggcttgttga 2700 ggggagaggt gtgtgacatg aaagagttgg aactgggcga ggcgagggac gtcgctgcaa 2760 cgttggaagc gatgccgatc caggaggtta tt 2792

Claims (4)

A Corynebacterium sp. Microorganism producing L-lysine with enhanced activity of the polypeptide of SEQ ID NO: 1.
The microorganism of claim 1, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum.
Culturing the microorganism of genus Corynebacterium according to claim 1 in a medium; And
And recovering L-lysine from the cultured microorganism, the culture medium or a mixture of the cultured microorganism and the culture medium.
4. The method according to claim 3, wherein the Corynebacterium sp. Microorganism produces Corynebacterium glutamicum, L-lysine.