Herein, the genus Corynebacterium (the genus of producing L- lysine Corynebacterium relates to) microorganisms, and production method of the L- lysine using the same.
BACKGROUND
[2]
L- lysine has been produced by the fermentation of animal feed, which is used in the pharmaceutical and cosmetic industry people, mainly the genus Corynebacterium or the genus Escherichia spp strains. For the production of L- lysine, a variety of studies for the efficient production strain and fermentation process technology it is being carried out. Specifically, the target substance-specific approaches such as increasing the expression of the gene coding for the enzyme involved in L- lysine biosynthesis, or to remove unwanted genes in the biosynthesis have been mainly used (Republic of Korea Patent No. 10-0838038 number).
[3]
[4]
The present inventors have found that by introducing an intrinsic gene of the genus Corynebacterium microorganism to search for effective transfection of increasing the lysine-producing ability at random were identify genes associated with high levels of production of lysine, wherein in Corynebacterium spp Check that L- lysine-producing ability increases when increasing the expression level of the gene, thereby completing the present application.
Detailed Description of the Invention
SUMMARY
[5]
One object of the present application is the genus Corynebacterium (the genus of producing L- lysine-containing protein consisting of the amino acid sequence of SEQ ID NO: 1 that has an increased activity compared to the endogenous activity Corynebacterium to provide a) the microorganism.
[6]
It is another object of the present application is to provide a method of producing L- lysine using the microorganisms.
Problem solving means
[7]
One aspect of the present application for achieving the above object, the genus Corynebacterium, which produce an L- lysine-containing protein consisting of the amino acid sequence of SEQ ID NO: 1 that has an increased activity compared to the endogenous activity (the genus Corynebacterium ) is a microorganism.
[8]
[9]
If it described in detail below. On the other hand, each of the description and the embodiments disclosed herein may be applied to other embodiments and description of each. That is, any combination of the various elements described herein is within the scope of the present application. In addition, to not by the specific techniques described see that the scope of the present limits.
[10]
[11]
As used herein, a protein consisting of the amino acid sequence of SEQ ID NO: 1 may be used interchangeably with "protein HM1524". In addition, it can be used interchangeably with "protein encoded by the gene HM1524". May also be used interchangeably with the term protein consisting of essential proteins, or amino acid sequence of SEQ ID NO: 1 consisting of the amino acid sequence of SEQ ID NO: 1.
[12]
In addition, the protein may comprise a polypeptide having at least 80% amino acid sequence of SEQ ID NO: 1, 90%, 95%, 97% or 99% homology. For instance, having this homologous, if the amino acid sequence shown efficacy corresponding to a protein consisting of the amino acid sequence of SEQ ID NO: 1, a part sequence deletions, modifications, even of a substituted or added in the amino acid sequences within the scope of the present included is obvious.
[13]
In addition, when having a protein with the corresponding activity consisting of the amino acid sequence of SEQ ID NO: 1, if excluding SEQ ID NO: 1 may occur as additional amino acid sequence meaningless sequence of back and forth, or natural mutation, or its potential mutations (silent mutation) in the it is not, within the scope of the present protein also has the amino acid sequence of SEQ ID NO: 1.
[14]
The term used herein, "homology" refers to the percent of identity between two polynucleotide or polypeptide moieties. Means the degree to match the given amino acid sequence or nucleotide sequence and can be expressed as a percentage. As used herein, a homologous sequence thereof having the same or similar activity with a given amino acid sequence or nucleotide sequence is represented by the "% homology". For example, the sequence by Southern hybridization experiment under the score (score), identities (identity) and similarity (similarity) standard software, stringent conditions specifically used, define the BLAST 2.0 calculating a parameter (parameter), such as appropriate hybridization conditions is to check, by comparing the definition is within the skill of a method well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989;. FM Ausubel et al, may be determined in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
[15]
[16]
Gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, but are not limited to, may be a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, at least 80% identical to the nucleotide sequence of SEQ ID NO: 2, 90% , it may be a polynucleotide having 95%, 97% or 99% homology. Codon degeneracy that (codon degeneracy) to be translated into a protein having a protein or its homology consisting of the amino acid sequence of SEQ ID NO: 1 polynucleotide may also be included by which it is apparent. Or which can be prepared from a known gene sequence probe, e.g., a protein having the complementary sequence and to under stringent conditions Hydride Chemistry, protein activity consisting of the amino acid sequence of SEQ ID NO: 1 for all or part of the nucleotide sequence If you encrypt the sequence may include, without limitation. Refers to conditions that permit the specific hybridization between the "stringent condition" is a polynucleotide. These conditions are described in detail in the literature (e.g., J. Sambrook et al., Above). For example, between highly homologous genes, more than 80%, specifically, by at least 90%, more specifically at least 95%, more specifically 97% or more, and particularly particularly genes having at least 99% homologous between the hybridization, rather homologous conditions do not hybridize low gene together, or the 60 ℃ ordinary washing condition of Southern hybridization, and 1 × SSC, 0.1% SDS, specifically 60 ℃, 0.1 × SSC, 0.1 % SDS, more specifically, at a salt concentration and temperature corresponding to 68 ℃, 0.1 × SSC, 0.1% SDS, 1 times,
[17]
Hybridization Although the mismatch (mismatch) between the base be possible depending on the stringency of hybridization, though, requires that the two nucleic acids having a complementary sequence. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also, as well as similar to the nucleic acid sequence substantially complementary to an isolated nucleic acid fragment comprising the sequence throughout.
[18]
Specifically, a polynucleotide having a homology can be detected using hybridization conditions comprising a hybridization step at Tm value of 55 ℃ using the above-described conditions. Further, the Tm value can be 60 ℃, 63 ℃ or 65 ℃. However, it is not limited to, it can be properly adjusted by those skilled in the art according to the purpose.
[19]
Appropriate stringency for hybridizing polynucleotide depends on the degree of complementarity and the length of the polynucleotide and variables are well known in the art.
[20]
The probe used for the hybridization, may be part of the complementary sequence of the base sequence. Such a probe is made up on the basis of the known sequence to the nucleotide as a primer, it can be also prepared by PCR, a gene fragment containing these sequences as the template. The gene fragment may be, for example, at least about 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, or at least 100 nucleotides. In addition, those skilled in the art can be adjusted, if necessary, in accordance with the temperature and wash solution salt concentration on factors such as length of the probe.
[21]
[22]
As used herein, the term, "intrinsic activity" is, in the case of the transformant microorganism of changes in genetic variation due to natural or artificial factors, transfection refers to the pre-change specific activity of the parent protein isolates that had originally.
[23]
That herein, the term "increased activity of the protein compared with the endogenous activity." It means that the enhanced activity compared to the endogenous activity or activity of the protein before modification with the microorganism. As the increased activity is the introduction of foreign HM1524, it may include both to enhance the intrinsic activity of the HM1524.
[24]
Specifically, the increased activity herein,
[25]
1) increasing the copy number of the polynucleotide encoding the protein,
[26]
2) transformation of the expression control sequences to increase the expression of the polynucleotide,
[27]
3) modification of the polynucleotide sequence on the chromosome so that the enhanced activity of the protein,
[28]
4) The introduction of a foreign polynucleotide or a codon optimized polynucleotide variants of the polynucleotide indicates the activity of the protein, or
[29]
5) but it can be carried out by such method that modified to enhanced by the combination thereof, but is not limited thereto.
[30]
[31]
Wherein 1) increasing the copy number of the polynucleotide is not particularly limited, be performed as operably linked to a vector form, it can be carried out by being inserted into the chromosome in the host cell. Specifically, a polynucleotide encoding the protein of the present application the vector to replicate independently of the host and the function is operably connected may be performed by being introduced into the host cell, to insert the polynucleotide into a chromosome in the host cell is the polynucleotide is operably linked to a vector introduced into a host cell capable of being can be carried out in a way to increase the copy number of the polynucleotide within the chromosome of the host cell.
[32]
Next, 2) modification of the polynucleotide expression control sequences so as to express the increase in the, particularly for but not limited to, the expression deletion of nucleic acid sequence so as to further enhance the activity of the regulatory sequence, insertion, Vivo wholly or conservative substitutions thereof performed in combination to induce a mutation on the sequence, or may be performed by replacing as a nucleic acid sequence having a stronger activity. The expression control sequence may comprise a particularly useful for but not limited to promoters, operator sequences, such as sequences that control the termination of the sequence, the transcription and translation coding for a ribosome binding site.
[33]
There are inherent promoter instead of a strong heterologous promoters upper portion of the polynucleotide expression units can be connected, examples of the strong promoter is CJ7 promoter (Patent No. 0,620,092 No. and WO2006 / 065095 Republic of Korea), lysCP1 promoter (WO2009 / 096689), EF Although the like -Tu promoter, groEL promoters, aceA or aceB promoter, and the like. In addition, 3) modification of the polynucleotide sequence on the chromosome is especially useful for but not limited to, deletion of a nucleic acid sequence so as to further enhance the activity of the polynucleotide sequence, expression control by insertion, Vivo wholly or conservative substitution or a combination of these sequences performed by inducing a mutation on, or can be performed by replacing as in the polynucleotide sequence improved so as to have a stronger activity.
[34]
Further, 4) introduction of a foreign polynucleotide sequence may be performed by introducing an exogenous polynucleotide, or a variant thereof codon optimized polynucleotide encoding a protein that represents the protein with the same / similar activity into a host cell. The exogenous polynucleotide may be used without limitation to the origin and a sequence representing the protein with the same / similar activity. May also be the said exogenous polynucleotide is introduced optimized in a host cell transcription, translation is to occur to optimize its codon be introduced into a host cell. The introduction can be carried out by any person skilled in the art to adequately select a known transformation method, the introduced polynucleotide are expressed in the host cell thereby it may be the activity increase the protein is produced.
[35]
Finally, 5) the above 1) to 4) how modified to enhanced by the combination of the increased copy number of the polynucleotide encoding the protein, modification of an expression control sequence such that its expression is increased, the polynucleotides on the chromosome of an exogenous polynucleotide, or a variant thereof codon optimized polynucleotide that represents a modification and activity of the protein variants of the sequence may be performed by applying with one or more ways.
[36]
The term "vector" as used herein refers to a DNA preparation containing the polynucleotide sequence encoding the desired protein operably linked to suitable control sequences so as to express the desired protein in a suitable host. The control sequences may include any operator sequence, sequences that control the termination of the sequence, and a transcription and translation encoding a suitable mRNA ribosome-binding site for regulating the promoter, such that transcription can initiate transcription. Vector may then be transformed into a suitable host cell, replicate independently of the host genome, or functions, may be integrated into the genome itself. With a polynucleotide via a vector for cell insertion mutations within the chromosome of a polynucleotide encoding a protein of interest in the chromosome in one example it can be replaced. Insertion into the chromosome of the above polynucleotides is any method known in the art, for example, but may be made by homologous recombination, but is not limited to this.
[37]
Vector as used herein is not particularly limited, it is possible to use any vector known in the art. Examples of the normal vector to be used may be a naturally occurring or recombinant plasmid of the state, cosmid, virus and bacteriophage. For example, the phage vector or course as mid vector may be used. PWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, etc. Charon4A, and Charon21A, pBR series, pUC system, pBluescriptII system as plasmid vector It may be used based pGEM, pTZ-based, such as pCL and pET-based system. Specifically, it may be used pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector or the like.
[38]
The term "transgenic" herein is meant to allow the protein to the polynucleotide encoding the expression in a host cell by introducing a vector comprising a polynucleotide encoding the target protein in the host cell. If the transformed polynucleotide can be as long as expression in a host cell, is inserted in the chromosome of the host cell may be located, or include both of these positions in addition to the chromosome, or no matter what. In addition, the polynucleotides include DNA and RNA encoding the target protein. The polynucleotide so long as it can be expressed is introduced into a host cell, it does not matter whether it is to be introduced in any form. For example, the polynucleotide may be introduced into a host cell in the form of there is expressed by itself in an expression cassette (cassette expression) gene construct containing all the elements required. The expression cassette may include a promoter that is normally operably linked to the polynucleotide (promoter), a transcription termination signal, ribosome binding site and translation termination signal. The expression cassette may be an expression vector form a self-replicable. In addition, the polynucleotide is introduced into a host cell in the form of itself, and may be, which is possibly connected with the operation sequence necessary for the expression in a host cell, and the like.
[39]
In addition, it means that the promoter sequence and the gene sequence to initiate and mediate the transcription of a polynucleotide encoding a protein of interest in terms rea herein "operably linked" in the above is operatively connected to.
[40]
A method of transforming a vector of the present application can be carried out, and includes any method of introducing the nucleic acid into a cell, by selecting suitable standard techniques as known in the art depending on the host cell. For example, electroporation method (electroporation), calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection (microinjection), polyethylene glycol (PEG) method, DEAE- dextran method, cationic liposome method, and Although the like -DMSO lithium acetate method, but is not limited thereto.
[41]
In the host cell has a high introduction efficiency of DNA, good to use a high expression efficiency of the introduced DNA host cell, it may be, for example, Corynebacterium spp.
[42]
[43]
Is one of the terms herein, is a basic amino acid α- "L- lysine" essential amino acid that is not synthesized by the body, has the formula NH 2 (CH 2 ) 4 CH (CH 2 means) COOH, a kind of L- amino acids. In addition, the L- lysine may be included in the scope of the present application, even if present in a salt form.
[44]
The term used herein, "microorganism producing L- lysine" is a microorganism strain capable of producing L- lysine purposes of the present application, and specifically refers to the strains which can produce L- lysine at a high concentration by an operation according to the present do. Thus, the microorganism is one capable of producing L- lysine, it is not particularly limited to the type of the parent strain. I.e. it can be present in the parent strain contains all the strains ability L- lysine-producing strain and the production of L- lysine-free capability. The L- lysine-producing ability can include everything that is naturally occurring or artificially manipulated. Microorganisms having artificially manipulated L- lysine-producing ability as is, for example, NTG (nitrosoguanidine) or the like is variation by mutagenic substances may be one having an L- lysine-producing ability, or the expression level or activity of a particular protein of interest but it may be adjusted by having an L- lysine-producing ability, but is not limited thereto. Specifically, the specific protein of interest may include all proteins that act to directly / indirectly to L- lysine biosynthetic pathways, to increase their expression level or activity, or may be reduced by the mutation have a L- lysine-producing ability may, or may be induced by their amino acid sequence or a mutation on the nucleotide sequence be one to have the L- lysine-producing ability. Random mutation induction and the like using the above-described NTG and human operation of a microorganism comprising the expression control of a particular target protein can be appropriately carried out by those skilled in the art as a known technique.
[45]
[46]
The microorganism, and specifically may be a microorganism of the genus Corynebacterium. Examples of Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes to Ness ( Corynebacterium ammoniagenes ), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Brevibacterium Plastic boom ( Brevibacterium flavum ), or Brevibacterium lactofermentum FER ( Brevibacterium fermentum ), but such can be used, but are not limited to. One example, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum may be used). However, not limited to these examples, In addition, there is a genus Corynebacterium microorganism may be used having a known L- lysine-producing ability.
[47]
Examples of the microorganism of the genus Corynebacterium having a known L- lysine-producing ability of the Republic of Korea Patent No. 10-0397322 call (or the US Patent Publication No. 2003-0124688 call), the Republic of Korea Patent No. 10-0924065 call (or US Publication Patent No. 2010-0143984), the Republic of Korea Patent No. 10-0073610 call, and / or the Binder et al, Genome Biology 2012, 13:., and the microorganism described in the R40, the entire contents of the above document is incorporated herein by reference this includes professional.
[48]
[49]
Another aspect, the genus Corynebacterium (for the genus, producing L- lysine-containing protein consisting of the amino acid sequence of SEQ ID NO: 1 that has an increased activity compared to the endogenous activity Corynebacterium culturing) the microorganism in a culture medium step; And a method of producing L- lysine, comprising the step of recovering the L- lysine from the cultured microorganism or its culture medium.
[50]
For the genus Corynebacterium microorganism producing L- lysine as described above.
[51]
The term herein, the term "culture" is meant that the growth in the environmental conditions of suitably adjusting the microorganism. Culturing process of the present application may be made in accordance with the appropriate culture medium and culture conditions known in the art. This culturing process may be used by those skilled in the art easily adjusted according to the selected strain. In the method, the method comprising culturing the microorganism, and may be particularly, but not limited thereto, carried out by the known batch culture method, the continuous culture method, a fed-batch culture method. At this time, the culture conditions, particularly for but not limited to, a basic compound to an appropriate pH using: (phosphoric acid or sulfuric acid for example) (for example, pH 5 to 9, in particular (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound It can be adjusted to pH 6 to 8, most specifically at pH 6.8). In addition, during the culture it can be suppressed and the bubbles generated using a defoaming agent such as fatty acid polyglycol ester, and, in order to maintain the culture of water aerobic conditions, the injection of oxygen or oxygen-containing gas into the culture, or the anaerobic and Miho group state or it may be injected with nitrogen, hydrogen or carbon dioxide gas, without injection of gas to maintain. The culture temperature is 20 to 45 ℃, specifically, can be maintained for 25 to 40 ℃, the incubation period can be continued until the yield the production of the desired useful substance, specifically, can be cultured for about 10 to 160 hours have. However, it is not limited thereto. L- lysine-cost by the production culture may be secreted into the culture medium or remains in the cell.
[52]
In addition, the medium for the culture to be used per a carbon source and a carbohydrate (such as glucose, sucrose trehalose, lactose, fructose, maltose, know three, starch and cellulose), maintenance, and fat (such as soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (for example, glycerol and ethanol) and organic acids (e.g. acetic acid) can be used by using individually or mixed, such as, but , but it is not limited thereto. The nitrogen source may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean bakbun and urea), or inorganic compounds (e.g., ammonium ammonium ammonium sulfate, chloride, phosphate, ammonium carbonate and ammonium nitrate), but it can be used by using individually or mixed, such as, but not limited thereto. Although the source of the phosphate can be individually used, or a mixture of monobasic potassium phosphate, potassium susoyi, such as the corresponding sodium-containing salts to, but is not limited thereto. In addition, the culture medium and other metal salts (such as magnesium sulfate or iron sulfate), essential amino acids, and growth, such as vitamins can include a promoting material.
[53]
The method for recovering an L- lysine-producing in the incubation step of the present application can be by using a suitable method known in the art according to the culture method to collect the desired amino acid from the culture broth. For example, this can be used centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, etc., it can be recovered in the L- lysine of interest from the medium or the microorganism by using the appropriate method known in the art. In addition, the recovery step may include a step of purification.
[54]
Effects of the Invention
[55]
By using a microorganism having an L- lysine-producing ability of the present application can produce L- lysine at a high efficiency.
[56]
Best Mode for Carrying Out the Invention
[57]
And in more detail described below through the embodiments herein. However, these examples are not intended to be limited to the scope of the present application to these examples are for explaining the present application by way of example.
[58]
[59]
Example 1: the genus Corynebacterium wild-type library of microbial production
[60]
[61]
Corynebacterium glutamicum handling restriction enzyme Sau3AI and then extracting the genomic DNA from the ATCC13032 and optionally to obtain as a DNA fragment of 3 ~ 4 kb by separating the DNA fragments according to size by electrophoresis on agarose gel. This restriction enzyme pECCG117 having a BamHI terminus (the Republic of Korea Patent No. 10-0057684), connect the vector introduced into E. coli DH5α and plated on the LB solid medium containing kanamycin (25 mg / L) are transformed colonies It was obtained. It was identified as performing PCR using from 100 colonies of random primers of SEQ ID NOS: 3 and 4, and the proportion of colonies containing the them is a 3 ~ 4 DNA fragments in kb about object through the insertion vector more than 90%. The inoculated that contain a kanamycin all the colonies obtained (25 mg / L) LB broth mixed culture and extracting the plasmid by using the conventional plasmid extraction method known to have completed the Corynebacterium glutamicum ATCC13032 genome DNA library .
[62]
[63]
SEQ ID NO: 3: 5'-ACGACGGGATCAGTACCGA-3 '
[64]
SEQ ID NO: 4: 5'-AGCTATCTGTCGCAGCGCC-3 '
[65]
[66]
Example 2: the introduction of the wild-type library lysine production and evaluation of microbial production
[67]
[68]
Embodied in Examples is by a genomic DNA library was created from the first using the electric pulse method lysine-producing strain Corynebacterium glutamicum KCCM11016P (the microorganism was published KFCC10881, the material deposited in the Budapest Treaty servant international deposition KCCM11016P after receiving the assigned an accession number, the introduction of the Republic of Korea Patent No. 10-0159812 call), kanamycin (25 mg / L) cultured colonies to about 2000 in a composite plate smeared on the medium, and 30 ℃ containing the dog for 24 hours It was obtained.
[69]
[70]

[71]
Glucose 20 g, (NH 4 ) 2 SO 4 50 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 5 g, K 2 HPO 4 10 g, MgSO 4 · 7H 2 O 0.5 g, ㎍ biotin 100, thiamin hydrochloride [1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 2000 ㎍, agar 20 g, kanamycin 25 mg (1L of distilled water basis)
[72]
[73]
96-well cell colony after the frequency division to obtain a composite liquid medium 200 μl to each well of a culture plate with each inoculated 30 ℃, were cultured for 24 hours with shaking under the conditions of 1200 rpm. Centrifuging the culture to separate the cells and supernatant and the supernatant was mixed with 50 μl reaction solution containing the lysine oxidase (lysine oxydase).
[74]
[75]

[76]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 · 7H 2 O 0.5 g, biotin 100 ㎍, thiamine HCl 1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 2000 ㎍, kanamycin 25 mg (per liter of distilled water)
[77]
[78]

[79]
Lysine oxidase (Sigma-Aldrich) 0.02 unit, peroxidase (peroxidase, Sigma-Aldrich) 0.2 unit, ABTS 2 mg (1 ml potassium phosphate buffer solution basis)
[80]
[81]
Then, 30 minutes OD 405 by analyzing the absorbance at were selected 15 kinds of experimental group showing a high absorbance than the control (KCCM11016P / pECCG117). To determine the lysine-producing ability of each transformants, kanamycin (25 mg / L) 250 ㎖ corner containing a seed medium 25 ㎖ include-inoculated with each strain in bapeul flask, 30 ℃, the conditions of 200 rpm the cultured with shaking for 20 hours. 250 ㎖ corner containing a kanamycin-24 production medium containing the ㎖ (25 mg / L) - inoculated with the seed culture in 1 ㎖ in bapeul flask, and cultured with shaking for 96 hours at 37 ℃, 200 rpm. After the end of the incubation, using the HPLC was analyzed L- lysine concentration (Table 1).
[82]
[83]

[84]
Glucose 20 g, (NH 4 ) 2 SO 4 5 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 · 7H 2 O 0.5 g, 100 ㎍ biotin, thiamine HCl 1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 2000 ㎍ (in 1 liter of distilled water)
[85]
[86]

[87]
Glucose 100 g, (NH 4 ) 2 SO 4 40 g, soy protein 2.5 g, corn steep solids (Corn Steep Solids) 5 g, urea 3 g, KH 2 PO 4 1 g, MgSO 4 · 7H 2 O 0.5 g, ㎍ biotin 100, thiamin hydrochloride [1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide ㎍ 3000, CaCO 3 30 g (per liter of distilled water)

WE Claims

[Claim 1]
The genus Corynebacterium, which produce an L- lysine-containing protein consisting of the amino acid sequence of SEQ ID NO: 1 that has an increased activity compared to the endogenous activity (the genus Corynebacterium) microorganism.
[Claim 2]
The method of claim 1, wherein the microorganism is Corynebacterium glutamicum (Corynebacterium glutamicum) which, genus Corynebacterium microorganism producing L- lysine.
[Claim 3]
Culturing a microorganism of the genus Corynebacterium, which produce the L- lysine-containing protein consisting of the amino acid sequence of SEQ ID NO: 1 that has an increased activity compared to the endogenous activity in the medium (Corynebacterium sp.); And, a method of producing L- lysine, comprising the step of recovering the L- lysine from the cultured microorganism or its culture medium.
[Claim 4]
4. The method of claim 3 wherein the microorganism is a method of Corynebacterium glutamicum which, L- lysine (Corynebacterium glutamicum) production.