JPH0412959B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for gene expression.
More specifically, the present invention uses a recombinant DNA that is a recombinant of a DNA fragment containing at least one gene and a vector DNA, and in which at least one of both DNAs is foreign to the host strain. The present invention relates to a method for expressing a gene, 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 to express 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, alanyl, 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 Each stage of purification of the purified clones. 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 system. When the Corynebacterium glutamicum strain L-22 or its derivative strain was transformed using this, the foreign gene was expressed in the host and could be used to increase the production of useful substances such as amino acids. They discovered what they could do and 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 culturing a gene to express the characteristics 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 the 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 pEthrl. 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 the collection. [Table] pCG11 and pCE54 are preferably 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 Bg in pCG1.
-250 (ATCC31830, FERM-P5939), streptomycin and/or spectinomycin resistance (Sm ^R /
This is a plasmid in which the BamHI fragment containing the Spec ^R ) gene is ligated using the same cohesive ends of both. pCG11 is a plasmid with a molecular weight of approximately 6.8 Kb and has Bg 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) By the method disclosed, pGA22 is concentrated and isolated from cultured cells of E. coli harboring it 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 harmoniously linked from a DNA mixture,
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 cells and proliferate 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 ml), or scrape them up after non-selectively reverting to normal cells on a regeneration medium, and use this resuspension to collect normal cells from the recipient bacteria. Drugs at concentrations that cannot be used (usually tetracycline 0.5-4 μg/ml,
It is obtained by isolating colonies growing on an agar medium containing 2-15 μg/ml of chloramphenicol and 2-25 μg/ml of kanamycin. Some of the transformed strains selected for tetracycline, chloramphenicol, or kanamycin resistance (referred to as Tc ^R , Cm ^R , and Km ^{R ,} respectively) have also acquired other drug resistance traits derived from pGA22. There is. The plasmid DNA possessed by the transformed strain obtained in this way was obtained by the present inventors and others in Japanese Patent Application No. 56-18101 (Japanese Patent Application No. 57-134500) and Japanese Patent Application No. 56-65777 (Japanese Unexamined Patent Publication 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 using 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 Xho.
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 cut by the same restriction enzyme have the same structure; some restriction enzymes give sticky ends with a single strand protruding, while other restriction enzymes give cohesive ends with one strand protruding. , 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 some kind of restriction enzyme cleavage site can be ligated and then internally cut to create cohesive ends before ligation. DNA fragments and vectors containing the gene of interest are created by these linking methods.
Recombinant DNA fragments are 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 a host-vector system of another microorganism, such as E. coli, and then the Corynebacterium or Brevibacterium species is transformed. A recombinant can be obtained by producing a recombinant with a vector of a bacterial species in a test tube, then transforming a Corynebacterium or Brevibacterium species, 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 U.S Patent 4237224, Gene manipulation experimental method [edited by Yasutaka Takagi, Kodansha Sun Enterprises (1980)], Method in Enzymology
68 ，Recombinant DNA edited by Rey 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 involves 1) preparation of protoplasts from cultured cells, 2)
This is carried out in a process consisting of transformation treatment of protoplasts with recombinant DNA, 3) return regeneration of protoplasts to normal cells, and selection of transformed strains. Examples of specific methods are shown below. 1 Preparation of protoplasts from cultured cells Protoplast formation involves growing microorganisms under conditions that make them sensitive to the cell wall lytic enzyme lysozyme.
This is carried out by subjecting the cultured cells to the action of lysozyme in a hypertonic solution to dissolve and remove the cell walls. 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-control 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 (containing 20 g of powdered broth and 5 g of yeast extract in 1 part of pure water, pH 7.2).
medium) or semi-synthetic medium SSM [glucose 10g, NH ₄ C 4g, urea 2g, yeast extract
1g, KH ₂ PO ₄ 1g, K ₂ HPO ₄ 3g, MgC ₂・
6H ₂ O 0.4g, FeSO ₄・7H ₂ O 10mg, MnSO ₄・
_4-6H2O 0.2mg, _ZnSO4.7H2O 0.9mg _,
CuSO ₄・5H ₂ O 0.4 mg, Na ₂ B ₄ O ₇・10H ₂ O
A medium containing 0.09 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O, 30 μg of biotin, and 1 mg of thiamine hydrochloride in 1 part of water and adjusted to pH 7.2] is used. Microorganisms are inoculated into this medium and cultured with shaking. Absorbance (OD) at 660nm by colorimeter
is measured, and penicillin such as penicillin G is added to the culture solution at a concentration of 0.1 to 2.0 units/ml at the beginning of the logarithmic growth phase (OD = 0.1 to 0.4). 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 cultured in a suitable hypertonic medium, such as PFM medium (0.4 M sucrose in 2-fold dilution of SSM, MgC ₂ 6H ₂
Medium containing O0.01M and adjusted to PH7.0-8.5) or RCG medium [glucose 5g, casein hydrolyzate 5g, yeast extract 25g, K ₂ HPO ₄ 3.5g,
KH ₂ PO ₄ 1.5g, MgC ₂・6H ₂ O 0.41g
_FeSO4・_7H2O 10mg, MnSO4・₄ ~ _6H2O2
mg, ZnSO ₄・7H ₂ O 0.9 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
Resuspend in a medium containing 0.04 mg of _O24.4H2O , 30 μg of biotin, 2 mg of thiamine hydrochloride _, and 1.35 g of disodium succinate in 1 part of water and adjusted to pH 7.0 to 8.5. Add this cell suspension to a final concentration of 0.2 to 10 mg/
ml of lysozyme and react at 30-37°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 the microscope varies depending on the concentration of penicillin added during cell culture and the concentration of lysozyme used, but 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 a nutrient medium, a semi-synthetic medium, or a synthetic medium supplemented with several types of amino acids, and 0.3-0.8M disodium succinate and 0.5-6
% polyvinylpyrrolidone (molecular weight 10000 or
40,000) is preferably used. Usually, semi-synthetic medium RCGP medium [RCG medium and 3
% polyvinylpyrrolidone (molecular weight 10000) and 1.4
% of agar, pH 7.2] 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. Regeneration of protoplasts in RCGP medium depends on bacterial species,
Although it varies depending on the concentration of penicillin added during the culture and the concentration of lysozyme treatment, 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.
Polyethylene glycol (PEG, average molecular weight 1540-6000) has a DNA uptake mediating effect.
Or polyvinyl alcohol (PVA, polymerization degree
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. PEG
The usable concentration ranges for PVA and PVA are 5-60% and 1-20%, respectively, in final concentration. Divalent metal cations include Ca ⁺⁺ , Mg ⁺⁺ ,
Mn ⁺⁺ , Ba ⁺⁺ , Sr ^++, etc. 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 is performed using hypertonic agar containing disodium succinate and polypyrrolidone, as in the regeneration of protoplasts described above. This is carried out by spreading protoplasts on a medium (for example, RCGP medium) and culturing them at a temperature at which normal cells can grow, generally 25 to 35°C. A transformed strain can be obtained by selecting for a trait imparted to the bacterium by a gene derived from donor 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 in step 1) except for directly treating the cells grown in culture with lysozyme without penicillin treatment. The same steps as 1) to 3) may be performed. 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, providing 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. That is, the threonine operon of Escherichia coli, the phosphoenol pinuvate 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)], a gene involved in lysine biosynthesis in Corynebacterium glutamicum, and an anthranilate synthase gene in Brevibacterium flavum were expressed in Corynebacterium spp. All of the exemplified genes were simply introduced in the form of a link 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, it is possible that Corynebacterium glutamicum also has transcription/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., Keneko, 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 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
DNA is stably retained in these bacterial species.
For expression to occur, slight differences in host bacterial properties such as DNA homology are not a problem, as long as these bacterial species have functions that enable autonomous replication of the plasmid and expression of the introduced gene. 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, the host bacteria to which the present invention can be applied are not limited to Corynebacterium glutamicum, but also include Corynebacterium and Brevibacterium species. Examples and reference examples of the present invention are shown below.Reference example 1 Corynebacterium glutamicum of genes involved in lysine biosynthesis of the lysine-producing bacterium Corynebacterium glutamicum ATCC21543. Production of lysine by Corynebacterium glutamicum using cloning and expression of the gene: (1) Corynebacterium glutamicum
Preparation of ATCC21543 chromosomal DNA and vector pCG11: Corynebacterium glutamicum
S-(2-aminoethyl)-cysteine, a lysine analog derived from ATCC13032 (hereinafter referred to as
Corynebacterium glutamicum, a lysine-producing mutant strain resistant to 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, MgC ₂・6H ₂ O 0.4g, FeSO ₄・7H ₂ O 10
mg, MnSO ₄・4~6H ₂ O 0.2mg, ZnSO ₄・7H ₂
O 0.9mg, CuSO ₄・5H ₂ O 0.4mg, Na ₂ B ₄ O ₇・
10H ₂ O 0.09 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 0.04
The seed culture was inoculated into a medium containing 1 mg of biotin, 30 μg of biotin, and 1 mg of thiamine hydrochloride in 1 part of water and adjusted to pH 7.2, supplemented with threonine to a concentration of 100 μg/ml, and cultured with shaking at 30°C. With Tokyo Photoden Colorimeter
Measure the absorbance (OD) at 660nm, OD0.2
When the concentration of penicillin G reaches 0.5 units/ml in the culture solution. 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, 0.05M NaC
: PH8.0], then suspended in 10ml of lysozyme solution (25% sucrose, 0.1M NaC, 0.05M Tris, 0.8mg/ml lysozyme: same below pH8.0) and incubated at 37℃ for 4 hours.
Allow time to react. The method of Saito et al. [Saito, H. et al : Biochim. Beiophys. Acta, 72 ,
619 (1963)]. On the other hand, pCG11 used as a vector plasmid
is the pCG11-bearing strain LA103/pCG11 of the derived strain LA103 of Corynebacterium glutamicum L-22 strain.
(ATCC39022) as follows. 400ml NB medium (20g powdered bouillon, yeast extract
5g in 1 part water (medium adjusted to PH7.2) at 30℃
Culture with shaking and grow until the OD reaches approximately 0.7. The cells are collected, washed with TES buffer, suspended in 10 ml of lysozyme solution, and reacted at 37°C for 2 hours. Reaction solution 5M NaC2.4ml, 0.5M EDTA (PH8.5) 0.6ml,
Add 4.4 ml of a solution consisting of 4% sodium laurisulfate and 0.7 M NaC in sequence, mix gently, and 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, then place in ice water. 10 hours later
Collect the pellet by centrifugation at 1500 xg for 10 minutes. Gently redissolve the pellet by adding 5 ml of TES buffer followed by 1.5 mg/ml ethidium bromide.
Add 2.0ml, add cesium chloride to this and gently dissolve to adjust the density to 1.580. This solution
Ultracentrifuge 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 irradiation with ultraviolet light. pCG11DNA is isolated by extracting this band from the side of the centrifuge tube with a syringe. Next, the fractionated solution was treated five times with an equal volume of isopropyl alcohol solution [volume percentage 90% isopropyl alcohol, 10% TES buffer (this mixture contains a saturated amount of cesium chloride)] to extract and remove ethidium bromide. and then TES
Dialyze against buffer. (2) Corynebacterium glutamicum
Cloning of genes involved in lysine biosynthesis of ATCC21543 Reaction solution for restriction enzyme Bg (10mM Tris-HCl, 7mM MgC ₂ , 60mM NaC, 7mM 2-
Mercaptoethanol PH7.5) 6 units in 60μ
Add Bg (manufactured by Takara Shuzo Co., Ltd.) and react at 37°C for 60 minutes, then heat at 65°C for 10 minutes to stop the reaction. On the other hand, Corynebacterium glutamicum
Restriction enzyme containing 8 μg of chromosomal DNA of ATCC21543
BamH reaction solution (10mM Trischlorine, 7mM
_MaC2 , 100mM NaC, 2mM 2-mercaptoethanol, 0.01% bovine serum albumin, PH8.0)
Add 4 units of BamH to 140μ and incubate at 37°C for 60
After reacting for a minute, the reaction is stopped by heating at 65°C for 10 minutes. Mix both digests and add 40μ of T4 ligase buffer (Tris-HCl 660mM, MaC ₂ 66mM, dithiothreitol 100mM, PH7.6), ATP (5mM).
40μ, T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1 unit/μ
) 0.3 μ and 120 μ H ₂ O at 12 °C.
Incubate for 16 hours. This mixture was extracted twice with 400 μl of phenol saturated with TES buffer and TES
Phenol is removed by dialysis against buffer. This ligase reaction mixture was applied to AEC derived from Corynebacterium glutamicum L-22 strain.
Use for transformation of susceptible LP4 strain. Transformation is
This is carried out using protoplasts of the LP4 strain. Inoculate the seed culture of LP4 strain into NB medium and culture with shaking at 30°C. When the OD reached 0.6, collect the cells and
RCGP medium [glucose 5g, casamino acids 5g, yeast extract 2.5g, K ₂ HPO ₄ 3.5g, KH ₂ PO ₄ 1.5g,
MgC ₂・6H ₂ O 0.41g, FeSO ₄・7H ₂ O 10mg,
MnSO ₄・4~6H ₂ O 2 mg, ZnSO ₄・7H ₂ O
0.9mg, (NH ₄ ) ₆ MO ₇ O ₂₄・4H ₂ O 0.04 mg, biotin 30 μg, thiamine hydrochloride 2 mg, disodium succinate 135 g, polyvinylpyrrolidone (molecular weight
10000) suspended in a medium containing 1 mg/ml of lysozyme (PH7.6) to approximately ¹⁰⁹ cells/ml, transferred to an L-shaped test tube, and incubated gently at 30°C for 5 hours. The cells are shaken and reacted to form protoplasts. Transfer 0.5 ml of this protoplast bacterial solution to a small test tube, centrifuge at 2500 xg for 5 minutes, and then 1 ml of TSMC buffer (10 mM magnesium chloride, 30 mM calcium chloride, 50 mM Tris, 40 mM sucrose, PH7.5).
After resuspending and washing by centrifugation, resuspend in 0.1ml of TSMC buffer. To this bacterial solution, add 100μ of a 1:1 mixture of 2x higher concentration TSMC buffer and the above ligase reaction DNA mixture and mix. Next, add 0.8ml of a solution containing 20% PEG6000 in TSMC buffer and mix. do. After 3 minutes, 2 ml of RCGP medium (PH7.2) was added, centrifuged at 2500
After suspending in RCGP medium, transfer 0.2 ml to RCGP agar medium containing 400 μg/ml spectinomycin (RCGP
Spread on a medium containing 1.4% agar (PH7.2),
Incubate 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 transferred to a minimal agar medium M1 containing the equivalent of 2 mg/ml threonine, 2 mg/ml AEC, and 12.5 μg/ml streptomycin [10 g glucose, NH ₄ H ₂ PO ₄
1g, KC 0.2g, MgSO ₄・7H ₂ O 0.2g,
_FeSO4・_7H2O 10mg, MnSO4・₄ ~ _6H2O
0.2mg, ZnSO ₄・7H ₂ O 0.9mg, CuSO ₄・5H ₂ O・
0.4mg, Na ₂ B ₄ O ₇・10H ₂ O 0.09mg, (NH ₄ ) ₆ Mo _7
O24・_4H2O 0.04mg, biotin 50μg, p-aminobenzoic acid 2.5mg, thiamine hydrochloride 1mg, agar
Reapply 16 g of the medium on a medium containing 1 part of water and adjust the pH to 7.2, and culture 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
It is isolated in the same manner as pCG11 was isolated above. Using 1 μg of these plasmid DNA,
After complete digestion with the restriction enzyme EcoR, which has a cleavage site on pCG11, it was analyzed by agarose gel electrophoresis, and the molecular weight was determined from the sum of the resulting fragments. The molecular weight is calculated based on a standard curve drawn by the migration distance of each fragment of known molecular weight generated by restriction enzyme Hind digestion of lambda phage DNA that was run simultaneously on the same agarose gel. Plasmid pAec5 obtained from one of the transformed strains has a molecular weight of 10.7 Kb and is similar to the Bg of pCG11.
This is a recombinant bent plasmid with a 3.9Kb DNA fragment inserted at the cleavage site. The protoplasts of the LP4 strain were transformed using pAec5 DNA in the same manner as above, and the transformed strains selected for spectinomycin resistance were simultaneously AEC.
It possesses a plasmid identical to pAec5, which is determined by the cleavage pattern of EcoR, which has been given a resistance trait. That is, it is clear that the gene governing the AEC resistance trait of Corynebacterium glutamicum ATCC21543 has been cloned into pAec5.
The pAec5-carrying strain is Corynebacterium in the American Type Culture Collection.
glutamicum K17ATCC39032. (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. Add one loopful of bacteria grown on NB agar medium to 5 ml of production medium P1 [glucose 100 g, (NH ₄ )]
₂ SO ₄ 24.5g, KH ₂ PO ₄ 1g, MgSO ₄・7H ₂ O
0.4g, _FeSO4・_7H2O 10mg, _MnSO4・4~
6H ₂ O 10mg, biotin 50μg, thiamine hydrochloride
200 μg of calcium pantothenate, 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] in a test tube 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 an acidic-copper ninhydrin reaction, and the results are shown in Table 1. [Table] Reference Example 2 Production of threonine by Corynebacterium glutamicum using cloning of genes involved in threonine biosynthesis in E. coli and 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.G., who created this plasmid.
et al: J. Bacteriol., 140, 400 (1979)],
The plasmid is isolated from cultured cells of Escherichia coli K-12 substrain carrying this plasmid. The high-molecular chromosomal DNA, which serves as the donor DNA, was extracted from cultured E. coli strain K-12 (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 MgC
₂ , 60mM NaC, PH7.5) 0.4 units in 60μ
Hind (manufactured by Takara Shuzo Co., Ltd., 6 units/μ) was added.
After reacting at 37°C for 30 minutes, stop the reaction by heating at 65°C for 10 minutes. There are two Hind cleavage sites in pGA22, but agarose gel electrophoresis of a Hind-digested sample under the same conditions confirms that the pGA22 is cleaved into one fragment. Separately, add 140μ of restriction enzyme Hind reaction solution containing 8μg of chromosomal DNA.
After adding 4 units of Hind to and reacting at 37℃ for 60 minutes,
Stop the reaction by heating at 65°C for 10 minutes. Mix both digests and add 40μ of T4 ligase buffer.
, 40μ of ATP (5mM), 0.3μ of T4 ligase, and 120μ of H ₂ O, and reacted 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 mixed with E. coli K-12 substrain.
GT-3 [J.Bacteriol・117, 133-143 (1974)]
(homoserine and diamomipimelic acid requirement)
used for transformation. Competent cells (bacterial cells 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 (Bactotryptone) supplemented with diaminopimelic acid to a concentration of 100 μg/ml.
Inoculate 10 g of yeast extract into 50 ml of a medium containing 5 g of yeast extract in 1 part water and adjust the pH to 7.2, and culture at 37°C until the OD reaches 0.6. Cool the culture solution with ice water for 10 minutes and collect the bacteria by centrifugation. 20ml chilled 0.1M calcium chloride
Resuspend at 0°C for 20 minutes. Resuspend cells in 0.5 ml of 0.1 M calcium chloride at 0°C for 18 min.
Give it time. Add and mix 200μ of the above ligase reaction mixture to 400μ of calcium chloride-treated bacterial solution, and heat to 0°C.
Let stand 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 minimal agar medium (glucose 2g, NH ₄ C 1g, Na ₂ HPO ₄ 6g,
KH ₂ PO ₄ 3g, MgSO ₄・7H ₂ O 0.1g, CaC
_{2.2H 2} O 15mg, thiamine hydrochloride 4mg and agar
Apply 15 g to a medium containing 1 part water and adjust the pH to 7.2, and culture at 37°C for 3 days. The only colony that appeared was either ampicillin 25μg/ml, chloramphenicol 25μg/ml or kanamycin.
It is confirmed that it grows even on L agar medium containing 25 μg/ml. pGA22 was extracted from the cultured cells of this transformed strain.
Plasmids were isolated by the same method as
Isolate the DNA. Analysis of this plasmid DNA by restriction enzyme digestion and agarose gel electrophoresis revealed that it had the structure shown as pGH2 in FIG. 1. The DNA fragment inserted into pGA22 was an E. coli opesun-containing DNA fragment that had already been cossunized [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 into Bg and
Completely digest with BamH under appropriate conditions. Mix digests containing 2 μg of each plasmid DNA, total volume
40μ of T4 ligase buffer to 200μ;
ATP (5mM) 40μ, T4 ligase 0.2μ and
Add 120μ of H ₂ O and react at 12°C for 16 hours.
This mixture was saturated with phenol in TES buffer.
Extract twice at 400μ and dialyze against TES buffer to remove phenols. followed by twice as high concentration
Corynebacterium glutamicum strain LA201 (derived strain of LA103, homoserine,
After transforming protoplasts of a leucine auxotroph, they are spread on an RCGP agar medium and cultured at 30°C for 6 days for regeneration and proliferation. The bacteria grown on the entire surface of the agar medium are collected, centrifugally washed with physiological saline, reapplied onto minimal agar medium M1 supplemented with 50 μg/ml of leucine, and cultured at 30°C for 3 days. Strains that can grow on NB agar medium containing 12 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 Reference 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. The structure characterized by the cleavage sites for the restriction enzymes pst, EcoR, and Xho is shown in Figure 3. pEthr1 is
pCG11 contains the threonine operon of pGH2,
It was found that it has a structure in which BamH cleavage fragments are combined. Using pEthr1DNA, Corynebacterium
As a result of re-transforming the C. glutamicum LA103 strain in the same manner as described above, homoserine non-auxotrophy and kanamycin and spectinomycin resistance traits were introduced in combination, and these transformed strains had pEthr1, which is characterized by various restriction enzyme cleavage patterns. It has the same plasmid. The homoserine auxotrophy of strain LA103 is caused by the deletion of homoserine dehydrogenase.
The return to homoserine non-requiring by pEthr1 is due to the expression of homoserine dehydrogenase on the E. coli threonine operon. (2) Creation of pEthr1-bearing strain Corynebacterium glutamicum LA-106 (methionine auxotrophic,
A pEthr1-bearing strain (resistant to AEC and resistant to α-amino-β-hydroxyvaleric acid) can be obtained by transforming protoplasts of LA-106 with pEthr1. For protoplasts, strain LA-106 is grown in semi-synthetic medium.
It is prepared by culturing cells in a medium containing SSM supplemented with methionine equivalent to 100 μg/ml and growing them to an OD of approximately 0.6, and treating them in the same steps as in Reference Example 1(2). Transformation is carried out in the same manner as in Reference Example 1(2), and a transformed strain is obtained by selection on an RCGP agar medium containing 400 μg/ml of spectinomycin. The pEthr1-carrying strain is Corynebacterium glutamicum in the American Type Culture Collection.
Deposited as K19 ATCC39034. (3) Production of threonine by pEthr1-carrying strain A threonine production test is performed on the pEthr1-carrying strain (ATCC39034) and the non-carrying strain of the LA-106 strain obtained as above. Bacteria produced on NB agar medium were added to each platinum loop of 5 ml of production medium P2 [glucose].
100g _, ( _{NH4 ) 2SO420g} , _{KH2PO40.5g , K2HPO4}
0.5g, _MgSO4・_7H2O1g , _FeSO4・_7H2O10mg ,
The cells were inoculated into a test tube containing a medium containing 10 mg _of MnSO4.4-6H2O, 100 μg of biotin, ₂₀ g of calcium carbonate, and 100 mg of methionine in 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 3 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 Reference Example 2
It is isolated from cultured cells of Escherichia coli K-12 substrain by the same method used to prepare pGA22 in (1). donation
The polymeric chromosomal DNA used as the DNA was prepared from Escherichia coli K-12 strain (ATCC23740) in Reference Example 2(1). pBR322 plasmid DNA 3μg and chromosome
Reaction solution for restriction enzyme Sal containing 9μg of DNA (10mM
Tris-HCl, 7mM _MgC2 , 100mM NaC,
7mM 2-mercaptoethanol, 0.01% bovine serum albumin, PH7.5) 10 units of Sal in 200μ
(manufactured by Takara Shuzo Co., Ltd.) and reacted at 37°C for 60 minutes.
Stop the reaction by heating at 65°C for 10 minutes. Add 40μ of T4 ligase buffer and ATP to this mixed digest.
(5mM) 40μ, T4 ligase 0.4μ and water 120μ
and react at 12℃ for 16 hours. This mixture was diluted with 400 μl of phenol saturated with TES buffer.
Extract twice and dialyze against TES buffer to remove phenols. This ligase reaction mixture was mixed with E. coli K-12 substrain.
PPC2 [Glansdorff, N., Genetics, 51 , 167
(1965)] (arg ^- , thr ^- , leu ^- , his ^- , Thi ^- ,
PPC ^- , ^STR ). Competent cells of strain PPC2 are cultured in L medium supplemented with 2 mg/ml glutamic acid, and prepared in the same manner as in Reference Example 2(1) to obtain competent cells of strain GT-3.
Transformation is carried out in the same manner as in Reference Example 2(1) using 200μ of the ligase reaction mixture. Add 9 ml of L medium and culture with shaking at 37°C for 2 hours to express the expression. Next, after centrifuging twice with physiological saline, arginine, threonine, leucine, and histidine were added.
Spread on M9 minimal agar medium supplemented with 50 μg/ml,
Incubate at ℃ for 3 days. Treat emerging colonies with ampicillin 25 μg/ml or tetracycline.
Replica onto L agar medium containing 25 μg/ml, 37
Culture at ℃ for 24 hours to select ampicillin-resistant and tetracycline-sensitive cells. 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 a recombinant plasmid with a genome size of 8.8 Kb had a 4.4 Kb DNA fragment inserted into the Sal1 cleavage site of pBR322. It turned out to be. This pPC1 plasmid was used to transform PPC2 strain in the same manner as above, and all of the transformed strains selected for ampicillin resistance were glutamate-nonrequiring and had the same structure as pPC1, which was characterized by the restriction enzyme cleavage pattern. is held. This indicates that the E. coli PPC gene has been cloned onto the pPC1 plasmid. To introduce the cloned PPC gene into Corynebacterium glutamicum, pCG11
and pPC1 recombinants are prepared in host E. coli.
Reaction buffer for restriction enzyme Pst containing 2 μg each of pCG11 and pPC1 plasmid DNA [20mM Tris-HCl, 10mM MgC ₂ , 50mM (NH ₄ ) ₂ SO ₄ , 0.01%
Bovine serum albumin, PH7.5] 4 units per 200μ
Add Pst (manufactured by Takara Shuzo Co., Ltd.) and react at 30°C for 60 minutes, then heat at 65°C for 10 minutes to stop the reaction.
Add 40μ of T4 ligase buffer to this reaction mixture.
ATP (5mM) 40μ, T4 ligase 0.2μ and water
Add 120μ and incubate at 12℃ for 16 hours. After phenol extraction in the same manner as above, phenol is removed by dialysis. Using 100μ of this ligase reaction mixture, strain PPC2 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. As a result of analysis of plasmid pEppc1 obtained from one of the previously transformed strains by restriction enzyme digestion and agarose gel electrophoresis, a recombinant plasmid with a genome size of 15.6 Kb in which pCG11 and pPC1 were conjugated and linked at their Pst cleavage sites was found. It was clearly shown that Using the pEppc1 plasmid DNA thus prepared in Escherichia coli, LP4 strain derived from Corynebacterium glutamicum L-22 strain is transformed. Transformation was carried out in the same manner as in Reference Example 1(2), and transformed strains were obtained from colonies growing on PCGP agar medium containing 400 μg/ml of spectinomycin. Plasmids isolated from these transformed strains were digested with the restriction enzyme Sal.
Alternatively, the possession of pEppc1 can be confirmed by single digestion of Pst or double digestion of both and analysis by agarose gel electrophoresis. The pEppc1-carrying strain is American type.
Corynebacteruim in the culture collection
glutamicum K-18ATCC39033. (2) Production of glutamic acid by pEppc1 strain: LP4 strain pEppc1 strain derived from Corynebacterium glutamicum L-22 strain (ATCC39033)
We conducted a glutamic acid production test on the non-possessed strain.
Collect the bacteria grown on the NB agar medium, wash with physiological saline, and add 5 ml of production medium P3 [glucose
50g, (NH ₄ ) ₂ SO ₄ 3g, Urea 3g, KH ₂ PO ₄
0.5g, _{K2HPO4 0.5g} , _MgSO4・_7H2O 0.5g,
_FeSO4・_7H2O 10mg, MnSO4・₄ ~ _6H2O10
3 μg of biotin, 500 μg of thiamine hydrochloride, and 10 mg of phenol red in 1 part pure water and adjusted to pH 7.2] in a test tube, and cultured with shaking at 30°C. During culture, add 0.2 ml of 20% urea solution three times and culture 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 4 Cloning and expression of the anthranilate synthase gene of Brevibacterium flavum ATCC14067 in Corynebacterium glutamicum: The chromosomal DNA of Brevibacterium flavum ATCC14067 was cloned in the same manner as in Reference Example 1(1). Prepared by method.
pCE53 used as a vector is as shown in Reference 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 Bg 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. Restriction enzyme Sal reaction solution containing 3 μg of pCE53 plasmid DNA and 9 μg of chromosomal DNA prepared above
Add 10 units of Sal to 200μ, 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 react at 12°C for 16 hours.
This mixture was saturated with phenol in TES buffer.
Extracted at 400μ, dialyzed against TES buffer,
Remove phenols. 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 created by selecting bacteria that could not grow on M1 agar medium through conventional mutation treatment and could 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 same procedure as in Reference Example 1(2) is carried out 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 LA105 strain was retransformed using pTrp2-3 in the same manner, tryptophan was found to be 100 μg/ml.
Colonies growing on RCGP agar containing 400 μg/ml of kanamycin and kanamycin simultaneously became anthranilic acid non-auxotrophic, and they carried a plasmid identical to pTrp2-3 as judged 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 K20ATCC39035
It has been deposited as. 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 plasmid pCG1 of Corynebacterium glutamicum [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)] previously disclosed by the present inventors and the plasmid pGA22 of Escherichia coli [An, G. et al . :J.Bacteriol.
140, 400 (1979)]. For details, there is only one place on pCG1.
BamH located at the Bg cleavage site and two locations on pGA22
Among the cleavage sites, the BamH cleavage site in the tetracycline resistance gene is ligated 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 strain L-22 harboring pCE52 in the same manner as in the method used to isolate pCG11 in Reference 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 carried out 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 added to 5 ml of production medium P4 [100 g of blackstrap molasses/
, (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 5 Production of pCB101: (1) Separation of CG11 and pUB110 pCG11 is Corynebacterium glutamicum LA103/pCG11 carrying this plasmid.
(ATCC39022) is grown in 400 ml NB medium until the OD reaches approximately 0.8, and the cultured cells are isolated by the same method as that used to isolate pCG2 in Reference Example 1 (1). pUB110 was obtained using the method of Gryczan et al. [Gryczan, T.
J. et al .: J. Bacteriol., 134, 318 (1978)]
Bacillus subtilis BR ¹⁵¹ /pUB ¹¹⁰ carrying this plasmid [Proc.Natl.Acad.Sci,
USA, 75 , 1423 (1978)]. (2) In vitro recombination of CG11 and pUB110 2μg of pCG11 plasmid DNA prepared above
Restriction enzyme Bg reaction buffer containing (10mM Tris-HCl, 7mM MgC ₂ , 60mM NaC,
7mM 2-mercaptoethanol, PH7.5)
Add 2 units of Bg (manufactured by Takara Shuzo Co., Ltd., 6 units/μ) to 100μ and react at 37°C for 60 minutes. In addition, 2 _units of BamH (manufactured by Takara Shuzo Co., Ltd., 6 units/μ) and react at 37°C for 60 minutes. Mix both restriction enzyme digests, add 40μ of T4 ligase buffer, 40μ of ATP (5mM) and add T4 ligase.
Add 0.2 μ and 120 μ of H ₂ O and incubate at 12 °C for 16
Allow time to react. This mixture was extracted twice with 400 μl of phenol saturated with TES buffer and
Exclude phenols dialyzed against TES buffer. (3) Obtaining pCB101 Add 100μ of a 1:1 mixture of twice as concentrated TSMC buffer and the above ligase reaction mixture to the donor DNA.
Corynebacterium glutamicum LA103 was transformed using the same method as in Reference Example 1(3), and a kanamycin-resistant strain was selected. The colonies that appeared were replicated onto NB agar medium containing 12.5 μg/ml of kanamycin or 100 μg/ml of spectinomycin, cultured at 30°C for 2 days, and 3 growing double-resistant transformants were randomly selected and identical. Purify on agar medium. 400μ of these three stocks
The cells are grown in NB 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 Reference Example 1 (1). 30-35 μg of plasmid DNA from any transformed strain
is obtained. These plasmid DNAs were analyzed by restriction enzyme digestion and agarose gel electrophoresis in the same manner as in Reference Example 1 (3), and the molecular weight and restriction enzymes Pst and EcoR were analyzed.
, identify the breakpoints of Hinc and Bg. All plasmids of the three strains are pCG11 and pUB110.
Two of them have the structure shown in Figure 2 for pCB101, but the other type has the opposite binding 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. The donated plasmid contains the same plasmid. Example 1 Cloning of a gene involved in L-histidine biosynthesis of Corynebacterium glutamicum strain C156 and the expression of the gene to produce Corynebacterium glutamicum, Corynebacterium, Hercullis, Brevibacterium flavum and Brevi Production of L-histidine by Bacterium lactofamentum: (1) Preparation of chromosomal DNA of Corynebacterium glutamicum strain C156 and plasmid pCG11: 1,2,4-triazole-3-alanine resistance and histidine production ability Chromosome of Bacterium glutamicum strain C156 (FERM BP-453)
DNA is prepared in the same manner as in Reference Example 1(1). On the other hand, pCG11 used as a vector plasmid
is the pCG11-bearing strain LA103/pCG11 of the derived strain LA103 of Corynebacterium glutamicum L-22 strain.
(ATCC39022) in the same manner as in Reference Example 1(1). (2) Cloning of genes involved in histidine biosynthesis of Corynebacterium glutamicum strain C156: Restriction enzyme Bg containing 3 μg of pCG11 plasmid DNA prepared above and 9 μg of the above chromosomal DNA
reaction solution [10mM Tris (PH7.5), 7mM MgC
_2. Add 10 units of Bg (manufactured by Takara Shuzo Co., Ltd.) to 200μ of 60mM NaC, 7mM 2-mercaptoethanol, react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. Add T4 ligase buffer (Tris 200mM, MgC ₂
66mM, dithiothreitol 100mM, PH7.6)
40μ, 5mM MATP solution 40μ, T4 ligase (Takara Shuzo Co., Ltd., 1 unit/μ) 0.3μ and water 120μ
and react at 12℃ for 16 hours. The T4 ligase reaction mixture was mixed with Corynebacterium glutamicum LH33 strain (histidine auxotrophic,
(susceptible to lysozyme). Transformation is performed using LH33 strain protoplasts. Protoplasts are prepared in the same manner as in Reference Example 1(2). Transfer 0.5ml of protoplast suspension to a small test tube.
Centrifuge at 2500 x g for 5 minutes and add TSMC buffer (10mM magnesium chloride, 30mM calcium chloride, 50mM Tris, 400mM sucrose, PH7.5) 1
After centrifugation and washing, resuspend in 0.1ml of TSMC buffer.
Resuspend in Add 2x concentration of TSMC to this suspension.
1:1 buffer and ligase reaction DNA mixture above
Add 100μ of the mixture and mix, then add 0.8ml of a solution containing 20% PEG6000 in TSMC buffer and mix. After 3 minutes, 2 ml of RCGP medium (PH7.2) was added, centrifuged at 2500
After suspending in RCGP medium, transfer 0.2 ml to RCGP agar medium containing 400 μg/ml spectinomycin (RCGP
Spread on a medium containing 1.4% agar (PH7.2),
Incubate at 30°C for 7 days. Scrape the spectinomycin-resistant colonies grown on the selection plate and incubate with saline for 2 hours.
After centrifugation and washing, the mixture is spread on minimal agar medium M1 containing 100 μg/ml of spectinomycin and cultured at 30° C. for 2 days to select transformants that are spectinomycin resistant and non-histidine auxotrophic. A plasmid is isolated from one of the transformed strains by ethidium bromide/cesium chloride density gradient centrifugation as described in Reference Example 1 (1). DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes are analyzed by agarose gel electrophoresis to identify the mode of restriction enzyme cleavage of this plasmid DNA. This plasmid was named pPH8. pPH8 is located approximately at the Bg cleavage site of pCG11.
This structure has a 10.6Kb DNA fragment inserted. Furthermore, using pPH8DNA, H33 strain [parent strain of LH33 strain (histidine auxotrophy, lysozyme resistance):
FERM BP-452], all of the transformed strains selected as spectinomycin-resistant strains were non-requiring for histidine.
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 cells harboring pPH8: Corynebacterium glutamicum LA-103 strain (FERM P-5947, ATCC31866) was transformed into pPH8DNA.
and select transformants resistant to the same drug on an RCGP agar medium containing 400 μg/ml of spectinomycin. After purifying the obtained transformed strain, plasmid isolation and structural analysis were performed in the same manner as above.
It was confirmed that the plasmid had the same structure as pPH8. pPH8-bearing strain Corynebacterium glutamicum LA103/pPH8 is a member of the American Type Culture Collection of Corynebacterium glutamicum.
glutamicumK32, 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 added to 5 ml of production medium P5 [molasses] supplemented with 200 μg/ml of arginine and methionine.
12% ₍ as sugar), _{KH2PO4 0.2} %, _K2HPO4
0.1%, _{MgSO4.7H2O0.05} %, NaC _0.25 %,
(NH ₄ ) ₂ SO ₄ 2.3%, urea 0.2%, CaCO ₃ 2%,
Inoculate at pH 7.4 (adjusted with ammonia). at 30℃
After 75 hours of culture, the amount of L-histidine produced in the medium was measured by 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
Plasmid pPH8 in ATCC14067 and Brevibacterium lactofamentum ATCC13869
Transformation is performed using each strain as a recipient strain in order to carry the . Each strain was grown in SSM medium and the OD660nm was 0.2.
When the concentration of penicillin G reaches 0.3 units/ml, add it. Continue culturing and OD660nm is 0.6
When the number of bacteria increases to 1.0, the bacteria are harvested, and protoplasts are formed in the same manner as described above in RCGP medium containing 1 mg/ml lysozyme. Transformation is performed using pPH8 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 and 57-134500, and it was confirmed by restriction enzyme cleavage that they have the same structure as pPH8. Based on the above, plasmid pPH8, which is a derivative of plasmid pCG1, can also be replicated in Corynebacterium herkyulis, Brevibacterium flavum, and Brevibacterium lactofamentum, and plasmid pCG11 is widely used in these bacterial species. I see that it is possible. Corynebacterium herkyulis K33 and Brevibacterium flavum, which are pPH8-carrying strains
K34, and Brevibacterium lactofamentum K35 are stored in the American Type Culture Collection ATCC39282 and Brevibacterium lactofamentum K35, respectively.
Deposited as 39283 and 39284. L-histidine production tests using these strains are conducted as follows. One platinum loopful of each of the pPH8-carrying strain and its parent strain, which were cultured overnight at 30°C on NB agar medium, is inoculated into 5 ml of production medium P5. After culturing with shaking at 30°C for 75 hours, the amount of L-histidine produced in the medium is determined colorimetrically by Pauly's method. The results are shown in Table 6. [Table] From the above, the 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 the protein was expressed in the cells and contributed to the production of histidine.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme map of plasmid pPGH2. 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. A DNA fragment that belongs to the genus Corynebacterium or Brevibacterium and contains a gene that is derived from a species of the genus Corynebacterium or Brevibacterium and has the activity of converting a microorganism that exhibits a histidine auxotrophy into a histidine non-auxotrophic strain. , a recombinant with vector DNA capable of autonomous replication in Corynebacterium or Brevibacterium species.
L-histidine is characterized by culturing a microorganism containing DNA in a medium, producing and accumulating L-histidine during the culture, and collecting L-histidine from the culture.
Method for producing histidine.