JPH0920791A - Chitin-synthesizing enzyme-inhibiting substance am-1 substance and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject substance exhibiting an antifungous action against fungous variants defected in chitin-synthesizing enzyme I which is a cellular wall-synthesizing enzyme, and useful for agriculture etc., by culturing a microorganism belonging to the genus Leptosphaeria sp. and having an ability for producing the AM-1 substance. SOLUTION: A new AM-1 substance having following characteristics. The mol. wt. (m/z): 337 (FAB-MS); molecular formula: C22 H43 ON; UV absorption spectrum has maximum absorption peaks at 248nm and 286(sh)nm; Rf value is 0.05 in a development solvent of benzene:acetone (7:3), 0.25 in chloroform- methanol (7:3), 0.28 in benzene:chloroform:ethyl ether (8:2:1:1) in a thin layer chromatography, and positive for an iodine reaction. The compound is obtained by culturing a microorganism belonging to the genus Leptosphaeria sp. and having an ability for producing the AM-1 substance and subsequently collecting the substance from the culture product.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel type of antifungal which exhibits antifungal activity against a mutant strain deficient in chitin synthase 1 which is a fungal cell wall synthase, and which does not exhibit antifungal activity in a wild strain. The present invention relates to an AM-1 substance that can be a fungal agent and a method for producing the same.

[0002]

2. Description of the Related Art In recent years,
There is a growing need for new fungal agents with selective toxicity that act only on fungi. There are various mechanisms that produce selective toxicity, such as activation and inactivation of drugs depending on cell type, permeability of cell membranes, etc. Among them, biosynthesis of cell wall constituents of microorganisms that do not exist in animal cells is involved. It is known that higher selective toxicity is expected by inhibiting. The structure of the cell wall of fungi differs greatly from that of bacteria, and consists of proteins, lipids, polysaccharides, inorganic salts, etc. Among them, β- (1 → 4) -binding product of N-acetylglucosamine, chitin, and glucose β-1,3 glucan, which is a β (1 → 3) binding product, is a major constituent of the cell wall in many fungi.

By the way, it is known that fungal chitin synthases are classified into four groups based on their predicted amino acid sequences, regardless of the species of organisms, and play a complex role in cell wall formation. Has been. Cabib is related to three yeast chitin synthases (CHS1, CHS2, CHS3). CHS1 is involved in chitin synthesis although it is not essential for yeast septum formation, and CHS2 is difficult to be originally inhibited by drugs. That, CHS3
Synthesizes most of the chitin in the cell wall, CHS2 and CHS
It has been reported to be fatal due to the double deletion of 3 [Proc. Natl. Acad. Sci. USA, 8
5 , 4735-4739 (1988) and Proc.
Natl. Acad. Sci. USA, 91 , 4727
-4730 (1990)].

At present, polyoxine is known as an agricultural antibiotic with a focus on the antibacterial activity against fungal chitin synthase-deficient mutants, but as described above, other new types of fungal agents are required. This is the current situation.

[0005]

The present inventors have tried to search for a chitin synthase inhibitor from a microbial culture by using the paper disk method using a chitin synthase 1 deficient strain and a wild strain. As a result, it can be a new type of antifungal agent that exhibits antifungal activity against a mutant strain lacking chitin synthase 1 in the culture of a strain belonging to the genus Leptosperia and does not exhibit antifungal activity in a wild strain. It was found that a new substance was produced, and the present invention was completed.

That is, the gist of the present invention resides in an AM-1 substance having the following physicochemical properties and a method for producing the same. (1) Molecular weight: m / z = 337 (FAB-MS) (2) Molecular formula: C ₂₂ H ₄₃ ON (3) Ultraviolet absorption spectrum: in methanol, 248 n
Absorption maxima at m and 286 (sh) nm (4) Thin layer chromatography: The following Rf values are shown under the following developing solvent conditions.

[0007]

[Table 2]

(5) Color reaction: positive for iodine reaction The present invention will be described in detail below. The AM-1 substance of the present invention (hereinafter sometimes abbreviated as “the compound of the present invention”) is
For example, a microorganism belonging to the genus Leptosphaeria capable of producing the compound of the present invention can be cultured and collected from the culture solution.
The microorganism is not particularly limited as long as it belongs to the genus Leptopheria and has the ability to produce the compound of the present invention. Specifically, by the present inventors, from the dicotyledonous plant collected in Keisatsugawa-mura, Okinawa Prefecture, Leptosperia sp.
eptosphereia sp. ) MCI2799
(Hereinafter "this strain", "MCI2799" or "MCI
It may be abbreviated as "2799 strain"). In addition, this strain was produced by the Institute of Biotechnology, National Institute of Industrial Science and Technology, Biotechnology Research Institute, No. 15016 (FERM
It has been deposited as P-15016). MCI279
The bacteriological properties of the 9 strains are as follows.

1) Morphological characteristics Pseudocysts are scattered on the host plant, are embedded in plant tissue, and then cause the neck to protrude onto the epidermis. Subglobular to slightly flattened, diameter 230 to 380 μm. The neck is markedly developed and papillary. Pseudocysts have a relatively thin shell wall, pseudosoft tissue,
The thickness is 8 to 11 μm. 120-132 μm with a long, stalk-shaped pedicle as a result
20.0-24.5 μm, clear double-walled structure, 8-sporic. False side yarns occur abundantly and have filaments and septa. Ascospores are arranged in 2 to 1 rows in the asci (two rows in the upper part of the ascos),
1 row at the bottom), 25.9-34.1 μm × 8.4 × 10
μm, oval to slipper shape. Dark dark brown, usually 3 septa, rarely 4 septa, slightly enlarged second cells, slightly constricted at septum. It lacks an appendage.

2) Characteristics on various media 24 on potato-glucose agar medium (PDA)
C., 2 weeks culture Colonies spread to 4.5 cm in diameter for 2 weeks. The color tone of the colony is initially white, then dark gray. Basal hyphae radially extend and branch, width 3.0-5. 6μ
m, having a partition wall. Aerial hyphae are abundantly formed. No formation of anamorph and teleomorph was observed on this medium. Cultivated on malt agar medium (MA) at 24 ° C. for 2 weeks. The colony spreads to a diameter of 3 to 4 cm in 2 weeks. The color tone is initially white, then grayish brown, and the basal hyphae extend radially and Branching, width 4.0-6.8μ
m, having a partition wall. Development of aerial hyphae is moderate. No formation of anamorph and teleomorph was observed on this medium.

3) Physiological properties Optimal growth conditions Optimum pH: 4 to 6 (in Miura medium (LCA) liquid medium, 2
Weekly culture) Optimum temperature: 20-27 ° C (on PDA medium for 2 weeks) Growth range pH range: pH 3-8 (in LCA liquid medium for 2 weeks) Temperature range: 15-30 ° C, 37 ° C Does not grow (cultivated on PDA medium for 2 weeks)

4) Taxonomic Consideration Higher taxonomic position This strain (MCI2799) 1) occurs on dicotyledonous plants, 2) forms a pseudospherical shell of subglobular to flat shape, 3) Ascos have clear double-walled structure, 4) Persistent pseudolateral yarn (ps)
euphoraphysis), and 5) ascospores are multicellular, L. Holm, Sym
b. Botan. Upsal. , 14 (3): 1-18
8 (1957), E.I. S. Lutrell, Locu
loacomycetes, The Fungi, v
ol. 4A (ed.GC Ainsworth et
al. ): 135-219 (1973), J. A. v
onArx & E. Muller, Stud. Myco
l. , 9 : 1-159 (1975), and A. Siva
nesan, The Bitunicate Asco
mycetes, 339-358 (1984), J. Am. C
loculospores in the classification table of Ramer (Loculo
ascomycetes) Pleospora (Pleo)
sporales) Pleospora
racea)).

Identification of genus level This strain (MCI2799) has 1) pseudocapsules that do not form ascidians, 2) pseudocapsules are buried in plant tissues, and 3) pseudocapsules Are not covered by bristles or fluffy hyphae. 4) Ascospores are oval to slipper-shaped, not filamentous spores. 5) Ascospores have only 3 or more transverse septa and longitudinal septa. It has the property of not having. Based on these features, R. W. G. FIG. Dennis, British
Ascomycetes, 382-385 (196)
8). Cramer, and A.A. Sivanesa
n, The Bitunicate Ascomyce
tes, 356-358 (1984), J. Am. Crame
A search of the genus of Pleosporaceae of r revealed that this strain was of the genus Leptosperia.
haeria).

Species Level Identification L. Holm, Taxonical notes
on Ascomycetes II. , Svensk,
Bot. Tidskr. 46 : 18-46 (1952)
Genus Leptosphaeria
According to the monograph of No. 23, 23 kinds of fungal species that adhere to monocotyledonous plants and 37 kinds of fungal species that adhere to dicotyledonous plants are described. These bacterial species differ in the host to which they settle, the morphology of the ascos, the properties of the ascospore, that is, the shape and size of the spores, the number of septa, the presence or location of swollen cells, the presence or absence of constriction at the septum insertion part, They are distinguished by the color tone and the presence or absence of attachments.
L. When the species of this strain (MCI2799) was searched according to the search table of Holm (1952), L.
clivensis was mentioned as a similar species. This strain and L. Clivensis has 1) a flexible pseudocapsule and a prominent cervix, 2) an ascidian with a long pedicle.
Being a club with 1), 3) ascospores have a dark dark brown color and are similar in that they have 3 septa. However, regarding the size of ascos and ascospores,
L. It was different from Clivensis. L. clive
The ascos of Nsis is 68 to 80 μm × 13 to 16 μm, the ascospore is of 18 to 24 μm × 6 to 8 μm, the ascos of this strain is 120 to 132 μm × 20 to 24.5 μm, and the ascospore is 25.9. The size is ˜34.1 μm × 8.4 to 10 μm. Furthermore, L. Clivensis is distributed in the subarctic region of Northern Europe, whereas this fungus is a species distributed in the subtropical region of the Ryukyu Islands. Therefore, this bacterium is L. cliven
Although it is considered to be a new species similar to sis, the identification of the level of the species is awaiting future taxonomic study.
Leptospheria sp.
ia sp. ) Identified as MCI2799.

The compound of the present invention can be obtained from the culture by culturing the compound-producing bacterium of the present invention belonging to the genus Leptosperia in a medium containing nutrients that can be utilized by ordinary microorganisms. As such a medium, glucose as a carbon source, starch syrup, dextrin, sucrose, starch, molasses, animal and vegetable oils can be used, and as a nitrogen source, soybean flour, corn steep liquor, fish meal, yeast extract,
Meat extract, peptone, and other inorganic nitrogen sources such as ammonium salts are used. In addition, heavy metal ions or other trace accelerating substances that generally promote the growth of bacteria or the production of antibiotics may be added.

Depending on the medium as described above, a pH of 5.0-
8.0, 15 to 30 ° C., preferably pH 6.0 to 7.
It is aerobically cultured at 0 to 20 to 27 ° C for several days. The production of the AM-1 substance of the present invention varies depending on the medium and culture conditions, but the maximum accumulation is usually reached within 2 to 5 days in both shaking culture and tank culture. When the accumulation of the AM-1 substance in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture.

Isolation of the AM-1 substance of the invention from the culture,
Purification is carried out by appropriately combining ordinary separation means utilizing the properties of the culture of the above-mentioned microorganism, for example, solvent extraction, adsorption or partition column chromatography, high performance liquid chromatography and the like. The compound of the present invention thus obtained does not show antibacterial activity in the wild strain, but has antibacterial activity in the chitin synthase 1-deficient strain, as shown in the test examples described later.

[0018]

As is apparent from the following test examples, the AM-1 substance of the present invention exhibits antifungal activity against mutant strains lacking fungal cell wall synthesizing enzyme chitin synthase 1, Is useful as a new type of antifungal agent that does not exhibit antifungal activity. It can also be expected as a lead compound for future effective fungal agents.

[0019]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1 1) Cultured fructose 3%, soybean flour 0.5%, potassium phosphate 1% 0.1%, magnesium sulfate (heptahydrate) 0.05%,
Medium containing 0.01% calcium chloride (pH 6.
0) was dispensed 7.0 ml each into a 500 ml flask,
It was autoclaved at 121 ° C. for 20 minutes. This strain (MCI2799) was inoculated in 1 platinum loop at 27 ° C for 3 hours.
The cells were cultured with shaking for days. The resulting culture contains malt extract 3.5%, corn starch 3.0%, corn steep liquor 1.5%, pharmacomedia 1.5%, sun grains 0.5%, calcium carbonate 0.3% 200 ml was inoculated into a 30-liter jar charged with 20 liter of the culture medium (pH 6.0) and cultured at 27 ° C. for 4 days with aeration and stirring.

2) Purification The obtained culture was centrifuged to separate into supernatant and cells, and the supernatant was adsorbed on Diaion HP-20 (manufactured by Mitsubishi Chemical), washed with water and washed with 50% methanol. , 70% methanol was eluted. The 70% methanol elution fraction was concentrated to dryness, and then applied to a silica gel column equilibrated with chloroform / methanol = 9/1, and the active fraction was eluted with chloroform / methanol = 85/15. The cells obtained by the above-mentioned culture centrifugation were extracted with n-butanol, the butanol layer was concentrated and dried, and then applied to a silica gel column equilibrated with chloroform / methanol = 9/1 to obtain an active fraction. Chloroform / methanol = 8
Elution was performed at 5/15, and the same fractions obtained from the supernatant were combined.

The fractions combined above were concentrated to dryness and again applied to a silica gel column equilibrated with chloroform / methanol = 9/1, and the active fraction was eluted with chloroform / methanol = 85/15. The active fraction was concentrated, dried, and subjected to preparative thin layer chromatography (Merck Ar).
t. 13895), benzene / acetone = 7/3
Unfolded. The active fraction is scraped off, extracted with methanol, concentrated to dryness, and subjected to preparative thin layer chromatography (Me.
rkArt. 13895) and developed with chloroform / methanol = 7/3. The active fraction was scraped off, extracted with methanol, concentrated to dryness, subjected to thin layer chromatography (Merk Art. 5744), and developed with benzene / methanol / chloroform = 8/2/1. The active fraction is scraped off, extracted with methanol, concentrated to dryness, and thin layer chromatography (Merk Art. 5744).
And developed with benzene / methanol / chloroform / ethyl ether = 8/2/1/1. The active fraction was scraped off, extracted with methanol and then dried to obtain AM-1 substance. The physicochemical properties of the obtained AM-1 substance were as described above.

Test Example 1 0.42 mg of the AM-1 substance was dissolved in 1 ml of methanol, and 60 μl thereof was soaked in a paper disk having a diameter of 8 mm and air dried. Wild-type Saccharomyces cerevisiae and mutant Saccharomyces cerevisiae deficient in chitin synthase 1 (available from Hokkaido University Faculty of Agriculture, Department of Applied Bacteriology) were each used at 10 ⁶ CFU (Col).
Bacterial fluid adjusted to ony Forming Unit) / ml, yeast extract 0.1%, polypeptone 0.2%,
An antibacterial activity measurement medium was prepared by mixing with an agar medium containing glucose 0.2% and agar 0.15%. Place a paper disc containing AM-1 on the surface of this medium, and
After culturing at ℃ for 18 hours, the resulting inhibition circle was measured.
A 16 mm growth inhibition circle was formed in the mutant strain, but no growth inhibition circle was observed in the wild strain. From the above results, the AM-1 substance of the present invention exhibits antifungal activity against a mutant strain lacking chitin synthase 1 which is a fungal cell wall synthase, and does not exhibit antifungal activity in a wild strain. Is clear.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 645) (C12P 1/04 C12R 1: 645)

Claims (3)

[Claims] 1. An AM-1 substance having the following physicochemical properties. (1) Molecular weight: m / z = 337 (FAB-MS) (2) Molecular formula: C ₂₂ H ₄₃ ON (3) Ultraviolet absorption spectrum: in methanol, 248 n
Absorption maxima at m and 286 (sh) nm (4) Thin layer chromatography: The following Rf values are shown under the following developing solvent conditions. (5) Color reaction: Positive in iodine reaction 2. A microorganism belonging to the genus Leptosperia, which has the ability to produce the AM-1 substance according to claim 1, is cultured, and the AM-1 substance is collected from the culture. A method for producing the AM-1 substance described. 3. Leptosperia sp. MCI279
9 (FERM P-15016).