Tacrolimus fermentation preparation process
By optimizing the tacrolimus fermentation medium and controlling dissolved oxygen, and adjusting the metabolic pathway of the strain, the problem of high ascomycin content during tacrolimus fermentation was solved, achieving efficient impurity removal and cost reduction.
Patent Information
- Application Number
- CN202511603106.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-01-30
AI Technical Summary
In existing technologies, the content of ascomycin, a byproduct of tacrolimus fermentation, is high and difficult to remove effectively, resulting in complex and inefficient extraction and purification.
By employing specific fermentation medium formulations and culture conditions, including the addition of L-arginine and trace element solutions, and controlling dissolved oxygen concentration, the metabolic pathways of the strains were adjusted to reduce the production of ascomycin.
It significantly reduced the ascomycin content at fermentation termination to no more than 1.5%, simplified the downstream extraction and purification process, and reduced production costs.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a tacrolimus fermentation preparation process. Background Technology
[0002] Ascomycin (FK520) is a byproduct produced during the fermentation process of tacrolimus (FK506). Due to its highly similar structure and physicochemical properties to tacrolimus, it is difficult to remove during the downstream extraction and purification stages of tacrolimus production. Therefore, ascomycin is listed as a major impurity and controlled in the tacrolimus monographs of the United States Pharmacopeia and the European Pharmacopeia.
[0003] The literature "Novel chemobiosynthetic approach for exclusive production of FK506" and "Origin of the allyl group in FK506 biosynthesis" discloses that the ascomycin content in the tacrolimus fermentation process ranges from 6% to 12%, and can even reach over 20%. The literature "Enhancement of FK506 production by engineering secondary pathways of Streptomycestsukubaensis and exogenous feeding strategies" mentions that by adding soybean oil, lactate, shikimic acid, cladonic acid, lysine, piperacillin, succinate, isoleucine, and valine exogenously, the final concentration of FK520, the main byproduct of FK506, decreased by 44% compared to the value without exogenous addition. In existing technologies, the ascomycin byproduct generated during tacrolimus fermentation is mainly removed through subsequent extraction and purification processes, either through preparative chromatography or chemical methods. These methods suffer from drawbacks such as multiple steps, complex processes, low removal efficiency, or low tacrolimus extraction yield.
[0004] This invention provides a fermentation process for preparing tacrolimus. By developing a fermentation process that can reduce the content of ascomycin, a byproduct of tacrolimus fermentation, the content of ascomycin, an impurity in tacrolimus, is reduced from the source. Summary of the Invention
[0005] The primary objective of this invention is to provide a tacrolimus fermentation preparation process that addresses the shortcomings of existing fermentation preparation methods, which result in products containing high levels of ascomycin, an impurity.
[0006] A second objective of this invention is to provide a tacrolimus.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] A tacrolimus fermentation preparation process includes the following steps:
[0009] (1) Inoculate Streptomyces tsukuba into seed culture medium and culture it to obtain a culture solution;
[0010] (2) The culture medium is inoculated into the fermentation medium for fermentation culture to obtain the fermentation broth;
[0011] The fermentation medium comprises the following components: sucrose 60-100 g / L, yeast extract 5-15 g / L, soybean peptone 2-8 g / L, L-arginine 2-4 g / L, K2HPO4·3H2O 1-3 g / L, MgSO4·7H2O 0.2-0.8 g / L, (NH4)2SO4 1-3 g / L, trace element solution 0.5-1.5 ml / L, and silica-based antifoaming agent 0.5-1.5 g / L.
[0012] In this invention, the seed culture medium comprises the following components: glucose 20-40 g / L, yeast extract 10-30 g / L, K2HPO4·3H2O 1-3 g / L, MgSO4·7H2O 0.2-0.8 g / L, and pH adjusted to 6.0-6.5.
[0013] Furthermore, the seed culture medium comprises the following components: glucose 30 g / L, yeast extract 20 g / L, K2HPO4·3H2O 2 g / L, and MgSO4·7H2O 0.5 g / L.
[0014] In this invention, the inoculum size of *Streptomyces tsukuba* on the seed culture medium is 1.0 × 10⁻⁶. 6 -1.0×10 7 spores / ml; the inoculum volume of culture medium added to the fermentation medium is 5-15%.
[0015] In this invention, the *Streptomyces tsukuba* is a strain deposited by the Agricultural Research Culture Collection (ARS) of the United States, under the accession number NRRL18488.
[0016] In this invention, the culture conditions in step (1) are to culture at 25-28°C for 40-60 hours.
[0017] Furthermore, the fermentation medium comprises the following components: 80 g / L sucrose, 10 g / L yeast extract, 5 g / L soybean peptone, 3 g / L L-arginine, 2 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 2 g / L (NH4)2SO4, 1.0 ml / L trace element solution, and 1.0 g / L silica-based antifoaming agent.
[0018] Furthermore, the pH of the fermentation medium is 7.0-7.2.
[0019] Further, the trace element solution composition is as follows: sodium molybdate dihydrate 0.2-0.4 g / L, boric acid 2.0-4.0 g / L, manganese sulfate 0.2-0.3 g / L, zinc sulfate heptahydrate 3.0-4.0 g / L, ferrous sulfate heptahydrate 1.5-2.0 g / L, calcium nitrate 5.0-6.5 g / L, citric acid 1.0-1.5 g / L, and copper sulfate 0.3-0.5 g / L.
[0020] In this invention, the culture conditions in step (2) are cultured at 26±2℃ for 7-13 days.
[0021] Furthermore, during the fermentation process, the tank pressure was 0.03±0.01 MPa, and the air flow rate was 0.5-1.5 m³ / h. 3 / h.
[0022] Furthermore, after 24 hours of fermentation, the dissolved oxygen (DO) was controlled at 30% ≤ DO ≤ 40%. The production strain, *Streptomyces tsukuba*, is an aerobic bacterium. Sufficient oxygen supply during cultivation can lead to an increase in ascomycin. Therefore, controlling the appropriate dissolved oxygen concentration can inhibit the production of ascomycin in the fermentation broth, thereby reducing the ascomycin impurity content in tacrolimus.
[0023] A tacrolimus was prepared using the above-mentioned fermentation process.
[0024] The present invention has the following beneficial effects:
[0025] (1) The tacrolimus fermentation preparation process of the present invention can adjust the metabolic pathway of the producing bacteria by adding L-arginine and trace element solution, so that the metabolism tends to produce tacrolimus and reduce the production of ascomycin.
[0026] (2) In this invention, dissolved oxygen is controlled during the fermentation of tacrolimus, which affects the metabolism of the strain and inhibits the production of ascomycin in the fermentation broth, thereby reducing the content of ascomycin impurities in tacrolimus.
[0027] Through the above process control, the ascomycin content at the end of tacrolimus fermentation is no higher than 1.5%, which is far lower than that of existing technologies. Detailed Implementation
[0028] The technical solution of the present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solution of the present invention.
[0029] This invention provides a tacrolimus fermentation preparation process that, compared with existing technologies, significantly reduces the ascomycin content at the end of fermentation. By employing a novel fermentation medium formulation and cultivation process, this invention influences strain metabolism, reducing ascomycin production and drastically lowering the ascomycin content in the fermentation broth, thereby reducing the cost of downstream refining and production.
[0030] In the following embodiments and comparative examples of this invention, *Streptomyces tsukuba* is a strain deposited at the Agricultural Research Culture Collection (ARS) in the United States, with the accession number NRRL18488. Other *Streptomyces tsukuba* strains can also achieve the technical objectives of this invention.
[0031] Example 1
[0032] A tacrolimus fermentation preparation process includes the following steps.
[0033] (1) Inoculate the seed culture medium with Streptomyces tsukuba (inoculation amount 5.0 × 10⁻⁶). 6 After measuring spores / ml, the culture medium was incubated at 26.5℃ for 50 hours to obtain the culture medium. Seed culture medium was prepared as follows: glucose 30g / L, yeast extract 20g / L, K₂HPO₄·3H₂O 2g / L, MgSO₄·7H₂O 0.5g / L, pH adjusted to 6.3.
[0034] (2) Inoculate the seed culture medium into the fermenter at a rate of 10%. Maintain the fermenter with a stirring speed of 250 rpm, a culture temperature of 26°C, a pressure of 0.03 MPa, and an air flow rate of 0.8 m³ / min. 3 / h, after culturing for 24 hours, the dissolved oxygen is controlled at 33%≤DO≤37%, and after culturing for 11 days, the fermentation broth is obtained.
[0035] Fermentation medium preparation: 80 g / L sucrose, 10 g / L yeast extract, 5 g / L soybean peptone, 3 g / L L-arginine, 2 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 2 g / L (NH4)2SO4, 1.0 ml / L trace element solution and 1.0 g / L silica-based antifoaming agent; adjust pH to 7.1.
[0036] The formula for the trace element solution (g / L) is as follows:
[0037]
[0038] (3) The fermentation broth obtained in step (2) was tested, and the ascomycin content was 1.02%. (The detection method of ascomycin and tacrolimus in this invention is in accordance with the method disclosed in the literature Novel chemobiosynthetic approach for exclusive production of FK506.)
[0039] Example 2
[0040] A tacrolimus fermentation preparation process includes the following steps.
[0041] (1) Inoculate the seed culture medium with Streptomyces tsukuba (inoculation amount 1.0 × 10⁻⁶). 7 After measuring the spores / ml, the culture medium was incubated at 25°C for 60 hours to obtain the culture medium. Seed culture medium was prepared as follows: glucose 20 g / L, yeast extract 10 g / L, K₂HPO₄·3H₂O 1 g / L, MgSO₄·7H₂O 0.2 g / L, pH adjusted to 6.0.
[0042] (2) Inoculate the seed culture medium into the fermenter at a rate of 5%. Maintain the fermenter with a stirring speed of 100 rpm, a culture temperature of 24°C, a pressure of 0.02 MPa, and an air flow rate of 1.0 m³ / min. 3 / h, after culturing for 24 hours, the dissolved oxygen is controlled at 30%≤DO≤33%, and after culturing for 13 days, it is placed in a tank to obtain the fermentation broth.
[0043] Fermentation medium preparation: sucrose 60 g / L, yeast extract 5 g / L, soybean peptone 2 g / L, L-arginine 2 g / L, K2HPO4·3H2O 1 g / L, MgSO4·7H2O 0.2 g / L, (NH4)2SO4 1 g / L, trace element solution 1.0 ml / L and silica-based antifoaming agent 1.0 g / L; adjust pH to 7.0.
[0044] The formula for the trace element solution (g / L) is as follows:
[0045]
[0046] (3) The fermentation broth obtained in step (2) was tested, and the ascomycin content was 1.23%.
[0047] Example 3
[0048] A tacrolimus fermentation preparation process includes the following steps.
[0049] (1) Inoculate the seed culture medium with Streptomyces tsukuba (inoculation amount 1.0 × 10⁻⁶). 6After measuring the spores / ml, the culture medium was incubated at 28℃ for 40 hours to obtain the culture medium. Seed culture medium was prepared as follows: glucose 40 g / L, yeast extract 30 g / L, K₂HPO₄·3H₂O 3 g / L, MgSO₄·7H₂O 0.8 g / L, pH adjusted to 6.5.
[0050] (2) Inoculate the seed culture medium into the fermenter at a rate of 15%. Maintain the fermenter with a stirring speed of 400 rpm, a culture temperature of 28°C, a pressure of 0.04 MPa, and an air flow rate of 1.5 m³ / min. 3 / h, after culturing for 24 hours, the dissolved oxygen is controlled at 37%≤DO≤40%, and after culturing for 7 days, the fermentation broth is obtained.
[0051] Fermentation medium preparation: sucrose 100g / L, yeast extract 15g / L, soybean peptone 8g / L, L-arginine 4g / L, K2HPO4·3H2O 3g / L, MgSO4·7H2O 0.8g / L, (NH4)2SO4 3g / L, trace element solution 1.0ml / L and silica-based antifoaming agent 1.0g / L; adjust pH to 7.2.
[0052] The formula for the trace element solution (g / L) is as follows:
[0053]
[0054] (3) The fermentation broth obtained in step (2) was tested, and the ascomycin content was 1.31%.
[0055] Comparative Example 1
[0056] A tacrolimus fermentation preparation process includes the following steps:
[0057] (1) The fermentation medium was not supplemented with L-arginine, and other conditions were the same as in Example 1.
[0058] (2) The fermentation broth obtained in step (1) was tested, and the ascomycin content was 6.25%.
[0059] Comparative Example 2
[0060] A tacrolimus fermentation preparation process includes the following steps:
[0061] (1) No trace element solution was added to the fermentation medium, and other conditions were the same as in Example 1.
[0062] (2) The fermentation broth obtained in step (1) was tested, and the ascomycin content was 3.22%.
[0063] Comparative Example 3
[0064] A tacrolimus fermentation preparation process includes the following steps:
[0065] (1) The fermentation medium was not supplemented with L-arginine and trace element solution, and other conditions were the same as in Example 1.
[0066] (2) The fermentation broth obtained in step (1) was tested, and the ascomycin content was 7.13%.
[0067] Comparative Example 4
[0068] A tacrolimus fermentation preparation process includes the following steps:
[0069] (1) Dissolved oxygen was controlled at DO≤30% throughout the fermentation process, and other conditions were the same as in Example 1.
[0070] (2) The fermentation broth obtained in step (1) was tested, and the ascomycin content was 2.64%.
[0071] Comparative Example 5
[0072] A tacrolimus fermentation preparation process includes the following steps:
[0073] (1) Dissolved oxygen was controlled at DO≥40% throughout the fermentation process, and other conditions were the same as in Example 1.
[0074] (2) The fermentation broth obtained in step (1) was tested, and the ascomycin content was 3.47%.
[0075] The above embodiments of the present invention are not intended to limit the scope of protection of the present invention. The implementation of the present invention is not limited thereto. All other modifications, substitutions or alterations made to the above structure of the present invention based on the above content of the present invention, in accordance with ordinary technical knowledge and common practice in the field, without departing from the basic technical idea of the present invention, shall fall within the scope of protection of the present invention.
Claims
1. A process for the fermentative production of tacrolimus, characterized in that, It comprises the following steps: (1) inoculating Streptomyces tsukubaensis into a seed culture medium for culture to obtain a culture solution; (2) inoculating the culture solution into a fermentation culture medium for fermentation culture to obtain a fermentation solution; The fermentation culture medium comprises the following components: sucrose 60-100 g / L, yeast extract 5-15 g / L, soybean peptone 2-8 g / L, L-arginine 2-4 g / L, K2HPO4·3H2O 1-3 g / L, MgSO4·7H2O 0.2-0.8 g / L, (NH4)2SO4 1-3 g / L, trace element solution 0.5-1.5 ml / L, and silicon-based antifoam agent 0.5-1.5 g / L.
2. The process for the fermentation preparation of tacrolimus according to claim 1, characterized in that, The seed culture medium comprises the following components: glucose 20-40 g / L, yeast extract 10-30 g / L, K2HPO4·3H2O 1-3 g / L, MgSO4·7H2O 0.2-0.8 g / L, and pH is adjusted to 6.0-6.
5.
3. The process for the fermentation preparation of Tacrolimus according to claim 2, characterized by, The inoculation amount of Streptomyces tsukubaensis to the seed culture medium was 1.0 x 10 6 -1.0 x 10 7 spores / ml; the inoculation amount of the culture solution to the fermentation medium was 5-15%.
4. The process for the fermentation preparation of Tacrolimus according to claim 1, characterized in that, The Streptomyces tsukubaensis is a strain preserved by the American Type Culture Collection (ATCC) with the collection number NRRL18488; the culture condition in step (1) is 25-28℃ for 40-60 hours.
5. The process for the fermentation preparation of Tacrolimus according to any of claims 1 to 4, characterized in that, The pH of the fermentation culture medium is 7.0-7.
2.
6. The process for the fermentation preparation of Tacrolimus according to claim 5, characterized by, The trace element solution comprises the following components: sodium molybdate 0.2-0.4 g / L, boric acid 2.0-4.0 g / L, manganese sulfate 0.2-0.3 g / L, zinc sulfate 3.0-4.0 g / L, ferrous sulfate 1.5-2.0 g / L, calcium nitrate 5.0-6.5 g / L, citric acid 1.0-1.5 g / L, and copper sulfate 0.3-0.5 g / L.
7. The process for the fermentation preparation of Tacrolimus according to claim 5, characterized by, The culture condition in step (2) is 26±2℃ for 7-15 days.
8. The process for the fermentation preparation of Tacrolimus according to claim 7, characterized by, During the fermentation culture process, the tank pressure is 0.03±0.01 Mpa, and the air flow is 0.5-1.5 m 3 / h.
9. The process for the fermentation preparation of Tacrolimus according to claim 7, characterized by, Dissolved oxygen (DO) control after 24 hours of fermentation culture: 30%≤DO≤40%.
10. Tacrolimus characterized in that, Prepared by the fermentation preparation process of any one of claims 1-9.