A method for producing rapamycin by fermentation

Abstract

The present invention relates to a method for producing rapamycin by fermentation. The fermentation method comprises using Actinoplanes sp., which has a preservation number of CGMCC NO.30440, as a fermentation strain. During the fermentation process, when the reducing sugar content in the fermentation broth is lower than 10 g / L, maltodextrin is added to the fermentation broth so that the reducing sugar content in the fermentation broth is 5-10 g / L, and soybean cake powder is added so that the amino nitrogen content in the fermentation broth is 200-300 mg / dL, and fermentation is performed. The rapamycin content in the fermentation broth obtained by the method for producing rapamycin by fermentation of the present invention reaches more than 2700 mg / L, and all operation steps are operated under normal conditions, which is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing rapamycin by fermentation. Background Art

[0002] Rapamycin (RAPA), also known as Sirolimus, is a nitrogen-containing triene macrolide antibiotic produced by Streptomyces hygroscopicus or Actinoplanes. Rapamycin has broad prospects in the pharmaceutical market, with antifungal, antiproliferative and antitumor effects. It is currently widely used in the treatment of organ transplant rejection and autoimmune diseases.

[0003] At present, the production of rapamycin mainly relies on microbial fermentation, generally using hygroscopic Streptomyces or motile actinomycetes for fermentation. During the fermentation process, there are many factors that affect the synthesis of rapamycin, such as pH, ventilation, stirring speed, temperature, culture medium composition and feeding process. Due to the long fermentation cycle, suitable carbon and nitrogen sources are generally added to promote the continuous and stable production of rapamycin by the bacteria.

[0004] Tamura et al. (SHINYA TAMURA, YONGSOO PARK, MASARU TORIYAMA, MISUYASU OKABE (1997) Change of Mycelial Morphology in Tylosin Production by Batch Culture of Streptomyces fradiae under Various Shear Conditions 83 (1997) 523-528) found that mycelial morphology is closely related to antibiotic production. The smaller the size of the bacterial ball, the more nutrients and oxygen enter, the more vigorous the secondary metabolism, and the higher the antibiotic production. Yen et al. (Hong-Wei Yen, Yung-Lin Li (2014) The effects of viscosity and aeration rate on rapamycin production in an airlift bioreactor by using Streptomyces hygroscopicus. Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 1149-1153) found that adding carboxymethyl cellulose to the culture medium limited the extension of mycelium, and the bacterial balls were small and compact, which led to an increase in the viscosity of the system and increased the fermentation level of rapamycin from 260 mg / L to 410 mg / L, but the titer level was still low, making it difficult to achieve industrial production. During the fermentation process, due to the aggregation of mycelium, the mycelium was entangled for a long time, resulting in the formation of bacterial balls. The degree of aggregation of mycelium determines the degree of tightness of the mycelium morphology from free filaments to bacterial balls. The mycelium morphology and bacterial ball size of filamentous fungi play a vital role in the production of rapamycin.

[0005] The prior art CN109486877A uses Streptomyces hygroscopicus as the fermentation strain, inoculates the seed liquid into a fermentation tank filled with liquid fermentation medium, controls the pH value of the fermentation liquid to 4.65-5.15 with ammonia water at 48 hours of fermentation, and adds glucose aqueous solution every day at 96 hours of fermentation until the fermentation is completed. The fermentation unit determined by HPLC can reach more than 1000 mg / L.

[0006] Prior art CN107365811A discloses a process for producing rapamycin by fermentation of Actinomycetes. After fermenting and culturing Actinomycetes for a period of time, glucose, fish meal and / or alanine are added to obtain rapamycin with a content of about 1000 mg / L. CN103740781A discloses a method for culturing Actinomycetes to obtain rapamycin fermentation liquid. By optimizing the culture medium and controlling the culture conditions, Actinomycetes can produce a higher concentration of rapamycin. In the fermentation experiment of a 30L fermenter, the yield of rapamycin can reach about 750 mg / L, and in the fermentation experiment of a 200L fermenter, the yield of rapamycin can reach about 840 mg / L. CN103740614A discloses a mutagenic strain of Actinomycetes with high rapamycin production. The yield of rapamycin obtained by fermenting and culturing the strain is 535-565 mg / L.

[0007] Therefore, the fermentation yield of rapamycin produced by fermentation in the prior art needs to be improved to better improve production efficiency. Prior art CN114381480A discloses a fermentation method for sirolimus, using Actinoplanes sp.N902-109 as the fermentation strain, accessing the seed liquid to a fermentation tank filled with a fermentation medium for cultivation, and adding glucose and phospholipids during the fermentation process, so that the optimal fermentation level of rapamycin reaches 2223.5 mg / L. However, there is no control standard for phospholipids in the fermentation process of the prior art, and the production process is difficult to unify and control.

[0008] Therefore, there is a need for a method for producing rapamycin by fermentation with high yield and easy-to-control operation process. Summary of the invention

[0009] The object of the present invention is to provide a method for producing rapamycin by fermentation. The fermentation method comprises using Actinoplanes sp., which has a preservation number of CGMCC NO.30440, as a fermentation strain. During the fermentation process, when the reducing sugar content in the fermentation broth is lower than 10 g / L, maltodextrin is added to the fermentation broth to control the reducing sugar content in the fermentation broth to be 5-10 g / L for fermentation.

[0010] In another preferred embodiment, during the fermentation process, when the amino nitrogen content in the fermentation broth is lower than 300 mg / dL, a nitrogen source is added to make the amino nitrogen content in the fermentation broth 200-300 mg / dL.

[0011] In another preferred embodiment, the nitrogen source is selected from yeast powder, fish meal, peptone, soybean cake powder, and ammonium sulfate.

[0012] In another more preferred embodiment, the nitrogen source is selected from soybean cake powder.

[0013] In another preferred embodiment, the nitrogen source is first prepared into a 100-200 g / L solution and then added to the fermentation broth.

[0014] In another preferred embodiment, maltodextrin is first prepared into a 250-350 g / L solution and then added to the fermentation broth.

[0015] In another preferred embodiment, the supplementation is performed intermittently.

[0016] In another preferred embodiment, the fermentation medium comprises the following components:

[0017] Corn starch 10-20g / L, glucose 30-50g / L, dry yeast 10-20g / L, corn syrup powder 5-15g / L, calcium carbonate 2-4g / L, potassium dihydrogen phosphate 3-5g / L, shikimic acid 4-6g / L and water.

[0018] In another more preferred embodiment, the fermentation medium comprises the following components:

[0019] Corn starch 15g / L, glucose 40g / L, dry yeast 15g / L, corn syrup powder 10g / L, calcium carbonate 3g / L, potassium dihydrogen phosphate 4g / L, shikimic acid 5g / L and water.

[0020] In another preferred embodiment, the water contained in the fermentation medium is tap water.

[0021] In another preferred embodiment, the fermentation process of the present invention is to culture actinomycetes in a fermenter at a fermentation temperature of 25-30°C and a pressure of 0.04-0.06Mpa; control the ventilation rate of 0.5-2.5vvm and the rotation speed of 100-400rpm according to the dissolved oxygen in the fermentation liquid, so that the dissolved oxygen in the culture liquid is >30%; when the reducing sugar content in the fermentation liquid is lower than 10g / L (for example, 1-9g / L, 2-8g / L or 3-7g / L, etc.), start to add maltodextrin to the fermentation liquid to maintain the reducing sugar concentration in the fermentation liquid at 5-10g / L, and when the amino nitrogen content in the fermentation liquid is lower than 300mg / dL, start to add liquid containing soybean cake powder to maintain the amino nitrogen content in the fermentation liquid at 200-300mg / dL, and the fermentation time is 9-12 days.

[0022] In another preferred embodiment, the fermentation time is 11 days.

[0023] In another preferred embodiment, the fermentation medium in the above fermenter comprises the following components:

[0024] Corn starch 10-20g / L, glucose 30-50g / L, dry yeast 10-20g / L, corn syrup powder 5-15g / L, calcium carbonate 2-4g / L, potassium dihydrogen phosphate 3-5g / L, shikimic acid 4-6g / L and water.

[0025] The invention also provides an actinomycete, which is Actinoplanes sp. and has a preservation number of CGMCC NO.30440.

[0026] The present invention also provides the use of the above-mentioned actinomycetes in the fermentation production of rapamycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the mycelial morphology observed after the fermentation culture of the actinomycetes obtained in Example 1 of the present invention lasted for 11 days.

[0028] Figure 2 This is the mycelial morphology observed after the fermentation culture of the actinomycetes obtained in Example 16 of the present invention lasted for 11 days.

[0029] Figure 3 It is the HPLC detection spectrum of rapamycin standard solution.

[0030] Figure 4 This is the HPLC detection spectrum of the final fermentation broth obtained in Example 16 of the present invention. DETAILED DESCRIPTION

[0031] In view of the problems existing in the fermentation method of producing rapamycin in the prior art, the inventor of the present application has conducted in-depth research on the fermentation method of producing rapamycin. Through a large number of experiments, it is surprisingly found that in the process of fermentation production of rapamycin, Actinoplanes sp. is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a deposit date of April 25, 2024 and a deposit number of CGMCC NO.30440. As a fermentation strain, the seed liquid is transferred to a fermentation tank containing a liquid culture medium, and after a period of fermentation, a sterilized maltodextrin solution and a nitrogen source (especially a liquid containing soybean cake powder) are added to produce rapamycin. The viscosity of the fermentation liquid gradually increases with the extension of the feeding time, and the accumulation of rapamycin product synthesis increases accordingly. For example, after 252-276 hours of fermentation culture, the rapamycin content can reach more than 2700 mg / L, which is much higher than the fermentation level of rapamycin currently reported at home and abroad. The possible reason is that the initial input of fast-acting carbon source glucose can promote the rapid growth of bacteria and obtain more bacteria in a short time. When the initial glucose is exhausted, the added maltodextrin aqueous solution can not only continuously provide the slow-release carbon source required for bacterial growth, but also limit the extension of hyphae, reduce the size of bacterial balls, and increase the viscosity of the system, which is beneficial to the production of rapamycin. Under the premise of adding maltodextrin, the added nitrogen source, especially soybean cake powder, can be used as a slow-release nitrogen source that can be used by the bacteria, so that the carbon-nitrogen ratio of the fermentation liquid will not change rapidly, stabilize the metabolism of the bacteria, and increase the viscosity of the system to promote the production of rapamycin.

[0032] In the method for producing rapamycin by fermentation of the present invention, the addition of carbon source and nitrogen source is a key step in the fermentation culture process. Preferably, when the reducing sugar content in the fermentation broth is lower than 10 g / L, maltodextrin is added to the fermentation broth so that the reducing sugar content therein is maintained at 5-10 g / L. At the same time, when the amino nitrogen content in the fermentation broth is lower than 300 mg / dL, a nitrogen source is added so that the amino nitrogen content in the fermentation broth is maintained at 200-300 mg / dL. The solution containing the carbon source and the solution containing the nitrogen source can be added to the fermentation broth intermittently or continuously, respectively. For example, they are added to the fermentation broth in a batch supplementation manner, that is, the solution containing the carbon source or the solution containing the nitrogen source is added in multiple batches, and the required amount is added each time.

[0033] The content of reducing sugar and the content of amino nitrogen in the fermentation broth can be determined by methods commonly used in the art.

[0034] In the method for producing rapamycin by fermentation of the present invention, during the fermentation culture process, the addition of carbon source and nitrogen source in combination with the fermentation medium of the present invention achieves a relatively ideal fermentation effect. The fermentation medium of the present invention provides sufficient carbon source and nitrogen source to the actinomycetes in the initial stage of fermentation. Preferably, the fermentation medium of the present invention comprises the components: 10-20 g / L corn starch, 30-50 g / L glucose, 10-20 g / L dry yeast, 5-15 g / L corn steep liquor powder, 2-4 g / L calcium carbonate, 3-5 g / L potassium dihydrogen phosphate, 4-6 g / L shikimic acid and water.

[0035] The inventors of the present application have also conducted in-depth research on substances used as carbon sources, and compared common carbon sources, such as glucose, liquefied corn starch, maltodextrin and sucrose, and found that maltodextrin is the best choice, which is most beneficial for the formation of compact and small-sized bacterial balls of actinomycetes. And it was found through experiments that when maltodextrin is used as a supplementary carbon source, when the reducing sugar content in the fermentation liquid is controlled to be maintained at 5-10g / L, the target product yield of the fermentation is the highest. The inventors of the present application have also conducted in-depth research on substances used as nitrogen sources, and compared common nitrogen sources, such as yeast powder, fish meal, peptone, soybean cake powder and ammonium sulfate, and found that soybean cake powder is the best choice, and it was found through experiments that when soybean cake powder is used as a supplementary nitrogen source, when the amino nitrogen content in the fermentation liquid is controlled to be maintained at 200-300mg / dL, the target product yield of the fermentation is the highest.

[0036] The inventors of the present application have studied the fermentation time and found that 9-12 days of fermentation is the best fermentation time. Through mycelium morphology observation and rapamycin content detection, it is found that before the optimal fermentation time, the mycelium morphology is complete and the rapamycin content continues to rise. After reaching the optimal fermentation time, the mycelium begins to break and the rapamycin content has no obvious upward trend. Therefore, the method for producing rapamycin by fermentation of the present invention uses the changes in mycelium morphology and the increase in the rapamycin content in the fermentation broth as the standard for whether the fermentation is stopped.

[0037] The fermentation temperature of the method for producing rapamycin by fermentation of the present invention is a conventional temperature in the art, such as 25-30° C., preferably 28° C. The tank pressure is a conventional pressure in the art, such as 0.04-0.06 MPa, preferably 0.05 MPa, and the dissolved oxygen in the fermentation broth is a conventional amount in the art, such as >30%.

[0038] In the present invention, there is no special limitation on the materials, and any material source known in the art can be used.

[0039] The main advantages of the present invention include:

[0040] 1. The present invention uses Actinoplanes sp. with a preservation number of CGMCC NO.30440 as a fermentation strain, and the preparation process of the method for producing rapamycin by fermentation is simple to operate, and the content of the target product in the obtained fermentation liquid is high, which is suitable for industrial production;

[0041] 2. The maltodextrin and soybean cake powder used for feeding in the method for producing rapamycin by fermentation of the present invention are both common carbon sources and nitrogen sources, which are easy to obtain, low in cost, and environmentally friendly; and the indicators (reducing sugar content and amino nitrogen content) controlled in actual production are detected quickly and accurately, and the entire feeding process can avoid deviation and is easy to control;

[0042] 3. Compared with the mycelial morphology obtained by adding glucose in the fermentation production of rapamycin (more and longer mycelia, larger ball size) (see Figure 1 , picture of the 11th day of fermentation culture), the method for producing rapamycin by fermentation with maltodextrin of the present invention obtains small and compact mycelium, that is, the mycelium length is short and the ball size is small, (see Figure 2 , picture of the 11th day of fermentation culture). In this state, the motile actinomycetes can fully absorb nutrients and oxygen, which is more conducive to the production of secondary metabolites; at the same time, shorter hyphae and smaller bacterial balls can reduce the negative impact of shear force, which is conducive to the production of rapamycin.

[0043] The present invention is further described in detail below in conjunction with the examples, which are explanations of the present invention and are not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0044] The microorganisms used in the following examples are derived from Actinoplanes sp. and are deposited in the General Microbiological Center of the China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a deposit date of April 25, 2024 and a deposit number of CGMCCNO.30440.

[0045] In the following examples, the "soybean cake powder" used refers to the powder obtained by crushing and drying soybean cake after the oil is squeezed out of soybeans, for example, the soybean cake powder of Yidu Mingliang Biotechnology Co., Ltd.

[0046] In the following examples, the rapamycin fermentation broth treatment and high performance liquid chromatography (HPLC) detection methods are as follows:

[0047] Fermentation broth treatment: Take 25 mL of fermentation broth into a shake flask, then add 50 mL of acetone into the shake flask. After sealing the shake flask, place it on a shaker at 200 rpm and shake for 2 hours. Take about 5 mL from the shake flask and put it into an Eppendorf tube for centrifugation. Take the supernatant after centrifugation for HPLC detection.

[0048] Standard preparation: Dilute 700 mg of rapamycin standard with purified water to 100 mL. See the standard test spectrum for details. Figure 3 .

[0049] High performance liquid chromatography detection method: chromatographic column Waters Symmetry C8 (150 mm×4.6 mm, 3.5 μm), mobile phase acetonitrile: water = 45:55; flow rate 1.0 mL / min, column temperature 60°C, detection wavelength 277 nm; injection volume 20 μL.

[0050] Viscosity detection method: Use the digital rotational viscometer (LVDV-1T) produced by Shanghai Fangrui Instrument Co., Ltd. to detect the viscosity value.

[0051] Reducing sugar detection method: Use SBA bioanalyzer from the Institute of Biology, Shandong Academy of Sciences to detect the reducing sugar content.

[0052] Amino nitrogen detection method: Take about 20mL of fermentation broth and centrifuge at 3000r / min for 15min. Accurately aspirate 5mL of supernatant and place it in a 250mL conical flask, add 50mL of purified water and 8 drops of methyl red indicator, shake well, add 1-2 drops of 1moL / L hydrochloric acid to adjust to red, and let it stand for 3min. Titrate with sodium hydroxide titrant (0.05moL / L) until the solution turns yellow, then add 4mL of 18% neutral formaldehyde solution and let it stand for 10min. Add 10 drops of phenolphthalein indicator and titrate with sodium hydroxide titrant (0.05moL / L) until the solution turns red. Record the volume of sodium hydroxide titrant consumed.

[0053]

[0054] Where: amino nitrogen is the content of amino acid nitrogen in the sample (mg / dL);

[0055] V is the volume of sodium hydroxide titration solution consumed by the sample after adding formaldehyde (mL);

[0056] V0 is the volume of blank sodium hydroxide titration solution consumed after adding formaldehyde (mL);

[0057] C is the concentration of sodium hydroxide titration solution (moL / L);

[0058] 14.01 is the amount (mg) of amino nitrogen equivalent to each milliliter of sodium hydroxide titration solution (0.05 mol / L).

[0059] Mycelial morphology observation method: Use Ningbo Sunny Optical Instrument Co., Ltd. E5 series biological microscope for observation.

[0060] Example 1

[0061] The method for producing rapamycin by fermentation using Actinoplanes sp. (Deposit Number CGMCC NO.30440) in this embodiment comprises the following steps:

[0062] ① Plate culture: First inoculate the actinomycetes into the newly prepared plate culture medium, spread it evenly with a spreading stick, and culture it in a biochemical incubator at 28°C for 7 days. When the slope is completely covered with yellow-brown spores, the plate culture is obtained and can be used immediately or placed in a 4°C environment for later use.

[0063] The plate culture medium of this embodiment comprises the following components: 18 g / L soluble starch, 6 g / L yeast extract, 10 g / L glucose, 1 g / L calcium carbonate, 1 g / L peptone, 16 g / L agar powder, pH 7.0, prepared with purified water, and sterilized with high pressure steam at 121°C for 20 min.

[0064] ② Primary seed culture: Use an inoculation spatula to scrape about 25 cm from the cultured plate culture medium. 2 The spores were inoculated into a 1L conical flask containing 200mL of fresh primary seed culture medium and cultured on a shaker at 28℃ for 3 days with a shaker speed of 250rpm.

[0065] The shake flask seed culture medium of this embodiment comprises the following components: 20 g / L soluble starch, 20 g / L glucose, 10 g / L dry yeast, 15 g / L corn steep liquor dry powder, 3 g / L calcium carbonate, 4 g / L potassium dihydrogen phosphate, pH 7.0, prepared with tap water, and sterilized with high pressure steam at 121°C for 20 min.

[0066] ③ Secondary seed culture: The cultured primary seed liquid is inoculated at a volume ratio of 2% into a 150L seed tank containing 85L fresh secondary seed culture medium, and cultured at 28°C for 3 days, with a flow rate of 0.5-2.5vvm, a rotation speed of 100-450rpm, a tank pressure of 0.05Mpa, and dissolved oxygen>30%.

[0067] The secondary seed culture medium of this embodiment comprises the following components: 30 g / L soluble starch, 25 g / L glucose, 10 g / L dry yeast, 15 g / L corn steep liquor powder, 3 g / L calcium carbonate, 4 g / L potassium dihydrogen phosphate, pH 7.0, prepared with tap water, and sterilized with high-pressure steam at 121°C for 30 min.

[0068] ④Third-level seed culture: Inoculate the cultured secondary seed liquid at a volume ratio of 10% into a 2000L seed tank containing 750L fresh third-level seed culture medium, and culture at 28°C for 3 days, with a flow rate of 0.5-2.5vvm, a rotation speed of 100-400rpm, a tank pressure of 0.05Mpa, and dissolved oxygen>30%.

[0069] The tertiary seed culture medium of this embodiment comprises the following components: 15 g / L corn starch, 40 g / L glucose, 15 g / L dry yeast, 10 g / L corn steep liquor powder, 3 g / L calcium carbonate, 4 g / L potassium dihydrogen phosphate, 5 g / L shikimic acid, pH 7.2, prepared with tap water, and sterilized with high pressure steam at 121°C for 30 min.

[0070] ⑤ Fermentation culture: The cultured tertiary seed liquid is inoculated at a volume ratio of 10% and inoculated into a 10000L fermentation tank filled with 7000L fresh fermentation medium. It is cultured at 28°C for 11 days, with a tank pressure of 0.05Mpa, a flow rate of 0.5-2.5vvm, a rotation speed of 100-350rpm, and dissolved oxygen>30%.

[0071] Feeding process: After the start of fermentation, the reducing sugar content in the fermentation broth was monitored every 12 hours. When the reducing sugar content was lower than 15 g / L, sterilized glucose aqueous solution was added in batches to control the reducing sugar content in the fermentation broth to 10-15 g / L; the concentration of glucose in the feeding solution was 300 g / L.

[0072] The fermentation medium of this embodiment comprises the following components: 15 g / L corn starch, 40 g / L glucose, 15 g / L dry yeast, 10 g / L corn steep liquor powder, 3 g / L calcium carbonate, 4 g / L potassium dihydrogen phosphate, 5 g / L shikimic acid, pH 7.2, prepared with tap water, and sterilized with high pressure steam at 121°C for 30 min.

[0073] The fermentation time was 3 days, the sample was tested for viscosity 475cp, and the rapamycin content was 254mg / L; the fermentation time was 7 days, the sample was tested for viscosity 1096cp, and the rapamycin content was 1024mg / L; the fermentation time was 11 days, and the mycelial morphology was observed. Figure 1 The hyphae are long and intertwined, the ball size is large, the viscosity is 1735cp, and the rapamycin content is 1627mg / L.

[0074] Example 2

[0075] The process of this embodiment is basically the same as that of embodiment 1, except that the feeding process of step ⑤ is different. The feeding process refers to the feeding process disclosed in the prior art CN114381480A.

[0076] Feed medium 1: 100 g / L glucose aqueous solution, 121 °C, sterilized for 30 min.

[0077] Feed medium 2: 30 g / L phospholipid suspension solution, 121°C, sterilization for 30 min.

[0078] At 72h and 120h after the start of fermentation, 350L of phospholipid aqueous solution was added respectively; during the fermentation process, the reducing sugar content of the fermentation system was monitored every 12h. When the reducing sugar content was lower than 1g / L, 116.7L of glucose aqueous solution was added each time until the fermentation was completed. The fermentation culture time was 3 days, the viscosity of the sample was 440cp, and the rapamycin content was 220mg / L; the fermentation culture time was 7 days, the viscosity of the sample was 962cp, and the rapamycin content was 722mg / L; the fermentation culture time was 11 days, the viscosity of the sample was 1425cp, and the rapamycin content was 1468mg / L.

[0079] From the test results, it can be seen that the feed-batch process disclosed in the prior art CN114381480A in this embodiment does not improve the titer compared with Example 1, and the process is complicated and the cost is high. Therefore, the feed-batch process is not suitable for this fermentation method.

[0080] Embodiment 3-5

[0081] The processes of Examples 3-5 are basically the same as those of Example 1, except that the carbon sources for feeding in the feeding process of step ⑤ are different, wherein the concentration of the carbon source in the feeding solution is 300 g / L, and all carbon sources are sterilized. The carbon sources for feeding in these Examples, the viscosity of the fermentation broth obtained at different time points, and the rapamycin content are specifically shown in Table 1.

[0082] Table 1

[0083]

[0084] It can be seen from Table 1 that when maltodextrin is used as the carbon source in the feed solution, the content of rapamycin in the final fermentation broth is the highest, so maltodextrin is the best choice.

[0085] Embodiment 6-9

[0086] The fermentation process of Examples 6-9 is basically the same as that of Example 4, except that the reducing sugar content in the fermentation broth is controlled differently in the feeding process of step ⑤. The reducing sugar content in the fermentation broth controlled in these Examples, the viscosity of the fermentation broth obtained at different time points, and the rapamycin content are specifically shown in Table 2.

[0087] Table 2

[0088]

[0089] It can be seen from Table 2 that when the reducing sugar content in the fermentation broth is controlled to be 5-10 g / L, the rapamycin content in the final fermentation broth is the highest. Therefore, a fermentation broth reducing sugar content of 5-10 g / L is the best choice.

[0090] Examples 10-14

[0091] The fermentation process of Examples 10-14 is basically the same as that of Example 7, except that in the feeding process of step ⑤, at the beginning of fermentation, a feed solution containing different nitrogen sources is fed every day, wherein the concentration of the nitrogen source in the feed solution is 150 g / L, and all nitrogen sources are sterilized, and the amino nitrogen content in the fermentation liquid is controlled to be 100-200 mg / dL. The feed nitrogen sources of these examples and the viscosity and rapamycin content in the fermentation liquid are specifically shown in Table 3.

[0092] Table 3

[0093]

[0094] It can be seen from Table 3 that when soybean cake powder is used as the nitrogen source for feed, the content of rapamycin in the final fermentation liquid is the highest. Therefore, soybean cake powder is the best choice.

[0095] Examples 15-18

[0096] The fermentation process of Examples 15-18 is basically the same as that of Example 13, except that the amino nitrogen content in the fermentation broth is controlled differently in the feeding process of step ⑤. The amino nitrogen content in the fermentation broth controlled in these Examples, the viscosity of the fermentation broth obtained at different time points, and the rapamycin content are specifically shown in Table 4.

[0097] Table 4

[0098]

[0099] It can be seen from Table 4 that when the amino nitrogen content in the fermentation broth is controlled to be 200-300 mg / dL, the rapamycin content in the final fermentation broth is the highest. The HPLC detection spectrum is shown in Figure 4 ; and the viscosity is the highest, the mycelium morphology is Figure 2 (Step ⑤, day 11 of fermentation culture), the mycelium is shorter and the pellet size is smaller, which is more conducive to secondary metabolism and produces more rapamycin. Therefore, the amino nitrogen content in the fermentation broth is 200-300 mg / dL as the best choice.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for producing rapamycin by fermentation, characterized in that: The fermentation method comprises using Actinoplanes sp., which is preserved as CGMCC NO.30440, as a fermentation strain. During the fermentation process, when the reducing sugar content in the fermentation liquid is lower than 10 g / L, maltodextrin is added to the fermentation liquid so that the reducing sugar content in the fermentation liquid is 5-10 g / L for fermentation.

2. The method for producing rapamycin by fermentation according to claim 1, characterized in that: During the fermentation process, when the amino nitrogen content in the fermentation broth is lower than 300 mg / dL, a nitrogen source is added to make the amino nitrogen content in the fermentation broth 200-300 mg / dL, and the nitrogen source is selected from soybean cake powder.

3. The method for producing rapamycin by fermentation according to claim 2, characterized in that: The nitrogen source is first prepared into a 100-200 g / L solution and then added into the fermentation liquid; the maltodextrin is first prepared into a 250-350 g / L solution and then added into the fermentation liquid.

4. The method for producing rapamycin by fermentation according to claim 1, characterized in that: The replenishment is done intermittently.

5. The method for producing rapamycin by fermentation according to claim 1, characterized in that: The fermentation medium contains the following components: Corn starch 10-20g / L, glucose 30-50g / L, dry yeast 10-20g / L, corn syrup powder 5-15g / L, calcium carbonate 2-4g / L, potassium dihydrogen phosphate 3-5g / L, shikimic acid 4-6g / L and water.

6. The method for producing rapamycin by fermentation according to claim 1, characterized in that: The fermentation process is as follows: culturing actinomycetes in a fermentation tank, the fermentation temperature is 25-30°C, the pressure is 0.04-0.06Mpa, the ventilation rate is controlled to be 0.5-2.5vvm and the rotation speed is 100-400rpm according to the dissolved oxygen situation in the fermentation liquid, so that the dissolved oxygen in the culture liquid is greater than 30%, when the reducing sugar content in the fermentation liquid is lower than 10g / L, maltodextrin is added to the fermentation liquid to maintain the reducing sugar concentration in the fermentation liquid at 5-10g / L, when the amino nitrogen content in the fermentation liquid is lower than 300mg / dL, liquid material containing soybean cake powder is added to maintain the amino nitrogen content in the fermentation liquid at 200-300mg / dL, and the fermentation time is 9-12 days. The fermentation medium in the fermenter contains the following components: Corn starch 10-20g / L, glucose 30-50g / L, dry yeast 10-20g / L, corn syrup powder 5-15g / L, calcium carbonate 2-4g / L, potassium dihydrogen phosphate 3-5g / L, shikimic acid 4-6g / L and water.

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