JPH0526467B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-histidine by fermentation. Among the oxygen involved in L-histidine production, histidinol phosphatase (hereinafter referred to as "HP") produces L-histidinol, which is the final intermediate metabolite in L-histidine production, from histidinol phosphate. It is oxygen that catalyzes the reaction. We focused on coryneform bacteria, which are known to produce large amounts of L-histidine, and
We have begun research to further increase productivity using DNA modification technology. Finally, they succeeded in isolating the gene encoding HP involved in L-histidine production (hereinafter referred to as "HP gene") from coryneform bacteria, and furthermore, they succeeded in isolating this gene into a plasmid vector capable of propagating within coryneform bacteria cells. By connecting it to a coryneform bacterium and introducing it into coryneform bacteria cells, we succeeded in significantly increasing the L-hiscytidine productivity of coryneform bacteria. That is, the present invention provides a vector plasmid in which a DNA fragment containing a gene encoding histidinol phosphaitase obtained from a DNA donor bacterium belonging to a coryneform glutamate producing bacterium can be autonomously replicated within the body of a coryneform glutamate producing bacterium. It is connected to 1,2,4-triazole-3, which belongs to coryneform glutamate producing bacteria.
- Production of L-histidine, which is characterized by culturing a microorganism capable of producing L-histidine obtained by introducing it into a DNA recipient microorganism that is resistant to alanine, and collecting L-histidine accumulated in the culture solution. It is the law. Corynebacteria are aerobic, gram-positive bacilli, non-acid-fast and classified as
It is described in Determinative Bacteriology, 8th edition, page 599 (1974). Among these, those that produce large amounts of L-glutamic acid are particularly known as shown below, and all of these are considered to belong to the same genus.

[Table] In addition to those that have glutamic acid productivity as described above, phyllum coryneform bacteria also include mutant strains that have lost glutamic acid productivity and other mutant strains. The method for isolating the HP gene is from coryneform bacteria.
First, extract the chromosomal gene from a strain that has the HP gene (for example, H.Saito and K.Miura,
The method of Biocham Biophys Acta 72 619 (1963) can be used), which is then cleaved with suitable limiting oxygen.
Then, it was connected to a plasmid vector capable of propagating in coryneform bacteria, and the obtained recombinant DNA was used to transform an HP-deficient mutant strain of coryneform bacteria,
A bacterial strain that has developed PH-generating activity can be isolated, and the HP gene can then be isolated. To cleave chromosomal genes, a wide variety of limiting oxygen species can be used by adjusting the cleavage reaction time and the degree of cleavage. 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) pAM330 See JP-A-58-67699 (2) pAM1519 See JP-A-58-77895 (3) pAJ655 (a) Host bacteria: Escherichia coli AJ11882
(FERM-P6517=FERM-BP136, etc.) (b) Molecular weight: 6.6 megadaltons (c) Limiting oxygen cleavage map: See Figure 1 (d) Properties: pAM330 and pBR325 (Gene 4 121
(1978) complex plasmid. Contributes to chloramphenicol resistance. (4) pAJ611 (a) Host bacteria: Escherichia coli AJ11884
(FERM-P6519=FERM-BP138, etc.) (b) Molecular weight: 6.6 megadaltons (c) Limiting oxygen cleavage map: See Figure 2 (d) Properties: Composite plasmid of pAM281 and pBR325. Contributes to chloramphenicol resistance. (5) pAJ440 (a) Host bacterium: Bacillus subtilis AJ11901
(FERM-BP140=ATCC39139, etc.) (b) Molecular weight: 6.0 megadaltons (c) Limiting oxygen cleavage map: See Figure 4 (d) Properties: pAM330 and pUB110 (J. Bacteriol.,
134318 (1978)) complex plasmid. Contributes to kanamycin resistance. (6) pAJ1844 (a) Host bacteria: Escherichia coli AJ11883
(FERM-P6518=FERM-BP137, etc.) (b) Molecular weight: 5.4 megadaltons (c) Limiting oxygen cleavage map: See Figure 3 (d) Properties: Composite plasmid of pHM1519 and pBR325. Contributes to chloramphenicol resistance. (7) pAJ3148 (a) Host bacteria: Corynebacterium glutamicum SR8203, ATCC39137, etc. (b) Molecular weight: 6.6 megadaltons (c) Limited oxygen cleavage map: See Figure 5 (d) Properties: Complex of pHM1519 and pUB110 Plasmid. Contributes to kanamycin resistance. Other examples of plasmids that can proliferate in coryneform bacterial cells include pCG1 (Japanese Patent Application Laid-Open No. 134500/1983);
pCG2 (Japanese Patent Publication No. 58-35197), pCG4, pCG11 (Japanese Patent Application Publication No. 1986-35197)
57-183799), and any of these could of course be used. The vector DNA is cut by the limiting oxygen used when cutting the chromosomal gene, or oligonucleotides having complementary base sequences are connected to both ends of the cut chromosomal DNA fragment and the cut vector DNA, The plasmid vector is then subjected to a ligation reaction with the chromosomal DNA fragment. In order to introduce the recombinant DNA of chromosomal DNA and vector plasmid thus obtained into a recipient bacterium belonging to coryneform bacteria, E.
As reported for E. coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53
159 (1970)) A method of treating recipient bacterial vacuoles with calcium chloride to increase DNA permeability, and
As reported for S. subtilis (Duncan, CH, Wilson, GA and Yaung, 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 into a protoplast or spheroplast, which readily takes up plasmid DNA, and introduce the prosmid into the DNA recipient. 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 carboxymethylcellulose, dextran, Ficoll, Pluronic F68 (Selva), etc. instead of polyethylene glycol or polyvinyl alcohol.
Similar results can be obtained by methods that promote DNA uptake. After transformation, gain HP productivity, or
Furthermore, a strain that expresses the marker properties of the plasmid vector is isolated as a desired transformed strain. Such a transformed strain is HP
Contains recombinant DNA into which the gene has been integrated. To isolate recombinant DNA, for example, a bacterial strain is lysed by lysozyme SDS treatment, treated with phenol, and then 2 volumes of ethanol are added.
Precipitate and collect DNA. The method for culturing the obtained L-histidine-producing bacteria is not particularly different from the conventional culture method for L-bistidine-producing bacteria. 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., 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-histidine substantially ceases. Thus, a significant amount of L-histidine is produced and accumulated during the culture. Conventional methods can be applied to collect L-histidine from the culture solution. Example (1) Regulation of chromosomal DNA containing HP gene Brevibacterium lactofamentum
Streptomycin-resistant mutant strain AJ12036 mutated from ATCC13869 in 1 CMG medium (containing peptone 1g/dl, yeast extract 1g/dl, glucose 0.5g/dl, and NaCl 0.5g/dl, adjusted to pH 7.2) ) and cultured with shaking at 30°C for about 3 hours, and the cells in the logarithmic growth phase were collected. After lysing the bacterial cells with lysozyme/SDS, using the usual phenol treatment method,
Chromosomal DNA was extracted and purified, and the final amount was 3.2 mg.
I got DNA. (2) Preparation of vector DNA Using pAJ1844 (molecular weight 5.4 megadaltons) as a vector, its DNA was prepared as follows. First, Brevibacterium lactofarmuntum harboring pAJ1844 as a plasmid.
AJ12037 (FERM P-7234, FERM BP-577)
was inoculated into 100 ml of CMG medium and cultured at 30°C until the late logarithmic growth phase, then lysed by lysozyme SDS treatment, and supernatant obtained by ultracentrifugation at 30,000 x g for 30 minutes. After the phenol treatment, 2 volumes of ethanol were added to precipitate and collect the DNA. This was mixed with a small amount of TEN buffer (20mM Tris-HCl,
After dissolving in 20mM NaCl, 1mM EDTA (PH8.0) and separating by agarose gel electrophoresis,
Approximately 15 μg of pAJ1844 plasmid DNA was obtained by cutting out. (3) Insertion of chromosomal DNA fragment into vector Treat 20 μg of chromosomal DNA obtained in (1) and 10 μg of plasmid DNA obtained in (2) with restriction endonuclease PstI at 37°C for 1 hour to completely cleave them. did. After heat treatment at 65℃ for 10 minutes, both reaction solutions were mixed and ligated with _T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol.
DNA strand ligation reaction was performed at 10°C for 24 hours. 65
After heat treatment at °C for 5 minutes, 2 times the volume of ethanol was added after the reaction, and the DNA after the ligation reaction was precipitated and collected. (4) Cloning of HP gene Brevibacterium with deletion of HP gene
Lactofermentum AJ12074 was used as the recipient strain. 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 log phase, and penicillin G was added at 0.6 units/ml.
After the addition, the cells were cultured with shaking for an additional 1.5 hours, and the cells were collected by centrifugation.
Washed with 0.5 ml of SMMP medium (PH6.5). then
The cells were suspended in SMMP medium containing 10 mg/ml lysozyme and incubated at 30°C for 20 hours to form protoplasts.
After centrifugation at 6000×g for 10 minutes, the protoplasts were washed with SMMP and resuspended in 0.5 ml of SMMP. Protoplasts obtained in this way and (3)
10 μg of the DNA prepared above was mixed in the presence of 5mM EDTA, polyethylene glycol was added to a final concentration of 30%, and the mixture was left at room temperature for 2 minutes to incorporate the DNA into protoplasts. Transfer these protoplasts to 1 ml of SMMP medium.
After washing with water, 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. Protoplast regeneration medium contains 12 parts of tris(hydroxymethyl)aminomethane per 1 part of distilled water.
g, KCl 0.5 g, glucose 10 g, MgCl ₂ .
6H ₂ O8.1g CaCal ₂・2H ₂ O2.2g, peptone 4
g, powdered yeast extract 4g, casamino acids (Difco
) 1g, K ₂ HPO ₄ 0.2g, sodium succinate 135g, agar 8g and chloramphenicol
Contains 3μg/ml. After culturing at 30°C for 2 weeks, approximately 10,000 chloramphenicol-resistant colonies appeared, which were then transferred to a histidine-free medium (His-deficient medium: 2% glucose, 1% ammonium sulfate,
0.25% urea, 0.1% potassium dihydrogen phosphate, 0.04
% magnesium sulfate heptahydrate, 2ppm iron ion,
2ppm manganese ion, 200μg/thiamine hydrochloride, 50μg/biotin, PH7.0, agar 1.8
%) to obtain 9 strains that were resistant to chloramphenicol and had lost histidine requirement. (5) Plasmid analysis of transformed strains A lysate was prepared from these strains using the method described in (2), and plasmid DNA was detected by agarose gel electrophoresis. A clearly large plasmid was detected. The representative strain among these was named AJ12075 (FERM-P7235). (6) Retransformation To confirm the presence of the HP gene on the plasmid (pAJ715) possessed by AJ12075,
Using this plasmid DNA, Brevibacterium lactofamentum AJ 12074 was grown again.
Transformed. When 10 of the resulting chloramphenicol-resistant colonies were picked up and tested for histidine auxotrophy, all of them had lost their auxotrophy, and no
It has become clear that the HP gene exists. (7) Histidine-producing ability of transformed strain Brevibacterium lactofa, a mutant strain having histidine-producing ability and resistant to 1,2,4-triazole-3-alanine, was obtained using the protoplast transformation method shown above. Meatum
pAJ715 was introduced into AJ12076. The transformed strain was selected for resistance to chloramphenicol, and the obtained
AJ12077 (FERM-P7236) and AJ12075 above
Histidine production medium (glucose 10g/dl,
(NH ₄ ) ₂ SO ₄ 5g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.2g/dl, FeSO ₄・7H ₂ O10
mg/, MnSO ₄ 7H ₂ O 10 mg/, Thiamine
Inoculate 20 ml of HCl 50 μg/, biotin 50 μg/, soy protein acid hydrolyzate (“taste liquid”) 128 mg/dl (as total nitrogen), PH 7.2, CaCO ₃ 5 g/dl),
Shaking culture was performed at 30°C for 72 hours. After culturing, L-histidine in the centrifuged supernatant was quantified by microbioassay. The results are shown in Table 1. Table 1 Strains L-histidine accumulation AJ12076 50mg/dl AJ12077 130mg/dl AJ12074 0mg/dl AJ12075 3mg/dl Brevibacterium lactofamentum
AJ12074 is a method of injecting Brevibacterium lactofamentum ATCC13869 into 2000 μg/ml N-methyl-N′-nitro-N-nitrosoguanidine.
Mutation treatment was performed by contacting for 20 minutes at ℃, and L-histidinol did not grow on His-deficient medium.
It was isolated as a strain that can grow in a medium supplemented with 10 mg/dl. Brevibacterium lactofamentum AJ12076 is Brevibacterium lactofamentum ATCC13869 at 1000μ
Mutation treatment was carried out by contacting with g/ml of N-methyl-N'-nitro-N-nitrosoguanidine at 0°C for 20 minutes.
g/dl, urea 0.25g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄ .7H ₂ O0.04g/dl, thiamine hydrochloride 200μ
DL-1,2,4-
It was isolated as a strain that can grow in a medium containing 1 mg/ml of triazole-3-alanine. AJ12075 and AJ12076 can be easily obtained from AJ12075 or AJ12077, respectively, by removing the complex plasmid in the host cell without damaging the host cell. That is, the plasmid may be lost naturally from the host, or it may be removed by a "removal" operation (Bact.Rev., 36 , p. 361-405).
(1972)). An example of a removal operation is as follows:
Concentrations that completely inhibit host growth (2-50μ
1 g/ml) in a medium containing acridine orange.
After inoculating a small amount of the bacterial strain to approximately ¹⁰⁴ cells per ml and completely inhibiting the growth of the host bacteria, 27-37
Incubate overnight at ℃ (J. Bacteriol., 88 , 261
(1964)). Spread the culture solution on agar medium and incubate at 27-37℃.
Incubate overnight. Among the colonies that appeared on the medium, the strains showing susceptibility to chloramphencol (10 μg/ml) were strains from which the plasmid had been removed, that is, AJ12074 and
It is AJ12076.

[Brief explanation of the drawing]

Figure 1: Restriction enzyme cleavage map of composite plasmid pAJ655, Figure 2: Restriction enzyme cleavage map of composite plasmid pAJ611, Figure 3: Restriction enzyme cleavage map of composite plasmid pAJ1844.
Restriction enzyme cleavage map, Figure 4: Complex plasmid
Restriction enzyme cleavage map of pAJ440, Figure 5: Restriction enzyme cleavage map of composite plasmid pAJ3148.

Claims (1)

[Claims] 1 Belongs to coryneform-glutamic acid producing bacteria
A DNA fragment containing the gene encoding histidinol phosphatase obtained from a DNA donor bacterium is
It is connected to a vector plasmid that can autonomously replicate within the cells of coryneform-glutamic acid producing bacteria.
Belongs to coryneform glutamate producing bacteria1.
The method is characterized by culturing a microorganism with the ability to produce L-histidine obtained by introducing it into a DNA recipient microorganism that is resistant to 2,4-triazole-3-alanine, and collecting L-histidine accumulated in the culture solution. A method for producing L-histidine.