KR20010039118A - A novel Streptomyces avermitilis mutant and a culture process therewith - Google Patents

Abstract

PURPOSE: Streptomyces avermitilis mutant strain useful for the production of avermectin and a method for producing avermectin are provided, thereby the avermectin having avermectin B1a can be mass produced. CONSTITUTION: Streptomyces avermitilis mutant strain (KCTC 0662BP) derived from Streptomyces avermitilis (ATCC 31271) produces more than 40 wt.% of avermectin B1a and less than 5 wt.% of avermectin B1b in fermented culture. The method for producing avermectin comprises the steps of: inoculating Streptomyces avermitilis mutant (KCTC 0662BP) on a spore-producing agar medium and incubating for 10 to 15 days; incubating the spores in a seed strain medium for 2 to 3 days; and inoculating the seed strain of Streptomyces avermitilis in a conventional avermectin producing medium containing vitamin B1(thiamine) and incubating them, in which the content of vitamin B1 is 5 to 2000 mg/l.

Description

A novel Streptomyces avermitilis mutant and a culture process therewith}

The present invention relates to a novel Streptomyces avermitilis mutant strain useful for producing avermectin and a method for producing avervetin using the same.

Avermectins have a wide range of antiparasitic effects on internal parasites, including nematodes, which cause severe economic losses to livestock and poultry. They have little antibacterial and antifungal activity but are useful as insecticides and acaricides due to their wide range of antiparasitic effects. Recently, the use of pesticides has been increasing rapidly, and it is reported that it is effective in treating tropical diseases in humans.

On the other hand, avermectin is a secondary metabolite present in Streptomyces avermitilis cells and biosynthesized into a mixture of eight subtypes. That is, four main components of A1, A2, B1, and B2 are obtained in the form of a mixture of eight types of subtypes, each divided into a and b. (See Formula 1 and Table 1)

Avermectin R ₁ R ₂ X-Y A1a CH ₃ C ₂ H ₅ CH = CH A1b CH ₃ CH ₃ CH = CH A2a CH ₃ C ₂ H ₅ CH ₂ -CH (OH) A2b CH ₃ CH ₃ CH ₂ -CH (OH) B1a H C ₂ H ₅ CH = CH B1b H CH ₃ CH = CH B2a H C ₂ H ₅ CH ₂ -CH (OH) B2b H CH ₃ CH ₂ -CH (OH)

Among the eight kinds of avermectins, the most effective in terms of biological effect is avermectin B1a, and avermectin as an animal repellent consists of avermectin B1a and B1b in a relatively small amount.

Avermectins are known to be obtained from the cultures of Streptomyces avermitilis ATCC31267, 31271, 31272 and mutant strains derived from these strains. A2a, B1a and B2a (US Pat. Nos. 4,310,519 and 4,429,042).

The avermectin production of Streptomyces avermitilis ATCC31271 is 485mg / L, the active ingredient avermectin B1 is 170mg / L and the composition ratio is 35% among the avermectin production strains (RWBurg et al., 1979). Antimicrob.Agents Chemother. 15: 361-367).

Variants of Streptomyces avermitilis include strains that produce only avermectin nuclei, strains that produce A2 and B2 components, strains that generate B components, and a component (A1a, A2a, B1a, and B2a). Strains have been developed (Ikeda et al., 1995. Control of Avermectin Biosynthesis in Streptomyces avermitilis for Selective Production of a useful Component. J. Antibiot. 48: 549-562). The total avermectin production reported so far is the highest at 2,300mg / L and the B1 component ratio is 48.5-50% (WO 98/56939). The productivity of the active ingredient, B1a, is the highest at 780 mg / L and the composition ratio is 39% (Zhinan Xu et al. 1999. Biotechnology Letters. 21: 91-95).

However, the above-mentioned conventional strains show a small amount of total avermectin production when the ratio of B1 is high, and a small amount of avermectin B1a when the ratio of avermectin B1a is high among the subtypes of avermectin.

Accordingly, the present inventors have repeatedly studied to develop strains that produce avermectins having both a large amount of avermectin in culture and a high ratio of avermectin B1a as an active ingredient, and surprisingly, specific streptomyces avermitili When the strain strain was cultured according to the method of the present invention, it was found that the above object can be obtained at the same time to complete the present invention.

Accordingly, an object of the present invention is to provide a Streptomyces avermitilis mutant that produces avermectin.

It is also an object of the present invention to provide a method of culturing the Streptomyces avermitilis mutant strain to produce avermectin from the culture.

1 is a result of analyzing the avermectin produced after culturing the parent strain and the mutant strain according to the present invention by silica gel thin film chromatography.

Column 1. Extract of Variation Wine according to the Present Invention

Heat 2. Extract of parent strain,

Column 3. oligomycin standard solution,

Column 4. Avermectin Standard

Figure 2 is a result of analyzing the avermectin produced by culturing the mutant strain according to the present invention by high-performance liquid chromatography.

Figure 3 is a view showing the growth of bacteria and productivity of avermectin B1a according to the culture time of the mutant strain according to the present invention.

-○-Growth of cells

-●-Avermectin B1a Produced Amount

The present invention provides Streptomyces avermitilis mutants that produce avermectins. The mutant strain according to the present invention was mutated from Streptomyces avermitilis ATCC 31271, which was deposited on September 28, 1999 in the Korean Collection for Type Cultures Deposited with KCTC 0662BP. Thus, the variant strain according to the present invention is Streptomyces avermitilis KCTC 0662BP.

The mutant strain according to the present invention has the characteristics that 40% by weight or more of Avermectin B1a and 5% by weight or less of Avermectin B1b in culture.

In order to obtain high titer of avermectin mutant strains, the present inventors have isolated numerous mutant strains through UV irradiation, NTG treatment, protoplast fusion, and the like, and surprisingly, streptomyces. It has been discovered that mutated strains from Streptomyces avermitilis ATCC 31271 produce high titers of avermectin with an avermectin B1a component ratio of at least 40% of the total avermectin subtypes resulting. This is not disclosed or implied at all by the prior art.

Morphological, Culture, Physiological, and Chemistry Characteristics of the Novel Streptomyces Avermitilis Variant (KCTC 0662BP) and Parental Known Streptomyces Avermitilis (ATCC 31271) According to the Invention Is shown in Table 2 below.

badge Growth Cell color Spore purple ATCC31271 KCTC0662BP ATCC31271 KCTC0662BP ATCC31271 KCTC0662BP ISP2 +++ ++ White White grey No spores ISP3 +++ ++ White White grey grey ISP4 +++ ++ White White No spores grey ISP5 +++ ++ beige beige No spores No spores ISP6 + + White yellow No spores No spores ISP7 + + White White grey No spores Bennett Agar +++ ++ White White White No spores Japek Agar + + White White No spores No spores Nutritional Agar ++ +++ beige yellow No spores No spores

Morphological characteristics

Unlike the parent strain (ATCC 31271), the mutant strain according to the present invention has the characteristic of making yellow cells in ISP6 medium and nutrient agar medium, and does not make wrinkled cells like the parent strain in ISP3 medium. Variants and parent strains of the present invention is different in the spore color. In addition, the parent strain forms gray spores in ISP 2 and 7 media, but the mutant strains of the present invention do not form spores. And, the parent strain did not form spores in ISP4 medium, but the mutant strain of the present invention is characterized by forming gray spores.

Culture characteristics

The growth temperature of the mutant and parent strains according to the present invention grows at 28 ℃ and 37 ℃ the same, but does not grow at 50 ℃. In addition, the mutant strains of the present invention are slower on various agar media than the parent strain, and the growth on the nutrient agar media has a faster characteristic than the parent strain.

Mutant strain and mother strain of the present invention is different in the spore color. The parent strain produces gray spores in ISP 2, 3, 7 medium, but the variant strain of the present invention produces white spores. And the parent strain did not form spores in ISP 4, 5, 6 medium and Zapeck agar medium (Czapeck solution agar), but the mutant strain of the present invention is characterized by forming white spores.

Physiological characteristics

Mutant strain and mother strain according to the present invention has a difference in the production of melanin-like pigment. The parent strain produces a black pigment in ISP 6 medium, but the mutant strain of the present invention is characterized by not producing a pigment. In addition, the characteristics of the carbon source availability of the mutant and parent strains of the present invention are shown in Table 3.

ATCC 31271 KCTC 0662BP glucose ++ ++ fruit sugar ++ +/- Lactose +++ + Maltose +++ + saccharose + + Starch +++ +

In the degree of growth of the two strains according to the carbon source, the parent strain is very excellent in the case of supplying lactose, maltose and starch to the carbon source at least agar medium, and growth is not active when the sucrose is supplied to the carbon source. However, the mutant strain of the present invention has a growth similar to that of the parent strain when the carbon source is glucose, and rarely grows when the fructose is specifically supplied to the carbon source.

Biochemical characteristics of the mutant and parent strains of the present invention are shown in Table 4 below.

ATCC 31271 KCTC 0662BP Starch hydrolysis ++ +++ Casein hydrolysis + ++ Nitrate reduction - - Iron sulfide generation + -

The mutant strains of the present invention are superior to the parent strain in the degradability of starch and casein. This indicates a difference between amylase and proteolytic enzyme, and it can be seen that the mutant strain of the present invention has superior hydrolytic ability of the polymer material than the parent strain. In addition, unlike the parent strain, the mutant strain of the present invention does not have the ability to produce iron sulfide.

Chemical classification

The mutant strain according to the present invention has diaminopimeric acid in the cell, and has the same LL-type diaminopimeric acid as the parent strain.

As described above, the novel Streptomyces avermitilis mutant strain (KCTC 0662BP) according to the present invention is a known Streptomyces avermitilius which is the parent strain in terms of morphological, culture, physiological and chemical taxonomic characteristics. It can be seen that the strain is completely different from the strain (ATCC 31271), and in particular, the strain is completely different from the known strain in terms of producing a high titer of avermectin having a high ratio of avermectin B1a.

The present invention also provides a method of culturing the above-described Streptomyces avermitilis KCTC 0662BP to prepare avermectin from the culture.

The culture method in the production method according to the present invention is as follows.

That is, Streptomyces avermitilis KCTC 0662BP was inoculated in spore-producing agar medium and incubated for 10-15 days, and the resulting spores were cultured in seed culture medium for 2-3 days, and then avermectin Inoculate the seed culture medium into the production medium.

As the medium for producing avermectin, a medium used for producing avermectin may be used (US Pat. Nos. 4,310,519 and 4,429,042). That is, glucose, fructose, maltose, sugar, soluble starch, or mixtures thereof may be used as the carbon source, and soybean meal, cottonseed meal, yeast extract, peptonized milk, peanut meal, gravy extract, or a mixture thereof as the nitrogen source. Can be used. In addition, inorganic salts such as potassium phosphate, zinc sulfate, magnesium sulfate, iron sulfate, and cobalt chloride, and soybean oil, cottonseed oil, and rapeseed oil may be included.

On the other hand, in the preparation of the high titer avermectin according to the present invention, the medium for producing avermectin preferably contains vitamin B1 (thiamine). According to the manufacturing method of the present invention

The produced avermectin has a higher production rate of avermectin B1a, which is a desired product, than other avermectin subtypes, but may also increase the amount of B1b component that is difficult to remove in the purification process. Therefore, when using the avermectin production medium containing vitamin B1, it is possible to selectively reduce the B1b ratio without affecting the avermectin B1a production amount. The content of vitamin B1 contained in the medium is preferably 5-2000 mg / L.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these are not intended to limit the scope of the present invention.

Example 1

After making agar pieces (diameter 10 mm) in solid medium (6% glucose, 2% soy flour, 0.3% yeast extract, 0.5% soybean oil, 2% agar), Streptomyces armitilis ATCC 31271 and Streptomyces arbor Inoculated with each of Mytiles KCTC 0662BP, and incubated at 28 ° C. for 10 days, and then extracted by stirring agar pieces with acetone for 2 hours. 10 μl of acetone solution from which avermectin was extracted was added to a silica gel thin film, and each component of avermectin was separated by chromatography using acetonitrile as a developing solvent. The result of comparing the amount of avermectin produced by irradiating ultraviolet (wavelength 254 nm) to the silica gel thin film separated from the avermectin component is shown in FIG. 1.

As can be seen in Figure 1, the extract obtained from the mutant strain according to the present invention can be seen that the productivity of the avermectin is much superior to the extract obtained from the parent strain.

Example 2

Streptomyces avermitilis Streptomyces avermitilis From the spores formed with the spores of KCTC 0662BP was inoculated into a 250 ml flask containing 50 ml of the following seed culture medium such that the spore concentration was 5 × 10 ⁶ spores / ml.

Glucose 20 g / L

Soy flour 15 g / L

Peanut powder 5 g / L

Yeast extract 3 g / L

Calcium Carbonate 1 g / L

Magnesium sulfate 10 mg / L

Zinc Sulfate 10 mg / L

Soybean oil 3 g / L

1000 ml of distilled water

7.0 After pH Sterilization

The flask was incubated for 48 hours at 220 rpm in a rotary shaker at 28 ℃, and then inoculated with 6 ml in a 500 ml flask containing 60 ml of avermectin production medium as follows.

Glucose 100 g / L

Soy flour 20 g / L

5 g / L of cotton thread

Yeast extract 3 g / L

Potassium Phosphate 1 g / L

Zinc Sulfate 10 mg / L

Magnesium Sulfate 100 mg / L

Ferrous Sulfate 10 mg / L

Cobalt chloride 10 mg / L

Soybean oil 5 g / L

1000 ml of distilled water

Before pH sterilization 7.6

The flask was incubated for 11 days at 150 rpm on a 28 ° C rotary shaker. 5 ml of the culture solution was taken, 10 ml of ethyl acetate was mixed and extracted for 2 hours. Avermectin extracted with ethyl acetate layer was measured by high performance liquid chromatography to determine the amount of avermectin produced in the culture. The result is shown in FIG.

The high performance liquid chromatography analysis conditions are as follows.

Device ; Hitachi models L-7100, 7200, 7300, 7400, D-7500

column ; COSMOSIL 5C18-AR-II, 4.6 X 250 mm

Mobile phase; Acetonitrile: Methanol: Distilled water (850: 50: 100)

Flow rate; 1ml per minute

Injection volume; 10 μl

The amount produced was calculated by comparing the peak area obtained from the culture extract with the peak area of the avermectin standard. The production amount was calculated for the eight components of avermectin, and the ratio corresponding to the whole avermectin was calculated by the area percentage method.

The amount of avermectin produced from the mutant strain according to the present invention was about 4,000 mg / L, and the active ingredient avermectin B1a showed a productivity of 1,786 mg / L, and the component ratio was 40 to 45% (average of three times). However, B1b components were produced at less than 5%.

As can be seen above, in the production amount of the avermectin B1a which is a useful component, it can be confirmed that the mutant strain according to the present invention is much superior to the parent strain.

Example 3

Seed cultures incubated in the same manner as in Example 2 using Streptomyces avermitilis KCTC 0662BP were inoculated 6 ml into a 500 ml flask containing 60 ml of the following avermectin production medium.

Glucose 100 g / L

Soy flour 20 g / L

5 g / L of cotton thread

Yeast extract 3 g / L

Potassium Phosphate 1 g / L

Zinc Sulfate 10 mg / L

Magnesium Sulfate 100 mg / L

Ferrous Sulfate 10 mg / L

Cobalt chloride 10 mg / L

Soybean oil 5 g / L

Vitamin B1 0-100 mg / L

1000 ml of distilled water

Before pH sterilization 7.6

The flask was incubated for 11 days at 150 rpm in a 28 ° C rotary shaker, and the amount of avermectin components present in the culture was analyzed in the same manner as in Example 2. The operation was repeated three times, and the results (average values) obtained are shown in Table 5 below.

Vitamin B1 Avermectin Total potency (ug / ml) B1a titer (ug / ml) B1b content ratio (%) 0 4026 1725 5.0 50 mg / L 4029 1678 2.8 100 mg / L 4087 1750 2.2

As can be seen in Table 5, when cultured by adding vitamin B1 to the medium, it can effectively lower the content of avermectin B1b.

Example 4

When cultured using a Streptomyces avermitilis KCTC 0662BP in a 7L fermenter with the same medium composition (vitamin B1 100 mg / L) at 28 ° C, 250 rpm, and 1.0 VVM As shown in Figure 3, the mutant strain of avermectin B1a according to the present invention had a much higher level of 2,800 mg / L.

As can be seen in the above embodiment, when producing avermectin using the mutant strain according to the present invention, it is possible to effectively improve the productivity of the avermectin B1a than the parent strain, the composition ratio can be maintained more than 40%. In addition, there is an advantage that can reduce the avermectin B1b component to less than 3%.

Claims (4)

Streptomyces avermitilis producing avermectin KCTC 0662BP. Streptomyces avermitilis A method for culturing KCTC 0662BP and preparing avermectin from the culture. The method of claim 2, wherein the medium comprises vitamin B1. The method according to claim 3, wherein the content of vitamin B1 in the medium is 5-2000 mg / L.