CN1367255A - Fermentation process for raising ebomycin A yield - Google Patents

Abstract

The invention discloses a method for increasing the output of secondary metabolites of myxobacteria, that is, a method for increasing the output of metabolite Epothilone A of Epothilone-producing bacteria Sorangium cellulosum in myxobacteria . The method includes 1) a domestication method for the uniform growth of myxobacteria growing in clusters, 2) selection of sweet potato starch, hydrolyzed milk protein, etc. suitable for epothilone A production, and 3) a mixed method for comprehensively eliminating the metabolic accumulation of products. Resin addition method and other steps, so that the yield of epothilone A product reached the level of 62.7mg/L.

Description

A kind of fermentation technology method that improves epothilone A output

(1) Technical field

The invention relates to a method for increasing the yield of metabolites of myxobacteria, in particular to a method for increasing the yield of epothilone A of S. cellulosum in myxobacteria.

(2) Background technology

At present, the most clinically successful anti-tumor chemotherapy drug is the microtubule polymerization-promoting natural compound paclitaxel (Taxol ^® ) and its analogue docetaxel (Taxotere ^® ), which are used for ovarian cancer, thymus cancer, colon cancer, lung cancer and liver cancer and other solid cancer treatment. The success of the inducible anti-tumor drug paclitaxel and its shortcomings in chemotherapy (low water solubility and cell drug resistance during chemotherapy, etc.) prompted researchers to further screen for drugs with better chemical, biological and pharmacological properties. microtubule stabilizer. However, after a long time and a lot of screening, it was not until recent years that four new natural compounds were discovered. Among them, in 1993, Hoeffler et al. reported that new 16-membered macrolide compounds Epothilone (Epothilone) A and B were isolated from the myxobacteria Sorangium cellulosum, and in 1995, Bolager After discovering the microtubule polymerization-promoting activity of epothilone in the large-scale screening of natural compounds with microtubule polymerization-promoting activity, it has attracted widespread attention. Like paclitaxel, epothilone induces tubulin to form microtubules at low temperature and in the absence of guanosine triphosphate or microtubule-associated proteins. Unlike paclitaxel, epothilones remain active against p-glycoprotein-expressing multidrug-resistant cell lines and tumors. On the other hand, the endotoxin-like activity of paclitaxel may cause non-hematological side effects in clinical chemotherapy, while epothilone does not trigger the endotoxin signaling pathway of cells. In addition, studies have also shown that microtubule stabilizers such as paclitaxel or epothilone A also have antimalarial activity, which can hinder the formation of merozoites.

Compared with paclitaxel, epothilone has a simpler structure, good water solubility and great medicinal potential, so that people have invested great enthusiasm in research, in order to develop it into an anti-tumor drug more quickly. Chemists have put a lot of energy into the synthesis and separation of epothilones and their analogues, but for the preparation of epothilones by biological fermentation, only the method of Goth et al. (Journal of Antibiotics, 1996, 49: 560 -563). The method comprises the So ce90 bacterial strain capable of producing epothilone, the fermentation vessel is a 300ml Erlenmeyer flask, and the fermentation conditions are: potato starch 8g/L, glucose 2g/L, defatted soybean protein 2g/L, yeast extract 2g/L, FeNaEDTA 8mg/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, HEPES 11.5g/L, add 20ml/L resin XAD-16. After inoculation, culture was carried out at 30° C. for 5 days. The yield of epothilone A is about 23mg/L.

The deficiency of the above-mentioned fermentation technology method is: 1) due to the agglomeration properties of myxobacteria growing in liquid, the improvement of the number of thalline cells is affected, thereby limiting the production of myxobacteria metabolite epothilones; 2) producing epothilones The fermentation conditions of bleomycin are too complicated and the cost is high, and potato starch, glucose, defatted soybean protein and yeast extract are not the most suitable carbon and nitrogen sources for bleomycin production; 3) to reduce the metabolism of myxobacteria Feedback inhibition of epothilone on the product, the XAD-16 resin added during the fermentation process is expensive, and this resin can only adsorb low-polarity metabolites, for other medium-polar or high-polarity metabolites Feedback inhibition has no effect, etc.

For above-mentioned deficiencies, the document or the patent of improving related technology and method have not been reported so far.

(3) Contents of the invention

The purpose of the present invention is to provide a method for improving the yield of epothilone A, which can effectively overcome the defects of the existing production process, and has the advantages of producing epothilones. The characteristics of large growth, simple culture conditions and low price provide a basis for the application in industrial production.

The object of the present invention is achieved through the following technical solutions, and the sequence of specific steps is as follows: (1) selection of fermentation strains: one of Sorangium cellulosum ATCC15384, ATCC25531 and ATCC25569 bacterial strains in myxobacteria strains; On the premise of maintaining the ability to synthesize metabolites, the myxobacteria strains can be domesticated in liquid culture for uniform growth: the above strains are inoculated on a solid slant medium, and the composition of the solid medium is: soybean peptone 5-15g/L, potato starch 15-25g/L L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, Agar powder 15g/L; The components of the fermentation medium are: soybean peptone 5~15g/L, potato starch 15~25g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L; 30℃ , 120 rpm shaker culture for 5-7 days, the above-mentioned cultured bacteria were reconnected to the solid medium for culture, cultivated according to the original conditions and then inoculated into the liquid fermentation medium, so repeated subculture and domestication, in 95-100 generations , the growth of the cultured strains in the liquid fermentation medium showed adaptive homogeneous growth, showing that the bacterial cells changed from the original aggregated state to the uniform growth state in the liquid, and the generation time of cell growth was shortened, and the growth The time to reach the maximum cell volume was shortened from 10 to 12 days to 5 to 7 days, and the maximum cell volume was increased from 1 to 4×10 ⁸ cells/ml to 5 to 10×10 ⁹ Cells/ml; (3) Optimization of fermentation culture conditions: inoculate the homogenized growth strain in step (2) into a solid medium, the composition of the solid medium is the same as above, at 30°C, after 5 days of cultivation, inoculate into a liquid fermentation culture containing resin base, the composition of the liquid fermentation medium is: the carbon source is one of sweet potato starch, potato starch, oligodextran, and xylan, and the concentration is 20g/L; the nitrogen source is hydrolyzed milk protein, soybean peptone, ammonium tartrate, One of ammonium citrate, the concentration is 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, the resin is XAD-16, and the addition amount is 20ml /L; the fermentation vessel is a 500ml Erlenmeyer flask containing 200ml liquid fermentation medium, the culture temperature is 30°C, the shaker speed is 120 rpm, and it is cultivated for 5 to 7 days; Influence of bleomycin: Inoculate the above-mentioned strains into a solid medium with the same composition as above, at 30°C, after culturing for 5 days, inoculate into a liquid fermentation medium containing resin, and the composition of the liquid fermentation medium is sweet potato starch 20g/L , hydrolyzed milk protein 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, choose one of XDA, AB-8 or mixed resin for the resin, the addition amount is 10-40ml/L; the fermentation container is a 500ml Erlenmeyer flask, containing 200ml liquid fermentation medium, the culture temperature is 30°C, the shaker speed is 120 rpm, and the culture is 6-7 days; (5) Epothilone A Separation, extraction and purification: after the fermentation, the resin added in the liquid fermentation medium was taken out, leached with methanol, and the extract was subjected to ion exchange, molecular sieve and high performance liquid phase extraction and purification to obtain epothilone A.

Wherein the fermentation strain described in the step (1) is selected from Sorangium cellulosum (Sorangium cellulosum) ATCC15384.

Wherein the solid medium composition described in step (2) is: soybean peptone 8g/L, potato starch 20g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg /L, agar powder 15g/L.

Wherein the liquid fermentation medium composition described in step (2) is: soybean peptone 10g/L, potato starch 21g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L.

Wherein the carbon source in the composition of the liquid fermentation medium described in step (3) is sweet potato starch.

Wherein the nitrogen source in the composition of the liquid fermentation medium described in step (3) is hydrolyzed milk protein.

Wherein the addition amount of the mixed resin described in step (4) is 20ml/L.

Wherein step (4) described mixed resin refers to CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 by weight ratio 0.8～1.2: 0.8～1.2: 1.8～2.2: 0.8～ 1.2: 2.8-3.2: 1.8-2.2 mixed.

Wherein the mixed resin in step (4) refers to the six resins CD180, CAD-40, XDA, S-8, NKA-II, and AB-8 mixed in a weight ratio of 1:1:2:1:3:2.

Take out the mixed resin added in the fermentation broth, add 10 times the volume of methanol to extract, and the extract is purified by ion exchange, molecular sieve and high performance liquid phase. The compound that purification obtains has measured mass spectrometry (see accompanying drawing 1) respectively, and ultraviolet spectrogram (seeing accompanying drawing 2) and NMR spectrogram (seeing accompanying drawing 3 and 4) have confirmed Epothilone (Epothilone) A structure (accompanying drawing 5).

The results in Table 1 show the epothilone A production of the strain after liquid uniform growth and acclimatization. The creativity of this technology lies in: 1) through the uniform growth and domestication method of the liquid, the agglomeration property of the growth of myxobacteria is changed, thereby increasing the growth cell density of the bacteria, and the growth generation time of the bacteria cells is shortened, shortening the fermentation cycle; 2 ) The method uses the fermentation medium as the domestication growth medium, so that the strains grown evenly in the liquid have better adaptability to the fermentation medium, and avoid the situation that the domestication strains lose the ability to produce epothilones. The result obtained by this technology is that the yield of epothilone A produced by the uniformly grown strain is three times that of the wild strain grown in clumps. Table 1

  Strain                                                                                              

Table 2 is the effect of different carbon sources on the epothilone A synthesis of liquid uniform growth acclimatized strains. Among them, sweet potato starch, potato starch, oligodextran, xylan, glucose, etc. can significantly promote the production of epothilone A. Table 2

Concentration Epothilone A Concentration Epothilone A

carbon source carbon source

(g/L) (mg/L) (g/L) (mg/L) sweet potato starch 20 49.5 glucose 8 33.6 Xylan 20 36.3 Oligodextran 20 42.9 Potato Starch 20 45.0 Soluble Starch 20 19.2 Xylose 08 18.9 Mannose 08 6.0 Arabinose 8 24.0 Maltose 8 8.4 Cellobiose 8 22.2 CF11 Cellulose Powder 20 0 Chitin 20 0 Inulin 20 0

Table 3 is the effect of different nitrogen sources on the epothilone A synthesis of liquid uniform growth acclimatized strains. Among them, hydrolyzed milk protein, soybean peptone, ammonium tartrate, ammonium citrate, skimmed milk powder, etc. can significantly promote the production of epothilone A. table 3 Nitrogen source concentration (g/L) Epothilone A (mg/L) Nitrogen source concentration (g/L) Epothilone A (mg/L) Skimmed milk powder 10 35,2 Casein peptone 10 11.5 Fish meal peptone 10 13.2 Soy peptone 10 43.3 Hydrolyzed milk protein 10 55.5 Beef extract 10 14.4 Ammonium carbonate 10 0 Ammonium nitrate 10 15.7 Ammonium tartrate 10 42.4 Ammonium tartrate 05 33.7 Ammonium citrate 10 39.8 Urea 10 8.5

The yield of sweet potato starch and hydrolyzed milk protein obtained from the results of Table 2 and Table 3 is 15-25% higher than that of literature culture medium. The creativity of this technology lies in that the obtained culture medium conditions are higher than the culture medium conditions in the literature to produce higher yields of epothilone A, and at the same time, the sources of the culture medium components are cheaper and easier to obtain, which lays the foundation for industrial fermentation.

Table 4 is the influence of different resins on the synthesis of epothilone A during fermentation. The results showed that these homemade resins could effectively adsorb epothilone A, among which XDA, AB-8 and several resins mixed in equal proportions had a better effect. Table 4 Resin dosage Epothilone A (ml/L) (mg/L) Resin dosage Epothilone A (ml/L) (mg/L) CD180 20 15.1CAD-40 20 0XDA 20 55.5 mixed resin 20 62.7 S-8 20 20.0NKA-II 20 12.3AB-8 20 45.5 Control XAD-16 20 53.6

Table 5 is the effect of the amount of mixed resin added on the synthesis of epothilone A. Among them, the effect of 10ml/L to 40ml/L is better.

table 5

  Mixed Resin (ml/L)     Epothilone A (mg/L)

0 12.5

10 43.7

20 63.2

40 37.7

70 31.0

The results of Table 4 and Table 5 not only obtained the equivalent or higher epothilone A yield with XAD-16, but also the resins used are cheap domestic resins, thereby reducing production costs and thus having better industrialization feasibility. In addition, the idea of using mixed resins not only reduces the feedback inhibition of epothilone on the metabolic pathway, but also reduces the feedback inhibition of other metabolites on the metabolism of the producing bacteria, thus improving the metabolic capacity of the bacteria as a whole , and provide the possibility to further increase the production of epothilone A.

(4) Description of drawings: the mass spectrogram of the epothilone A that Fig. 1 purifies

As shown in the figure, the sample is purified epothilone A, the (M+H) ⁺ of the component is 494.2561, the molecular formula of the fitted compound is C ₂₆ H ₃₉ O ₆ NS, and the relative deviation is 1.922e-06 ; in perfect agreement with the theoretical value of epothilone A. Fig. 2 The UV spectrum of the purified epothilone A Fig. 3 The PMR spectrum of the purified epothilone A Fig. 4 The ¹³ C-NMR spectrum of the purified epothilone A Fig. 5 Epothilone Chemical Structure of Epothilone A

(5) Specific implementation methods

Example 1: (1) Fermentation strain Sorangium cellulosum (Sorangium cellulosum) ATCC15384 among myxobacteria strains was selected. (2) Under the premise of maintaining the synthesis ability of metabolites, the myxobacteria strains are domesticated by liquid culture and uniform growth: the above-mentioned strains are inoculated on a solid slant medium, and the solid medium components are: soybean peptone 8g/L, potato starch 20g/L , CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, agar powder 15g/L; 30°C, after 5 days of cultivation, inoculated into liquid fermentation medium, liquid fermentation The medium components are: soybean peptone 10g/L, potato starch 20g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L; 30°C, 120 rpm Cultivate in a shaking table for 7 days, reconnect the above-mentioned cultured bacteria to the solid medium for cultivation, cultivate them according to the original conditions and then inoculate them into the liquid fermentation medium. The growth in the medium showed adaptive homogeneous growth, which showed that the bacterial cells changed from the original aggregated state to the uniform growth state in the liquid, and the time to reach the maximum cell mass was shortened from the original 11 days to 6 days. The maximum cell amount increased from the original 4×10 ⁸ cells/ml to 10×10 ⁹ cells/ml; (3) Optimization of fermentation culture conditions: Inoculate the homogenized strains grown in step (2) into solid medium, The composition of the solid medium is the same as above, 30°C, after 5 days of cultivation, inoculate into the liquid fermentation medium containing resin, the composition of the liquid fermentation medium is: the carbon source is sweet potato starch, the concentration is 20g/L, and the nitrogen source is hydrolyzed milk protein , the concentration is 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, the resin is D-16, the addition amount is 20ml/L; the fermentation vessel is 500ml Erlenmeyer flask, containing 200ml liquid fermentation medium, culture temperature is 30 ℃, shaking table speed is 120 rev/min, cultivates for 7 days; (4) comprehensively eliminates the influence of metabolites on bacterial strain producing epothilone: combine the above The strain was inoculated into a solid medium, the composition of which was the same as above, at 30°C, and after 5 days of cultivation, it was inoculated into a liquid fermentation medium containing resin. The composition of the liquid fermentation medium was sweet potato starch 20g/L, hydrolyzed milk protein 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, resin selection mixed resin, the addition amount is 20ml/L; mixed resin consists of CD180, CAD-40, XDA, S -8, NKA-II, AB-8 six kinds of resins are mixed by weight ratio 1: 1: 2: 1: 3: 2, and the fermentation vessel is a 500ml triangular flask containing 200ml liquid fermentation medium, and the cultivation temperature is 30 ℃, shaker speed is 120 rev/min, cultivated for 7 days; (5) Separation, extraction and purification of epothilone A: after the fermentation, the resin added in the liquid fermentation medium was taken out, and 10 times the volume of methanol was added to extract , the extract was purified by ion exchange, molecular sieve and high-performance liquid phase extraction, and the yield of epothilone A was 66.7mg/L.

Example 2: (1) Sorangium cellulosum (Sorangium cellulosum) ATCC15384 among the myxobacteria strains was selected for fermentation. (2) Under the premise of maintaining the synthesis ability of metabolites, the myxobacteria strains are domesticated by liquid culture and uniform growth: the above-mentioned strains are inoculated on a solid slant medium, and the solid medium components are: soybean peptone 10g/L, potato starch 22g/L , CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, agar powder 15g/L; 30°C, after 5 days of cultivation, inoculated into liquid fermentation medium, liquid fermentation The medium components are: soybean peptone 12g/L, potato starch 23g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L; 30°C, 120 rpm Cultivate in a shaking table for 5 days, reconnect the above-mentioned cultured bacteria to the solid medium for cultivation, cultivate them according to the original conditions, and then inoculate them into the liquid fermentation medium, repeat the subculture and acclimatization in this way, and at the 96th generation, the cultured strains were cultured in the liquid fermentation culture. The growth in the medium showed adaptive homogeneous growth, which showed that the bacterial cells changed from the original aggregated state to the uniform growth state in the liquid, and the time to reach the maximum cell mass was shortened from the original 10 days to 5 days. The maximum cell amount increased from 2×10 ⁸ cells/ml to 7×10 ⁹ cells/ml; (3) Optimization of fermentation culture conditions: inoculate the homogenized strains grown in step (2) into solid medium, The composition of the solid medium is the same as above, at 30°C, after cultivating for 5 days, inoculate into the liquid fermentation medium containing resin, the composition of the liquid fermentation medium is: the carbon source is potato starch, the concentration is 20g/L, the nitrogen source is soybean peptone, The concentration is 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, the resin is D-16, the addition amount is 20ml/L; the fermentation vessel is 500ml Erlenmeyer flask, containing 200ml liquid fermentation medium, culture temperature is 30 ℃, shaker speed is 120 rev/min, cultivate 5 days; (4) comprehensively eliminate the influence of metabolites on bacterial strain producing epothilone: the above bacterial strain Inoculate into a solid medium with the same composition as above, at 30°C. After culturing for 5 days, inoculate into a liquid fermentation medium containing resin. The components of the liquid fermentation medium are sweet potato starch 20g/L, hydrolyzed milk protein 10g/L, CaCl _{2.2H 2} O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, choose XDA as the resin, add 20ml/L; the fermentation vessel is a 500ml Erlenmeyer flask containing 200ml liquid fermentation medium , the culture temperature is 30°C, the shaker speed is 120 rev/min, and cultivated for 6 days; (5) Separation, extraction and purification of epothilone A: after the fermentation, the resin added in the liquid fermentation medium is taken out, and 10 Double the volume of methanol leaching, the extract was purified by ion exchange, molecular sieve and high performance liquid phase extraction, and the yield of epothilone A was 66.3mg/L.

Example 3: (1) Fermentation strain Sorangium cellulosum (Sorangium cellulosum) ATCC15384 among myxobacteria strains was selected. (2) Under the premise of maintaining the synthesis ability of metabolites, the myxobacteria strains are acclimated to uniform growth in liquid culture: inoculate the above-mentioned strains on a solid slant medium, and the components of the solid medium are: soybean peptone 6g/L, potato starch 17g/L , CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, agar powder 15g/L; 30°C, after 5 days of cultivation, inoculated into liquid fermentation medium, liquid fermentation The medium components are: soybean peptone 7g/L, potato starch 17g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L; 30°C, 120 rpm Cultivate in a shaking table for 7 days, reconnect the above-mentioned cultured bacteria to the solid medium for cultivation, cultivate them according to the original conditions, and then inoculate them into the liquid fermentation medium, repeat the subculture and acclimatization in this way, and at the 98th generation, the cultured strains will be cultured in the liquid fermentation culture. The growth in the medium showed adaptive homogeneous growth, which showed that the bacterial cells changed from the original aggregated state to the uniform growth state in the liquid, and the time to reach the maximum cell mass was shortened from the original 12 days to 7 days. The maximum cell amount increased from 2×10 ⁸ cells/ml to 5×10 ⁹ cells/ml; (3) Optimization of fermentation culture conditions: inoculate the homogenized strains grown in step (2) into solid medium, The composition of the solid medium is the same as above, 30°C, after culturing for 5 days, inoculate into the liquid fermentation medium containing resin, the composition of the liquid fermentation medium is: the carbon source is dextran oligosaccharide, the concentration is 20g/L, and the nitrogen source is tartaric acid Ammonium, the concentration is 10g/L, CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, resin is D-16, the addition amount is 20ml/L; fermentation vessel It is a 500ml Erlenmeyer flask containing 200ml liquid fermentation medium. The culture temperature is 30°C, the shaking table speed is 120 rpm, and cultivated for 7 days; (4) comprehensively eliminate the influence of metabolites on the production of epothilones by The above-mentioned strains were inoculated into a solid medium with the same composition as above, at 30°C, cultured for 5 days, and then inoculated into a liquid fermentation medium containing resin. The composition of the liquid fermentation medium was sweet potato starch 20g/L, hydrolyzed milk protein 10g/L , CaCl ₂ .2H ₂ O 1g/L, MgSO ₄ .7H ₂ O 1g/L, Fe-EDTA 8mg/L, resin selection AB-8, the addition amount is 20ml/L; the fermentation vessel is a 500ml Erlenmeyer flask containing 200ml Liquid fermentation medium, culture temperature is 30 ℃, shaker speed is 120 rev/min, cultivate for 5 days; (5) Separation, extraction and purification of epothilone A: after the fermentation, the resin added in the liquid fermentation medium Take it out, add 10 times the volume of methanol to extract, and the extract is purified by ion exchange, molecular sieve and high-performance liquid phase extraction, and the yield of epothilone A is 65.4mg/L.

Claims (9)

1. fermentation technology that improves ebomycin A yield, this method concrete steps order is as follows: the selection of (1) fermentation strain: sorangium cellulosum in the slime bacteria bacterial strain (Sorangium cellulosum) ATCC15384, one of ATCC25531 and ATCC25569 bacterial strain; (2) the slime bacteria bacterial strain is keeping under the meta-bolites synthesis capability prerequisite, liquid culture is the domestication of growth evenly: above-mentioned bacterial strains is seeded on the solid slant culture base, the solid culture based component is: soy peptone 5～15g/L, yam starch 15～25g/L, CaCl ₂.2H ₂O 1g/L, MgSO ₄.7H ₂O 1g/L, Fe-EDTA 8mg/L, agar powder 15g/L; 30 ℃, cultivate after 5 days, be seeded to liquid fermentation medium, the liquid fermentation medium composition is: soy peptone 5～15g/L, yam starch 15～25g/L, CaCl ₂.2H ₂O 1g/L, MgSO ₄.7H ₂O 1g/L, Fe-EDTA8mg/L; 30 ℃, 120 rev/mins of shaking tables were cultivated 5～7 days, above-mentioned cultivation bacterium taken back again to solid medium cultivate, according to inoculating to liquid fermentation medium after the old terms cultivation, so repeat passage and attenuation, when 95～100 generations, the growth of bacterial strain in liquid fermentation medium of cultivating presents adaptive homogenizing growth, show as somatic cells and in liquid, become even growth conditions by original poly-bulk attitude, cell growth simultaneously for the time shorten, increment improves, the time that reaches the maximum cell amount was by original 10～12 days, foreshorten to 5～7 days, the maximum cell amount of acquisition is by original 1～4 * 10 ⁸Cell/ml is increased to 5～10 * 10 ⁹Cell/ml; (3) optimization of fermentation culture conditions: with the inoculation of step (2) homogenizing growth to solid medium, the solid culture based component is the same, 30 ℃, cultivate after 5 days, be seeded to the liquid fermentation medium that contains resin, the composition of liquid fermentation medium is: carbon source is one of ground melon starch, yam starch, ligoglucoside, xylan, concentration is 20g/L, nitrogenous source is one of lactoalbumin hydrolysate, soy peptone, ammonium tartrate, ammonium citrate, and concentration is 10g/L, CaCl ₂.2H ₂O 1g/L, MgSO ₄.7H ₂O 1g/L, Fe-EDTA 8mg/L, Choice of Resin XAD-16, addition are 20ml/L; Fermenting container is the 500ml triangular flask, includes the 200ml liquid fermentation medium, and culture temperature is 30 ℃, and shaking speed is 120 rev/mins, cultivates 5～7 days; (4) comprehensively eliminate meta-bolites produces ebormycine to bacterial strain influence: above-mentioned bacterial strains is seeded to solid medium, the solid culture based component is the same, 30 ℃, cultivate after 5 days, be seeded to the liquid fermentation medium that contains resin, the composition of liquid fermentation medium is ground melon starch 20g/L, lactoalbumin hydrolysate 10g/L, CaCl ₂.2H ₂O 1g/L, MgSO ₄.7H ₂O1g/L, Fe-EDTA 8mg/L, Choice of Resin XDA, one of AB-8 or hybrid resin, addition are 10～40ml/L; Fermenting container is the 500ml triangular flask, includes the 200ml liquid fermentation medium, and culture temperature is 30 ℃, and shaking speed is 120 rev/mins, cultivates 6～7 days; (5) the separation and Extraction purifying of ebomycin A: after the fermentation ends, the resin that adds in the liquid fermentation medium is taken out, through the methyl alcohol lixiviate, extractive substance passes through ion-exchange again, and molecular sieve and high performance liquid phase extract purifying, promptly obtain ebomycin A.

2. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, the described fermentation strain of step (1) is selected sorangium cellulosum (Sorangium cellulosum) ATCC15384.

3. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, the described solid culture based component of step (2) is: soy peptone 8g/L, yam starch 20g/L, CaCl ₂.2H ₂O1g/L, MgSO ₄.7H ₂O 1g/L, Fe-EDTA 8mg/L, agar powder 15g/L.

4. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, the described liquid fermentation medium composition of step (2) is: soy peptone 10g/L, yam starch 21g/L, CaCl ₂.2H ₂O 1g/L, MgSO ₄.7H ₂O 1g/L, Fe-EDTA 8mg/L.

5. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, carbon source is the ground melon starch in the composition of the described liquid fermentation medium of step (3).

6. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, nitrogenous source is a lactoalbumin hydrolysate in the composition of the described liquid fermentation medium of step (3).

7. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1 is characterized in that, the addition of the described hybrid resin of step (4) is 20ml/L.

8. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 1, it is characterized in that, the described hybrid resin of step (4) is meant CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 mix by weight 0.8～1.2: 0.8～1.2: 1.8～2.2: 0.8～1.2: 2.8～3.2: 1.8～2.2.

9. a kind of fermentation technology that improves ebomycin A yield as claimed in claim 8 is characterized in that, the described hybrid resin of step (4) is meant CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 were by weight 1: 1: 2: mix at 1: 3: 2.

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