JPH0732711B2 - Method for producing L-isoleucine - Google Patents

Description

The present invention has a recombinant DNA of a DNA fragment containing a gene encoding a gene encoding aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase of a microorganism belonging to the genus Escherichia and a vector DNA, and A method of culturing a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium having L-isoleucine-producing ability in a medium, allowing L-isoleucine to be produced and accumulated in the culture, and collecting L-isoleucine from the culture. The present invention relates to a characteristic method for producing L-isoleucine.

As a method for producing L-isoleucine by a direct fermentation method using a so-called glutamic acid-producing bacterium such as Corynebacterium or Brevibacterium, a method using an L-isoleucine-producing mutant strain derived from a wild strain is known. ing. Examples of L-isoleucine-producing mutant strains include, for example, Japanese Patent Publication No. 47-38995, Japanese Patent Publication No.
As described in, for example, 54-32070, auxotrophic mutations of amino acids, resistance mutations to analogs of amino acids, or strains sharing those mutations are known.

In the L-isoleucine production method using a bacterium of the genus Corynebacterium or the genus Brevibacterium, the present inventors have succeeded in producing L-isoleucine by a recombinant DNA technique which is completely different from the conventional breeding by imparting an L-isoleucine-producing mutation. As a result of repeated studies on the production method, the strain containing the recombinant of the gene relating to the biosynthesis of threonine, which is a precursor of L-isoleucine, and the vector plasmid of these strains is superior to the non-recombinant strain. -The inventors have found that they have the ability to produce isoleucine, and have completed the present invention.

When a recombinant DNA containing a gene related to threonine biosynthesis was introduced into an L-isoleucine non-producing strain belonging to the genus Corynebacterium or Brevibacterium,
The bacterium became an L-isoleucine-producing strain, and already L-
L when introduced into a strain having isoleucine productivity
The fact that the isoleucine productivity is improved is the first finding found by the present inventors. Tokuban Sho 58-893 describes a method for producing L-isoleucine by a bacterium of the genus Corynebacterium or Brevibacterium in which a chromosomal gene region involved in isoleucine antagonist resistance is incorporated, but the gene according to the present invention is described. Is different from.

Hereinafter, the present invention will be described in detail.

According to the present invention, a recombinant DNA of a DNA fragment containing a gene encoding aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase of a microorganism belonging to the genus Escherichia and a vector DNA is retained, and L-isoleucine producing ability is obtained. L-isoleucine is produced by culturing a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium, which has L, in the medium to produce and accumulate L-isoleucine, and collecting L-isoleucine from the culture. it can.

As the microorganism belonging to the genus Corynebacterium or the genus Brevibacterium used as the host microorganism, all known so-called glutamic acid-producing microorganisms can be used, but the following strains are preferably used.

Corynebacterium glutamicum ATCC13032 Corynehagterium acetoacidophilum ATCC13870 Corynebacterium herculis ATCC13868 Corynebacterium Lilium ATCC15990 Brevibacterium dibaricatum ATCC14020 Brevibacterium flavum TCC14067 Brevibacterium CC-Imariophylium AT68 Umbo lactofermentum ATCC13869 Brevibacterium thiogenitalis ATCC19240 As a host, a wild strain having no isoleucine-producing ability can be used, but a strain having already isoleucine-producing ability can also be used. As isoleucine-producing strains, known strains such as amino acid-requiring mutant strains and amino acid analog-resistant mutant strains can be applied.

Enzymes involved in threonine biosynthesis from aspartate to threonate include aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase. (Agr. Biol. Chem., 38 , 993 (197
Four)〕.

In the present invention, genes relating to threonine biosynthesis encoding aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase of microorganisms belonging to the genus Escherichia are desirable, and particularly derived from threonine or isoleucine-producing mutant strains derived from the microorganism. Genes are preferred. A specifically preferable example is the threonine operon of Escherichia coli K-12.

Vectors for incorporating the DNA include pCG1, pCG2, pCG4, pCG11, pCE54 and pCB101, which were developed by the present inventors.
Etc. are preferably used, and the method for producing these vectors is described in JP-A-57-134500, JP-A-57-183799, JP-A-58-35197 and JP-A-58-105999.

Donor DNA and vector DNA for enzymes related to threonine biosynthesis
Recombinant DNA was digested with restriction enzymes in vitro and then religated with DNA ligase, and using this ligation product, the gene encoding the enzyme involved in threonine biosynthesis was deficient in Corynebacterium. It can be obtained by a recombinant DNA technique in which a mutant strain of the genus or Brevibacterium is transformed and a transformant complemented with a defective trait is selected. This recombinant DNA technique can be performed according to the method described in JP-A-57-186492 and JP-A-657-186489.

Instead of direct selection of recombinant DNA in Corynebacterium or Brevibacterium strains, it is also possible to use a host-vector system in which genetic recombination techniques have already been established, such as E. coli. That is, an in vitro ligation reaction product of a donor DNA of an enzyme involved in threonine biosynthesis and a vector DNA was used to transform a mutant strain of Escherichia coli deficient in a gene encoding an enzyme involved in threonine biosynthesis, and a defective trait was complemented. The selected transformant was selected, and the DNA cloned from this transformant and the vector DNA of the genus Corynebacterium or Brevibacterium were digested with a restriction enzyme in a test tube, and then a DNA ligase recombination reaction was performed. Using this ligation reaction, a mutant strain of Corynebacterium or Brevibacterium deficient in a gene encoding an enzyme involved in threonine synthesis is transformed, and a transformant complemented with the defective trait is selected. The recombinant DNA can be obtained in the same manner.

Includes genes related to threonine biosynthesis used in the present invention
A specifically preferable example of DNA is the threonine operon of Escherichia coli K-12. The present invention will be described in more detail with reference to the recombinant plasmid pEthr1 containing the E. coli threonine operon DNA.

A DNA fragment containing the E. coli threonine operon can be cloned in advance using the E. coli host vector form. Methods for cloning genes in host E. coli are described, for example, in Methods in Enzymology, Volume 68, Ray Wu (Ed) Academic.
It is described in Press New York (1979). Specifically, the procedure is as follows.

Chromosomal DNA extracted from Escherichia coli containing the wild-type threonine operon was restricted with Escherichia coli vector plasmid pGA22.
After digestion with HindIII, T4 phage ligase is allowed to act. E. coli K-12 was prepared by a known method using this mixed solution.
Strain Substrain GT-3 (mutant strain lacking three types of aspartokinase, homoserine diaminopimelic acid auxotrophic) is transformed, and a trait that grows on a minimal medium containing diaminopimelic acid and kanamycin which is a selectable marker for pGA22. Select a convertible strain. The plasmid possessed by the transformant that appeared can be isolated from the cultured bacterial cells by a conventional method, and then the DNA fragment produced by treatment with various restriction enzymes can be analyzed by agarose gel electrophoresis to obtain its structure. it can. One of the plasmids thus obtained is pGH2 having the structure shown in FIG. It has already been cloned into a DNA fragment containing Escherichia coli threonioperon, and the restriction enzyme cleavage site has also been located [Cossart, P. et al .: M
olec.Gen.Genet, 175, 39 (1979)]. pGH2 has a matching DNA fragment and certainly contains the threonine operon. The aspartokinase deficiency of the GT-3 strain is complemented by the aspartokinase present on the threonine operon of pGH2, and becomes homoserine and diaminopimelic acid non-auxotrophic.

pEthr1 can be obtained as a recombinant of pGH2 and vector plasmid pCG11 of the genus Corynebacterium and the genus Brevibacterium. pCG11 is a plasmid that was previously invented by the present inventors and applied for a patent (Japanese Patent Application Laid-Open No. 57-134500), and is a Corynebacterium glutamicum 225-57 strain (ATCC31808, FER
Corynebacterium glutamicum 225-250 strain (ATCC31830, FERM-P5939) at the only cleavage site of the restriction enzyme Bgl II in the plasmid pCG1 isolated from M-P5865).
It is a plasmid obtained by ligating the restriction enzyme BamH I fragment containing the streptomycin and / or spectinomycin resistance gene of the plasmid pCG4 isolated from the above using the same cohesive ends of both. The pCG11 is concentrated and isolated from the cultured strain of Corynebacterium glutamicum ATCC39022 which carries it by the method disclosed by JP-A-57-186492. BamHI for pGH2 and BglII for pCG11 by standard method
After digestion with, mix and let T4 ligase act. Next, this DNA mixed solution is used to transform Corynebacterium glutamicum LA201 (homoserine dehydrogenase gene-deficient mutant strain). Corynebacterium glutamicum LA201 is Corynebacterium glutamicum ATCC318
A homoserine (or threonine and methionine) or leucine-auxotrophic mutant strain derived from the lysozyme-sensitive mutant strain L-22 derived from 33 (JP-A-57-186492) by a commonly used mutation treatment. Is based on a defect in the homoserine dehydrogenase gene that metabolizes aspartose semialdehyde to homoserine on the threonine biosynthetic pathway.

For the transformation, a transformation method using protoplasts of the genus Corynebacterium or Brevibacterium, which the present inventors previously invented and applied for a patent (JP-A-57-1864).
92 and JP-A-57-186489, specifically shown in Examples).
Can be carried out. This method is used to transform protoplasts of Corynebacterium glutamicum LA201 strain, and then non-selectively repopulate normal cells on a regeneration medium. Thicken the regenerated bacteria, wash with sterilized physiological saline, and reapply on the minimal medium supplemented with leucine.
Separate the growing colonies.

Among the homoserine-non-requiring strains thus obtained, there are those that simultaneously acquired the pGH2-derived kanamycin resistance trait and the pCG11-derived spectinomycin resistance trait. The plasmid DNA carried by these transformants is the pCG11
It can be isolated and purified from the cultured bacterial cells by the same method as for the isolation of 1., and the structure can be known by analyzing them with various restriction enzymes and agarose gel electrophoresis. The plasmid obtained from one transformant strain is pEthr1, and its restriction enzyme digestion map and its construction process are shown in FIG. pEthr1 is pGH2
It is clear that the BamHI fragment containing the threonine operon of E. coli is a plasmid integrated into pCG11.

From another transformant, a plasmid in which the threonine operon-containing BamHI fragment cleaved from pCG11 was reversed from pEthr1 was obtained. Corynebacterium glutamicum LA20 is retransformed with either recombinant plasmid
1 was associated with the kanamycin and spectinomycin resistance traits and complemented for homoserine auxotrophy, confirming that these transformants carried donor plasmids characterized by various restriction enzyme cleavage sites.

The homoserine auxotrophic phase function is derived from the expression of the Escherichia coli homoserine dehydrogenase gene located on the Escherichia coli threonine operon carried by the recombinant plasmid. The E. coli threonine operon encodes the genes for aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase, which are transcribed as a single transcription unit [Theze, J. and I.
Saint-Girons: J. Bacteriol., 118 , 990 (1974)], and the expression function of all the genes is shown in FIG.
Hind III site of scale 5.4Kb and scale 11.3K in pGH2
It has been revealed that it exists in the region between the BamH I sites of b [Cossart, P. et al .: Molec. Gen. Genet., 175, 39.
(1979)]. Therefore, it is certain that pEthr1 retains the expression function of all genes and that not only the homoserine dehydrogenase gene but also the aspartokinase, homoserine kinase and threonine synthase genes are expressed in Corynebacterium glutamicum carrying it. Is.

An L-isoleucine-producing bacterium belonging to the genus Corynebacterium or the genus Brevibacterium and carrying pEthr1 transforms protoplasts of the genus Corynebacterium or Brevibacterium using pEthr1 in the same manner as described above.
It is obtained as a transformant selected with the spectinomycin and / or streptomycin resistance marker possessed by r1. It can be confirmed that the transformant possesses pEthr1 by analyzing the structure of the plasmid extracted and isolated from it by various restriction enzymes and agarose gel electrophoresis as described above. As a specifically produced L-isoleucine producing strain, Corynebacterium glutamicum K40 (F
ERM P-7160) (FERM BP-455) and Brevibacterium flavum ATCC14067 carrying pEthr1 can be mentioned (see Examples). Corynebacterium glutamicum K40 is Corynebacterium glutamicum ATCC318
An L-isoleucine-producing strain derived from 33 as an S- (2-aminoethyl) -cysteine resistant mutant strain, which is a pEthr1-carrying strain K41 (FERM P-7161) (FERM BP-45).
6) and pE of Brevibacterium flavum ATCC 14067
It has been deposited with the Institute for Microbial Technology, Institute of Industrial Science and Technology, together with thr1 strain K42 (FERM BP-355).

Production of L-isoleucine by the pEthr1-carrying transformant can be carried out by a culture method used for producing L-isoleucine by a conventional fermentation method. That is, if the transformed strain is cultured in an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance, an amino acid, a vitamin, etc. under aerobic conditions while controlling temperature, pH, etc., Since isoleucine is purified and accumulated, it is collected.

Carbon sources include glucose, glycerol, fructose, sucrose, maltose, mannose, starch,
Various kinds of carbohydrates such as starch hydrolyzate, molasses and various organic acids such as polyalcohol, pyruvic acid, fumaric acid, lactic acid and acetic acid can be used. Furthermore, depending on the assimilation ability of the bacterium, hydrocarbons, alcohols, etc. may be used. Especially, molasses is preferably used.

As the nitrogen source, ammonia or ammonium chloride,
Various inorganic and organic ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium acetate, urea and other nitrogen-containing substances, peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or Various substances such as digested products thereof, defatted soybean meal or digested products thereof, and nitrogen-containing organic substances such as pupa hydrolysates can be used.

Further, as inorganic substances, potassium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate,
Manganese sulfate and calcium carbonate are used. The sources of vitamins and amino acids required for the growth of microorganisms are
It may not be added if it is supplied to the medium according to the other medium components as described above.

The culture is performed under aerobic conditions such as shaking culture or aeration-agitation culture. Generally, the culture temperature is preferably 20 to 40 ° C. It is desirable to maintain the pH of the medium during culture at around neutral.
The culture period is usually 1 to 5 days, and L-isoleucine accumulates in the medium.

After completion of the culture, the cells are removed and L-isoleucine is recovered from the culture broth by a known method such as activated carbon treatment or ion exchange resin treatment.

Thus, by using the strains of the genus Corynebacterium and the genus Brevibacterium in which pEthr1 is retained, L-isoleucine can be produced in a higher yield as compared with the non-retained strain.

The utility of the present invention is that a recombinant DNA obtained by recombinantly expressing a gene related to threonine biosynthesis and a vector DNA of Corynebacterium spp. Or Brevibacterium spp. Can be expressed in Corynebacterium spp. Or Brevibacterium spp. When introduced into a species of the genus Urum, it is possible to impart or enhance L-isoleucine-producing ability. Although an example of using the threonine operon of Escherichia coli has been shown in the present specification, the object can be achieved by using a gene involved in threonine biosynthesis of other organisms instead. Therefore, the gene involved in threonine biosynthesis is not limited to the E. coli threonine operon exemplified in the present specification. In addition, the vector plasmid only provides the autonomous replication ability for stably inheriting the gene related to threonine biosynthesis linked as a recombinant. Therefore, not only pCG11 exemplified in the present specification but also a plasmid capable of autonomous replication in the genus Corynebacterium or the genus Brevibacterium is included in the plasmid of the method of the present invention.

The so-called glutamic acid-producing bacterium having a high glutamic acid-producing ability is given various microbial names by each researcher due to its industrial importance, despite having the same main mycological properties. There are various genus names such as Corynebacterium and Brevibacterium. However, these bacterial groups consist of cell wall amino acid composition and DNA.
It was pointed out that they are the same bacterial species because their base compositions are uniform. Furthermore, it has recently been revealed that 70-80% or more of DNA homology exists between these bacterial species, and it is clear that they are very closely related microorganisms.

(Komatsu Y .: Report of the Fermentative Research I
nstitute, No.55, 1 (1980) and Suzuki, K., Kaneko, T.,
and Komagata, K.: Int.J Syst.Baoteriol., 31 ,, 131 (198
1)].

As used herein, Corynebacterium glutamicum K40
And a Brevibacterium flavum ATCC14067, a recombinant of a gene related to threonine synthesis was introduced, and an example of improving L-isoleucine productivity based on the phenotypic expression of the gene was shown, but in view of the above facts, glutamic acid-producing bacteria in general The effect in can be easily inferred. The presence or absence of the effect depends on whether or not the recombinant DNA replicates autonomously in all glutamic acid-producing bacteria and can express a gene involved in threonine synthesis, and there are some differences in the DNA homology between the glutamic acid-producing bacteria. It doesn't matter. However, the fact that these bacterial strains equally retain the functions involved in plasmid replication and gene expression is due to the fact that the present inventors previously filed a patent application (Japanese Patent Laid-Open No. 57-183799) on Corynebacterium glutamicum 225-250 strain. And a plasmid pCG4 having a spectinomycin and / or streptomycin resistance gene can also be replicated in a glutamic acid-producing bacterium such as Corynebacterium spp. And Brevibacterium spp. 57-
186492). Therefore, the bacterial species to which the method for producing an L-isoleucine-producing bacterium by introducing the recombinant DNA containing the gene related to threonine biosynthesis of the present invention can be applied is Corynebacterium glutamicum K40.
And not only Brevibacterium flavum ATCC14067 but also all glutamic acid-producing bacteria including Corynebacterium spp. And Brevibacterium spp.

Examples of the present invention will be shown below.

Example 1. (1) Cloning of E. coli threonine operon-containing DNA fragment Cloning is carried out in an E. coli host vector system. PGA22 used as a vector was prepared by the method of Ann et al. [An, G. et al .: J. Bacteriol., 140, 400 (19
79)], it was isolated from the cultured bacterial cells of Escherichia coli K-12 strain substrains carrying this plasmid. Chromosome D to be the donor DNA
NA is a method for extracting Smith's phenol [Smith, MG: Methods i from the cultured bacterial cells of Escherichia coli K-12 strain Hfr strain (ATCC23740).
n Enzymology, 12 , partA, 545 (1967)].

Restriction enzyme Hind III reaction solution containing 4 μg of pGA22 plasmid DNA (10 mM Tris-HCl, 7 mM MgCl ₂ , 60 mM NaCl, pH 7.5) 60
0.4 units of Hind III for μ (Takara Shuzo 16 units / μ)
Was added and reacted at 37 ° C for 30 minutes, and then heated at 65 ° C for 10 minutes to stop the reaction. Although there are two Hind III cleavage sites in pGA22, a sample digested with Hind III under the same conditions was examined by agarose gel electrophoresis, and it was confirmed that the fragment was cleaved into one fragment. Separately, a restriction enzyme containing 8 μg of chromosomal DNA
Add 4 units of Hind III to 140μ of Hind III reaction mixture,
After reacting at 7 ° C for 60 minutes, the reaction was stopped by heating at 65 ° C for 10 minutes. Both reaction digests were mixed, and T4 ligase buffer [Tris (hydroxymethyl) aminomethane (abbreviated as Tris hereinafter) 660 mM, MgCl ₂ 60 mM, dithiothreitol 100 mM, pH
7.6] 40μ, ATP (5mM) 40μ, T4 ligase (Takara Shuzo, 1 unit / μ) 0.3μ and H ₂ O 120μ
The reaction was carried out at 16 ° C for 16 hours. The reaction mixture was extracted twice with 400 μl phenol saturated with TES buffer (0.03 M Tris, 0.005 M EDTA, 0.05 M NaCl, pH 8.0) and dialyzed against TES buffer to remove phenol.

This ligase reaction mixture was used for E. coli K-12 strain substrain GT-3.
[J. Bacteriol., 117, 133 (1974)] (homoserine and diaminopimelic acid auxotrophic) were used for transformation. GT-
Competent cell 3 is the method of Dagert et al. [Dagert,
M. et al .: Gene 6 , 23 (1979)]. That is, 100
GT-3 strain in 50 ml of L medium supplemented with diaminopimelic acid to μg / ml (medium containing bactotryptone 10 g, yeast extract 5 g, butter sugar 1 g and sodium chloride 5 g in water 1 and adjusted to pH 7.2) 50 ml 660nm with Tokyo photoelectric colorimeter
The cells were cultured at 37 ° C until the absorbance (OD) at 0.5 was 0.5. The culture is cooled in ice water for 10 minutes and then collected by centrifugation. The cells were resuspended in 20 ml of chilled 0.1 M calcium chloride and placed at 0 ° C. for 20 minutes. The cells were re-centrifuged, suspended in 0.5 ml of 0.1 M calcium chloride, and placed at 0 ° C. for 18 hours. 200 μ of the ligase reaction mixture was added to and mixed with 400 μl of the calcium chloride-treated bacterial solution, and the mixture was allowed to stand at 0 ° C. for 10 minutes, then 37
Warm at 5 ° C for 5 minutes. Next, add 9 ml of L medium, 37 ℃
The cells were cultivated with shaking for 2 hours. After centrifuging and washing twice with physiological saline,
M9 minimal agar medium supplemented with 12.5 μg / ml kanamycin (glucose 2 g, NH ₄ Cl 1 g, Na ₂ HPO ₄ 6 g, KH ₂ PO ₄ 3 g, Mg
SO ₄・ 7H ₂ O 0.1g, CaCl ₂・ 2H ₂ O 15mg, thiamin hydrochloride
4 mg and agar 15 g were applied to a medium containing 1 of water and adjusted to pH 7.2, and cultured at 37 ° C. for 3 days. Appeared, only 1
It was confirmed that one colony grows on an agar medium containing 25 μg / ml of ampicillin, 25 μg / ml of chloramphenicol or 25 μg / ml of kanamycin, which are selective markers for pGA22.

Plasmid DNA was isolated from the cultured cells of this transformant by the same method as that used to isolate pGA22. Analysis of this plasmid DNA by restriction enzyme digestion and agarose gel electrophoresis revealed that it had the structure shown as pGH2 in FIG. The DNA fragment inserted into pGA22 is a cloned E. coli operon-containing DNA fragment [Cossart, P. et al .:
Molec. Gen. Genet., 175, 39 (1979)], it has been confirmed that pGH2 contains the threonine operon because it has the same restriction enzyme cleavage site.

(2) In vitro recombination of pCG11 and pGH2 Corynebacterium glutamicum LA1 harboring pCG11
03 / pCG11 (ATCC39022) 400ml NB medium (powder broth)
20 g and yeast extract 5 g were added to water 1 and the pH was adjusted to 7.2), and the OD was grown to about 0.8. After collecting the cells from the culture solution and washing with TES buffer, lysozyme solution (25% sucrose, 0.1M NaCl, 0.05M Tris, 0.8mg / ml lysozyme: pH
8.0) was suspended in 10 ml and reacted at 37 ° C. for 4 hours. 2.4 ml of 5M NaCl, 0.6 ml of 0.5M EDTA (pH8.5), 4.4 ml of a solution containing 4% sodium ularyl sulfate and 0.7M NaCl in the reaction solution.
Were sequentially added, mixed gently, and then placed in ice water for 15 hours. Transfer all the lysate to a centrifuge tube and transfer to 69,400 at 4 ℃ for 60 minutes.
The supernatant was collected by centrifugation at × g. Polyethylene glycol (PEG) 6,000 with a weight percentage of 10%
(Made by Hanai Chemical Co., Ltd.), gently mix and dissolve,
It was placed in ice water. After 10 hours, the pellet was recovered by centrifugation at 1,500 xg for 10 minutes. 5 ml of TES buffer was added to gently redissolve the pellet, then 2.0 ml of 1.5 mg / ml ethidium bromide was added, to which cesium chloride was added and gently dissolved to adjust the density to 1.580. 105,0 this solution
It was subjected to ultracentrifugation at 00 × g and 18 ° C. for 48 hours. The circular DNA covalently closed by this density gradient centrifugation was found as a high density band in the lower part of the centrifuge tube upon irradiation with ultraviolet light. By removing this band from the side of the centrifuge tube with a syringe, plasmid pCG1
1 was separated. Then, the separated liquid is treated with an equal volume of isopropyl alcohol solution [volume percentage 90% isopropyl alcohol, 10% TES buffer solution (containing a saturated dissolved amount of cesium chloride in this mixed solution)] five times to extract and remove ethidium bromide. Then, it was dialyzed against TES buffer. Thus, pCG11 plasmid DNA was obtained.

Using this pCG11 DNA, DNA fragments generated by single digestion with various restriction enzymes and multiple digestion with multiple restriction enzymes were analyzed by agarose gel electrophoresis, and the result of determining the molecular weight and the cleavage site for each restriction enzyme in the plasmid molecule, It was found to have the restriction enzyme cleavage map shown in FIG.

Restriction enzyme Bgl II reaction buffer containing ₂ μg of pCG11 plasmid DNA (10 mM Tris-HCl, 7 mM MgCl ₂ , 60 mM NaCl, 7 mM 2
-Mercaptoethanol, pH 7.5) 2 units of Bgl per 100μ
II (Takara Shuzo Co., Ltd., 6 units / μ) was added and reacted at 37 ° C. for 60 minutes. Separately, a restriction enzyme BamHI reaction buffer containing 2 μg of pGH plasmid DNA (10 mM Tris-HCl, 7 mM MgCl ₂ ,
100 units of 100 mM NaCl, 2 mM mercaptoethanol, 0.01% bovine serum albumin, pH 8.0 was added with 2 units of BamHI (Takara Shuzo Co., Ltd., 6 units / μ) and reacted at 37 ° C. for 60 minutes. After heating both digests at 65 ° C for 10 minutes, mix and add T4 ligase buffer 40μ, ATP (5mM) 40μ, T4 ligase (Takara Shuzo, 4 units / μ) 0.2μ and H ₂ O 120μ. The mixture was reacted at 12 ° C for 16 hours. The mixture was extracted twice with 400 μl phenol saturated with TES buffer and dialyzed against TES buffer to remove phenol.

(3) Acquisition of pEthr1 Transformation was carried out using protoplasts of Corynebacterium glutamicum LA201 strain (homoserine leucine-requiring strain). Corynebacterium glutamicum LA201
The seed culture of the strain was inoculated into NB medium and cultured at 30 ° C. with shaking. OD
At 0.6, the cells were placed in RCGP medium [glucose 5 g,
Casamino acid 5g, yeast extract 2.5g, K ₂ HPO ₂ 3.5g, KH ₂ PO ₄
1.5g, MgCl ₂・ 6H ₂ O 0.41g, FeSO ₄・ 7H ₂ O 10mg, MnSO ₄・
_{4~6H 2 O 2mg, ZnSO 4 ·} 7H 2 O 0.9mg, (NH 4) 6 Mo 7 O 24 · 4
H ₂ O 0.04 mg, biotin 30 μg, thiamine hydrochloride 2 mg, disodium succinate 135 g, polyvinylpyrrolidone (molecular weight 1
0,000) 30g in water 1] in 1mg / ml lysozyme solution (pH 7.6) at about 10 ⁹ cells / ml, transfer to L-type test tube and 30 ° C for 5 hours The reaction was gently shaken to form protoplasts.

Transfer 0.5 ml of this protoplast bacterium solution to a small test tube and
Centrifuge at xg for 5 minutes and then use TSMC buffer [10 mM magnesium chloride, 30 mM calcium chloride, 50 mM Tris, 400 mM sucrose, p.
H7.5] was resuspended in 1 ml, centrifuged and washed, and then resuspended in 0.1 ml of TSMC buffer. To this bacterial solution was added 2 times the concentration of TSMC buffer and 100 μ of 1: 1 mixture of the above-mentioned ligase reaction DNA mixture and mixed, and then a solution containing 20% PEG6,000 in TSMC buffer 0.8m
l was added and mixed. After 3 minutes, RCGP medium (pH7.2) 2ml
Was added, the supernatant was removed by centrifugation at 2,500 × g for 5 minutes, and the precipitated protoplasts were suspended in 1 ml of RCGP medium, and then gently shaken at 30 ° C. for 2 hours. Then 0.1 ml of this protoplast suspension was added to kanamycin 4
It was smeared on RCGP agar medium containing 00 μg / ml (RCGP medium containing 1.4% agar, pH 7.2), and cultured at 30 ° C. for 6 days.

Kanamycin-resistant transformants grown on the entire surface of the agar medium were scraped off, washed with physiological saline by centrifugation, and then leucine 50 μg /
Minimal agar medium M1 (glucose 10 g, NH ₄ H ₂ PO _{4
1g, KCl 0.2g, MgSO 4 ·} 7H 2 O 0.2g, FeSO 4 · 7H 2 O 10mg, M
nSO 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 0.09mg, (NH 4) 6 Mo 7 O 24 · 4
H ₂ O 0.04 mg, biotin 50 μg, p-aminobenzoic acid 2.5 mg,
Thiamine hydrochloride 1mg, agar 16g in water 1 pH7.2
The culture medium prepared in step 1) was applied again and cultured at 30 ° C. for 3 days. From the emerged colonies, strains capable of growing on NB agar medium containing 20 μg / ml of kanamycin and 100 μg / ml of spectinomycin were obtained.

Three arbitrarily selected strains were grown in 400 ml NB medium until the OD reached about 0.8, and after collecting the cells, the plasmid was isolated from the cultured cells by ethidium bromide cesium chloride density gradient centrifugation as described in Example 1 (2). . From any stock
40-55 μg of plasmid DNA was obtained.

These plasmid DNAs were analyzed by restriction enzyme digestion and agarose gel electrophoresis, and the molecular weight and restriction enzymes Pst I, EcoR I,
And Hho I breakpoints were identified. The plasmid obtained from one strain was named pEthr1 and its structure is shown in FIG.
pEthr1 is BamH containing pGH2 threonine operon in pCG11
It was found to have a structure in which I cut pieces were bound. Of the remaining two strains, one strain possesses the same plasmid as pEthr1, but the other strain has a plasmid in which the binding direction of the BamHI fragment containing the threonine operon of pGH2 is inserted in the opposite direction to pEthr1. I have it.

Using these plasmid DNAs, Corynebacterium
As a result of retransformation of the glutamicum LA201 strain in the same manner as described above, homoserine non-auxotrophy was obtained at a high frequency (about 10 ^-3 per regenerated live bacterium), and they all acquired the kanamycin and spectinomycin resistance traits. , And possessed the same plasmid as the donor plasmid characterized by various restriction enzyme cleavage modes.

(4) Production of L-isoleucine by pEthr1-carrying strain pEthr1 was introduced by transforming protoplasts of Corynebacterium glutamicum K40 and Brevibacterium flavum ATCC14067. Corynebacterium glutamicum K40 and Brevibacterium flavum ATCC140
67 was cultured in NB medium at 30 ° C. for 16 hours with shaking, and its seed culture was 0.
1 ml to 10 ml of SSM medium (glucose 10 g, NH ₄ Cl 4 g, urea 2 g,
Yeast extract 1g, KH ₂ PO ₄ 1g, K ₂ HPO ₄ 3g, MgCl ₂ 6H ₂ O 0.
4g, FeSO ₄ / 7H ₂ O 10mg, 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
0.09mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 0.04mg, biotin 30μg,
A L-shaped test tube containing 1 mg of thiamine hydrochloride in purified water 1 and adjusted to pH 7.2] was inoculated and shake-cultured at 30 ° C. in a mono-type culture tank. Penicillin G was added to 0.5 unit / ml when the OD reached 0.15. Continue culturing and collect the cells when the OD reaches about 0.6.
The P medium was suspended in 2 ml of a solution (pH 7.6) containing 1 mg / ml of lysozyme, transferred to an L-shaped test tube, and gently shaken at 30 ° C. for 14 hours to form protoplasts.

1 ml of this protoplast bacterium solution is put in a small test tube and 2,500 xg
The precipitate was resuspended in 1 ml of TSMC buffer and washed by centrifugation (2,500 × g), and 0.1 ml of TSMC buffer was added to resuspend. To this was added 100 μl of a 1: 1 mixture of the TSMC buffer solution having a double concentration and the pEthr1 DNA solution isolated above, and the mixture was mixed, and transformation was carried out via PEG6,000 in the same manner as in Example 1 (3). After the phenotype, 0.1 ml was spread on RCGP agar medium containing 400 μg / ml spectinomycin at 30 ℃.
The cells were cultured for 10 days. From the emerged colonies, strains capable of growing on NB agar medium containing 100 µg / min of spectinomycin and 20 µg / ml of kanamycin were obtained.

A transformant strain simultaneously resistant to spectinomycin and kanamycin was shake-cultured in 400 ml SSM medium, and OD was 0.
Penicillin G was added to reach 0.5 unit / ml at 15 and further cultured to an OD of 0.65. From the collected cells, a method similar to the method for isolating pCG11 in Example 1 (2) was used. The plasmid was isolated. As a result of analyzing these plasmids by restriction enzyme digestion and agarose gel electrophoresis, it was found that some plasmids have the same structure as pEthr1 characterized by various restriction enzyme cleavage modes. Such a transformant is Corynebacterium glutamicum K41 (FERM BP-4
56), Brevibacterium flavum K42 (FERM BP-3
55).

The L-isoleumine production test of Corynebacterium glutamicum K40, Brevibacterium flavum and their pEthr1 carrying strains was performed. Seed culture 0.5 ml that had been shake-cultured in NB medium for 16 hours at 30 ° C was added to production medium [glucose 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
1g, FeSO ₄・ 7H ₂ O 10mg, MnSO ₄・ 4-6H ₂ O 10mg, biotin 100μg, calcium carbonate 30g in water 1, pH 7.2
To the test tube containing the prepared medium] and cultured at 30 ° C. for 72 hours with shaking. After culturing, the culture filtrate was subjected to paper chromatography to develop ninhydrin, and then colorimetrically determined to measure the amount of L-isoleucine produced.

The results are shown in Table 1.

Escherichia coli-derived aspartokinase, homoserine dehydrogenase, homoserine kinase and a gene encoding threonine synthase were introduced into a microorganism belonging to the genus Corynebacterium or Brevibacterium, and expressed to give about 2.3 in Corynebacterium glutamicum. Double (2.7mg / ml), Brevibacterium
In Flavum, the one in which a detectable amount of L-isoleucine could not be excreted out of the cells showed a marked improvement in productivity of 0.8 mg / ml.

Japanese Unexamined Patent Publication (Kokai) No. 58-893 discloses the production amount of L-isoleucine by a coryneform-type transformed microorganism in which a gene region involved in isoleucine antagonist resistance is incorporated, but the maximum production amount is 1.45 mg / ml. The present invention has shown a production result superior to that of JP-A-58-893.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme digestion map of pEthr1 and the construction process thereof. Bgl II / BamH I indicated by a broken line is a ligation site at the same sticky ends generated by both restriction enzyme cleavages. The restriction enzymes used to construct the restriction map were Pst I, EcoR I and Xho I.
Is. The molecular weight of plasmids is expressed in kilobases (Kb).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location (C12N 15/09 C12R 1:15) (C12N 15/09 C12R 1:13) (C12P 13/06 C12R 1:15) (C12P 13/06 C12R 1:13) C12R 1:15) (C12N 15/00 A C12R 1:13) (56) Reference JP-A-58-893 (JP, A) JP-B 37-47954 (JP, B1) Japanese Patent Publication Sho 43-14938 (JP, B1) Amino Acid / Nucleic Acid Roundtable "Amino Acid Fermentation Each" (Sho 47.4.5) Kyoritsu Shuppan P. 170

Claims (3)

[Claims] 1. A recombinant DNA of a vector DNA and a DNA fragment containing a gene encoding aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase derived from a microorganism belonging to the genus Escherichia.
A microorganism belonging to the genus Corynebacterium or Brevibacterium that possesses L-isoleucine and has the ability to produce L-isoleucine is cultured in a medium, and L-isoleucine is produced and accumulated in the culture, and L-isoleucine is produced from the culture. A method for producing L-isoleucine, which comprises: 2. The method according to claim 1, wherein the vector is a vector derived from a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium, and derivatives thereof. 3. The vector is pCG1, pCG2, pCG4, pCG11, p
The manufacturing method according to claim 1, wherein the manufacturing method is selected from CE54 and pCB101.