The title of the invention: L- valine capability enhanced strain and L- valine production method using the production
Art
[1]
The present invention relates to L- valine production method using L- valine improved strain and strain capability art produced through genetic engineering.
BACKGROUND
[2]
One branch chain amino acid L- valine in microbial biosynthesis is by starting from pyruvate via the lactic acid acetonide (acetolactic acid), di-hydroxy isovaleric acid (dihydroxy isovaleric acid), Kerala Small toys acid (ketoisovaleric acid) . This intermediate metabolites are acetonitrile hydroxy acid synthase (acetohydroxy acid synthase), acetonitrile hydroxy acid isopropyl booties reductase kinase (acetohydroxy acid isomeroreductase), dihydroxy-acid-dihydroxy Alpharetta claim (dihydroxy acid dehydratase), transaminase B the reaction to produce the catalysts by (transaminase B). However, enzymes are the keto acid (ketobutyric acid) and is involved in L- isoleucine biosynthesis starting from pyruvic acid, in Kane junggandae things toy cows from 2-isopropyl-malic acid (2-isopropylmalic acid), 3- isopropyl malate (3-isopropylmalic acid), Kerala via a small toy aminocaproic acid (ketoisocaproic acid) and also by the L- leucine biosynthesis. Thus, in one thing There is difficult to manufacture because of the branched-chain amino acid using the same enzyme branched-chain amino acids as described above, that is, L- valine, L- isoleucine, L- leucine biosynthesis is that industrially through fermentation as known, there is the further end product of L- valine, or derivatives thereof by feedback inhibition is an issue which has caused the hagieneun industrial mass production constraints as follows.
[3]
[4]
The inventors of this invention discovered the L- valine production capability is excellent compared with the control region in which the parent strain strain transformed to inactivate the working end of the shell height, L- valine operon in order to develop a superior ability L- valine producing strain It was completed.
[5]
[Prior Art Document]
[6]
[Patent Document]
[7]
(Patent Document 1) KR 10-1990-0007948 B1
[8]
(Patent Document 2) KR 10-2008-0025355 A1
Detailed Description of the Invention
SUMMARY
[9]
An object of the present invention, L- full nucleotide sequence coding for the leader peptide in the control region of the defect or valine operon is partially deficient or replaced, L- valine, L- valine production in the transformed to express a novel reinforced operon to provide a strain.
[10]
[11]
It is another object of the present invention to provide a method for producing L- valine using the valine-producing strain of the new L-.
[12]
[13]
It is also an object of the present invention is to provide a control region of the mutant L- valine operon.
Technical Solution
[14]
In order to achieve the above object, the present invention is full nucleotide sequence coding for the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon or a defect is a defect portion or a substituted, L- valine operon and the expression provides the L- valine producing strain of transformed so that new enhanced.
[15]
[16]
In addition, the present invention provides a method for producing L- valine using the valine-producing strain of the new L-.
[17]
[18]
The present invention also provides a variant of L- valine operon control region having an amino acid sequence represented by SEQ ID NO: 3 or 4.
Effects of the Invention
[19]
The novel L- valine-producing strain of the present invention, as is the entire nucleotide sequence coding for the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon or a defect is a defect portion or a substituted, L - because of the increased expression of the enzyme acetoacetate valine hydroxy acid synthase (acetohydroxy acid synthase) involved in the biosynthesis, the ability of the excellent effects produced L- valine.
[20]
[21]
Further, according to the production method of the present invention L- valine using the valine-producing strain of the new, there is an effect that can be produced L- valine in high efficiency and high yield.
Brief Description of the Drawings
[22]
1 is a control region and a nucleotide sequence of the entire defect variants encoding the leader peptide control region (DvalL), variants of the regulatory region of the stop part of the leader peptide loss of the ilvBN operon of the wild type Corynebacterium glutamicum ATCC13032 (DvalP), the ilvBN operon containing the last amino acid of the still region of the leader peptide is mutated in the stop codon mutation type control region (DvalS) and the leader peptide and the attenuation region are all deficient mutant regulatory region (DvalA) Approximate a schematic diagram.
[23]
2 is a schematic detail of the control region of the ilvBN operon of the wild type Corynebacterium glutamicum ATCC13032.
[24]
3 is a schematic detail of a variant nucleotide sequence regulatory region entirety defect (DvalL) coding for the leader peptide of Figure 1;
[25]
4 is a schematic detail of the variant control region (DvalP) the stop region of the leader peptide of Figure 1 deficiency.
[26]
5 is a schematic detail of the variant control region (DvalS) variation to the last amino acid is the termination codon of the leader peptide region of the stop in Fig.
[27]
Figure 6 is a schematic detail of the variant control region (DvalA) of the leader peptide and the defect both damping regions of FIG.
Mode for the Invention
[28]
The present invention has been transformed with the entire nucleotide sequence coding for the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon or a defect is a defect portion or a substituted, L- valine operon such that the expression of enhanced It relates to a novel strain of L- valine production.
[29]
[30]
In the present invention, the L- valine operon (hereinafter referred to as "ilvBN operon") is from 2 pyruvic acid (pyruvate) in the molecule as an enzyme involved in the first step of the biosynthesis of L- valine-valine-producing microorganism in the L- lactic acid acetonide (acetolactate ) to include the enzyme acetoacetate hydroxy acid synthase (acetohydroxy acid synthase), the encoding gene (ilvBN) for the synthesis.
[31]
[32]
In more detail, the ilvBN operon promoter (promotor), the regulatory region including the leader peptide (leader peptide) stop parts (pause site) and the damping portion (attenuator) positioned in the base sequence, the leader peptide coding for and acetonitrile Hyde and a structural gene (structural gene) that encrypts the hydroxy acid synthase (acetohydroxy acid synthase) (see Figs. 1 and 2).
[33]
[34]
In the present invention, the ilvBN operon, the entire of the nucleotide sequence coding for the of the control region leader peptide defect or it is part of the defect or substituted enhance the expression of the structural gene coding for the acetonide hydroxy acid synthase (acetohydroxy acid synthase) sikimeuroseo, characterized in that the improved production performance of L- valine. Preferably some defect or substitution site of the base sequence encoding the leader peptide may be stopped part tomorrow, it may be more preferably the nucleotide sequence of the end stop part substitution site region is replaced by a termination codon.
[35]
[36]
In the present invention, it is the whole of a base sequence coding for the leader peptide of the regulatory region of the ilvBN operon deletion or some defect or substituted as above, L- valine, L- valine production of transformed so that new expression of the operon is enhanced to provide a strain, first, the entire nucleotide sequence coding for the leader peptide of the regulatory region of the ilvBN operon deletion, or may partially be produced a recombinant vector comprising a deletion or substitution of the nucleotide sequence.
[37]
For the production of the vector, in the embodiments of the present invention First, the genus Corynebacterium in the chromosome of the microorganism is a mold through the Polymerase Chain Reaction encoding the leader peptide of the regulatory region of the ilvBN operon sequences entire defect or a portion defect or control regions having the substituted nucleotide sequence to amplify the (DvalL, DvalP, DvalS), and then the amplified control region and inserted into the vector making a recombinant vector, which was obtained, respectively transformed with the parental strain with the recombinant vector.
[38]
[39]
The vector used in the construction of the recombinant vector may be a naturally occurring or recombinant state of a plasmid, cosmid, virus and bacteriophage. For example, phage vectors, or cosmid vectors as are available to pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and Charon21A etc., pDZ vector as a plasmid vector, pBR series, pUC series , you can use the pBluescriptII series, pGEM series, pTZ system, pCL pET-based system and the like. The available vector may be an expression vector known not particularly limited.
[40]
In the specific embodiment of the present invention it was used pDZ. The pDZ vector is produced according to the process disclosed in Corynebacterium with a vector for chromosomal insertion of a microorganism of the genus Solarium, Republic of Korea Laid-Open Patent Publication No. 2008-0025355.
[41]
[42]
The amplified control region (DvalL, DvalP, DvalS) to the vector pDZ were produced respectively inserted into a recombinant vector pDZDvalL, pDZDvalP, pDZDvalS to.
[43]
[44]
In the present invention, the parent strain is transformed by the recombinant vector is not particularly limited as long as the microorganism can produce L- valine, preferably Escherichia microbes and in Corey and in Corynebacterium microorganism, in particular Corynebacterium Tommy glue can kumil. Corey or ATCC 13032 Corynebacterium glutamicum, preferably may be in the L- valine producing strain which is resistant to an L- valine, or derivatives thereof, it may be used for Corynebacterium glutamicum KCCM11201P more preferably .
[45]
The Corynebacterium glutamicum KCCM11201P is the inventors of the preceding Patent No. 10-1117022 disclosed a call as a strain, L-glutamic acid production for Corynebacterium glutamicum KFCC 10661 (Corynebacterium glutamicum KFCC 10661, Republic of Korea Patent Application Nos. 1988-0016543; 1990-0007948 Publication No. No.) after performing a random mutation in the parent strain, isoleucine derivative of alpha-amino acid (α-Aminobutyric acid, ABA), valine derivative of alpha-hydroxy valine ( α-hydroxyvaline, AHV), thiazol-alanine (thiazole alanine, TA) and norvaline (norvaline, NV) 20 mM for each and plated on minimal medium is supplemented with, 20 mM, 40 mM, and resistance to 50 mM concentration as a common resistant mutant strain having the mutant strain is a common resistance L- valine production strain is increased more than four times superior as compared to the parent strain, Corynebacterium glutamicum KFCC 10661.
[46]
[47]
Corey said yes to the tumefaciens glutamicum parent strain was confirmed by KCCM11201P L- valine producing excellent ability compared to transgenic strains that KCCM11336P, KCCM11337P, KCCM11338P that the parent strain Corynebacterium glutamicum KCCM11201P with the recombinant vector .
[48]
[49]
The Corynebacterium glutamicum KCCM11336P is the parent strain, stiffness in the inserted recombinant vector pDZDvalL variant control region, the entire nucleotide sequence encoding a leader peptide having an amino acid sequence of SEQ ID NO: 1 in the pDZ vector deficient (DvalL) as the strain was transformed Corynebacterium glutamicum KCCM11201P, was it Corynebacterium glutamicum as KCJ-456 named and given the accession No. KCCM11336P to deposit 19 dated November 2012 in Korea Culture Center of microorganisms.
[50]
[51]
The Corynebacterium glutamicum KCCM11337P is the parent strain, stiffness in the inserted recombinant vector pDZDvalP the stop of the leader peptide region has the amino acid sequence of SEQ ID NO: 1 in the pDZ vector deficient mutant regulatory region (DvalP) Corynebacterium KCCM11201P as a glutamicum strain was transformed, it was called Corynebacterium glutamicum named KCJ-457, and given the accession number of the deposit KCCM11337P dated 19 November 2012 the Korea Culture Center of microorganisms.
[52]
[53]
The Corynebacterium glutamicum KCCM11338P is the parent to the variant control region last amino acid of the stop of the leader peptide region has the amino acid sequence of SEQ ID NO: 1 in the pDZ vector is replaced with the termination codon (DvalS) is inserted into a recombinant vector pDZDvalS a strain of Corynebacterium glutamicum strains were transformed KCCM11201P, it Corynebacterium glutamicum KCJ-458 as naming, and the deposit of 19 on January 11 2012 in Korea Culture Center of microorganisms an accession number KCCM11338P It was granted.
[54]
[55]
In this invention, by L- valine biosynthesis control region is produced by adding the fire switch L- valine biosynthesis overexpression vector activated and, with this, the transformed wild-type strain of Corynebacterium glutamicum ATCC13032 known, L- It provides a control region valine biosynthesis is inactivated L- valine biosynthesis overexpressing strains.
[56]
[57]
In the present invention, the L- valine biosynthesis regulatory region is the four corridor for the production of the non-L- valine biosynthesis overexpression vector activated tumefaciens glutamicum KCCM11201P, KCCM11336P Corynebacterium glutamicum, Corynebacterium glutamicum KCCM11337P, Corynebacterium glutamicum KCCM11338P, Corynebacterium glutamicum were amplified the vector insertion sequence for by PCR method with the chromosome as a template for KCCM11201P_DvalA, which respectively insert on pECCG117 vector, each L - prepare a valine biosynthesis overexpression vector pECCG117-DvalW, pECCG117-DvalL, pECCG117-DvalP, pECCG117-DvalS and pECCG117-DvalA
[58]
[59]
Then, the L- valine biosynthesis by overexpression vector pECCG117-DvalW, pECCG117-DvalL, pECCG117-DvalP, pECCG117-DvalS and transforming the parent strain, Corynebacterium glutamicum ATCC13032 by pECCG117-DvalA, L- valine biosynthesis gene regulatory region is inactivated L- valine biosynthesis overexpression strain Corynebacterium glutamicum ATCC13032_DvalW, Corynebacterium glutamicum ATCC13032_DvalL, Corynebacterium glutamicum ATCC13032_DvalP, Corynebacterium glutamicum Corey and four ATCC13032_DvalS the tumefaciens ATCC13032_DvalA glutamicum were produced.
[60]
The strain of Corynebacterium glutamicum ATCC13032_DvalL, Corynebacterium glutamicum ATCC13032_DvalP, Corynebacterium glutamicum ATCC13032_DvalS strains of Corynebacterium glutamicum the L- valine biosynthesis gene regulation of the area of ​​KCCM11201P It confirmed the ability to transformed strains of Corynebacterium glutamicum L- valine production compared to ATCC13032_DvalW excellent.
[61]
[62]
The novel L- valine-producing strain of the present invention, as is the entire nucleotide sequence coding for the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon or a defect is a defect portion or a substituted, L - because of the increased expression of the enzyme acetoacetate valine hydroxy acid synthase (acetohydroxy acid synthase) involved in the biosynthesis, the ability of the excellent effects produced L- valine.
[63]
[64]
In addition, the present invention is the full base sequence is some defect or deficiency, or substitution that encodes the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon, enhanced expression of the L- valine operon to a method for producing L- valine using L- valine-producing strain of the transformed novel.
[65]
[66]
To produce L- valine in the present invention, a method for the culture of the new L- valine-producing strain of the present invention can be carried out using a culture method of Corynebacterium genus Corey widely known in the art binary microorganism. Specifically, in the example of the culture method, including, but batch culture (batch culture), continuous culturing (continuous culture), and fed-batch culture (fed-batch culture) is not limited to this. These various methods, for example, "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp138-176, 1991) or the like has been disclosed.
[67]
[68]
The medium used to culture should meet the requirements of the particular strains in a suitable manner. Culture medium for a microorganism of the genus Corynebacterium is a known (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981). In that can be used glycogen is glucose, saccharose, lactose, paroxetine lactose, maltose, starches, sugars and carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, such as coconut oil and fat, palmitic acid, stearic acid, , fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, include organic acids such as acetic acid. These materials may be used individually or as a mixture. With which can be used nitrogen source include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean, for wheat, and the element or the inorganic compound, for example, may include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used separately or as a mixture. By personnel that may be used are phosphoric acid or the corresponding sodium, potassium hydrogen phosphate or potassium susoyi-containing salts can be included. In addition, the culture medium has to contain a metal salt such as magnesium sulfate or iron sulfate needed for growth. In addition, in addition to the above material it may include essential growth substances such as amino acids and vitamins.
[69]
In addition, suitable precursors may be used in the culture medium. The raw materials can be added to a batch or continuously by any suitable way to the culture in the culture process.
[70]
Using a base or an acid compound or phosphoric acid compound such as sulfuric acid, such as sodium hydroxide, potassium hydroxide, and ammonia in a suitable manner it is possible to adjust the pH of the culture. In addition, by using an antifoaming agent such as fatty acid polyglycol ester can suppress foam generation. Inject-containing gas (eg, air), oxygen or oxygen into the culture in order to maintain aerobic conditions. Culture temperature of water is usually 20 ℃ to 45 ℃. Culture is continued until the maximum obtainable amount of the desired L- valine. For this purpose is normally accomplished in 10 to 160 hours. L- valine or may be released into the culture medium, is contained in the cell.
[71]
[72]
L- valine production method of the present invention may further comprise the step of recovering from L- valine, or cell culture. Method for recovering from cells or L- valine cultures methods known in the art, for example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, but may be used such as, but is not limited to these examples.
[73]
According to one embodiment of the invention, it was isolated from the culture by low-speed centrifugation to remove the biomass and separation through the ion exchange chromatography of the obtained supernatant.
[74]
[75]
Hereinafter, the present invention in more detail by examples. However, the following examples are for the purpose of illustrating the invention by way of example and are not the scope of the present invention be limited to these embodiments.
[76]
[77]
Example 1: L- valine leader peptides for the deactivation of the control region of the biosynthesis gene vector to some defect or the whole base sequence coding for the base sequence comprises a deletion or substitution production
[78]
1 and 2 shown in the promoter (Pro), the nucleotide sequence (Leader peptide), stop region coding for the leader peptide (Pause site), attenuation regions consisting of control region and a structural gene (gene) containing the (Attenuator), acetonitrile Hyde control region of the L- valine operon (ilvBN) to synthesize a hydroxy-acid synthase, is 1 and the total base sequence encoding the leader peptide deficient mutant regulatory region like (hereinafter referred to as 'DvalL'), the reader adjusting the peptides of the stop portion deficient variant region (hereinafter referred to as 'DvalP'), and the last amino acid of the still region of the leader peptide is replaced by a termination codon mutation type control region (hereinafter 'DvalS' d) and the leader peptide attenuation region are all defect mutated type control region (hereafter 'DvalA' quot;) amplification to order the United States biological resource Center (American type Culture Collection: ATCC) from purchased from Corynebacterium glutamicum ATCC13032 strain in the chromosomal DNA of the as a template was amplified by Polymerase Chain Reaction (hereafter referred to as "PCR method").
[79]
[80]
Specifically, to make the DvalL, first, the Corynebacterium glutamicum ATCC13032 strain in the chromosomal DNA using the primers of the mold to the SEQ ID NO: 5 and 6 through the PCR 1 bungan denaturation at 94 ℃, 30 seconds at 58 ℃ annealing, the conditions for polymerization for 30 seconds in 72 ℃ Pfu was amplified a fragment of about 700 base pairs with the BamHI restriction enzyme located in the 5 'region by PCR method by repeating 30 times polymerase.
[81]
Second, by using the SEQ ID NO: 7 and 8 of the primers amplify a fragment of about 700 base pairs with the restriction enzyme XbaI located in the 3 'region through the above-described PCR methods. The resulting DNA fragments are GeneAll R Expin TM GEL SV kit (Seoul, Korea), after the separation, crossover PCR was used as a template for.
[82]
Then, using the primers of SEQ ID NOS: 5 and 8, a crossover PCR was carried out by the two DNA fragments obtained above as a template. Specifically it was amplified a fragment of about 2000 base pairs through the above described PCR method. The amplified fragment is treated with restriction enzymes BamHI and XbaI and then to prepare a pDZDvalL through ligation with the pDZ treated with the same enzyme .
[83]
[84]
To make DvalP, first, and amplifying the Corynebacterium SEQ ID NO: 5 and 9 of about 700 fragments of base pairs having a BamHI restriction enzyme located in the 5 'region using the primer of the chromosome of glutamicum ATCC13032 strain as a template .
[85]
Second, using the sequence number 10 and 8 of the primers amplify a fragment of about 700 base pairs with the restriction enzyme XbaI located in the 3 'region through the above-described PCR methods.
[86]
It was then amplified a fragment of about 2000 base pairs through the crossover PCR method described by the two DNA fragments amplified as a template. The amplified fragment is treated with restriction enzymes BamHI and XbaI and then to prepare a pDZDvalP through ligation with the pDZ treated with the same enzyme.
[87]
[88]
To make DvalS, it was amplified a fragment of about 700 base pairs with the restriction enzyme BamHI Corynebacterium a 5 'region of the chromosome as a template glutamicum ATCC13032 strain using the primers of SEQ ID NOS: 5 and 11.
[89]
Second, SEQ ID NO: 12 using primers and 8 were amplified fragment of about 700 base pairs with the restriction enzyme XbaI located in the 3 'region through the above-described PCR methods.
[90]
It was then amplified a fragment of about 2000 base pairs through the crossover PCR method described by the two amplified DNA fragments as a template. The amplified fragment is treated with restriction enzymes BamHI and XbaI and then to prepare a pDZDvalS through ligation with the pDZ treated with the same enzyme.
[91]
[92]
To make DvalA, first, and amplifying the Corynebacterium SEQ ID NO: 5 and 13 approximately 700 fragments of base pairs having a BamHI restriction enzyme located in the 5 'region using the primer of the chromosome of glutamicum ATCC13032 strain as a template .
[93]
Second, SEQ ID NO: 14 using the primer of the 8 through the above-described method was PCR amplified a fragment of about 700 base pairs with the restriction enzyme XbaI located in the 3 'region.
[94]
It was then amplified a fragment of about 2000 base pairs through the crossover PCR method described by the two amplified DNA fragments as a template. The amplified fragment is treated with restriction enzymes BamHI and XbaI and then to prepare a pDZDvalA through ligation with the pDZ treated with the same enzyme.
[95]
[96]
The leader sequence of the peptide and amino acid level in the region of the stop is equal to the SEQ ID NOs: 1, 2, and amino acid sequence of DvalP DvalS are as SEQ ID NO: 3 and 4, respectively.
[97]
In addition, the control region full nucleotide sequence, leader peptide sequences, and still region nucleotide sequence of valine operon ilvBN are the same as those of SEQ ID NOs: 21, 22 and 23, DvalL, DvalP, DvalS and the base sequence of DvalA is SEQ ID NOs: 24 to 27 and the same.
[98]
[99]
Example 2: L- valine producing strain Corynebacterium glutamicum-derived KCCM11201P L- valine production in the control region is an inactivated strain of the biosynthetic genes
[100]
To produce a control region of the L- valine biosynthesis gene is inactivated strain, L- valine producing strain stiffness as the parent strain was used for Corynebacterium glutamicum KCCM11201P.
[101]
A Corynebacterium glutamicum KCCM11201P in pDZDvalL, pDZDvalP, and pDZDvalS pDZDvalA vector produced in Example 1 by electroporation was converted each transfection.
[102]
The second cross-process via the Corynebacterium glutamicum were obtained L- valine producing strains control region of L- valine biosynthetic genes on the chromosome, which contains a base sequence of an inactivated mutant KCCM11201P, respectively.
[103]
Whether or not the base substitution promoter for DvalL SEQ ID NO: 15, in the case of DvalP SEQ ID NO: 16, in the case of DvalS SEQ ID NO: 17, a gene through the PCR using the case of DvalA primers of SEQ ID NO: 18 as primers and a combination of SEQ ID NO: 8 end was confirmed by nucleotide sequence analysis of the amplified region, and whether the purpose.
[104]
The pDZDvalL, pDZDvalP, pDZDvalS vector in transformed strains, each of Corynebacterium glutamicum KCJ-456, Corynebacterium glutamicum KCJ-457, Corynebacterium glutamicum as KCJ-458 named, and by deposit date of 19 November 2012 on the Korea Culture Center of microorganisms been given the accession number of each KCCM11336P, KCCM11337P, KCCM11338P.
[105]
In addition, the transformed strain in pDZDvalA vector was named as Corynebacterium glutamicum KCCM11201P_DvalA.
[106]
[107]
[108]
Example 3: The control region L- valine biosynthesis of L- valine biosynthetic gene inactivated production of over-expression vector
[109]
L- valine producing strains were produced Corey Valine biosynthesis of L- overexpression vector from Corynebacterium glutamicum KCCM11201P.
[110]
[111]
In addition, each of the one prepared in Example 2 KCCM11336P Corynebacterium glutamicum, Corynebacterium glutamicum KCCM11337P, KCCM11338P Corynebacterium glutamicum and Corynebacterium glutamicum KCCM11201P_DvalA from L- valine biosynthesis the control region of the gene was produced L- valine biosynthesis fire overexpression vector activated.
[112]
[113]
For the production of the vector, using the primers of SEQ ID NOS: 19 and 20 with a combination of Corynebacterium glutamicum KCCM11201P, Corynebacterium glutamicum KCCM11336P, Corynebacterium glutamicum KCCM11337P, Corynebacterium glutamicum KCCM11338P and Corynebacterium glutamicum were amplified gene by PCR method with the chromosomal DNA as a template for KCCM11201P_DvalA, by inserting the pECCG117 vector treated with the same enzymes and then treated with the restriction enzymes BamHI and XbaI, respectively L- valine biosynthesis overexpression vector pECCG117-DvalW, pECCG117-DvalL, pECCG117-DvalP, pECCG117-DvalS and was manufactured pECCG117-DvalA.
[114]
[115]
Example 4: L- valine biosynthesis gene is controlled areas of the fire L- valine biosynthesis activating production of a strain overexpressing
[116]
One prepared in Example 3 L- valine biosynthesis overexpression vector-pECCG117 DvalW, pECCG117-DvalL, pECCG117-DvalP, pECCG117 DvalS-and-pECCG117 DvalA the Corynebacterium glutamicum by electroporation, respectively inserted into the ATCC13032 Corey the Corynebacterium glutamicum ATCC13032_DvalW, Corynebacterium glutamicum ATCC13032_DvalL, Corynebacterium glutamicum ATCC13032_DvalP, Corynebacterium glutamicum ATCC13032_DvalS, Corynebacterium glutamicum ATCC13032_DvalA strains were produced, respectively. If the vector is to be transformed, so have the kanamycine-resistant transformants was confirmed through the kanamycine whether growth in medium containing a concentration of 25㎍ / ㎖.
[117]
[118]
Example 5: L- to the control region in the inactivated strain of L- valine biosynthesis gene lean manufacturing
[119]
Each of the L- valine-producing strain of Corynebacterium was prepared in Example 2 Solarium KCCM11336P glutamicum, Corynebacterium glutamicum KCCM11337P, KCCM11338P Corynebacterium glutamicum and Corynebacterium glutamicum KCCM11201P_DvalA from to for L- valine production and cultured in the same way. At this time, the control Examples were used to culture the host cells of Corynebacterium glutamicum KCCM11201P.
[120]
[121]
One also prepared in Example 4 from Corynebacterium glutamicum ATCC13032_DvalW, Corynebacterium glutamicum ATCC13032_DvalL, Corynebacterium glutamicum ATCC13032_DvalP, Corynebacterium glutamicum ATCC13032_DvalS, Corynebacterium for the L- valine production from ATCC13032_DvalA glutamicum strain was cultured in the same way.
[122]
[123]
250ml 25ml of production medium containing a composition as shown in Table 1 below corner-to bapul flask inoculated with one platinum loop of the strain, which was produced with 200rpm for 72 hours at 30 ℃.
[124]
After the completion of culture it was measured the production of L- valine by HPLC. Experiments L- valine concentration in the culture medium for each strain was as Table 2 and 3 below.
[125]
Table 1 [Table 1]
Production Medium
5% glucose, 2% ammonium sulfate, potassium phosphate first 0.1%, magnesium sulfate heptahydrate 0.05%, CSL (corn steep liquor), 2.0%, Biotin 200 ug / L, pH7.2

[126]
Table 2 [Table 2] Corynebacterium glutamicum KCCM11201P, KCCM11336P, KCCM11337P, KCCM11338P and valine productivity KCCM11201P_DvalA
Strain L- Valine concentration (g / L)
KCCM11201P 2.8
KCCM11336P 3.1
KCCM11337P 3.2
KCCM11338P 3.5
KCCM11201P_DvalA 0.5

[127]
Table 3 Table 3 Corynebacterium glutamicum ATCC13032_DvalW, ATCC13032_DvalL, ATCC13032_DvalP, ATCC13032_DvalS, valine productivity ATCC13032_DvalA
Strain L- Valine concentration (g / L)
ATCC13032_DvalW 0.1
ATCC13032_DvalL 0.9
ATCC13032_DvalP 1.1
ATCC13032_DvalS 1.3
ATCC13032_DvalA 0.1

[128]
As shown in Table 2, L- valine control region so that the strain transformed inactivation of biosynthetic genes, with the exception of Corynebacterium glutamicum KCCM11201P_DvalA the parent strain, L- valine-producing strain of Corynebacterium compared to glutamicum KCCM11201P confirmed the improved L- valine productivity. In particular Corynebacterium glutamicum was KCCM11338P indicate the L- valine production result improved by 25% compared to the parent strain.
[129]
[130]
In addition, L- valine biosynthesis gene control region overexpressing L- valine biosynthesis inactivated strain of Corynebacterium glutamicum ATCC13032_DvalL, Corynebacterium glutamicum and Corynebacterium ATCC13032_DvalP As shown in Table 3 Tommy ATCC13032_DvalS Qom strains of Corynebacterium glutamicum transformed strains Corey to have an L- valine biosynthesis gene regulatory region of the KCCM11201P Corynebacterium glutamicum L- valine production capability nine-fold to 13-fold compared to the Great ATCC13032_DvalW It was confirmed.
[131]
[132]
Reference Numerals
[133]
Pro: Promoter (Promoter),
[134]
Leader Peptide: nucleotide sequence encoding a leader peptide
[135]
Pause site: Stop area
[136]
Attenuator: Attenuation site
[137]
Gene: gene structure
[138]
[139]
[Accession number;
[140]
Depositary Organization: Korea Culture Center of Microorganisms (offshore)
[141]
Accession number: KCCM11336P
[142]
Commissioned Date: 20,121,119
[143]
[144]
Depositary Organization: Korea Culture Center of Microorganisms (offshore)
[145]
Accession number: KCCM11337P
[146]
Commissioned Date: 20,121,119
[147]
[148]
Depositary Organization: Korea Culture Center of Microorganisms (offshore)
[149]
Accession number: KCCM11338P
[150]
Commissioned Date: 20,121,119
[151]
[152]
[153]

Claims
[Claim 1]
Full nucleotide sequence coding for the leader peptide of the amino acid sequence described in SEQ ID NO: 1 in the control region of the L- valine operon or a defect is a defect portion or a substituted, the transgenic expression of the L- valine operon to enhance L- valine producing strains.
[Claim 2]
According to claim 1, wherein the leader peptide is a strain characterized in that it comprises a stop portion (pause site) of the amino acid sequence represented by SEQ ID NO: 2.
[Claim 3]
According to claim 2, wherein the whole or part of the defect or strain characterized in that a part of the nucleotide sequence coding for the still area of ​​substitution of the nucleotide sequence coding for the still area.
[[4]
According to claim 3, characterized in that the strain 13 to 15th nucleotide of the nucleotide sequence of SEQ ID NO: 23 coding for the still area is replaced with the termination codon.
[Claim 5]
According to claim 1, wherein the strains are strains, it characterized in that the microorganism of the genus Corynebacterium.
[6.]
The method of claim 5, wherein the strain is Corynebacterium glutamicum strain that is characterized.
[7.]
Claim 1 to any one of the culturing of the 6 strains and recovering L- valine from the culture broth of the strains; L- valine production method comprising a.
[8.]
L- valine operon control region of the mutant has an amino acid sequence represented by SEQ ID NO: 3 or 4.