Improved promoter and method for producing L-lysine using the same

Abstract

The present invention discloses a nucleic acid molecule derived from Corynebacterium glutamicum and having improved promoter activity and being operably connected to a gene encoding diaminopimelate dehydrogenase, a vector comprising the nucleic acid molecule, a transformant transformed with the vector, and a method for producing L-lysine using the transformant.

Description

[0001] The present application is a divisional application. The application date of the original application is January 23, 2009, the application number is 200980103315.6 (PCT / KR2009 / 000382), and the name of the invention is “Improved promoter and method for producing L-lysine using the same”. Technical Field

[0002] The present invention relates to an improved promoter and a method for producing L-Lysine therewith. More specifically, the present invention relates to Corynebacterium glutamicum ( Corynebacteriumglutamicum ) derived from a nucleic acid molecule showing improved promoter activity and operably linked to a gene encoding diaminopimelate dehydrogenase; a vector containing the nucleic acid molecule; a transformant into which the vector is introduced; and a method for producing L-lysine using the transformant. Background Art

[0003] Rod-shaped bacteria are traditional industrial microorganisms, which are most widely used in the production of various useful chemicals in animal feed, medicine and food industries, including amino acids, such as L-lysine, L-threonine, L-arginine, L-threonine and glutamic acid, and nucleic acid-related substances. These microorganisms are Gram-positive and require biotin for their growth. They split by cleavage (snapping), and their weak degradation power of the metabolites they produce can be beneficially utilized. Representative examples of rod-shaped bacteria include Corynebacterium such as Corynebacterium glutamicum, such as Brevibacterium flavum ( Brevibacteriumflavum ) of the genus Brevibacterium and certain species of Arthrobacter ( Athrobacter spp. ) and some species of Microbacterium ( Microbacterium spp. )wait.

[0004] L-Lysine is a commercially important L-amino acid that is used as a feed additive for animal nutrition because it helps the body absorb other amino acids to improve feed quality. For the body, L-Lysine is used as an injection component and is also found in the pharmaceutical field. Therefore, the industrial production of L-Lysine is an economically important industrial process.

[0005] The productivity of Methionin is relevant with the enzymic activity in the biosynthetic pathway, and this activity is generally enhanced by one or more genes in the amplification Methionin biosynthetic pathway or by the promoter of the modification of the application gene.It is well known that the coryneform bacteria strain that Lysine biosynthesis related gene is enhanced and utilize it to carry out Methionin production therein.For example, U.S. Patent number 6,746,855 discloses the method that the coryneform bacteria of the Methionin production with the enhanced lysE gene (Methionin export carrier gene) by fermentation produces Methionin, wherein additionally, the gene selected from dapA gene, lysC gene, pyc gene and dapB gene is enhanced.U.S. Patent number 6,221,636 discloses the coryneform bacteria transformed with the recombinant DNA of the nucleotide sequence that comprises the nucleotide sequence of coding aspartokinase and the nucleotide sequence of coding diaminopimelate decarboxylase, and in the nucleotide sequence of coding aspartokinase, the feedback inhibition of Methionin and L-Threonine has substantially reduced sensitivity (desentitized).

[0006] Korean Patent No. 10-0345592 describes an Escherichia strain in which dapA and lysC modified to eliminate feedback inhibition of L-lysine are introduced and dapB and ddh genes are amplified, and a method for producing L-lysine using the same.

[0007] In order to develop coryneform bacteria into variants capable of producing target products in high titers, genetic or metabolic engineering techniques that can selectively control genes involved in metabolism are required. To this end, it is important to modify the activity of promoters - regulatory DNA regions that provide a reliable initial binding site for RNA polymerase to control the transcription of the regulated gene.

[0008] Modified promoters derived from coryneform bacteria are found in many patents. For example, U.S. Patent No. 5,700,661, entitled "Gene expression regulatory DNA", U.S. Patent No. 5,965,391, entitled "DNA which regulates gene expression in coryneform bacteria", U.S. Patent No. 7,141,388, entitled "Nucleotide sequences for transcriptional regulation in corynebacterium glutamicum", and Korean Patent No. 10-0653742, entitled "Novel L-lysine-inducible promoter", describe promoters of coryneform bacteria, and Korean Patent Publication No. 10-2006-0068505, entitled "Novel promoter nucleic acid originating from corynebacterium genus bacteria, expression cassette comprising the promoter and vector comprising the cassette, host cell comprising the vector and method for expressing a gene using the cell", describe promoters of coryneform bacteria ( Corynebacteriumammoniagenes ) promoter.

[0009] However, there has been no disclosure to date of a coryneform bacterium which is improved in the activity of ddh (diaminopimelate dehydrogenase) which plays an important role in the lysine biosynthesis pathway and has the ddh-enhanced promoter to replace the endogenous promoter on the host cell genome. Summary of the invention

[0010] Technical issues

[0011] The present invention has conducted a thorough and in-depth study on the production of L-lysine and found that when a ddh gene promoter modified at a specific base is transformed in the genome of coryneform bacteria, coryneform bacteria microorganisms show diaminopimelate dehydrogenase activity improved over the endogenous activity.

[0012] Technical Solution

[0013] One of the objects of the present invention is to provide nucleic acid molecules derived from Corynebacterium glutamicum which show improved promoter activity.

[0014] Another object of the present invention is to provide a vector containing a nucleic acid molecule which exhibits improved promoter activity.

[0015] Another object of the present invention is to provide a transformant having a vector anchored therein.

[0016] Still another object of the present invention is to provide a method for producing L-lysine by fermenting the transformant.

[0017] Beneficial effects

[0018] When the nucleic acid molecule of the promoter activity that is derived from the improved performance of Corynebacterium glutamicum was operably connected to the ddh gene, Corynebacterium glutamicum according to the present invention showed higher promoter activity than wild-type promoter, and therefore can improve the level of diaminopimelate dehydrogenase.Therefore, the product lysine bacterial strain that transforms with nucleic acid molecule can produce Methionin in high yield. Sequence Listing <110> CJ CheilJedang Co., Ltd. <120> Improved promoter and method for producing L-lysine using the same <130> T15C11478CN <150> KR10-2008-0010073 <151> 2008-01-31 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 603 <212> DNA <213> Corynebacterium glutamicum <220> <221> gene <222> (1)..(603) <223> ddh promoter and part of ddh gene <400> 1 gccgtgcgtg gcgagtttta caaagaaccc cacatcatca atgcctaaat ggcgggtatt 60 ttcatccaaa cccaaccgcg catcattcca atgctgatcc accccatccg gataaaccac 120 catgaacggc aacggatcaa aagtcctgtt ggtgaagctg cgccccacag atcctgactg 180 ctgggagcca tgaaaataga tcagcgcatc cgtggtggaa ccaaaaggct caacaatacg 240 aaacgttcgc tttcggtcct gatgaaagag atgtccctga atcatcatct aagtatgcat 300 ctcggtaagc tcgaccagga cagtgccacc acaattttgg aggattacaa gaacatgacc 360 aacatccgcg tagctatcgt gggctacgga aacctgggac gcagcgtcga aaagcttatt 420 gccaagcagc ccgacatgga ccttgtagga atcttctcgc gccgggccac cctcgacaca 480 aagacgccag tctttgatgt cgccgacgtg gacaagcacg ccgacgacgt ggacgtgctg 540 ttcctgtgca tgggctccgc caccgacatc cctgagcagg caccaaagtt cgcgcagttc 600 gcc 603 <210> 2 <211> 603 <212> DNA <213> Corynebacterium glutamicum <220> <221> gene <222> (1)..(603) <223> Part of the ddhP1 promoter and the ddh gene <400> 2 gccgtgcgtg gcgagtttta caaagaaccc cacatcatca atgcctaaat ggcgggtatt 60 ttcatccaaa cccaaccgcg catcattcca atgctgatcc accccatccg gataaaccac 120 catgaacggc aacggatcaa aagtcctgtt ggtgaagctg cgccccacag atcctgactg 180 ctgggagcca tgaaaataga tcagcgcatc cgtggtggaa ccaaaaggct caacaatacg 240 aaacgttcgc tttcggtcct gatgaaagag atgtccctga atcatcatct aagtatgcat 300 tgtggtaagc tcgaccagga cagtgccacc acaattttgg aggattacaa gaacatgacc 360 aacatccgcg tagctatcgt gggctacgga aacctgggac gcagcgtcga aaagcttatt 420 gccaagcagc ccgacatgga ccttgtagga atcttctcgc gccgggccac cctcgacaca 480 aagacgccag tctttgatgt cgccgacgtg gacaagcacg ccgacgacgt ggacgtgctg 540 ttcctgtgca tgggctccgc caccgacatc cctgagcagg caccaaagtt cgcgcagttc 600 gcc 603 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer ddh / PF <400> 3 ccggggatcc tctagagtgc gtggcgagtt ttacaaag 38 <210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer ddh / PR <400> 4 gcaggtcgac tctagaggcg aactgcgcga actttgg 37 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer ddh / P1F <400> 5 tatgcattgt ggtaagctcg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer ddh / P1R <400> 6 cgagcttacc acaatgcata 20 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer ddh / P1mut <400> 7 ctaagtatgc attgt 15 BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram showing the genetic map of the vector pDZ integrated into the genome of Corynebacterium.

[0020] Figure 2 is a schematic diagram showing the genetic map of the vector pDZ-ddhP1 used for promoter replacement.

[0021] Best Mode for Carrying Out the Invention

[0022] According to one aspect thereof, the present invention relates to a nucleic acid molecule derived from Corynebacterium glutamicum having the nucleotide sequence of SEQ ID NO. 2, which is operably linked to a gene encoding diaminopimelate dehydrogenase and exhibits improved promoter activity.

[0023] The term "promoter" as used herein refers to a DNA region that contains an initial binding site for RNA polymerase and promotes transcription of a specific gene downstream thereof. That is, a promoter is a non-translated nucleotide sequence, upstream of a coding region, to which RNA polymerase binds to initiate gene transcription, and is usually located near the gene it regulates, upstream on the same strand (close to the 5' region of the sense strand).

[0024] The nucleic acid molecule of the present invention with promoter activity derived from Corynebacterium glutamicum is operably linked to a gene encoding diaminopimelate dehydrogenase. The ddh gene encodes diaminopimelate dehydrogenase, which plays an important role in the lysine biosynthetic pathway of Corynebacterium.

[0025] Term " operably connected ", as used herein, means to have according to the connection between the nucleotide sequence of promoter activity described in the present invention and the promoter sequence, can carry out the initiation or stopping of the gene transcription of encoding diaminopimelate dehydrogenase with such functional relationship promoter.That is, when being operably connected to the ddh gene, have according to the nucleotide sequence of promoter activity described in the present invention can control the transcriptional activity of the ddh gene.

[0026] Have according to the nucleotide sequence of promoter activity of the present invention, it derives from the wild-type ddh gene promoter of Corynebacterium glutamicum, is modified to guarantee the enzymic activity that is better than endogenous activity.Endogenous activity means the enzymic activity in the wild-type rod-shaped bacterium.Guarantee that the modification of higher promoter activity can utilize technology well known in the art to realize, preferably by deletion, insertion, non-conservative or conservative substitution or its combination to induce the nucleotide sequence sudden change of the promoter of ddh gene.

[0027] Have according to the nucleic acid molecule of promoter activity of the present invention and can utilize standard biological technology to separate or prepare.For example, can carry out PCR to separate in the presence of suitable primer.Alternatively, can utilize standard biological technology to use automatic DNA synthesizer to synthesize.In one embodiment, based on nucleotide sequence (SEQ ID NO.1)---it contains the promoter region of the ddh gene (NCBI gene ID: NCgl2528) available from NIH GenBank data, synthesized 4 primers (SEQ ID NOS.3~6).In the presence of primer, carry out PCR as template so that the nucleic acid molecule that contains promoter is provided with with the genomic DNA of Corynebacterium glutamicum KFCC10881, and this nucleic acid molecule has modification at specific base position (SEQ ID NO.2).

[0028] Preferably, the nucleic acid molecule having the promoter activity of Corynebacterium glutamicum according to the present invention can be used as a promoter in prokaryotes, especially in Escherichia coli ( E. coli ) or promoters for the expression of coryneform bacteria genes. As used herein, the term "coryneform bacteria" refers to microorganisms belonging to the genus Corynebacterium or Brevibacterium. Examples of coryneform bacteria that can be used in the present invention include, but are not limited to: Corynebacterium glutamicum ATCC13032, Corynebacterium thermoammonigenicum ( Corynebacteriumthermoaminogenes )FERMBP-1539, Brevibacterium flavum ( Brevibacteriumflavum )ATCC 14067, Brevibacterium lactofermentum ( Brevibacteriumlactofermentum ) ATCC 13869 and L-amino acid-producing mutants or strains derived therefrom, such as Corynebacterium glutamicum KFCC10881 and Corynebacterium glutamicum KFCC11001, preferably Corynebacterium glutamicum KFCC10881.

[0029] According to another of its aspects, the present invention relates to a vector, in which a nucleic acid molecule with improved promoter activity is located.

[0030] As used herein, the term "vector" refers to a DNA construct in which a gene of interest is operably linked to regulatory elements so that the gene can be expressed in a suitable host in which the vector is anchored. Regulatory elements include promoters that initiate transcription, operator genes that control transcription, sequences encoding mRNA ribosome binding sites, and sequences that control transcription and translation termination.

[0031] As long as it can be replicated in the host, any vector known in the art can be used in the present invention without particular limitation. For example, the vector that can be used in the present invention can be a plasmid, a phage particle or simply a possible genome insert, but the present invention is not limited thereto. A preferred vector is pACYC177 (New England Biolab, GenBank accession number X06402). After transformation into a suitable host, the vector can replicate or function independently of the host genome or integrate itself into the genome.

[0032] More specifically, when being introduced into host cell according to vector of the present invention, the nucleic acid molecule with promoter activity in the vector can carry out homologous recombination with the promoter region of endogenous ddh gene on the host genome, causes the vector to be integrated in the host cell chromosome.Therefore, can further comprise selective marker with the insertion of indicator vector in the host chromosome according to vector of the present invention.Be suitable for indicating the cell transformed with the vector, whether promptly gene of interest inserts the host cell genome, and selective marker can make cell have following ability: drug resistance, cytotoxic agent resistance, auxotrophy or as the selectable phenotypic expression of surface protein expression.In the presence of selective agent, transformed cell can be selected, because only have the cell survival of expression selective marker or show another phenotype.Preferably, carrier can comprise lacZ gene as selective marker.

[0033] In embodiments of the present invention, construct a vector to contain the modified ddh promotor of improved activity, which can replace the endogenous ddh promotor of Corynebacterium glutamicum by homologous recombination.For this reason, at first, the vector pACYC177 that will be used for Escherichia coli cloning is digested with restriction enzymes and blunt-ended with Klenow fragments.Respectively, the nucleotide sequence that comprises the lacZ gene and its promoter is amplified by PCR from the genomic DNA of Escherichia coli K12W3110.The two DNA fragments thus obtained are interconnected to obtain the circular nucleic acid molecule, and then the ligation sequence that comprises a plurality of restriction enzyme sites in it is inserted in the circular nucleic acid molecule, so that the vector pDZ ( Figure 1 ), for insertion into the chromosome of coryneform bacteria. Thereafter, the ddh gene promoter modified at a specific base to exhibit high activity was inserted into the linker sequence of the pDZ vector to provide a vector pDZ-ddhP1 ( Figure 2 ).

[0034] According to another aspect thereof, the present invention relates to a transformant in which a vector is anchored.

[0035] The term "conversion" as used herein refers to that foreign DNA material is introduced into host cell, wherein the foreign DNA material can be used as an element replication that is independent of the host genome or is integrated into the host genome. Because the vector is transformed into the host cell, the transformant is anchored with the form of plasmid with the vector, or after the endogenous promoter of the ddh gene on the nucleotide sequence with promoter activity and the host cell genome carries out homologous recombination, the vector is integrated into the host cell chromosome.

[0036] Any technique can be used in the present invention as long as it is used to introduce the vector described in the present invention into the host cell. Depending on the host cell, appropriate standard techniques can be selected, such as electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology and lithium acetate-DMSO technology.

[0037] It is useful to use a host cell that efficiently takes up and expresses foreign DNA material, which can be applicable to all microorganisms including prokaryotes and eukaryotes.Preferably, Escherichia coli or coryneform bacteria can be used, more preferably Corynebacterium glutamicum KFCC10881.

[0038] In the cell transformed with carrier of the present invention, the improved promoter with improved activity has replaced endogenous promoter by homologous recombination, has strengthened the mRNA level of ddh gene.Therefore, transformant has higher diaminopimelate dehydrogenase activity than wild type.

[0039] In one embodiment of the present invention, the vector pDZ-ddhP1, which comprises a promoter having the nucleotide sequence SEQ ID NO. 2 of the present invention, is transformed into Corynebacterium glutamicum KFCC10881 to obtain a transformant (KFCC10881-ddhP1), designated as CA01-0136, which exhibits improved diaminopimelate dehydrogenase activity, and then the transformant is deposited in the Korean Culture Center of Microorganisms (hereinafter referred to as "KCCM") on January 18, 2008 under the number KCCM10920P.

[0040] According to another aspect thereof, the present invention relates to a lysine production method comprising fermentation of the transformant.

[0041] Compared with the wild type, the transformant according to the present invention has improved diaminopimelate dehydrogenase activity. Since diaminopimelate dehydrogenase is the most important enzyme in the lysine biosynthesis pathway, fermentation of the transformant results in the production of a higher yield of lysine.

[0042] In the present invention, the fermentation of the transformant can be carried out using known methods, and the fermentation conditions, including temperature, time, pH, etc., can be appropriately controlled. The following documents provide a detailed description of the fermentation [Chmiel; Bioprozesstechnik 1.Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und periphereEinrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)]. Fermentation can be achieved by batch culture, continuous culture or fed-batch culture. Preferably, a fed-batch or repeated fed-batch method is used for the continuous mode of fermentation, but the present invention is not limited thereto.

[0043] For fermentation, the culture medium must meet the needs of the strain used. Culture mediums suitable for culturing various microorganisms are well known in the art (e.g., from the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981)). The culture medium may include carbon source sugars (saccharides) and carbohydrates (carbohydrates) (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), lipids and fats (e.g., soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, ricinoleic acid), alcohols (e.g., glycerol and ethanol) and organic acids (e.g., acetic acid). These substances can be used separately or in combination. As a nitrogen source, nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, wort, corn steep liquor, soybean meal and urea) or nitrogen-containing inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) can be used separately or in combination. Examples of phosphorus sources used in the culture medium include dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and corresponding sodium salts.

[0044] In addition, the culture medium may contain metal salts (such as magnesium sulfate or iron sulfate) necessary for cell growth, and may be supplemented with essential nutrients such as amino acids and vitamins to stimulate growth. In addition, appropriate precursors may be added to the culture medium. Nutrients and additives may be added all at once or separately during the fermentation process.

[0045] The pH of the culture medium can be adjusted with alkaline compounds (such as sodium hydroxide, potassium hydroxide or ammonia) or acidic compounds (such as phosphoric acid or sulfuric acid). The generation of foam in the culture medium can be inhibited by defoamers such as polyethylene glycol fatty acid esters. The culture medium can be kept under aerobic conditions by introducing oxygen or oxygen-containing gas mixtures therein. For the culture temperature, usually between 20°C and 45°C, preferably between 25°C and 40°C. Fermentation is carried out continuously until the maximum amount of L-amino acid is produced. In this regard, it can be completed in 10 to 160 hours. After L-Lysine is produced, L-Lysine can be exported to the culture medium or can be kept in the cell.

[0046] Alternatively, the method for producing lysine of the present invention may further include collecting the produced lysine. L-Lysine can be isolated from culture medium or cells using known methods. Examples of collection methods for the present invention include filtration, anion exchange chromatography, crystallization and HPLC, but are not limited thereto.

[0047] A better understanding of the present invention may be obtained through the examples described below, but they are not intended to limit the present invention.

[0048] Invention method

[0049] In the following examples, made up recombinant vector to comprise the promotor of Corynebacterium glutamicum ddh gene, it is modified and has improved activity.Recombinant vector is transformed into Corynebacterium glutamicum KFCC10881, and wherein the ddh promotor of modification is integrated into cytogenomic group by homologous recombination with endogenous promoter immediately, produces the new bacterial strain that can produce Methionin in high yield.

[0050] The Corynebacterium glutamicum KFCC10881 used in the present invention has resistance to S-(2-aminoethyl)cysteine ​​(hereinafter referred to as "AEC") and homoserine leakage (Korean Patent Nos. 0159812 and 0397322) from a wild-type strain of Corynebacterium glutamicum (ATCC13032) through artificial mutagenesis.

[0051] Example 1: Construction of a recombinant vector containing an improved promoter

[0052] (1) Construction of vector for integration into the genome (pDZ)

[0053] In this example, pDZ, a vector to be integrated into the genome of Corynebacterium, was constructed based on the vector pACYC177 (New England Biolab, GenBank accession # X06402) for Escherichia coli.

[0054] After treatment with AcuI and BanI, the pACYC177 vector was blunt-ended with the Klenow fragment. The preparation of the lacZ gene derived from Escherichia coli, used as a selection marker, was carried out by PCR amplification of the genomic DNA of Escherichia coli K12 W3110 including the gene and its promoter, followed by treatment of the PCR product with T4 DNA polymerase and polynucleotide kinase, respectively, to phosphorylate the 5' end and blunt-end the opposite end. The two DNA fragments thus obtained were ligated to each other to obtain a circular nucleic acid molecule, and then an artificially synthesized linker sequence containing multiple restriction enzyme sites was inserted to provide a vector pDZ for insertion into the chromosome of coryneform bacteria. Figure 1 In FIG. 1 , the pDZ vector for integration into the chromosome of coryneform bacteria is schematically illustrated.

[0055] (2) Construction of a vector containing the improved promoter of the ddh gene

[0056] In this example, a recombinant vector was constructed to contain the improved promoter of the ddh gene of the lysine-producing strain Corynebacterium glutamicum.

[0057] On the basis of NIH GenBank data, first obtain the nucleotide sequence (SEQ ID NO. 1) that comprises the ddh gene promoter region (NCBI ID No.NCgl2528).Thus nucleotide sequence is prepared at the DNA fragmentation of specific base position mutation.The promoter sequence of each modification is designed based on the common consensus promoter sequence found in the microorganism.

[0058] For the preparation of modified promoter sequences, four primers were synthesized based on the base sequence (SEQ ID NOS. 3 to 6, Table 1).

[0059] Table 1

[0060]

[0061] By PCR, using the primer set shown in Table 1, PfuUltra TMIn the presence of high-fidelity DNA polymerase (Stratagene), the promoter sequence of the ddh gene of Corynebacterium glutamicum was prepared using the genomic DNA of Corynebacterium glutamicum KFCC10881 as a template. PCR was performed with 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 30 seconds. Thus, the PCR product was a 300 bp long DNA fragment, and the replacement part was located at one terminal region. ddhP1-1 was amplified with a set of primers SEQ ID NOS.3 and 6, and ddhP1-2 was amplified with a set of primers SEQ ID NOS. 5 and 4. The PCR product was digested with XBaI and cloned into pDZ using In-fusion Cloning Kit (TAKARA) to obtain the recombinant vector pDZ-ddhP1.

[0062] Figure 2 The map is a vector pDZ-ddhP1 containing the promoter sequence SEQ ID NO. 2 integrated into the genome of the coryneform bacterium.

[0063] Example 2: Introduction of recombinant vectors into Corynebacterium glutamicum strains

[0064] In this embodiment, the recombinant vector prepared above is introduced into the bacterial strain Corynebacterium glutamicum KFCC-10881 of producing lysine, so that the ddh promoter sequence modified on the carrier is integrated into the cell genome by homologous recombination with the endogenous ddh promoter on the genome.

[0065] To this end, the recombinant vector pDZ-ddhP1 containing the DNA fragment corresponding to the modified promoter sequence was transformed into Corynebacterium glutamicum KFCC10881 using the electroporation method (based on Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then transformants were selected on a selection medium containing 25 mg / L of kanamycin, in which the modified promoter was integrated into the genome by homologous recombination with the endogenous promoter.

[0066] Successful insertion of the vector into the genome was identified by the appearance of a blue color on plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Single crossovers with the vector integrated into the genome were grown in nutrient broth with agitation (30°C, 8 hours) and then diluted to a concentration of 10 before plating on plates containing X-gal. -4 to 10 -10. When most of the clones growing on the plate appear blue, only a few clones remain white. The white clones are selected as double exchange clones, which are anchored with ddh promoters mutated at specific base positions. For confirmation, base substitutions of the selected strains are detected by PCR and base sequencing. Promoter base substitution detection is performed on strains transformed with pDZ-ddhP1 using a set of primers SEQ ID NOS. 4 and 7 by PCR and base sequencing.

[0067] Finally, the lysine-producing strain Corynebacterium glutamicum KFCC10881-ddhP1 was determined by double crossover, in which the ddh promoter mutated at a specific base position was integrated into the genome.

[0068] Example 3: Detection of diaminopimelate dehydrogenase activity in ddh promoter-modified strains

[0069] The mother strain Corynebacterium glutamicum KFCC10881 and the L-lysine-producing strain Corynebacterium glutamicum KFCC10881-ddhP1 finally prepared in Example 2 were cultured, and proteins were separated from the cultures and the diaminopimelate dehydrogenase activity was detected as follows.

[0070] Inoculate each culture grown to logarithmic phase into 50 mL of the following seed medium (I) to reach OD 600 The value was 0.3, and then cultured until the optical density at 600nm reached about 15. After centrifugation and collection (5,000rpm, 15 minutes), the cells were washed twice with 20mMTrisHCl (pH 8.0) and suspended in the same buffer so that the light absorption at 610nm reached a turbidity of 160. The cells were broken for 6 minutes in a glass stirrer with glass beads added at 1.25 g / 1.5 ml suspension. After centrifugation (15,000rpm, 20 minutes), the protein content of the supernatant was quantitatively determined by the Bradford method (Bradford, MM 1976. Anal. Biochem. 72:248-254), and the supernatant was used as a crude protein solution for determining the activity of diaminopimelate dehydrogenase.

[0071] To quantify diaminopimelate dehydrogenase activity, approximately 0.01 mL of crude protein solution was mixed with a reaction solution containing 0.2 M glycine / NaOH (pH 10.5), 2 mM NADP, and 4 mM meso-diaminopimelate to a total volume of 1 mL and allowed to react at 25°C for 10 min, during which the absorbance at 340 nm was monitored. Diaminopimelate dehydrogenase activity was defined as the number of micromoles (μmoles) of NADPH reduced per minute by 1 mg of protein and was expressed in units (U).

[0072] It was observed that Corynebacterium glutamicum KFCC10881-ddhP1 had a diaminopimelate dehydrogenase activity 23.2 times higher than that of the mother strain KFCC10881 (Table 2).

[0073] Table 2

[0074]

[0075] Seed medium (Ⅰ) (pH 7.0)

[0076] Glucose 20 g, peptone 10 g, yeast extract 5 g, (NH4)2SO4 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4 7H2O 0.5 g, biotin 150 μg, thiamine hydrochloride 1500 μg, calcium pantothenate 3000 μg, nicotinamide 3000 μg (per liter of distilled water)

[0077] Example 4: Production of lysine in ddh promoter-modified strains

[0078] The mother strain Corynebacterium glutamicum KFCC10881 and the L-lysine-producing strain Corynebacterium glutamicum KFCC10881-ddhP1 prepared in Example 2 were fermented to produce L-lysine as follows.

[0079] Corynebacterium glutamicum KFCC10881 and KFCC10881-ddhP1 were inoculated into 250 mL elbow-baffle bottles, each containing 25 mL of the following seed culture medium (II), and cultured at 30°C with shaking at 200 rpm for 20 hours. 1 mL of the seed culture was added to 24 mL of the following production medium in a 250 mL elbow-baffle bottle, followed by culture at 30°C with shaking (200 rpm) for 120 hours.

[0080] After the cultivation was completed, HPLC analysis was performed to determine the amount of L-lysine produced by the strain. The concentration of L-lysine in the culture of Corynebacterium glutamicum KFCC10881 and KFCC10881-ddhP1 is summarized in Table 3, as follows.

[0081] Table 3

[0082]

[0083] Seed medium (Ⅱ) (pH 7.0)

[0084] Raw sugar 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO47H2O 0.5 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (per liter of distilled water)

[0085] Production medium (pH 7.0)

[0086] Glucose 100 g, (NH4)2SO4 40 g, soy protein 2.5 g, corn syrup solids 5 g, urea 3 g, KH2PO4 1 g, MgSO4 7H2O 0.5 g, biotin 100 μg, thiamine chloride 1000 μg, calcium pantothenate 2000 μg, nicotinamide 3000μg, CaCO3 30 g (per liter of distilled water)

[0087] As shown in Table 3, it was found that the lysine productivity of Corynebacterium glutamicum KFCC-ddhP1, in which the diaminopimelate dehydrogenase activity was increased by about 23 times, was increased by about 3% compared with the mother strain KFCC10881.

[0088]

Industrial Applicability

[0089] As mentioned above, can improve the activity of diaminopimelate dehydrogenase according to the nucleic acid molecule with higher promoter activity than wild type in Corynebacterium glutamicum source of the present invention, thereby increase the biosynthetic efficiency of Methionin.Therefore, the bacterial strain that is anchored with improved promoter can generate industrially important amino acid L-Methionine in high yield.

Claims

1. A transformant having a vector comprising a nucleic acid molecule derived from Corynebacterium glutamicum ( Corynebacterium glutamicum ) has an improved promoter activity, has a modification at a specific base position of the nucleotide sequence SEQ ID NO: 1, and is modified to the nucleotide sequence shown in SEQ ID NO: 2, wherein the transformant belongs to Corynebacterium glutamicum ( Corynebacterium glutamicum ). 2 . The transformant according to claim 1 , wherein the modification is tgt in the nucleic acid sequence tgtGGTAAGCTCGA.

3. The transformant according to claim 1, wherein the nucleic acid molecule is operably linked to a gene encoding diaminopimelate dehydrogenase (ddh).

4. The transformant according to any one of claims 1 to 3, wherein the nucleic acid molecule is integrated into the genome of the transformant by homologous recombination.

5. A method for producing lysine, comprising fermenting the transformant according to any one of claims 1 to 4 in a culture solution, and recovering lysine from the transformant or the culture solution.

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