JPH055480B2 - - Google Patents

Description

Detailed Description of the Invention Industrial Field of Application The present invention comprises a recombinant DNA into which 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase (hereinafter referred to as "DS") is incorporated. The present invention relates to coryneform bacteria and a method for producing L-tryptophan using the same. Prior Art DS is an enzyme that catalyzes the synthesis of 3-deoxy-D-arabino-heptulonic acid-7-phosphate (hereinafter referred to as "DAHP") from phosphoenolpyruvate and erythrose 4-phosphate. , DAHP is converted to phenylalanine, tyrosine or tryptophan via chorismate. In coryneform bacteria, DS activity is known to be synergistically inhibited by the final products phenylalanine and tyrosine, and when breeding L-tryptophan-producing bacteria, a strong It is important to select strains that retain DS activity. On the other hand, the breeding of L-tryptophan-producing coryneform bacteria using recombinant DNA method was reported in JP-A-59-156292.
It does not contain the gene encoding DS. In addition, in coryneform bacteria that have recombinant DNA containing the gene encoding DS, L-
An example of breeding phenylalanine or L-tyrosine producing bacteria has been reported in Japanese Patent Application No. 59-245700, but there is no example of breeding L-tryptophan producing bacteria. That is, the present invention provides 3-deoxy-D-arabino-heptyuronic acid-7 obtained from a DNA donor bacterium belonging to coryneform glutamic acid producing bacteria and expressed in the restriction enzyme map of the inserted gene portion in Fig. 1 or Fig. 2. -Contains phosphate synthase gene
A DNA fragment, or a DNA fragment containing the 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase gene obtained by shortening the fragment, autonomously replicates within the cells of coryneform-glutamate producing bacteria. A microorganism having the ability to produce L-tryptophan obtained by being connected to a vector plasmid and introduced into a DNA recipient bacterium belonging to coryneform-glutamic acid-producing bacteria is cultured, and L-tryptophan accumulated in the culture solution is collected. This is a method for producing L-tryptophan, which is characterized by: Furthermore, the present invention provides the above-mentioned method for producing L-tryptophan, characterized in that the DNA donor bacteria belonging to coryneform glutamic acid producing bacteria are resistant to m-fluorophenylalanine. Problems to be Solved by the Invention The present invention aims to obtain microorganisms with higher productivity of L-tryptophan, and thereby to obtain L-tryptophan.
The goal is to find a more efficient method for producing tryptophan. Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors conducted research and found that the gene encoding DS, which is expressed in coryneform bacterial cells, can proliferate within coryneform bacterial cells. Recombinant connected to plasmid vector
We have successfully isolated coryneform bacteria having DNA and found that the obtained coryneform bacteria have high productivity of L-tryptophan. Coryneform bacteria (Coryneform) according to the present invention
bacteria) is the Burgess Manual of
Bargeys Manual of Determinative
Bacteriology), 8th edition, p. 599 (1974), is a group of microorganisms that are aerobic, Gram-positive, non-acid-fast, and have no spore-forming ability. Among these coryneform bacteria, the following coryneform glutamate-producing bacteria are particularly preferred in the present invention. Examples of wild strains of coryneform glutamate-producing bacteria include the following. [Table] E
[Table] Near Philum
The coryneform glutamate-producing bacteria of the present invention include not only wild strains having glutamate productivity as described above but also mutant strains having glutamate productivity or having lost glutamate productivity. The method for isolating the DS gene is based on coryneform bacteria.
First, extract the chromosomal gene from a strain that has the DS gene (for example, H. Saito and K. Miura
The method of Biochem, Biophys. Acta 72, 619, (1963) can be used. ), which is then cut with an appropriate restriction enzyme. It is then connected to a plasmid vector that can propagate within coryneform bacterial cells, and the resulting recombinant
A DS-deficient mutant strain of a coryneform bacterium is transformed using DNA, and a strain that has developed DS-producing activity is isolated, thereby allowing the DS gene to be isolated. In addition, we transformed a wild strain of coryneform bacteria using the same recombinant DNA, and isolated a strain that was resistant to aromatic amino acid antagonists that have an inhibitory effect on DS activity. It is also possible to isolate genes. Examples of aromatic amino acid antagonists include o-fluorophenylalanine, m-fluorophenylalanine, p-fluorophenylalanine, p-aminophenylalanine, β-3-thienylalanine, 3-aminotyrosine, Examples include tyrosine hydroxamate and 5-methyltryptophan. As the DNA-donor bacterium, it is even better to use a mutant strain in which the biosynthetic activity of aromatic amino acids or their precursors is increased by imparting mutations such as resistance to the aromatic amino acid antagonists described above. What is the aromatic amino acid precursor mentioned here?
DAHP3-dehydroquinic acid, 3-dehydroshikimic acid, shikimic acid, shikimic acid-3-phosphoric acid, 5-enolpyruvylshikimic acid-3-phosphoric acid, chorizmic acid, prephenic acid, phenylpyruvic acid, anthranilic acid, indole 3-glycerol phosphate, etc. Examples of such mutant strains include the U.S. patent
No. 3,660,235, U.S. Pat. No. 3,759,970, and Japanese Patent Application Laid-open No. 56-64793. As the DS gene, a wild type gene can be used, and a mutant strain gene can also be used. As a mutant gene, the degree of synergistic inhibition by phenylalanine and tyrosine was reduced.
Particularly preferred are those mutated to encode DS. To obtain a mutant gene, a DNA donor bacterium may be subjected to mutation treatment, or the DS gene may be inserted into a vector plasmid, and the resulting recombinant DNA may be introduced into a DNA recipient bacterium to create a transformed strain. Mutation processing may be performed. Furthermore, a mutant gene can also be obtained by mutating the recombinant DNA itself in vitro. In order to cleave chromosomal genes, a wide variety of restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time and the like. The plasmid vector used in the present invention is
Any substance that can grow within coryneform bacterial cells may be used. Specific examples include the following. (1) pAM 330 See JP-A-58-67699 (2) pHM 1519 See JP-A-58-77895 (3) pAJ 655 See JP-A-58-192900 (4) pAJ 611 Same as above (5) pAJ 1844 Same as above (6) pCG 1 See JP-A-57-134500 (7) pCG 2 See JP-A-58-35197 (8) pCG 4 See JP-A-57-183799 (9) pCG 11 Same as above Cleavage of plasmid vector DNA is as follows: the DNA concerned
This is done by cutting using a restriction enzyme that cuts at one site or partially cutting using a restriction enzyme that cuts at multiple sites. The vector DNA is cleaved with the restriction enzyme used to cleave the chromosomal gene, or oligonucleotides having complementary base sequences are connected to both ends of the chromosomal DNA cleavage fragment and the cleaved vector DNA. The plasmid vector is then subjected to a ligation reaction with the chromosomal DNA fragment. In order to introduce the thus obtained recombinant DNA of chromosomal DNA and vector plasmid into a recipient bacterium belonging to coryneform bacteria, E.
As reported for coli K-12 (Mandel, M. and Higa, A., J. Mol., Biol.,
53, 159 (1970) Treatment of recipient bacterial cells with calcium chloride to increase DNA permeability, or as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, F.
E., Gene, 1 , 153 (1977)) This is possible by introducing cells into a proliferation stage (so-called compatible cells) where they can take up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chang, S. and
Choen, SN, Molec. Gen., Genet., 168 . 111
(1979); Bibb, M.J., Ward, J.Mand
Hopwood, OA, Nature, 274 398 (1978);
Hinnen, A., Hicks, JBand Fink, G.R.
Proc. Natl. Acad. Sci. USA, 75 1929 (1978)),
It is also possible to transform the DNA recipient bacteria into protoplasts or spheroplasts, which readily take up plasmid DNA, and to introduce the plasmid into the DNA recipient bacteria. In the protoplast method, a sufficiently high frequency can be obtained using the method used for Bacillus subtilis mentioned above, and polyethylene glycol or Of course, a method of incorporating DNA in the presence of polyvinyl alcohol and divalent metal ions can also be used. By adding carboxymethyl cellulose, dextran, Ficoll, Brulonik F68 (Selva), etc. instead of polyethylene glycol or polyvinyl alcohol.
Similar results can be obtained by methods that promote DNA uptake. As L-tryptophan-producing bacteria, strains transformed with wild-type strains or DS-deficient strains as hosts can be used, but strains with higher L-tryptophan productivity can be obtained by using the following hosts. There is. Specifically, a mutant strain of Brevibacterium that requires phenylalanine and tyrosine and is resistant to 5-methyltryptophan (I. Shiio, H. Sato, M.
Nakagawa, Agric.Biol.Chem. 36 , 2315
(1972)), require phenylalanine from Brevibacterium, m-fluorophenylalanine,
Mutant strain resistant to 5-fluorotryptophan (I.Shiio, S.Sugimoto, M.Nakagawa,
Agric.Biol.Chem., 39 , 627 (1975)), a mutant strain of Brevibacterium that requires tyrosine and is resistant to 5-fluorotryptophan and azaserine, phenylalanine of Corynebacterium,
requires tyrosine, 5-methyltryptophan,
4-methyltryptophan, 6-fluorotryptophan, tryptophan hydroxamate, p
- Mutant strains resistant to fluorophenylalanine, tyrosine hydroxamate, and phenylalanine hydroxamate (H. Hagino, K.
Nakayama, Agric.Biol.Chem., 39 , 345
(1975)) etc. The method of culturing the thus obtained coryneform bacteria capable of producing L-tryptophan to produce and accumulate L-tryptophan is different from the method conventionally used for producing L-tryptophan using coryneform bacteria. There are no major differences.
That is, the culture medium is a usual one containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins. As the carbon source, glucose, sucrose, lactose, etc., and starch hydrolyzate containing these, whey, molasses, etc. are used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. can be used. Cultivation is carried out under aerobic conditions while adjusting the pH and temperature of the medium as appropriate until the production and accumulation of L-tryptophan substantially ceases. Example (1) Preparation of chromosomal DNA containing the DS gene As a source for preparing chromosomal DNA, the inhibition of DS by phenylalanine is released,
Brevibacterium lactofermentum AJ 11957 (FERM-P6673), a phenylalanine-producing bacterium that has developed resistance to m-fluorophenylalanine, a phenylalanine analogue
was used. This bacterium was grown in 1 CMG medium (peptone 1g/dl,
(containing 1 g/dl of yeast extract, 0.5 g/dl of glucose, and 0.5/dl of NaCl, adjusted to PH 7.2) and cultured with shaking at 30°C for about 3 hours until the bacteria were in the logarithmic growth phase. I gathered my body. After lysing the bacterial cells with lysozyme/SDS, the chromosomes were treated with normal phenol.
The DNA was extracted and purified, and 3.5 mg of DNA was finally obtained. (2) Preparation of vector DNA pAJ 1844 (molecular weight 5.4 megadaltons) was used as a vector, and its DNA was prepared as follows. First, Brevibacterium lactofermentum harboring pAJ 1844 as a plasmid.
AJ12037 (FERM BP-577) was inoculated into 100 ml of CMG medium and cultured at 30°C until the late logarithmic growth phase, and then lysed by lysozyme SDS treatment.
The supernatant was obtained by ultracentrifugation at ×g for 30 minutes. After phenol treatment, add 2 volumes of ethanol to remove DNA.
was collected as a precipitate. Add this to a small amount of TEN buffer (20mM Tris-HCl, 20mM NaCl, 1mM
After dissolving in EDTA (PH8.0), the mixture was subjected to agarose gel electrophoresis for separation and excised to obtain approximately 15 μg of pAJ 1844 plasmid DNA. (3) Insertion of chromosomal DNA fragment into vector 10 μg of chromosomal DNA obtained in (1) and 5 μg of plasmid DNA obtained in (2) were injected with restriction endonuclease Pst.
1, each was held at 37°C for 1 hour and cut.
After heating to 65 °C for 10 min, both reactions were mixed;
In the presence of ATP and dithiothreitol, the DNA strands were ligated by holding at 10°C for 24 hours using DNA ligase derived from _T4 phage. Then, add the reaction solution to
The mixture was heated at 65° C. for 5 minutes, and 2 volumes of ethanol was added to the reaction mixture to collect the ligated DNA precipitate. (4) Cloning of DS gene The DNA recipient bacteria include Brevibacterium lactofermentum AJ12036 (FERM P-7559,
FERM BP-734) was used. The protoplast transformation method was used as the transformation method. First, the strain was cultured in 5 ml of CMG liquid medium until the early logarithmic growth phase, and after adding 0.6 units/ml of penicillin G,
After culturing with shaking for an additional 1.5 hours, the bacterial cells were collected by centrifugation, and the bacterial cells were transferred to SMMP medium (PH
6.5) Washed with 0.5ml. The cells were then suspended in SMMP medium containing 10 mg/ml of lysozyme to form protoplasts at 30°C for 20 hours. After centrifugation at 6000×g for 10 minutes, wash the protoplasts with 0.5 ml of SMMP.
resuspended in SMMP. The protoplasts obtained in this way and 10 μg of the DNA prepared in (3) were
After mixing in the presence of 5mM EDTA and adding polyethylene glycol to a final density of 30%,
The DNA was left at room temperature for 2 minutes to incorporate into the protoplasts. This protoplast
After washing with 1 ml of SMMP medium, the cells were resuspended in 1 ml of SMMP medium and cultured at 30°C for 2 hours for expression. This culture solution was spread on a protoplast regeneration medium at pH 7.0. The protoplast regeneration medium contains 12 g of tris(hydroxymethyl)aminomethane, 0.5 g of KCl, 10 g of glucose,
_MgCl2・_6H2O8.1g , _CaCl2・_2H2O2.2g , peptone 4g, powdered yeast extract 4g, casamino acid (Difco) 1g, _{K2HPO4 0.2g} , sodium succinate 135g, agar 8g, and Contains chloramphenicol 3μg/ml. After culturing for 2 weeks at 30°C, approximately 25,000 chloramphenicol-resistant colonies appeared, which were then transferred to a minimal medium containing 500 μg/ml of m-fluorophenylalanine (2% glucose, 1% ammonium sulfate, 0.3% urea). , 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2ppm iron ion, 2ppm manganese ion, 200μg/thiamine hydrochloride, 50μg/biotin, chloramphenicol 10μg/ml, PH7.0, agar 1.8% ) to obtain five strains resistant to chloramphenicol and m-fluorophenylalanine. (5) Plasmid analysis of transformed strains A lysate was prepared from these strains by the method described in (2), and plasmid DNA was detected by agarose gel electrophoresis. A clearly larger plasmid was detected. When the plasmids of the five strains were each digested with the restriction enzyme Pst 1 used for recombination, a common 6.7 kb DNA insert fragment was found in four of the strains. In addition, one other strain has 3.7kb, 3.0kb, 1.7kb,
9 of 1.6kb, 1.4kb, 1.3kb, 0.8kb, 0.7kb, 0.4kb
DNA insertion fragments were observed. Vector pAJ
The recombinant plasmid containing the former 6.7 kb Pst 1 fragment at the 1844 Pst 1 breakpoint was named pAR-2, and the latter recombinant plasmid containing 9 Pst 1 DNA fragments was named pAR-1, pAR- AJ 12181 (FERM-P 7948), pAR
AJ 12182 (FERM P-7947,
FERM BP-917). Note that pAR-2 has the restriction enzyme map shown in FIG. (6) Retransformation In order to confirm the presence of the gene conferring m-fluorophenylalanine resistance on the recombinant plasmid containing the DNA fragment detected in (5), this plasmid DNApAR-1 and Using pAR-2,
Brevibacterium lactofermentum AJ
12036 was transformed again. When 50 of the resulting chloramphenicol-resistant colonies were picked up and tested for sensitivity to m-fluorophenylalanine, all of them were found to be resistant to m-fluorophenylalanine. It has become clear that there is a gene that confers enilalanine resistance. (7) DS activity of transformed strain The test strain was grown in a phenylalanine production medium (130 g of glucose, 10 g of (NH ₄ ) ₂ SO ₄ , 1 g of KH ₂ PO ₄ ,
_MgSO4・_7H2O1g , fumaric acid 12g, acetic acid 3ml,
Soybean protein acid hydrolyzate “Ajiri” 50ml, FeSO ₄ .
_7H2O10mg , _MnSO4・_4H2O10mg , Bithion 50μ
g, 2000 μg of thiamine hydrochloride and 50 g of CaCO ₃ in 1 part of water. A lysate was prepared from the bacterial cells cultured in 20 ml (PH7.0) at 30° C. for 15 hours by ultrasonication, and this was centrifuged at 32,000×g for 20 minutes to obtain a supernatant. This supernatant was used as a crude enzyme solution,
50mM Tris-HCl buffer (PH7.5), 0.5mM phosphoenolpyruvate, 0.5mM erythrose
In a 1.0 ml reaction mixture consisting of 4-phosphoric acid at 30°C for 10
After the reaction is complete, add 0.2 ml of trichloroacetic acid to inactivate the enzyme. After removing the denatured protein by centrifugation, add 0.25 ml of 0.025M periodic acid to 0.25 ml of the resulting supernatant and incubate at 37°C for 30 minutes.
After adding 0.5 ml of 2% sodium arsenite to stop the oxidation reaction, 2.0 ml of 0.3% 2-thiobarbituric acid was added and heated at 100°C for 8 minutes. of reaction solution
The absorbance at 549 nm was measured, and the molecular extinction coefficient of DAHP was 4.5×10 ⁴ (Jensen, RA and Nester, EW, J.
Biol, Chem 241 , 3365 (1966)) was used to determine enzyme activity. Additionally, phenylalanine, tyrosine, or both may be added to the reaction solution.
The activity was also measured when 1mM was added and expressed as 100% when no addition was made. Table 1 shows the measurement results. [Table] By introducing the obtained recombinant plasmid, DS activity increased 2.6 to 1.4 times, and the activity inhibition pattern by each amino acid also changed from the wild strain type to the mutant strain type, indicating that pAR- 1. It is clear that the m-fluorophenylalanine resistance gene encoded by pAR-2 is a DS gene. Also,
Since the Pst 1 fragments of pAR-1 and pAR-2 are completely different, it is thought that there are two or more types of genes encoding proteins with DS activity. (8) Subcloning of DS gene Cut pAR-1 with restriction enzyme Pst 1, 65℃,
After heating for 10 minutes, the DNA strands were allowed to stand overnight at 4°C using T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol to ligate the DNA strands.
The reaction solution was then heated at 65° C. for 10 minutes, and twice the volume of ethanol was added to the reaction solution to collect the ligated DNA precipitate. A small amount of TEN buffer was added to this and used for transformation. Brevibacterium lactofermentum AJ 12036 was transformed using the transformation method shown in (4). Chloramphenicol for 2 weeks at 30℃
After regeneration in regeneration medium containing 3 μg/ml, m-
A large number of strains that were resistant to chloramphenicol and m-fluorophenylalanine were obtained by replicating them into a minimal medium containing 500 μg/ml of fluorophenylalanine. Some of these stocks are
It had a smaller plasmid group than pAR-1.
The most compact plasmid of these was pAR−
The strain carrying pAR-112 was named AJ 12183.
(FERM-P 7946). Furthermore, pAR−112
has the restriction enzyme map shown in FIG. (9) Tryptophan production ability of transformed strain Brevibacterium lactofermentum resistant to m-fluorophenylalanine and 5-fluorotryptophan using the above pAR-112.
M247 was transformed by the method described in (4), and transformed strains were selected using chloramphenicol resistance as an indicator. FERM P-8414 thus obtained,
FERM BP-918 and AJ 12251 were cultured and their tryptophan production ability was examined, and the results shown in Table 2 were obtained. Culture is performed using tryptophan production medium (glucose
130g, (NH ₄ ) ₂ SO ₄ 25g, fumaric acid 12g, acetic acid 3
ml, KH ₂ PO ₄ 1g, MnSO ₄・7H ₂ O 10mg,
MgSO ₄ 7H ₂ O 1g, d-bithione 50μg, thiamine hydrochloride 2000μg, methionine 400mg, tyrosine 650mg, soybean protein acid hydrolyzate "Ajiri" 50ml,
Contains 50g of CaCO ₃ in 1 part of water, PH6.5. ) 20ml to 500
The test bacterial strain was inoculated into a ml Sakaguchi flask and incubated at 30°C for 72 hours with shaking. After culturing, L-tryptophan in the centrifuged supernatant was extracted from Leuconostoc mecentroides.
mesentoroides) ATCC 8042 as a quantitative strain. [Table] In order to obtain M247, the deposited FERM
P-8414AJ 12251 allows removal of complex plasmids in host cells without damaging the host cells. That is, the plasmid may be naturally lost from the host, or it may be removed by a "removal" operation (Bact.Rev., 36 , p. 361-405).
(1972)). Examples of other removal operations are as follows.
Inoculate AJ 12195 into CMG liquid medium, culture it overnight at 37℃ (high temperature treatment), dilute the culture solution appropriately, and prepare it with or without chloramphenicol.
Spread on CMG agar medium and culture at 30°C for 1 to 3 days. The strain thus isolated as a chloramphenicol-sensitive strain is M247.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme map of pAR-2. Figure 2 shows a restriction enzyme map of pAR-112.

Claims (1)

[Claims] 1 Belongs to coryneform glutamic acid producing bacteria
Contains the 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase gene obtained from the DNA donor and expressed in the restriction enzyme map below.
A DNA fragment, or a DNA fragment containing the 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase gene obtained by shortening the fragment, autonomously replicates within the cells of coryneform-glutamate producing bacteria. A microorganism having the ability to produce L-tryptophan obtained by being connected to a vector plasmid and introduced into a DNA recipient bacterium belonging to coryneform-glutamic acid-producing bacteria is cultured, and L-tryptophan accumulated in the culture solution is collected. A method for producing L-tryptophan, characterized by: 2 Belongs to coryneform-glutamic acid producing bacteria
Contains the 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase gene obtained from the DNA donor and expressed in the restriction enzyme map below.
A DNA fragment, or a DNA fragment containing the 3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase gene obtained by shortening the fragment, autonomously replicates within the cells of coryneform-glutamate producing bacteria. A microorganism having the ability to produce L-tryptophan obtained by being connected to a vector plasmid and introduced into a DNA recipient bacterium belonging to coryneform-glutamic acid-producing bacteria is cultured, and L-tryptophan accumulated in the culture solution is collected. A method for producing L-tryptophan, characterized by: 3 Belongs to coryneform-glutamic acid producing bacteria
3. The method for producing L-tryptophan according to claim 1 or 2, wherein the DNA donor bacterium is resistant to m-fluorophenylalanine. 4. Claims 1 or 2
3-deoxy-D-arabino-heptyuronic acid-7-phosphate synthase encoded by the DNA fragment is characterized in that synergistic feedback inhibition by L-tyrosine and L-phenylalanine has been released. A method for producing L-tryptophan according to claim 1 or 2.