阅读说明：本技术 一种天维菌素产生菌及其应用 (Protovermectin producing strain and application thereof ) 是由 林甲壇 常宗明 黄隽 于 2018-05-28 设计创作，主要内容包括：本发明涉及一株产天维菌素的除虫链霉菌(Streptomyces avermitilis),其产天维菌素B的质量占天维菌素A+B总质量的86％～92％。本发明的除虫链霉菌(Streptomyces avermitilis)B107-169-21于2018年03月09日保藏于“中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)”,其保藏编号为CGMCC No.15433。本发明的除虫链霉菌(Streptomyces avermitilis)B107-169-21是以天维菌素产生菌MA220为出发菌株,通过NTG诱变结合ARTP诱变筛选得到的,其产天维菌素(包括天维菌素A和天维菌素B)的摇瓶效价可达7000mg/L,5T罐的效价达6000mg/L。(The invention relates to Streptomyces avermitilis (Streptomyces avermitilis) capable of producing oryzanol, wherein the mass of the Streptomyces avermitilis capable of producing the oryzanol B accounts for 86-92% of the total mass of the oryzanol A and the Streptomyces avermitilis. The Streptomyces avermitilis (Streptomyces avermitilis) B107-169-21 is preserved in China general microbiological culture Collection center (CGMCC) on 09.03.2018, and the preservation number is CGMCC No. 15433. The Streptomyces avermitilis B107-169-21 is obtained by taking a Streptomyces avermitilis producing strain MA220 as an initial strain and performing NTG mutagenesis and ARTP mutagenesis screening, wherein the shake flask titer of the Streptomyces avermitilis producing strain (including the Streptomyces avermitilis A and the Streptomyces avermitilis) can reach 7000mg/L, and the titer of a 5T tank can reach 6000 mg/L.)

1. A Streptomyces avermitilis (Streptomyces avermitilis) B107-169-21 has a preservation number of CGMCC NO. 15433.

2. The streptomyces avermitilis B107-169-21 of claim 1, wherein the mass of the tianweimycin B in the fermentation product of the streptomyces avermitilis B107-169-21 is 86 to 92% of the total mass of the tianweimycin a + B, preferably the mass of the tianweimycin B is 88 to 92% of the total mass of the tianweimycin a + B.

3. Use of streptomyces avermitilis B107-169-21 according to claim 1 or 2 for the production of ivermectin or a pharmaceutical composition comprising ivermectin.

4. A method for producing oryzanol by fermentation of S.avermitilis B107-169-21 of claim 1 or 2 in a medium containing assimilable carbon and/or nitrogen sources.

5. The method according to claim 4, wherein the assimilable carbon source is one or more of corn starch, dextrin, glucose, sucrose, sorbitol, mannitol, amylase, glycerol, fructose, lactose, maltose, industrial molasses, glycine, rhamnose, salicin, inositol, raffinose, xylose, arabinose, galactose, trehalose, xylan, and inulin.

6. the method of claim 4 or 5, wherein the assimilable nitrogen source is one or more of soybean meal, yeast powder, urea, yeast extract, cottonseed meal, peanut meal, corn steep liquor dry powder, ammonium salt, peptone, tryptone, gluten meal, bran, yeast extract, beef extract, and nitrate.

7. The method according to any one of claims 4 to 6, wherein the medium further comprises inorganic salts, the inorganic salts being one or more of FeCl3, FeSO4, ZnSO4, MnSO4, CoCl2, Na2MoO4, MgSO4, MgCl2, CaCO3, (NH4)2SO4, CuSO4, NiSO4, KNO3, K2HPO4, KH2PO4, NaCl, KCl.

8. The method according to any one of claims 4 to 7, wherein the culture medium comprises 120 to 160g/L of corn starch, 0.02 to 0.2g/L of amylase, 10 to 40g/L of soybean cake powder, 0 to 10g/L of peanut cake powder, 5 to 20g/L of yeast powder, (NH4)2SO 40 to 4g/L, MnSO40 to 0.024g/L, Na2MoO 40 to 0.024g/L, CoCl20.01 to 0.04g/L, and CaCO 33 to 7 g/L.

9. The method of any one of claims 4-8, wherein the medium comprises 140g/L corn starch, 0.2g/L amylase, 20g/L soybean meal, 5g/L peanut meal, 10g/L yeast powder, (NH4)2SO 42 g/L, mnso40.024g/L, na2moo 40.024g/L, cocl20.02g/L, CaCO 37 g/L.

10. The method according to any one of claims 4 to 9, wherein the temperature of the fermentation is 20 ℃ to 40 ℃; the pH value is 6.0-8.0; the fermentation time is 288-312 hours; the ventilation volume is 0.6-1.1 vvm; wherein the temperature of the fermentation is preferably 25-30 ℃, and more preferably 28 ℃; the pH is preferably 6.5 to 7.5, more preferably 7.0 to 7.2.

Technical Field

The invention belongs to the field of biological medicine, and relates to Streptomyces avermitilis with high natamycin yield and a method for producing natamycin by using the strain.

background

The avermectin is a sixteen-element macrolide compound with insecticidal, acaricidal and nematicidal activities, which is firstly developed by Dacun Zhi and the like of North Rily university of Japan and Merck company in America, and is produced by fermenting Streptomyces avermitilis in Streptomyces griseus. Abamectin was marketed as a veterinary drug in 1981 and abamectin as a pesticide in 1985, and then ivermectin and doramectin were successively developed and marketed. Up to now, in China, abamectin and ivermectin are agricultural and veterinary insecticidal antibiotics which occupy the largest market share.

The abamectin belongs to high-toxicity insecticidal antibiotics, and with the continuous popularization of abamectin application, the drug resistance of pests to abamectin products is increased, the dosage of the abamectin is increased, and the harm to the environment is continuously deepened.

At present, the global policy and regulation leads the development of pesticides in the direction of high efficiency, low toxicity, low residue and little environmental pollution. Therefore, it is urgent to develop new agricultural chemicals with high efficiency, low toxicity and low residue.

WO2015135242A1 and WO2015135467A1 disclose the construction process of genetically engineered bacterium MA 220. CN201610211064.5 and WO2017173957 disclose that oryzanol has a significant pesticidal effect against harmful insects in agricultural and forestry crops, such as bemisia tabaci, thrips occidentalis, laodelphax striatellus, brown planthopper, sogatella furcifera, cnaphalocrocis medinalis, rice stem borer and terex formosana, and against parasites in aquatic organisms, such as sporozoites, tapeworms, nematodes, eimeria, lung fluke, schistosome, dactylogyrus or anchor carp louse. CN201610213645.2 and WO2017173956 disclose that tianweimycin has an extremely strong killing effect on common animal parasite diseases such as whipworm, roundworm, acarid and the like. Therefore, the development and application of the tianweimycin have very important significance for improving the quality of agricultural products in China and improving the safety of grains.

However, the existing fermentation process of the ivermectin has the outstanding problem of low titer, and the titer of the ivermectin produced by the genetic engineering bacteria MA220 disclosed in WO2015135242A1 and WO2015135467A1 is 1500mg/L, which cannot meet the requirement of industrial production, so that the problem of improving the titer of the ivermectin is urgently needed to be solved.

Disclosure of Invention

The invention aims to provide a high-yield strain Streptomyces avermitilis (Streptomyces avermitilis) B107-169-21 capable of producing oryzanol so as to solve the problems.

One aspect of the present invention is to provide a high-producing microbial strain of ivermectin. The strain is Streptomyces avermitilis (Streptomyces avermitilis) B107-169-21, which is preserved in China general microbiological culture Collection center (address: Beijing, Chaoyang district, North Xilu No.1, institute of microbiology, China academy of sciences) in 2018, 03.09, and the preservation number is CGMCC NO.15433, is classified and named as Streptomyces avermitilis, and is registered in a book to prove survival. In the fermentation product of the streptomyces avermitilis B107-169-21, the mass of the ivermectin B accounts for 86-92% of the total mass of the ivermectin A and B, and preferably the mass of the ivermectin B accounts for 88-92% of the total mass of the ivermectin A and B.

Another aspect of the invention provides the use of S.avermitilis B107-169-21 in the manufacture of a ivermectin or pharmaceutical composition comprising a ivermectin.

Another aspect of the present invention is to provide a method for producing oryzanol using S.avermitilis B107-169-21, comprising fermenting S.avermitilis B107-169-21 in a medium comprising an assimilable carbon and/or nitrogen source.

Preferably, the assimilable carbon source is one or more of corn starch, dextrin, glucose, sucrose, sorbitol, mannitol, amylase, glycerol, fructose, lactose, maltose, industrial molasses, glycine, rhamnose, salicin, inositol, raffinose, xylose, arabinose, galactose, trehalose, xylan and inulin.

Preferably, the assimilable nitrogen source is one or more of soybean cake powder, yeast powder, urea, yeast extract, cottonseed cake powder, peanut cake powder, corn steep liquor dry powder, ammonium salt, peptone, tryptone, gluten powder, bran, yeast extract, beef extract and nitrate.

Preferably, the culture medium further comprises inorganic salts, wherein the inorganic salts are one or more of FeCl3, FeSO4, ZnSO4, MnSO4, CoCl2, Na2MoO4, MgSO4, MgCl2, CaCO3, (NH4)2SO4, CuSO4, NiSO4, KNO3, K2HPO4, KH2PO4, NaCl and KCl.

Preferably, the culture medium contains 120-160 g/L of corn starch, 0.02-0.2 g/L of amylase, 10-40 g/L of soybean cake powder, 0-10 g/L of peanut cake powder, 5-20 g/L of yeast powder, (NH4)2SO 40-4 g/L, MnSO 40-0.024 g/L, Na2MoO 40-0.024 g/L, CoCl20.01-0.04 g/L and CaCO 33-7 g/L.

Preferably, the culture medium contains 140g/L of corn starch, 0.2g/L of amylase, 20g/L of soybean cake powder, 5g/L of peanut cake powder, 10g/L of yeast powder, (NH4)2SO 42 g/L, MnSO40.024g/L, Na2MoO40.024 g/L, CoCl20.02g/L and CaCO 37 g/L.

Preferably, the temperature of the fermentation is 20 ℃ to 40 ℃, more preferably 25 ℃ to 30 ℃, and most preferably 28 ℃; a pH of 6.0 to 8.0, more preferably 6.5 to 7.5, most preferably 7.0 to 7.2; the fermentation time is 288-312 hours; the aeration rate is 0.6-1.1 vvm.

In the present invention, "aeration amount" means the amount of sterile air sterilized by filtration per minute per unit volume of culture solution.

In the present invention, the ivermectin is a compound disclosed in CN201410208660.9 and shown as the following formula (I):

Wherein R is CH3 or C2H5, when R is CH3, it is oryzanol A, when R is C2H5, it is oryzanol B.

in the invention, the ivermectin is detected by the following HPLC method:

The chromatographic column is C18 column, 5 μm, 4.6 × 250 mm;

The mobile phase is methanol: acetonitrile: water 81:7:12 (volume ratio);

Flow rate: 1 ml/min;

Detection wavelength: 240 nm;

Sample introduction amount: 10 μ l.

Compared with the prior art, the invention has the following advantages:

1. In the fermentation product of the streptomyces avermitilis B107-169-21 obtained by screening, the mass of the oryzanol B accounts for 86-92% of the total mass of the oryzanol A and the oryzanol B, so that the preparation process of high-purity oryzanol B is avoided, and the production cost is greatly reduced.

2. The titer of the natamycin produced by the genetic engineering bacteria MA220 disclosed in WO2015135242A1 is 1500mg/L, and the titer of the impurity ivermectin B1B is 200 mg/L. The titer of the streptomyces avermitilis B107-169-21 screened by the invention for producing the oryzanol in the shake flask can reach 7000mg/L, and the titer in the 5T fermentation tank can reach 6000 mg/L; furthermore, the titer of the ivermectin B1B in the fermentation liquor of the streptomyces avermitilis B107-169-21 is 100mg/L, and the impurities are few. The streptomyces avermitilis B107-169-21 produces oryzanol with high titer and few impurities, so that the purification process of the oryzanol is simpler.

Description of the drawings:

FIG. 1: a comparison graph of fermentation products of the streptomyces avermitilis B107-169-21 and the starting strain MA220 is shown, wherein the upper graph shows an HPLC (high performance liquid chromatography) graph of fermentation liquid generated by the starting strain MA220 in example 1, and the lower graph shows an HPLC graph of the fermentation liquid generated by the streptomyces avermitilis B107-169-21 in example 1. Wherein A is tianweimycin A, B is tianweimycin B, and C is ivermectin B1B.

FIG. 2: example 1 shows the selection spectrum of S.avermitilis B107-169-21 of the present invention.

Detailed Description

in the following examples, unless otherwise specified, the reagents and instruments used are those commonly used in the art and may be purchased from chemical or biological products/preparations; the procedures used in the following examples are conventional in the art, and the procedures of these experiments can be unambiguously known by those skilled in the art from the prior art or the operation manual provided by the manufacturer and the corresponding results can be obtained.