JPS58162290A - Novel microorganism, its separation, and preparation of pyrrolenitrin using and microorganism - Google Patents

Abstract

PURPOSE:To improve the productivity of pyrrolenitrin, remarkably, by using a bacterial strain belonging to Pseudomonas genus and containing plasmid integrated with deoxyribonucleic acid carrying the gene information participating in the tryptophan synthesis of Escherichia coli. CONSTITUTION:A bacterial strain capable of producing pyrrolenitrin (e.g. Pseudomonas pyrrocinia ATCC159858 strain) is used as the plasmid-receptor strain. The deoxyribonucleic acid carrying the gene information participating in the tryptophan synthesis of Escherichia coli e.g. Escherichia coli W3110 (FERM-P No.6439) is integrated into a plasmid, and the plasmid is introduced into the above receptor strain. The introduction is carried out by contacting the bacteria with the plasmid. The pyrrolenitrin productivity of Pseudomonas pyrrocinia can be remarkably increased by the introduction of the plasmid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism capable of producing virolnidoline, a method for obtaining the same, and a method for producing pyrrolnidoline using the microorganism.

Regarding the production method of the antifungal substance virolnidoline by fermentation method, refer to Pseudomonas vilocinia ATC01595.
A method using eight strains is known.

The present inventors used Escherichia coli, which has a plasmid containing deoxyribonucleic acid (hereinafter referred to as the tryptophan operon) that carries genetic information involved in tryptophan synthesis, as a plasmid donor, and transferred this plasmid to a pyrrolnidoline-producing bacterium belonging to the genus Pseudomonas. They succeeded in introducing this plasmid and found that the pyrrolenidoline productivity of pyrrolenidoline-producing bacteria containing this plasmid was significantly increased.

The present invention is based on this knowledge, and is a pyrrolnidoline-producing microorganism containing a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis.

The microorganism of the present invention is cultured, and the culture is obtained from the microorganism (pyrrolni).
By collecting IJ, it is possible to produce IJ at a much higher yield than conventional methods.

The microorganism of the present invention is produced by contacting a microorganism having a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis with a pyrrolnidoline-producing bacterium,
It can be obtained by introducing the above-mentioned plasmid into pyrrolnidoline-producing bacteria.

As a plasmid-donor microorganism having a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis, for example, Escherichia coli, particularly N. coli W5110 strain, is preferable.

All microorganisms can be used as plasmid recipients as long as they have the ability to produce pyrrolnidoline.
For example, Pseudomonas pyrosinia ATCC15958
Strain, Pseudomonas aureofaciens ATCC1
5926 strain etc. are preferred. These strains are collected from the American Type-Culture Collection (ATCC) in the United States.
) and are listed in the ATCC catalog (list of bacterial strains), and can be easily obtained by those skilled in the art. Among the pyrrolnidoline-producing bacteria of the genus Pseudomonas, strains that require specific substances for growth, such as tryptophan-requiring strains, and drug-resistant bacteria, such as strains that are resistant to chloramphenicol, are selected after transformation.
It is commonly used because it is convenient for separation. In order to obtain such resistant bacteria and tryptophan-requiring strains, known means such as treatment with N-methyl-■-nitro-N-nitrosoguanidine (hereinafter referred to as NTG) can be used as appropriate.

Known transformation methods, such as conjugative transfer, can be used to bring the plasmid donor and recipient bacteria into contact and introduce the plasmid into the pyrrolnidoline-producing bacteria.

Screening and isolation of the thus obtained transformed strain does not require any specific method; it is sufficient to select and separate the transformed strain using the properties of the donor and recipient bacteria as indicators.

The pyrrolenidoline-producing microorganism of the present invention is Pseudomonas vilosini 7NK157 strain (RP4-
trp) (Feikoken Bacteria Deposit No. 6458). The mycological properties of this microorganism are as follows: Pseudomonas vilocinia ATC0159 used as a plasmid recipient bacterium
It is the same as the 58 strain, but in addition, E. coli (RP4-t
rp) plasmid and is resistant to ampicillin, tetracycline, and kanamycin. The mycological properties of Pseudomonas pyrosinia ATC015958 strain are described in Japanese Patent Publication No. 1983-25876.

As a method for culturing the IJ-producing microorganism of the present invention, a conventional method for culturing pyrrolnidoline-producing microorganisms can be used. That is, as a medium, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients is used.

Cultivation is carried out under aerobic conditions by shaking culture, aerated stirring culture, etc., while adjusting the pH and temperature of the medium as appropriate, until the production and accumulation of pyrrolenidoline substantially reaches its maximum.

The pH of the medium is preferably 5 to 6, and the culture temperature is generally 25
-65°C, preferably around 60°C.

To collect pyrrolenidoline from the culture, methods commonly used for separating and purifying pyrrolenidoline can be applied.

When the microorganism of the present invention is used, not only is the production yield of bepirol nitrin higher than when known bacteria such as Pseudomonas pyrosinia ATCC 15958 strain are used, but the amount of by-products of substances other than pyrrol nitrine is small, and It is also convenient for the separation and purification of

Example (1) Obtaining chloramphenicol-resistant bacteria of Pseudomonas φ pyrosinia 5 Pseudomonas pyrosinia ATCC15958 strains
ml Noshi medium (1% peptone, 0.5% yeast extract,
Shaking culture was performed overnight at 60°C in 0.5% NaCl, pH 7.0). Next, after collecting the bacterial cells in the logarithmic growth phase, 100
L containing μfl/ml chloramphenicol
When it was spread on an agar medium (L medium with 1.5% agar added) and cultured at 30°C for 48 hours, the formation of colonies of spontaneous mutant strains resistant to chloramphenicol was observed. Isolation of a single colony was repeated on this chloramphenicol-containing agar medium to obtain a purified bacterial strain resistant to chloramphenicol (hereinafter this strain will be referred to as Pseudomonas pyrosinia Cmr).

(2) Pseudomonas pyrosinia N 15 (Cmr
Obtaining Pseudomonas pyrosinia strain (Trp-) Strain was cultured overnight with shaking at 30° C. in 5 ml of L medium. Then, add this culture solution to 1
00μ stones were collected, added to 5 ml of L medium, and cultured at 30° C. for 5 hours. The culture solution was then centrifuged at 300 rpm for 10 minutes to collect bacteria.

Add 1rnl of this bacterial cell to ONTG solution (0.05 M of NTG)
200μy-7m in Tris-maleate buffer of
Dissolved at a concentration of 1 ml, suspended in pH 6.5),
After shaking at 0°C for 30 minutes, the cells were collected by centrifugation at 6000 rpm for 10 minutes. The isolated bacterial cells were washed with 1 ml of Tris-maleate buffer, and then washed again at 50%
Bacteria were collected by centrifugation at 0 Orpm for 10 minutes.

Kono bacterial cells were grown in minimal medium (K2HP0.10 t/13, N
aNH, HPO, -4H203,5P/-e, glucose 2.5 V13, citric acid-H2O27, Mg5O
,・7H200,2P/43) with 0.5 casamino acids
The cells were suspended in 5 ml of a medium supplemented with y-/-e and Tryptocia/100 μyyrnl, cultured overnight at 30°C, and then 10% inoculated into a medium with the same composition and cultured at 60°C for 4 hours. This culture solution is used as a minimal medium containing 0.5 casamino acids!
i! 10% of the cells were inoculated into a medium supplemented with -/-e, and cultured at 60°C for 1.5 hours.

Next, 2 x 10-2 M of D-cycloserine was added to this culture solution.
After further culturing at 30°C for 3 hours, 3000
Bacteria were collected by centrifugation at rpm for 10 minutes. The cells were cultured overnight at 30° C. in a minimal medium supplemented with 0.5 ft# of casamino acid and 7 ml of tryptophan 100 μP. After diluting this culture solution 107 times, it was inoculated onto an agar medium with the same composition and cultured for 48 hours to form colonies. Next, tryptophan auxotrophs were selected by replicating on a minimal agar medium and a minimum agar medium to which 100 μf/ml of tryptophan was added.

Furthermore, single colony isolation was repeated to obtain Pseudomonas pyrosinia N15 (amrtrp-) (hereinafter this strain will be referred to as Pseudomonas pyrosinia N15 strain).

(3) Pseudomonas pyrosinia N by conjugative transfer
Transformation of 15 strains of N. coli W3110 (trpAEl)
(RP4-trp) (Feikoken Bacteria Deposit No. 6439) was cultured in L medium at 60°C with shaking overnight, and 10% of this culture
0 μl was added to L medium with 5 ml of KNO30.4%.
The cells were inoculated and cultured statically at 60°C for 6 hours.

On the other hand, after culturing Pseudomonas keyaki vilocinia N i S strain overnight with shaking at 130°C, this culture solution 2
00 μm in L medium plus KNO30.4% 5
ml, and statically cultured at 60°C for 3 hours.

Next, 0.5 ml of the above-mentioned static culture of N. ko1JW311D strain was applied onto a medium prepared by adding 20 μJ/ml of chloramphenicol to a Pseudomonas vilocinia small agar medium and kept at 30°C. A single colony was isolated from the colonies generated two days later to obtain a purified strain. This is Pseudomonas vilocinia NK157.
strain (RP4-trp).

It was confirmed that E. coli RP4-trp had been transferred to this microorganism because E. coli could be transformed using the plasmid extracted from this microorganism. The plasmid DNA extraction method, Escherichia coli transformation method, agarose gel electrophoresis method, restriction enzyme treatment, etc. were performed using known methods. In addition, E. coli W3110 (trp AE 1 ) (RP4) was used as a plasmid donor.
trp) acquisition method and mycological properties in Applied Environmental Microbiology Vol. 46, No. 2, 19
It was written in 1982.

(4) Production of virolnidoline The above-mentioned Pseudomonas pilonnia strain NK15 (RP
4-trp) was cultured with shaking at 30°C for 4 days in the following medium.

Medium composition: 1% glucose, 1% pezoton, 1% meat extract, 0.3% salt, 2.6% KH2P○4.0, 5% N
a2HPQ4, pH 5,4 The product was then extracted by the method described in the Journal of Antibiotics, Vol. 18, pp. 201-204 (1965).

Similarly, for comparison, Pseudomonas pyrosinia A
Culture and extraction were performed using the TC015958 strain and the N15 strain.

The titer test is Penicillium chrysogenum 111i'04
626 (Q-176) by the cup method. The results are as follows.

ATCo 15958 (wild strain) 4 days
0.96N 15 (Cmr t;rp-)
4 days Undetectable NK157 (RP4-t;r
p) 3rd 5.8 Applicant Kaken Kagaku Co., Ltd. Agent Patent Attorney Masao Kobayashi

Claims (1)

[Scope of Claims] 1. A pyrrolnidoline-producing microorganism containing a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis. 2. The microorganism according to claim 1, which is a planitrin-producing bacterium that has incorporated deoxyribonucleic acid that carries genetic information involved in tryptophan synthesis in E. coli. 3. A method for obtaining a pyrrolnidoline-producing microorganism, which comprises introducing a plasmid into a pyrrolenidoline-producing microorganism from a microorganism having a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis. 4. Contact Escherichia coli containing a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis with a pyrrolnidoline-producing bacterium of the genus Pseudomonas, and introduce the plasmid from E. coli into the pyrrolnidoline-producing bacterium. The method according to claim 6, characterized in that: 5. The plasmid donor is N. coli W61
The method according to claim 4, wherein 10 strains are used. 6. The method according to claim 4, wherein the plasmid recipient strain is Pseudomonas pyrosinia ATCC' 15958 strain. 7.11 A method for producing pyrrolenidoline, which comprises culturing a pyrrolenidoline-producing microorganism containing a plasmid incorporating deoxyribonucleic acid carrying genetic information involved in butophane synthesis, and collecting pyrrolenidoline from the culture. Manufacturing method. 8. The method according to claim 7, which uses a vilolnidoline-producing microorganism of the genus Pseudomonas into which deoxyribonucleic acid carrying genetic information involved in tryptophan synthesis in E. coli is incorporated.