Method for acquiring chlamydomonas reinhardtii source recombinant hirudin algae powder by means of fermentation culture method
The fermentation culture method was used to obtain recombinant hirudin powder from Chlamydomonas reinhardtii, which solved the problems of high production cost, long cycle and batch instability of recombinant hirudin. It provided a low-cost, biosafe long-acting anticoagulant, avoided the risk of infection and achieved stable production.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- WUHAN YUANQI HE SHENG TECHNOLOGIAE SOCIETAS LTD
- Filing Date
- 2024-11-20
- Publication Date
- 2026-05-15
AI Technical Summary
Existing recombinant hirudin has high production costs, long growth cycles, and unstable batches. Frequent injections also pose risks of infection and bleeding side effects. Furthermore, commercially available products lack post-translation modification functions.
The fermentation culture method was adopted, using TAP medium and sodium acetate and acetic acid to adjust the pH, adding corn oil as an antifoaming agent, and controlling the temperature, dissolved oxygen and stirring speed. The fermentation culture was carried out by stepwise gradient scale-up, and the recombinant hirudin algal powder from Chlamydomonas reinhardtii was obtained by low temperature vacuum freeze drying.
It achieves low cost, biosafety, and stable production, avoids infection risks, provides long-lasting anticoagulants, does not occupy arable land, and has a short production cycle.
Smart Images
Figure CN2024133289_15052026_PF_FP_ABST
Abstract
Description
A method for obtaining recombinant hirudin algal powder derived from Chlamydomonas reinhardtii via fermentation culture. Technical Field
[0001] This invention relates to the field of fermentation production, and specifically to a method for obtaining recombinant hirudin algal powder from Chlamydomonas reinhardtii via fermentation culture. Background Technology
[0002] Chlamydomonas reinhardtii is a single-celled eukaryotic organism that normally photosynthesizes using light as its energy source. However, in the dark, it can grow heterotrophically using acetate as its sole carbon source. Its cultivation process is as simple as that of yeast and other microorganisms, earning it the nicknames "green yeast" and "photosynthetic yeast." Chlamydomonas reinhardtii is rich in nutrients, with a protein content as high as 46.9% by dry weight, comparable to superfoods like Chlorella (45.3%) and Spirulina (50.4%). It also contains 12.5% crude polysaccharides, 11.9% dietary fiber, and contains all eight essential amino acids, various trace elements, unsaturated fatty acids, and various forms of biotin. Chlamydomonas reinhardtii is considered a safe food ingredient in the United States, the European Union, Singapore, Hong Kong, and other countries and regions. In May 2022, the National Health Commission of China officially approved Chlamydomonas reinhardtii as a new food ingredient, allowing for "unrestricted consumption." Due to its high nutritional value, it has broad development prospects in fields such as alternative protein raw materials, medicine, brewing, food additives, and feed.
[0003] Hirudin is a polypeptide extracted from the salivary glands of leeches. It can specifically bind to thrombin to prevent thrombus formation and has been developed as an effective anticoagulant. The FDA has successively approved recombinant hirudin and its derivatives, such as bivalirudin, lepirudin, and desirudin, mainly for the prevention of complications in thromboembolic diseases, the prevention of arterial thrombosis after surgery, and the prevention of thrombus formation after thrombolysis or angiogenesis. However, as a polypeptide-based biological drug, conventional recombinant hirudin and its derivatives have a short half-life in vivo after being administered via gastrointestinal injection. Frequent injections are required to achieve the anticoagulant effect in clinical patients, which carries a risk of infection. Furthermore, the clinical application of anticoagulants commonly results in bleeding as a side effect. Therefore, the development of a long-acting formulation is extremely important. In addition, since commercially available recombinant hirudin products are mostly derived from engineered E. coli or yeast, they lack post-translational modification capabilities. Chlamydomonas reinhardtii, as a novel chassis organism, possesses this capability, undergoing sulfation modification on 87Tyr, resulting in higher activity than recombinant hirudin products derived from other yeasts, E. coli, etc.
[0004] Recombinant hirudin derived from Chlamydomonas reinhardtii is cultivated through fermentation, which has a lower production cost than other similar drugs or recombinant hirudin from other sources. It also has a shorter growth cycle and more stable production batches (currently, plant-derived recombinant protein drugs on the market rely on rice cultivation, which requires arable land, has a long growth cycle, and has unstable batch sources). Summary of the Invention
[0005] To address the aforementioned problems in the existing technology, the present invention aims to provide a fermentation and cultivation method for obtaining recombinant hirudin-based algal powder from Chlamydomonas reinhardtii. To solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] A method for obtaining recombinant hirudin-derived Chlamydomonas reinhardtii strains by fermentation culture is characterized in that the main body of the fermentation broth is TAP medium, the supplementary culture medium is 50×TAP concentrate without Tirs, and sodium acetate and acetic acid are added.
[0007] In a preferred embodiment, the concentration of added sodium acetate is 0.5 mol / L; the concentration of acetic acid is 20%.
[0008] As a preferred embodiment, the pH value is adjusted using 1 mol / L sodium hydroxide and 20% acetic acid.
[0009] As a preferred embodiment, corn oil is used as the defoamer during the cultivation process.
[0010] As a preferred embodiment, the culture conditions are: temperature: 23-28℃, pH: 6.0-8.0, dissolved oxygen: >15%, stirring speed: >100rpm.
[0011] As a preferred embodiment, the fermentation cultivation method is characterized by using a gradient-scaled fermentation cultivation method, as shown in Figure 1 / 2.
[0012] As a preferred embodiment, the fermentation cultivation method is characterized in that the recombinant hirudin algal powder is obtained by low-temperature vacuum freeze-drying at a temperature of -20℃ to -80℃. Beneficial effects:
[0013] To achieve efficient expression of recombinant hirudin, this invention designs a method for obtaining recombinant hirudin algal powder from *Chlamydomonas reinhardtii* via fermentation. Compared with existing recombinant protein drug production systems, this expression system has the following main advantages:
[0014] 1. Low production cost; 2. Biosafety (obtained safety certification and approval from the U.S. Food and Drug Administration and the National Health Commission of China in 2019 and 2022 respectively, allowing for direct and unlimited consumption), metabolites do not contain endotoxins or other toxic substances, and cells do not carry pathogenic microorganisms; 3. High-density fermentation culture is used, which does not occupy arable land and ensures product quality stability; 4. Cell culture operation is simple, production cycle is short, saving a lot of time. Attached Figure Description
[0015] Figure 1 Microalgae fermentation process flow
[0016] Figure 2. Algal powder obtained from fermentation culture of recombinant hirudin-expressing algal strains.
[0017] Specific implementation methods
[0018] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.
[0019] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.
[0020] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.
[0021] 1. Preparation of fermentation medium: The main fermentation medium is TAP medium; the pH is adjusted with 1 mol / L sodium hydroxide and 20% acetic acid; corn oil is used as an antifoaming agent; the fed medium is 50×TAP concentrate without Tirs, with the addition of 0.5 mol / L sodium acetate and 20% acetic acid.
[0022] 2. Seed culture: The mutant strain activated by TAP plates was inoculated into a triangular shaker flask of liquid TAP medium (1 / 10 of the seed tank) and placed in a shaker for culture at 200 rpm / min, 24℃, and a photoperiod (light:dark = 14:10h) until the late logarithmic growth phase.
[0023] 3. Sterilize the empty fermentation tank and pipelines at 121℃ for 30 minutes.
[0024] 4. Electrode calibration: The dissolved oxygen electrode is calibrated at zero point in saturated sodium sulfite, and the pH electrode is calibrated in standard buffer solution.
[0025] 5. Inoculation: Inject anhydrous ethanol into the inoculation port, ignite it, and then insert the seed through the inoculation port.
[0026] 6. Fermentation tank parameter settings: Transfer the Chlamydomonas reinhardtii culture from step 2, which has reached the late logarithmic growth stage, into a 5L fermentation tank for fermentation culture. Set A. Temperature: 23-28℃; B. pH: 6.0-8.0; C. Dissolved oxygen: >15%; D. Stirring speed: >100rpm. Control the stirring speed and aeration rate. Start checking for foam and defoaming 3 days after inoculation.
[0027] 7. Fermentation status: Biomass is measured every 12 hours 3 days after inoculation. When the biomass growth rate is less than 10% every 12 hours, the fermentation can be stopped and the fermentation can be terminated.
[0028] 8. Fermentation cycle: The entire cycle is controlled at around 15 days, and around 7 days for a 2T tank;
[0029] 9. Collect algal powder: Collect the centrifuged algal mud and freeze-dry it at -20℃~-80℃ under low temperature and vacuum to obtain Chlamydomonas reinhardtii recombinant hirudin algal powder.
[0030] Related formulas
[0031] 1. TAP medium should be prepared in the following order:
[0032] 2. Prepare Phosphate Buffer II (for 100ml) in the following order:
[0033] 3. Solution A (for 500ml) should be prepared in the following order:
[0034] 4. Dissolve and mix the TAP trace elements (for 1L) separately in the following order:
[0035] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
Claims
1. A method for obtaining a recombinant hirudin-derived Chlamydomonas reinhardtii strain through fermentation culture, characterized in that, The main body of the fermentation broth was TAP medium, and the supplemental medium was 50×TAP concentrate without Tirs, with the addition of sodium acetate and acetic acid.
2. The fermentation cultivation method according to claim 1, characterized in that, The added sodium acetate concentration was 0.5 mol / L, and the acetic acid concentration was 20%.
3. The fermentation cultivation method according to claim 1, characterized in that, pH was adjusted using 1 mol / L sodium hydroxide and 20% acetic acid.
4. The fermentation cultivation method according to claim 1, 2, or 3, characterized in that, The defoaming agent used during the cultivation process is corn oil.
5. The fermentation cultivation method according to claim 1, 2, or 3, characterized in that, The cultivation conditions are: temperature: 23-28℃, pH: 6.0-8.0, dissolved oxygen: >15%, stirring speed: >100rpm.
6. The fermentation cultivation method according to claim 1, 2, or 3, characterized in that, Fermentation culture was carried out using a gradient-scale-step-by-step approach.
7. The fermentation cultivation method according to claim 1, 2, or 3, characterized in that, The recombinant hirudin algal powder was obtained by low-temperature vacuum freeze drying at a temperature of -20℃ to -80℃.