Fermentation technology for producing rapamycin using actinomycetes

Abstract

The present invention discloses a process for producing rapamycin by fermentation of actinomycetes, which comprises the following steps: 1. inoculating actinomycetes into a slant culture medium, culturing the slant culture medium, obtaining slant strains; 2. inoculating the slant strains into a primary seed culture medium, culturing the primary seeds, obtaining primary seeds; 3. inoculating the primary seeds into a secondary seed culture medium, culturing the secondary seeds, obtaining secondary seeds; 4. inoculating the secondary seeds into a basic fermentation culture medium, adding one or two of 0.1-0.5% fish meal and 0.01-0.05% alanine and 0.1-0.2% glucose after fermentation and culture for 48h or 96h, continuing fermentation and culture to the 10th day, and obtaining a fermentation liquid containing rapamycin. The present invention can synthesize more rapamycin by adding glucose, fish meal and / or alanine after fermentation and culture for a period of time, and the content can reach up to 1000μg / mL or more.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a process for producing rapamycin by fermenting actinomycetes. Background Art

[0002] Rapamycin, now known as Sirolimus, is a macrolide antibiotic produced by Streptomyces hygroscopicus isolated by Veniza et al. in 1975. After years of research, rapamycin has been developed into a potent immunosuppressant for clinical use. At the same time, some novel derivatives synthesized by chemical modification based on rapamycin have been found to have new therapeutic effects in immunosuppression, anti-cancer, anti-Parkinson's disease and AIDS. Among them, its synthetic compounds Temsirolimus, Everolimus and AP23573 have been clinically studied as new anti-tumor targeted drugs. Rapamycin has broad prospects in the field of medical applications.

[0003] Currently, rapamycin is mainly produced by microbial fermentation, including fermentation with Streptomyces hygroscopicus (see Chinese patent documents CN101486976A, CN103555785A, etc.) and fermentation with Actinomycetes (see Chinese patent documents CN103740614A, CN103740781A).

[0004] The journal article "Improvement of the fermentation process of rapamycin production by actinomycetes", Chen Changfa et al., Chinese Journal of Pharmaceutical Industry, 45 (10), pp. 925-928, 2014" discloses a process for improving the yield of rapamycin by optimizing the components and contents of the fermentation medium using Actinoplanes sp. SIPI-8011 as the starting strain. The basic fermentation medium is: 1.4% corn starch, 3.5% glucose, 0.8% corn syrup powder, 0.8% cottonseed protein powder, 0.4% potassium dihydrogen phosphate, 0.4% soybean oil, 0.3% sodium pyrophosphate, 0.3% calcium carbonate, and the pH value is 6.9. By adding 0.5% glycerol, 1.5% mannitol and 0.1% betaine to the basic fermentation medium, an optimized fermentation medium is obtained, which can increase the content by 31.7%, and the maximum content can reach 920 μg / mL. Summary of the invention

[0005] The object of the present invention is to solve the above problems and provide a process for producing rapamycin with a higher content by fermentation using actinomycetes.

[0006] The technical solution to achieve the purpose of the present invention is: a process for producing rapamycin by fermentation using actinomycetes, comprising the following steps:

[0007] ① Inoculate the actinomycetes into the slant culture medium and culture them on the slant to obtain the slant strain;

[0008] ② Inoculate the slant strain obtained in step ① into the primary seed culture medium, and culture the primary seeds to obtain the primary seeds;

[0009] ③ Inoculating the primary seeds obtained in step ② into the secondary seed culture medium, and culturing the secondary seeds to obtain the secondary seeds;

[0010] ④ The secondary seeds obtained in step ③ are inoculated into the basic fermentation medium, and after fermentation culture for 48 hours or 96 hours, one or two of 0.1-0.5% fish meal and 0.01-0.05% alanine and 0.1-0.2% glucose are added, and the fermentation culture is continued until the 10th day to obtain a fermentation liquid containing rapamycin.

[0011] The basic fermentation medium described in the above step ④ comprises the following components in weight percentage: 1.0-2.0% corn starch, 0.1-1.0% corn steep liquor powder, 1.5-4.0% glucose, 0.1-1.0% cottonseed protein powder, 0.1-0.5% calcium carbonate, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% soybean oil, 0.1-0.5% dapoxetine, and the rest is purified water; the pH value of the basic fermentation medium is 6.5-7.0.

[0012] The actinomycetes described in step ① above are Actinoplanes sp.SIPI-8011.

[0013] The slant culture medium in step ① above comprises the following components in weight percentage: 1.5-2.5% soluble starch, 0.1-1.0% yeast extract, 0.5-2.0% glucose, 0.1-0.5% calcium carbonate, 1.5-2.5% agar powder, and the remainder is purified water; the pH value of the slant culture medium is 7.0-7.2.

[0014] The specific method of the slant culture described in the above step ① is as follows: inoculate the actinomycetes into a newly prepared plate, spread it evenly with an inoculation spatula and then scrape it evenly with a toothpick, and culture it in a biochemical incubator at 25-35°C for 7-9 days. When the slant is completely covered with spores and the color is light red or red, the slant strain is obtained and used immediately or stored at 4°C.

[0015] The primary seed culture medium described in the above step ② comprises the following components in weight percentage: 1.5-2.5% soluble starch, 0.1-1.0% corn steep liquor powder, 0.5-2.0% glucose, 0.1-1.0% dry yeast powder, 0.1-0.5% glycerol, 0.1-1.0% peanut meal, 0.1-0.5% calcium carbonate, and the rest is purified water; the pH value of the primary seed culture medium is 7.0-7.2.

[0016] The specific method of the primary seed culture described in step ② is as follows: use an inoculation spatula to scrape thumb-sized spores from the cultured slant strain and inoculate them into a 250 mL shake flask containing 20 to 50 mL of the primary seed culture medium, place it in a shaker at 25 to 35° C. for 3 to 5 days, and the shaker speed is 220 to 250 rpm. When the appearance is red and thick and the wall is well attached, the mycelium is net-shaped, darkly colored, has many branches, and the mycelium is thick and long under microscopic examination.

[0017] The secondary seed culture medium described in the above step ③ includes the following components in weight percentage: 1.0-2.0% corn starch, 0.1-1.0% corn steep liquor powder, 1.5-4.0% glucose, 0.1-1.0% cottonseed protein powder, 0.1-0.5% calcium carbonate, 0.1-0.5% potassium dihydrogen phosphate, and the rest is purified water; the pH value of the secondary seed culture medium is 6.5-7.0.

[0018] The specific method for culturing the secondary seeds described in step ③ above is as follows: inoculate the obtained primary seeds into a 250mL shake flask containing 20-50mL of secondary seed culture medium at an inoculation rate of 3%-10%, and culture on a shaker at 25-35°C for 2-4 days with a shaker speed of 220-250rpm to obtain secondary seeds.

[0019] The present invention has the following positive effects: (1) The present invention surprisingly found through a large number of experiments that adding glucose and fish meal and / or alanine after a period of fermentation culture can obtain a higher content of rapamycin, wherein the glucose in the early stage can make the bacteria grow rapidly and reach a higher bacterial density, and the added glucose can effectively control the pH value of the fermentation liquid, which is more conducive to the production of rapamycin, and the addition of fish meal and / or alanine can effectively regulate the growth and metabolism of microorganisms, making the growth of microorganisms slow, but the secondary metabolism becomes vigorous, thereby synthesizing more rapamycin. (2) In addition, the time of addition also has a certain effect on the content of rapamycin. The effect of adding too early is relatively poor. It is best to add after 96h of fermentation culture, which can obtain a content of more than 1000μg / mL. DETAILED DESCRIPTION

[0020] (Example 1)

[0021] The process of producing rapamycin by fermentation using actinomycetes in this embodiment comprises the following steps:

[0022] ① Slant culture: Inoculate the motile actinomycetes into a newly prepared plate, spread it evenly with an inoculation spatula and then scrape it evenly with a toothpick. Place it in a biochemical incubator at 28°C and culture it for 8 days. When the slant is completely covered with spores and the color is light red or red, the slant strain is obtained and can be used immediately or stored at 4°C.

[0023] The actinomycetes used in this example are Actinoplanes sp. SIPI-8011.

[0024] The slant culture medium of this embodiment includes the following components in weight percentage: 2.0% soluble starch, 0.5% yeast extract, 1.0% glucose, 0.1% calcium carbonate, 1.6% agar powder, and the rest is purified water; the pH value of the slant culture medium is 7.0.

[0025] ② Primary seed culture: Use an inoculation spatula to scrape thumb-sized spores from the cultured slant and inoculate them into a 250mL shake flask containing 30mL of primary seed culture medium. Incubate at 28℃ in a shaker for 4 days at 220rpm until the culture appears red and thick and hangs on the wall well. Microscopic examination of the hyphae shows a network, dark color, many branches, and thick and long hyphae.

[0026] The primary seed culture medium of this embodiment includes the following components in weight percentage: 1.5% soluble starch, 0.5% corn syrup powder, 1.0% glucose, 0.5% dry yeast powder, 0.5% glycerol, 0.5% peanut meal, 0.2% calcium carbonate, and the rest is purified water; the pH value of the primary seed culture medium is 7.0.

[0027] ③ Secondary seed culture: Inoculate the obtained primary seeds into a 250 mL shake flask containing 30 mL of secondary seed culture medium at a 6% inoculation rate, and culture on a shaker at 28°C for 3 days at a shaking speed of 220 rpm to obtain secondary seeds.

[0028] The secondary seed culture medium of this embodiment includes the following components in weight percentage: 1.4% corn starch, 1.0% corn steep liquor powder, 3.5% glucose, 0.8% cottonseed protein powder, 0.3% calcium carbonate, 0.4% potassium dihydrogen phosphate, and the rest is purified water; the pH value of the secondary seed culture medium is 6.9.

[0029] ④ Fermentation culture: The obtained secondary seeds were inoculated at a rate of 6% into a 250 mL shake flask containing 30 mL of basic fermentation medium, and cultured on a shaker at 28°C with a shaker speed of 220 rpm. After 96 hours, 0.1% glucose, 0.05% fish meal and 0.05% alanine were added, and the fermentation culture was continued for 6 days to obtain a fermentation broth containing rapamycin.

[0030] The basic fermentation medium of this embodiment includes the following components in weight percentage: 1.4% corn starch, 1.0% corn steep liquor powder, 3.5% glucose, 0.8% cottonseed protein powder, 0.3% calcium carbonate, 0.4% potassium dihydrogen phosphate, 0.4% soybean oil, 0.2% sodium dapoxetine, and the rest is purified water; the pH value of the basic fermentation medium is 6.9.

[0031] The pH value of the fermentation liquid obtained in step ④ was measured and the result was 6.63.

[0032] Another appropriate amount of fermentation broth was added with an equal volume of acetone, and the mixture was soaked at room temperature for 24 h. The mixture was centrifuged, the supernatant was collected, and the mixture was filtered. The filtrate was used to determine the content of rapamycin by HPLC, and the result was 1065 μg / mL.

[0033] The HPLC chromatographic conditions are shown in Table 1.

[0034] Table 1

[0035] Chromatographic columns C18 column (4.6 mm × 150 mm, 5 μm) Mobile phase 65% acetonitrile Flow rate 1mL / min Column temperature 50℃ Detection wavelength 278nm Injection volume 20μL

[0036] (Example 2 to Example 6)

[0037] The processes of each embodiment are basically the same as those of embodiment 1, except for the components added during the fermentation process of step ④ and the time of addition, as shown in Table 2 for details.

[0038] Table 2

[0039] Example 1 Example 2 Example 3 Example 4 Example 5 Example 5 Add time 96h 96h 96h 48h 48h 48h Adding components Glucose 0.1% + fish meal 0.05% + alanine 0.05% Glucose 0.1% + fish meal 0.05% Glucose 0.1% + Alanine 0.05% Glucose 0.1% + fish meal 0.05% + alanine 0.05% Glucose 0.1% + fish meal 0.05% Glucose 0.1% + Alanine 0.05% content 1123 μg / mL 1065 μg / mL 1088 μg / mL 950 μg / mL 892 μg / mL 922 μg / mL

[0040] (Comparative Example 1)

[0041] The process of this comparative example is basically the same as that of Example 1, except that no component is added during the fermentation process of step ④, as shown in Table 3 for details.

[0042] (Comparative Example 2 to Comparative Example 7)

[0043] The processes of the comparative examples are basically the same as those of Example 1, except for the components added during the fermentation process of step ④ and the time of addition, as shown in Table 3 for details.

[0044] Table 3

[0045] Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Add time No Addition 96h 96h 96h 48h 48h 48h Adding components No Addition Glucose 0.1% Fishmeal 0.05% Alanine 0.05% Glucose 0.1% Fishmeal 0.05% Alanine 0.05% content 515 μg / mL 712 μg / mL 624 μg / mL 617 μg / mL 637 μg / mL 604 μg / mL 608 μg / mL

Claims

1. A process for producing rapamycin by fermentation using actinomycetes, characterized in that The following steps are involved: ① Inoculate the actinomycetes into the slant culture medium and culture them on the slant to obtain the slant strain; ② Inoculate the slant strain obtained in step ① into the primary seed culture medium, and culture the primary seeds to obtain the primary seeds; ③ Inoculating the primary seeds obtained in step ② into the secondary seed culture medium, and culturing the secondary seeds to obtain the secondary seeds; ④ The secondary seeds obtained in step ③ are inoculated into the basic fermentation medium, and after fermentation culture for 48 hours or 96 hours, one or two of 0.1-0.5% fish meal and 0.01-0.05% alanine and 0.1-0.2% glucose are added, and the fermentation culture is continued until the 10th day to obtain a fermentation liquid containing rapamycin.

2. The process for producing rapamycin by fermentation using actinomycetes according to claim 1, characterized in that: The slant culture medium in step ① above comprises the following components in weight percentage: 1.5-2.5% soluble starch, 0.1-1.0% yeast extract, 0.5-2.0% glucose, 0.1-0.5% calcium carbonate, 1.5-2.5% agar powder, and the remainder is purified water; the pH value of the slant culture medium is 7.0-7.

2.

3. The process for producing rapamycin by fermentation using actinomycetes according to claim 1, characterized in that: The primary seed culture medium described in the above step ② comprises the following components in weight percentage: 1.5-2.5% soluble starch, 0.1-1.0% corn steep liquor powder, 0.5-2.0% glucose, 0.1-1.0% dry yeast powder, 0.1-0.5% glycerol, 0.1-1.0% peanut meal, 0.1-0.5% calcium carbonate, and the rest is purified water; the pH value of the primary seed culture medium is 7.0-7.

2.

4. The process for producing rapamycin by fermentation using actinomycetes according to claim 1, characterized in that: The secondary seed culture medium described in the above step ③ includes the following components in weight percentage: 1.0-2.0% corn starch, 0.1-1.0% corn steep liquor powder, 1.5-4.0% glucose, 0.1-1.0% cottonseed protein powder, 0.1-0.5% calcium carbonate, 0.1-0.5% potassium dihydrogen phosphate, and the rest is purified water; the pH value of the secondary seed culture medium is 6.5-7.

0.

5. The process for producing rapamycin by fermentation using actinomycetes according to any one of claims 1 to 4, characterized in that: The basic fermentation medium described in the above step ④ comprises the following components in weight percentage: 1.0-2.0% corn starch, 0.1-1.0% corn steep liquor powder, 1.5-4.0% glucose, 0.1-1.0% cottonseed protein powder, 0.1-0.5% calcium carbonate, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% soybean oil, 0.1-0.5% dapoxetine, and the rest is purified water; the pH value of the basic fermentation medium is 6.5-7.0.