JPS58175493A - Preparation of guanosine by fermentation process - Google Patents

Abstract

PURPOSE:To improve the productivity of guanosine, by adding a vector integrated with a genetic field of purine analogue resistance or dechoinine resistance to a variant belonging to the genus Bacillus, having adenine demanding properties. CONSTITUTION:Chromosome DNA taking part in purine analogue resistance or dechoinine resistance is extracted from variant belonging to the genus Bacillus, having purine analogue resistance or dechoinine resistance. The chromosome DNA is connected to vector DNA, and the prepared recombined vector DNA integrated with a chromosome DNA piece is introduced to a variant belonging to the genus Bacillus, having adenine demanding properties. The transformed substance thus obtained is cultivated in a liquid medium containing a carbon source, nitrogen source, inorganic salt, adenine, etc., and guanosine is accumulated in the culture.

Description

[Detailed description of the invention] The present invention relates to a method for producing guanosine by fermentation.

Regarding the production of guanosine by fermentation methods, microorganisms of the genus Bacillus with adenine auxotrophy or resistance to various purine analogs (Japanese Patent Publication No. 39-16347),
Microorganisms of the genus Brevibacterium (Japanese Unexamined Patent Publication No. 51-12),
History of sulfa drugs (Japanese Patent Application Laid-open No. 50-70592), decoinine or psychofuranine (Japanese Patent Application Laid-open No. 5(1973) 1-126886)
) or methionine or azaserine (Japanese Patent Application Laid-Open No. 1973 =
It is known that microorganisms of the genus Bacillus that have been made resistant to 1358 (J) produce guanosine.

In contrast to the conventional guanosine production method as described above, we have developed purine analog resistance or decoinine resistance obtained from Bacillus chromosomes that have purine analog resistance or decoinine resistance! It has been found that a guanosine-producing microorganism of the genus Bacillus, in which a mutant strain V of the genus Bacillus auxotrophic for adenine contains a vector into which a gene region relating to 1'1 has been incorporated, accumulates a significant amount of guanonone.

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 the inhibition inhibits the growth of hypoxanthine, inosine, 51-inosinic acid, guanine, guanosine, 51-guanylic acid, adenine, adenone, 5-adenylic acid, etc. When added to the culture medium, Gi' is completely or partially released. For example, 8-azaguanine, 8-
Examples include azahypoxanthia, 8-azaadenine, 2,6-diamitpurine, 6-mercaptopurine, 6-mercaptobulin riboside, and 8-mercaptoguanosine.

The donor strain for the chromosomal gene related to purine analog resistance or decoinine resistance may be any mutant strain of the genus Chirus that has purine analog resistance or decoinine resistance, but one with a higher degree of resistance is preferable. or,
Adenine9. If a mutant strain with purine analog resistance or decoinine resistance is induced using a permanent resident strain with guano7-producing ability as a parent strain, a mutant strain with higher guanosine-producing ability can be obtained. Better results can be obtained if the strain is used as a genetic f“contributor.

In addition, as genetic bacterium, adenine auxotrophic and purine analog or decoinine resistant mutant strains, Saratsuko, etc. By inducing and using strains with added resistance, azaserine resistance, saifufuranine resistance, sulfaguanidine resistance, etc., it is possible to obtain strains with higher guanosine production ability, and if such strains are used as chromosomal genetic pediatric bacteria. , more favorable results are obtained.

For example, J. Racteriol, +89+]
This can be carried out by conventional methods such as those described in J. O. 65 (1'965).

The vector DNA may be any plasmid or phage that is produced in the cells of the genus Bacillus. For example, pT+ derived from microorganisms of the genus Staphylofthucus
2'7, pc]94, pc221°pc223. p
UBII2 (hereafter "-1Proc, Na1l.

Acad, Sci, U, S, A, +74. 16
80 (+977)), p U B 1. I O
(J, Bacteriol.

134, 318 (1978)), pTP4+pT
P5 (Microbiol Letters+
5 + 55 (+978)), p derived from Bacillus subtilis
Ls+5゜I) LS28 (hereinafter, J, Bacter
iol, + I 31 .

699 (+977)), pLsI3 (J, Bact
eriol, +129. 1487 (+977
), pPLl, pPL2 (see above, J, Bac
teriol, +124, 484 (1975))
rho l, also known as temperate phage
l (Gene, + upper, 89 (+9'79)')
+phi+05(Gene,.

5, 87 (1979)), 5PO2 (Gene, Yu.

5] (+979)) etc. 1, more? Naturally, a composite plasmid constructed based on the plasmid described above can also be used as a vector DIAG.

Chromosomal DNA and vector DNA are each cut using restriction endonucleases. each vector 1
There is a restriction enzyme enzyme suitable for moss, but it is −
There are documents etc. that describe the problem of vector 1 [hereinafter referred to]. For chromosomal DNA, many species and 11 restriction threads can be used if the reaction conditions are adjusted so that cleavage by restriction endonucleases is performed only partially.

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

The recipient strain of the recombinant vector DNA by integrating the chromosomal DNA fragment thus obtained 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. Furthermore, if a strain having purine analog resistance or decoinine resistance and a higher guanosine-producing ability is used as a recombinant DNA recipient, a transformed strain with a higher guano7-producing ability can be obtained. Of course, the receptor bacteria is guano 7.
A product with a lower production capacity must be used.

DNA recipient 1Y41 mix of chromosomal DNA and vector
For example, Malec, Gene, G
enet, +168. Conventional transformation methods can be used, such as those described in 111 (+979).

For example, an adenine auxotrophic mutant strain can be used as a vector recipient to select a transformed strain that has the ability to produce guanosine and contains a gene region that is involved in purine analog resistance or decoycin resistance. A strain that grows in a medium containing a purine analog or decoisin may be selected. Also, vector DNA...l.

Selection will be easier if a culture medium is used that allows selection of bacterial strains with properties such as antibiotic resistance.

In this way, the once selected recombinant vector DNA into which the gene region for purine analog resistance, etc. By introducing it into a strain capable of producing guanosine, it is possible to further increase the amount of guanosine accumulated.

The method for producing guanosine by culturing the guanosine-producing bacteria thus obtained is not particularly different from conventionally known methods.

That is, the medium in which such microorganisms are cultivated is a conventional liquid medium containing a carbon source, a nitrogen source, inorganic salts, adenium/and, if necessary, other micronutrients. Carbohydrates such as glucose, molasses, and starch hydrolyzate are preferred as carbon sources. Nitrogen sources include ammonium sulfate, ammonium nitrate,
Ammonium salts such as ammonium phosphorus, nitric acid salts such as ammonium nitrate, urea, ammonia gas, etc. can be used. In addition, adenine as a substance required for education is adenine, adenine mineral II a<
, adenosine, adenylic acid, etc. can be used. Furthermore, it is often possible to increase the amount of guanosine accumulated by adding micronutrients such as vitamins, amino acids, and nucleobases other than adenine as needed.

The culture method is preferably carried out under aerobic conditions, and the culture humidity is preferably in the range of 27 to 38C. Depending on the case, the trowel culture temperature may be changed during the culture. At the start of culture and during culture, the p lf of the culture solution is set to 5.0 to 9.01.
It is desirable to adjust and culture this.

For adjustment of pII, an inorganic acid, an organic acid or an alkali, urea, calcium carbonate, aqueous ammonia, ammonia gas, etc. can be used. Thus, after 2 to 5 days of culture, a significant amount of guano/7 accumulates in the culture medium.

Guanosine may be collected from the culture solution by a conventional method such as using an ion exchange resin.

Example: N-methyl-N-nitro-N from Chillus subtilis AJ'11711 (Arginino, Leucine multi-requiring strain)
-Nitrosoguanine mutation treatment (5001+ y/lr
A bacterial strain AJ11862 having the ability to produce guanosine from adenine auxotrophs was obtained by contacting 10''/-t bacterial cells with 1 OC of N-methyl-N-nitrosoguanidine for 30 minutes. (FpRM-p 64
'll-) (arginine/auxotrophy, leucine auxotrophy, adenine auxotrophy) was obtained. Zara1' Guano/7 producing bacteria A derived from this adenine auxotrophic strain by similar mutation treatment
J I I 863 (FERM-P et al. 4A'E) (
arginisine requirement, leucine requirement, adenine requirement, 8-azagua-requirement), AJ11864 (FERM-PG price 9@
) (alkinine auxotrophy, leucine auxotrophy, adenine auxotrophy, definine resistance), A J l + 865 (
-f luginine requirement, leucine requirement, adenine requirement, 8-azaguanine resistance, decoycine resistance).

(1) Preparation of chromosomal DNA AJl'+863, AJl! 864, AJ +1865
Bacto-Penassay B
Shaking culture was performed in 30C1 for about 2 hours in roth 1 (manufactured by Difco), and after collecting the cells in the logarithmic growth phase, they were cultured in a normal D
NA extraction method (J, Bacteriol, +89+
1065 (+965)), chromosomal DNA was extracted and purified from the final AJ11863. c+mg, A
Jl 1864-3.6my, AJ11865-3
．． I got 3 ■.

+21 Insertion of a chromosomal DNA fragment into a vector In order to clone the gene region governing purine analog resistance or decoinino resistance, an autonomously replicating plasmid is used as the vector 1) L, I B l I O (expressing kanamayone resistance, neomynon resistance) ) was used.

5 μf each of the chromosomal DNA used in UL and 3 μf each of the plasmid pUB11057if and 3 μf of the restriction endonuclease EcoRI. The r) NA chain was cut by using an rt trowel for 60 minutes. After heat treatment at 65C for 10 minutes,
Both reaction solutions were mixed to obtain ATP and dithi.

In the presence of Oslytol, DNA strands were ligated using T4 phage-derived DNA ligase using a 10C1 trowel for 24 hours.

(;1) Transforming Bacillus subtilis AJII862 with recombinant plasmid DNAt, IP, ena
Ssay-Broth j (manufactured by Difco) was inoculated and cultured with shaking overnight at 30R, and the first culture medium (Glucose sy/l, (N114), 5O
42? / L1KH2PO469/2HP to 'tS'
O4149711Mg504・7H,OO,2f/
t, sodium citrate + y/lS yeast extract 2
t/1SL-Arginine 250 doors9. /1%L-Roy・
7750 y/t.

containing 5011 g/l of adenine) and cultured with shaking at 37R for 4 hours, and the first culture medium (f
Luco 75, f/l − (Nf4<) 250
42? /l 1 KH2P0. 6 f/
l, °<,,HPo, 6 ta/l,”:'
, HPO, I 4 f/1% MS, ), a7H20
+, 2y/l, sodium citrate If/'tsfl
f-P4 extract 0.2f/l, L-az+; 4::,
50 mg/l sI,-leucine 5my/z, 7
'>', including z750my/1), 37rt
trowel] Incubate with shaking for 5 hours 1. This results in so-called competent cells (having the ability to take in DNA).
(Reference J, BacLeriol, +
81 + 741 (+961)). The DNA solutions obtained in Step 21 were separately added to this competent cell suspension, and the cells were further cultured with shaking at 37C for 2 hours to complete the transformation reaction.

Next, a suspension containing the DNA and A transformants of strain AJ11862 was added to a minimal medium (minimum medium 10
), minimal medium containing Kanamaishifu 5μ97ml and decoinino (minimum medium ■), Kanamainone 5 ltf7
A plate of minimal medium (minimal medium ■) containing ml and 8-azaguanine OO1t9/me and 1000 pf,/d gold was plated and cultured at 37C.

(The minimal medium is glucose 59/l, (NH4)2S
o42 f/l, KH, Po, 6? /l.

K2HPO4149711MgSO4・7H20・o
, zy/11 sodium citrate ly/l, L-arginine 1100 tn/l, L-leucine 100/1° adenine 50/l and agar 2 oy/l.

(pH 7.2). ) After 3 days of culture, 4 colonies were found on the minimum medium (Lv 5), 3 colonies were found on the minimum medium (1), and 3 colonies were found on the minimum medium (Lv 5).
As individual colonies appeared, these were harvested and each clone was isolated.

The properties of the obtained transformed strain are as follows: arginine auxotrophy, leunon auxotrophy, adenine auxotrophy, kanamayone resistance, and the traits obtained from the minimal medium ■...ζ
The transformed strain was resistant to 8-7zaguanine, the transformed strain obtained from minimal medium (■) was resistant to defuicin, and the transformed strain obtained from minimal medium (■) was resistant to 8-azaguanine and decoysif. Ta.

In this way, clone AJ+ 186 on minimal medium ■
6 (FERM-P), minimal medium h clone AJ I + 867 (, 12ERM-P 4)
97), clone AJ11868 (FE
RM-P (l,<'ig) was obtained.

(4) Extraction of recombinant plasmid DNA AJ1186
8 using the method of C, 1, Kada et al. (J, Ba
cLeriol, + Yu 4, 1365 (1981
)) DNA extraction method based on the method 1 Extract the DNA of each bacterial cell separately, separate the plasmid DNA and chromosomal DNA by agarose electrophoresis, collect 4 minutes each of the plasmid DNA, and purify. did. The obtained plasmid was reintroduced into AJ11865 by a transformation reaction using a method similar to that described in (3), and the corresponding kanamycin-resistant strain AJ + 1.869 (FE
RM-P et al. 499).

(5) Guano 7 production AJ+1862, ΔJ11863, AJl+864, A
J11865, AJ+1866, AJ11867, ΔJ
The results of culturing I+868 and AJ11869 were
Table V is shown here. The culture method is to use guanone production medium (glucose 80?/l, NH,N
o3151/l, KH2PO40,2? / L,
Mg5O, 7H200, 4y/t 1FeSO
4・7H2010m9/L, MnSO4・7H
2010m9/ASCaC+2 ・2H202f,
/ t1 adenine 20otng/l, soy protein hydrolyzate 4011/, /11 arginine 100g/110 isine 100mg/1 and! +-glutamic acid yot
/l, pH 6,5 In KOH. ) was dispensed at 20m/each, sterilized under pressure at 115C for 10 minutes, and then inoculated with various bacterial bodies previously cultured on slant culture medium.
Shaking culture was performed at 4C for h days.

Table 1 arg + arginine, auxotrophy 8AGr; 8-7 zaguanine resistance leu; leucine auxotrophy Decr; decoysif resistance ade; 7-F' auxotrophy Kmr; Kanayainone resistance patent applicant Ajinomoto Co., Inc.

Claims (1)

[Claims] A vector incorporating a gene region for purine analog resistance or decoinine resistance obtained from the chromosomal gene of a mutant strain of Bacillus genus that has purine analog resistance or depuinine resistance is used as an adenine auxotrophic mutant strain of Bacillus genus. A method for producing guanosine, which comprises culturing the guanosine-producing microorganism containing the guanosine and collecting guanosine accumulated in the medium.