JPH0422557B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing guanosine-5'-monophosphate (hereinafter referred to as "GMP") by a fermentation method. Regarding the production of GMP by the fermentation method, microorganisms of the genus Bacillus (Special Publication No. 1973-
6158) Microorganisms of the genus Brevibacterium (Special Publication 1977)
-6158), microorganisms of the genus Escherichia (Special Publication 1977-
11760), microorganisms of the genus Bacillus that are auxotrophic for adenine and resistant to decoinine or methionine sulfoxide (Japanese Patent Publication No. 12438/1983), etc. are known to produce GMP. The present inventors developed a vector incorporating a gene region involved in purine analog resistance or decoinine resistance obtained from the chromosome of a Bacillus genus that has purine analog resistance or decoinine resistance, in contrast to the conventional GMP production method described above. We found that GMP-producing Bacillus microorganisms containing adenine auxotrophic Bacillus microorganisms accumulated a significant amount of GMP. The present invention was completed based on this knowledge. Purine analogs as used in the present invention inhibit the growth of microorganisms belonging to the genus Bacillus, and can be inhibited completely by adding hypoxanthine, inosine, 5-inosinic acid, guanine, guanosine, 5'-guanylic acid, etc. to the medium. It shall be such that it may be terminated in its entirety or in part. Examples include 8-azaguanine, 8-azahypoxanthine, 8-azaadenine, 2,6-diaminobulin, 6-mercaptopurine, 6-mercaptopurimboside, and 8-mercaptoguanosine. The donor strain of the chromosomal gene involved in purine analog resistance or decoinine resistance may be any mutant strain of the genus Bacillus as long as it has purine analog resistance or decoinine resistance, but one with a higher degree of resistance is preferable. Also, it is adenine-requiring.
If a mutant strain with purine analog resistance or decoinine resistance is induced using a strain with GMP-producing ability as a parent strain, a mutant strain with higher GMP-producing ability can be obtained, and such a mutant strain can be used as a gene donor. This will give you better results. or,
As gene-donating bacteria, we have added to the adenine auxotrophic and purine analog or decoinine resistant mutant strains, properties that improve the GMP production ability, such as methionine sulfoxide resistance, cycofuranine resistance, sulfaguanidine resistance, etc. By inducing and using a strain with a high level of GMP production, it is possible to obtain a strain with a higher GMP production ability, and if such a strain is used as a chromosomal gene donor,
More favorable results are obtained. Chromosomal DNA can be extracted from gene-donating bacteria by a conventional method such as that described in J. Bacteriol., 89 , 1065 (1965). The vector DNA may be any plasmid or phage that replicates in the cells of the genus Bacillus. For example, pT127, pC194, pC221, pC223 derived from Staphylococcus microorganisms,
pUB112 (above, Proc. Natl. Acad. Sci. USA, 74 ,
1680 (1977)), pUB110 (J. Bacteriol., 134 ,
318 (1978)), pTP4, pTP5 (and above,
Microbiol Letters, 5 , 55 (1978)), pLS15 and pLS28 derived from Bacillus subtilis (see J. Bacteriol.
131, 699 (1977)), pLS13 (J. Bacteriol.
129, 1487 (1977)), pPL1, pPL2 (see above, J.
Bacteriol., 124 , 484 (1975)), rho11, also known as temperate phage (Gene., 5 ,
89 (1979)), phi105 (Gene., 5 , 87 (1979)),
There are SPO2 (Gene., 7 , 51 (1979)), etc. Furthermore, a complex plasmid constructed based on the above-mentioned plasmid can also be used as a vector DNA. The chromosomal DNA and vector DNA are each cut using restriction endonucleases. There is a suitable restriction endonuclease for each vector, which is indicated in the literature describing the vector. For chromosomal DNA, many types of restriction enzymes can be used by adjusting the reaction conditions so that the restriction endonuclease can partially cut the DNA. A conventional method using ligase can be used to link the chromosomal NDA fragment thus obtained and the cut vector NDA. On the other hand, using terminal transferase, chromosome
A method may also be used in which deoxyadenylic acid and deoxycytidylic acid are added to the NDA fragment and the cleaved vector DNA, respectively, and the mixture is annealed and ligated. The recipient bacteria of the recombinant vector NDA incorporating the thus obtained chromosomal NDA fragment may be any mutant strain of the genus Bacillus that has adenine auxotrophy, but strains that do not have purine analog resistance or decoinine resistance may be used. If used, it is convenient when selecting transformed strains. Further recombination
If a strain having purine analog resistance or decoinine resistance and higher GMP production ability is used as a DNA receptor, a transformed strain with higher GMP productivity can be obtained. Of course as a recipient bacterium
A lower GMP resolution must be used. To introduce a mixture of chromosomal NDA and vector into NDA recipient bacteria, e.g. Molec.Gene.Genet., 168 ;
111 (1979) are available. To select a transformant strain containing a vector that has GMP-producing ability and incorporates a gene region involved in purine analog resistance or decoinine resistance, for example, an adenine auxotrophic mutant strain is used as a vector recipient. A strain that can be converted and grows in a medium containing a purine analog or decoinine may be selected. In addition, selection can be made easier if a medium is used that allows selection of strains that have the characteristics of vector NDA such as antibiotic resistance. In this way, the once selected recombinant vector NDA incorporating a gene region involved in purine analog resistance, etc., is extracted from the transformed strain and then transferred to other recombinant vector NDA recipient bacteria, e.g.
By introducing it into a strain with GMP production ability.
The amount of GMP accumulated can be further increased. The GMP-producing bacteria thus obtained are cultured to produce GMP.
The manufacturing method is not particularly different from conventionally known methods. That is, the medium for culturing such microorganisms is a conventional liquid medium containing a carbon source, a nitrogen source, inorganic salts, adenine and, if necessary, further micronutrients. Carbohydrates such as glucose, molasses, and starch hydrolyzate are preferred as carbon sources. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorous, nitrates such as ammonium nitrate, urea, ammonia gas, etc. can be used. Further, as the adenine as a nutritional substance, any of adenine, adenine mineral salt, adenosine, adenylic acid, etc. can be used. Furthermore, if necessary, it is often possible to increase the amount of GMP accumulated by adding micronutrients such as vitamin acids, amino acids, and nucleobases other than adenine. The culture method requires aerobic conditions, and the culture temperature is
A range of 27 to 38°C is preferred. In some cases, the culture temperature may be changed during the culture. At the start of culture and during culture, adjust the pH of the culture solution to 5.0 or above.
It is desirable to adjust the temperature to 9.0 and culture. To adjust the pH, use inorganic acids, organic acids or alkalis, as well as urea,
Calcium carbonate, ammonium water, ammonia gas, etc. can be used. Thus, after 2 to 5 days of culture, a significant amount of GMP accumulates in the medium. GMP may be collected from the culture solution by any conventional method such as using an ion exchange resin. Example Bacillus subtilis AJ11711 (arginine,
Leucine multi-request strain) was treated with N-methyl-N'-nitro-nitrosoguanine mutation (500 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine at 0°C for 30 minutes at ¹⁰⁸ cells/ml). GMP is extracted from adenine auxotrophs by contacting bacterial cells).
Strain AJ11853 (FERM-P6484) with production ability
(arginine requirement, leucine requirement, adenine requirement) were obtained. Furthermore, GMP-producing bacteria derived from this adenine auxotroph strain by similar mutation treatment.
AJ11854 (FERM-P6485) (arginine requirement,
leucine requirement, adenine requirement, 8-agaguanine resistance), AJ11855 (FERM-P) (arginine requirement, leucine requirement, adenine requirement, decoinine resistance), AJ11856 (arginine requirement, leucine requirement, adenine requirement) , 8-azaguanine resistance, and decoinine resistance). (1) Adjustment of chromosomal DNA 1 each of AJ11854, AJ11855 and AJ11856
"Bacto-Penassay Broth" (manufactured by Difco)
After culturing with shaking at 35°C for about 2 hours and harvesting the strains in the logarithmic growth phase, they were extracted using the usual NDA extraction method (J.
Bacteriol., 89 , 1065 (1965))
Extract and purify NDA 3.1 from final AJ11854
mg, 2.6 mg from AJ11855, and 3.6 mg from AJ11856. (2) Insertion of chromosomal DNA fragment into a vector In order to clone the gene region governing purine analog resistance or decoinine resistance, use an autonomously replicating plasmid as the vector.
pUB110 (expressing kanamycin resistance and neomycin resistance) was used. Chromosome obtained in (1)
5 μg of each DNA and 5 μg of plasmid pUB110
Each restriction endonuclease EcoRI
was applied at 37°C for 60 minutes to cleave the DNA strand.
After heat treatment at 65℃ for 10 minutes, both reaction solutions were mixed,
DNA strand ligation reaction was carried out at 10°C for 24 hours using _T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol. (3) Transformation with recombinant plasmid NDA Bacillus subtilis AJ11853 was contacted with "Penassay-Broth" (manufactured by Difco).
Culture with shaking was carried out overnight at 30°C, and the first culture medium (glucose 5g/, (NH ₄ ) ₂ SO ₄ 2g/,
KH ₂ PO ₄ 6g/, K ₂ HPO ₄ 14g/,
(contains 0.2 g/MgSO ₄ 7H ₂ O/, 1 g/ sodium citrate, 2 g/ yeast extract, 250 mg/L-arginine, 50 mg/ L-leucine, and 50 mg/ adenine) and shaken at 37°C for 4 hours. After culturing, a second culture medium (glucose 5g/, (NH ₄ ) ₂ SO ₄ 2g/,
KH ₂ PO ₄ 6g/, K ₂ HPO ₄ 14g/,
(containing 1.2 g of MgSO ₄ 7H ₂ O, 1 g of sodium citrate, 0.2 g of yeast extract, 50 mg of L-arginine, 5 mg of L-leucine, and 50 mg of adenine) and shaken at 37°C for 1.5 hours. By culturing, so-called competent cells (having the ability to take in NDA) were prepared (Reference J. Bacteriol., 81 , 741
(1961)). The NDA solutions obtained in (2) were separately added to this competent cell suspension, and the mixture was further shaken at 37°C for 2 hours to complete the transformation reaction. Next, a suspension containing the DNA transformant of strain AJ11853 was mixed into a minimal medium (minimal medium) containing 5 μg/ml of kanamycin and 100 μg/ml of 8-azaguanine (minimum medium) containing 5 μg/ml of kanamycin and 1000 μg/ml of decoinine ( The cells were spread on a plate of minimal medium (minimum medium) containing 5 μg/ml of kanamycin, 100 μg/ml of 8-azaguanine, and 1000 μg/ml of decoinine, and cultured at 37°C. (The minimum medium is glucose 5g/, (NH ₄ ) ₂ SO ₄ 2g/,
KH ₂ PO ₄ 6g/, K ₂ HPO ₄ 14g/,
_MgSO4・_7H2O0.2g /, sodium citrate 1g/, L-arginine 100mg/, L-
It has a composition of 100 mg of leucine, 50 mg of adenine, and 20 g of agar, pH 7.2). ) After 3 days of culture, 6 colonies appeared on the minimal medium, 5 colonies appeared on the minimal medium, and 3 colonies appeared on the minimal medium, so these were harvested and each clone was isolated. Purely isolated. The properties of the obtained transformants were arginine auxotrophy, leucine auxotrophy, adenine auxotrophy, and kanamycin resistance, and the transformant obtained from minimal medium was resistant to 8-azaguanine, and the transformed strain obtained from minimal medium was resistant to 8-azaguanine. The transformed strain obtained from the minimal medium was a strain having both 8-azaguanine resistance and decoinine resistance. (4) Extraction of recombinant plasmid NDA Among the clones obtained in (3), clone AJ11857 (FERM-P6488) on minimal medium and clone AJ11858 (FERM-P6488) on minimal medium
P6489), using clone AJ11859 on double ground
The method of CIkado et al. (J. Bacteriol., 145 , 1368
(1981)), bacterial cell DNA was extracted separately, plasmid DNA and chromosomal DNA were separated by agarose electrophoresis, and each plasmid DNA fraction was fractionated, collected, and purified. The resulting plasmid, i.e. the strain
Using the same method as described in (3), the plasmids obtained from AJ11857, AJ11858, and AJ11859 were used to transform each original strain, GMP-producing strain AJ11854,
Kanamycin-resistant strains corresponding to each strain were reintroduced into AJ11855 and AJ11856 by the transformation method.
AJ11860, AJ11861 (FERM-P6490) and
AJ11870 (FERM-P6501) was obtained. (5) GMP production; AJ11853, AJ11854,
AJ11855, AJ11856, AJ11857, AJ11858,
Table 1 shows the results of culturing AJ11859, AJ11860, AJ11861, and AJ11870. Culture method is 500ml
GMP production medium (glucose) in a shoulder flask.
80g/, NH ₄ Cl 15g/, KH ₂ PO ₄ 10g/
, K ₂ HPO ₄ 14g/, MgSO ₄・7H ₂ O 5g/
, FeSO ₄・7H ₂ O10mg/, MnSO ₄・
7H ₂ O 10mg/, CaCl ₂・2H ₂ O 2g/, adenine 200mg/, soy protein hydrolyzate 40mg/
, arginine 100mg/, leucine 100mg/
Contains 10g/L-glutamic acid and has a pH of 6.5.
Prepared with KOH. ) was dispensed in 20 ml portions and heated to 115°C.
After heating and sterilizing the cells for 10 minutes, the cells were brought into contact with various bacterial cells obtained by culturing them in advance on a slant medium, and cultured with shaking at 34° C. for 3 days. [Table] [Table] [Table]

Claims (1)

[Claims] 1. A vector incorporating a gene region involved in purine analog resistance or decoinine resistance obtained from the chromosomal gene of a mutant strain of Bacillus having purine analog resistance or decoinine resistance was contained in an adenine auxotrophic mutant strain of Bacillus genus. Guanosine-5'-monophosphate-producing microorganisms are cultivated and guanosine-5'-monophosphate accumulated in the culture medium is collected.
Method for producing 5'-monophosphoric acid.