WO2000056858A1

WO2000056858A1 - Process for producing l-lysine

Info

Publication number: WO2000056858A1
Application number: PCT/JP2000/001654
Authority: WO
Inventors: Masakazu Sugimoto; Hisao Ito; Osamu Kurahashi
Original assignee: Ajinomoto Co., Inc.
Priority date: 1999-03-19
Filing date: 2000-03-17
Publication date: 2000-09-28
Also published as: JP2003135066A; AU3194200A

Abstract

L-Lysine productivity is enhanced by transferring a gene encoding diaminopimelic acid epimerase into a coryneform bacterium capable of producing L-Lysine so as to strengthen the diaminopimelic acid epimerase activity in the cells.

Description

Shiran

L-Lysine production method

The present invention relates to a method for producing L-lysine by a fermentation method. L-Lysine is widely used as a feed additive. Background art

Heretofore, L-lysine has been industrially produced by a fermentation method using a coryneform bacterium which is capable of producing L-lysine and which succumbs to the genus Brevipacterium and Corynebacterium. As these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.

In addition, various techniques for increasing L-lysine producing ability by enhancing the activity of L-lysine biosynthetic enzymes by recombinant DNA technology have been disclosed. For example, in a coryneform bacterium capable of producing L-lysine, a gene (mutant lysC) encoding aspartokinase that has been freed of feedback efficiency by L-lysine and L-threonine; Yuichize gene (dapB), dihydroxypicolinate synthase gene (dapA), diaminobimephosphate decarboxylase gene (lysA) gene, and diaminopimephosphate dehydrogenase gene (ddh) (W096) / 40934), lysA and DDH (JP-A 9, item three hundred twenty-two thousand seven hundred and seventy-four), lysCヽlysA及beauty Hosuhoeno one Rubirubin carboxylase gene (pp _C) (JP-A-10- 165180), mutant lysC, dapB, dapA, lysA and Introduction of the aspartate aminotransferase gene (aspC) (Japanese Patent Publication No. 10-215883) may improve the L-lysine production ability of the bacterium. Are known.

In the Eshierihia厲細{1, da _P A, varying type 5 lys dapB, Jiaminobi Mellin acid de arsenide Dorogena Ichize Zodenko (ddh) (say yes Te Torahi Dorojipikori phosphate disk Shiniraze遗伝Ko (dapD) And succinyldiaminopimelate deacylase gene (dapE)) are known to increase L-lysine activity when secondary ^γ- enhancement is enhanced (W095 / 16042). ι '), succinyl tetrahidrodipicolinate in W0 95/16042 Ze is described as succinyl diaminopimelate transaminase. However, the gene encoding diaminopimelate epimerase is not known in coryneform bacteria, and the relationship between the activity of the enzyme and the ability of microorganisms to produce L-lysine is not known. Disclosure of the invention

An object of the present invention is to provide a method for producing L-lysine by a fermentation method which has been further improved than before, and a microorganism used therefor.

The present inventors conducted intensive studies in order to solve the above-mentioned problems, and introduced a gene encoding diaminopimelate epimerase (hereinafter, also referred to as “dapF”) into a coryneform bacterium having an ability to produce L-lysine. However, they have found that the L-lysine producing ability of the bacterium is improved, and completed the present invention.

That is, the present invention is as follows.

(1) A coryneform bacterium having enhanced diaminopimelate evimelase activity in cells and L-lysine producing ability.

(2) The coryneform bacterium according to (1), wherein the enhancement of diaminopimelate epimerase activity is due to an increase in the number of copies of a gene encoding diaminopimelate epimerase in the bacterial cells.

(3) The coryneform bacterium according to (2), wherein the gene encoding diaminopimelate epimerase is derived from a coryneform bacterium.

(4) The coryneform bacterium according to (1), wherein at least one of the activity of aspartole kinase, dihydropicolinate reductase activity, dihydrodipicolinate synthase activity, and diaminobimelate decarboxylase activity is enhanced.

(5) The coryneform bacterium described in any of (1) to (4) above is cultured in a medium, L-lysine is produced and accumulated in the width of the culture, and L-lysine is collected from the culture. A method for producing L-lysine. Screaming in the drawing

FIG. 1 is a diagram showing the process of gross construction of plasmid P299LYSA, which shows lysA. FIG. 2 is a diagram showing a process of constructing a plasmid pCAB having mutant lysC, dapA, dapB and Brevi._ori.

FIG. 3 is a view showing a process of constructing a plasmid pC ABL having mutant lysC, dapA, dapB, lysA and Brevi.-ori. BEST MODE FOR CARRYING OUT THE INVENTION

<1> Coryneform bacterium of the present invention

The coryneform bacterium of the present invention is a coryneform bacterium having L-lysine-producing ability and having enhanced diaminobimelinate epimerase activity in cells.

The coryneform bacteria referred to in the present invention include a group defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974). A bacterium that is aerobic, Gram-positive, non-acid-fast, and has no sporulating ability. It is a bacterium that was previously classified as Brevibacterium, but is now integrated as Corynebacterium. Includes (Int. J. Syst. Bacteriol., 41, 255 (1981)) and also includes bacteria of the genus Brevibacterium and Micropaterium which are very closely related to the genus Corynebacterium. Examples of the strain of coryneform bacterium suitably used for the production of L-lysine include the following.

Corynebacterium acetate acetate film ATCC13870

Corynepaterum Acetoglu evening Mikum ATCC15806

Corynebacterium carnae ATCC15991

Corynebacterium glutamicum ATCC13032

(Brevibacterium divaricatum) ATCC14020

(Brevibacterium lactofamentum) ATCC13869

(Corynepactium realm) ATCC15990

(Brevibacterium flavum) ATCC14067

Corynebacterium meracecola ATCC17965

Brevi Pacterium Saccharolyticum ATCC14066

Brevi Pacterium Inmario Film ATCC14068 Brevibacterium rosem ATCC13825 Brevibacterium choge squirrel ATCC19240

Microbacterium '' Ammonia Filum ATCC15354

Corynebacterium salmonaminogenes AJ12340 (FERM BP-1539) These can be obtained from, for example, American Type Calcia Collection. That is, a registration number corresponding to each microorganism is assigned, and the microorganism can be ordered by referring to this registration number. The registration number corresponding to each microorganism is listed in the Amerikan 'Type, Culture, and Collection Power Logs. AJ12340 strain has been deposited with the Ministry of International Trade and Industry of the National Institute of Advanced Industrial Science and Technology under the Budapest Treaty.

In addition to the above strains, mutants having L-lysine-producing ability derived from these strains can also be used in the present invention. Such artificial mutants include the following. S- (2-aminoethyl) one-cysteine (hereinafter abbreviated as "AEC") resistant mutant (brevipacterium lactophormentamentum AJ11082 (NRR LB-11470), if it is cold, Japanese Patent Publication No. 56-1914) , JP-B-56-1915, JP-B-57-14157, JP-B-57-14158, JP-B-57-30474, JP-B-58-10075, JP-B-59-4993 Bow, JP-B-61-35840, (See Japanese Patent Publication No. 62-24074, Japanese Patent Publication No. 62-36673, Japanese Patent Publication No. 5-11958, Japanese Patent Publication No. 7-112437, and Japanese Patent Publication No. 7-112438), and their growth requires amino acids such as L-homoserine. Mutants (JP-B-48-28078, JP-B-56-6499), which are resistant to AEC, and furthermore, L-lipid isin, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, Mutants requiring amino acids such as L-parin (US Patent Nos. 3,708,395 and 3,825,472), DL-Hamino Lactam, hyamino-lauryl lactam, aspartic acid-analogue, sulfa drugs, quinoids, five L-lysine-producing variants resistant to N-lauroylleucine, Oxaguchi acetic acid decarboxylase

L-lysine-producing mutants that are resistant to M (decarboxylase) or respiratory system mM '' m (Japanese Patent Publication No. 50-53588, Japanese Patent Application No. 50-31093, Japanese Patent Application No. 52-102498, Japanese Patent Application Laid-Open No. 53-9394) No.53-86089, No.55-9783, No.55-9759, No.56-32995, No.56-39778, No.56-39778, No.53-43591-, No.53-1833 , Lysine-producing mutants that require inositol or acetic acid (JP-A-55-9784, JP-A-56-86) No. 92), L-lysine-producing mutants that are sensitive to fluoropyruvate or a temperature of 34 ° C. or higher (JP-A-55-9783 and JP-A-53-86090), exhibit resistance to ethylene glycol, Brevibacterium flexi or Corynebacterium II mutant producing L-lysine (US Patent No. 4411997)

As used herein, the term “L-lysine-producing ability” refers to the ability of a coryneform bacterium to accumulate a significant amount of L-lysine in a medium when cultured in the medium, or the L-lysine in the cells. The ability to increase content.

<2> Enhancement of diaminobimelinate epimerase activity

In order to enhance diaminopimelate epimerase activity in coryneform bacterium cells, a gene fragment encoding diaminopimelate epimerase is ligated to a vector that functions in the bacterium, preferably a multicopy vector, to produce recombinant DNA. It may be transformed into a coryneform bacterium having the ability to produce L-lysine and transformed. As a result of an increase in the copy number of the gene encoding diaminopimelate epimerase in the cells of the transformed strain, diaminopimelate epimerase activity is enhanced.

As the gene encoding diaminopimelate epimelase, a gene of a coryneform bacterium or a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.

The nucleotide sequence of the gene encoding the diaminopimelate epimerase of Escherichia coli (dagF gene) has already been elucidated (Nucleic Acid Research, 16 (21), 10367 (1988)). What primers have been created, for example, an array of arrays? !! No. 1 and 2, PCR method using Escherichia coli color chromosome DNA as type III (PCR: polymerase chain reaction; White, TJ et al; Trends Genet. 5, 185 (1989)) See) to get the dapF 遗 gene. Genes encoding diaminopimelate epimelase of other microorganisms such as corynebacteria can be obtained in a similar manner.

DNA from bacteria that are DNA donors can be obtained from the DNA donor bacteria by the method of H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963). Science Experiment Book, edited by the Biotechnology Society of Japan, 97-98] 3, Baifukan, 1992) Can be prepared.

The dapF 遗 gene amplified by the PCR method is connected to vector DNA capable of autonomous replication in the cells of Escherichia coli and / or coryneform bacteria to prepare recombinant DNA, which is then added to Escherichia coli. If introduced into cells, subsequent operations will be difficult. As a vector capable of autonomous replication in Escherichia coli cells, a plasmid vector is preferable, and a vector capable of autonomous replication in a host cell is preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSFIOIO and the like.

The vector one autonomously replicable in cells of coryneform bacteria (see Japanese Patent Laid HirakiAkira 58-67699) PAM330, _P HM1519 (see JP-A-58- 77895) and the like et be. In addition, a DNA fragment having the ability to autonomously replicate plasmid in coryneform bacteria is extracted from these vectors, and inserted into the above-mentioned vector for Escherichia coli. It can be used as a so-called shuttle vector that cannot be replicated autonomously by both.

Such shuttle vectors include the following. The accession number of the international depositary organization for the microorganisms carrying the respective vectors is shown in parentheses.

PAJ655 I Shierihia] Re AJ11882 (FERM BP-136)

Coryneha, 'Caterum' 'C' 'Tamicum SR820 ATCC39135)

PAJ1844 I Shierihia 'Kori AJ11883 (FERM BP-137)

Coryneha ,, Cterium 'C ,, Luyu Mikum SR8202 (ATCC39136)

PAJ611 I Shih X Richia 'Kori AJ 11884 (FERM BP-138)

PAJ3148 IP n ,,

pAJ440 'Chills, Chillis, AJ11901 (FERM BP-140)

pHC4 Escherichia coli E. coli AJ 12617 (FE BP-3532)

These vectors are obtained from the deposited microorganism as follows. The cells collected during the logarithmic growth phase are washed with lysozyme and SDS; centrifuged at 300,000 X g; polyethylene glycol is added to the supernatant obtained from the lysate; Separate and purify by mouth-mould equilibrium density gradient centrifugation. Recombinant DNA by prescribing diaminobimelinate epimerase gene and vector To prepare E. coli, cut the vector with a restriction enzyme that matches the end of the DNA fragment containing the diaminopimelate epimerase gene. Ligation is usually performed using a ligase such as T4 DNA ligase.

In order to introduce the recombinant DNA prepared as described above into a coryneform bacterium, it may be carried out according to a transformation method reported so far. For example, Escherichia coli

Methods for increasing DNA permeability by treating recipient cells with calcium chloride as reported for K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159). (1970)), and a method for preparing a competent cell from a cell at the growth stage and introducing DNA as described in Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene , 1, 153 (1977)). Alternatively, the recombinant DNA is introduced into the DNA recipient by transforming the cells of the DNA recipient into protoplasts or spheroplasts, as is known for Bacillus subtilis, actinomycetes and yeast, to facilitate the uptake of recombinant DNA. (Chang, S. and Choen, SN "Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ" Ward, JM and Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A ., Hicks, JB and Fink, GR, Pro Natl. Acad. Sci. USA, 75 1929 (1978)). Transformation of a coryneform bacterium can also be performed by the air pulse method (Sugimoto et al., JP-A-2-207791).

Enhancement of diaminobimelinate epimerase activity can also be achieved by allowing the dapF gene to be present in multiple copies on the chromosomal DNA of the above host. In order to introduce multiple copies of the dapF gene into the DNA of a microorganism belonging to a coryneform bacterium, homologous recombination is performed using a sequence present on the chromosomal DNA in multiple copies as a target. As a sequence present in multiple copies on the chromosome DNA, a repetitive DNA and an inverted repeat present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is also possible to load the dapF gene into a transposon, transfer it, and introduce multiple copies into DNA i :. Either method increases the number of copies of the dapF promoter in the Form H transformant, resulting in enhanced diaminopimelate epimerase activity.

Enhancement of diaminobimelinate epimerase activity is not due to the width of the gene Alternatively, it can be achieved by replacing the expression control sequence such as the promoter of the dapF gene on chromosomal DNA or plasmid with a strong one (see Japanese Patent Application Laid-Open No. 1-215280). For example, lac promoter one, trp promoter Isseki one, trc promoter one evening one, tac promoter, [rho _kappa promoter of lambda phage, the P _L promoter evening, First known as a powerful promoter Isseki scratch. The substitution of these promoters all at once enhances the expression of the dapF gene, thereby enhancing the activity of diaminopimelate epimelase. Modification of the expression control sequence may be combined with increasing the copy number of the dapF gene.

To confirm the enhancement of diaminopimelate epimerase activity, measure the diaminopimelate epimerase activity of the cell extract by a known method (for example, see White, PJ et al., Biochem. J., 113, 589 (1969)). Can be performed.

The coryneform bacterium of the present invention may have enhanced activity of other L-lysine biosynthetic pathways or glycolytic enzymes in addition to diaminopimelate epimerase activity. Examples of such an enzyme and a gene encoding the enzyme include an aspartokinase-substituted protein and / or a subunit protein from which synergistic feedback inhibition by L-lysine and L-threonine has been released. (W094 / 2 5605 international publication pamphlet), wild-type phosphoenolpyruvyl carboxylase derived from coryneform bacterium (Japanese Patent Application Laid-Open No. 60-87788), coryneform bacterium There are known a gene encoding a wild type dihydrodipicolinate synthase (Japanese Patent Publication No. 6-55149), a dihydrodipicolinate reductase gene (Japanese Patent Application Laid-Open No. 7-75578), and the like. Enhancement of these enzyme activities can be carried out in the same manner as enhancement of the activity of diaminobimelinate epimelase.

L-Lysine biosynthetic enzymes As described above, the coryneform bacterium which has been strongly enhanced includes a gene encoding aspartokinase, which is free from the L-lysine and L-threonine-induced feedback damage. Mutant lysC), dihydrodipicolinate reductase gene (dapB), dihydrodipicolinate synthase gene (dapA), diaminonopimelate decarboxylase gene (lysA), and diaminopimelate dehydrogenase Zes gene (ddh) (W096 / 40934), LysA and DDH (Japanese Patent Publication No. 9-322774), LysC, LysA and phosphoenorubyruvate carboxylase gene (ppc) (Kaihei 10-165180), mutant lysC, dapB, dapA, lysA and aspartate aminotransferase gene (aspC) (Japanese Patent Application Laid-Open No. 10-215883). It is shown.

Brevipacterium strain AJ12691, a strain in which the plasmid p399AK9B containing the mutant lysC was introduced into the AJ12036 strain (FERM BP-734), which is a wild type strain of Lactofamentum, was traded on April 10, 1992. Deposited with the Research Institute of Biotechnology, Industrial Technology Institute (Ministry of Industry) (zip code: 30-5-8566, 1-3-1, Tsukuba, Higashi, Ibaraki, Japan) under the accession number FERM P-12918. It was transferred to the International Deposit under the Budapest Treaty on October 10 and deposited under the accession number FERM BP-4999.

The transformed AJ13107 strain obtained by introducing the plasmid pCRDAPB containing dapB into the Escherichia coli JM109 strain has been used since May 26, 1995 by the Institute of Life Science and Industrial Technology, the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry. (Postal code 305-8566, 1-3-1 Tsukuba, Ibaraki, Japan) with accession number FERM ΒΡ-5Π4, deposited internationally under the Budapest Treaty.

The transformed AJ13106 strain obtained by introducing the plasmid pCRDAPA containing dapA into the Escherichia coli JM109 strain has been used since May 26, 1995 by the Institute of Life Science and Industrial Technology, the Ministry of Economy, Trade and Industry of the Ministry of International Trade and Industry, Japan. It has been deposited internationally under the Budapest Treaty under the postal code 305-8566, under the accession number ^ FERM BP-5113 13 in Tsukuba, Ibaraki Prefecture, Japan.

Mutant lysC, da _P B, each gene dapA can it to prepare by conventional methods from the deposited strains. In addition, lysA is a primer obtained from a chromosomal DNA of a coryneform bacterium, for example, Brevipacterium 'Lactofamentum wild ATCC13869 strain, having an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 or 4 in the sequence listing. As a result, the DNA fragment can be obtained as a DNA fragment containing argS and lysA encoding arginyl-tRNA synthase and a promoter of an operon containing these.

The above-mentioned L-lysine synthesis enzyme gene and dapF 遗 gene may be carried on one vector and may be carried on two or more vectors separately.

In addition, the coryneform bacterium of the present invention diverges from the L-lysine biosynthesis pathway to produce L-lysine. The activity of an enzyme that catalyzes a reaction for producing a compound other than gin may be reduced or lacking. Homoserine dehydrogenase is an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway (see WO95 / 23864).

In the present specification, “enhanced activity” of an enzyme generally means that the enzyme activity in a cell is higher than that of a wild-type strain. When a strain whose enzyme activity is enhanced by the modification is obtained, it means that the enzyme activity in the cell is higher than that of the strain before the modification. The term “reduced activity” of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain, and the enzyme activity has been reduced by modification by genetic recombination technology or the like. When a strain is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.

3> L-Lysine production

When a coryneform bacterium having enhanced diaminopimelate epimelase activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine accumulates in the medium. The medium used for producing L-lysine using the microorganism of the present invention is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, other organic micronutrients. Carbon sources include carbohydrates such as glucose, lactose, galactose, fructos, sucrose, molasses, starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, and citric acid. And organic acids such as succinic acid.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, and other inorganic ammonium salts, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'stip' liquor, soybean hydrolysis Organic nitrogen, ammonia gas, aqueous ammonia, etc., can be used.

As inorganic ions, potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. are added in small amounts. It is preferable to add the required ingredients such as vitamins and / or the extract as necessary as a special ingredient II.

Cultivation is performed under aerobic conditions such as shaking culture and aeration and stirring culture under cows for 16 to 72 hours. The culture temperature is controlled at 30 ° C; ~ 45, and the pH is controlled at 5 ~ 9 during the culture. For pH adjustment, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used.

The collection of L-lysine from the fermentation broth can usually be carried out by a combination of an ion exchange resin method, a precipitation method and other known methods. Example

Hereinafter, the present invention will be described more specifically with reference to examples.

1> Isolation of dapF gene from coryneform bacterium and production of dapF gene transfer plasmid

Based on the known dapF gene sequence of Escherichia coli (Nucleic Acid Research, 16 (21), 10367 (1988)), the primers shown in SEQ ID NOs: 1 and 2 were prepared, and the chromosomal DNA of the Escherichia coli JM109 strain was prepared. PCR was performed using the DNA as a type II to obtain a dapF gene fragment. The DNA was synthesized using a DNA synthesizer model 380B manufactured by Applied Biosystems, using the phosphoramidite method (see Tetrahedron Letters (1981), 22, 1859) according to a conventional method. The PCR reaction was carried out using a DNA thermal cycler model PJ2000 manufactured by Takara Shuzo Co., Ltd., using Taq DNA polymerase according to the method specified by the supplier.

To introduce the amplified gene fragment into a coryneform bacterium, the DNA fragment was connected to a shuttle vector pVK7 (see JP-A-10-215883). The amplified fragment was ligated with pVK7, which had been digested with BamHI, treated with T4 polymerase and blunt-ended. The DNA connection was performed by ffling a DNA reige kit (Takara Shuzo Co., Ltd.). The constructed plasmid was named pVKdapF. pVKdapF retains the kanamycin resistance gene as a marker.

pVK7 was introduced into PHSG299 (Km '; Takeshita, S. et al., Gene, 61. 63-74, (1987)), a vector for Escherichia coli, as follows. It was constructed by linking pAM330, Mentum's cryptoplasmid. PAM330 was prepared from Blepino teratium lactofermentum ATCC13869 strain. Avail (Takara Shuzo Co., Ltd.) And then blunt-ended with T4 DNA polymerase, then cut with Hindlll (Takara Shuzo Co., Ltd.), and connected to PAM330 blunt-ended with T4 DNA polymerase. . The two kinds of generated plasmids were named pVK6 and pVK7 according to the insertion direction of pAM330 into pHSG299. pVK7 is capable of autonomous replication in cells of E. coli and Brevipacterium 'Lactofamentum, and has a multiple cloning site derived from pHSG299 and lacZ'.

2> Introduction of plasmid with dapF into Brevibacterium lactofermentum L-lysine-producing bacteria

(1) Acquisition of lysA and production of plasmid containing it

Chromosomal DNA was prepared from Brevibacterium 'lactofermentum wild strain ATCC13869 according to a conventional method. From the chromosomal DNA, a DNA fragment containing argS, lysA and the promoter of the operon containing them was amplified by PCR. DNA primers used for amplification include sequences known from Corynebacterium gullica micam (Molecular Microbiology 4 (11), 1819-1830 (1990), Molecular and General Genetics 212, 112-119 (1988)) to amplify a region of about 3.6 kb encoding arginyl-tRNA synthase and diaminopimelic acid decarboxylase, each having the nucleotide sequence of SEQ ID NO: 3 or 4 in the sequence listing. 23mer synthetic DNA was used. The DNA was synthesized by a phosphoramidite method using a DNA synthesizer model 380B manufactured by Applied Biosystems (see Tetrahedron Letters (1981), 22, 1859) according to a conventional method. In addition, the PCR reaction was performed using a DNA thermal cycler PJ2000 manufactured by Takara Shuzo Co., Ltd., using Taq DNA polymerase according to the method specified by the supplier. PHSG399 was used as a vector for cloning the amplified 3579 bp gene fragment. pHSG399 was digested with restriction enzyme Smal (Takara Shuzo Co., Ltd.) and ligated with the amplified DNA fragment containing lysA. The plasmid having lysA derived from ATCC13869 thus obtained was named p399LYSA.

Furthermore, lysA-containing DNA fragment was extracted by cutting p399LYSA with Kpnl (manufactured by Shushou Co., Ltd.) and BamHI (manufactured by Shuzo Co., Ltd.). This DNA fragment was ligated with a fragment obtained by cutting pHSG299 with Kpnl and BamHI. The resulting plasmid was named p299LYSA. Figure 1 shows the process of construction of p299LYSA. (2) Preparation of plasmid having both mutant lysC, dapA and dapB

W096 / 40934 International publication pamphlet Brevibacterium 'plasmid containing dapA derived from lactofamentum pCRDAPA is digested with Kpnl and EcoRI, a DNA fragment containing dapA is extracted, and vector plasmid pHSG399 is extracted. Was cut with Kpnl and EcoRI. The obtained plasmid was designated as P399DPS. p399D PS was digested with EcoRI and Sphl and blunt-ended, and then a dapA fragment was extracted. This fragment was ligated with blunt-ended plasmid p399AK9 containing mutant lysC derived from Brevipacterium lactofermen described in W096 / 40934 International Publication, which was cut with Sail and blunt-ended. A plasmid P399CA in which mutant lysC and dapA coexist was constructed.

The above plasmid pCRDAPA can be prepared from Escherichia coli AJ13106 (FERM BP-5113) by a conventional method. P399AK9 cuts p399AK9B with BamHI, cuts out a DNA fragment capable of autonomously replicating plasmid in coryneform bacteria (hereinafter referred to as “Brevi.-ori”), and performs self-ligation. In some cases, it can be obtained. P399AK9B can be prepared from Escherichia coli AJ12691 (FERM BP-4999) by a conventional method.

Next, the plasmid pCRDAPB having the dapB from Brevipacterium / Lactofamen described in W096 / 40934 International Publications was cut with EcoRI, blunt-ended, and cut with SacI. A 2.0 kb DNA fragment containing dapB was extracted. Plasmid p399CA containing dap A and mutant lysC is cut with Spel, blunt-ended, cut with Sacl, and connected to the extracted dapB fragment to obtain a plasmid containing mutant lysC, dapA and dapB Was. This plasmid was named P399CAB. pCRDAPB can be prepared from Escherichia coli AJ13107 (FERM BP-5114) by a conventional method. Next, Brevi .-ori was introduced into p399CAB. A plasmid pHK4 having Brevi. -ori derived from pHM1519 was cut with a restriction enzyme BamHI (manufactured by Shuzo (ft)), and the cut surface was blunt-ended. 'The blunt ends were prepared using the DNA Blunting kit (' ίϊShinzo Co., Ltd.) according to the designated method. After blunting, phosphorus? ! A modified Kpnl linker (Takara Shuzo Co., Ltd.) was connected, and modified from PHK4 so that the DNA fragment of the Brevi. -Ori portion was cut out only by cutting with Kpnl. This plasmid was cut with Kpnl and the resulting Brevi. The -ori DNA fragment was ligated to p399CAB also digested with Kpnl, and a plasmid capable of autonomous growth in coryneform bacteria and having mutant lysC, dapA and dapB was prepared, and named pCAB. Figure 2 shows the process of pCAB construction.

(3) Preparation of plasmid having both mutant lysC, dapA, dapB and lysA

Plasmid P299LYSA containing lysA was cut with Kpnl and BamHI, blunt-ended, and then a lysA fragment was extracted. This fragment was ligated with pCAB cut with Hpal (manufactured by Takara Shuzo Co., Ltd.) and blunt-ended. The plasmid was capable of autonomous growth in coryneform bacteria and had mutant lysC, dapA, dapB, and lysA. And was named pCABL. pCABL carries the chloramphenicol resistance gene as a marker. Figure 3 shows the process of pCABL construction. In pCABL, the lysA fragment was inserted into the Hpal site in the DNA fragment containing dapB, but this Hpal site was located upstream from the dapB promoter, and dapB was fragmented. Absent.

(4) Preparation of coryneform bacterium L-lysine-producing bacterium transformed with dapF gene The plasmid pCAB or pCABL prepared as described above is used for brevipacterium.

AJ11082 (NRRL B-11470), which is a lactofarmentum L-lysine producing bacterium, was transformed to obtain AJ11802 / pCAB and AJ11802 / pCABL. The AJ11082 strain has AEC resistance.

Next, the transformants AJ11802 / pCAB and AJ11802 / pCABL were transformed with pVKdapF. While pCAB and pCABL use the PHM1519-derived origin as the replication origin in Brevibacterium 'lactofermentum cells and use the chloramphenicol resistance gene as a marker gene, pVKdapF uses Brevibacterium • Both plasmids are stable in Brevibacterium 'lactofermentum cells because the pAM330-derived origin is used as the origin of replication in lactofermentum cells and the kanamycin resistance gene is used as a marker. Is held. In this way, AJ11082 / pCAB / pVKdapF and AJ11082 / pCABL / pVKdapF in which a plasmid containing the L-lysine biosynthetic gene and a plasmid containing the dapF gene coexisted were obtained. <3> Determination of diaminopimelate epimerase activity of each transformant;

The diaminopimelinate epimeridase activity of AJ11082, AJ11082 / pCABL and AJ11082 / pCABL / pVKdapF was measured. Measurement of diaminopime phosphate epimerase activity White, PJ et al., Biochem. J .. 113, 589 (1969)). The flask culture solution about 10 ml, after removing the 20-second centrifugation CAC0 ₃ at 1500 rpm, cells were harvested by centrifugation 6 min the supernatant with 3000 rpm. The obtained cells were washed twice with 200 mM potassium phosphate buffer (pH 6.9) (P Buf fer), suspended in KP Buffer 300-1 and sonicated. After the crushed solution was centrifuged at 3000 rpm for 10 minutes, the supernatant was used as a crude enzyme solution, and the diaminobimellinate epimerase activity was measured. Table 1 shows the results. In the table, lysC * represents a mutant lysC gene. In addition, the diaminopime phosphate epimerase activity was shown as a relative value when the activity of AJ11082 was set to 1. Table 1. Strain / plasmid transgenes Merinjech. Melase, 'active

(Relative value)

AJ11082 1.0

AJ11082 / pCAB lysC *, dapA, dapB, lysA 1.2

AJ11082 / pCABL lysC *, dapA, dapB, lysA 0.9

AJ11082 / pCAB / pVKdapF lysC *, dapA, dapB, lysA, dapF 12.5

AJ11082 / pCABL / pVKdapF lysC *, dapA, dapB, lysA, dapF 14.8

4) Production of L-lysine

AJ11082, AJ11082 / pCAB, AJ11082 / pCABL, AJ11082 / pCAB / pVKdapF and AJ11082 / pCABL / pVKdapF were cultured in an L-lysine production medium, and the L-lysine production ability was evaluated. The composition of the L-lysine production medium is as shown below.

[: L-lysine production medium]

Dissolve the following components (in 1 L) except calcium carbonate, adjust to pH 8.0 with KOH, sterilize at 115 ° C for 15 minutes, and add 50 g of dry acid-killed ^ acid calcium. .

100 g glucose

(ΝΗ,) .SO. 55 g KH 2 P 0.1

Piotin 500 j g

Thiamine 2000 jug

Nicotinamide 5 m

Protein hydrolyzate (bean concentrate) 30 ml

50 g calcium carbonate

Each transformant and the parent strain were inoculated into a medium having the above composition, subjected to reciprocal shaking culture at 31.5 C, and cultured for 72 hours to measure the amount of L-lysine accumulated in the medium. Table 1 shows the results. It was confirmed that L-lysine production ability was improved in the amplified strain of diaminobimelinate epimelase activity. Table 2 Strain / plasmid transgene L-lysine production (g / L)

AJ11082 29.8

AJ11082 / pCAB lysC *, dapA, dapB 44.9

AJ11082 / pCAB / pVKdapF lysC *, dapA, dapB, lysA, dapF 46.4

AJ11082 / pCABL lysC *, dapA, dapB, lysA 46.3

AJ11082 / pCABL / pVKdapF lysC *, dapA, dapB, lysA, dapF 48.2

Industrial applicability

According to the present invention, the ability of coryneform bacteria to produce L-lysine can be improved, and a method for producing L-lysine with high efficiency can be provided.

Claims

The scope of the claims

1. Coryneform bacterium having enhanced diaminopimelate epimerase activity in cells and L-lysine-producing ability.

2. The coryneform bacterium according to claim 1, wherein the enhancement of diaminopimelate epimerase activity is caused by increasing the copy number of a gene encoding diaminopimelate epimerase in the bacterial cell.

3. The coryneform bacterium according to claim 2, wherein the gene encoding diaminopimelate shrimp is derived from a coryneform bacterium.

4. The coryneform bacterium according to claim 1, wherein at least one of aspartase kinase activity, dihydrodipicolinate reductase activity, dihydrodipicolinate synthase activity, and diaminobimelate decarboxylase activity is enhanced.

5. The coryneform bacterium according to any one of claims 1 to 4 is cultured in a medium, L-lysine is produced and accumulated in the culture, and L-lysine is collected from the culture. A method for producing L-lysine.