JPH0511959B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-lysine. More specifically, using a recombinant DNA that is a recombinant of a DNA fragment containing at least one gene of the present invention and a vector DNA, and in which at least one surface DNA is foreign to the host strain, Corynebacterium spp. The present invention relates to a method for producing L-lysine, which comprises culturing a transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Brevibacterium or Brevibacterium in a medium, and expressing the trait of the gene. Recombinant gene technology has been established using Escherichia coli as a host, and to date, somatostatin, insulin,
human growth hormone, human interferon-α,
It was shown that it is possible to produce peptides and vaccines such as human interferon-β and foot-and-mouth disease vaccines. Although Escherichia coli is considered to be sufficient as a host for the expression of these highly bioactive peptides and vaccines in many cases, higher productivity, extracellular secretion, and glycosylation are required, or the contamination of intracellular toxins is avoided. Therefore, yeast and Bacillus subtilis have also been developed as hosts. When producing biologically active substances such as peptides and proteins, it is sufficient to use strains for which recombinant DNA techniques have already been established or the foundations for such techniques are in place. When using recombinant DNA technology to improve the industrial productivity of substances, it is necessary to devise ways to apply the same technology to each of the conventionally used producing bacteria. Corynebacterium glutamicum was the first microorganism used for the industrial production of amino acids, and since then Coryneform bacteria including Corynebacterium have been used to produce glutamic acid, lysine, alanine, histidine, tryptophan, tyrosine, phenylalanine, Industrial production of amino acids such as threonine, isoleucine, valine, leucine, glutamine, proline, and arginine has been developed, and today most amino acids are produced by microorganisms. Therefore, the establishment of recombinant DNA techniques in these microorganisms is considered to be extremely important for improving amino acid production in the future. Recombinant DNA techniques include, for example, (1) fragmentation of DNA containing the target gene with restriction enzymes, (2) linearization by single cleavage of vector DNA with the same restriction enzyme, and (3) above (1) and (2). Creation of recombinant DNA by annealing by mixing the products and ligation using DNA ligase (4) Introduction of the above recombinant DNA into the host strain (transformation) (5) Selection of recombinant containing the target gene and each step of purification of the selected clone. The efficiency of creating a recombinant strain obtained in this way can be said to be the product of each of the above steps, so it is important to prepare a means to verify each step, know the efficiency of each step, and improve it. It is not possible to obtain a transformed strain capable of expressing the target gene. Furthermore, even if a transformed strain containing a recombinant DNA containing the target gene is obtained in this way, there are various barriers to expressing the gene if the gene is foreign to the host strain. It is known that [“Chemistry and Biology” 18 ,
110-118 (1978)] It is extremely difficult to induce its expression. A microorganism belonging to the genus Corynebacterium or Brevibacterium is used as a host, and a recombinant DNA containing a target gene or vector that is foreign to the host is introduced into the host to express the trait of the target gene. No such example is known until now. In recombinant DNA techniques using microorganisms belonging to the genus Corynebacterium or Brevibacterium as hosts, vector systems that replicate autonomously in these microorganisms, have selectable phenotypes, and can be used for cloning many genes. It is necessary to construct a transformer and establish an efficient transformation system. Furthermore, it is necessary to establish a method to eliminate the above-mentioned barriers. The present inventors have previously constructed a plasmid vector that autonomously replicates in microorganisms belonging to the genus Corynebacterium or Brevibacterium and has a selectable phenotype and an appropriate cloning site, while developing a highly efficient transformation system. Developed [Special application 1984-
58186 (Japanese Patent Publication No. 57-183799), No. 56-58187 (Japanese Patent Publication No.
57-186492), 56-65777 (Japanese Patent Publication No. 57-186489)].
Therefore, the present inventors used the already known in vitro DNA recombination technique (US Patent 4237224) to ligate a DNA fragment containing a foreign gene involved in amino acid biosynthesis to the plasmid vector, and developed a transformation method. When Corynebacterium glutamicum strain L-22 or its derivative strain was transformed using the system, the foreign gene was expressed in the host, and amino acids, especially L-
The present inventors have discovered that the present invention can be used to increase the production of lysine, and have completed the present invention. The present invention will be explained in detail below. The present invention relates to DNA containing at least one gene.
A recombinant of the fragment and vector DNA, and both
A transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using a recombinant DNA in which at least one of the DNAs is foreign to the host strain is cultured. The present invention provides a method for producing L-lysine by culturing L-lysine and expressing the trait of the gene. DNA fragments containing genes used in the present invention include DNA fragments derived from eukaryotes, prokaryotes, viruses, bacteriophages, or plasmids and containing at least one complete gene.
Examples of genes derived from eukaryotes include genes encoding peptides such as interferon, insulin, and growth hormone in mammals, particularly humans. Genes derived from prokaryotes include those derived from strains of bacteria, particularly those belonging to the genera Etschierichia, Corynebacterium, Brevibacterium, Bacillus, or Staphylococcus, and are important for cell metabolism, especially synthetic activity. Examples include genes involved. Cellular metabolic or synthetic activity refers to the synthesis of amino acids, vitamins, nucleic acids, or antibiotics, as well as metabolic systems involved in the synthesis. Activity is preferably mentioned. In addition, when the amino acid composition of the target peptide, protein, etc. is known, the corresponding DNA
can also be synthesized and used. Examples of DNA synthesis methods include K. Itakura et al, Science 198 ,
1056 (1977). The vector used in the present invention must be compatible with the host bacteria and capable of autonomous replication. As a specific example, the present inventors have collected from a microorganism belonging to the genus Corynebacterium, or created by inducing the collected material.
pCG1 [Patent application 1984-18101 (JP 57-134500)],
pCG2 [Patent application 1984-133557 (Japanese patent application 1982-35197)],
pCG4 [Patent application 1986-58186 (Japanese patent application 1983-183799)],
Examples include pCE53, pCE54, pCG11, pCB101, and pEthr1. Bacterial strains carrying these plasmids have been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and the American Type Culture Institute, USA, with the following deposit numbers.
Deposited in a collection. Plasmid FERM-P ATCC pCG1 5865 31808 pCG2 5954 31832 pCG4 5939 31830 pCE54 − 39019 pCG11 − 39022 pCB011 − 39020 pEthr1 − 39021 Preferably, pCG11 and pCE54 are used. pCG11
is a plasmid previously disclosed by the present inventors [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)], and is a plasmid that was previously disclosed by the present inventors, and is a plasmid that was previously disclosed by the present inventors.
Plasmid isolated from FERM-P5865)
Corynebacterium glutamicum 225 appears at the only cleavage site of the restriction enzyme Bgl in pCG1.
-250 (ATCC31830, FERM-P5939) of streptomycin and/or spectinomycin resistance (Sm ^R /
This is a plasmid in which the BamH fragment containing the Spec ^R ) gene is joined using the same cohesive ends of both. pCG11 is a plasmid with a molecular weight of approximately 6.8 Kb and has Bgl and Pst as single restriction sites ^.
Gives the Spec ^R phenotype. pEC54 can be created as follows. First, pCG2 was extracted from Corynebacterium glutamicum strain 225-218 (FERM-P5954,
Patent application No. 56-133557 from cultured bacterial cells of ATCC31832)
(Japanese Unexamined Patent Publication No. 58-35197)
is concentrated and isolated from cultured E. coli cells by a commonly used method. Both plasmid DNAs are cut with a restriction enzyme that has one cut point in each molecule, such as Pst.
After complete digestion and linearization with
T4 phage DNA ligase is applied to generate a conjugated molecule of DNA molecules. this
To obtain a recombinant plasmid in which both plasmid molecules are joined together from a DNA mixture, once
This is achieved by isolating transformed strains of Corynebacterium or Brevibacterium species selected for drug resistance derived from pGA22, and analyzing the plasmids possessed by these transformed strains. Transformation with a DNA hybrid is carried out by the transformation method using protoplasts of Corynebacterium and Brevibacterium species previously disclosed by the present inventors [Patent Application No. 58187/1983
186492) and patent application No. 186492) and patent application No. 186492
186489)]. The drugs used for selection are tetracycline (Tc), which corresponds to the drug resistance genes derived from pGA22, excluding the ampicillin resistance gene, which is inserted and inactivated because it serves as a connection site with pGA22;
Chloramphenicol (Cm) or kanamycin (Km) may be used. The transformed strain is
Drugs at concentrations that do not allow recipient protoplasts to revert to normal cell proliferation in a DNA-free system (usually tetracycline 0.4-1.6 μg/ml, chloramphenicol 2.5-5 μg/ml, and kanamycin 100-800 μg/ml)
Either isolate the reverting colonies on a hypertonic agar medium containing 100 g/ml), or scrape them after non-selectively reverting them to normal cells on a regeneration medium, and use this resuspension to collect the recipient normal cells. drug at a concentration that does not allow for growth (usually tetracycline 0.5-4μ)
g/ml, 2-15 μg/ml of chloramphenicol and 2-25 μg/ml of kanamycin). Tetracycline, chloramphenicol or kanamycin resistance (Tc ^R , Cm ^R ,
Some of the transformed strains selected by Km ^R ) have also acquired other drug resistance traits derived from pGA22. The plasmid DNA possessed by the thus obtained transformed strain was obtained by the present inventors in Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Application No. 57-134500) and Japanese Patent Application No. 56-65777 (Unexamined Japanese Patent Application No. 56-65777)
It can be isolated and purified from cultured bacterial cells by the method disclosed in 57-186489), and its structure can be determined by a conventional method of analyzing DNA fragments produced by digestion with various restriction enzymes by agarose gel electrophoresis. The plasmid pCE54 was isolated from one of the transformed strains. pCE54 is a plasmid approximately 14.5 Kb in size with single restriction sites including EcoR, Sal, Sma, and
etc., giving the Tc ^R , Cm ^R , and Km ^R phenotypes. Xho is located in the Km ^R gene, and selection by so-called insertional inactivation (a phenomenon in which expression of the relevant phenotype is prevented by insertion of a DNA fragment) is also possible. Collection of plasmids from plasmid-carrying strains is described in, for example, Japanese Patent Application No. 56-18101
134500), No. 56-58186 (Japanese Unexamined Patent Publication No. 57-183799) and No. 56-133557 (Japanese Unexamined Patent Publication No. 58-35197). Preparation of a recombinant between a DNA fragment containing a gene and vector DNA can be carried out by making full use of known in vitro recombinant DNA techniques. In vitro DNA recombination is usually performed by cutting and recombining donor DNA containing the gene of interest and vector DNA. DNA can be easily cut using restriction enzymes. Restriction enzymes used for in vitro recombination can be used for all double-stranded enzymes, regardless of biological species.
Recognizes and cleaves specific base sequences on DNA.
The base sequence differs depending on the type of restriction enzyme. Therefore, by using appropriate restriction enzymes, the gene of interest can be isolated without impairing its expression function.
It is excised as a DNA fragment. The end structures of donor DNA and vector DNA fragments cut by the same restriction enzyme have the same structure, with some restriction enzymes giving a cohesive end with a single strand protruding, and other restriction enzymes giving a cohesive end with a protruding single strand. , giving blunt ends. As long as both ends are cut with the same restriction enzyme, donor DNA cut fragments and vector DNA cut fragments are
Ligation can be performed using T4 phage DNA ligase. Even when both DNAs are cut with different restriction enzymes,
For example, the sticky ends can be repaired with DNA polymerase to make double strands, and the ends can be blunted before ligation, or a complementary homopolymer can be added with terminal transferase to create sticky ends before ligation, or Synthetic oligonucleotide linkers containing certain restriction enzyme cleavage sites can be ligated and then internally cut to create cohesive ends before ligation. Through these linking methods, DNA fragments containing the gene of interest and vector DNA are
A recombinant of the cut fragment is generated. In addition to the desired recombinant, other recombinants are generated by the ligase reaction, but in order to obtain the desired recombinant, this DNA mixture is used to directly incubate Corynebacterium or Brevibacterium species. This can be achieved by transforming, selecting and separating a transformed strain endowed with genetic traits derived from the genetic information of the gene of interest, and extracting and isolating it from the cultured bacterial cells. Instead of directly transforming the Corynebacterium or Brevibacterium species, the gene of interest is once cloned in the host vector system of another microorganism, such as E. coli, and then transformed into Corynebacterium or Brevibacterium species. A recombinant can be obtained by producing a recombinant with Pecter in a test tube, then transforming a Corynebacterium or Brevibacterium, and then selecting and separating the transformed strain in the same manner as above. can. For recombinant production, the descriptions in the following documents can be widely applied. SNCohen, et al, USPatent 4237224, Genetic Manipulation Experimental Method [edited by Yasutaka Takagi, Kodansha Sun Enteifutsuk (1980)], Method in
Enzymology 68 , Recombinant DNA edited
by Ray Wu, Academic Press 1979 The host microorganism of the present invention includes DNA belonging to the genus Corynebacterium or Brevibacterium.
Any strain may be used as long as it has uptake ability. Preferably, the present inventors first filed a patent application in 1983.
-151464 (Japanese Unexamined Patent Publication No. 58-56678) uses a lysozyme-sensitive microorganism. Specific examples of bacterial strains include the following strains.

[Table] Transformation of host microorganisms with recombinant DNA
(1) Preparation of protoplasts from cultured cells, (2) Transformation treatment of protoplasts with recombinant DNA,
(3) It is carried out in a process consisting of regeneration of protoplasts into normal cells and selection of transformed strains. Examples of specific methods are shown below. (1) Preparation of protoplasts from cultured cells Protoplast formation is achieved by growing microorganisms under conditions that make them sensitive to the cell wall lytic enzyme lysozyme, and then treating the cultured cells with lysozyme in a hypertonic solution to dissolve and remove the cell wall. It will be done. Various cell wall synthesis inhibitors are used to convert microorganisms into lysozyme-sensitive cells. For example, microbial cells can be made sensitive to lysozyme by adding penicillin at a concentration that does not inhibit or semi-inhibit growth in the middle of the logarithmic growth phase of microbial culture, and then propagating the culture for several generations. The medium used at this time may be any medium that allows microorganisms to grow, such as nutrient medium NB (medium containing 20 g of powdered bouillon and 5 g of yeast extract in 1 part of pure water, adjusted to pH 7.2) or semi-synthetic medium SSM [glucose 10g, NH ₄ Cl4g, urea 2
g, yeast extract 1g, KH ₂ PO ₄ 1g, K ₂ HPO ₄ 3
g, _MgCl2・_6H2O0.4g , _FeSO4・_7H2O10mg ,
MnSO ₄・4～6H ₂ O0.2mg, ZnSO ₄・7H ₂ O0.9
mg, CuSO ₄・5H ₂ O 0.4 mg, Na ₂ B ₄ O ₇・
10H ₂ O 0.09 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 0.04 mg,
30 μg of biotin, 1 mg of thiamine hydrochloride in 1 water
A medium containing 100% of the total amount of phthalate and adjusted to a pH of 7.2] is used. Microorganisms are inoculated into this medium and cultured with shaking. The absorbance (OD) at 660 nm was measured using a colorimeter to determine the initial phase of logarithmic growth (OD=0.1~
0.4), add penicillin such as penicillin G to a concentration of 0.1 to 2.0 units/ml in the culture solution. The culture is continued further, and when the OD increases to 0.3 to 0.5, the cells are harvested and washed with SSM medium. The cells are then placed in a suitable hypertonic medium, e.g.
PFM medium (sucrose 0.4M in 2-fold dilution of SSM,
Medium containing 0.01M MgCl ₂ 6H ₂ O and adjusted to pH 7.0 to 8.5) or RCG medium [glucose 5
g, casein hydrolyzate 5g, yeast extract 25g
g, K ₂ HPO ₄ 3.5 g, KH ₂ PO ₄ 1.5 g,
_MgCl2・_{6H2O0.41gFeSO4}・_{7H2O10mg ,}
MnSO ₄・4～6H ₂ O2mg, ZnSO ₄・7H ₂ O0.9mg,
CuSO ₄・5H ₂ O0.4mg, Na ₂ B ₄ O ₇・10H ₂ O0.09
mg _{, (NH4)6Mo7O24 ・ 4H2O0.04mg , biotin}
30 μg, 2 mg of thiamine hydrochloride, and 1.35 g of disodium succinate in 1 part water and resuspended in a medium adjusted to pH 7.0 to 8.5. Lysozyme is added to this cell suspension at a final concentration of 0.2 to 10 mg/ml and reacted at 30 to 37°C. Protoplast formation progresses as the reaction time progresses, and its progress can be observed using an optical microscope. The time required for most cells to become protoplasts under a microscope varies depending on the concentration of penicillin added during cell culture and the concentration of lysozyme used.
It is 3 to 24 hours under the above conditions. Since the generated protoplasts rupture and die under hypotonic conditions, the degree of protoplast formation can be indirectly determined by the degree of survival of normal cells that survive under hypotonic conditions. Normally, normal cells can be suppressed to a residual level of about 10 ^-4 of normal cells treated with lysozyme. The protoplasts prepared in this manner have the ability to form colonies (regenerate) on an appropriate hypertonic agar medium. This agar medium is preferably a nutrient medium, a semi-synthetic medium, or a synthetic medium supplemented with several types of amino acids containing 0.3-0.8M disodium succinate and 0.5-6% polyvinylpyrrolidone (molecular weight 10,000 or 40,000). used. Usually, semi-synthetic medium RCGP medium [RCG medium with 3% polyvinylpyrrolidone (molecular weight 10,000)]
and 1.4% agar-added medium, PH7.2] can be used. Cultivation is preferably carried out at 25-35°C. The number of culture days required for the appearance of regenerated colonies varies depending on the strain, but it takes 10 to 14 days for the colonies to reach a size that can be harvested. The regeneration of protoplasts in RCGP medium varies depending on the bacterial species, the concentration of penicillin added during the culture, and the concentration of lysozyme treatment, but the efficiency is 10 ^-2 to 10 ^-4 per normal cell sample treated with lysozyme. (2) Transformation of protoplasts with recombinant DNA In order to incorporate recombinant DNA into protoplasts, protoplasts and recombinant DNA are mixed in a hypertonic solution that allows cells to maintain a protoplast state, and then the recombinant DNA is mediated by DNA uptake. Active polyethylene glycol (PEG, average molecular weight
1540-6000) or polyvinyl alcohol (PVA, degree of polymerization 500-1500) and divalent metal cations. Stabilizers that provide hypertonic conditions may be those commonly used to maintain protoplasts of microorganisms, such as sucrose or disodium succinate. Usable concentration ranges for PEG and PVA are 5-60% final concentration and 1-60%, respectively.
It is 20%. Divalent metal cations have a final concentration of 1~
100mM Ca ⁺⁺ , Mg ⁺⁺ , Mn ⁺⁺ , Ba ⁺⁺ , Sr ⁺⁺
These are effective and can be used alone or in combination. The temperature of the treatment is preferably 0 to 25°C. (3) Regeneration of protoplasts back to normal cells and selection of transformed strains Regeneration of protoplasts transformed with recombinant DNA contains disodium succinate and polypyrrolidone, as in the regeneration of protoplasts described above. This is carried out by spreading protoplasts on a hypertonic agar medium (for example, RCGP medium) and culturing them at a temperature at which normal cells can grow, generally 25 to 35°C. The transformed strain is the donor
It can be obtained by selecting for traits imparted to bacteria by genes derived from DNA. Selection based on the acquisition of characteristic traits may be performed simultaneously with regeneration on a hypertonic agar medium, or it may be performed once on a non-selective regeneration and then the regenerated normal cells are collected and performed on an ordinary hypotonic agar medium. . When using a lysozyme-sensitive strain shown as a specifically preferred host strain in the present invention, the transformation is performed as described above except for directly treating cells that have been cultured and grown without penicillin treatment in step (1) above. It may be performed in the same manner as steps (1) to (3). When using lysozyme-sensitive microorganisms, transformed strains can be obtained at a high frequency of 10 ^-4 to 10 ^-6 per regenerating microorganism. The transformed strain can express the traits of the introduced recombinant DNA by culturing it in an ordinary nutrient medium. If the recombinant DNA has properties derived from gene DNA or vector DNA, the culture medium may be supplemented with drugs depending on the properties. Collection of useful substances such as amino acids produced by the method for expressing the characteristics of the present invention is carried out by a conventional method of collecting these substances from a fermentation liquid. The present invention has made it possible to increase or provide new productivity for amino acids, nucleic acids, vitamins, antibiotics, enzymes, peptides, and proteins in microorganisms of the genus Corynebacterium and Brevibacterium. It has also become possible to improve the production method by enhancing the metabolic activity of microorganisms, increasing their ability to utilize substrates, imparting new metabolic activities, and providing new substrate utilization. A further feature of the present invention is that a heterologous gene or exogenous recombinant DNA was successfully expressed in a microorganism of the genus Corynebacterium or Brevibacterium. Specifically, the threonine operon of Escherichia coli, the phosphoenolpyrupine carboxylase (PPC) gene, and the gene pUB110 expressed in Bacillus subtilis and Staphylococcus [Keggins KM, et al.,
Proc. Natl Acad. Sci., USA 75 , 1423 (1978)]
The kanamycin resistance gene of Corynebacterium glutamicum, the gene involved in lysine biosynthesis of Corynebacterium glutamicum, and the anthranilate synthase gene of Brevibacterium flavum were expressed in Corynebacterium spp. All of the exemplified genes were simply introduced in the form of a ligation to a Corynebacterium glutamicum plasmid, and no special manipulation was performed to express them in Corynebacterium glutamicum. Furthermore, no matter which direction the DNA fragment containing the gene is ligated to the Corynebacterium glutamicum plasmid, it will be expressed within Corynebacterium glutamicum. It is clear that it has the ability to accurately recognize the transcription/translation start point of a given gene and carry out transcription/translation. As is well known, all genes are accurately transcribed and
Considering that Corynebacterium glutamicum has similar sites at the base sequence level necessary for initiation of translation, Corynebacterium glutamicum also has transcription and translation initiation sites for genes other than the exemplified genes. It is easily inferred that it can be recognized and expressed. Although the so-called glutamate-producing bacteria that have a high glutamate production ability have the same main mycological properties, each researcher has given them various names due to their industrial importance, and the genus name is different. Even the genus Corynebacterium and Brevibacterium are diverse. However, it has been pointed out that these bacterial groups are the same bacterial species because the amino acid composition of their cell walls and the base composition of their DNA are uniform. Furthermore, it has recently been revealed that there is more than 70-80% DNA homology between these bacterial species, making it clear that they are very closely related microorganisms [Komatsu, Y.: Report of
the Fermentative Research Institute, No.55,
1 (1980), and Suzuki, K., Kaneko, T.,
and Komagata, K.: Int.J.Syst.Bacteriol., 31 ,
131 (1981)]. In this specification, since the hosts that can be used for recombinant DNA experiments are regulated, the usefulness of the present invention is limited to Corynebacterium glutamicum L.
-22 derivative strain was shown as a host, but based on the above facts, it can be easily inferred that this method can be applied to all glutamic acid producing bacteria as is. recombinant
In order for DNA to be stably retained and expressed in these bacterial species, slight differences in host bacterial properties such as DNA homology are not a problem, and these bacterial species are capable of autonomously replicating the plasmid and expressing the introduced gene. It suffices if it has a function that enables it. However, the fact that these bacterial species share both of these functions was previously disclosed by the present inventors [Patent Application No. 1983-
Plasmid pCG4, which is isolated from Corynebacterium glutamicum 225-250 and has a streptomycin and/or spectinomycin resistance gene, is used to isolate Corynebacterium and Brevibacterium species, etc. It can also be replicated within glutamate-producing bacteria,
In addition, the resistance gene is expressed [Patent Application 1986-
58187 (Japanese Unexamined Patent Publication No. 57-186492)]. Therefore, host bacteria to which the present invention can be applied include not only Corynebacterium glutamicum but also all glutamic acid-producing bacteria including Corynebacterium and Brevibacterium species. Examples of the present invention are shown below. Example 1 Cloning of a gene involved in lysine biosynthesis of the lysine-producing bacterium Corynebacterium glutamicum ATCC21543 in Corynebacterium glutamicum and production of lysine by Corynebacterium glutamicum using the expression of the gene: ( 1) Corynebacterium glutamicum
Preparation of ATCC21543 chromosomal DNA and vector pCG11: Corynebacterium glutamicum
Lysine-producing mutant Corynebacterium glutamicum derived from ATCC13032 and resistant to the lysine analog S-(2-aminoethyl)-cysteine (hereinafter abbreviated as AEC)
The chromosomal DNA of ATCC21543 is extracted and isolated as follows. 400ml semi-synthetic medium SSM [glucose 20g,
(NH ₄ ) ₂ SO ₄ 10g, urea 3g, yeast extract 1g,
KH ₂ PO ₄ 1g, MgCl ₂・6H ₂ O 0.4g, FeSO ₄・
_7H2O10mg , MnSO4・₄ ～ _6H2O0.02mg ,
ZnSO ₄・7H ₂ O0.9mg, CuSO ₄・5H ₂ O0.4mg,
Na ₂ B ₄ O ₇・10H ₂ O 0.09 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・
Medium containing 0.04 mg of 4H ₂ O, 30 μg of biotin, and 1 mg of thiamine hydrochloride in 1 part water and adjusted to pH 7.2]
The seed culture was inoculated into a medium supplemented with threonine at 100 μg/ml, and cultured with shaking at 30°C. The absorbance (OD) at 660 nm is measured using a Tokyo Photoden colorimeter, and when the OD reaches 0.2, penicillin G is added to the culture solution at a concentration of 0.5 units/ml. The culture is continued until the OD reaches approximately 0.6. Collect bacteria from the culture solution and add TES buffer [0.03M tris(hydroxymethyl)aminomethane (hereinafter abbreviated as Tris), 0.005M EDTA
After washing with 0.05M NaCl: PH8.0], lysozyme solution (25% sucrose, 0.1M NaCl, 0.05M Tris, 0.8
mg/ml lysozyme: PH8.0 or less) Suspend in 10 ml and react at 37°C for 4 hours. The method of Saito et al. [Saito, H. et al: Biochim.
Biophys. Acta, 72 , 619 (1963)]. On the other hand, used as a vector plasmid
pCG11 is a pCG11-carrying strain of the derived strain LA103 of Corynebacterium glutamicum L-22 strain.
It is isolated from LA103/pCG11 (ATCC39022) as follows. 400ml NB medium (medium containing 20g of powdered bouillon and 5g of yeast extract in 1 part water and adjusted to pH 7.2)
Culture with shaking at 30°C until the OD reaches approximately 0.7. After collecting the bacteria and washing with TES buffer,
Suspend in 10 ml of lysozyme solution and react at 37°C for 2 hours. 5M NaCl2.4ml, 0.5M to reaction solution
Add 0.6 ml of EDTA (PH 8.5), 4.4 ml of a solution consisting of 4% sodium lauryl sulfate and 0.7 M NaCl in sequence, mix gently, and then place in ice water for 15 hours. Transfer the entire lysate to a centrifuge tube and incubate at 4°C for 60 minutes.
Centrifuge at 69400×g and collect the supernatant.
Add polyethylene glycol (PEG) 6000 (manufactured by Hanui Chemical Co., Ltd.) equivalent to 10% by weight to this, mix gently to dissolve, and then place in ice water. Ten
After that time, collect the pellet by centrifugation at 1500 xg for 10 minutes. Add 5 ml of TES buffer to gently redissolve the pellet, then add 2.0 ml of 1.5 mg/ml ethidium bromide, add cesium chloride, and gently dissolve to adjust the density to 1580. This solution is subjected to ultracentrifugation at 105,000 xg and 18°C for 48 hours. The circular DNA covalently closed by density gradient centrifugation is found as a high-density band in the lower part of the centrifugation tube by UV irradiation. pCG11DNA is isolated by extracting this band from the side of the centrifuge tube with a syringe. The fractionated solution was then diluted with an equal volume of isopropyl alcohol solution [volume percentage 90% isopropyl alcohol, 10%
Ethidium bromide is extracted and removed by treatment five times with TES buffer (this mixture contains a saturated amount of cesium chloride), and then dialyzed against the TES buffer. (2) Corynebacterium glutamicum
Cloning of genes involved in lysine biosynthesis of ATCC21543 3 μg of pCG11 plasmid DNA prepared above
Reaction solution for restriction enzyme Bgl containing (10mM Tris-HCl, 7mM _MgCl2 , 60mM NaCl, 7mM
Add 6 units of Bgl (manufactured by Takara Shuzo Co., Ltd.) to 60μ of M2-mercaptoethanol, PH7.5, and hold at 37°C.
After reacting for 60 minutes at 65℃, stop the reaction by heating at 65℃ for 10 minutes. On the other hand, a restriction enzyme BamH reaction solution containing 8 μg of chromosomal DNA of Corynebacterium glutamicum ATCC21543 (10 mM Tris-HCl, 7
Add 4 units of BamH to 140μ of mM MgCl ₂ , 100mM NaCl, 2mM 2-mercaptoethanol, 0.01% bovine serum albumin, PH8.0), react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to react. to stop. Mix both digests, add 40μ of T4 ligase buffer (Tris-HCl 660mM, MgCl ₂ 66mM, dithiothreitol 100mM, PH7.6), ATP (5
Add 40μ of T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1 unit/μ) and _120μ of H2O,
Incubate for 16 hours at °C. The mixture is extracted twice with 400 μl of phenol saturated with TES buffer and dialyzed against TES buffer to remove the phenol. This ligase reaction mixture was derived from Corynebacterium glutamicum strain L-22.
Use for transformation of AEC-susceptible LP4 strain. Transformation is performed using protoplasts of the LP4 strain. Inoculate the seed culture of LP4 strain into NB medium and culture with shaking at 30°C. Collect bacteria when the OD reaches 0.6,
The cells were cultured in RCGP medium [glucose 5g, casamino acids 5g, yeast extract 2.5g, K ₂ HPO ₄ 3.5g,
KH ₂ PO ₄ 1.5g, MgCl ₂・6H ₂ O 0.41g,
_FeSO4・_7H2O10mg , MnSO4・₄ ~ _6H2O0.2
mg, ZnSO ₄・7H ₂ O 0.9 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・
4H ₂ O 0.04mg, biotin 30μg, thiamine hydrochloride 2mg, disodium succinate 135g, polyvinylpyrrolidone (molecular weight 10000) 30g in water 1
Suspend the cells in a solution containing 1 mg/ml lysozyme (PH7.6) to approximately ¹⁰⁹ cells/ml,
Transfer to an L-shaped test tube and perform a gentle shaking reaction at 30°C for 5 hours to form protoplasts. Transfer 0.5 ml of this protoplast bacterial solution to a small test tube, centrifuge at 2500 x g for 5 minutes, and add TSMC buffer (10 mM magnesium chloride, 30 M calcium chloride, 50 mM Tris, 400 mM sucrose, PH
7.5) Resuspend in 1 ml, centrifuge and wash, then resuspend in 0.1 ml of TSMC buffer. To this bacterial solution, add 100μ of a 1:1 mixture of twice as concentrated TSMC buffer and the above ligase reaction DNA mixture and mix.
Next, 0.8 ml of a solution containing 20% PEG6000 in TSMC buffer is added and mixed. 3 minutes later, RCGP
Add 2 ml of medium (PH7.2), centrifuge at 2500 xg for 5 minutes to remove the supernatant, suspend the precipitated protoplasts in 1 ml of RCGP medium, and then add 0.2 ml to spectinomycin at 400 μg/ml.
(RCGP medium containing 1.4% agar, pH 7.2) and cultured at 30°C for 7 days. Collect all the bacteria grown on the agar medium, wash with physiological saline, and suspend in 1 ml of physiological saline. This bacterial solution was mixed with threonine 2mg/ml, AEC2
mg/ml and streptomycin 12.5 μg/ml
Minimal agar medium M1 containing the equivalent of glucose 10
g, NH ₄ H ₂ PO ₄ 1 g, KCl 0.2 g, MgSO ₄ .
_7H2O0.2g , _FeSO4・_7H2O10mg , _MnSO4・4
~ _6H2O0.2mg , _ZnSO4・_7H2O0.9mg , _CuSO4・
5H ₂ O 0.4mg, Na ₂ B ₄ O ₇・10H ₂ O 0.09mg,
( _NH4 ) _6Mo7O24・_4H2O0.04mg , _{biotin 50μ}
2.5 mg of p-aminobenzoic acid, 1 mg of thiamine hydrochloride, and 16 g of agar in 1 part water and adjusted to pH 7.2] and cultured at 30°C for 3 days. Among the colonies that appear, strains resistant to AEC, spectinomycin, and streptomycin are obtained. The plasmids possessed by these transformed strains are isolated by the same method as that used to isolate pCG11 described above. Using 1 μg of these plasmid DNA, use a restriction enzyme with a cutting site on pCG11.
After complete digestion with EcoR, it was analyzed by agarose gel electrophoresis, and the molecular weight was determined from the sum of the generated fragments. The molecular weight is the restriction enzyme Hind of lambda phage DNA that was run simultaneously on the same agarose gel.
The calculation is based on a standard curve drawn by the migration distance of each fragment of known molecular weight produced by digestion. Plasmid pAec5 obtained from one of the transformed strains has a molecular weight of 10.7 Kb and is located at the Bgl cleavage site of pCG11.
This is a recombinant plasmid with a 3.9Kb DNA fragment inserted. LP4 using pAec5DNA in the same manner as above.
The protoplasts of the strain were transformed and the transformed strains were selected for spectinomycin resistance at the same time.
It possesses a plasmid identical to pAec5, which is determined by the cleavage pattern of EcoR, which confers AEC resistance. That is, pAec5 has Corynebacterium
It is clear that the gene governing the AEC resistance trait of C. glutamicum ATCC21543 has been cloned. The pAec5-carrying strain is included in the American Type Culture Collection.
Corynebacterium glutamicum K17
Deposited as ATCC39032. (3) Lysine Production by pAec5-Holding Strain A lysine production test was conducted using a pAec5-harboring strain (ATCC39032) and a non-harboring strain of LP4 strain derived from Corynebacterium glutamicum L-22 strain. Bacteria grown on NB agar medium were added to 5 ml of production medium P1 [glucose 100 g,
(NH ₄ ) ₂ SO ₄ 24.5g, KH ₂ PO ₄ 1g, MgSO ₄・
_7H2O0.4g , _FeSO4・_7H2O10mg , _MnSO4・4
~6H ₂ O 10mg, biotin 50μg, thiamine hydrochloride 200μg, calcium pantothenate 500μg,
The cells were inoculated into a test tube containing a medium containing 500 μg of nicotinic acid, 10 g of soybean hydrolyzate, and 30 g of calcium carbonate in 1 part water and adjusted to pH 7.2, and cultured with shaking at 30°C for 75 hours. After culturing, the amount of L-lysine produced in the medium was measured by a colorimetric method using a fermentative-copper ninhydrin reaction, and the results are shown in Table 1.

[Table] Reference example 1 Cloning of genes involved in threonine biosynthesis in E. coli and production of threonine by Corynebacterium glutamicum using the expression of the genes: (1) DNA containing the E. coli threonine operon
Cloning of the fragment and introduction into Corynebacterium glutamicum: Cloning is performed in the E. coli host vector system. pGA22 used as a vector was prepared by the method used by An et al. who produced this plasmid [An.G. et al: J. Bacteriol., 140 , 400 (1979)].
According to E. coli K-12 carrying this plasmid,
Isolate from cultured bacterial cells of substrains. The high-molecular chromosomal DNA serving as the donor DNA was extracted from cultured cells of Escherichia coli K-12 strain (ATCC23740) using Smith's phenol extraction method [Smith.MG: Method in Enzy
-mology, 12, part A, 545 (1967)]. Restriction enzyme containing 4 μg of pGA22 plasmid DNA
Hind reaction solution (10mM Tris-HCl, 7mM
Add 0.4 units of Hind (manufactured by Takara Shuzo Co., Ltd., 6 units/μ) to 60μ of MgCl ₂ , 60mM NaCl, PH7.5), react at 37°C for 30 minutes, and then heat at 65°C for 10 minutes to stop the reaction. pGA22 has two Hind locations.
Although there is a cleavage site, Hind under the same conditions
Examination of the digested sample by agarose gel electrophoresis confirms that it has been cleaved into one fragment. Separately, restriction enzyme containing 8 μg of chromosomal DNA
Add 4 units of Hind to 140μ of Hind reaction solution, react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. Mix both digests and add 40μ of T4 ligase buffer.
, ATP (5mM) 40μ, T4 ligase 0.3μ
and 120μ of H ₂ O and react at 12°C for 16 hours. The mixture is extracted twice with 400 μl of phenol saturated with TES buffer and dialyzed against TES buffer to remove the phenol. This ligase reaction mixture was used as Escherichia coli K-12 substrain GT-3 [J. Bacteriol. 117 , 133-143 (1974)].
(homoserine and diaminopimelic acid requirement)
used for transformation. Competent cells (a strain capable of taking up DNA) of the GT-3 strain are prepared by the method of Dagert et al. [Dagert.M., et al: Gene. 6 , 23 (1979)]. That is, L medium supplemented with diaminopimelic acid to give a concentration of 100 μg/ml (containing 10 g of Bactotryptone and 5 g of yeast extract in 1 part of water, PH
Inoculate 50 ml of culture medium (adjusted to 7.2) and culture at 37°C until OD 0.6. Cool the culture solution with ice water for 10 minutes and collect the bacteria by centrifugation. 0.1M cooled
Resuspend in 20 ml of calcium chloride and place at 0°C for 20 minutes. Resentrifuge the cells and add 0.1M calcium chloride.
Suspend in 0.5 ml and leave at 0°C for 18 hours. 200μ of the ligase reaction mixture was added to 400μ of the calcium chloride-treated bacterial solution, and mixed.
Leave at ℃ for 10 minutes, then warm at 37℃ for 5 minutes.
Next, 9 ml of L medium is added and cultured with shaking at 37°C for 2 hours. After centrifugal washing twice with physiological saline,
M9 supplemented with kanamycin equivalent to 12.5μg/ml
Minimal agar medium (2 g glucose, 1 g NH ₄ Cl,
Na ₂ HPO ₄ 6g, KH ₂ PO ₄ 3g, MgSO ₄・
_7H2O0.1g , _CaCl2・_2H2O15mg , thiamine hydrochloride 4mg and agar 15g in 1 part water, PH
7.2) and culture at 37℃ for 3 days. The only colony that appeared was ampicillin 25μg/ml and chloramphenicol 25μg/ml.
It has been confirmed that the cells grow on L agar medium containing 25 μg/ml or 25 μg/ml of kanamycin. From the cultured cells of this transformed strain, in Reference Example 1 (1)
Isolate plasmid DNA by the same method used to isolate pGA22. This plasmid DNA
As a result of analysis by restriction enzyme digestion and agarose gel electrophoresis using pGH2, it had the structure shown in Figure 1 as pGH2. DNA inserted into pGA22
The fragment is a previously cloned E. coli operon-containing DNA fragment [Cossart, P., et al: Molec.
Gen., Genet., 175 , 39 (1979)].
It is confirmed that pGH2 contains a threonine operon. Next, we will create a recombinant of pCG11 and pGH2.
First, pCG11 and pGH2 were converted to Bgl and
Completely digest with BamH under appropriate conditions. Mix the digests containing 2 μg of each plasmid DNA, add 40 μg of T4 ligase buffer to a total volume of 200 μg,
Add 40μ of ATP (5mM), 0.2μ of T4 ligase and 120μ of H ₂ O, and react at 12°C for 16 hours.
The mixture is extracted twice with 400 μl of phenol saturated with TES buffer and dialyzed against TES buffer to remove the phenol. Subsequently, Corynebacterium glutamicum strain LA201 (LA103) was induced in the same manner as in Example 1 (1) using 100μ of a 1:1 mixture of 2 medium high concentration TSMC buffer and the above ligase reaction mixture as donor DNA. After transforming the protoplasts of the strain, homoserine, and leucine auxotrophs, they are spread on an RCGP agar medium and cultured at 30°C for 6 days to regenerate and proliferate.
Bacteria grown on the entire surface of the agar medium were collected, centrifugally washed with physiological saline, reapplied onto minimal agar medium M1 supplemented with 50 μg/ml of leucine, and incubated at 30°C.
Culture for 3 days. Strains that can grow on NB agar medium containing 12.5 μg/ml of kanamycin or 100 μg/ml of spectinomycin are obtained from the colonies that appear. Plasmids are isolated from these transformed strains by ethidium bromide and cesium chloride density gradient centrifugation as described in Example 1 (1). Using 0.5 μg of these plasmid DNA, the DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes were analyzed by agarose gel electrophoresis, and the molecular weight and each restriction enzyme cleavage site in the plasmid molecule were determined. identify The plasmid obtained from one strain was named pEthr1. Restriction enzymes Pst, EcoR, and
The structure characterized by the cleavage site of Xho is
As shown in the figure. pEthr1 was found to have a structure in which a BamH cleavage fragment containing the threonine operon of pGH2 was joined to pCG11. As a result of re-transforming Corynebacterium glutamicum strain LA103 using pEthr1DNA in the same manner as described above, homoserine non-auxotrophy and kanamycin and spectinomycin resistance traits were introduced in combination, and these transformed strains were transformed into various strains. It carries a plasmid identical to pEthr1, which is characterized by its restriction enzyme cleavage pattern. The reason why the homoserine auxotrophy of strain LA103, which was caused by the deletion of homoserine dehydrogenase, is restored to homoserine non-auxotrophy by pEthr1 is due to the expression of homoserine dehydrogenase located on the E. coli threonine operon. (2) Creation of pEthr1-bearing strain Corynebacterium glutamicum LA-106, a threonine-producing bacterium derived from Corynebacterium glutamicum L-22 strain (methionine auxotrophy, AEC resistance, α-amino-β-hydroxyvaleric acid resistance) ) pEthr1 holding strain is LA-106
can be obtained by transforming protoplasts with pEthr1. For protoplasts, strain LA-106 is grown in semi-synthetic medium.
Cells are cultured in a medium containing SSM supplemented with methionine equivalent to 100 μg/ml and grown to an OD of approximately 0.6, and then treated in the same manner as in Example 1(2). Transformation was carried out in the same manner as in Example 1 (2) using RCGP containing 400 μg/ml of spectinomycin.
Obtain transformed strains by selecting on an agar medium.
The pEthr1-carrying strain is American type.
Corynebacterium in the culture collection
glutamicum K19 has been deposited as ATCC39034. (3) Production of threonine by pEthr1-carrying strain A threonine production test is conducted on the pEth1-carrying strain (ATCC39034) and the non-pEth1-carrying strain of the LA-106 strain obtained as described above. A platinum loop of 5 ml of the bacteria produced on NB agar medium was added to production medium P2 [100 g of glucose, 20 g of (NH ₄ ) ₂ SO ₄ , 0.5 g of KH ₂ PO ₄ ,
K ₂ HPO ₄ 0.5g, MgSO ₄・7H ₂ O1g, FeSO ₄・
7H ₂ O 10mg, MnSO ₄・4~6H ₂ O 10mg, biotin 100μg, calcium carbonate 20g, methionine
The cells were inoculated into a test tube containing 100 mg of culture medium containing 1 part water and adjusted to pH 7.2, and cultured with shaking at 30°C for 75 hours. After culturing, the culture solution was subjected to paper chromatography to develop ninhydrin color, and the amount of L-threonine produced was measured by colorimetry. The results are shown in Table 2.

[Table] Reference Example 2 Production of glutamic acid by Corynebacterium glutamicum carrying a recombinant plasmid containing the E. coli phosphoenolpyruvate carboxylase (PPC) gene: (1) E. coli PPC gene (this gene is
Cloning of a DNA fragment containing a gene involved in glutamate biosynthesis known to convert Glu ^- to Glu ⁺ and introduction into Corynebacterium glutamicum: Cloning was carried out using the E. coli host-vector system. It will be carried out at pBR322 used as a vector is isolated from cultured cells of Escherichia coli K-12 substrain by the same method used to prepare pGA22 in Reference Example 1 (1). The polymeric chromosomal DNA used as the donor DNA was prepared from E. coli K-12 strain (ATCC23740) in Example 2 (1). pBR322 plasmid DNA 3 μg and chromosome
Reaction solution for restriction enzyme Sal containing 9μg of DNA (10
mM Tris-HCl, 7mM MgCl ₂ , 100mM
NaCl, 7mM 2-mercaptoethanol,
0.01% bovine serum albumin, PH7.5) to 200μ
Added 10 units of Sal (manufactured by Takara Shuzo Co., Ltd.) and heated to 37°C.
After reacting for 60 minutes at 65℃, stop the reaction by heating at 65℃ for 10 minutes. To this mixed digest, 40μ of T4 ligase buffer, 40μ of ATP (5mM), 0.4μ of T4 ligase, and 12μ of water are added and reacted at 12°C for 16 hours. This mixture was extracted twice with 400 μl of phenol saturated with TES buffer and TES
Dialyze against buffer to remove phenol. This ligase reaction mixture was mixed with E. coli K-12 substrain PPC2 [Glansdorff, N., Genetics, 51 , 167
(1965)] (arg ^- , thr ^- , Ieu ^- , his ^- , Thi ^- ,
^PPC- , ^STR ). Competent cells of PPC2 strain were cultured in L medium supplemented with 2 mg/ml glutamic acid, and GT-
Prepare in the same manner as for obtaining 3 strains of competent cells. Transformation is performed using the ligase reaction mixture described above.
Using 200μ, conduct in the same manner as in Reference Example 1 (1).
Add 9 ml of L medium and culture with shaking at 37°C for 2 hours to express the expression. After centrifugal washing twice with physiological saline, the mixture is spread on M9 minimal agar medium supplemented with 50 μg/ml each of arginine, threonine, leucine, and histidine, and cultured at 37° C. for 3 days. The colonies that appear are replicated onto L agar medium containing 25 μg/ml of ampicillin or 25 μg/ml of tetracycline, and cultured at 37° C. for 24 hours to select those that are resistant to ampicillin and sensitive to tetracycline. Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. Analysis of plasmid pPC1 obtained from one of the transformed strains by restriction enzyme digestion and agarose gel electrophoresis revealed that there was a 4.4Kb fragment at the Sal1 cleavage site of pBR322.
It turned out to be a recombinant plasmid with a genome size of 8.8Kb into which a DNA fragment had been inserted. This pPC1 plasmid was used to transform the PPC2 strain in the same manner as above, and all of the transformed strains selected for ampicillin resistance were glutamic acid non-auxotrophic and were characterized by their restriction enzyme cleavage pattern.
Contains a plasmid with the same structure as pPC1.
This indicates that the E. coli PPC on the pPC1 plasmid
Indicates that the gene has been cloned. In order to introduce the cloned PPC gene into Corynebacterium glutamicum,
Prepare recombinant pCG11 and pPC1 in host E. coli. Reaction buffer for restriction enzyme Pst containing 2 μg each of pCG11 and pPC1 plasmid DNA [21
mM Tris-HCl, 10mM MgCl ₂ , 50mM
( _NH4 ) _2SO4 , 0.01% bovine serum albumin _, PH
7.5] Add 4 units of Pst (manufactured by Takara Shuzo Co., Ltd.) to 200μ, react at 30°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. In this reaction mixture
T4 ligase buffer 40μ, ATP (5mM)
Add 40μ of T4 ligase, 0.2μ of T4 ligase, and 120μ of water, and incubate at 12°C for 16 hours. After phenol extraction in the same manner as above, phenol is removed by dialysis. Using 100μ of this ligase reaction mixture,
PPC2 strain was transformed in the same manner as above. Plasmids were isolated from the resulting colonies by the method described above, and their sizes were examined by agarose gel electrophoresis. A plasmid with a size of approximately 15 to 16 Kb was selected, and the PPC2 strain was transformed again with this plasmid to confirm the presence of the PPC gene. Plasmid obtained from one of the previously transformed strains
Analysis of pEppc1 by restriction enzyme digestion and agarose gel electrophoresis revealed that pCG11 and pPC1 were
Genome size merged at the Pst cleavage site
It was clearly identified as a 15.6Kb recombinant plasmid. thus prepared with E. coli
was derived from Corynebacterium glutamicum L-22 strain using pEppc1 plasmid DNA.
Transform the LP4 strain. Transformation was carried out in Example 1 (2).
Transformants were obtained from colonies growing on RCGP agar medium containing 400 μg/ml of spectinomycin. Plasmids isolated from these transformed strains are digested with restriction enzymes.
Digestion of Sal or Pst alone or both
The presence of pEppc1 is confirmed by heavy digestion and analysis by agarose gel electrophoresis. The pEppc1 strain is included in the American Type Culture Collection.
Corynebacterium glutamicum K-18
Deposited as ATCC39033. (2) Production of glutamic acid by a pEppc1-carrying strain: A glutamic acid production test was conducted on a pEppc1-carrying strain (ATCC39033) and a non-pEppc1-carrying strain of the LP4 strain derived from the Corynebacterium glutamicum L-22 strain. Collect the bacteria grown on the NB agar medium, wash with physiological saline, and add 5 ml of production medium P3 [glucose 50 g, (NH ₄ ) ₂ SO ₄ 3 g, urea 3 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5g,
_MgSO4・_7H2O0.5g , _FeSO4・_7H2O10mg ,
MnSC ₄・4～6H ₂ 0.10mg, biotin 3μg, thiamine hydrochloride 500μg, phenol red 10mg
inoculated into a test tube containing a medium containing 1 part of pure water and adjusted to pH 7.2, and cultured with shaking at 30°C.
During culture, add 0.2 ml of 20% urea solution three times.
Incubate for 40 hours. After culturing, the culture solution was subjected to paper chromatography, and after coloring with ninhydrin, the amount of L-glutamic acid produced was measured by colorimetry. The results are shown in Table 3.

[Table] Reference Example 3 Cloning and expression of the anthranic acid synthase gene of Corynebacterium flavum ATCC14067 in Corynebacterium glutamicum: The chromosomal DNA of Brevibacterium flavum ATCC14067 was cloned in the same manner as in Example 1(1). Prepared by method. pCE53 used as a vector was as described in Example 1 (1).
It is isolated from cultured cells of Corynebacterium glutamicum strain L-22, which harbors it, in the same manner as pCG11 was isolated. pCE53 is the plasmid pCG1 of Corynebacterium glutamicum [Patent Application No. 18101 (Japanese Unexamined Patent Publication No. 57-198]) previously disclosed by the present inventors.
134500)] and E. coli plasmid pGA22 [An, G.
et al.: J. Bacteriol., 140 , 400 (1979)]. For more information
There is only one Bgl cleavage site on pCG1 and
Of the two BamH cleavage sites on pGA22, BamH is not located within the tetracycline resistance gene.
The cleavage site is ligated using the same cohesive ends of both restriction enzymes. pCE53 has selection markers such as the kanamycin resistance gene derived from pGA22, and has one cleavage site for the restriction enzyme Sal. Add 10 units of Sal to 200 μg of the restriction enzyme Sal reaction solution containing 3 μg of pCE53 plasmid DNA and 9 μg of chromosomal DNA prepared above, react at 37° C. for 60 minutes, and then heat at 65° C. for 10 minutes to stop the reaction. Add 40μ of T4 ligase buffer to this mixed digest.
, 40μ of ATP (5mM), 0.4μ of T4 ligase and 120μ of H ₂ O, and reacted at 12°C for 16 hours. The mixture is extracted with 400 μl of phenol saturated with TES buffer and dialyzed against TES buffer to remove the phenol. This ligase reaction mixture is subjected to transformation.
Corynebacterium spp. as a recipient bacterium for transformation.
Anthranilic acid auxotrophic mutant strain LA105 (anthranilic acid synthase-deficient mutant strain) derived from C. glutamicum L-22 strain is used. Anthranilic acid auxotrophic mutants were obtained by selecting bacteria that cannot grow on M1 agar medium by conventional mutation treatment, but can grow on M1 agar medium supplemented with anthranilic acid (equivalent to 30 μg/ml). be obtained. Preparation and transformation of protoplasts of strain LA105 were carried out using growth medium.
The procedure is carried out in the same manner as in Example 1 (2) except that a medium containing NB supplemented with anthranilic acid equivalent to 100 μg/ml is used.
Transformants are selected as colonies growing on RCGP agar medium containing the equivalent of 200 μg/ml of kanamycin. A transformed strain that can grow on M1 agar medium is obtained from the colonies that appear. Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. As a result of analyzing the plasmid pTrp2-3 obtained from one of the transformed strains by digestion with various restriction enzymes and agarose gel electrophoresis, it was found that approximately 7.1 Kb was found at the unique Sal cleavage site of pCE53.
It was found that this was a plasmid into which a SalDNA fragment had been inserted. When the LA105 strain was retransformed using pTrp2-3 in the same manner, 100 μg of tryptophan/
Colonies growing on RCGP agar containing ml and 400 μg/ml of kanamycin simultaneously become anthranilic acid nonauxotrophic, and they carry a plasmid identical to pTrp2-3 as determined by the Sal cleavage mode. The above results show that approximately 7.1Kb of cloned
This shows that the SalDNA fragment contains a gene encoding the anthranilate synthase of Brevibacterium flavum ATCC14067, and that it is expressed in Corynebacterium glutamicum LA105 strain. The pTrp2-3 strain is included in the American Type Culture Collection.
Corynebacterium glutamicum K20
Deposited as ATCC39035. The same treatment as above was performed using plasmid pCE52, and Brevibacterium flavum
Plasmid pTrp4-3 containing the gene encoding the anthranilate synthase of ATCC14067 is obtained. pEC52 is the Corynebacterium glutamicum plasmid pCG1 [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)] previously disclosed by the present inventors and the Escherichia coli plasmid pGA22 [An, G. et al.: J .Bacteriol. 140 ,
400 (1979)] are conjugated together. For details, there is only one Bgl on pCG1.
Of the two BamH cleavage sites on pGA22, there is one in the tetracycline resistance gene.
It is ligated to the BamH cleavage site using the same cohesive ends of both restriction enzymes. pCE52 is
It has a selection marker such as the kanamycin resistance gene derived from pGA22, and has one cleavage site for the restriction enzyme Sal. pCE52 is isolated from cultured cells of Corynebacterium glutamicum L-22 strain harboring pCE52 in the same manner as that used to isolate pCG11 in Example 1 (1). Similarly to the above, tryptophan-producing Corynebacterium glutamicum K36 strain (FERM BP-
451) with pTrp4-3. The obtained transformed strain was transferred to the American Type Culture Collection of Corynebacterium.
glutamicum K31, deposited as ATCC39280. An L-tryptophan production test using Corynebacterium glutamicum K20, ATCC39035, which possesses pTrp2-3, and Corynebacterium glutamicum K31, ATCC39280, which possesses pTrp4-3, is conducted as follows. The bacterial strain was cultured in NB liquid medium at 30℃ for 16 hours with shaking, and 0.5 ml of the bacterial suspension was mixed with 5 ml of production medium P4 [blackstrap molasses 100
g/, (NH ₄ ) ₂ SO ₄ 20g/, KH ₂ PO ₄ 0.5g/
, K ₂ HPO ₄ 0.5g/, MgSO ₄・7H ₂ O 0.25g/
, CaCO ₃ 20g/, PH7.2] and cultured with shaking at 30°C for 96 hours. After culturing, the culture solution was subjected to paper chromatography, and after coloring with ninhydrin, it was determined colorimetrically.
Measure the amount of L-tryptophan produced. As a control, LA-105 strain and LAR-1 strain are treated in the same manner. The results are shown in Table 4.

[Table] Reference Example 4 Production of pCB101: (1) Separation of pCG11 and pUB110 pCG11 is Corynebacterium glutamicum LA103/pCG11 carrying this plasmid.
(ATCC39022) was grown in 400 ml NB medium until the OD was about 0.8, and from the cultured cells, Example 1
Isolate pCG2 using the same method as in (1). pUB110 was obtained by the method of Gryczan et al. [Gryczan, TJet al.: J. Bacteriol., 134 ,
318 (1978)], Bacillus subtilis BR ¹⁵¹ /pUB ¹¹ carrying this plasmid [Proc.
Natl. Acad. Sci, USA, 75 , 1423 (1978)]. (2) In vitro recombination of pCG11 and pUB110 2 μg of pCG11 plasmid DNA prepared above
Restriction enzyme Bgl reaction buffer containing (10mM Tris-HCl, 7mM _MgCl2 , 60mM NaCl, 7mM
mM 2-mercaptoethanol, PH7.5)
Add 2 units of Bgl (manufactured by Takara Shuzo Co., Ltd., 6 units/μ) to 100μ, and react at 37°C for 60 minutes. In addition, restriction enzyme BamH reaction buffer (10mM Tris-HCl, 7mM MgCl ₂ , 100mM NaCl, 2μg of pUB110 plasmid DNA,
2 units of BamH in 100μ (mM mercaptoethanol, 0.01% bovine serum albumin, PH8.0)
(manufactured by Takara Shuzo Co., Ltd., 6 units/μ) at 37°C.
Incubate for 60 minutes. Both restriction enzyme digests are mixed, 40μ of T4 ligase buffer, 40μ of ATP (5mM), 0.2μ of T4 ligase and 120μ of H ₂ O are added and reacted at 12°C for 16 hours. This mixture was extracted twice with 400 μl of phenol saturated with TES buffer and
Exclude phenols dialyzed against TES buffer. (3) Obtain pCB101 Provide 100 μg of a 1:1 mixture of twice as concentrated TSMC buffer and the above ligase reaction mixture.
Used as DNA, in the same manner as in Example 1 (3),
Transform Corynebacterium glutamicum LA103 and select a kanamycin-resistant strain. Contain emerging colonies with 12.5 μg/ml of kanamycin or 100 μg/ml of spectinomycin.
Replicas were placed on NB agar medium, cultured at 30°C for 2 days, and three double-resistant transformants grown were arbitrarily selected and purified on the same agar medium. These three stocks
The cells are grown in a 400 μNB medium until the OD reaches approximately 0.8, and after harvesting, plasmids are isolated from the cultured cells by ethidium bromide-cesium chloride density gradient centrifugation as described in Example 1 (1). 30 to 35 μg of plasmid DNA can be obtained from each transformed strain. These plasmid DNAs were analyzed by restriction enzyme digestion and agarose gel electrophoresis in the same manner as in Example 1 (3), and the molecular weight and restriction enzymes Pst and EcoR were analyzed.
, identify the breakpoints of Hinc and Bgl.
The plasmids of the three strains all have a structure in which pCG11 and pUB110 are bonded together, and two of them have the structure shown for pCB101 in FIG. 2, but the other type has the binding direction in the opposite direction. Transformants with either plasmid
Spectinomycin resistance trait derived from pCG11 and
It has kanamycin resistance trait derived from pUB110. The kanamycin-resistant transformant obtained by re-transforming Corynebacterium glutamicum LA103 using these plasmid DNAs also acquired spectinomycin resistance, and was characterized using various restriction enzyme cleavage methods. Contains the same plasmid as the donated plasmid. Reference Example 5 Cloning of a gene involved in L-histidine biosynthesis of Corynebacterium glutamicum strain C156 and expression of the gene to produce Corynebacterium glutamicum, Corynebacterium herkyulis, Brevibacterium flavum and Brevi Production of L-histidine by Bacterium lactofamentum: (1) Preparation of chromosomal DNA of Corynebacterium glutamicum strain C156 and plasmid pCG11: Resistant to 1,2,4-triazole-3-alanine and capable of producing histidine Corynebacterium glutamicum strain C156 (FERM BP−
453) is prepared in the same manner as in Example 1(1). On the other hand, used as a vector plasmid
pCG11 is a pCG11-carrying strain of the derived strain LA103 of Corynebacterium glutamicum L-22 strain.
Example 1 (1) from LA103/pCG11 (ATCC39022)
Isolate in the same manner. (2) Cloning of genes involved in histidine biosynthesis of Corynebacterium glutamicum strain C156: 3 μg of pCG11 plasmid DNA prepared above
and restriction enzyme containing 9 μg of the above chromosomal DNA
Reaction solution for Bgl [10mM Tris (PH7.5), 7m
M _MgCl2 , 60mM NaCl, 7mM2-mercaptoethanol] 10 units of Bgl (manufactured by Takara Shuzo Co., Ltd.) was added to 200μ, and after reacting at 37°C for 60 minutes, 65
Stop the reaction by warming at °C for 10 min. Add T4 ligase buffer (Tris 200ml) to this mixed digest.
M, _MgCl2 66mM, dithiothreitol 100mM,
Add 40μ of PH7.6), 40μ of 5m MATP solution, 0.3μ of T4 ligase (manufactured by Takara Shuzo, 1 unit/μ) and 120μ of water, and react at 12°C for 16 hours. The T4 ligase reaction mixture is used to transform Corynebacterium glutamicum strain LH33 (histidine auxotrophic, lysozyme sensitive). Transformation is performed using LH33 strain protoplasts. Protoplasts are prepared in the same manner as in Example 1(2). Transfer 0.5 ml of the protoplast suspension to a small test tube, centrifuge at 2500
7.5) Resuspend in 1 ml, centrifuge and wash, then resuspend in 0.1 ml of TSMC buffer. To this suspension, add 100μ of a 1:1 mixture of double concentration TSMC buffer and the above ligase reaction DNA mixture and mix.
Next, 0.8 ml of a solution containing 20% PEG6000 in TSMC buffer is added and mixed. 3 minutes later, RCGP
Add 2 ml of medium (PH7.2), centrifuge at 2500 xg for 5 minutes to remove the supernatant, suspend the precipitated protoplasts in 1 ml of RCGP medium, and then add 0.2 ml to spectinomycin at 400 μg/ml.
(RCGP medium containing 1.4% agar, pH 7.2) and cultured at 30°C for 7 days. Scrape the spectinomycin-resistant colonies grown on the selection plate, centrifugally wash them twice with physiological saline, and add 100μ of spectinomycin.
Spread on minimal agar medium M1 containing g/ml and incubate at 30°C.
The cells were cultured for 2 days, and transformed strains that were spectinomycin resistant and non-histidine auxotrophic were selected. A plasmid is isolated from one of the transformed strains by ethidium bromide/cesium chloride density gradient centrifugation as described in Example 1 (1). DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes were analyzed by agarose gel electrophoresis, and this plasmid was
Identify the restriction enzyme cutting mode of DNA. This plasmid was named pPH8. pPH8 is pCG11
This structure has a DNA fragment of approximately 10.6 Kb inserted into the Bgl cleavage site of . Furthermore, when the H33 strain [parent strain LH33 (histidine auxotrophic, lysozyme resistant): FERM BP-452] was re-transformed using pPH8DNA, all of the transformed strains selected as spectinomycin-resistant strains were non-histidine resistant. It had become a sexual thing. From these results, it is clear that the genes involved in histidine biosynthesis of the histidine-producing bacterial strain C156 have been cloned. Cloning of genes involved in histidine production can also be performed from the beginning using strain H33 as a host strain. (3) Production of L-histidine by Corynebacterium glutamicum strain harboring pPH8: Corynebacterium glutamicum LA-103
Stock (FERM P-5947, ATCC31866)
Transform with pPH8 DNA, and select a transformant resistant to the drug on an RCGP agar medium containing 400 μg/ml of spectinomycin. After purifying the obtained transformed strain, plasmid isolation and
Structural analysis was performed and it was confirmed that the plasmid had the same structure as pPH8. The pPH8 strain Corynebacterium glutamicum LA103/pPH8 is included in the American Type Culture Collection.
Corynebacterium glutamicum K32,
Deposited as ATCC39281. Corynebacterium glutamicum LA103/
pCG11 (ATCC39022) and LA103/pPH8
(ATCC39281) L-histidine production test is conducted as follows. One platinum loop of each of the above bacteria was cultured overnight at 30°C on NB agar medium and 5 ml of production medium supplemented with 200 μg/ml of arginine and methionine.
P5 [molasses 12% (as sugar), KH ₂ PO ₄ 0.2%,
_{K2HPO4 0.1} %, _MgSO4・_7H2O0.05 %,
NaCl 0.25%, (NH ₄ ) ₂ SO ₄ 2.3%, Urea 0.2%,
Inoculate in CaCO ₃ 2%, pH 7.4 (adjusted with ammonia). After culturing at 30°C for 75 hours, the amount of L-histidine produced in the medium was measured using a colorimetric method using sulfanilic acid (Pauly) reagent [H. Pauly, Hoppe].
Seylers; Z.Physiolo.Chem., 42 , 508 (1904),
94 , 284 (1915)]. The results are shown in Table 5.

[Table] (4) Production of L-histidine by Corynebacterium herkyulis, Brevibacterium flavum and Brevibacterium lactofamentum carrying pPH8: Corynebacterium herkyulis
ATCC13868, Brevibacterium flavum
Plasmids in ATCC14067 and Brevibacterium lactofamentum ATCC13869
In order to carry pPH8, each strain is transformed as a recipient strain. Each strain was grown in SSM medium, and the OD660nm was
0.3 units/ml of penicillin G when it reaches 0.2
Add so that Continue culturing, OD660n
Bacteria were collected when m increased to 0.6, and 1mg/
Protoplasts are formed as described above in RCGP medium containing ml lysozyme. pPH8
Transformation is performed according to the method described above, and transformed strains are selected as colonies growing on RCGP agar medium containing 400 μg/ml of spectinomycin. Plasmid DNA was extracted from the cultured cells of the purified spectinomycin-resistant transformant.
183799, prepared according to the description of 57-134500,
It is confirmed from the restriction enzyme cleavage pattern that these have the same structure as pPH8. From the above, plasmid pPH8, which is a derivative of plasmid pCG11, can be replicated in Corynebacterium herkyulis, Brevibacterium flavum, and Brevibacterium lactofamentum, and plasmid pCG11 is widely used in these bacterial species. It turns out that it is possible. Corynebacterium herkyulis K33, Brevibacterium flavum K34, and Brevibacterium lactofamentum K35, which carry pPH8, are respectively
Type Culture Collection
Deposited as ATCC 39282, 39283 and 39284. L-histidine production tests using these strains are conducted as follows. pPH8 grown overnight at 30°C on NB agar medium
Inoculate one platinum loopful of each of the retained strains and their parent strains into 5 ml of production medium P5. 75 at 30℃
After shaking culture for a period of time, the amount of L-histidine produced in the medium is determined colorimetrically by Pauly's method. The results are shown in Table 6.

【table】

[Table] From the above, genes involved in histidine production derived from Corynebacterium glutamicum are found in Corynebacterium herkyulis, Brevibacterium flavum, and Brevibacterium lactofamentum in addition to Corynebacterium glutamicum. It was clear that this gene was expressed in this species and contributed to the production of histidine.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme map of plasmid pGH2. Figure 2 shows the restriction enzyme map of plasmid pCB101. Figure 3 shows a flowchart for the construction of plasmid pEthr1.

Claims (1)

[Claims] 1 Microorganisms that belong to the genus Corynebacterium or Brevibacterium and are derived from species of the genus Corynebacterium or Brevibacterium and are sensitive to S-(2-aminoethyl)-cysteine are referred to as S-
(2-Aminoethyl)-A recombinant with a DNA fragment containing a gene having the activity of converting into a cysteine-resistant strain and a vector DNA capable of autonomous replication in Corynebacterium or Brevibacterium species.
A microorganism containing DNA is cultured in a medium, L-lysine is produced and accumulated in the culture, and L-lysine is produced and accumulated in the culture.
- A method for producing L-lysine, which comprises collecting lysine.