Method for producing sirolimus through streptomyces fermentation
By optimizing the Streptomyces fermentation process, including seed-liquid volume ratio, temperature and dissolved oxygen management, and continuous feeding strategy, the problems of low potency and long cycle in sirolimus production have been solved, achieving efficient fermentation and cost reduction.
Patent Information
- Application Number
- CN202511991623.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-06
AI Technical Summary
The existing sirolimus production process suffers from low potency and long production cycles, resulting in low production efficiency.
The method for producing sirolimus by Streptomyces fermentation includes controlling the seed liquid volume ratio, managing fermentation temperature and aeration ratio, linking dissolved oxygen and stirring speed, continuously fed culture medium, and substrate feedback strategy to optimize the fermentation process.
It significantly improved the fermentation unit of sirolimus, increased the fermentation potency by 90.02% to 120.63%, reduced production costs, and ensured the stability of large-scale production.
Smart Images
Figure CN121472346A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of antibiotic fermentation technology, and more specifically to a method for producing sirolimus by fermentation with Streptomyces. Background Technology
[0002] Sirolimus, also known as rapamycin, is an immunosuppressant used to prevent rejection in organ transplants; it is particularly useful in kidney transplants. Sirolimus is a macrolide antibiotic originally discovered as a product of *Streptomyces hygroscopicus* found in soil samples. Sirolimus was initially developed as an antifungal agent.
[0003] The main uses of sirolimus are: Medical applications: Sirolimus is an immunosuppressant used to prevent rejection after kidney and liver transplants.
[0004] Research Applications: Sirolimus inhibits cell motility by suppressing the mTOR-mediated S6K1 and 4E-BP1 pathways. Rapamycin inhibits the activation / proliferation of T lymphocytes in response to antigens and cytokines (interleukins IL-2, IL-4, and IL-15), and its mechanism differs from other immunosuppressants. Sirolimus also inhibits antibody production.
[0005] The current production process for sirolimus suffers from bottlenecks such as low potency and long production cycles.
[0006] Currently, domestic sirolimus fermentation units generally maintain a bioavailability level of 200-300 u / ml. The production method basically adopts no feeding or intermittent feeding of the whole material at fixed cycles. If high viscosity occurs after feeding, water is generally added to control the viscosity. The fermentation process is relatively simple to control.
[0007] Therefore, there is an urgent need for a fermentation medium and cultivation method that can effectively improve the fermentation unit of sirolimus. Summary of the Invention
[0008] This invention provides a method for producing sirolimus by Streptomyces fermentation, which can effectively improve the fermentation medium and culture method of sirolimus fermentation units.
[0009] Specifically, this invention discloses a method for producing sirolimus by fermentation with Streptomyces. The method includes the following steps: activating and seeding the sirolimus-producing strain; inoculating the seed culture medium with a liquid volume ratio of 8-15% v / v; controlling the fermentation temperature at 28-30℃; the aeration ratio at 0.6-1.4 vvm; linking dissolved oxygen and stirring speed, controlling the dissolved oxygen concentration at ≥30% and the stirring speed at 200-600 rpm; fermenting for 48-90 hours; when the glucose content of the fermentation broth is lower than 1 g / L, continuously feeding culture medium is started; the pH is controlled at 5.0-6.5 throughout the fermentation process; fermentation ends after 150-168 hours.
[0010] Preferably, the seed culture preparation method is as follows: the sirolimus producing strain is inoculated into the primary seed culture medium at an inoculation rate of 1% v / v for primary seed culture in shake flasks. After culturing for 40-48 hours, it is transferred to the secondary seed culture medium at an inoculation rate of 5-10% v / v for secondary seed culture. After culturing for another 40-48 hours, it is inoculated into a fermenter at an inoculation rate of 8-15% v / v. The seed culture temperature is 28-30℃.
[0011] Preferably, the seed culture medium, basal culture medium, and supplemental culture medium do not contain animal-derived components.
[0012] Preferably, the basal culture medium is formulated with the following components (g / 100mL): dextrin 2.0–3.0, glucose 5.0–10.0, glycerol 1.0–2.0, soybean meal 3.0–5.0, dipotassium hydrogen phosphate 0.2–0.5, yeast extract 0.5–1.0, sodium chloride 0.3–0.5, cobalt chloride hexahydrate 0.0005–0.0025, lysine hydrochloride 0.5–1.0, maltose 50–100, calcium carbonate 0.2–0.5; pH 6.8–7.0. Preferably, the feed culture medium is formulated with the following components in g / 100mL: 0-1.0 g of dextrin, 0-5.0 g of glucose, 0-1.0 g of soybean meal, 0.5-1.0 g of lysine hydrochloride, 0-5 g of maltose, and pH 6.8-7.5.
[0013] Preferably, the seed culture medium includes: The formulation of the primary seed culture medium (g / 100mL) is as follows: dextrin 2.0–3.0, glucose 0.5–1.0, glycerol 1.0–2.0, soybean meal 1.0–2.0, dipotassium hydrogen phosphate 0.2–0.5, light calcium carbonate 0.1–0.5, pH 6.8–7.5; The formulation of the secondary seed culture medium (g / 100mL) is as follows: dextrin 2.5–3.5, glucose 0.5–1.0, glycerol 1.0–2.0, soybean meal 1.5–2.0, dipotassium hydrogen phosphate 0.2–0.5, peptone 0.3–0.8, light calcium carbonate 0.1–0.5, pH 6.8–7.5.
[0014] Preferably, a substrate feedback strategy is used for continuous feeding, and the timing and speed of feeding are determined by detecting the glucose content of the substrate during fermentation.
[0015] Preferably, the timing and rate of feeding are as follows: no feeding is performed from 0 to 48 hours; after 48 hours, when the glucose content of the fermentation broth is lower than 1 g / L, feeding medium is started; the glucose content is controlled at 1 to 5 g / L by adjusting the feeding rate; the feeding rate is controlled at 1 to 5 ml / L / h throughout the fermentation process.
[0016] Preferably, the dissolved oxygen control level varies accordingly at different fermentation stages. During the initial fermentation stage (0-48h), the dissolved oxygen concentration is controlled at ≥40%, while during the middle and later stages (48-168h), the dissolved oxygen concentration is controlled at ≥30%. This is achieved by adjusting the stirring speed and aeration rate.
[0017] Compared with the prior art, the beneficial effects of the present invention are: The method for producing sirolimus by Streptomyces fermentation provided by this invention not only improves the potency stability of different batches, but also ensures the normal operation of large-scale production.
[0018] In a preferred embodiment of the present invention, the fermentation potency of sirolimus is increased by 90.02% to 120.63% compared with the traditional process, which significantly reduces production costs. Attached Figure Description
[0019] Figure 1 The liquid phase spectrum of the fermentation broth prepared in Example 2 of this invention; Figure 2 This is a liquid phase spectrum of the fermentation broth prepared in Comparative Example 2 of the present invention. Detailed Implementation
[0020] The present invention will now be described in detail with reference to various embodiments. However, it should be noted that these embodiments are not intended to limit the present invention. Equivalent changes or substitutions in function, method, or structure made by those skilled in the art based on these embodiments are all within the protection scope of the present invention.
[0021] The seed culture medium, basal culture medium and supplemental culture medium in this invention do not contain animal-derived components, such as fish meal, feather meal, silkworm pupa meal, animal-derived peptone, etc.
[0022] Example 1 This embodiment provides a method for producing sirolimus by Streptomyces fermentation. The specific formulation of the culture medium and the fermentation process are as follows.
[0023] Culture medium formulation: The primary seed culture medium consists of: 2.0 g of dextrin, 0.5 g of glucose, 1.0 g of glycerol, 1.0 g of soybean meal, 0.2 g of dipotassium hydrogen phosphate, and 0.1 g of light calcium carbonate. The pH is 6.8–7.5.
[0024] The secondary seed culture medium consists of: 2.5g dextrin, 0.5g glucose, 1.0g glycerol, 1.5g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.3g peptone, and 0.1g light calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 6.8–7.5.
[0025] The basal culture medium is formulated with the following components: 2.0g dextrin, 5.0g glucose, 1.0g glycerol, 3.0g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.5g yeast extract, 0.3g sodium chloride, 0.0005g cobalt chloride hexahydrate, 0.5g lysine hydrochloride, 50g maltose, and 0.2g calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 7.0.
[0026] The feed culture medium is formulated with the following components: 0.5 g dextrin, 5.0 g glucose, 0.5 g soybean meal, 0.5 g lysine hydrochloride, and 5 g maltose. The concentrations of these components are in g / 100 mL. The pH is 7.5.
[0027] Fermentation process: After activation, the sirolimus-producing bacteria were inoculated into primary seed culture medium at a rate of 1% (v / v) for primary seed culture in shake flasks. After 42 hours of culture, the culture was transferred to secondary seed culture medium at a rate of 5% (v / v) for secondary seed culture in shake flasks, and cultured for another 40 hours. The seed culture temperature was 30℃±1℃, and the shaker speed was 230 rpm.
[0028] The secondary seed culture was inoculated into the basal medium of a 15L fermenter at an inoculation rate of 8% (v / v), and the fermentation temperature was controlled at 30℃±1℃; the aeration ratio was 0.8~1.2vvm. Dissolved oxygen and stirring speed were linked, and the stirring speed was 200~600rpm.
[0029] During the initial fermentation stage (0-48h), the dissolved oxygen concentration should be controlled at ≥40%, and during the middle fermentation stage (48-168h), the dissolved oxygen concentration should be controlled at ≥32%. This can be achieved by adjusting the stirring speed and aeration rate.
[0030] No feeding was performed for 0-30 hours. After 30 hours, if the glucose content was below 2 g / L, continuously fed culture medium was initiated, with the feeding rate adjusted to control the glucose content between 1 and 5 g / L. The feeding rate varied from 1 to 5 ml / L / h during fermentation. The pH was maintained between 5.5 and 6.5. Fermentation was terminated after 168 hours. At the end of fermentation, the fermentation titer was 820 g / mL as measured by liquid chromatography.
[0031] Example 2 This embodiment provides a method for producing sirolimus by Streptomyces fermentation. The specific formulation of the culture medium and the fermentation process are as follows.
[0032] Culture medium formulation: The formulations of the primary and secondary seed culture media are the same as those in Example 1.
[0033] The basal culture medium is formulated with the following components: 2.0g dextrin, 5.0g glucose, 1.0g glycerol, 3.0g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.5g yeast extract, 0.3g sodium chloride, 0.0005g cobalt chloride hexahydrate, 0.5g lysine hydrochloride, 55g maltose, and 0.2g calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 6.8.
[0034] The feed culture medium is formulated with the following components: 0.5 g dextrin, 5.0 g glucose, 0.5 g soybean meal, 0.5 g lysine hydrochloride, and 5 g maltose. The concentrations of these components are in g / 100 mL. The pH is 7.2.
[0035] Fermentation process: After activation, the sirolimus-producing bacteria were inoculated into primary seed culture medium at a rate of 1 v / v% for primary seed culture in shake flasks. After 46 h of culture, the culture was transferred to secondary seed culture medium at a rate of 8 v / v% for secondary seed culture in shake flasks, and cultured for another 45 h. The seed culture temperature was 30℃±1℃, and the shaker speed was 220 rpm.
[0036] The secondary seed culture was inoculated into the basal medium of a 15L fermenter at an inoculation rate of 12% (v / v), and the fermentation temperature was controlled at 30℃±1℃; the aeration ratio was 0.8~1.2vvm. Dissolved oxygen and stirring speed were linked, and the stirring speed was 200~600rpm.
[0037] During the initial fermentation stage (0-42 hours), the dissolved oxygen concentration should be controlled at ≥40%, and during the middle fermentation stage (42-168 hours), the dissolved oxygen concentration should be controlled at ≥35%. This is achieved by adjusting the stirring speed and aeration rate.
[0038] No feeding was performed for 0-40 hours. After 40 hours, the glucose content was below 1 g / L, at which point a continuously fed culture medium was initiated. The feeding rate was adjusted to control the glucose content at 1-5 g / L. The feeding rate varied from 1-5 ml / L / h during fermentation. The pH was maintained between 5.0 and 6.0. Fermentation was terminated after 165 hours. At the end of fermentation, the concentration of the fermentation broth was 809 mg / L according to liquid chromatography.
[0039] Liquid phase spectrum as follows Figure 1 As shown, the peak with a retention time of 23.5 is sirolimus.
[0040] Example 3 This embodiment provides a method for producing sirolimus by Streptomyces fermentation. The specific formulation of the culture medium and the fermentation process are as follows.
[0041] Culture medium formulation: The formulations of the primary and secondary seed culture media are the same as those in Example 1. The formulation of the seed tank is the same as that of the secondary seed culture media.
[0042] The basal culture medium is formulated with the following components: 2.0g dextrin, 5.0g glucose, 1.0g glycerol, 3.0g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.5g yeast extract, 0.3g sodium chloride, 0.0005g cobalt chloride hexahydrate, 0.5g lysine hydrochloride, 60g maltose, and 0.2g calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 7.48.
[0043] The feed culture medium is formulated with the following components: 0.5g dextrin, 5.0g glucose, 0.5g soybean meal, 0.55g lysine hydrochloride, and 5g maltose. The concentrations of these components are in g / 100mL. The pH is 7.28.
[0044] Fermentation process: The cultivation methods for primary and secondary seeds were the same as in Example 1. The secondary seed culture was inoculated into a 15L seed tank at an inoculation rate of 9% (v / v) and cultured for another 42 hours. The seed tank culture temperature was 30℃±1℃, the rotation speed was 220 rpm, the aeration was 0.8~1.0 vvm, and the tank pressure was 0.02~0.03 MPa.
[0045] The seed culture from a 15L seed tank was inoculated into a 50L fermenter containing 30L of basal culture medium. The fermentation temperature was controlled at 30℃±1℃, and the aeration ratio was 0.7~1.0vvm. The tank pressure was 0.03~0.05MPa. Dissolved oxygen and stirring speed were linked, with the stirring speed set at 200~600rpm.
[0046] During the initial fermentation stage (0-40 hours), the dissolved oxygen concentration should be controlled at ≥40%, and during the middle fermentation stage (40-168 hours), the dissolved oxygen concentration should be controlled at ≥30%. This is achieved by adjusting the stirring speed and aeration rate.
[0047] No feeding was performed for 0-40 hours. After 40 hours, when the glucose content was below 1 g / L, continuously fed culture medium was introduced, with the glucose content controlled at 1-5 g / L by adjusting the feeding rate. The feeding rate varied from 1-5 ml / L / h during fermentation. The pH was maintained between 5.0 and 6.5. Fermentation was completed after 168 hours. The fermentation broth volume was 36 L. Liquid chromatography analysis showed a concentration of 815 mg / L.
[0048] Compare with Example 1 Culture medium formulation: The primary seed culture medium consists of: 2.0 g of dextrin, 0.5 g of glucose, 1.0 g of glycerol, 1.0 g of soybean meal, 0.2 g of dipotassium hydrogen phosphate, and 0.1 g of light calcium carbonate. The pH is 6.8–7.5.
[0049] The secondary seed culture medium consists of: 2.5g dextrin, 0.5g glucose, 1.0g glycerol, 1.5g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.3g peptone, and 0.1g light calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 6.8–7.5.
[0050] The basal culture medium is formulated with the following components: 2.0g dextrin, 5.0g glucose, 1.0g glycerol, 3.0g soybean meal, 0.2g dipotassium hydrogen phosphate, 0.5g yeast extract, 0.3g sodium chloride, 0.0005g cobalt chloride hexahydrate, 0.5g lysine hydrochloride, 50g maltose, and 0.2g calcium carbonate. The concentrations of these components are in g / 100mL. The pH is 7.1.
[0051] No feed was added during fermentation; sterile water was only added later when the fermentation broth viscosity became very high. The fermentation cycle was 165 hours. Other fermentation control parameters were similar to those in the previous example. Liquid chromatography analysis showed the concentration at the end of fermentation to be 235 mg / L.
[0052] Compare with Example 2: The formulations of the seed culture medium and basal culture medium were the same as those in Control Example 1, but the entire material was added intermittently three times during fermentation, with a fermentation cycle of 169 hours. Other fermentation control parameters were similar to those in the previous example. Liquid chromatography analysis showed that the concentration at the end of fermentation was 225 mg / L.
[0053] Liquid phase spectrum as follows Figure 2 As shown.
[0054] In summary, the liquid phase yield of the fermentation broth in Examples 1-3 was increased by 244.26% to 264.44%. The method for producing sirolimus by Streptomyces fermentation provided by this invention not only improves the potency stability of different batches but also ensures the normal operation of large-scale production.
[0055] The above descriptions are merely some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. A method for producing sirolimus by fermentation with Streptomyces, characterized in that, The method includes the following steps: activating and seed culture of the sirolimus-producing strain; inoculating the seed culture medium with a liquid volume ratio of 8-15% v / v; controlling the fermentation temperature at 28-30℃; aeration ratio of 0.6-1.4 vvm; linking dissolved oxygen and stirring speed, controlling the dissolved oxygen concentration to ≥30% and the stirring speed to 200-600 rpm; fermentation time is 48-90 h; when the glucose content of the fermentation broth is lower than 1 g / L, continuously feeding culture medium is started; the pH is controlled at 5.0-6.5 throughout the fermentation process; fermentation is completed after 150-168 h.
2. The method according to claim 1, characterized in that, The method for preparing the seed culture is as follows: the sirolimus producing strain is inoculated into the primary seed culture medium at an inoculation rate of 1% v / v for primary seed culture in shake flasks. After culturing for 40-48 hours, it is transferred to the secondary seed culture medium at an inoculation rate of 5-10% v / v for secondary seed culture. After culturing for another 40-48 hours, it is inoculated into the fermenter at an inoculation rate of 8-15% v / v. The seed culture temperature is 28-30℃.
3. The method according to claim 2, characterized in that, The seed culture medium, basal culture medium, and supplemental culture medium do not contain any animal-derived components.
4. The method according to claim 3, characterized in that, The basal culture medium is formulated with the following components (g / 100mL): dextrin 2.0–3.0, glucose 5.0–10.0, glycerol 1.0–2.0, soybean meal 3.0–5.0, dipotassium hydrogen phosphate 0.2–0.5, yeast extract 0.5–1.0, sodium chloride 0.3–0.5, cobalt chloride hexahydrate 0.0005–0.0025, lysine hydrochloride 0.5–1.0, maltose 50–100, calcium carbonate 0.2–0.5; pH 6.8–7.
0.
5. The method according to claim 4, characterized in that, The feed culture medium is formulated with the following components in g / 100mL: 0-1.0g of dextrin, 0-5.0g of glucose, 0-1.0g of soybean meal, 0.5-1.0g of lysine hydrochloride, 0-5g of maltose, and pH 6.8-7.
5.
6. The method according to claim 5, characterized in that, The seed culture medium includes: The formulation of the primary seed culture medium (g / 100mL) is as follows: dextrin 2.0–3.0, glucose 0.5–1.0, glycerol 1.0–2.0, soybean meal 1.0–2.0, dipotassium hydrogen phosphate 0.2–0.5, light calcium carbonate 0.1–0.5, pH 6.8–7.5; The formulation of the secondary seed culture medium (g / 100mL) is as follows: dextrin 2.5–3.5, glucose 0.5–1.0, glycerol 1.0–2.0, soybean meal 1.5–2.0, dipotassium hydrogen phosphate 0.2–0.5, peptone 0.3–0.8, light calcium carbonate 0.1–0.5, pH 6.8–7.
5.
7. The method according to claim 6, characterized in that, A substrate feedback strategy was adopted for continuous feeding, and the timing and speed of feeding were determined by detecting the glucose content of the substrate during fermentation.
8. The method according to claim 7, characterized in that, The timing and rate of feeding are as follows: no feeding is performed from 0 to 48 hours; after 48 hours, when the glucose content of the fermentation broth is lower than 1 g / L, feeding medium is started; the glucose content is controlled at 1 to 5 g / L by adjusting the feeding rate; the feeding rate is controlled at 1 to 5 ml / L / h throughout the fermentation process.
9. The method according to claim 8, characterized in that, The dissolved oxygen control level varies at different fermentation stages. During the initial fermentation stage (0-48h), the dissolved oxygen concentration is controlled at ≥40%, while during the middle and later stages (48-168h), the dissolved oxygen concentration is controlled at ≥30%. This is achieved by adjusting the stirring speed and aeration rate.