JP2007228849A - Method for creating mutant filamentous fungus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for creating mutant filamentous fungi by which variation can be induced in high frequency in a range in which many survival fungi exist. <P>SOLUTION: The method for creating the mutant filamentous fungi comprises irradiating conidium of filamentous fungi with heavy ion beam and selecting mutated filamentous fungi from among filamentous fungi formed by culturing the conidium subjected to irradiation treatment. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for efficiently producing mutant filamentous fungi by irradiation with a heavy ion beam.

  Aspergillus such as Monascus anca, Monascus pirosus, Aspergillus oryzae, Aspergillus awamori, fermented foods such as soy sauce and miso, and alcoholic beverages such as sake and shochu In addition, it is an industrially useful microorganism that occupies an important position in industries such as enzymes, pharmaceuticals, and chemical products. In particular, Monascus is known to produce various secondary metabolites such as monacholine K, a cholesterol-lowering substance, γ-aminobutyric acid (GABA), which is a blood pressure-lowering substance, and monascus pigment. Is one of the functional food ingredients. Increase the production of bioactive substances and enzymes by inducing mutations in such industrially useful microorganisms using chemical substances, ultraviolet rays, X-rays and γ-rays as radiation sources. Moreover, it has been performed to provide characteristics that meet new demands.

  So far, several examples of mutagenesis against koji mold have been known (Patent Document 1, Non-Patent Documents 1 and 2), and the mutation means are mainly drug treatment, ultraviolet irradiation, combined use of drug treatment and ultraviolet irradiation. is there. Most of the traits used as an index for selection of mutant strains are abnormal pigment production (high pigment production or albino), and some are based on low production of citrinin (toxin). Other reports on mutagenesis of filamentous fungi include drug treatment for Aspergillus sp. (Non-patent document 3), γ-irradiation for Aspergillus sp. (Non-patent document 4), and γ-irradiation for Enokitake (Patent document 2). )and so on. However, the conventional mutagenesis methods such as drug treatment and ultraviolet irradiation generally have a problem that the mutation rate is low and ancestoring easily occurs.

  On the other hand, in recent years, a new mutagenesis method for irradiating a heavy ion beam (heavy particle beam) in which ion atoms such as carbon are accelerated at high speed using an accelerator has been developed. For Patent Document 6) and yeast (Non-Patent Document 5), mutants produced by heavy ion beam irradiation have been produced. Both heavy ion beam irradiations have a high mutagenesis rate, have higher energy than γ rays, and cause large changes in chromosomes, so there is an expectation that mutations that cannot be obtained with γ rays can be induced. In addition, as for filamentous fungi, there is a report on the production of mutant strains by irradiation with heavy ion beams against black koji mold (Non-patent Document 6), but even when mutations are induced, the survival rate is as low as about 0.1%, and the efficiency Is very bad.

JP 2004-073199 A Japanese Patent Application Laid-Open No. 07-274749 Japanese Patent Laid-Open No. 09-28220 Japanese Patent Laid-Open No. 09-220035 JP 2002-12596 A JP 2001-95423 A J. Ferment. Technol. (1973) 51 (10), 757-759 J. Agric. Food Chem. (2004) 52, 6977-6982 J. Ind. Microbiol. (1991) 8: 113-120 Agr. Biol. Chem. (1973) 37 (4), 789-798 JAERI-Review (2004-025) 565-569 Food Sci. Technol. Res. (1999) 5 (2), 153-155

  Accordingly, an object of the present invention is to provide a method for producing mutant filamentous fungi that is more efficient than conventional methods.

  As a result of intensive studies to achieve the above-mentioned problems, the inventors of the present invention irradiate filamentous fungal conidia with a heavy ion beam, thereby causing mutations in filamentous fungi at a very high frequency within a large number of viable bacteria. We have found that it can be triggered and have completed the present invention.

That is, the present invention includes the following inventions.
(1) Irradiating a filamentous fungus conidia with a heavy ion beam, and selecting the filamentous fungus with mutation from the filamentous fungus formed by culturing the irradiated conidia, suddenly A method for producing mutant filamentous fungi.
(2) The method according to (1), wherein the heavy ion beam is an argon ion beam or a carbon ion beam.
(3) The method according to (1), wherein the filamentous fungus is a red koji mold.
(4) The method according to any one of (1) to (3), wherein the selection is performed using productivity of useful substances by filamentous fungi as an index.
(5) A mutant filamentous fungus obtained by the method according to any one of (1) to (4).
(6) Mutant fungus Monascus pilosus K-69 strain (FERM P-20784).

  According to the method of the present invention, mutation can be induced at a very high frequency in a range where the number of surviving bacteria is large with respect to the filamentous fungus, and the induced mutation is genetically stable. Therefore, the method of the present invention makes it possible to produce mutant filamentous fungi more efficiently than the conventional method. In addition, mutant filamentous fungi obtained by the method of the present invention and having improved productivity of useful substances such as physiologically active substances, antibiotics and enzymes are very useful in various industrial fields such as pharmaceuticals, foods and fermentation. .

  Hereinafter, the present invention will be described in detail.

  In the method for producing a mutant filamentous fungus of the present invention, a filamentous fungus having a mutation is selected from filamentous fungi formed by irradiating the conidia of the filamentous fungus with a heavy ion beam and culturing the irradiated conidia. It is characterized by being selected.

  In this specification, the filamentous fungus includes all of flagella, zygomycetes, ascomycetes, basidiomycetes, and incomplete fungi. The so-called “mold” that proliferates. Examples of the filamentous fungus used in the present invention include, for example, the genus Monascus, the genus Aspergillus, the genus Penicillium, the genus Cephalosporium, the genus Acremonium, and the genus Humicola. And microorganisms belonging to the genus Trichoderma, Mucor, Rhizopus, Fusarium, and Emericella. Of these, industrially useful koji molds used in the pharmaceutical, food, fermentation, and enzyme industries are preferable. For example, Monascus pilosus, Monascus purpureus, Monascus anka, Aspergillus Examples include Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus sojae, and the like.

  Filamentous conidia (asexual spores) are organs formed by extending mycelia into the air and constricting the tip of the filamentous fungus. In order to induce mutations frequently by heavy ion beam irradiation, Although it is preferable to use conidia in a fresh state immediately after growth, the present invention is not limited to this.

  The type of heavy ion beam is not particularly limited as long as it can induce mutations in filamentous fungi. For example, argon (Ar) ion beam, carbon (C) ion beam, nitrogen (N) ion beam, neon ( Ne) ion beam, iron (Fe) ion beam, and the like can be used. Preferred heavy ion beams include argon (Ar) ion beams and carbon (C) ion beams.

  LET (Linear Energy Transfer) in heavy ion beam irradiation is preferably in the range of 20 to 100 keV / μm. The irradiation dose of the heavy ion beam may be determined according to the type of ion beam used, the type of filamentous fungus to be irradiated, etc., and is not particularly limited as long as it does not damage the filamentous fungus and can induce mutation. For inducing mutation at a high frequency in a range with a large number of surviving bacteria, the range of 5 to 30Gray, preferably 10 to 20Gray for Ar ion beam, 50 to 200Gray for C ion beam, preferably 100 to 150Gray it can.

  Next, the conidia irradiated with the heavy ion beam is cultured to form a colony, and a filamentous fungus having a mutation is selected therefrom. The culture may be performed according to a conventional method, for example, by seeding conidia on an agar medium and incubating at 15 to 35 ° C. for 2 to 7 days.

  Any selected filamentous fungus may be used, but industrially useful substances (for example, physiologically active substances and antibiotics that can be active ingredients of drugs, functional food materials, enzymes involved in substance production, etc.) ) Having high productivity is preferred. For example, in the case of red koji mold, those having high productivity such as monacholine K (cholesterol lowering substance), γ-aminobutyric acid (blood pressure effect substance), and red koji pigment (monascus dye) are preferable. The selection method is not particularly limited. For example, when monacolin K productivity is used as an index, monacolin K has antibacterial activity (Journal of microbiological Methods 2000, 40, 99-104), and will be described in the examples below. Such a circle of inhibition assay can be performed. In addition, if it is red yeast pigment production, it can also be selected by visual observation.

  Among the filamentous fungi obtained by the above method, the Monascus pilosus K-69 strain, a mutant strain with high production of monacolin K, was patented by the National Institute of Advanced Industrial Science and Technology as of February 3, 2006. Deposited under the deposit number FERM P-20784 at the Biological Deposit Center (IPOD) (1-1-3 East Tsukuba, Ibaraki).

  The form of the strain on the agar medium is white or light red, and has conidia in which a few are connected under a microscope. The range of growth of this strain is pH 4-8 and temperature 15-35 ° C. This strain produces about twice the amount of monacolin K as compared to the wild strain (parent strain) before the mutation treatment, as shown in the Examples below.

  The mutant filamentous fungus obtained by the method of the present invention may be cultured and propagated by the same method as that for normal wild-type filamentous fungi. As the medium, any natural or synthetic medium may be used as long as it is a medium normally used for culturing filamentous fungi and appropriately contains an assimitable carbon source, nitrogen source, inorganic salts and necessary nutrient sources. . In addition, the culture conditions may be appropriately set and changed depending on the type of filamentous fungus. For example, in the case of red yeast, the pH of the medium is 4 to 8, the culture temperature is 15 ° C to 35 ° C, preferably 20 ° C to 30 ° C. Examples of the number of days are 2 days to 15 days, preferably 5 days to 8 days.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.

(Example 1) Production of mutant strain of koji mold by irradiation with argon ion beam
(1) Irradiation with an argon ion beam Conidia of Monascus pilosus IFO 4520 were collected in sterilized 0.7% physiological saline and dispensed into a plastic tube. This was irradiated with an argon ion beam (LET 281.5 keV / μm; irradiation doses 5, 10, 20, 50, 100 (Gy)).

  The conidia after irradiation was cultured on a potato dextrose agar medium, and the number of formed colonies was counted. On the other hand, unirradiated conidia were similarly cultured on a potato dextrose agar medium, and the number of colonies formed at this time was defined as 100%, and the survival rate was calculated from the number of colonies.

(2) Screening by Productivity of Monacolin K Akapan mold (Neurospora crassa NBRC6067) cultured on a Sabouraud dextrose agar medium for 1 week was scraped together with mycelia with platinum ears and recovered in 0.85% physiological saline. After vortexing the suspension, the mycelium was filtered off with a funnel filled with absorbent cotton to obtain a conidial liquid. A Sabouraud dextrose agar medium having a low agar concentration was autoclaved and cooled to about 40 ° C., and then the above conidial liquid was added and hardened in a petri dish. On the other hand, on the paper disk, the surviving colony by the argon ion beam irradiation of (1) was subjected to liquid culture, and the culture filtrate obtained by filtering the cells was dropped and dried. The paper disk was placed on the solidified agar medium and cultured at 28 ° C. overnight. The area of the inhibition circle formed at this time was calculated by image analysis software, and a strain having a larger area than that of the wild strain (WT) was selected as a mutant strain candidate (this is regarded as a primary selection). Each mutant was subcultured, and the same operation as in the primary selection was performed (this is referred to as secondary selection) to obtain a mutant. FIG. 1 shows the survival rate and mutation rate (high production of monacholine K) of argon ion beam irradiation. In general, in the case of artificial mutagenesis, it is said that the frequency of occurrence of mutant strains is high within the range of survival rate of 0.1 to 1% (revised new edition Experimental Agricultural Chemistry Vol. 268, Asakura Shoten) In this mutation method, the mutation rate increased in the range where the survival rate was almost 100% (almost not dead).

(3) Monacholine K productivity of mutant strain of koji mold Baffle with a medium consisting of glycerin 7%, glucose 3%, corn steep liquor 3%, soy protein 0.8%, magnesium sulfate 0.1%, sodium nitrate 0.2% (pH 6.4) 50 mL each was dispensed into an Erlenmeyer flask (300 mL) and sterilized at 120 ° C. for 20 minutes. This medium was inoculated with a potato dextrose agar medium on a slant culture (2) screened with koji mold mutant (K-69) or, as a comparison, wild strain (WT) and cultured at 25 ° C for 14 days with shaking. did. The bacterial cells were separated from the culture solution, and the filtrate was subjected to the HPLC analysis shown below.

A sample obtained by diluting 1.0 g of the filtrate 10-fold with a solution of water / acetonitrile = 50/50 (v / v) and filtering through a filter having a pore diameter of 0.45 μm was used as a sample. As the mobile phase, 0.1% (w / v) phosphoric acid / acetonitrile = 40/60 (v / v) was used. The column was silica C ₁₈ (Wakosil-II 5C18AR 4.6 × 250 mm), the column temperature was controlled around 40 ° C., and the analysis was performed at a flow rate of 1.5 mL / min. Detection was performed with a PDA detector and monitored at 236 nm.

  Under the above conditions, the retention time of acid type monacolin K was 4-5 minutes. A calibration curve was created from the peak area of a standard product of Monacolin K (trade name: Lovastatin; manufactured by Wako Reagent Industries Co., Ltd.), and acid type Monacolin K in the sample was quantified. As a result, the production amount of monacolin K was 88 mg / L for WT, whereas it was 150 mg / L for the Monascus mutant (K-69 strain), and a marked improvement in production was observed. As for the mutant of Koji mold (K-69 strain), the accession number of the National Institute of Advanced Industrial Science and Technology (IPOD) (1-1-3 East, Tsukuba, Ibaraki) on February 3, 2006. Deposited as FERM P-20784.

(Example 2) Production of mutant strain of koji mold by carbon ion beam irradiation
(1) Irradiation with a carbon ion beam Conidia of Monascus pilosus IFO 4520 were collected in sterilized 0.7% physiological saline and dispensed into a plastic tube. This was irradiated with a carbon ion beam (LET 22.5 keV / μm, irradiation dose 50, 100, 150, 200, 300 (Gy)).

  The conidia after irradiation was cultured on a potato dextrose agar medium, and the number of formed colonies was counted. On the other hand, unirradiated conidia were similarly cultured on a potato dextrose agar medium, and the number of colonies formed at this time was defined as 100%, and the survival rate was calculated from the number of colonies.

(2) Screening by pigment productivity Liquid colonies of surviving colonies by carbon (C) ion beam irradiation, and measuring the absorbance at 450nm of the culture filtrate obtained by filtering the cells, compared to the wild strain (WT) Strains with high absorbance were selected as mutant strain candidates. In the same manner as in Example 1, secondary selection was performed to obtain a mutant strain. FIG. 2 shows the survival rate and mutation rate (high pigment production) of bacteria by carbon ion beam irradiation. In this treatment, the mutation rate increased in the range of 20 to 40% survival rate.

FIG. 1 shows the survival rate and mutation rate of Aspergillus niger by irradiation with an argon ion beam. FIG. 2 shows the survival rate and mutation rate of Monascus by carbon ion beam irradiation.

Claims (6)

  A mutant filamentous fungus characterized by selecting a filamentous fungus mutated from filamentous fungi formed by irradiating a conidia of a filamentous fungus with a heavy ion beam and culturing the irradiated conidia. How to make   The method according to claim 1, wherein the heavy ion beam is an argon ion beam or a carbon ion beam.   The method according to claim 1, wherein the filamentous fungus is a red koji mold.   The method according to any one of claims 1 to 3, wherein the selection is performed using productivity of a useful substance by a filamentous fungus as an index.   A mutant filamentous fungus obtained by the method according to claim 1.   Mutant fungus Monascus pilosus K-69 strain (FERM P-20784).

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