JPS58158195A - Preparation of 5'-inosic acid by fermentation process - Google Patents

Abstract

PURPOSE:To prepare 5'-inosic acid in high yield, by cultivating a bacterium belonging to the genus Bacillus containing a vector integrated with a genetic range taking part in purine analog resistance. CONSTITUTION:A bacterium such as Bacillus subtilis AJ11818(FERM-P6340), capable of producing 5'-inosic acid, obtained by adding a vector integrated with a genetic range participating in purine analog resistance obtained from the chromosome of a variant belonging to the genus Bacillus having purine analog resistance to an adenine damanding variant belonging to the genus Bacillus is inoculated into a nutritive medium, cultivated under aerobic conditions at 4- 8pH for 1-5 days, and 5'-inosic acid is collected from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 51-inosinic acid by fermentation. 51-inosinic acid is widely used as a seasoning, and a method is known in which 51-inosinic acid is produced by culturing mutant strains of Bacillus or Frinebacterium (Japanese Patent Publication No. 1982- 32918. JP 55-150899).

The present inventors developed a vector incorporating a gene region involved in purine analog resistance obtained from the chromosome of a Bacillus mutant strain having resistance to purine analogs, in contrast to the conventional method for producing 51-inosinic acid as described above. It has been found that a microorganism containing 51-inosinic acid in an adenine-auxotrophic Bacillus mutant strain accumulates 51-inosinic acid at a high yield.

Although it is not necessary to use a specific purine analog resistant strain of the genus Bacillus, one with a higher degree of resistance is desirable. In addition, those resistant to purine analogs and those resistant to other drugs such as sulfa drugs can also be used. More desirable results can often be obtained by using purine analog-resistant strains that have a higher ability to produce 51-inosinic acid.

Many such strains of Bacillus that are resistant to purine analogs are already known;
It can be obtained by a conventional method such as mutating a Bacillus microorganism with N-nitrosoguanidine and isolating a mutant strain that has acquired resistance to growth inhibition by purine analogs.

Purine analogs as used in the present invention inhibit the growth of microorganisms of the genus Bacillus, and this inhibition can be completely or partially relieved by adding hypoxanthine, inosine, or 51-ynonic acid to the culture medium. It is like

For example, 8-azaguanine, 8-azahypoxanthine,
Examples include 8-azaadenine, 2,6-diamitpurine, 6-mercaptopurine, 6-mercaptopurine riboside, and 8-mercaptoguanone.

A method for extracting chromosomal DNA from purine analog resistant strains is described in J. Bacteriol, 83, 1065 (1).
The vector DNA that can be used in the conventional method as described in 965) may be any plasmid or phage that replicates in the cells of the genus Bacillus. For example, pT127 derived from a microorganism of the genus Staphylofuncus. pc194. pc221. pC
223, pUB112 (Proc, Natl,
Acad, 8ci, U, S, A,, 74.1
680 (1977)), pUBl 10 (J,
Bacteriol, 134.318 (1978)
), pTP4. p'rps (more than Microbi
ol Letters.

5.55 (1978)), pLsls from Bacillus subtilis
, pL828 (J, Bacterio1..13
1.699 (1977)), pLs13 (J, Ba
cteriol, 129.1487 (1977)), pPLl.

pPL2 (J, Bacteriol, 12
4, 484 (1975)), rho 11, also known as the temperate phage (Gene, 5
．． 89 (1979)), phi105 (Gene,...
5.87 (1979)), SPO2 (Gene,...
Yu, 51 (1979)). Furthermore, a complex plasmid constructed based on the above-mentioned plasmid can also be used as a vector DNA.

Chromosomal riNA and vector DNA are each cut using restriction endonucleases. There is a suitable restriction endonuclease for each vector, as 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 endonucleases partially cut the DNA.

A conventional method using ligase can be used to link the chromosomal DNA fragment thus obtained and the cut vector DNA. On the other hand, it is also possible to use a method in which deoxycytidylic acid and deoxycytidylic acid are respectively added to a chromosomal DNA fragment and cleaved vector DNA using terminal transferase, mixed, and then annealed to link them.

The recipient bacterium for the mixture of chromosomal DNA fragment and vector obtained by extraction may be any mutant strain of the genus Bacillus that has adenine auxotrophy, but if a strain that does not have purine analog resistance is used, the This is advantageous when selecting convertible stocks.

Of course, it is better to use DNA recipient bacteria that have a lower ability to degrade 51-inosinic acid. Therefore, more desirable results can be obtained by using a compound with a higher ability to produce and accumulate 5'-ynonic acid.

To introduce a mixture of chromosomal DNA and vector into a recipient DNA strain, e.g.
et,, IJII.

111 (1979) can be used.

To select a transformed strain that has the ability to produce 5'-inosinic acid and contains a vector into which a gene region related to purine analog resistance has been integrated, for example, an adenine auxotrophic mutant strain is used as a DNA recipient. A strain that can be converted and grows in a medium containing a purine analog may be selected. In addition, if a medium is used that allows selection of bacterial strains that have vector DNA properties such as antibiotic resistance, selection will be easier.

In this way, the once selected recombinant beta that incorporates the gene region involved in purine analog resistance is extracted from the transformed strain and then transferred to other DNA recipient bacteria, such as a strain capable of producing 51-inosinic acid. to be introduced into
This makes it possible to further increase the amount of 51-inosinic acid accumulated.

Using the thus obtained 5'-inosinic acid producing bacteria, 5
The method for producing 1-inosinic acid is not particularly different from the conventional culture method for 51-inosinic acid producing bacteria. That is, the medium is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and inorganic micronutrients. Carbohydrates such as glucose are preferred as carbon sources. As the nitrogen source, organic nitrogen sources such as aqueous ammonia, ammonia gas, ammonium salts, and amino acids can be used. As an inorganic ion, phosphorus
In addition to the 1 ion required, potassium ions, magne/lamb ions, iron ions, manogan ions, etc. are added to the medium as appropriate. A substance that should satisfy adenine requirement as an organic micronutrient, such as adenine, adenosine, or RNA hydrolyzate, is added. In addition, vitamins, amino acids, etc. are appropriately used as organic e-nutrients.

Cultivation may be carried out under aerobic conditions, preferably with pH controlled at 4 to 8, for 1 to 5 days. A significant amount of 51-inosinic acid is produced and accumulated in the culture solution thus obtained. 51-inosinic acid may be collected from the culture solution by any conventional method such as using an ion exchange resin.

Example Adenine auxotrophic mutant strain AJ11813 (FERMP 6,000) induced from Bacillus subtilis AJ11711 (arginine, leucine auxotrophic) by N-methyl-Nl-nitro-N-nitrosoguanidine mutation treatment
) was obtained. Furthermore, 51-inosinic acid producing bacteria AJ were derived from this adenine auxotrophic strain by the same mutation treatment.
11814 (FERMPA+z6) (Arginine
auxotrophy, leucine auxotrophy, adenine auxotrophy, 8-azaguanine resistance), AJ 11 '815 (FERMP
6 λ7 ) (arginino auxotrophy, leucine auxotrophy,
Adenine auxotrophic, 8-7zaguanine resistant, sulfaguanidine resistant) was used as the original strain, from which a new 51-inosinic acid producing bacterium was constructed by the following method.

+11 Preparation of chromosomal DNA 1t rB of each of AJ11814 and AJ11815
Shaking culture was carried out at 30°C for about 2 hours in acto-Penassay Broth J (trade name, manufactured by Difco), and after collecting the cells in the logarithmic growth phase, the normal DNA extraction method (J, Bac-teriol, 89.1065
(1965) ) to extract and purify chromosomal DNA,
Final results were 3.9~ from AJ11814 and 3.2~ from AJ11815.

(2) Insertion of chromosomal DNA fragment into vector In order to clone the gene region governing purine analog resistance and 5'-inosinic acid production ability, we used the autonomously replicating plasmid pUB110 (Kanamai 7) as a sonovector.
(expressing resistance to neomycin). 5μ2 each of the chromosomal DNA obtained in (1) and plasmid pUB
1105μ2 each of restriction endonuclease E
The DNA strand was cleaved with coRI at 37°C for 60 minutes. After heat treatment at 65°C for 10 minutes, both reaction solutions were mixed, and DNA was ligated with T4 phage-derived DNA ligase at 10°C for 24 hours in the presence of ATP and dithiothreitol.
A chain ligation reaction was performed.

Transformation of Bacillus subtilis AJ] 1813 (arginine, leucine double auxotrophic strain, adenine auxotrophic mutant strain) with a plasmid carrying sulfa drug resistance and inosinic acid production inheritance.
Inoculate assay Broth J (manufactured by Difco) and culture with shaking overnight at 30°C, first culture medium (glucose 5 l/A, (NHI\5042 f/l
1 M, PO46t/l.

K, HPO414f/L 1Mg804・7H, OO,
2f/l 1 sodium citrate lt/l, yeast extract 29/l, L-arginine 250 H2/1% L-
Leucine (containing 50~%4Adenine5o #v/l) was inoculated and shaken at 37°C for 4 hours in MgSO4.7H2O1,
2f/l, sodium z7ate 1f/11 yeast-like kiss 0.2f/l, L-fulginine 50q/1%L-
A so-called competent (DN
Cells with A uptake ability were prepared (References, J, B
acteriol,, 81.741 (1961)
). The DN obtained in a2) was added to this competent cell suspension.
Solution A was added separately to each and cultured with shaking at 37°C for an additional 2 hours to complete the transformation reaction.

Next, the suspension containing the transformed strain of AJ11814 DNA was added to Kanamai Schift 5 μ97ml 1.8-7 Zaguanin 50
Minimal plate containing 0 r/me, medium ■ (glucose 59/l), (NH4), 5o4s+f/z, KH
, PO46t/l, K2HPO414Ill,
Mg804・7H, 00, 2f/1. Sodium citrate 1f/l, L-arginine 100η/l, L-leucine 100III/11azo = 750ml/l.

The mixture was spread on agar (20 f/l, pH 7.2) and cultured at 37°C. In addition, a suspension containing a transformed strain of AJ1111115 DNA was treated with kanamycin (5) tf/ml, 8-7
Zaguanine 100r/at, Sulfaguanidine 10
Spread on the above minimal plate medium containing 0r74/3.
Cultured at 7°C. After 3 days of culture, 6 colonies appeared on the Cm) medium and 5 colonies appeared on the medium (■), which were harvested and each clone was isolated.

The properties of the transformed strain obtained from the medium (III) were as follows: arginine auxotrophy, leucine auxotrophy, adenine auxotrophy, 8-7 zaguanine resistance, kanamycin resistance,
The properties of the transformed strain obtained from the medium (■) are arginine auxotrophy, leucine auxotrophy, adenine auxotrophy,
It showed resistance to 8-azaguanine, sulfaguanidine, and kanamycin.

+41 Extraction of plasmid pUB 1 to carrying purine analog resistance or sulfa drug resistance region Among the clones obtained in 13), clone A on medium (III)
Jl1816 (vwRM-p IE, ??>, clone A J 11 Ill 7 (F
ERM-Pj/2wo) using the method of C,1, Kado et al. (J.

Bacteriol, 145.1365 (1981
) The DNA of each bacterial cell was extracted separately using a DNA extraction method based on
A fraction of each fraction was collected and purified.

The new plasmid thus obtained, namely strain AJ118
The plasmid obtained from No. 16 was transformed into the original strain of 51-inosinic acid producing bacterium AJ1 using the same method as described in (3).
1814 by the transformation method, and the kanamycin-resistant strain AJ 11818 (FERM-p jlj
l) was obtained. In addition, the plasmid obtained from AJ1'1817 was reintroduced into 5'-inosinic acid producing bacterium AJ11815 by the transformation method, and a kanamycin-resistant strain AJ11 was obtained.
819 (FERMP-jguj/) was obtained.

(5) 51-ino produced by a novel 51-inosinic acid producing bacterium.

Each strain A J 11813 obtained as above,
AJ 11814, AJ 11815, AJ 118
16, AJ11817, AJ11818, AJ 118
Table 1 shows the results of culturing No. 19. The culture was carried out using 51-inosinic acid production medium (glucose 80f/NH4No315y7tlIKH, PO
410q/l s Mg5o4-7H,, o 5 Vt
sFe S 04 ・7 HqO10'f/L, Mn
SO4・7 H,010W/l, CaC1,2*2
H2O2Ill, adenine 0,2Ill, soybean protein hydrolyzate 40sl/l, pH 6,5 (K
Prepared with OH)) was dispensed into 2°IIe portions and incubated at 115℃
After pressure sterilization for 0 minutes, various bacterial cells previously cultured on a slant medium were inoculated, followed by shaking culture at 34° C. for 72 hours.

Table 1 Patent applicant: Ajinomoto Co., Inc.

Claims (1)

[Claims] 51-inosinic acid production in which adenine auxotrophic mutant strain of Bacillus genus contains a vector incorporating a gene region involved in purine analog resistance obtained from the chromosome of a mutant strain of Bacillus genus that is resistant to purine analogs. 1. A method for producing 51-inosinic acid, which comprises culturing a sexually active microorganism and collecting 51-inosinic acid accumulated in a culture medium.