Patent Application
20200131552
The present invention belongs to the field of biomedicine, and relates to a strain of Streptomyces avermitilis with high yield of ivermectin B1b and a method for producing ivermectin B1b using the strain.
Ivermectin has an effective killing effect on both internal and external parasites, especially nematodes and arthropods. It has stronger permeability and safety for mammalian body tissues and has a longer duration. It is especially suitable for the control of parasites in muscles, organs and special tissues that are difficult to be reached by general oral anthelmintic. It has a special effect on the gastrointestinal nematodes and parasites. It is recognized as a multi-component antibiotic with broad spectrum, high efficiency, safety, little residue and no drug resistance.
So far, there are two main methods for producing ivermectin according to domestic and foreign reports. One of the methods is to chemically reduce avermectin B1 by hydrogenation to obtain ivermectin. The chemical method needs to separate and purify avermectin B1 from the fermentation broth of Streptomyces avermitilis and then change it to ivermectin by reduction reaction. The production cost of the process is relatively high, and the yield of ivermectin B1b is only 15%. There are many impurities in the chemical synthesized ivermectin B1b obtained from avermectin B1, and the purification process is complicated, and it is difficult to isolate a large amount of ivermectin B1b. The other method is to use genetically engineered strain to produce ivermectin. For example, CN200510074936.X discloses a method of obtaining a engineered strain producing ivermectin by replacing the coding region of the avermectin polyketide synthase DH2-KR2 domain of Streptomyces avermitilis with the coding region of polyketide synthase DH-ER-KR domain, and the yield of ivermectin B1a is about 1-4 ng/mL, but the yield of ivermectin B1b is not mentioned. CN200910089970.2 discloses the expression plasmid of malEFG gene encoding maltose transporter. The plasmid was transformed into Streptomyces avermitilis and the maltose transporter overexpression recombinant strain was constructed. The recombinant strain is used for the overexpression of a maltose transport system gene to improve the utilization ratio of maltose. This method increases the yield of ivermectin from about 10 ng/mL to about 35 ng/mL. PCT Applications WO2015135242A1 and WO2015135467A1 disclose the constructing process of a genetically engineered strain MA220, which is obtained by a two-step genetic modification from Streptomyces avermitilis MA-4680: first, the aveD gene in MA4680 is inactivated by PCR targeting technology to obtain a genetically engineered strain AD28 which does not produce avermectin A component but only produces avermectin B component; then, the aveA1 gene is replaced with the milA1 gene of Streptomyces milbemycinicus by genetic recombination technology to obtain recombinant Streptomyces MA220. The fermentation product of recombinant Streptomyces MA220 contains a very small amount of ivermectin B1b component in addition to the main components of tenvermectin A (TEVA) and tenvermectin B (TEVB). The above-mentioned method for producing ivermectin by genetic engineering to construct a new strain either has relatively high requirements on experimental conditions and skill level of researchers, or the fermentation yield of the genetic engineering strain is unsatisfactory, so this method basically remains at the laboratory stage, which is not conducive to the formation of industrialization.
In summary, the yield of ivermectin in the conventional art is low, and the product is either a mixture of ivermectin B1a and B1b, or ivermectin B1a, and no attention has been paid to the production of ivermectin B1b as the single component. Therefore, there is an urgent need to find new microbial strains for producing ivermectin, especially ivermectin B1b, and a preparation method thereof, which can give a satisfactory yield.
The inventors unexpectedly screened and obtained a high-yield strain which can be directly used for fermenting ivermectin B1b, and further provide a method for producing ivermectin B1b using the strain. On one hand, ivermectin B1b can be effectively produced, and on the other hand, it can lay a foundation for further research on the biological activity of ivermectin B1b and structural modification.
One of the objects of the present disclosure is to provide a novel high-yield strain capable of producing ivermectin B1b to solve the above-mentioned problems.
One aspect of the present disclosure is to provide a high-yield microorganism strain for ivermectin B1b. The strain is Streptomyces avermitilis strain C63-51, which was deposited on Dec. 22, 2016 in the China General Microbiological Culture Collection Center (address: No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences) with an accession number of CGMCC NO. 13370 and registered and confirmed to be viable.
Another aspect of the present disclosure is to provide a use of Streptomyces avermitilis strain C63-51 in the manufacture of ivermectin B1b or a pharmaceutical composition containing ivermectin B1b.
Another aspect of the present disclosure is to provide a method of producing ivermectin B1b using Streptomyces avermitilis strain C63-51. The method comprises performing fermentation in a culture medium containing an assimilable carbon source and/or nitrogen source by using Streptomyces avermitilis strain C63-51.
The present disclosure is also directed to a composition comprising Streptomyces avermitilis strain C63-51 of the present disclosure. The composition may be a strain composition comprising a strain other than the strain C63-51; preferably, the other strain may be a strain for producing ivermectin B1b, such as Streptomyces avermitilis, Streptomyces hygroscopicus, genetically engineered strain MA220, etc., especially genetically engineered strain MA220.
The present disclosure is also directed to a use of the above-mentioned composition in the manufacture of ivermectin B1b or a pharmaceutical composition comprising ivermectin B1b.
In another aspect, the present disclosure is also directed to an antiparasitic composition comprising ivermectin B1b produced by the method of the present disclosure.
Preferably, the above-mentioned 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.
Preferably, 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.
Preferably, the culture medium further comprises an inorganic salt, and the inorganic salt is one or more of MnSO4, Na2MoO4, CoCl2, CaCO3, ZnSO4, FeSO4, (NH4)2SO4, FeCl3, KNO3, KH2PO4, K2HPO4, MgCl2, MgSO4, NaCl, CuSO4, NiSO4 or KCl.
Preferably, the culture 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, (NH4)2SO4 0-4 g/L, MnSO4 0.024 g/L, Na2MoO4 0.024 g/L, CoCl2 0.01-0.04 g/L, CaCO3 3-7 g/L.
Preferably, the culture 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, (NH4)2SO4 2 g/L, MnSO4 0.024 g/L, Na2MoO4 0.024 g/L, CoCl2 0.02 g/L, CaCO3 7 g/L.
Preferably, the temperature of the fermentation is 20 to 40° C., more preferably 25 to 30° C., most preferably 28° C. Preferably, the pH condition of the fermentation is controlled in the fermentation method of the present disclosure, and the pH is 6.0-8.0, more preferably 6.5-7.5, most preferably 7.0-7.2. The duration of the fermentation of the method of the present disclosure may be 288-312 hours; the ventilation of the method of the present disclosure may be 0.6-1.1 vvm.
In the present disclosure, the “ventilation” is the amount of filter-sterilized air passing through each unit volume of culture medium every minute.
In the present disclosure, the “Streptomyces avermitilis strain C63-51”, “strain C63-51”, and “mutagenized strain C63-51” have the same meaning.
In the present disclosure, the “genetically engineered strain MA220”, “strain MA220”, “tenvermectin producing strain MA220”, “recombinant Streptomyces MA220”, and “original strain MA220” have the same meaning.
In the present disclosure, ivermectin B1b may be detected by the following HPLC method:
The present disclosure has at least the following advantages over the conventional art:
1. Streptomyces avermitilis strain C63-51 obtained by screening in the present disclosure can be directly used for the fermentation and production of ivermectin B1b. Compared with the chemical preparation of ivermectin, it avoids complicated process steps of separation and purification and chemical reduction, so that greatly reduces production cost.
2. In the conventional art, for example, in the method of CN200510074936.X, the yield of ivermectin B1a is about 1-4 μg/mL, but the yield of ivermectin B1b is not mentioned; in the method of CN200910089970.2, the yield of ivermectin is about 35 μg/mL, the product is a mixture of ivermectin B1a and B1b, and it is relatively difficult to separate ivermectin B1b; in the methods of WO2015135242A1 and WO2015135467A1, the titer of ivermectin B1b produced by the original strain MA220 is 200 mg/L. In the present disclosure, the ivermectin B1b produced by Streptomyces avermitilis strain C63-51 in shake flask fermentation can reach 6500 mg/L, and the titer in 50 L fermenter can reach 4000 mg/L. The yield of the ivermectin B1b of the present disclosure is much higher than that of the conventional art; moreover, the content of ivermectin B1b in the fermentation broth of Streptomyces avermitilis strain C63-51 exceeds 60% with little impurities, high titer, and the purification process is simple.
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.
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.
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 μl 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
Seed medium formula (g/L): corn starch 25, soybean meal 8, peanut meal 10, yeast powder 9.5, CoCl2 0.03, pH 7.2, sterilized at 121° C. for 20 minutes. About 1 cm2 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.
Fermentation medium formula (g/L): corn starch 120, amylase 0.2, soybean meal 10, yeast powder 5, MnSO4 0.024, Na2MoO4 0.024, CoCl2 0.01, CaCO3 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.
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 4.6×250×5 (Dalian Elliott); mobile phase, methanol:acetonitrile:water=81:7:12 (volume ratio); flow rate, 1 ml/min; detection wavelength, 240 nm.
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.
(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.
(2) Utilization of nitrogen source: the culture medium with a formula of KH2PO4 1.36 g/L, Na2HPO4 2.13 g/L, MgSO4 0.2 g/L, CaCl2 0.005 g/L, glucose 10 g/L, FeSO4 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.
(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.
(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.
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.
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).
Streptomyces avermitilis
Streptomyces
griseochromogenes
Streptomyces fimbriatus
Streptomyces plumbeus
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.
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, (NH4)2SO4 0-4, MnSO4 0.024, Na2MoO4 0.024, CoCl2 0.01-0.04, CaCO3 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.
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 4.6×250×5 (Dalian Elite); mobile phase, methanol:acetonitrile:water=81:7:12 (volume ratio); flow rate, 1 ml/min; absorption wavelength, 240 nm.
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, (NH4)2SO4 2, MnSO4 0.024, Na2MoO4 0.024, CoCl2 0.02, CaCO3 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.
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.
Seed medium formula (g/L): corn starch 25, soybean meal 8, peanut meal 10, yeast powder 9.5, CoCl2 0.03, pH 7.2, sterilized at 121° C. for 20 minutes. About 1 cm2 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%.
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 28±1° 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).
The formula of the fermentation medium was: corn starch 140, amylase 0.2, soybean meal 20, peanut meal 5, yeast powder 10, (NH4)2SO4 2, MnSO4 0.024, Na2MoO4 0.024, CoCl2 0.02, CaCO3 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 28±1° 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.
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 mm×9.4 mm)
Eluent: methanol:acetonitrile:water=46:46:8 (volume ratio)
Flow rate: 1.5 ml/min
Detection wavelength: 2=240 nm
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
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201710145887.7 | Mar 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/078773 | 3/13/2018 | WO | 00 |
