Method for synthesizing calcitriol through biological fermentation of pichia guilliermondii GXDK6 and application
Through the biofermentation of Pichia jiyemon's GXDK6 strain, the use of cheap carbon sources and conventional culture media to optimize the fermentation conditions, the problems of high production costs, complex processes and limited material sources are solved, and low-cost, environmentally friendly large-scale production is achieved.
Patent Information
- Application Number
- CN202510182501.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, calcitriol has high production costs, complex processes, high environmental hazards, limited material sources and long production cycles, making it difficult to achieve large-scale production.
The Pichia citrus citrus GXDK6 strain was used to bioferment using cheap carbon sources such as glucose and conventional culture media, and the fermentation conditions such as temperature, pH and salt concentration were optimized to achieve efficient synthesis of calcitriol.
It provides a low-cost and environmentally friendly calcitriol production method, which is suitable for large-scale production, simplifies the process flow and reduces production costs.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of microbial fermentation technology and biopharmaceuticals, and particularly relates to a method for synthesizing calcitriol by microbial fermentation. Technical Background
[0002] In 1971, DeLuca, Kodicek and Norman jointly discovered calcitriol, the active form of vitamin D3. Calcitriol is also known as 1α,25-dihydroxyvitamin D3, caltriol, dihydroxycholecalciferol, Rocaltrol, and its chemical name is (5Z,7E)-(1S,3R)-9,10-secocholesta-5,7,10(19)-triene-1,3,25-triol. Calcitriol is the activated form of vitamin D in the human body, the most intuitive and effective indicator for evaluating the vitamin D nutritional status in vivo, and has many important physiological activities. First, after being formed in the kidneys, calcitriol reaches the intestines and bones through the blood, promotes calcium and phosphorus absorption and bone calcium mobilization, and increases the blood calcium level, which is used to treat rickets, osteoporosis, etc. Secondly, calcitriol is an immunomodulator that acts on a variety of immune cells and is clinically used in the research of leukemia, systemic lupus erythematosus, rheumatoid arthritis, etc. At present, calcitriol has also been found to regulate the growth and differentiation of leukemia cells, tumor cells, and skin cells, and has been used in the treatment research of various cancers. In addition to animals being able to naturally synthesize calcitriol, it has been found that some plants can also synthesize it, with the highest content in Salix liukiuensis, but no calcitriol biosynthesized from Pichia guilliermondii has been found.
[0003] At present, the large-scale production methods of calcitriol mainly include two categories: chemical total synthesis and semi-synthesis. Total synthesis uses small molecule fragments as starting materials, while semi-synthesis directly modifies compounds with similar structures. According to the reports in the literature, calcitriol can be disassembled into three synthons, namely A-ring, CD-ring, and side chain. Most total synthesis routes first combine the CD-ring and the side chain, and then condense the combination with the A-ring to form a trans-diene. Such methods have complex processes, long flowcharts, and low total product yields. Semi-synthesis refers to starting from suitable monomers and synthesizing them in a flexible manner. Among them, the route using natural steroids as monomers is the most characteristic, including vitamin D2, stigmasterol, 7-dehydrocholesterol, diosgenin, etc. Each monomer synthesis has its own advantages and disadvantages, and the hydroxyl group must be introduced into C1 or C25 of the substrate carbon chain in a stereospecific and regioselective manner. In addition, there are currently many studies using the ability of microorganisms to hydroxylate steroids. By adding precursors of calcitriol to the culture medium, they are transformed into calcitriol through biological fermentation. These microorganisms include Nocardia, Mycobacterium, Streptomyces, Pseudonocardia, and filamentous fungus Rhizopus, etc. However, the current microbial transformation still requires expensive raw materials as fermentation substrates and has high requirements for the process parameters of the culture process, resulting in difficult production, low economic benefits, and difficulty in large-scale production. Therefore, if calcitriol can be directly synthesized through microbial fermentation using ordinary and inexpensive carbon sources (glucose, sucrose) and conventional culture media as fermentation substrates, it is expected to solve important bottleneck problems such as limited material sources, high costs, complex processes, large environmental hazards, and long production cycles. Summary of the Invention
[0004] In view of this, the object of the present invention is to use the previously preserved Pichia guilliermondii GXDK6 as the starting strain, and use inexpensive carbon sources (such as glucose, sucrose) and conventional culture medium components (peptone, yeast extract, potassium chloride, sodium chloride) as fermentation substrates for the production, preparation and application of calcitriol. The present invention has created a new method for synthesizing calcitriol, providing technical support for the subsequent large-scale production of calcitriol by microbial fermentation method.
[0005] A method for synthesizing calcitriol by biological fermentation of Pichia guilliermondii GXDK6, characterized in that the optimal biological fermentation conditions are: glucose 2.0%, peptone 2.0%, yeast extract 1.0%, sodium chloride 7.5%, fermentation temperature 30 °C, pH 7.5, and the concentration of calcitriol synthesized by fermenting Pichia guilliermondii GXDK6 in a 5L fermenter for 6 days can reach 20.85 mg / L.
[0006] Pichia guilliermondii GXDK6 is currently deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 16007.
[0007] Advantages of the present invention:
[0008] The Pichia guilliermondii strain provided by the present invention can efficiently biosynthesize calcitriol, laying a foundation for the industrialization of calcitriol biosynthesis.
[0009] The present invention is the first reported method for directly using simple and inexpensive carbon sources for biosynthesis of calcitriol by biological fermentation.
[0010] Using the method of the present invention to prepare calcitriol has the advantages of environmental protection in the production process, simple operation, low cost, and suitability for large-scale production. Description of the Drawings
[0011] Figure 1 Is the qualitative analysis of calcitriol in GXDK6 cultured for 48 hours in 0%-10% NaCl.
[0012] Figure 2 Is the ELISA analysis of calcitriol in Pichia guilliermondii GXDK6 under different salt concentrations.
[0013] Figure 3 Is the yield of calcitriol at different fermentation times (shake flask).
[0014] Figure 4 Is the yield of calcitriol at different fermentation times (5L fermenter).
[0015] Specific implementation cases
[0016] The following specific embodiments are used to further elaborate the present invention in detail, and the description is an explanation rather than a limitation of the present invention.
[0017] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0018] The materials and reagents used in the following embodiments can be obtained from commercial sources unless otherwise specified. Example 1 Qualitative analysis of calcitriol biosynthesized by Pichia guilliermondii GXDK6 using gas chromatography-mass spectrometry
[0019] (1) Biosynthesis of calcitriol by Pichia guilliermondii GXDK6 through biological fermentation.
[0020] The seed liquid of Pichia guilliermondii GXDK6 was inoculated into the fermentation medium at an inoculation amount of 1% (V / V) (glucose 2.0%, peptone 2.0%, yeast powder 1.0%), the loading volume was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 30 °C, the initial pH was natural, and the shaking culture was carried out at a rotation speed of 200 rpm for 48 h. The fermentation broth was collected by centrifugation at 10000 rpm.
[0021] (2) Treatment of the fermentation broth sample.
[0022] 1) Take 1 mL of the bacterial liquid of the sample under each condition and place it in a 1.5 mL centrifuge tube (in triplicate), centrifuge at 10000 rpm for 10 min, and take the supernatant.
[0023] 2) Filter the supernatant with a 0.8 μm filter membrane and place it in a new 1.5 mL centrifuge tube.
[0024] 3) Take 500 μL of the above filtrate respectively and place it in a new 1.5 mL centrifuge tube, then add 10 μL of ribitol solution with a concentration of 0.1 mg / mL. Subsequently, all the samples after adding ribitol are frozen and centrifuged to concentrate to powder.
[0025] 4) Derivatization treatment of the sample. (Add 80 μL of 20 mg / mL methoxyamine hydrochloride - pyridine solution and react for 2 h)
[0026] 5) Alkylation treatment of the sample. (Add 80 μL of trichloroacetamide and react for 2 h, then centrifuge at 10000 rpm for 10 min.)
[0027] 6) Gas chromatography - mass spectrometry (GC - MS) detection and analysis.
[0028] 7) Detection conditions of the gas chromatography - mass spectrometer (GC / MS): The chemical analysis of the sample is carried out using the DSQII single quadrupole gas chromatography - mass spectrometer (GC / MS) of Thermo company. The inlet temperature of the instrument is maintained at 250 °C. 1 μL of the derivatized sample is injected in splitless mode into a DBS (Dodecyl Benzene Sulfonic Acid) column (length 30 m × inner diameter 250 μm × thickness 0.25 μm). The temperature of the mass spectrometry source is maintained at 250 °C in the direct ionization mode with an ionization energy of 70 eV and an acceleration voltage of 8000 V. In the experiments of full scan and selected ion recording, the temperature of the mass spectrometry quadrupole is kept constant at 150 °C. The initial temperature of the gas chromatography oven is set at 85 °C for 5 minutes, and then increased to 300 °C at a rate of 15 °C / min. The carrier gas of the gas chromatography is helium, and the flow rate is kept constant at 1 mL / min. The operating range of the mass spectrometry is 50 - 600 m / z.
[0029] The GC - MS detection results show that a characteristic peak appears at about 28.88 min for calcitriol, indicating that GXDK6 has the ability to biosynthesize calcitriol de novo using low - cost carbon sources. The results are as Figure 1 shown. Further analysis reveals that the NaCl concentration is a synthetic factor affecting calcitriol. Therefore, it is expected that using Pichia pastoris fermentation to synthesize calcitriol and carry out large - scale production will become a new source of calcitriol.
[0030] Example 2 Implementation Case: Quantifying Calcitriol Biosynthesized by Pichia guilliermondii GXDK6 under Different NaCl Concentrations Using the ELISA Method
[0031] (1) Biosynthesis of calcitriol by Pichia guilliermondii GXDK6 through bioreactor fermentation.
[0032] The seed liquid of Pichia guilliermondii GXDK6 was inoculated into fermentation media containing 0.0%, 2.5%, 5.0%, 7.5%, and 10.0% NaCl (2.0% glucose, 2.0% peptone, 1.0% yeast extract) at an inoculation amount of 1% (V / V). The liquid volume was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 30 °C, the initial pH was natural, and shaking culture was carried out at 200 rpm for 48 h. The fermentation broth was collected by centrifugation at 10,000 rpm.
[0033] (2) Treatment of the fermentation broth samples.
[0034] 1 mL of the supernatant fermentation broth was taken from each flask and concentrated and dried using a centrifugal concentrator. After being concentrated and dried into powder, 1 mL of methanol (4 °C) was added to each tube of the sample for dissolution. After mixing evenly, it was shaken and impregnated at 200 rpm for 4 hours, then centrifuged at 12,000 rpm for 10 min. After taking the supernatant, it was filtered using a 0.45 μm filter membrane and then detected using an ELISA calcitriol detection kit (Kit number: JL52676; purchased from Shanghai Jianglai Biotechnology Co., Ltd.).
[0035] Using the ELISA method, the standard curve of calcitriol was found to be: A = -0.56 Ln(C) + 3.5502 (R2 = 0.9998) (where "A" represents the absorbance at 450 nm and "C" represents the concentration of calcitriol). Subsequently, the fermentation broths under different NaCl concentrations were detected and it was found that as the NaCl concentration increased (0% - 10%), the production of calcitriol showed a trend of first increasing and then decreasing ( Figure 2 ). Without NaCl, the production of calcitriol was only 0.27 mg / L. However, when the NaCl concentration increased to 7.5%, the production of calcitriol reached as high as 1.24 mg / L, which was 359.26% higher than that under the condition without NaCl, indicating that 7.5% NaCl is the optimal concentration for calcitriol biosynthesis in GXDK6.
[0036] Example 3 Implementation Case: Effects of Different Fermentation Times on Calcitriol Biosynthesized by Pichia guilliermondii GXDK6
[0037] (1) Biosynthesis of calcitriol by Pichia guilliermondii GXDK6 through bioreactor fermentation.
[0038] The seed liquid of Pichia guilliermondii GXDK6 was inoculated into the fermentation medium at an inoculation amount of 1% (V / V) (glucose 2.0%, peptone 2.0%, yeast powder 1.0%, 7.5% NaCl). The liquid loading amount was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 30 °C, and the shaking culture was carried out at a rotation speed of 200 rpm for 16 h, 48 h, 72 h, 96 h, and 120 h respectively. The fermentation broth was collected by centrifugation at 10000 rpm.
[0039] (2) Treatment of the fermentation broth sample.
[0040] 1 mL of the supernatant fermentation broth was taken from each bottle of fermentation broth and concentrated and dried by a centrifugal concentrator. After being concentrated and dried into powder, 1 mL of methanol (4 °C) was added to each tube of the sample to dissolve it. After mixing evenly, it was shaken and impregnated at 200 rpm for 4 hours, and then centrifuged at 12000 rpm for 10 min. After taking the supernatant, it was filtered through a 0.45 μm filter membrane and then detected using an ELISA detection kit for calcitriol (kit number: JL52676; purchased from Shanghai Jianglai Biotechnology Co., Ltd.).
[0041] The results showed that the production of calcitriol showed an increasing trend at the fermentation times of 16 h, 48 h, 72 h, 96 h, and 120 h, which were 1.24 mg / L, 2.63 mg / L, 10.24 mg / L, 17.84 mg / L, and 18.67 mg / L ( Figure 3 ). It indicated that GXDK6 required a longer fermentation time to promote the production of calcitriol and could synthesize this substance more effectively in a longer time.
[0042] Example 4 Scale-up fermentation of the production process of calcitriol in Pichia guilliermondii GXDK6
[0043] (1) Biosynthesis of calcitriol by Pichia guilliermondii GXDK6 fermentation.
[0044] The seed liquid of Pichia guilliermondii GXDK6 was inoculated into the fermentation medium at an inoculation amount of 1% (V / V) (glucose 2.0%, peptone 2.0%, yeast powder 1.0%, 7.5% NaCl). The liquid loading amount was 5 L fermenter, the culture temperature was 30 °C, and the on-tank fermentation was carried out at a stirring speed of 120 rpm for 1 - 7 d. The fermentation broth was collected by centrifugation at 10000 rpm.
[0045] (2) Treatment of the fermentation broth sample.
[0046] Absorb 1 mL of the supernatant fermentation broth from each bottle of fermentation broth and concentrate and dry it using a centrifugal concentrator. After concentrating and drying it into a powder, add 1 mL of methanol (4 °C) to each tube of sample for dissolution. After mixing evenly, shake and soak it at 200 rpm for 4 hours, then centrifuge at 12,000 rpm for 10 min. After taking the supernatant, filter it using a 0.45 μm filter membrane and then detect it using an ELISA detection kit for calcitriol (kit number: JL52676; purchased from Shanghai Jianglai Biotechnology Co., Ltd.).
[0047] The results show that the production of calcitriol under fermentation times of 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d shows a trend of increasing first and then decreasing, which are 2.36 mg / L, 3.93 mg / L, 12.40 mg / L, 17.53 mg / L, 19.03 mg / L, 20.85 mg / L, and 17.59 mg / L( Figure 4 ). It indicates that in the initial stage of fermentation, as time goes by, the microbial activity increases. However, due to the depletion of nutrients or the accumulation of metabolic by-products, the production decreases after 6 d.
[0048] Application fields:
[0049] (1) In pharmaceutical research: The calcitriol produced by the method of the present invention can be used as a source of medical raw materials and excipients for further separation and purification to prepare pure calcitriol or its derivative drugs;
[0050] (2) In clinical applications: The calcitriol produced by the method of the present invention, after being formulated to the corresponding pharmacological concentration, can be used as a crude product for clinical treatment as a supplementary source for diseases such as osteoporosis, rickets, and renal dystrophy;
[0051] (3) In health products: The calcitriol produced by the method of the present invention belongs to the fermentation product of probiotics. For people with calcitriol synthesis disorders or deficiencies, it can be used as a source of health products to prevent the occurrence of related diseases; (4) In livestock and poultry breeding: The calcitriol produced by the method of the present invention can be directly used to feed livestock and poultry animals, which helps to improve the intestinal health of animals, promote calcium nutrition absorption, and improve the immune function and growth and development speed of animals.
Claims
1. A method for synthesizing calcitriol (1α,25-dihydroxyvitamin D3) by bioconversion using Pichia guilliermondii GXDK6, characterized in that, Using the microorganism Pichia guilliermondii GXDK6 as the starting strain, and using inexpensive carbon sources (glucose, fructose, sucrose) and conventional culture medium components (peptone, yeast extract, potassium chloride, sodium chloride) as fermentation substrates, in a fermentation system of 100 - 5000 mL, inoculum size of 0.5% - 5.0%, temperature of 30 - 37 °C, pH of 4.0 - 10.0, and stirring speed of 0 - 300 rpm, ferment for 16 - 120 h to produce and apply calcitriol.
2. A method for biosynthesizing calcitriol using Pichia guilliermondii GXDK6 by biological fermentation according to claim 1, characterized in that, Under more preferred biological fermentation conditions, the method is as follows: glucose 2.0%, peptone 2.0%, yeast extract 1.0%, sodium chloride 7.5%, fermentation temperature 30 °C, pH 7.5, fermentation time 48 h, and the concentration of calcitriol synthesized by Pichia guilliermondii GXDK6 fermentation can reach 0 - 20.85 mg / L.
3. The Pichia guilliermondii GXDK6 according to claim 1, characterized in that, This strain can tolerate a NaCl concentration of up to 12% and can directly ferment in a co - stress medium of strong acid (pH = 3.0) and high salt (10% NaCl) without sterilization, and other miscellaneous bacteria will not contaminate the sealed fermentation process.
4. The application of Pichia guilliermondii GXDK6 as claimed in claim 1 as a fermentation strain for preparing calcitriol.
5. Use of Pichia guilliermondii GXDK6 as claimed in claim 5 as a fermentation strain for preparing calcitriol, characterized in that, The said application includes the formula, steps, and culture conditions for inoculating Pichia guilliermondii GXDK6 into the culture medium. The formula of the culture medium uses inexpensive carbon sources (such as glucose, fructose, sucrose, etc.) and conventional culture medium components (peptone, yeast extract) as fermentation substrates.
6. Use of Pichia guilliermondii GXDK6 as claimed in claim 5 as a fermentation strain for preparing calcitriol, characterized in that, The said inexpensive carbon sources include one or more of glucose, fructose, and sucrose, and the said conventional culture medium components include one or more of peptone, yeast extract, potassium chloride, and sodium chloride.
7. The method for biosynthesizing calcitriol by using Pichia guilliermondii GXDK6 according to claim 6, characterized in that, The said sodium chloride concentration is 0.0%, 2.5%, 5.0%, 7.5%, 10.0%.
8. The method for biosynthesizing calcitriol by using Pichia guilliermondii GXDK6 according to claim 6, characterized in that, The said fermentation time is 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d.
9. Use of Pichia guilliermondii GXDK6 according to claim 6 as a fermentation strain for preparing calcitriol, characterized in that, After collecting and concentrating the fermentation broth, extract and separate calcitriol to obtain its pure product, and use gas chromatography - mass spectrometry to qualitatively analyze calcitriol, and a characteristic peak appears at about 28.88 min.
10. Use of Pichia guilliermondii GXDK6 as claimed in claim 6 as a fermentation strain for preparing calcitriol, characterized in that, Directly inoculate Pichia guilliermondii GXDK6 into the culture medium for fermentation, and use it as a microbial preparation in the fields of pharmaceutical product preparation, clinical treatment, health product development, agricultural breeding, etc.