JP3100785B2 - Novel avermectin derivative, method for producing the same and microorganism producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel avermectin derivative, a method for producing the same, and a microorganism producing the same.

[0002]

2. Description of the Related Art Evermectin
s) is already known as a 16-membered macrolide antiparasitic antibiotic obtained from a culture of Streptomyces evermitilis.

[0003]

SUMMARY OF THE INVENTION Evermectin is widely used as an antiparasitic antibiotic. For this reason, finding new avermectin compounds is extremely significant.

[0004]

The compounds of the novel avermectin derivatives of the present invention include Streptomyces avermitilis.
lis) is a series of macrolides isolated from the culture of one strain. The novel avermectin derivatives are characterized by having a 16-membered skeleton substituted by a mono- or disaccharide and having a bicyclic spiroketal fused on the skeleton.

The present invention provides a novel avermectin derivative, a method for producing a novel avermectin derivative from a culture solution of a mutant of the avermectin derivative producing Streptomyces avermitilis, and a microorganism belonging to Streptomyces avermitilis having a novel avermectin derivative-producing ability. is there.

Accordingly, it is an object of the present invention to obtain such novel avermectin derivatives, and another object is to provide Streptomyces avermitilis.
esavermitilis), K205
7 and its transformant K2057 / pKU109 :: a
Another object of the present invention is to provide a method for producing a novel avermectin derivative from a culture solution and a microorganism producing the same.

[0007] The novel avermectin derivative, which is a novel compound of the present invention, is represented by the following five structural formulas [I], [II], [III], [IV] and [V]. .

[0008]

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[0009]

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[0010]

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[0011]

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[0012]

Embedded image Microorganisms that produce the novel avermectin derivatives [I], [II], [III] and [VI], which are novel compounds described above, are Streptomyces evermitilis (Strep).
tomyces averagelitis) K2038
A mutant of (FERM BP-2775),
This strain is the parental strain Streptomyces evermitilis K20
38 (FERM BP-2775) with N-
It is obtained by treating with methyl-N'-nitro-N-nitrosoguanidine, then growing on agar medium and selecting colonies at random.

The above mutant strain was designated as Streptomyces avermitilis K2057 by the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.
2829).

The microorganism producing the novel avermectin derivative [V] is Streptomyces avermitilis K20.
This is a strain obtained by introducing a gene involved in avermectin biosynthesis (evermectin B 5-O-methyltransferase gene) into 57 strains. The above transformant was Streptomyces avermitilis K2057 / pKU10
9 :: aveD1 was sent to the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.
830).

[0015] Mutation induction itself can be relatively easily carried out by a usual microorganism mutation treatment method. That is, the parent strain is irradiated with ultraviolet rays, X-rays or gamma rays, or a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosourea,
Ethyl methanesulfonic acid, nitrogen mustard,
After treatment with nitrous acid, hydroxylamine, 2-aminopurine or the like, the obtained colonies are each cultured in an appropriate production medium, and after completion of the culture, the culture solution is extracted with an organic solvent. The extracted culture may be analyzed by thin-layer chromatography or high-performance liquid chromatography to obtain a mutant strain producing a novel avermectin compound.

The novel avermectin derivative-producing mutant strain of the present invention, Streptomyces avermitilis K2057,
Although a mutant of Streptomyces evermitilis K2038, it has the same morphological characteristics as the parent strain from which it was derived, and the morphological properties of the parent strain are described in US Pat. No. 4,310,519. I have.

Further, a transformant K2057 / pKU obtained by introducing a gene (avermectin B 5-O-methyltransferase gene) involved in avermectin biosynthesis into Streptomyces avermitilis strain K2057.
The 109 :: aveD1 strain is a compound 5 of the formula [I]
Methylated form can accumulate.

Accordingly, the Streptomyces avermitilis K2057 and K2057 / pKU10 of the present invention
9 :: aveD1 is a compound of the above formula [I], formula [II], [III
], Having different properties from the previous Streptomyces avermitilis K2038 and other homologous strains in having the ability to produce the novel avermectin derivatives represented by [IV] and [V].

The novel avermectin derivatives of the present invention include Streptomyces avermitilis strain K2057 and K2
057 / pKU109 :: aveD1 strain is produced by aerobic fermentation in a suitable liquid nutrient medium under the conditions described below. Liquid media similar to those used for the production of many antibiotics are suitable for use in the production of these novel avermectin derivatives.

Such nutrient broths contain carbon and nitrogen sources assimilated by the microorganism and generally low levels of inorganic salts. Furthermore, the fermentation medium can contain trace amounts of metals essential for the growth of the present microorganism. These trace metals are generally present in sufficient amounts in the complex carbon and nitrogen sources used as nutrient sources, but can be added separately to the medium if necessary.

In general, carbohydrates such as sugars, such as glucose, sucrose, maltose, lactose, dextrin, starch, cellulose, and the like, are suitable assimilable carbon sources in the nutrient medium. The exact amount of carbon source used in the medium will depend on the other components in the medium, but will depend on the medium weight of 0.5%.
Satisfactory results have usually been found with carbohydrates between 5% and 5%. These carbon sources can be used together in the same medium.

Yeast hydrolyzate, yeast autolysate, soybean flour, casein hydrolyzate, yeast extract, soybean hydrolyzate, corn steep liquor, Daisailers solble, cottonseed flour,
Nitrogen sources such as meat extracts are readily assimilated by Streptomyces avermitilis in the production of novel avermectin derivatives. Various nitrogen sources can be used alone or in combination within a range of 0.2% to 6% of the weight of the medium.

Examples of the nutrient inorganic salts that can be added to the medium include ordinary salts capable of releasing ions such as sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride, and carbonate. Further, what may be contained are trace metals such as cobalt, manganese and iron.

The culture is performed under aerobic conditions. During the cultivation, the pH of the medium is adjusted to 5 to 9 and the temperature to 25 to 35 ° C., and shaking or aeration culture for 120 to 240 hours can obtain favorable results. If the purpose is to collect a novel compound in the above culture, it goes without saying that a production strain and a production medium that produce and accumulate at least a novel avermectin derivative in the medium are used.

The novel avermectin derivatives of the present invention have important parasiticidal activity as anthelmintics, insecticides and acaricides in human and animal health and agriculture.

For isolation of the compound of the present invention, well-known standard extraction and purification techniques are employed. Techniques such as solvent extraction, column chromatography, thin-layer chromatography, preparative thin-layer chromatography, and high-performance liquid chromatography are useful for isolating and purifying the compound of the present invention.

Example 1 Acquisition of Streptomyces avermitilis strain K2057: Spores of Streptomyces avermitilis strain K2038 were treated with N-methyl-N'-nitro-N-nitrosoguanidine (1 mg / ml, pH 9.0) by a conventional method. 30 ° C 90
After that, the spores were diluted with sterile water to grow 200 colonies per plate, spread on a YMS plate, and cultured at 30 ° C. for 5 days.

The resulting colonies were separated by a fungus and transplanted on a YMS plate in the form of a 1 cm ² patch, which was used as a master plate. Replicate the master plate on a production agar medium,
For 8 days. After the cultivation, each patch-shaped cell on the production agar medium was cut out together with the agar medium, put into a plastic tube, added with 0.5 ml of acetone, and left at room temperature for 15 minutes to extract the culture.

The extract was concentrated to dryness except for the cells and agar fragments. The obtained extract was dissolved in 0.025 ml of acetone, and 0.005 ml of the extract was dissolved in a silica gel thin plate (Merk).
Was spotted on Kiesel gel 60F _254), 1
Developed with 5% v / v isopropanol / hexane. After the development was completed, the components of the culture were observed under ultraviolet light at 254 nm by chromatography to obtain Streptomyces evermitilis strain K2057 producing a novel compound.

Example 2 Fermentation of Streptomyces evamitilis strain K2057 0.1 ml of a spore suspension of Streptomyces evamitilis strain K2057 was added to 500 ml containing 100 ml of seed medium.
The flask was inoculated into a Erlenmeyer flask, and the flask was cultured on a rotary shaker at 200 rpm for 2 days at 28 ° C. Each 2 ml of this seed culture is added to 50 ml of 100 ml production medium.
The mixture was transferred to 65 0 ml Erlenmeyer flasks, and the flasks were cultured on a rotary shaker at 28 ° C. for 8 days at 180 rpm.

Example 3 Production of a New Compound from the Culture Solution of Streptomyces evermitilis K2057 Strain 6000 ml of the culture solution after fermentation obtained in Example 2 was filtered, and the filtrate was removed. After suspending the cells in 1000 ml of ion-exchanged water, the cells were filtered again to collect the cells. The washed cells were extracted by stirring with 1000 ml of acetone for 1 hour. After extraction, filtration was performed, and the filtrate was collected. The cells were again thoroughly washed with 1000 ml of acetone, filtered, combined with the filtrate to obtain an acetone extract.

The acetone extract (2000 ml) was concentrated under reduced pressure to 500 ml, and ion-exchanged water (500 ml) was added.
Extracted twice with 0 ml of methylene chloride. The methylene chloride extracts were combined, treated with 100 g of anhydrous sodium sulfate, filtered and dried. This solution was concentrated under reduced pressure.

The concentrated extract was dissolved in 5 ml of ethyl acetate,
The mixture was passed through a column packed with 25 g of silica gel, and further 50 ml of ethyl acetate was passed. The liquid passed through was collected and concentrated under reduced pressure to obtain 2.43 g of a viscous oil containing the compounds represented by the formulas [I], [II] and [III].

Example 4 Isolation of the compound represented by the formula [I] 2.43 g of the oily substance obtained in Example 3 was dissolved in 5 ml of hexane / methylene chloride / methanol (10: 10: 1). The mixture was passed through a Sephadex LH-20 (120 g) column equilibrated with the same mixed solvent. The mixture was eluted with the above mixed solvent, and fractionated in 4 ml portions. Fractions 41-54 were collected and concentrated under reduced pressure to give 1.15 g of extract.

1.15 g of the obtained extract was dissolved in 2 ml of hexane / isopropanol (85:15), and a medium-pressure silica gel column (35φ ×
(300 mm). Eluted with hexane / isopropanol (85:15) and fractionated in 14 ml portions. Fraction 4
3-70 was collected and concentrated under reduced pressure to dryness to obtain 46 mg of a pale yellow powder.

Further, 46 mg of the obtained pale yellow powder was dissolved in a small amount of chloroform, and the silica gel plate for preparative separation (2
Five spots (0 × 20 cm, 0.25 mm thick) were spotted in strips and developed with hexane / isopropanol (85:15). The band corresponding to an Rf value of 0.31 was scraped off, eluted with chloroform / methanol (5: 1), and the eluate was concentrated under reduced pressure to obtain 13 mg of a compound represented by the formula [I].

Characterization data λ max (CH ₃ OH) 242 nm and 236 nm [α] D + 52.80 (c = 1.00 in methyl alcohol) m / z = 737 (M + Na) IR: ¹ H-NMR as shown in FIG. : As shown in FIG.

Example 5 Isolation of the compound represented by the formula [II] Sephadex performed in Example 4
LH-20 column chromatography fractions 36-40
Was collected and concentrated under reduced pressure to obtain 50 mg of a pale yellow powder. 50 mg of the obtained pale yellow powder was dissolved in a small amount of chloroform, spotted in a strip on five preparative silica gel plates (20 × 20 cm, 0.25 mm thick), and developed with hexane / isopropanol (85:15). did. Rf value 0.5
The band corresponding to No. 1 was scraped off, eluted with chloroform / methanol (5: 1), and the eluate was concentrated under reduced pressure to give the formula [I
13 mg of the compound of the formula [I] was obtained.

Characterization data λmax (CH ₃ OH) 235 nm [α] D + 7.00 (c = 1.00 in methyl alcohol) m / z = 735 (M + Na) IR: ¹ H-NMR as shown in FIG. As shown in 4

Example 6 Isolation of the compound represented by the formula [III] A band corresponding to an Rf value of 0.36 on the silica gel plate for preparative separation performed in Examples 4 and 5 was scraped off, and chloroform / methanol (5: The eluate was eluted in 1), and the eluate was concentrated under reduced pressure. 20 mg of the compound from the plate from Example 4 and 14 mg of a white powder compound of the formula [III] were obtained from the plate of Example 5.

Characterization data λmax (CH ₃ OH) 236 nm [α] D + 1.00 (c = 1.00 in methyl alcohol) m / z = 753 (M + Na) IR: ¹ H-NMR as shown in FIG. As shown in 6

Example 7 Fractions 28-35 of Sephadex LH-20 column chromatography performed in Example 4 were collected, concentrated under reduced pressure,
1.04 g of a pale yellow oil was obtained. 1.04 g obtained
70 mg of the oily substance was dissolved in a small amount of chloroform, spotted in a strip on four preparative silica gel plates (20 × 20 cm, 0.25 mm thick), and developed with hexane / isopropanol (85:15). Rf value 0.
The band corresponding to 39 was scraped off, eluted with chloroform / methanol (5: 1), and the eluate was concentrated under reduced pressure.
2 mg of the white powder was dissolved in a small amount of methanol, and the solution was separated by preparative HPLC (ODS 5 μm, 10 mm × 300 mm, solvent: acetonitrile / methanol / water (62: 18: 2).
0), flow rate 3 ml / min, detection 246 nm).
The fraction eluted at 8.7 minutes was collected and concentrated under reduced pressure to obtain the formula [I
V] of 4.0 mg of the compound was obtained.

Characterization data λ max (CH ₃ OH) 249 nm m / z = 879 (M + Na) IR: as shown in FIG. 7 ¹ H-NMR: as shown in FIG.

Example 8 Streptomyces avermitilis K2057 / pKU
Acquisition of Strain 109 :: aveD1 Spores of Streptomyces avermitilis 2057 strain were transformed with Y containing 30% sucrose and 5 mM magnesium chloride.
Transfer to a 500 ml Erlenmeyer flask containing 50 ml of EME medium and place on a rotary shaker at 28 ° C. for 3 days for 200 days.
Cultured at rpm. 3000 rpm of the obtained culture solution,
The cells were centrifuged for 10 minutes to collect the cells.

The obtained cells were suspended in 20 ml of P10 medium, and washed cells were obtained by centrifugation. 1 mg / ml of washed cells
Suspended in 10 ml of P10 medium containing lysozyme of
The mixture was kept at 7 ° C. for 60 minutes to obtain protoplasts. The obtained protoplasts were added to 10 ml of P20 medium and mixed well, and then passed through a cotton plug to remove undigested hypha. Collect protoplasts by centrifugation at 3000 rpm for 10 minutes,
P20 medium was added, and suspended so as to be 10 ⁹ protoplasts / ml.

To 50 μl of the obtained protoplasts, 1 ng of the recombinant plasmid pKU109 :: aveD1 was added, and a 500 μl polyethylene glycol solution (25% polyethylene glycol MW1000, 0.1%) was further added.
mM potassium dihydrogen phosphate, 0.1 M calcium chloride,
50 mM Tris-maleate buffer pH 8.0)
Left at room temperature for 1 minute.

After adding 500 μl of P20 medium and mixing well, 100 μl of each was placed on RM14 agar medium and spread with 2.5 ml of soft agar RM14 medium. After culturing at 30 ° C. for 21 hours, 2.5 ml of soft agar medium containing 200 μg / ml thiostrepton was overlaid. After culturing at 30 ° C. for 10 days, a transformant resistant to thiostrepton was obtained.

Example 9 Streptomyces evermitilis K2057 / pKU
109 :: Fermentation of aveD1 strain Streptomyces evermitilis K2057 / pKU
5 μg of a 0.1 ml spore suspension of the 109 :: aveD1 strain
A 500 ml Erlenmeyer flask containing 100 ml of seed medium containing thiostrepton / ml was inoculated, and the flask was cultured on a rotary shaker at 28 ° C. for 2 days at 200 rpm. Each 2 ml of this seed culture was mixed with 500 ml of 100 ml of production medium containing 5 μg / ml thiostrepton.
The mixture was transplanted to seven Erlenmeyer flasks, and the flasks were cultured on a rotary shaker at 28 ° C. for 8 days at 180 rpm.

Example 10 Streptomyces avermitilis K2057 / pKU
Production of Compound from Culture Solution of 109 :: aveD1 Strain 600 ml of the culture solution after fermentation obtained in Example 9 was filtered,
The filtrate was removed. After washing the cells with 500 ml of ion-exchanged water, the washed cells were extracted twice with 100 ml of acetone.
The acetone extract was concentrated under reduced pressure to 50 ml, 50 ml of ion-exchanged water was added, and the mixture was extracted twice with 100 ml of methylene chloride. The methylene chloride extracts were combined, treated with 10 g of anhydrous sodium sulfate, filtered and dehydrated. This solution was concentrated under reduced pressure. The concentrated extract was dissolved in 2 ml of ethyl acetate,
g of silica gel and passed through the column for another 20 g.
Ethyl acetate was flushed out. Collect the flow-through, concentrate under reduced pressure,
730 mg of a viscous oil containing the compound of the formula [IV] was obtained.

Example 11 Isolation of the compound represented by the formula [V] 730 mg of the oily substance obtained in Example 10 was dissolved in a small amount of chloroform, and the mixture was separated on a silica gel plate (20 × 20).
(cm, 1.0 mm in thickness) were strip-shaped and spread with hexane / isopropanol (85:15). A band corresponding to an Rf value of 0.67 was scraped off, and chloroform /
Elution was performed with methanol (5: 1). After the eluate was concentrated to dryness, it was dissolved in a small amount of methanol.
DS 5 μm, 10φmm × 300 mm, solvent: acetonitrile / methanol / water (62:18:20), flow rate 3 ml /
The fraction eluted at 60.5 minutes was collected and concentrated under reduced pressure to obtain 2.03 mg of the compound represented by the formula [V].

Characterization data λmax (CH ₃ OH) 242 nm m / z = 751 (M + Na) IR: as shown in FIG. 9 ¹ H-NMR: as shown in FIG. 10

The compositions of the various media used in the above Examples are as shown below. Composition of YMS medium Malt extract (Difco) 10 g Yeast extract (Difco) 4 g Soluble starch (Difco) 4 g Agar 20 g Distilled water 1000 ml After adjusting the pH to 7.2 with 2N potassium hydroxide, 121 ° C. 15
Subject to autoclaving for minutes. After the sterilization, magnesium chloride and calcium nitrate are added to 10 mM and 8 mM, respectively.

Production agar medium glucose 46 g peptone milk (Oxoid) 24 g yeast autolysate (Difco) 2.5 g agar 20 g distilled water 1000 ml After adjusting the pH to 7.2 with 2N sodium hydroxide, 121 ° C.1
Subject to autoclaving for 5 minutes.

Seed medium lactose 20 g Destilers Soluble 15 g Yeast autolysate (Difco) 2.5 g Distilled water 1000 ml After adjusting the pH to 7.2 with 2N potassium hydroxide, the mixture was adjusted to 121 ° C. 15
Subject to autoclaving for minutes.

Production medium Glucose 46 g Peptone milk (Oxoid) 24 g Yeast autolysate (Difco) 2.5 g Polypropylene glycol # 2000 2.5 ml Distilled water 1000 ml After adjusting the pH to 7.2 with 2N potassium hydroxide, 121 ° C. 15
Subject to autoclaving for minutes.

Trace element solution Ferric chloride hexahydrate 200 mg Zinc chloride 40 mg Cupric chloride dihydrate 10 mg Manganese chloride tetrahydrate 10 mg Sodium borate decahydrate 10 mg Ammonium molybdate tetrahydrate 10 mg Distilled water 1000 ml After dissolving the above components, store at 4 ° C.

YEM medium 10 g glucose 10 g peptone (Difco) 5 g yeast extract (Difco) 3 g malt extract (Oxoid) 3 g distilled water 1000 ml No pH adjustment, 121 ° C. for 15 minutes, high pressure steam sterilization.

P10 medium Sucrose 103 g Potassium sulfate 0.25 g Magnesium chloride hexahydrate 2.03 g Trace element solution 2.0 ml The above components were dissolved in distilled water 800 ml, and subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. Add the following ingredients.

0.5% potassium dihydrogen phosphate 10 ml 3.68% calcium chloride dihydrate 100 ml 0.25M * MES buffer pH 6.5 100 ml * 2-morpholinoethane sulphonic acid

P20 medium Sucrose 205 g Potassium sulfate 0.25 g Magnesium chloride hexahydrate 2.03 g Trace element solution 2.0 ml The above components were dissolved in distilled water 800 ml, steam sterilized at 121 ° C for 15 minutes, and cooled, Add the ingredients.

100 ml of 0.5% potassium dihydrogen phosphate 100 ml of 3.68% calcium chloride dihydrate 100 ml of 0.25 M MES buffer pH 6.5

RM14 medium Sucrose 205 g Potassium sulfate 0.25 g Magnesium chloride hexahydrate 10.12 g Glucose 10.0 g Casamino acid (Difco) 0.1 g L-proline 3.0 g Yeast extract (Difco) 2.0 g Trace elements Solution 2.0 ml Oatmeal agar medium (Difco) 3.0 g Agar 20.0 g The above components were dissolved in 870 ml of distilled water, subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes, and then the following components were added.

10 ml of 0.5% potassium dihydrogen phosphate 80 ml of 3.68% calcium chloride dihydrate 40 ml of 0.25 M MES buffer pH 6.5

Soft agar RM14 medium Sucrose 205 g Potassium sulfate 0.25 g Magnesium chloride hexahydrate 10.12 g Glucose 10.0 g Casamino acid (Difco) 0.1 g L-proline 3.0 g Yeast extract (Difco) 2.0 g Trace element solution 2.0 ml Oatmeal agar medium (Difco) 3.0 g Agar 5.0 g The above components are dissolved in 870 ml of distilled water, subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes, and then the following components are added.

10 ml of 0.5% potassium dihydrogen phosphate 80 ml of 3.68% calcium chloride dihydrate 40 ml of 0.25 M MES buffer pH 6.5

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of a compound represented by the formula [I].

FIG. 2 is a proton nuclear magnetic resonance spectrum of the compound represented by the formula [I].

FIG. 3 is an infrared absorption spectrum of the compound represented by the formula [II].

FIG. 4 is a proton nuclear magnetic resonance spectrum of the compound represented by the formula [II].

FIG. 5 is an infrared absorption spectrum of the compound represented by the formula [III].

FIG. 6 is a proton nuclear magnetic resonance spectrum of the compound represented by the formula [III].

FIG. 7 is an infrared absorption spectrum of the compound represented by the formula [IV].

FIG. 8 is a proton nuclear magnetic resonance spectrum of the compound represented by the formula [IV].

FIG. 9 is an infrared absorption spectrum of the compound represented by the formula [V].

FIG. 10 is a proton nuclear magnetic resonance spectrum of the compound represented by the formula [V].

FIG. 11. Streptomyces evermitilis K205
7 is a map of the recombinant plasmid pKU109 :: aveD1 used to construct the 7 / pKU109 :: aveD1 strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 465) (C12P 19/62 C12R 1: 465)

Claims (3)

(57) [Claims] 1. The compound of the following formula [I], [II], [III],
A novel avermectin derivative represented by [IV] or [V]. Embedded image Embedded image Embedded image Embedded image Embedded image 2. A compound of the formula [I] belonging to the genus Streptomyces,
A microorganism having the ability to produce an avermectin compound represented by [II], [III], [IV] or [V] is cultured in a medium, and the cultures are subjected to formulas [I], [II], [III],
Isolating an avermectin compound represented by [IV] or [V], wherein the compound of formula [I], [II] or [I
A method for producing an avermectin compound represented by [II], [IV] or [V]. Embedded image Embedded image Embedded image Embedded image Embedded image 3. A compound of the formula [I] belonging to the genus Streptomyces,
A microorganism having the ability to produce an avermectin compound represented by [II], [III], [IV] or [V] is Streptomyces evermitilis (Streptomyc).
es avermitilis) K2057 ( FERM
P-12829 ) or K2057 / pKU10
9 :: aveD1 ( FERM P-12830 ).