JPH0523752B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing tyrosine by recombinant DNA techniques. More specifically, the present invention provides a combination of a DNA fragment containing a gene encoding at least one enzyme among 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, chorismate miutase, and prephenate dehydrogenase, and vector DNA. replacement body
A transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using DNA is cultured in a medium, and tyrosine is produced and accumulated in the culture. The present invention relates to a method for producing tyrosine, which comprises collecting tyrosine from. As a method for directly producing L-tyrosine by fermentation, a method using mutant strains of bacteria such as Corynebacterium and Brevibacterium such as phenylalanine auxotrophs and tyrosine-resistant strains is known. [Journal of Agricultural Chemistry 50 (1) R79-R87,
(1976)] 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 a suitable cloning site, while also having high efficiency. Developed a transformation system and filed a patent application (Japanese Unexamined Patent Publications No. 57-183799, No. 57-186492, No. 57-
186489). In addition, the present inventors have used the plasmid vector to perform known in vitro recombination.
Using DNA technology (US Patent 4237224), we link DNA fragments containing exogenous genes involved in the biosynthesis of amino acids such as glutamic acid, lysine, tryptophan, histidine, and phenylalanine;
When Corynebacterium glutamicum strain L-22 or its derivative strain was transformed using the developed transformation system, the foreign gene was expressed in the host and used to increase the production of the amino acid. He discovered that it was possible to do this and filed a patent application (Japanese Patent Applications 1987-126789, 1986-25397, 58-
25398 and 58-94392). As a result of further research, the present invention was completed by discovering that a strain obtained by the same method regarding tyrosine can accumulate a significant amount of tyrosine in the culture medium. The present invention will be explained in detail below. The present invention provides a recombinant DNA comprising a DNA fragment containing a gene encoding at least one enzyme among 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, chorismate miutase, and prephenate dehydrogenase, and vector DNA. Provided is a method for producing tyrosine by culturing a transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium in a medium. Examples of DNA fragments containing genes (target genes) used in the present invention include DNA fragments containing genes derived from prokaryotes, viruses, bacteriophages, or plasmids and involved in tyrosine production. The biosynthetic pathway of aromatic amino acids and its regulation in microorganisms have been studied in detail in glutamate-producing bacteria such as Escherichia coli, Bacillus subtilis, Corynebacterium, and Brevibacterium [Journal of the Japanese Society of Agricultural Chemistry 50 (1 ), R79〜
R87, (1976) and Ann.Rev.Biochemistry
47, 533 (1978)]. The gene related to tyrosine biosynthesis in the present invention is DNA that carries genetic information for at least one enzyme among the enzymes directly or indirectly involved in the biosynthesis of these aromatic amino acids.
In particular, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (3-deoxy-D-arabino-
It carries the genetic information for enzymes such as heptulosonate 7-phosphate (hereinafter referred to as DAHP) synthase (hereinafter referred to as DAHPase), chorismate miutase (hereinafter referred to as CMase), prephenate dehydrogenase (hereinafter referred to as PDGase), and pretyrosine aminotransferase. DNA is preferred.
Furthermore, among these genes, genes in which feedback inhibition is not present (including cases in which it is not present in the first place) or genes in which feedback inhibition is not present can also be used. As the target gene of the present invention, any gene that is expressed in a microorganism belonging to the genus Corynebacterium or Brevibacterium and acts to enhance the activity of at least one enzyme in the tyrosine biosynthesis system can be used. can. Sources of this gene include bacterial species used for fermentative production of amino acids, especially aromatic amino acids, bacterial species whose biosynthesis of aromatic amino acids and its regulatory mechanism have been elucidated, and bacterial species whose genetics have not been sufficiently elucidated. Bacterial species such as those of the genus Ethscherichia, Corynebacterium, Brevibacterium, Microbacterium, Bacillus, Staphylococcus, Streptococcus, or Serratia are preferred. Among the genes derived from the bacterial strains to which it belongs, preferred examples include genes related to the production of tyrosine and the metabolic system involved in its production. In the examples of the present invention, Escherichia coli strain JA194 [Proc.Natl.
Acad. Sci., 74 , 487-491 (1977)] was used. The vector used in the present invention must be capable of autonomous replication within host bacterial cells. As a specific example, pCG1 (Japanese Patent Laid-Open Publication No. 1989-1995), which the present inventors created by collecting or deriving from a microorganism belonging to the genus Corynebacterium,
134500), pCG2 (Japanese Patent Application Laid-Open No. 1982-35197), pCG4 (Japanese Patent Application Publication No. 1837-183799), pCE51, pCE52 (Japanese Patent Application Publication No. 1983-18379)
126789), pCE53 (patent application 1982-25398), pCE54,
Examples include pCG11 and pCB101. Bacterial strains carrying these plasmids have been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and the American Type Culture Institute, USA, with the following deposit numbers.
Deposited in a collection. Plasmid FERM-P ATCC pCG1 5865 31808 pCG2 5954 31832 pCG4 5939 31830 pCE54 - 39019 pCG11 - 39022 pCB101 - 39020 Plasmid pCE51 is preferably used. pCE51 can be created as follows. First, pCG1 was extracted from Corynebacterium glutamicum strain 225-57 (FERM-P5865,
ATCC31808) by the method disclosed in the above patent application specification (Japanese Patent Application Laid-open No. 134500/1983).
pGA22 is obtained from cultured cells of E. coli harboring pGA22 using a commonly used method [An, G. etal., J. Bacteriol., 140 ,
400 (1979)]. Plasmid pCG1 is linearized with restriction enzyme Bg1, and the fragment containing the kanamycin resistance (Km ^R ) gene obtained by digesting pGA22 with restriction enzyme BamH is ligated to this using the same cohesive ends of both. . Selection of pCE51 from this DNA mixture was derived from pGA22 with ^KmR
This can be accomplished by isolating transformed strains of Corynebacterium or Brevibacterium species that have only the following, and analyzing the plasmids possessed by these transformed strains. Transformation using a DNA hybrid is carried out using a transformation method using protoplasts of Corynebacterium and Brevibacterium species, for which the present inventors previously applied for a patent (Japanese Patent Application Laid-Open No. 57-186492 and Japanese Patent Application Publication No. 186492).
57-186489). Kanamycin (Km) is used to select transformed strains. Transformants can be obtained by isolating colonies formed on a hypertonic agar medium containing a drug (usually Km: 100-800 μg/ml) in a DNA-free system that does not allow recipient protoplasts to revert to normal cell proliferation, or , once non-selectively revert to normal cells on a regeneration medium, the suspension is collected, and this suspension is treated with a drug (usually
Km: 2-25 μg/ml) by isolating colonies growing on an agar medium containing Km: 2-25 μg/ml). Some of the transformed strains selected for kanamycin resistance have also acquired other drug resistance traits derived from pGA22, but these are excluded because they do not seem to contain the desired plasmid. The plasmid DNA possessed by the thus obtained transformed strain can be isolated and purified from the cultured bacterial cells by the method disclosed by the present inventors in JP-A-57-134500 and JP-A-57-186489, and further purified with various restriction enzymes. The structure can be determined by the standard method of analyzing the DNA fragments produced by digestion using agarose gel electrophoresis.
The plasmid isolated from one transformed strain
It is pCE51. pCE51 is a plasmid approximately 6 Kb in size with restriction sites Hinc, Hind, SmaI, XhoI, and EcoR.
etc., giving the Km ^R phenotype. Plasmids can be collected from plasmid-carrying strains, for example, according to the methods described in JP-A-57-134500, JP-A-57-183799, and JP-A-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 joining (ligase reaction) donor DNA containing the target gene and vector DNA (Japanese Patent Laid-Open No. 1983-
No. 126789, see USP 4237224). 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, USPatent 4237224, Genetic manipulation experimental methods [edited by Yasutaka Takagi, Kodansha Scientific (1980)], Methods in
Enzymology 68 , Recombinant DNA, edited
by Ray Wu, Academic Press 1979, Japanese Patent Publication No. 1983
-126789. 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. The following strains are preferred. Deposit number FERM-P ATCC Corynebacterium glutamicum L-15
5946 31834 Corynebacterium glutamicum K-387087 Corynebacterium glutamicum K-437162 Corynebacterium herkyulis 13868 Corynebacterium herkyulis L-103
5947 31866 Brevibacterium deivaricatum L-204
5948 31867 Brevibacterium lactofamentum
13869 Brevibacterium lactofamentum L
-312 5949 31868 Brevibacterium flavum 14067 Tyrosine-producing strains mutated from these strains can be used as more preferred hosts. As described above, the productive mutant strain can be obtained as a strain that requires amino acids, is resistant to amino acid analogs, or has both of these. 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 of protoplasts with recombinant DNA, 3) regeneration of protoplasts back to normal cells, and selection of transformed strains. The specific method is disclosed in Japanese Patent Application Laid-Open No. 186492, 57-
186489, 58-105999, etc. may be followed. Tyrosine can be produced by culturing the thus obtained transformed strain using a culture method conventionally used for producing tyrosine by fermentation. That is, if the transformed strain is cultured in a normal medium containing carbon sources, nitrogen sources, inorganic substances, amino acids, vitamins, etc. under aerobic conditions while controlling temperature, pH, etc., Tyrosine is produced and accumulated, so it is collected. As carbon sources, carbohydrates such as glucose, fructose, sucrose, maltose, mannose, sorbitol, mannitol, sugar alcohols, glycerol, starch, starch hydrolyzate, molasses, etc. can be used, and pyruvic acid, lactic acid, acetic acid,
Various organic acids such as fumaric acid and gluconic acid, and lower alcohols such as ethanol can also be used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate,
Various inorganic and organic ammonium salts such as ammonium acetate or urea and other nitrogen-containing substances as well as peptones, NZ-amines, meat extracts,
A variety of nitrogenous organic substances such as yeast extract, corn steep liquor, casein hydrolyzate, fruit meal or its digested product, and pupa hydrolyzate can be used. Furthermore, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate,
Sodium chloride, ferrous sulfate, manganese sulfate and calcium carbonate are used. Vitamins, amino acid sources, etc. necessary for the growth of microorganisms do not need to be added as long as they are supplied to the medium in accordance with the other medium components as described above. Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Generally, a culture temperature of 20 to 40°C is suitable. It is desirable to maintain the pH of the medium during culture near neutrality. A significant amount of tyrosine accumulates in the medium during the culture period, which usually lasts from 2 to 5 days. After the culture is completed, the bacterial cells are removed and tyrosine is recovered from the culture solution by a known method such as activated carbon treatment or ion exchange resin treatment. Although the so-called glutamate-producing bacteria that have a high glutamate production ability share the same main mycological properties, various researchers have given them different names due to their industrial importance, and the genus name has been changed. Even the genus Corynebacterium and Brevibacterium are diverse. However, it has been pointed out that these bacterial groups are the same bacterial species because the amino acid composition of their cell walls and the base composition of their DNA are uniform. Furthermore, it has recently been revealed that there is more than 70-80% DNA homology between these bacterial species, making it clear that they are very closely related microorganisms [Komatsu, Y.: Report of
the Fermentative Research Institute, No.55,
1 (1980), and Suzuki, K., Kaneko, T.,
and Komagata, K.: Int.J.Syst.Bacteriol., 31 ,
131 (1981)]. Although the usefulness of the present invention was demonstrated using a Corynebacterium glutamicum-derived strain as a host, in light of the above facts, it is easily inferred that the present invention can be applied to all glutamic acid producing bacteria as is. In order for recombinant DNA to be stably retained and expressed in these bacterial species, slight differences in host bacterial properties such as DNA homology are not a problem, and these bacterial species are capable of autonomously replicating the plasmid and transgene It is sufficient if it has a function that enables expression. However, the fact that these bacterial species share both of these functions was demonstrated by the present inventors, which was isolated from Corynebacterium glutamicum 225-250, for which we had previously applied for a patent (Japanese Patent Laid-Open No. 183799). and/or plasmid pCG4 carrying a spectinomycin resistance gene can be similarly replicated in glutamate-producing bacteria such as Corynebacterium and Brevibacterium species, and the resistance gene can be expressed (JP-A-57-1989-1).
186492). Furthermore, as shown in the patent filed by the present inventor (Japanese Unexamined Patent Publication No. 58-126789), the histidine-producing plasmid is compatible with Corynebacterium herkyulis, Brevibacterium
flavum, and Brevibacterium lactofamentum. Therefore, host bacteria to which the present invention can be applied include not only Corynebacterium glutamicum but also all glutamic acid-producing bacteria including Corynebacterium and Brevibacterium species. Examples of the present invention are shown below. Example 1 (1) Preparation of chromosomal DNA and plasmid DNA: Escherichia coli (hereinafter referred to as E.coli) JA194 strain (Proc.
Natl.Acad.Sci., 94 , 487-491 (1977)] chromosomal DNA was prepared by the following method. 400ml of L-broth (10g of bacto-tryptone,
Bacto yeast extract 5g, NaCl 5g to 1 water
The seed culture was inoculated into a medium (hereinafter referred to as LB) containing 100 ml of seed culture and adjusted to pH 7.0, cultured with shaking at 37°C, and grown to late logarithmic phase. Collect bacteria from the culture solution, and use the method of Saito et al.
Biochim. Biophys. Acta, 72 619 (1963)]. pBR322 used as a vector was isolated from cultured cells of the E. coli JA194 strain by the following method. The seed culture was inoculated into 400 ml LB containing 100 μg/ml ampicillin, cultured with shaking at 37° C., and grown to late logarithmic phase. After collecting bacteria from the culture solution, the bacterial cells were lysed according to the method of Tanaka et al. [J. Bacteriol. 121 354-362 (1975)]. The obtained lysate was heated at 28,000 rpm and 4°C.
The mixture was centrifuged for 1 hour and the supernatant was collected. To the supernatant, 1/5
A 50% (w/v) polyethylene glycol (PEG) 6000 aqueous solution was added thereto, mixed gently, and then left at 4°C overnight. The resulting precipitate was heated at 4°C.
Collect by centrifugation at 3000 rpm for 5 minutes, add 5 ml of TE buffer (10mM Tris・HCl, 1mM EDTA・Na ₂ PH
7.5), add 1 ml of ethidium bromide at a concentration of 1.5 mg/ml, and add exactly 7.5
ml. After adding 7.875 g of CsCl to this solution and completely dissolving it, it was centrifuged at 105,000 xg and 20°C for 40 hours. The plasmid band detected under ultraviolet irradiation was removed with a syringe, the ethidium bromide was extracted three times with isopropanol containing 15% TE (V/V) buffer, and then the plasmid band was extracted with TE buffer overnight at 4°C. The cells were dialyzed and the dialysate was used as plasmid DNA. (2) Cloning of DNA fragments containing genes encoding DAHPase, CMase, and PDGase: 3μg of pBR322 plasmid DNA prepared above
Add 5 units each of EcoR and Hind (manufactured by Takara Shuzo Co., Ltd.) to 100 μl of a restriction enzyme Hind reaction solution containing After each reaction was carried out at 37°C for 60 minutes, the reaction was stopped by heating at 65°C for 10 minutes. After mixing both reaction solutions, add 40μl of T4 ligase buffer (Tris 660mM, _MgCl2 66mM, dithiothreitol 100mM, Ph7.6), ATP to this mixture.
(5mM) 40μl, T4 ligase (Takara Shuzo Co., Ltd., 1
Add 0.4 μl (units/μl) and 120 μl of H ₂ O,
The reaction was allowed to take place at ℃ for 16 hours. This ligase reaction mixture was subjected to transformation.
E.coli strain AB3248 lacking DAHPase [J.Bact. 93 237-244] was used as the recipient strain for transformation.
(1967)] or E. coli AT2273 strain lacking the tyrA gene (CMase) (J. Bact. 91 1494
(1966)] was used. The seed culture was inoculated into LB and M.
Competent cells were prepared according to the method of Dagert et al. [Gene, 623-28 (1979)]. 50 μl of the ligase reaction mixture was added to 0.2 ml of a solution containing 10 ⁹ competent cells/ml, and the mixture was left on ice for 10 minutes. Then, after heat treatment at 37℃ for 5 minutes, 2ml of LB
was added and allowed to stand at 37°C for 90 to 120 minutes. after that,
After centrifugally washing the bacterial cells twice with physiological saline, each
50 μg/ml histidine, proline, arginine,
M9 plate medium containing isoleucine and valine [NH ₄
Cl 1g, Na ₂ HPO ₄ 6g, KH ₂ PO ₄ 3g, NaCl
5g, _MgSO4・_7H2O 0.1g, _CaCl2・_2H2O
0.05g, glucose 3g, vitamin B ₁ 4mg in 1 water
J. Bact. 121 354-362 (1975)] was applied to a medium containing 1.5% agar and adjusted to pH 7.0. Colonies grown on M9 plate medium were each treated with ampicillin 100 μg/ml and tetracycline 20 μg/ml.
Colonies that grew on the ampicillin-containing medium and did not grow on the tetracycline-containing medium were selected. Containing histidine, proline, arginine, isoleucine, and valine selected in this way
Plasmid DNA was isolated from the ampicillin-resistant, tetracycline-sensitive transformant grown on M9 plate medium in the same manner as described above. The plasmid pEaroF1 obtained from one of the transformed strains was digested with various restriction enzymes and analyzed by agarose gel electrophoresis. As a result, the larger EcoR-
About 4.2 Kb of EcoR-Hind is added to the Hind cleavage fragment.
It turned out to be a plasmid into which cut DNA fragments had been inserted. Using the obtained pEaroF1, perform the same procedure as above.
When E.coli AB3248 and E.coli AT2273 strains were transformed, both strains contained histidine, proline, arginine, isoleucine, and valine.
They were found to have the same plasmid as pEaroF1, growing on M9 medium and simultaneously becoming resistant to ampicillin. The above results demonstrate that the approximately
On the 4.2Kb DNA fragment, DAHPase, CMase,
This indicates the existence of a gene encoding PDGase. Furthermore, pEaroF1 is an EcoR protein as shown in Figure 1.
, BamH, Hind, etc., and compared with the plasmid pKB45 reported by G. Zurawski et al. [Proc. Natl. Acad. Sci. USA 75 4271 (1978)], pEaroF1 The approximately 4.2Kb inserted DNA fragment contains E.coli
The aroF, tyrA, and pheA genes were found to exist. (3) Subcloning of aroFtyrA gene: Plasmid pEaroF1DNA prepared above
A DNA fragment of the aroFtyrA gene portion was obtained and ligated to the common vector pCE51 for E. coli and Corynebacterium glutamicum. pCE51 is a Corynebacterium glutamicum plasmid for which the present inventors previously applied for a patent.
pCG1 (JP 57-134500) and E. coli plasmid pGA22 [An, G. et al: J. Bacterikl.) 140 , 400
(1979)]. For details, there is only one Bgl on pCG1.
The cleavage site and the BamH fragment containing the kanamycin resistance gene of pGA22 were ligated using the same cohesive ends of both restriction enzymes. In fact, it can be produced by the method for which the present inventors previously applied for a patent (Japanese Patent Laid-Open Nos. 58-1105999 and 58-126789). 5 units of Hinc (manufactured by Takara Shuzo Co., Ltd.) were added to 100 μl of a restriction enzyme Hinc reaction solution containing 3 μg of plasmid pEaroF1 DNA, and the mixture was reacted at 37° C. for 60 minutes. Also,
Add 0.3 units to 100 μl of restriction enzyme Hinc reaction solution containing 3 μg of plasmid pCE51 DNA prepared as above from E. coli JA194 strain carrying pCE51.
Add Hinc and incubate at 37℃ for 60 minutes, pCE51
Of the two Hinc cleavage sites on the top, cleavage was performed at only one site. After the reaction, mix both reaction solutions and add T4 ligase buffer (Tris) to this mixture.
660mM, MgCl ₂ 66mM, dithiothreitol
100mM, PH7.6) 40μl, ATP (5mM) 40μl, T4 ligase (Takara Shuzo Co., Ltd., 1 unit/μl) 0.4μl and
120 μl of H ₂ O was added, and the mixture was reacted at 12° C. for 16 hours.
After the reaction, the reaction was stopped by heating at 65°C for 10 minutes. This ligase reaction mixture is subjected to transformation.
E. coli AB3248 as a recipient strain for transformation.
Use stocks. Transformation was performed in the same manner as above,
Colonies growing on M9 plate medium containing histidine, proline, arginine, isoleucine, and valine were obtained. From the obtained colonies, kanamycin
Colonies growing on LB plates containing 20 μg/ml were selected. Histidine, proline, obtained in this way,
Plasmid DNA was isolated from a kanamycin-resistant transformant grown on M9 plate medium containing arginine, isoleucine, and valine in the same manner as described above. Plasmid pKm1aroF1 obtained from one of the transformed strains was digested with various restriction enzymes and analyzed by agarose gel electrophoresis. As a result, approximately 3.8 Kb of pEaroF1 was found at one Hinc cleavage site of pCE51.
It turned out to be a plasmid into which a Hinc-cleaved DNA fragment containing aroF.tyrA was inserted. Plasmid obtained as above
pKm1aroF1 has cleavage patterns of various restriction enzymes as shown in FIG. (4) Tyrosine and tyrosine analog resistance plasmid pKm1aroF1 from pKm1aroF1 carrying strain
Obtaining -m-18: AB3248 strain carrying pKm1aroF1 was grown in LB medium containing 20 μg/ml of kanamycin until late logarithmic growth. After centrifuging and washing the bacterial cells twice with 50mM Tris-maleate buffer (PH6.0), N-methyl-N'-nitro-N-nitrosoguanidine was added.
It was treated for 30 minutes at room temperature in 50mM Tris-maleate buffer (PH6.0) containing 400μg/ml. Treated bacterial cells in 50mM Tris-maleate buffer (PH6.0)
After centrifugal washing twice, wash the washed bacterial cells with kanamycin.
The cells were cultured in LB medium containing 20 μg/ml at 30° C. for 16 hours, and plasmid DNA was isolated in the same manner as described above. Using the isolated plasmid, E. coli strain AB3248 was transformed in the same manner as described above. Selection of transformed strains was performed on M9 plate medium containing 0.25 mg/ml of tyrosine and 50 μg/ml each of histidine, proline, arginine, isoleucine, and valine. From the colonies that appeared, 0.25 mg/ml of tyrosine, histidine, proline, arginine, isoleucine,
Colonies that could grow on M9 plates each containing 50 μg/ml of valine and LB plates containing 20 μg/ml of kanamycin were selected. The colonies obtained in this way also contain the tyrosine analog 3-aminotyrosine (3AT).
(manufactured by Sigma) 0.2mg/ml and each of histidine, proline, arginine, isoleucine, and valine
Can be grown on M9 plate medium containing 50μg/ml, 3AT
It turned out to be resistant. Plasmids obtained from transformed strains thus obtained can confer tyrosine or tyrosine analog resistance to bacteria. E. coli carrying one such plasmid, pKm1aroF-m-18.
AB3248 strain is tyrosine 1mg/ml or 3AT0.5
mg/ml and 50μg/ml histidine, proline,
Can grow on M9 plate medium containing arginine, isoleucine, and valine. (5) Transformation of Corynebacterium glutamicum K43 with pKm1aroF1, pKm1aroF1-m-18: Semi-synthetic medium SSM containing 50 μg/ml phenylalanine [glucose 20 g, (NH ₄ ) ₂ SO ₄ 10 g, urea 3 g, Yeast extract 1g, KH ₂ PO ₄ 1g, MgCl 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 0.4mg, Na ₂ B ₄ O ₇・10H ₂ O0.09
mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 0.04 mg, biotin
30 μg, 1 mg of thiamine hydrochloride in 1 part of water, pH
Corynebacterium glutamicum K43 (FERM P-7162) seed culture was inoculated into a medium adjusted to 7.2 and cultured with shaking at 30°C. Seed culture is in NB medium (20 g of powdered bouillon, 5 g of yeast extract in 1 part water, PH
A medium adjusted to 7.2) was used. Measure the absorbance (OD) at 660 nm with a Tokyo Photoden colorimeter,
When the OD reached 0.2, penicillin G was added to the culture solution at a concentration of 0.5 units/ml. The culture was further continued until the OD reached approximately 0.6. When the OD reached 0.6, collect the bacteria and transfer the cells to RCGP medium [glucose 5g, casamino acids 5g, yeast extract
2.5g, K ₂ HPO ₄ 3.5g, KH ₂ PO ₄ 1.5g,
MgCl ₂・6H ₂ O 0.41g, 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 at 30°C for 50 minutes.
Protoplasts were formed by a gentle shaking reaction for a period of time. Place 0.5 ml of this protoplast suspension in a small test tube, centrifuge at 2500 x g for 5 minutes, and add 1 TSMC buffer (10 mM magnesium chloride, 30 mM calcium chloride, 50 mM Tris, 400 mM sucrose, PH7.5).
After centrifugation and washing, resuspend in 0.1ml of TSMC buffer.
resuspended in. Add 2x concentration of TSMC to this suspension.
A 1:1 mixture of buffer solution and the above plasmid DNA
Add 100 μl and mix, then add to TSMC buffer.
0.8 ml of a solution containing 20% PEG6000 was added and mixed.
Plasmid DNA pKm1aroF1, pKm1aroF1−
m-18 was prepared from E. coli AB3248 strain harboring these as described above. After 3 minutes, add RCGP medium (PH
7.2) Add 2 ml of kanamycin and centrifuge for 5 minutes to remove the supernatant. Suspend the precipitated protoplasts in 1 ml of RCGP medium, then transfer 0.2 ml to RCGP agar medium containing 200 μg/ml of kanamycin (RCGP medium). The cells were plated on a medium containing 1.4% agar (PH7.2) and cultured at 30°C for 7 days. In this way,
Kanamycin resistant colonies grown on selection plates were obtained. (6) Production of tyrosine by transformed strain: pKm1aroF1 obtained as above,
The transformed strain carrying pKm1aroF1-m-18 is
Corynebacterium glutamicum K44,
FERM P-7163 FERM BP-458 and
Corynebacterium glutamicum K45, FERM P
-7164 FERM BP-460. An L-tyrosine production test using pKm1aroF1 and pKm1aroF1-m-18 strains will be conducted as follows. The bacterial strain was cultured in NB liquid medium at 30℃ for 16 hours with shaking, and 0.5 ml of the bacterial suspension was added to 5 ml of production medium P4 [100 g of blackstrap molasses/
l, (NH ₄ ) ₂ SO ₄ 20g/l, KH ₂ PO ₄ 0.5g/,
K ₂ HPO ₄ 0.5g/, MgSO ₄・7H ₂ O 0.25g/
, CaCO ₃ 20g/, PH7.2] was inoculated into a test tube containing a medium containing 0.25% NZ amine.
The cells were cultured with shaking at ℃ for 96 hours. After culturing, add 50μ of 6N NaOH solution to 1ml of culture solution.
After adding and heating at 65℃ for 5 minutes to completely dissolve the precipitated tyrosine, the culture filtrate was subjected to paper chromatography, and after coloring with ninhydrin,
The amount of L-tyrosine produced was measured by colorimetry. As a control, Corynebacterium glutamicum K43 strain was treated in the same manner. The results are shown in Table 1. Table 1 L-Tyrosine strain (mg/ml) Corynebacterium glutamicum K43 4.8 〃 K44 5.3 〃 K45 7.7

[Brief explanation of the drawing]

FIG. 1 shows the restriction enzyme cleavage pattern of plasmid pEaroF-1. Figure 2 shows the plasmid
The restriction enzyme cleavage pattern of pKm1aroF1 is shown. In Figures 1 and 2, the horizontal arrow indicates the direction in which the gene is transcribed.

Claims (1)

[Scope of Claims] 1. 3- derived from a microorganism belonging to the genus
Deoxy-D-arabino-heptulosonic acid 7-
A host microorganism selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium is transformed using recombinant DNA of vector DNA and a DNA fragment containing genes encoding phosphate synthase, chorismate miutase, and prephenate dehydrogenase. A method for producing tyrosine, which comprises culturing the resulting transformed strain in a medium, producing and accumulating tyrosine in the culture, and collecting tyrosine from the culture. 2. The method according to claim 1, wherein the DNA fragment confers tyrosine or tyrosine analog resistance to the microorganism. 3. The method according to claim 2, wherein the tyrosine analog is 3-aminotyrosine. 4. The vector is capable of autonomous replication in a microorganism of the genus Corynebacterium or Brevibacterium;
Claim 1, characterized in that it is a plasmid, phage, or a derivative thereof derived from a microorganism.
The method described in section. 5. The plasmid and its derivatives are pCG1, pCG2, and pCG2 derived from microorganisms belonging to the genus Corynebacterium.
pCG4, pCE51, pCE52, pCE53, pCE54,
5. The method according to claim 4, wherein the plasmid is named pCG11 or pCB101. 6. Claim 1, characterized in that the host microorganism belongs to the genus Corynebacterium or the genus Brevibacterium and is sensitive to lysozyme.
The method described in section. 7. The method according to claim 1, wherein the host microorganism is Corynebacterium glutamicum, Corynebacterium herkyulis, Brevibacterium flavum, Brevibacterium lactofamentum, or a derivative thereof. . 8. The method according to claim 1, wherein the transformed strain is Corynebacterium glutamicum K44 or Corynebacterium glutamicum K45.