IVERMECTIN B1B PRODUCING STRAIN AND USE THEREOF

Abstract

Provided are a Streptomyces avermitilis strain C63-51 with a high yield of ivermectin B1b and a method for producing ivermectin B1b by using this strain. By using the above-mentioned strain and method, efficient production of single ivermectin B1b can be achieved.

Claims

1. A Streptomyces avermitilis strain C63-51, deposited on Dec. 22, 2016 in the China General Microbiological Culture Collection Center (CGMCC) with an accession number CGMCC NO. 13370.

2. A composition comprising the Streptomyces avermitilis strain C63-51 according to claim 1.

3. A method of manufacturing ivermectin B1b or a pharmaceutical composition containing ivermectin B1b comprising using the Streptomyces avermitilis strain C63-51 according to claim 1.

4. A method of producing ivermectin B1b comprising performing fermentation in a medium containing an assimilable carbon source and/or nitrogen source using the Streptomyces avermitilis strain C63-51 according to claim 1.

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

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

7. The method according to claim 4; wherein the medium further comprises an inorganic salt, and the inorganic salt is one or more of MnSO.sub.4, Na.sub.2MoO.sub.4, CoCl.sub.2, CaCO.sub.3, ZnSO.sub.4, FeSO.sub.4, (NH.sub.4).sub.2SO.sub.4, FeCl.sub.3, KNO.sub.3, KH.sub.2PO.sub.4, K.sub.2HPO.sub.4, MgCl.sub.2, MgSO.sub.4, NaCl, CuSO.sub.4, NiSO.sub.4 or KCl.

8. The method according to claim 4, wherein the medium contains corn starch 120-160 g/L, amylase 0.2 g/L, soybean meal 10-40 g/L, peanut meal 0-10 g/L, yeast powder 5-20 g/L, (NH.sub.4).sub.2SO.sub.4 0-4 g/L, MnSO.sub.4 0.024 g/L, Na.sub.2MoO.sub.4 0.024 g/L, CoCl.sub.2 0.01-0.04 g/L and CaCO.sub.3 3-7 g/L.

9. The method according to claim 4, wherein the medium contains corn starch 140 g/L, amylase 0.2 g/L, soybean meal 20 g/L, peanut meal 5 g/L, yeast powder 10 g/L, (NH.sub.4).sub.2SO.sub.4 2 MnSO.sub.4 0.024 g/L, Na.sub.2MoO.sub.4 0.024 g/L, CoCl.sub.2 0.02 g/L and CaCO.sub.3 7 g/L.

10. The method according to claim 4, wherein the temperature of the fermentation is 20 to 40 C.

11. The method according to claim 4, wherein the pH of the medium is 6.0-8.0.

12. The method according to claim 4, wherein the duration of the fermentation is 288-312 hours, and/or with a ventilation of 0.6-1.1 vvm.

13. The method according to claim 4, wherein the temperature of the fermentation is 25 to 30 C., preferably 28 C.; the pH of the medium is 6.5-7.5, preferably 7.0-7.2.

14. An pesticidal composition comprising ivermectin B1b produced by the method according to claim 4.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 shows the comparison of fermentation products of Streptomyces avermitilis strain C63-51 and original strain MA220: A is the HPLC chromatogram of the fermentation broth produced by the original strain MA220 in Example 1; B is the HPLC chromatogram of the fermentation broth produced by Streptomyces avermitilis strain C63-51 in Example 1.

[0032] FIG. 2 shows the mass spectrum of ivermectin B1b prepared in Example 5.

DETAILED DESCRIPTION

[0033] In the following examples, unless otherwise specified, the used reagents and instruments are all commonly used in the art, and can be purchased from chemical or biological products/preparation companies; the methods used in the following examples are all conventional methods in the art, and those having ordinary skill in the art can know the operation of these experiments and obtain corresponding results without any doubt according to the conventional art or the operation manual provided by the manufacturer.

Example 1 Screening of Streptomyces avermitilis Strain C63-51

1. Nitrosoguanidine (NTG) Mutagenesis of Strain MA220

[0034] The tenvermectin producing strain MA220 (the preparation method thereof is described in WO2015135242A1 and WO2015135467A1) was the original strain. A fresh culture slant of the original strain MA220 was taken, and a spore suspension was prepared with 0.1 mol/L phosphate buffer solution of pH 6.0. The spore suspension was treated with NTG with a final concentration of 1 mg/ml, and shaken at 34 C. for 1 hour. The spores were washed three times with saline, diluted and then applied to YMS solid medium (yeast extract 4 g/L, glucose 4 g/L, malt extract 10 g/L, trace element solution 5 ml/L, agar 20 g/L), and cultured at 28 C. for 4 days. A single colony with relatively large diameter and high spore production was picked and transferred to YMS solid culture medium plate, and cultured at 28 C. for 7 days. According to the fermentation verification method described below, about 600 mutagenized strains were screened and mutant strain 58-46 was obtained, which has an ivermectin B1b titer of 1600 mg/L.

2. Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis of Mutant Strain 58-46

[0035] The above-mentioned mutant strain 58-46 mutagenized by NTG was used as the starting strain, and the mature spores on the fresh culture were picked, washed with saline, and the spore suspension was prepared. 10 l of spore suspension was added to a sterilized microscope slide and placed in a plasma chamber for irradiation. The irradiation conditions were set as follows: helium gas as the generator, flow rate: 12.5 L/min, the distance between the plasma torch nozzle exit and the sample plate: 2 mm, the irradiation power: 100 W, and the irradiation time: 30 seconds. After the irradiation, the sample slide was taken out with sterile tweezers, placed in a 2 ml sterile EP tube containing saline, shaken vigorously for 2 minutes using a vortex mixer, and the sample on the slide was thoroughly washed and suspended in saline. After dilution by gradient, the sample was applied to YMS solid culture medium plate and cultured at 28 C. for 4 days. A single colony with relatively large diameter and high spore production was picked and transferred to YMS solid culture medium plate, and cultured at 28 C. for 7 days. According to the fermentation verification method described below, about 600 mutagenized strains were screened and a high-yield strain capable of producing ivermectin B1b was obtained and named Streptomyces avermitilis strain C63-51. The titer of ivermectin B1b produced by the original strain MA220 was 200 mg/L with lots of impurities; the titer of ivermectin B1b produced by Streptomyces avermitilis strain C63-51 was increased to 3000 mg/L with less impurities. The result is shown in FIG. 1, wherein A is the HPLC chromatogram of the fermentation broth of the original strain MA220, and B is the HPLC chromatogram of the fermentation broth of Streptomyces avermitilis strain C63-51.

3. Fermentation Verification Method

(1) Seed Medium Preparation and Cultivation

[0036] Seed medium formula (g/L): corn starch 25, soybean meal 8, peanut meal 10, yeast powder 9.5, CoCl.sub.2 0.03, pH 7.2, sterilized at 121 C. for 20 minutes. About 1 cm.sup.2 of the lawn were scraped with an inoculating shovel to a seed culture medium from the cultured spores, and the culture temperature was 28 C., 250 rpm, cultured for 40 hours in a shaker. At this time, the culture solution had a pH of 6.86 and a mycelium concentration of 35% by volume.

(2) Fermentation Medium Preparation and Cultivation

[0037] Fermentation medium formula (g/L): corn starch 120, amylase 0.2, soybean meal 10, yeast powder 5, MnSO.sub.4 0.024, Na.sub.2MoO.sub.4 0.024, CoCl.sub.2 0.01, CaCO.sub.3 3, pH 7.2, sterilized at 121 C. for 20 minutes. The inoculum size was 6% by volume. The culture temperature was 28 C., 250 rpm, cultured for 12 days in a shaker. At this time, the culture solution had a pH of 6.60 and a mycelium concentration of 33% by volume.

(3) Determination of the Titer of the Fermentation Broth by High Performance Liquid Chromatography (HPLC)

[0038] Extraction of fermentation broth: 1 ml of fermentation broth was collected, 4 ml of anhydrous methanol was added, subjected to ultrasonication for 1 h and then filtered. The filtrate was used directly for HPLC analysis. The HPLC analysis conditions were: chromatographic column, C18 Hypersil ODS2 4.62505 (Dalian Elliott); mobile phase, methanol:acetonitrile:water=81:7:12 (volume ratio); flow rate, 1 ml/min; detection wavelength, 240 nm.

Example 2 Morphology and Cultivation Characteristics of Streptomyces avermitilis Strain C63-51

[0039] Experiments were carried out with reference to the contents of the Streptomyces Identification Manual, the Classification and Identification of Actinomycetes, and the Manual for Identification of Common Bacterial. The seven culture media ISP2, ISP3, ISP4, Gauze's No. 1, Glucose Aspartate, LB and YMS were used for the test of the morphological and cultivation characteristics of the strains. After incubation at 28 C. for 5 to 7 days, the color and pigment of the mycelium were observed. The result is shown in Table 1.

TABLE-US-00001 TABLE 1 Culture characteristics of strain C63-51 on 7 different media Growth Substrate Aerial Soluble Medium Situation Mycelium Mycelium Pigment ISP2 4 Light Cinnamon Light Brown Yellow Brown ISP3 3 Light Yellow None Yellow White ISP4 3 Yellow Light Light Yellow Yellow Brown Gauze's 4 Beige Milky Yellow No. 1 Glucose 4 Yellow Yellow Light Aspartate Orange Orange Yellow Tan LB 2 Beige Beige Yellow YMS 4 Yellow Yellow Yellow

Example 3 Physiological and Biochemical Characteristics of Streptomyces avermitilis Strain C63-51

[0040] (1) Utilization of carbon source: ISP9 was used as the basic medium, and the final concentration of each carbon source was 1.0% (mass percent concentration). The result is shown in Table 2.

[0041] (2) Utilization of nitrogen source: the culture medium with a formula of KH.sub.2PO.sub.4 1.36 g/L, Na.sub.2HPO.sub.4 2.13 g/L, MgSO.sub.4 0.2 g/L, CaCl.sub.2 0.005 g/L, glucose 10 g/L, FeSO.sub.4 0.0005 g/L was used as the basic medium. The concentrations of potassium nitrate and ammonium sulfate were both 0.1% (mass percent concentration). The result is shown in Table 2.

TABLE-US-00002 TABLE 2 Utilization of carbon and nitrogen sources of strain C63-51 Inorganic Carbon Growth Carbon Growth Carbon Growth Nitrogen Growth Source Situation Source Situation Source Situation Source Situation D-Glucose 4 Maltose 4 Inositol 3 Ammonium + sulfate D-Raffinose 3 D-Fructose 4 Mannitol 4 Potassium nitrate D-Xylose 3 D-Sucrose 3 Glycine 0 D-Sorbitol 4 Salicin 3 Xylan 3 L-Arabinose 4 D-Lactose 4 Inulin 2 Glycerol 4 Galactose 3 Rhamnose 4

[0042] (3) Degradation test and NaCl tolerance test: the basic medium used in the degradation test was GYEA (pH 6.8), and the results of various degradation tests are shown in Table 3. The NaCl tolerance test result showed that the strain has good tolerance to NaCl, and it could grow in the presence of 7% NaCl.

TABLE-US-00003 TABLE 3 Degradation test results of strain C63-51 Concentration of Degradation Product Degradation Product Result Adenine 0.5% 4, + Guanine 0.5% 4, Hypoxanthine 0.4% 4, Casein 1.0% 4, + Tyrosine 1.0% 4, + Tween-40 1.0% 3, Tween-80 1.0% 4,

[0043] (4) Physiological and biochemical tests, pH test and temperature test: the result of physiological and biochemical test is shown in Table 4. Both the pH test and the temperature test were carried out using Gauze's No. 1 medium. The results showed that the strain could grow between 14 C. and 37 C., and the optimum growth temperature was 28 C.; and it could grow between pH of 6.0 to 8.0, and the optimum range was 6.5 to 7.5.

TABLE-US-00004 TABLE 4 Main physiological and biochemical characteristics of strain C63-51 Test Item Result Gelatin Liquefaction Starch Hydrolysis + Milk Solidification + Milk Peptonization + Nitrate Reduction + Hydrogen Sulfide Production + Cellulose Utilization Note: The numbers and symbols in Tables 1-4 represent: 0: no growth; 1: weak growth; 2: growth; 3: good growth; 4: best growth; +: positive; : negative.

Example 4 16S rDNA Sequence Analysis and Strain Identification

[0044] Fresh cells of the test strain C63-51 were collected, and the total DNA template was extracted by the lysozyme-modified Pitcher method (Letters in Applied Microbiology, 1989, 8: 151-156), and 16S rDNA gene amplification was carried out using universal primers (27F and 1495R). The PCR product was tested and purified, and subjected to sequencing directly (sequencing was carried out by Genscript Biotechnology (Nanjing) Co., Ltd.). The determined 16S rDNA sequence was compared with the sequences of related species and genus in the GenBank database to determine the taxonomic status of the strain.

[0045] The 16S rDNA sequence of strain C63-51 (CGMCC NO. 13370) was compared with related sequences in GenBank by BLAST. The results are shown in Table 5 (only the strains with relatively high homology are listed in the table).

TABLE-US-00005 TABLE 5 Homology of strain C63-51 with related strains GenBank Number of base Homology Species No. differences (%) Streptomyces avermitilis NR_074747.1 1491/1491 100% MA-4680 Streptomyces CP016279.1 1476/1491 99% griseochromogenes strain ATCC 14511 Streptomyces fimbriatus AB045868.1 1459/1476 99% Streptomyces plumbeus HQ850409.1 1468/1490 99% strain S14

[0046] Strain C63-51 (CGMCC NO. 13370) was found to have 100% homology with Streptomyces avermitilis via 16S rDNA region sequencing. In addition, phenomenal characteristics test of strain C63-51 (CGMCC NO. 13370) was carried out. The strain was found to be very similar to the classification parameters of Streptomyces avermitilis, so the strain C63-51 (CGMCC NO. 13370) was identified as Streptomyces avermitilis.

Example 5 Optimization of Fermentation Medium

(1) Fermentation Medium Optimization and Cultivation

[0047] The fermentation medium in Example 1 was used as the basic medium. The concentration of each component in the formula was adjusted, the fermentation medium was optimized, and the formula was optimized to be (g/L): corn starch 120-160, amylase 0.2, soybean meal 10-40, peanut meal 0-10, yeast powder 5-20, (NH.sub.4).sub.2SO.sub.4 0-4, MnSO.sub.4 0.024, Na.sub.2MoO.sub.4 0.024, CoCl.sub.2 0.01-0.04, CaCO.sub.3 3-7, pH 7.2, sterilized at 121 C. for 20 minutes. The inoculum size was 6% by volume. The culture temperature was 28 C., 250 rpm, and the culture was carried out for 12 days in a shaker.

(2) Determination of the Titer of the Fermentation Broth by High Performance Liquid Chromatography (HPLC)

[0048] Extraction of fermentation broth: 1 ml of fermentation broth was taken, 4 ml of anhydrous methanol was added, and the mixture was filtered after a one-hour ultrasonication. The filtrate was used directly for HPLC analysis. The HPLC analysis conditions were: chromatographic column, C18 Hypersil ODS2 4.62505 (Dalian Elite); mobile phase, methanol:acetonitrile:water=81:7:12 (volume ratio); flow rate, 1 ml/min; absorption wavelength, 240 nm.

[0049] The titer of the fermentation broth of different fermentation media was determined as described above, and the preferred fermentation medium formulation was determined according to the titer of the fermentation broth. The most preferred fermentation medium formula is (g/L): corn starch 140, amylase 0.2, soybean meal 20, peanut meal 5, yeast powder 10, (NH.sub.4).sub.2SO.sub.4 2, MnSO.sub.4 0.024, Na.sub.2MoO.sub.4 0.024, CoCl.sub.2 0.02, CaCO.sub.3 7, pH 7.2, sterilized at 121 C. for 20 minutes. The culture temperature was 28 C., 250 rpm, and the culture was carried out for 12 days in a shaker. At this time point, the pH of the culture broth was 6.65 and the mycelium concentration was 37% by volume. The titer of ivermectin B1b produced by Streptomyces avermitilis strain C63-51 was increased to 6500 mg/L, which was 2 times higher than the original formulation.

Example 6 Fermentation, Isolation and Preparation of Ivermectin B1b

(1) The Slant Strain Cultivation

[0050] YMS medium was used for preparing the agar slant (g/L): yeast extract 4, glucose 4, malt extract 10, trace element solution 5 ml/L, agar 20, distilled water 1000 ml, pH 7.2. The mixture was sterilized at 121 C. for 20 minutes, cooled to 50-60 C. and used to prepare the culture slant. The strain C63-51 was inoculated on the slant, cultured at 28 C. for 7 days.

(2) Seed Medium Preparation and Cultivation

[0051] Seed medium formula (g/L): corn starch 25, soybean meal 8, peanut meal 10, yeast powder 9.5, CoCl.sub.2 0.03, pH 7.2, sterilized at 121 C. for 20 minutes. About 1 cm.sup.2 of the culture with spores were scraped with an inoculating shovel to a seed culture medium, and the culture was carried out at temperature 28 C., 250 rpm for 40 hours in a shaker. At this time, the pH of the culture broth was 6.86 and the mycelium concentration was 35%.

(3) Preparation of Seed Medium in a Tank

[0052] 10 L seed medium was placed in a 15 L tank, sterilized by steam at 121 C. for 30 minutes. 300 ml seed culture from a shake flask was inoculated into the tank. The culture temperature was 281 C., the stirring speed was 100-250 rpm, the ventilation was 1.0 vvm and the culture was carried out for 18 hours. At this time, the pH of the culture broth was 6.80 and the mycelium concentration was 30% (volume percentage).

(4) Fermenter Culture Medium Preparation and Cultivation

[0053] The formula of the fermentation medium was: corn starch 140, amylase 0.2, soybean meal 20, peanut meal 5, yeast powder 10, (NH.sub.4).sub.2SO.sub.4 2, MnSO.sub.4 0.024, Na.sub.2MoO.sub.4 0.024, CoCl.sub.2 0.02, CaCO.sub.3 7, fermenter volume 50 L, feed volume 30 L, pH 7.2, sterilized by steam at 121 C. for 30 minutes and then cooled. 3 L of seed culture from a shake flask was inoculated into the fermenter. The temperature for culture was 281 C., the stirring speed was 101-308 rpm, the ventilation was 0.6-1.1 vvm and the culture was carried out for 12 days. The fermenter was discharged, and the fermentation unit of ivermectin B1b was measured to be 4000 mg/L.

(5) Fermentation Broth Extraction

[0054] The obtained fermentation broth was filtered with a filter cloth to obtain a filter cake, and the filter cake was extracted twice with ethanol and combined to obtain an ethanol extract. The ethanol extract was concentrated until dry via vacuum concentration and extracted with ethyl acetate to obtain an extract containing ivermectin B1b. After mixed with silica gel, the extract was applied to a silica gel column and eluted with a gradient of petroleum ether/acetone at volume ratios of 90:10, 80:20, 70:30, 60:40, and the eluted fractions were collected respectively, detected by TLC, to obtain a component containing ivermectin B1b. The component was concentrated until dry via vacuum concentration to obtain a sample containing ivermectin B1b. The sample was subjected to reversed-phase chromatography separation under the following conditions:

Liquid phase system: Agilent 1100 semi-preparative high pressure liquid chromatography
Column: ZORBAXEclipse XDB-C18 (250 mm9.4 mm)
Eluent: methanol:acetonitrile:water=46:46:8 (volume ratio)
Flow rate: 1.5 ml/min
Detection wavelength: 2=240 nm

[0055] A compound with a peak at a time of 26.79 minute was collected. The compound was subjected to mass spectrographic analysis, and the spectrum is shown in FIG. 2. The result showed that the molecular weight of the compound was 860, and it was determined to be ivermectin B1b, and the structure was as follow:

##STR00001##

[0056] In summary, Streptomyces avermitilis strain C63-51 of the present disclosure is a high-yield strain for ivermectin B1b, and the shake flask fermentation unit can reach 6500 mg/L, the fermentation unit in 50 L fermenter can reach 4000 mg/L.