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WO2016155568A1 - 一种链霉菌及其生产米尔贝霉素a3的方法 - Google Patents

一种链霉菌及其生产米尔贝霉素a3的方法 Download PDF

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WO2016155568A1
WO2016155568A1 PCT/CN2016/077357 CN2016077357W WO2016155568A1 WO 2016155568 A1 WO2016155568 A1 WO 2016155568A1 CN 2016077357 W CN2016077357 W CN 2016077357W WO 2016155568 A1 WO2016155568 A1 WO 2016155568A1
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milbemycin
streptomyces
powder
fermentation
medium
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PCT/CN2016/077357
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French (fr)
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WO2016155568A8 (zh
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滕云
徐美冬
莫美依
何志慧
陈正杰
江连清
白骅
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浙江海正药业股份有限公司
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Priority to EP16771323.9A priority Critical patent/EP3275996B1/en
Priority to US15/562,155 priority patent/US10287545B2/en
Priority to JP2017549455A priority patent/JP6550144B2/ja
Publication of WO2016155568A1 publication Critical patent/WO2016155568A1/zh
Publication of WO2016155568A8 publication Critical patent/WO2016155568A8/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C12R2001/55—Streptomyces hygroscopicus

Definitions

  • the present invention relates to a novel Streptomyces, and a method for producing milbemycin A3 by fermentation of the bacterium.
  • Milbemycin is a microbial natural product pesticide, and it has been shown to be one of the best acaricides in the world today.
  • the US Environmental Protection Agency identified it as a low-risk pesticide, and the Netherlands approved it as “GNO” (a natural product in crop production). It is an eco-friendly pesticide and is suitable for the comprehensive prevention and control of organic agricultural pests and diseases. It has become a popular insecticide and acaricide in developed countries.
  • Milbemycin is a metabolite with insect resistance activity selected from the fermentation broth of microorganisms by the Japanese Sankyo Company as a test insect (US 3,950,360).
  • 1% of milbeknock was used as an acaricide in tea and eggplant in Japan.
  • 1% of milbemycin emulsifiable concentrate was used as an insecticide for pears, peaches, watermelons, strawberries, eggplants, and flowers.
  • milbemycin has been registered in Japan, Europe and the United States, and is recommended as a safe, environmentally friendly insecticide and acaricide by the US Environmental Protection Agency.
  • milbemycin Due to the complex composition of milbemycin fermentation broth and the difficulty of separation, milbemycin is usually used and reported as a mixture of A3 and A4. The production and use of single components are rarely reported. The main reason is that A3 is separated from the fermentation broth with similar ratio of A3 and A4, and the single component of A4 is difficult, which affects the final yield. Taking A3 as an example, the content of A3 in the fermentation broth is less than 30%. To obtain a single component of A3, a large amount of A4 needs to be lost. The realization of single component production of A3 or A4 can further promote the application of milbemycin single component in other fields. The most effective way to obtain a single component is to obtain a new strain that is capable of producing a single component.
  • One of the objects of the present invention is to provide a microbial strain capable of increasing the yield of milbemycin A3, which is characterized in that the content of milbemycin A3 component in the fermentation broth accounts for milbemycin A3 and The percentage of the total content of milbemycin A4 is greater than 70%, and the unit yield of milbemycin A3 can be greater than 3000 ug/ml, and the impurity content is low.
  • the microbial strain of Streptomyces sp. HS7523 of the present invention was deposited on September 16, 2014 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (Address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), The accession number is CGMCC No.9672, and the classification is named Streptomyces hygroscopicus, and it is registered and proved to be alive.
  • the main biological characteristics of Streptomyces sp. HS7523 of the present invention are: ISP1, ISP2, ISP3 medium colony color white, round, slightly convex in the middle, wrinkled, medium-sized (about 6mm), with a small amount of spores, The mycelium is developed, and the hyphae are tightly combined with the culture medium, which is not easy to provoke. No pigment was produced on ISP1, ISP2 medium, and beige brown pigment was produced on ISP3 medium.
  • the present invention describes the morphological and molecular level characteristics of Streptomyces sp. HS7523. It can be concluded that Streptomyces sp. HS7523 belongs to Streptomyces hygroscopicus by comparison with morphological and molecular levels of the known milbemycin-producing bacteria. Streptomyces HS7523 has 99% homology with Streptomyces sp. NRRL 5739 16s rRNA and 99% homology with Streptomyces bingchenggensis BCW-1. The most morphological difference between the Streptomyces sp. HS7523 strain and other milbemycin-producing bacteria is that colonies of other strains such as Streptomyces sp.
  • CGMCC No. 7677 secrete golden teardrops on the surface of the colony on the production plate, and pigmentation occurs. Streptomyces HS7523 was grayish in color on the same medium, with no tears on the surface and no pigmentation (see Figure 2).
  • the present invention also provides a method for preparing milbemycin by using Streptomyces HS7523 (CGMCC No. 9672).
  • the method comprises the process of aerobic fermentation in a nutrient medium containing assimilable carbon and nitrogen sources using Streptomyces HS7523 (CGMCC No. 9672).
  • the above assimilable carbon source is preferably derived from starch, dextrin, glucose, industrial molasses, glycerin, sucrose, lactose, maltose, trehalose, xylan, mannitol, sorbitol or the like. combination.
  • the assimilable nitrogen source is preferably selected from the group consisting of yeast extract, yeast powder, beef extract, tryptone, peptone, skim milk powder, whole milk powder, soybean cake powder, cottonseed cake powder, peanut cake powder, A combination of gluten meal, corn syrup dry powder, bran, urea, ammonium salt or a combination thereof.
  • the nutrient medium contains yeast extract, yeast extract or peptone 2-10 g/L, sucrose or molasses 20-200 g/L, skim milk powder or corn syrup 2-11 g/L, soy cake powder 2-11g/L, cottonseed cake powder or gluten meal 5-15g/L, dipotassium hydrogen phosphate 0.5-1g/L, ferrous sulfate heptahydrate 0.05-0.1g/L, zinc sulfate 0.005-0.02g/L, Calcium carbonate 1-5 g/L, copper sulfate 0.01-0.05 g/L and/or sodium molybdate 0.1-0.5 g/L.
  • the temperature of the fermentation culture is preferably from 20 ° C to 40 ° C, more preferably from 25 ° C to 35 ° C, the pH of the medium is between 6.0 and 8.0, preferably about 7.0; and the culture time is from 300 to 360 hours;
  • the dissolved oxygen is not less than 35%; the aeration is 0.5-1.0 vvm.
  • the fermentation mode is liquid submerged fermentation.
  • Milbemycin can be detected by the following methods:
  • UV absorption wavelength 240nm
  • HPLC mobile phase conditions as shown in Table 9.
  • Injection volume 10 ⁇ l.
  • the milbemycin-producing bacteria used in the present invention are spontaneous mutant strains of Streptomyces HS7523 (CGMCC No. 9672) and Streptomyces HS7523 (CGMCC No. 9672) or mutant strains which are conventionally induced.
  • the present invention provides a novel strain of Streptomyces sp. HS7523 for producing milbemycin and a method for producing milbemycin.
  • the strain Streptomyces sp. HS7523 is characterized in that the content of milbemycin A3 in the fermentation broth accounts for more than 70% of the total content of milbemycin A3 and milbemycin A4, and the unit yield of milbemycin A3 can be It is greater than 3000ug/ml and has low impurity content.
  • Streptomyces HS7523 of the present invention increases the proportion of milbemycin A3 in the fermentation product by the fermentation method, the difficulty of preparing the single component of milbemycin A3 is reduced, which is advantageous for reducing the production cost and expanding the milbe The range of applications of the single component of mycin A3.
  • Figure 1 Breeding map of Streptomyces sp. HS7523 of the present invention.
  • Figure 2 Comparison of colony morphology of Streptomyces HS7523 strain of the present invention with Streptomyces milbemycinicus CGMCC No. 7677, wherein A is a colony form of Streptomyces milbemycinicus CGMCC No. 7677, and B is a colony form of Streptomyces HS7523 of the present invention.
  • Figure 3 HPLC chromatogram of milbemycin A3, A4 standard.
  • Figure 4 HPLC chromatogram of the fermentation broth of Example 5, the percentage of milbemycin A3 in the total content of A3 and A4 was 72%.
  • Figure 5 Fermentation curve of Streptomyces sp. HS7523 of the present invention for producing milbemycin A3 and A4 in the 50 L fermentor of Example 6.
  • Figure 6 HPLC chromatogram of milbemycin A3 produced by Streptomyces sp. HS7523 of the present invention under the conditions of Example 6, the content of milbemycin A4 is 1246 mg / L, the content of A3 is 3050 mg / L, the milbemycin A3 The percentage of the total content of A3 and A4 was 71%.
  • Sucrose is a product of Guangxi Dongmen Nanhua Sugar Co., Ltd.
  • the yeast extract is a product of Zhejiang Dongcheng Pharmaceutical Co., Ltd.
  • Yeast paste is a product of Hefei Rice Bioengineering Co., Ltd.
  • Peptone is a product of Huzhou Huihe Biotechnology Co., Ltd.
  • Molasses is a product of Guangdong Jiangmen Biotechnology Co., Ltd.
  • Skim milk powder is a product of Hulunbeier Sanyuan Dairy Co., Ltd.
  • Corn syrup is a product of Shandong Shouguang Giant Energy Golden Corn Development Co., Ltd.
  • Soybean cake powder is a product of Ningbo Beilun Jiangnan Oil Co., Ltd.
  • Cottonseed cake powder is a product of Beijing Kangmingwei Medium Technology Co., Ltd.
  • the gluten powder is a product of Beijing Kangmingwei Medium Technology Co., Ltd.
  • the Streptomyces hygroscopicus HS7523 of the present invention is bred by multiple rounds of mutagenesis on the basis of the milbemycin-producing bacterium Streptomyces milbemycinicus CGMCC No. 7677 (see Chinese patent CN103789339A, the holder of the bacterium is the applicant of this patent). (including NTG, EMS, UV and other mutagenic means) obtained milbemycin A3 high-yield strains. Compared with the original strains, the strain has a large change in appearance and morphology, and belongs to the morphological mutant strain.
  • Streptomyces milbemycinicus CGMCC No. 7677 strain was cultured in ISP3 slant medium at 28 ° C for 10-12 days, then the mycelium was scraped off under sterile conditions with a inoculating shovel, and the mycelium was ground and suspended in a ground tube.
  • the bacterial suspension is prepared by NTG (nitrosoguanidine), EMS (ethyl methanesulfonate), UV (ultraviolet) mutagenesis treatment, the specific method is as follows:
  • EMS 1 ml of EMS was taken up in 2 ml of absolute ethanol, and 22 ml of a 0.1 mol/L phosphate buffer of pH 7.2 was added. Take a 10 -1 or 10 -2 gradient of single-cell suspension 5ml into a plate with a paper clip, add 5ml, 4.0% EMS solution, and finally the EMS solution concentration is 2.0%, set on a magnetic stirrer, stir treatment 20 Minutes - 60 minutes. The reaction was stopped by adding 10 ml of 5% sodium thiosulfate to the mutagenesis plate, and diluted 10 -2 -10 -7 with physiological saline, and the ISP3 plate was used to obtain the mutagenesis group.
  • ISP1, ISP2, ISP3, ISP4, ISP5, Gaoshi No.1, calcium malate, nutrient agar, YMS and Shi's ten kinds of medium after 7 to 10 days of culture at 28 °C, observe the color and pigment of mycelium ( The culture characteristics are shown in Table 1).
  • ISP9 inorganic nitrogen source
  • YMS medium was used for the oxidase and catalase tests (see Table 4 for the results), the pH test (see Table 5 for the results), and the temperature test (see Table 6 for the results).
  • Carbon source growing situation Carbon source growing situation
  • Inorganic nitrogen source growing situation D-glucose 4 Salicin 4 Ammonium sulfate + D-cotton sugar 4
  • D-Lactose 3 Potassium nitrate - D-xylose 0
  • Galactose 4 D-sorbitol 2 Inositol 2 L-arabinose 0 Mannitol 3
  • Degradant Degradant concentration result Adenine 0.5% 4,+ Casein 1.0% 4,- Guanine 0.5% 4,- Tyrosine 1.0% 4,- Huang Wei 0.4% 4,- Tween-40 1.0% 2,+ Xylan 0.4% 4,- Tween-60 1.0% 3,+ Hypoxanthine 0.4% 4,- Tween-80 1.0% 4,+
  • Example 4 16S rDNA sequence analysis and comparison with known milbemycin-producing bacteria
  • the mycelium of the present invention grown on ISP2 was collected, placed in a TSB liquid medium containing glass beads, placed in an incubator at 28 ° C, and shaken at 250 r / min for 2-4 d.
  • the mycelium was collected by centrifugation, washed twice with sterile water, and stored at 4 ° C until use.
  • the mycelium was centrifuged at 1000 rpm for 1 min.
  • the extracted genome was used as a template, and a universal primer was used for PCR amplification.
  • the upstream primer 27F is 5'-GAGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1).
  • the downstream primer 1495R is 5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID No. 2).
  • the reaction was carried out on a PCR instrument. The procedure was: pre-denaturation at 95 ° C for 5 min, denaturation at 94 ° C for 45 s, renaturation at 55 ° C for 45 s, extension at 72 ° C for 90 s, 30 cycles, and extension at 72 ° C for 10 min.
  • the reaction system is as follows:
  • the PCR product was detected by 0.8% agarose gel electrophoresis. Purification is carried out using a clear product. The amplified product was recovered by gel electrophoresis and ligated into the T vector and sequenced to obtain the sequence of the primary structure of the 16S rDNA of the strain. By performing a similarity search (blast) in the Genebank database, the results showed 99% homology with Streptomyces sp. NRRL 5739 16s rRNA and 99.5% homology with Streptomyces bingchenggensis BCW-1, and the results are shown in Table 8.
  • the milbemycin-producing strain Streptomyces NRRL NO.5739 is aerially hyphae gray on the ISP2 medium, yellowish brown on the back, many yellow teardrops on the surface of the colony, yellow pigment production; airborne bacteria on ISP4 medium It is silky gray and has a yellowish back. There are also many yellow teardrops on the surface of the colony. It has a bright olive green pigment and can use arabinose and xylose.
  • HS7523 of the present invention has a white hyphae on the SP2 medium, a beige color on the back, no teardrops on the surface of the colony, and no pigmentation;
  • the aerial hyphae on the ISP4 medium is white, the back is ivory, and the surface of the colony has no tears, no Pigments are produced and arabinose and xylose cannot be used.
  • the milbemycin-producing strain Streptomyces bingchengsis sp. nov CGMCC No.1734 has a gray surface on the Gao's No.1 medium (with black water-absorbing spots), and the colony has a yellow-gray back and yellow Brown pigment is produced.
  • the Streptomyces sp. HS7523 of the present invention has a white background on the surface of the colony on the high-grade medium, a surrounding TV ash 4, and a TV ash 4 on the back of the colony, and no pigment is produced.
  • Streptomyces sp. HS7523 of the present invention belongs to the genus Streptomyces, but it is different from the known milbemycin-producing bacterium Streptomyces NRRL NO. 5739, Streptomyces bingchengsis sp. nov. CGMCC No. 1734 Streptomyces HS7523 is a brand new strain.
  • Inclined spore medium formula yeast extract 2, malt extract 2, sucrose 8, skim milk powder 1, agar 20, pH 7.0-7.2 before disinfection, test tube 30 ⁇ 200 mm, loading 20 mL, After sterilization at 121 ° C for 20 minutes, Cool to 50-60 ° C pendulum slope, access to a loop of mycelium cultured at 28 ⁇ 1 ° C for 10 days, the hyphae mature.
  • the inoculum size of the cells was 10 5 -10 6 cfu/mL, the culture temperature was 28 ⁇ 1 ° C, 250 rpm, and shaking culture was carried out for 48 hours.
  • HPLC detection method of milbemycin is as follows:
  • UV absorption wavelength 240nm
  • HPLC mobile phase conditions are as follows:
  • Injection volume 10 ⁇ l.
  • HPLC profiles of the milbemycin A3 and A4 standards under the same conditions are shown in Fig. 3 (HPLC detection of the fermentation broth of the following examples includes the HPLC detection steps of the milbemycin A3 and A4 standards).
  • the retention time of the target product was compared by HPLC chromatogram under the same conditions as the milbemycin A3 and A4 standards, and the target product in the fermentation broth was determined to be milbemycin A3, A4 (the fermentation broth of the following examples)
  • the HPLC map was also subjected to this alignment to determine the target product as milbemycin A3, A4).
  • the content of milbemycin A4 in the fermentation broth was 1,206 mg/L, the content of A3 was 3,100 mg/L, and the percentage of milbemycin A3 in the total content of A3 and A4 was 72%.
  • the formulation of the fermentation medium was the same as that of the above Example 5, except that 0.25% of the foaming agent was added as the defoaming agent, the fermenter was 50 L, the feeding volume was 35 L, the pH before disinfection was 7.2-7.6, and the steam sterilization was carried out at 121 ° C for 25 minutes. After cooling, about 3.5L seed tank culture solution is added, the fermentation temperature is 28 ⁇ 1°C, the stirring paddle rotation speed is 150 rpm, and the dissolved oxygen is not less than 35%. The aeration rate was 0.6 vvm, fermented for 14 days, and the can was placed.
  • the fermentation broth was determined by HPLC as shown in Example 5.
  • the content of milbemycin A4 in the fermentation broth was 1246 mg/L, and the A3 content was 3050 mg/L.
  • the content of milbemycin A3 as a percentage of the total content of A3 and A4 was 71%.
  • 8T seed medium was added to the 15T seed tank (see Table 5 for the ratio of seed medium, and 0.25% foaming agent was added as defoamer). Sterilization was carried out by steam sterilization at 121 °C for 35 minutes. After disinfection, the volume was 10T. After cooling, 2L shake flask seed solution was added, the culture temperature was 28 ⁇ 1 °C, the stirring speed was 100 rpm, the aeration amount was 0.8 vvm, and the culture was carried out for 48 hours.
  • the formulation of the fermentation medium was the same as that of the above Example 5, except that 0.3% of the foaming agent was added as the defoaming agent, the fermenter 70T, the feeding volume was 55T, the pH before disinfection was 7.2-7.6, and the steam sterilization was carried out at 121 ° C for 35 minutes. After cooling, about 6T seed tank culture solution is connected, the fermentation temperature is 28 ⁇ 1° C., the stirring paddle rotation speed is 50 rpm, and the dissolved oxygen is not less than 35%. The aeration rate was 0.5 vvm, fermented for 14 days, and the can was placed.
  • the fermentation broth was determined by HPLC as shown in Example 5.
  • the content of milbemycin A4 in the fermentation broth was 1413 mg/L, and the A3 content was 3300 mg/L.
  • the content of milbemycin A3 is 70% of the total content of A3 and A4.
  • the volume of the shake flask was 25 mL in a 250 mL triangular flask and sterilized at 121 ° C for 20 minutes.
  • a 1 ⁇ 2 cm area of the bacterium was excavated from the inclined surface of Example 5 and inserted into a seed bottle, and cultured at 28 ⁇ 1° C. for 45 hr.
  • yeast extract 10 molasses 200, skim milk powder 11, soy cake powder 11, cottonseed cake powder 11, dipotassium hydrogen phosphate 1, ferrous sulfate heptahydrate 0.1, zinc sulfate 0.02, calcium carbonate 5, copper sulfate 0.05, sodium molybdate 0.5, shake flask volume of 250mL triangle bottle 30mL, sterilized at 121 ° C for 20 minutes, shaken at 28 ⁇ 1 ° C, 250 rpm shaker for 14 days
  • the fermentation broth was determined by HPLC shown in Example 5, and the content of milbemycin A4 in the fermentation broth was 1054 mg/L, the content of A3 was 3000 mg/L, and the content of milbemycin A3 was determined. The percentage of total A3 and A4 content is 74%.
  • the preparation and culture of the seed liquid was the same as in Example 8.
  • 2.5 mL of seed liquid was taken into the fermentation medium (g/L): peptone 10, sucrose 140, corn syrup 10, soybean cake powder 10, gluten powder 15, and hydrogen phosphate II.
  • Potassium 1, ferrous sulfate heptahydrate 0.1, zinc sulfate 0.02, calcium carbonate 5, copper sulfate 0.05, sodium molybdate 0.5, shake flask volume 250 mL of a 250 mL triangular bottle was sterilized at 121 ° C for 20 minutes, shake cultured at 28 ⁇ 1 ° C, shaking at 250 rpm for 14 days.
  • the fermentation broth was determined by HPLC as shown in Example 5, and the mil in the fermentation broth was measured.
  • the content of betrimycin A4 was 1146 mg/L
  • the content of A3 was 3100 mg/L
  • the content of milbemycin A3 was 73% of the total content of A3 and A4.
  • Comparative Example 1 Comparative experiment of producing milbemycin with Streptomyces sp. HS7523 and original strain CGMCC No. 7677
  • Example 5 The composition and culture conditions of the slope, seed, and fermentation medium were the same as those in Example 5.
  • the strain was cultured with the original strain CGMCC No. 7677, and five groups of parallel fermentation cultures were carried out. After the fermentation, the fermentation broth was determined by HPLC and the milbean was measured.
  • the average content of mycin A4 was 748 mg/L
  • the average content of A3 was 251 mg/L
  • the content of milbemycin A3 was 25% of the total content of A3 and A4.
  • Example 5 Slope, seed, fermentation medium composition and culture conditions are the same as in Example 5, and the strain is subjected to five parallel fermentation cultures using the Streptomyces HS7523 of the present invention. After the fermentation, the fermentation broth is determined by HPLC, and the milbemycin is measured. The average content of A4 was 1114 mg/L, the average content of A3 was 3012 mg/L, and the content of milbemycin A3 was 73% of the total content of A3 and A4.

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Abstract

提供了一种链霉菌(Streptomyces hygroscopicus)HS7523，以及通过培养它制备米尔贝霉素A3的方法。链霉菌(Streptomyces hygroscopicus)HS7523于2014年9月16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"，保藏登记号为CGMCC No.9672。

Description

一种链霉菌及其生产米尔贝霉素A3的方法 技术领域
本发明涉及一种新的链霉菌、以及通过发酵培养该菌生产米尔贝霉素(milbemycin)A3的方法。
背景技术
米尔贝霉素是微生物天然产物农药，已有数据表明它是当今世界上最优良的杀螨剂之一。美国环保署认定其为低危险性杀虫剂，荷兰批准其为“GNO”(作物生产中的天然产物)。它属生态友好型农药，适用于有机农业病虫害的综合防治，在发达国家已成为一种受欢迎的杀虫杀螨剂。
米尔贝霉素是日本三共公司以二斑叶螨作为试虫，从微生物的发酵液中筛选出的具有抗虫活性的代谢产物(US3,950,360)。经过大量基础研究，1983年米尔贝霉素A3和A4组分的混合物(A3:A4＝3:7)被用作杀螨剂，米尔贝霉素A3和A4结构如式I所示。1990年，1％的米尔贝霉素乳油(milbeknock)在日本作为杀螨剂用于茶叶、茄子。1993年，1％的米尔贝霉素乳油用作梨、桃、西瓜、草莓、茄子、花卉的杀虫剂也在日本登记。当前，米尔贝霉素已在日本、欧美等多个国家登记，并且作为安全、对环境友好的杀虫杀螨剂被美国环保局推荐使用。
由于米尔贝霉素发酵液组分复杂，分离难度较大，因此米尔贝霉素通常情况下是以A3和A4混合组分使用和申报，单一组分的生产与使用则鲜有报导。其最主要的原因在于从A3，A4比例相近的发酵液中单独分离出A3，A4单组分难度较大，影响最终收率。以A3为例，发酵液中A3的含量低于30％，欲获得A3单组分，则需要损失大量的A4。实现A3或者A4的单一组分生产，可进一步推动米尔贝霉素单组分在其他领域的应用。而得到单组分最有效的方法是获得能够生产单一组分的新菌种。
Figure PCTCN2016077357-appb-000001
发明内容
本发明的目的之一在于提供一种能提高米尔贝霉素A3单位产量的微生物菌种，该菌种的特点在于其发酵液中米尔贝霉素A3组分的含量占米尔贝霉素A3和米尔贝霉素A4总含量的百分比大于70％，且米尔贝霉素A3的单位产量可以达到大于3000ug/ml，杂质含量低。
本发明的链霉菌HS7523的微生物菌种于2014年9月16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(地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所)，保藏号为CGMCC No.9672，分类命名为吸水链霉菌Streptomyces hygroscopicus，并登记入册，证明存活。
本发明的链霉菌HS7523主要生物学特征为：ISP1，ISP2，ISP3培养基上的菌落颜色白色，圆形，中间稍凸起，多皱，直径大小为中型(约6mm左右)，有少量孢子，基内菌丝发达，菌丝与培养基结合紧密，不易挑起。在ISP1，ISP2培养基上无色素产生，在ISP3培养基上有米褐色色素产生。
本发明描述了链霉菌HS7523的形态学和分子水平上的特征，经过和已知米尔贝霉素生产菌进行形态学和分子水平的比较，可以认定链霉菌HS7523属吸水链霉菌。链霉菌HS7523与Streptomyces sp.NRRL 5739 16s rRNA同源性99％，与Streptomyces bingchenggensis BCW-1同源性99％。链霉菌HS7523菌株与其它米尔贝霉素生产菌在形态上最大的差异在于其他菌种如Streptomyces sp.CGMCC No.7677的菌落在生产平板上会分泌金黄泪珠体于菌落表面，且有色素产生。而链霉菌HS7523在同样的培养基上颜色灰白，表面无泪珠，无色素产生(见图2)。
本发明还提供了一种采用链霉菌HS7523(CGMCC No.9672)制备米尔贝霉素的方法。该方法包括采用链霉菌HS7523(CGMCC No.9672)在含有可同化的碳、氮源的营养培养基里，进行有氧发酵的过程。
优选的实施方案中，上述可同化的碳源优选自淀粉、糊精、葡萄糖、工业糖蜜、甘油、蔗糖、乳糖、麦芽糖、海藻糖、木聚糖、甘露醇、山梨醇之一或上述物质的组合。
优选的实施方案中，上述可同化的氮源优选自酵母抽提物、酵母粉、牛肉浸膏、胰蛋白胨、蛋白胨、脱脂奶粉、全脂奶粉、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮、尿素、铵盐之一或上述物质的组合。
优选的实施方案中，所述营养培养基含有酵母抽提物、酵母膏或蛋白胨2-10g/L，蔗糖或糖蜜20-200g/L，脱脂奶粉或玉米浆2-11g/L，黄豆饼粉2-11g/L，棉籽饼粉或麸质粉5-15g/L，磷酸氢二钾0.5-1g/L，七水硫酸亚铁0.05-0.1g/L，硫酸锌0.005-0.02g/L，碳酸钙1-5g/L，硫酸铜0.01-0.05g/L和/或钼酸钠0.1-0.5g/L。
优选的实施方案中，所述发酵培养的温度优选为20℃-40℃，更优选25℃-35℃，培养基pH为6.0-8.0之间，优选7.0左右；培养时间为300-360小时；溶氧不低于35％；通气量为0.5-1.0vvm。
所述发酵方式为液体深层发酵。
米尔贝霉素可以通过以下方法进行检测：
取发酵液0.5ml，加入4.5ml 75％乙醇，混合均匀，3000rpm、15分钟离心，取上层液进样。
HPLC柱：Zorbex RX-C8；150mm*4.6mm；5μm
紫外吸收波长：240nm
温度控制：摄氏22度
HPLC流动相条件：如表9所示
进样量：10μl。
本发明采用的米尔贝霉素产生菌为链霉菌HS7523(CGMCC No.9672)、链霉菌HS7523(CGMCC No.9672)自发的突变株或通过常规的诱变得到的突变株。
本发明的主要优点在于：
1.本发明提供了一种生产米尔贝霉素的新菌链霉菌HS7523及其生产米尔贝霉素的方法。本发明菌链霉菌HS7523的特点在于其发酵液中米尔贝霉素A3的含量占米尔贝霉素A3和米尔贝霉素A4总含量的百分比大于70％，且米尔贝霉素A3的单位产量可以达到大于3000ug/ml，杂质含量低。
2.由于本发明所述的链霉菌HS7523通过发酵方法提高米尔贝霉素A3在发酵产物中的比例，降低了制备米尔贝霉素A3单一组分的难度，有利于降低生产成本和扩大米尔贝霉素A3单组分的应用范围。
3.提高了米尔贝霉素A3发酵单位。本发明所述的链霉菌HS7523生产米尔贝霉素A3的效价较原始菌Streptomyces milbemycinicus CGMCC No.7677大幅提高，有利于实现产业化生产。
附图说明：
图1：本发明链霉菌HS7523的选育图谱。
图2：本发明链霉菌HS7523菌株的菌落形态与链霉菌Streptomyces milbemycinicus CGMCC No.7677对比图，其中A为链霉菌Streptomyces milbemycinicus CGMCC No.7677菌落形态，B为本发明链霉菌HS7523菌落形态。
图3：米尔贝霉素A3，A4标准品HPLC图谱。
图4：实施例5发酵液HPLC图谱，米尔贝霉素A3的含量占A3和A4总含量的百分比为72％。
图5：本发明链霉菌HS7523在实施例6的50L发酵罐中生产米尔贝霉素A3和A4的发酵曲线。
图6：本发明链霉菌HS7523在实施例6条件下产生的米尔贝霉素A3的HPLC图谱，米尔贝霉素A4的含量为1246mg/L，A3含量为3050mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为71％。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
米尔贝霉素A3、A4标准品的制备参考美国专利US3950360实施例1。
蔗糖为广西东门南华糖业有限责任公司产品。
酵母抽提物为浙江东成药业有限公司产品。
酵母膏为合肥莱斯生物工程有限公司产品。
蛋白胨为湖州汇合生物科技有限公司产品。
糖蜜为广东江门生物科技有限公司产品。
脱脂奶粉为呼伦贝尔三元乳业有限责任公司产品。
玉米浆为山东寿光巨能金玉米开发有限公司产品。
黄豆饼粉为宁波市北仑江南油脂有限公司产品。
棉籽饼粉为北京康明威培养基技术有限责任公司产品。
麸质粉为北京康明威培养基技术有限责任公司产品。
实施例1：菌株来源
本发明链霉菌Streptomyces hygroscopicus HS7523系在米尔贝霉素产生菌Streptomyces milbemycinicus CGMCC No.7677(见中国专利CN103789339A，该菌的保藏人为本专利的申请人)的基础上，通过多轮的诱变选育(包括NTG，EMS，UV等诱变手段)得到的米尔贝霉素A3高产菌种。该菌种与原始菌种相比，其外观形态发生了较大的变化，属于形态突变株。
链霉菌Streptomyces milbemycinicus CGMCC No.7677菌株在ISP3斜面培养基中28℃培养10-12天后，在无菌条件下用接种铲将菌丝体刮下，在磨口试管中磨碎菌丝体并悬浮于无菌水中，制得菌悬液，采用NTG(亚硝基胍)，EMS(甲基磺酸乙酯)，UV(紫外线)诱变处理，具体方法如下：
称取NTG晶体10mg，溶解在10ml无菌Tris缓冲液(pH8.0)中，再用移液管加入1ml菌悬液，置于28℃培养基中旋转式或往复式摇床上振荡处理30min。将处理过的菌悬液经适当稀释涂布于ISP3平板上。未作诱变处理的菌悬液亦经适当稀释涂布于ISP3平板作为对照。28℃培养10天后，检查菌落数，并计致死率。
取10-1或10-2梯度的单细胞菌悬液5ml-10ml到带一根回形针的平板内(直径9cm)，置UV诱箱内，置磁力搅拌器上，开盖于UV15W，30cm处照射数分钟(一般2-5分钟)，边照边搅拌，照后用黑布包好，然后用生理盐水(0.9％氯化钠溶液)稀释10-2-10-7，分别涂ISP3平板得诱变组。
吸取1ml EMS溶于2ml无水乙醇中，再加入22ml，pH7.2的0.1mol/L磷酸缓冲液。取10-1或10-2梯度的单细胞菌悬液5ml到带一根回形针的平板内，加入5ml，4.0％EMS溶液，最后EMS溶液浓度为2.0％，置磁力搅拌器上，搅拌处理20分钟-60分钟。诱变平板内加入10ml 5％的硫代硫酸钠即可中止反应，用生理盐水依次稀释10-2-10-7，涂ISP3平板得诱变组。
挑选经过多轮上述单个或组合诱变后的单菌落不少于10000株，进行摇瓶发酵，HPLC检测米尔贝霉素的产量。经过图1所示的多轮诱变，筛选出突变菌株Streptomyces hygroscopicus HS7523。
实施例2：链霉菌HS7523菌种培养特征
参照《链霉菌鉴定手册》、《放线菌的分类与鉴定》、《常见细菌系统鉴定手册》中的有关内容进行实验。
采用ISP1、ISP2、ISP3、ISP4、ISP5、高氏一号、苹果酸钙、营养琼脂、YMS和查氏十种培养基，28℃培养7～10天后，观察菌丝体的颜色及色素情况(培养特征见表1)。
表1链霉菌HS7523在10种培养基上的培养特征
Figure PCTCN2016077357-appb-000002
Figure PCTCN2016077357-appb-000003
备注：表1中“/”为不产生色素。
实施例3：链霉菌HS7523生理生化试验
参照《链霉菌鉴定手册》、《放线菌的分类与鉴定》、《常见细菌系统鉴定手册》中的有关内容进行实验。除温度实验外，均为28℃培养7～10天。
1)碳源的利用：采用ISP9作为基础培养基，各种碳源的终浓度均为1.0％。结果见表2。
2)无机氮源的利用：采用ISP9作为基础培养基，硝酸钾和硫酸铵的浓度均为0.1％。结果见表2。
3)降解试验和NaCl耐受实验(结果见表7)采用基础培养基为GYEA(pH6.8)，各种降解物的浓度见表3。结果见表3。
4)氧化酶和过氧化氢酶试验(结果见表4)、pH试验(结果见表5)和温度试验(结果见表6)均采用YMS培养基。
表2链霉菌HS7523菌株的碳源和氮源的利用情况
碳源 生长情况 碳源 生长情况 无机氮源 生长情况
D-葡萄糖 4 水杨苷 4 硫酸铵 +
D-棉子糖 4 D-乳糖 3 硝酸钾 -
D-木糖 0 半乳糖 4    
D-山梨醇 2 肌醇 2    
L-阿拉伯糖 0 甘露醇 3    
甘油 4 甘氨酸 0    
麦芽糖 4 木聚糖 4    
D-果糖 1 菊粉 4    
D-蔗糖 4 鼠李糖 2    
表3链霉菌HS7523菌株的降解试验结果
降解物 降解物浓度 结果* 降解物 降解物浓度 结果
腺嘌呤 0.5％ 4，+ 酪蛋白 1.0％ 4，-
鸟嘌呤 0.5％ 4，- 酪氨酸 1.0％ 4，-
黄嘌呤 0.4％ 4，- Tween-40 1.0％ 2，+
木聚糖 0.4％ 4，- Tween-60 1.0％ 3，+
次黄嘌呤 0.4％ 4，- Tween-80 1.0％ 4，+
表4链霉菌HS7523菌株主要的生理生化特征
试验项目 结果 试验项目 结果 试验项目 结果
明胶液化 + 牛奶胨化 - 纤维素利用 -
淀粉水解 + 硝酸盐还原 + 过氧化氢酶 -
牛奶凝固 - 硫化氢产生 -    
V.P实验 - M.R实验 -    
表5链霉菌HS7523菌株生长的pH试验
pH 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
生长情况 0 0 4 4 4 4 4 4 4
表6链霉菌HS7523菌株生长的温度试验
温度(℃) 7 14 28 37 45
生长情况 0 3 3 2 0
表7链霉菌HS7523菌株对NaCl的耐受性
NaCl浓度 1％ 4％ 7％ 10％
菌株生长情况 3 0 0 0
备注：表2-7中：0：无生长；1：生长很弱；2：能生长，有少量孢子；3：生长良好，有大量孢子；4：生长最好，有丰富孢子；+：阳性；-：阴性。
实施例4：16S rDNA序列分析及与已知的米尔贝霉素产生菌的比较
收集在ISP2上生长好的本发明菌丝体，接于含玻璃珠的TSB液体培养基中，置于28℃的培养箱中，250r/min震荡培养2-4d。离心收集菌丝体，用无菌水洗涤2次后保存于4℃备用。
(1)菌丝体1000rpm离心1min。
(2)加终浓度为3-4mg/ml的溶菌酶溶液(菌丝体体积:溶菌酶溶液体积＝1:5-10)，37℃水浴1-3hr。
(3)加50-100ug/ml蛋白酶K及1％SDS，37℃水浴0.5-3h。
(4)加等体积的中性酚/氯仿，振荡30sec，12000rpm离心5min。
(5)上清加1/10体积的3M NaAc溶液和等体积的异丙醇，混匀。室温放置5min，12000rpm离心5min。
(6)沉淀用70％乙醇洗涤2遍，干燥后溶于TE/RNase。
(7)将提取的基因组作为模板，采用通用引物进行PCR扩增。
上游引物27F为5’-GAGAGTTTGATCCTGGCTCAG-3’(SEQ ID No.1)
下游引物1495R为5’-CTACGGCTACCTTGTTACGA-3’(SEQ ID No.2)
反应在PCR扩增仪上进行。其程序是：95℃预变性5min，94℃变性45s，55℃复性45s，72℃延伸90s，进行30个循环，72℃延伸10min。反应体系如下：
Figure PCTCN2016077357-appb-000004
用0.8％琼脂糖凝胶电泳检测PCR产物。选用条带清晰的产物进行纯化。扩增产物经凝胶电泳回收，并连接到T载体后进行测序，获得了该菌株16S rDNA一级结构的序列。通过在Genebank数据库中进行相似性搜索(blast)，结果显示与Streptomyces sp.NRRL 5739 16s rRNA同源性99％，与Streptomyces bingchenggensis BCW-1同源性99.5％，结果见表8。
表8链霉菌HS7523和相关菌株的同源性
Figure PCTCN2016077357-appb-000005
链霉菌HS7523与已知的米尔贝霉素产生菌的比较
据US3950360报道，米尔贝霉素产生菌Streptomyces NRRL NO.5739在ISP2培养基上气生菌丝灰色，背面黄褐色，菌落表面有许多黄色泪珠，有黄色色素产生；在ISP4培养基上气生菌丝灰色，背面土黄，菌落表面也有许多黄色泪珠，有明亮的橄榄绿色素产生，能够利用阿拉伯糖和木糖。而本发明链霉菌HS7523在SP2培养基上气生菌丝白色，背面米色，菌落表面无泪珠，无色素产生；在ISP4培养基上气生菌丝白色，背面象牙色，菌落表面无泪珠，无色素产生，不能利用阿拉伯糖和木糖。
据CN101100651A报道，米尔贝霉素产生菌冰城链霉菌(streptomyces bingchengsis sp.nov)CGMCC No.1734 在高氏一号培养基上菌落表面灰色(有黑色吸水斑)，菌落背面黄灰色，有黄褐色色素产生。而本发明链霉菌HS7523在高氏一号培养基上菌落表面中间白，周围电视灰4，菌落背面电视灰4，无色素产生。
综上所述，本发明链霉菌HS7523属于链霉菌属，但它不同于已知的米尔贝霉素产生菌Streptomyces NRRL NO.5739，菌冰城链霉菌(streptomyces bingchengsis sp.nov)CGMCC No.1734，链霉菌HS7523是一株全新的菌种。
本发明链霉菌HS7523菌株的菌落形态与链霉菌Streptomyces milbemycinicus CGMCC No.7677对比图如图2所示。
实施例5：制备米尔贝霉素A3
(1)斜面菌丝体的制备与培养
斜面孢子培养基配方(g/L)：酵母抽提物2，麦芽抽提物2，蔗糖8，脱脂奶粉1，琼脂20，消毒前pH 7.0-7.2，试管30×200mm，装量20mL，经121℃灭菌20分钟后， 冷却到50-60℃摆斜面，接入一环菌丝体经28±1℃培养10天后，菌丝成熟。
(2)种子液的制备与培养
种子培养基配方(g/L)：酵母抽提物5，蛋白胨5，蔗糖20，脱脂奶粉2，磷酸氢二钾0.5，消毒前pH7.0-7.2，摇瓶装液量为250mL三角瓶装30mL，经121℃灭菌20分钟。菌体接种量为105-106c.f.u./mL，培养温度28±1℃，250rpm，摇床振荡培养48小时。
(3)米尔贝霉素A3发酵培养基的制备与培养
发酵培养基配方(g/L)：酵母抽提物5，蔗糖120，脱脂奶粉10，黄豆饼粉10，棉籽饼粉14，磷酸氢二钾1，七水硫酸亚铁0.1，硫酸锌0.02，碳酸钙5，硫酸铜0.05，钼酸钠0.5，摇瓶装液量为250mL三角瓶装30mL，经121℃灭菌20分钟。将种子液以10％(体积百分比)的接种量接入，在28±1℃，250rpm摇床振荡培养14天，发酵结束后，发酵液经HPLC测定。
米尔贝霉素的HPLC检测方法如下：
取发酵液0.5ml，加入4.5ml 75％乙醇，混合均匀，3000rpm、15分钟离心，取上层液进样。
HPLC柱：Zorbex RX-C8；150mm*4.6mm；5μm
紫外吸收波长：240nm
温度控制：摄氏22度
HPLC流动相条件如下：
表9梯度洗脱
Figure PCTCN2016077357-appb-000006
进样量：10μl。
米尔贝霉素A3、A4标准品同样条件下的HPLC图谱如图3所示(下述实施例的发酵液的HPLC检测均包含米尔贝霉素A3、A4标准品的HPLC检测步骤)。
发酵液的HPLC图谱如图4所示。
经过与米尔贝霉素A3、A4标准品同样条件下的HPLC图谱进行目的产物保留时间的比对，确定发酵液中的目的产物为米尔贝霉素A3、A4(下述实施例的发酵液的HPLC图谱同样经过该比对，确定目的产物为米尔贝霉素A3、A4)。
测得发酵液中的米尔贝霉素A4的含量为1206mg/L，A3含量为3100mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为72％。
实施例6：制备米尔贝霉素A3
(1)种子罐种子液的制备
在15L种子罐中投入10L的种子培养基(种子培养基的配比见实施例5，同时添加0.25％的泡敌作消泡剂)，灭菌采用蒸汽灭菌，121℃条件下30分钟，待冷却后，接入200ml摇瓶种子液，培养温度28±1℃，搅拌转速150rpm，通气量1vvm，培养48小时。
(2)发酵罐培养基的配制与培养
发酵培养基的配方与前述实施例5相同，但要添加0.25％泡敌作为消泡剂，发酵罐50L，投料体积为35L，消毒前pH7.2-7.6，于121℃下蒸汽灭菌25分钟，冷却后，接入约3.5L种子罐培养液，发酵温度28±1℃，搅拌桨转速底限150rpm，与溶氧关联，溶氧不低于35％。通气量为0.6vvm，发酵培养14天，放罐，发酵液经实施例5中所示的HPLC测定，测得发酵液中米尔贝霉素A4的含量为1246mg/L，A3含量为3050mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为71％。
发酵罐中生产米尔贝霉素A3和A4的发酵曲线如图5所示。
发酵液中米尔贝霉素A3(F075-A3)的HPLC图谱如图6所示。
实施例7：制备米尔贝霉素A3
(1)种子罐种子液的制备
在15T种子罐中投入8T的种子培养基(种子培养基的配比见实施例5，同时添加0.25％的泡敌作消泡剂)，灭菌采用蒸汽灭菌，121℃条件下35分钟，消毒后体积10T，待冷却后，接入2L摇瓶种子液，培养温度28±1℃，搅拌转速100rpm，通气量0.8vvm，培养48小时。
(2)发酵罐培养基的配制与培养
发酵培养基的配方与前述实施例5相同，但要添加0.3％泡敌作为消泡剂，发酵罐70T，投料体积为55T，消毒前pH7.2-7.6，于121℃下蒸汽灭菌35分钟，冷却后，接入约6T种子罐培养液，发酵温度28±1℃，搅拌桨转速底限50rpm，与溶氧关联，溶氧不低于35％。通气量为0.5vvm，发酵培养14天，放罐，发酵液经实施例5中所示的HPLC测定，测得发酵液中米尔贝霉素A4的含量为1413mg/L，A3含量为3300mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为70％。
实施例8：制备米尔贝霉素A3
种子培养基配方(g/L)：酵母膏10，蛋白胨5，蔗糖40，脱脂奶粉2，磷酸氢二钾0.5，消毒前pH7.0-7.2。摇瓶装液量为250mL三角瓶装25mL，经121℃灭菌20分钟。从实施例5中的斜面上挖取1×2cm面积的菌块接入种子瓶中，28±1℃下培养45hr。取上述种子培养液2.5mL接入发酵培养基(g/L)：酵母膏10，糖蜜200，脱脂奶粉11，黄豆饼粉11，棉籽饼粉11，磷酸氢二钾1，七水硫酸亚铁0.1，硫酸锌0.02，碳酸钙5，硫酸铜0.05，钼酸钠0.5，摇瓶装液量为250mL三角瓶装30mL，经121℃灭菌20分钟，在28±1℃，250rpm摇床振荡培养14天，发酵结束后，发酵液经实施例5中所示的HPLC测定，测得发酵液中米尔贝霉素A4的含量为1054mg/L，A3含量为3000mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为74％。
实施例9：制备米尔贝霉素A3
种子液的制备与培养同实施例8，取2.5mL种子液接入发酵培养基(g/L)：蛋白胨10，蔗糖140，玉米浆10，黄豆饼粉10，麸质粉15，磷酸氢二钾1，七水硫酸亚铁0.1，硫酸锌0.02，碳酸钙5，硫酸铜0.05，钼酸钠0.5，摇瓶装液量为 250mL三角瓶装30mL，经121℃灭菌20分钟，在28±1℃，250rpm摇床振荡培养14天，发酵结束后，发酵液经实施例5中所示的HPLC测定，测得发酵液中米尔贝霉素A4的含量为1146mg/L，A3含量为3100mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为73％。
对比实施例1：本发明链霉菌HS7523与原始菌CGMCC No.7677生产米尔贝霉素的对比实验
(1)斜面，种子，发酵培养基组成和培养条件同实施例5，菌种采用原始菌CGMCC No.7677，进行五组平行发酵培养，发酵结束后，发酵液经HPLC测定，测得米尔贝霉素A4的平均含量为748mg/L，A3的平均含量为251mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比25％。
(2)斜面，种子，发酵培养基组成和培养条件同实施例5，菌种采用本发明链霉菌HS7523，进行五组平行发酵培养，发酵结束后，发酵液经HPLC测定，测得米尔贝霉素A4的平均含量为1114mg/L，A3的平均含量为3012mg/L，米尔贝霉素A3的含量占A3和A4总含量的百分比为73％。

Claims (10)

  1. 一种链霉菌(Streptomyces hygroscopicus)HS7523，其保藏编号为CGMCC No.9672且具有生产米尔贝霉素的能力。
  2. 根据权利要求1所述的链霉菌HS7523在生产米尔贝霉素中的应用。
  3. 一种发酵生产米尔贝霉素的方法，其特征在于：包括采用链霉菌HS7523在含有可同化的碳源和氮源的营养培养基里，进行有氧发酵的步骤。
  4. 根据权利要求3所述的方法，其特征在于：所述可同化的碳源选自淀粉、糊精、葡萄糖、工业糖蜜、甘油、蔗糖、乳糖、麦芽糖、海藻糖、木聚糖、甘露醇、山梨醇之一或上述物质的组合。
  5. 根据权利要求3所述的方法，其特征在于：所述可同化的氮源选自酵母抽提物、酵母粉、牛肉浸膏、胰蛋白胨、蛋白胨、脱脂奶粉、全脂奶粉、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮、尿素、铵盐之一或上述物质的组合。
  6. 根据权利要求3-5任一项所述的方法，其特征在于：其中所采用的米尔贝霉素产生菌为链霉菌HS7523(CGMCC No.9672)、链霉菌HS7523(CGMCC No.9672)自发的突变株或通过常规的诱变得到的突变株。
  7. 根据权利要求3-5任一项所述的方法，其特征在于：所述营养培养基含有酵母抽提物、酵母膏或蛋白胨2-10g/L，蔗糖或糖蜜20-200g/L，脱脂奶粉或玉米浆2-11g/L，黄豆饼粉2-11g/L，棉籽饼粉或麸质粉5-15g/L，磷酸氢二钾0.5-1g/L，七水硫酸亚铁0.05-0.1g/L，硫酸锌0.005-0.02g/L，碳酸钙1-5g/L，硫酸铜0.01-0.05g/L和/或钼酸钠0.1-0.5g/L。
  8. 根据权利要求3所述的方法，其特征在于：所述发酵培养的温度为20℃-40℃，优选25℃-35℃；培养基pH为6.0-8.0，优选7.0；培养时间为300-360小时。
  9. 根据权利要求3-5任一项所述的方法，其特征在于：所述发酵时的溶氧不低于35％。
  10. 根据权利要求3-5任一项所述的方法，其特征在于：所述发酵时的通气量为0.5-1.0vvm。
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