CN1769468A - Method for highly-effective producing epothilone using myxobacteria sorangium cellulosum - Google Patents

Abstract

The invention discloses a method for efficiently producing epothilone by using S. cellulosus. The present invention improves the fermentation medium of S. cellulosus, and adds cyclodextrin or cyclodextrin derivatives to the fermentation medium, so that the fermentation medium is more suitable for cell growth, and the epothilone Yield is higher.

Description

A method for efficient production of epothilone by using S. cellulosus

technical field

The invention relates to the field of microorganisms, in particular to a high-efficiency production method of epothilone.

Background technique

Myxobacteria (Myxobacteria) are the highest Gram-negative prokaryotic group, with complex multicellular behavior, occupying an important position in the study of cell differentiation, development and biological evolution. Myxobacteria can produce extremely rich secondary metabolites, and the diversity of biological activity of the products can be compared with the famous Streptomyces group, which is an excellent strain resource in the development and research of new microbial drugs.

Sorangium cellulosum belongs to the cellulolytic group of myxobacteria, and the macrocyclic ester compound epothilone (epothilone) produced by it has the activity of promoting microtubule polymerization. carcinogen. Epothilone can combine with the microtubule skeleton in eukaryotic cells, resulting in blocked chromosome segregation and inability to divide the nucleus. The effective concentration of the effect is 10-20ng/ml.

Epothilone was first discovered in Sorangium cellulosum90 strain. It is a class of macrocyclic lactone compounds secreted by Sorangium cellulosum. Two natural derivatives have been found, which are called epothilone A (molecular formula C ₂₆ H ₃₉ NO ₆ S) and Epothilone B (molecular formula is C ₂₇ H ₃₉ NO ₆ S), the molecular structure is shown in formula (I). They appear as colorless oils which crystallize in ethyl acetate to form powders.

Epothilones have a similar mechanism of action to Taxol, and have a better curative effect on cancer treatment. But Epothilones has the following advantages compared with paclitaxel.

1. The structure of Epothilones is simpler than that of paclitaxel, and its inhibitory effect in vitro is 2000-5000 times stronger than that of paclitaxel, and the effect of B with a methyl group is basically 2 times greater than that of A.

2. Epothilones have better water solubility and are easier to obtain through fermentation.

3. Epothilones are not substrates of P2 glycoprotein, and have strong cytotoxicity to multidrug-resistant cells. Therefore, the anti-tumor performance of epothilone is not only much stronger than that of paclitaxel, but also its anti-tumor spectrum may be much wider. Epothilones have significant selective activity against breast tumor cells and colon tumor cells.

4. The administration of paclitaxel will be accompanied by some clinical side effects (such as neutropenia, peripheral neuropathy, alopecia and allergic reaction).

At present, there are some reports about the experiments of the cellulocomplexus strains fermenting and synthesizing Epothilones in fermenters, and the research on the nutritional control affecting the synthesis of Epothilones, but the production methods of the existing Epothilones generally have unstable yields and low yields, etc. question.

In order to achieve the purpose of Epothilones being used in medicine, it is necessary to obtain a sufficient amount of fermentation broth mixture containing Epothilones. So far, the extraction of Epothiliones in myxobacteria, especially its Soce90 strain, has been reported in the literature. In order to extract relatively high concentrations of Epothilones, resins made of polystyrene, such as AmberliteXAD-1180, were often added to the fermentation medium to be extracted. However, the disadvantage of this method is that it will cause a series of problems in large-scale applications. The valves of the fermenter can be damaged by resin pellets; the pipes can become clogged and the instruments can experience greater wear due to mechanical friction. Resin is a porous material, so it will have a large internal surface area (about 825m2/g). During the autoclaving process, the internal air cannot be sterilized together. Therefore, in actual large-scale applications, the method of adding resin cannot be used. However, if no resin is added, it is difficult to extract higher concentrations of Epothiliones in the fermentation medium.

So far, the method of obtaining Epothiliones from myxobacteria and applying it to the field of industrial technology production without the above-mentioned disadvantages is still in the seeking stage.

Contents of the invention

The purpose of the present invention is to provide a method for efficient production of epothilones by using S. cellulosus to solve various problems in the existing epothilone production methods.

S. cellulosus was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Brauuschweig, Germany), by improving its fermentation medium and adding cyclodextrin or cyclodextrin derivatives to the fermentation medium, The fermentation medium is more suitable for cell growth, and the yield of Epothiliones is higher. Since there are hydrophobic cavities inside the cyclodextrin molecule, while the outside of the molecule is hydrophilic, it can not only adsorb the product without affecting the growth of the bacteria, but also weaken the phenomenon of product inhibition. In addition, Epothiliones can be extracted from the fermentation broth.

The present invention utilizes S. cellulosus to produce the method for epothilone, specifically comprises the following steps:

(1) Pre-cultivation: Add the sterilized medium G52 into the shake flask, and then insert S. cellulosus into the shake flask for cultivation. The cultivation time is 3-4 days. The cultivation conditions are: 120-250rpm, 29 ~31°C;

The culture medium G52 is:

                                                                                        

MgSO ₄ 1g/L

CaCl ₂ 1g/L

                                                                         

    Potato Starch                                

Dextrose Anhydrous 2g/L

EDTA-Fe(III)-Na 1ml/L

Adjust the pH to 7.4, and sterilize at 120°C for 20 minutes;

The purpose of the pre-cultivation is to activate the strains and cultivate the strains.

(2) Intermediate cultivation: Add the sterilized medium G52 into the fermenter, and then put the pre-culture into the fermenter for cultivation. The cultivation time is 3 to 4 days. The cultivation conditions are: 29～31°C, 200°C ～300rpm, the ventilation rate is 0.5 liters of air per liter of liquid medium per minute, 120～122℃, pH7.4; the purpose of the intermediate culture is to make the growth of the bacteria enter the logarithmic growth phase and prepare for the next fermentation culture .

(3) Fermentation culture: Add sterilized 1B12 improved medium into the fermenter, then add sterilized cyclodextrin or its derivatives, then insert the intermediate culture into the fermenter for cultivation, and the cultivation time is After 6-7 days, the culture conditions are: 20-35°C, 120-250rpm, and the fermentation broth containing epothilone can be obtained; cyclodextrin and its derivatives are added in the form of its aqueous solution, dextrin The amount of dextrin and its derivatives added is: 1 gram of dextrin or its derivatives/per 100ml fermentation broth. The volume of the added dextrin or its derivative solution is 10% of the volume of the whole fermentation broth. The volume of the fermentation broth includes medium volume, dextrin solution volume and intermediate culture volume.

The 1B12 improved medium is:

Potato starch 20g/L

                                             

MnCl ₄ 1mg/L

K ₂ HPO ₄ 0.06g/L

_ZnCl2 1mg/L

CuSO ₄ 1mg/L

EDTA-Fe(III)-Na 8mg/L

  Adjust the pH to 7.8 and sterilize at 120°C for 20 minutes.

For the fermentation culture described in the above step (3), 4 mg/L amino acid and 4 mg/L growth factor were added on the third day of fermentation. The best amino acid is alanine, and the best growth factor is potassium acetate.

The optimal culture conditions for the pre-cultivation in the above step (1) are: 180 rpm, 30° C., and a shaker flask displacement of 50 mm.

The optimal culture conditions for the intermediate culture in the above step (2) are: 30° C., 250 rpm, ventilation rate of 0.5 liter of air per minute per liter of liquid medium, 121° C., pH 7.4.

The optimal culture conditions for the fermentation culture in the above step (3) are: 25° C., 180 rpm, pH 7.8.

The above-mentioned method for producing epothilones also includes the separation and purification of epothilones. The method is as follows: (1) the fermentation broth is centrifuged and filtered, and the obtained filtrate and resin are mixed at a volume ratio of 65:1, stirred and mixed, centrifuged, and the obtained resin First wash with deionized water, then wash with isopropanol, add water to the washing liquid obtained, extract the isopropanol therein, and then extract with ethyl acetate in the remaining water phase, the ethyl acetate extract obtained is in Concentrate and dry in a vacuum rotary evaporator to obtain crude crystals. Gained crude crystals are added in isopropanol, vibrated, filtered, and then dried in a vacuum desiccator to obtain epothilones B white crystals; the resulting white crystals are further purified by reverse-phase chromatographic column RP-18, and acetonitrile is used as eluent. Finally, crystallize with ethyl acetate:toluene=2:3 mixture to obtain epothilone A.

Compared with the prior art, the present invention has the following beneficial effects:

1. In the present invention, cyclodextrin is added to the fermentation broth. Because there are hydrophobic cavities in the cyclodextrin molecule, while the outside of the molecule is hydrophilic, it can not only adsorb the product without affecting the growth of the bacteria, but also weaken the Phenomenon of product inhibition.

2. The present invention improves the composition of the fermentation medium of Epothilone, and improves the fermentation condition liquid, makes it more suitable for the production of Epothilone, and improves the output.

3. In the present invention, the resin XAD-16 adsorption product is added only when the fermentation is completed and the extraction is carried out, and a higher concentration of Epothilone is effectively obtained.

Detailed ways

Generally, the inoculation amount of the seed liquid is 10% (V/V), and that of the fermentation liquid is 20% (V/V). It should be noted that the composition of the fermentation medium is calculated according to the final volume. For example, 20L of culture includes: 18L of 1B12 modified medium + 2L of G52 seed liquid, that is to say, 20L of fermentation broth only contains 18L of fermentation medium.

Example

1. Activation of strains: Take out S. cellulosus DSM11999 from the -80°C refrigerator, first place the strains in ice water to slowly heat up and dissolve, take 1ml and transfer to 10ml of G52 medium (put in a 50ml shake flask) Cultivate for 3 days, 180 rpm, 30°C, shake flask displacement 25mm. Then take 5 ml of this G52 culture and transfer it to 45 ml of G52 medium (put in a 200 ml shake flask) and cultivate for 3 days, 180 rpm, 30° C., and the shake flask is displaced by 25 mm. Then take 50 ml of G52 culture and transfer it to 450 ml of G52 medium (in a 2L shake flask) for 3 days, 180 rpm, 30° C., and the displacement of the shake flask is 50 mm. Medium G52 is: dry yeast powder 2g/L, MgSO ₄ 1g/L, CaCl ₂ 1g/L, skimmed milk powder 2g/L, potato starch 8g/L, anhydrous glucose 2g/L, EDTA-Fe(III)- Na 1ml/L, adjust the pH to 7.4, and sterilize at 120°C for 20 minutes;

2. Maintenance culture: Take 50 ml of the above-mentioned culture and add it to 450 ml of G52 medium (in a 2-liter shake flask) for culture.

3. Pre-cultivation in shake flasks: 1 × 450ml of G52 medium (placed in a 2-liter shake flask) was inserted into 50ml of maintenance culture, cultured for 4 days, 180rpm, 30°C, pH7.4, shake flask displacement 50mm. The purpose is to activate the strains and cultivate the strains.

4. Intermediate culture: 90 liters of G52 medium is placed in a 150 liter fermenter, and 10 liters of pre-culture is inserted, and cultivated for 4 days. The culture conditions are: 30 ° C, 250 rpm, and the ventilation rate is per liter of liquid medium 0.5 liters of air per minute, 121°C, pH 7.4. The purpose is to make the strain reach the logarithmic growth phase and prepare for the next fermentation.

5. Fermentation culture: Fermentation is carried out in a 750L fermenter, and 350 liters of sterilized 1B12 improved medium and 50 liters of sterilized 2-hydroxypropyl-β-cyclodextrin solution (containing 2-hydroxypropyl - β-cyclodextrin 5000 g), then inoculate 100 liters of intermediate culture. The culture lasted for 6 days, and the culture conditions were: 25°C, 180 rpm, pH 7.8. On the third day of fermentation, 4 mg/L alanine and 4 mg/L potassium acetate were added. 1B12 modified medium is: potato starch 20g/L, tryptone 11g/L, MnCl ₄ 1mg/L, K ₂ HPO ₄ 0.06/L; ZnCl ₂ 1mg/L, CuSO ₄ 1 mg/L, EDTA-Fe(III )-Na 8mg/L, adjust the pH to 7.8, and sterilize at 120°C for 20 minutes.

6. Separation and purification of the product: ① 500 liters of fermented product was centrifuged and filtered, and the obtained fermentation broth and resin were mixed at a volume ratio of 65:1, stirred for 2 hours, and the product was transferred from the cyclodextrin to the resin, centrifuged, and the obtained resin First wash and desorb with 12 liters of deionized water, then wash twice with isopropanol, transfer the product to the organic phase, stir with 30 liters of isopropanol for 30 minutes each time, add water to the obtained washing solution, and extract it isopropanol, the remaining water phase was extracted with ethyl acetate three times, and the obtained ethyl acetate extract was concentrated and dried in a vacuum rotary evaporator to obtain crude crystals.

②The crude crystals obtained above were suspended in 1ml of isopropanol, shaken at 25°C for 24 hours, filtered, and dried in a vacuum desiccator to obtain white crystals of epothilones B. Epothilone B also contains a small amount of epothilone A, which can be further purified by reverse-phase chromatography column RP-18, with acetonitrile as the eluent. Finally, crystallize with ethyl acetate:toluene=2:3 mixture to obtain epothiloneA.

comparative example

Also adopt S. cellulosus DSM11999 to produce epothilones according to the production method of the examples. There are three differences: 1. Instead of using 1B12 modified medium, 1B12 medium is used instead; 2. Dextrin is not added; 3. Alanine and potassium acetate are not added.

The product of embodiment and comparative example is analyzed:

Add 2% (V/V) XAD-16 resin to each shake flask, shake at 180rpm for 2h. Allow the resin to fully absorb the product in the culture. Then use a 150um nylon sieve to filter, and the filtered resin is washed with a little water. Water is a polar solvent. Washing with water can desorb the product in the resin, and the washing solution is loaded into a container.

Then wash the resin with 60ml of isopropanol (>99%), put the filtrate into a sealed tube, and shake it on a Rota-Mixer (Labinco BV, Netherlands) for 3min at room temperature, then get 2ml of liquid, centrifuge, and get the supernatant for use in HPLC.

HPLC result: the thick crystal that the embodiment is obtained is dissolved in 1ml isopropanol, concentrates, adopts HPLC analysis to know that epothilones output is 16.2mg/L; The thick crystal that comparative example obtains is dissolved in 1ml isopropanol, concentrates, adopt HPLC analysis showed that the yield of epothilones was 11mg/L. It can be seen that the yield of epothilones obtained by the production method of the present invention is 47.3% higher than that of the original method.

Claims (9)

1. A method for producing epothilone is to utilize S. cellulosus to ferment and produce, and it is characterized in that by improving the fermentation medium of S. cellulosus. 2. The method for producing epothilone according to claim 1, further comprising adding cyclodextrin to the fermentation medium. 3. The method for producing epothilone as claimed in claim 2, characterized in that it comprises the steps of: (1) Pre-cultivation: Add the sterilized medium G52 into the shake flask, and then insert S. cellulosus into the shake flask for cultivation. The cultivation time is 3-4 days. The cultivation conditions are: 120-250rpm, 29 ~31°C; the culture medium G52 is: Dry Yeast Powder 2g/L MgSO ₄ 1g/L CaCl ₂ 1g/L Skimmed milk powder 2g/L Potato starch 8g/L Anhydrous glucose 2g/L EDTA-Fe(III)-Na 1ml/L Adjust the pH to 7.4, and sterilize at 120°C for 20 minutes; (2) Intermediate cultivation: Add the sterilized medium G52 into the fermenter, and then put the pre-culture into the fermenter for cultivation. The cultivation time is 3 to 4 days. The cultivation conditions are: 29～31°C, 200°C ～300rpm, the ventilation rate is 0.5 liters of air per minute per liter of liquid medium, 120～122°C, pH7.4; (3) Fermentation culture: Add sterilized 1B12 improved medium into the fermenter, then add sterilized cyclodextrin or its derivatives, then insert the intermediate culture into the fermenter for cultivation, and the cultivation time is After 6-7 days, the culture conditions are: 20-35°C, 120-250rpm, and the fermentation broth containing epothilone can be obtained; The 1B12 improved medium is: Potato starch 20g/L Tryptone 11g/L MnCl ₄ 1mg/L K ₂ HPO ₄ 0.06g/L _ZnCl2 1mg/L CuSO ₄ 1mg/L EDTA-Fe(III)-Na 8mg/L Adjust the pH to 7.8, and sterilize at 120°C for 20 minutes. 4. The method for producing epothilone according to claim 3, characterized in that 4 mg/L amino acid and 4 mg/L growth factor are added to the fermentation culture in step (3) on the third day of fermentation. 5. The method for producing epothilone according to claim 3, characterized in that the culture conditions of the pre-cultivation in step (1) are: 180 rpm, 30°C. 6. The method for producing epothilone as claimed in claim 3, characterized in that the culture conditions of the intermediate culture in step (2) are: 30° C., 250 rpm, and the ventilation rate is 0.5 liter per minute per liter of liquid medium Air, 121°C, pH 7.4. 7. The method for producing epothilone according to claim 3, characterized in that the culture conditions of the fermentation culture in step (3) are: 25°C, 180 rpm, pH 7.8. 8. The method for producing epothilones as claimed in claims 3 to 7, which is characterized in that it also includes the separation and purification of epothilones. The method is as follows: ① the fermentation broth is centrifuged and filtered, and the obtained filtrate and resin are mixed by volume Ratio of 65:1, stirred and mixed, centrifuged, the obtained resin was first washed with deionized water, then washed with isopropanol, water was added to the obtained washing liquid, and the isopropanol was extracted, and then used in the remaining water phase Ethyl acetate extraction, the obtained ethyl acetate extract was concentrated and dried in a vacuum rotary evaporator to obtain crude crystals. 9. The method for producing epothilone as claimed in claim 8, characterized in that said separation and purification method also comprises the following steps: adding the obtained crude crystals to isopropanol, vibrating, filtering, and then desiccating in a vacuum desiccator Dry to obtain epothilones B white crystals; the resulting white crystals are further purified by reverse-phase chromatographic column RP-18, acetonitrile is used as eluent, and finally crystallized with ethyl acetate: toluene=2: 3 mixed solution to obtain epothilone A.