Preparation method of cyclic diguanylate
By constructing a restructuring E. coli to express the cycloidase of the two -birdotide and perform high -temperature treatment during the fermentation process, the problem of high cost and cumbersome steps of the existing cyclic two -bird dharma system is solved. , Economic preparation and large -scale industrialized production.
Patent Information
- Application Number
- CN202310933184.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-27
- Publication Date
- 2025-01-28
AI Technical Summary
The existing preparation methods for cyclic diguanylic acid are costly and cumbersome, making it difficult to achieve a simple, economical and efficient preparation process.
By constructing a reorganization of E. coli, the two -bird dioxide cycle enzyme that expresses the source of subcutaneous fake thermal bacteria, and is treated at high temperature during the fermentation process, destroys the bacteria wall and releases enzymes, and then catalyze the reaction under specific conditions to prepare high purity with high purity Ring two birds.
The efficient and economic preparation of the two -bird dioxide is achieved, simplified the process of process, reduced equipment investment and resource consumption, and can industrialize large -scale industrialization.
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Figure CN119372274A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug preparation, and in particular to a method for preparing cyclic diguanylate. Background Art
[0002] Cyclic diguanylate (c-di-GMP) is a cyclic dinucleotide synthesized by two molecules of guanosine triphosphate (GTP) through diguanylate cyclase (DGC). c-di-GMP is a conserved second messenger molecule in bacteria, which is involved in regulating a variety of physiological functions, including bacterial movement, fixation, differentiation, biofilm formation, pathogenic factor production, cell-to-cell communication, etc. In recent years, studies have found that c-di-GMP can act on eukaryotic cells as an immunomodulator to produce a good immunomodulatory effect. Experimental studies have shown that it may become a potential vaccine adjuvant.
[0003]
[0004] The reaction formula of DGC catalyzing GTP to generate c-di-GMP
[0005] c-di-GMP can stimulate the body to produce immune responses, and research on its use as a new type of immunopotentiator has been initiated. Whether c-di-GMP can become an effective adjuvant must be evaluated in more aspects, but its expensive market price limits its use and the development of many studies. At present, the preparation of cyclic diguanylate is mainly prepared by chemical synthesis and enzyme synthesis, but the chemical synthesis method requires the use of multiple protective groups, which is time-consuming, has many steps and is not environmentally friendly, while the enzyme synthesis method requires the use of the more expensive substrate GTP and efficient and pure enzyme proteins. Therefore, the current market price of cyclic diguanylate is 4000-6000 yuan / mg.
[0006] Even if recombinant microorganisms are used to prepare DGC, the enzyme produced still needs to be extracted from the complex culture to improve the purity and titer of the enzyme, and then a suitable enzyme catalytic reaction system is prepared to obtain the final product, cyclic dinucleotide. The steps are still cumbersome and the cost is high. Therefore, a simple, economical and efficient method for preparing cyclic dinucleotide is needed. Summary of the invention
[0007] The invention provides a method for preparing cyclic diguanylate with simple process, low cost and high conversion efficiency.
[0008] The method constructs a recombinant Escherichia coli to express diguanylate cyclase from Pseudothermotoga hypogea (DSM11164=NBRC 106472), the molecular weight of which is 40.3 kDa, and the level of exogenous soluble expression and catalytic activity are very high. More importantly, after the protein is expressed in vitro, it can be guaranteed not to be denatured at 85°C or even 90°C, and still maintains high biological activity. After the fermentation of the recombinant bacteria is completed, the temperature is directly raised to 85-95°C to destroy the wall membrane of the recombinant bacteria and release the expressed diguanylate cyclase; at the same time, other enzymes or proteins are inactivated, while the activity of the high-temperature resistant diguanylate cyclase is retained, and then magnesium chloride, buffer salt and catalytic substrate are directly added to the fermentation liquid, and the catalytic reaction is carried out at 70-75°C to obtain a high-purity cyclic diguanylate clear liquid.
[0009] The technical solution provided by the present invention is to first carry out high-density fermentation of genetically engineered bacteria. The fermentation process aims to increase the expression amount and activity of the target product, and finely controls each stage of the fermentation. The process includes: seed liquid preparation stage, cell rapid propagation stage, and induction recombinant protein expression stage.
[0010] Seed liquid preparation stage: genetically engineered glycerol bacteria are inoculated into liquid culture medium for activation culture, the activation culture temperature is 35-37°C, the time is 8-16h; cell rapid propagation stage: the activated seed liquid is inoculated into the fermentation medium, cultured for 6-10h, the temperature is 33-37°C, the dissolved oxygen content is 25-75%, and the pH value is 6.5-7.0; enzyme production stage: the cell concentration reaches OD6 00 =20-30, add IPTG to induce the engineered bacteria to produce enzymes, the culture temperature is 22-28°C, the dissolved oxygen content is 25-55%, and the pH value is 6.5-7.0; add 80% glycerol, the addition speed is 20-30g / L / H, the cell concentration reaches OD600=110-140, and the fermentation ends. The carbon source is added by constant rate flow addition, so that the main physiological metabolic activities of the genetically engineered bacteria are suitable for the efficient expression of cyclic diguanylate enzymes, and the fermentation density is further improved.
[0011] The fermentation medium in the preferred technical solution is: 10g / L glycerol, 12g / L peptone, 24g / L yeast extract, 12.5g / LK2HPO4·12H20, 3.0g / L KH2PO3, 0.2g / L MgSO4·7H20, 3-5ml / L trace elements, and the kanamycin concentration is 50μg / mL; wherein the trace elements are: 6g / L FeCl3, 0.58g / L ZnSO4·7H20, 0.2g / L LiCl2, 0.2g / L COCl2, and 0.3g / L MnSO4.
[0012] The process of the present invention artificially divides the fed-batch fermentation into two stages and controls the culture conditions of each stage, so that the cyclic diguanylate enzyme genetic engineering bacteria can achieve high-density fermentation, which not only increases the bacterial concentration, but also increases the enzyme activity of cyclic diguanylate enzyme.
[0013] Then, the catalytic reaction of enzymatic conversion of cyclic diguanylate is carried out. After the fermentation culture is completed, the fermentation liquid is diluted to a state containing 5% of the bacteria, and the fermentation tank is directly heated to 85-95°C, preferably 90°C, and kept warm for 10-15 minutes, and then cooled to 70-75°C. At this time, magnesium chloride, phosphate buffer salt and substrate are added, fully mixed, and the pH value is adjusted to 6.0-7.5. The catalytic reaction is completed in 90-120 minutes. In the preferred technical scheme, the concentration of magnesium chloride in the catalytic reaction system is 10-20mM; the buffer salt is 50mM phosphate, pH7.5; the catalytic substrate is guanosine triphosphate, and the concentration is 2-4g / L. After adding the flocculant, a high-purity cyclic diguanylate clear liquid can be obtained by plate and frame filtration. This cyclic diguanylate preparation process is simple and easy, and almost no impurities are generated in this conversion process, which is convenient for subsequent efficient purification work.
[0014] The process of the present invention realizes high-density fermentation through fine control of the entire fermentation process, which not only improves the bacterial concentration of the recombinant bacteria (to 70-100 g / L on a dry weight basis), but also improves the enzymatic activity of cyclic diguanylate. When the conversion reaction is carried out with 5% bacterial dosage, a reaction solution with a cyclic diguanylate content of more than 2.7 mg / ml can be obtained, so that the production of cyclic diguanylate reaches a kilogram-level output and large-scale industrialization is realized.
[0015] The preparation process of the present invention does not require purification of the recombinant enzyme, and the treated fermentation liquid can be used to prepare the catalytic reaction system, the process is simple, and the cost is saved; at the same time, the equipment investment is greatly reduced, and the enzyme production of a small-tonnage fermentation tank can meet the large-scale industrial production of cyclic diguanylate. The process of the present invention has obvious energy-saving and emission-reduction effects, reduces the demand for water, electricity, and steam, and greatly reduces the amount of three wastes generated. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 : Example 2 SDS-PAGE electrophoresis after IPTG-induced expression: 1. Soluble protein after 6 hours of fermentation induction; 2. Soluble protein after 8 hours of fermentation induction; 3. Soluble protein after 10 hours of fermentation induction; 4. Soluble protein after 14 hours of fermentation induction; 5. Soluble protein after 18 hours of fermentation induction (the arrow indicates the recombinantly expressed diguanylate cyclase);
[0018] Figure 2 : Liquid phase detection spectrum after the catalytic reaction of Example 4 (the arrow indicates the cyclic diguanylate generated by the catalysis). DETAILED DESCRIPTION
[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0020] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.
[0021] Example 1 Construction and cultivation of genetically engineered bacteria
[0022] The present invention obtains a diguanylate cyclase gene sequence from Pseudothermotoga hypogea (DSM 11164=NBRC 106472) by using methods such as genome database mining and homologous sequence comparison, combined with molecular biological methods, as shown in SEQ ID NO: 1, and the amino acid sequence encoding the diguanylate cyclase is shown in SEQ ID NO: 2;
[0023] S1: The nucleotide sequence shown in SEQ ID NO: 1 was artificially synthesized (commissioned to Nanjing Qingke Biotechnology Co., Ltd.) to obtain the pET-28a(+)-dgc recombinant plasmid;
[0024] S2: The pET-28a(+)-dgc recombinant plasmid carrying the dgc gene was transformed into BL21(DE3) host Escherichia coli (BL21(DE3) host Escherichia coli was purchased from Nanjing Qingke Biotechnology Co., Ltd.) and spread on a plate;
[0025] S3: Pick a single colony and inoculate it into a triangular shake flask containing LB liquid medium (containing Kana resistance), and culture it at 37°C, 180rpm overnight as a seed solution (glycerol-retaining bacteria after verification). Then take 2ml of the seed solution and transfer it to a triangular shake flask containing 100ml LB medium containing Kana resistance, culture it at 37°C, 180rpm for 1-2h, and when OD600 reaches about 0.6, add IPTG with a final concentration of 0.2mM, and induce it at 26°C, 180rpm for about 8h;
[0026] S4: After induction, centrifuge at 10000r / min and 4℃ for 5min to collect the cells. Resuspend the cells with cells / water (1:10) and use an ultrasonic cell disruptor to break the cells. Take 5ml of the bacterial suspension, 200W, start for 5S, stop for 7S, and cycle 15 times in an ice bath;
[0027] S5: Take the cell lysate and centrifuge it at 4°C, 10000r / min for 5min, collect the supernatant and precipitate respectively. Then analyze the expression of the target protein by SDS-PAGE (see Figure 1The seed solution with normal expression was mixed with 40% glycerol (1:1) to preserve the glycerol bacteria and stored in a -80°C refrigerator.
[0028] Example 2 Catalytic production of cyclic diguanylate (10L tank)
[0029] (1) Seed culture: Inoculate the expression-verified genetically engineered bacteria glycerol bacteria into 50 ml of liquid culture medium for activation culture: the seed culture temperature is 35° C. and the time is 15 h;
[0030] The seed culture medium was LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 M sodium hydroxide solution was used to adjust the pH value to 7.0, sterilized at 121°C for 20 min, and kanamycin was added at a concentration of 50 μg / mL before inoculation.
[0031] (2) Rapid cell propagation stage: 6 L of basal culture medium was placed in a fermentation tank, with an initial pH of 6.56, sterilized at 121°C for 20 min, cooled to 35°C, with a pH of 6.21, and adjusted to pH 7.0 with ammonia water. 50 ml of seed inoculation liquid was connected to the fermentation tank for fermentation.
[0032] The above-mentioned basic fermentation medium: 10g / L glycerol, 12g / L peptone, 24g / L yeast extract, 12.5g / LK2HPO4·12H20, 3.0g / L KH2PO3, 0.2g / L MgSO4·7H20, trace elements 3ml / L (trace elements: 6g / LFeCl3, 0.58g / LZnSO4·7H20, 0.2g / L LiCl2, 0.2g / L COCl2, 0.3g / L MnSO4), kanamycin concentration is 50μg / mL.
[0033] (3) Enzyme production stage: After 6 hours of fermentation, the OD600 of the fermentation broth was measured to be 20, the temperature of the fermentation broth was lowered to 26°C, and the inducer IPTG 0.1mM was added, and the fermentation was continued for 22 hours. During this period, the dissolved oxygen was maintained at 25-55%, and the feed rate was 20g / L / H. 600 =120, cell dry weight is 50g / L.
[0034] The feed was: 80% glycerol.
[0035] (4) Enzyme catalysis process: After the fermentation culture is completed, the fermentation liquid is diluted to a state containing 5% bacterial cells, and then the temperature of the fermenter is raised to 90°C and kept warm for 10 minutes, then cooled to 70°C, and 10 mM magnesium chloride, 50 mM pH 7.5 phosphate buffer and 4 g / L substrate GTP are added to the fermenter. The mixture is thoroughly mixed, and the pH value is adjusted to 6.0-7.5. The temperature is controlled at 70°C to start the conversion reaction, and the conversion reaction is completed after 100 minutes.
[0036] At the end of the conversion, a sample was taken for liquid phase detection. The HPLC chromatographic conditions were: C18 (4.6×250 mm), 0.1 mol / L phosphate buffer (17.9 g disodium hydrogen phosphate and 6.8 g potassium dihydrogen phosphate were dissolved in 900 ml of water, the pH was adjusted to 7.0 with phosphoric acid, 0.805 g tetrabutylammonium bromide was added, water was added to 1000 ml, and shaken)-acetonitrile (95:5) as the mobile phase, the flow rate was 1.0 ml / min, the detection wavelength was 253 nm, and the column temperature was 30°C.
[0037] After adding the flocculant, the reaction solution was filtered through a plate and frame filter, and the cyclic diguanylic acid content in the obtained reaction solution was 2800ug / ml.
[0038] Example 3 Catalytic production of cyclic diguanylate (50L tank)
[0039] (1) Seed culture: inoculate pure genetically engineered bacteria with verified expression into 150 ml of liquid culture medium for activation culture: the seed culture temperature is 35° C. and the time is 14 h;
[0040] The seed culture medium was LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 M sodium hydroxide solution was used to adjust the pH value to 7.0, sterilized at 121°C for 20 min, and kanamycin was added at a concentration of 50 μg / mL before inoculation.
[0041] (2) Rapid cell propagation stage: 30 L of basal culture medium was placed in a fermenter, with an initial pH of 6.60, sterilized at 121°C for 20 min, cooled to 36°C, with a pH of 6.21, and adjusted to pH 7.0 with ammonia water. 300 ml of seed inoculation liquid was connected to the fermenter for fermentation.
[0042] The above-mentioned basic fermentation medium: 10g / L glycerol, 12g / L peptone, 24g / L yeast extract, 12.5g / LK2HPO4·12H20, 3.0g / L KH2PO3, 0.2g / L MgSO4·7H20, trace elements 3ml / L (trace elements: 6g / LFeCl3, 0.58g / L ZnSO4·7H20, 0.2g / L LiCl2, 0.2g / L COCl2, 0.3g / L MnSO4), kanamycin concentration is 50μg / mL.
[0043] (3) Enzyme production stage: After 6 hours of fermentation, the OD600 of the fermentation broth was measured to be 26. The fermentation broth temperature was lowered to 27°C, and the inducer IPTG 0.2mM was added. The fermentation was continued for 22 hours, during which the dissolved oxygen was maintained at 25-55%; the feed rate was 22g / L / H. After the fermentation was completed, the OD 600 =130, cell dry weight is 58g / L.
[0044] The feed was: 80% glycerol.
[0045] (4) Enzymatic conversion of cyclic diguanylate: After the fermentation culture is completed, the fermentation liquid is diluted to a state containing 5% bacterial cells, and then the fermentation tank temperature is raised to 90°C, kept warm for 10 minutes, and then cooled to 70°C. At this time, 10mM magnesium chloride, 50mM pH7.5 phosphate buffer and 3g / L substrate GTP are added, and the mixture is fully mixed. The pH value is adjusted to 6.0-7.5, and the temperature is controlled at 70°C to start the conversion reaction, which ends in 100 minutes. HPLC liquid phase detection is the same as in Example 2.
[0046] After adding the flocculant, the reaction solution was filtered through a plate and frame filter, and the cyclic diguanylate content in the obtained reaction solution was 2900ug / ml.
[0047] Example 4 Catalytic production of cyclic diguanylate (700L tank)
[0048] (1) Seed culture: Inoculate pure genetically engineered glycerol bacteria that have been verified for expression into 150 ml of liquid culture medium for activation culture: the temperature of the seed culture is 35° C. and the time is 15 h;
[0049] 30L LB seed culture medium was placed in a seed tank, pH was natural, sterilized at 121℃ for 20min, cooled to 37℃, kanamycin was added at a concentration of 50μg / ml before inoculation, and then 300ml seed inoculation liquid was added to the seed tank for secondary seed culture. The temperature of the secondary seed culture was 37℃ and the time was 4h.
[0050] The LB seed culture medium formula is shown in Examples 2 and 3.
[0051] (2) Rapid cell propagation stage: 210L of basal culture medium was placed in a fermentation tank, with an initial pH of 6.56, sterilized at 121°C for 20 min, cooled to 35°C, with a pH of 6.21, and adjusted to pH 7.0 with ammonia water. 20L of seed inoculation liquid was connected to the fermentation tank for fermentation.
[0052] The above-mentioned basic fermentation medium is: 10g / L glycerol, 12g / L peptone, 24g / L yeast extract, 12.5g / LK2HPO4·12H20, 3.0g / L KH2PO3, 0.2g / L MgSO4·7H20, trace elements 3ml / L (trace elements: 6g / LFeCl3, 0.58g / L ZnSO4·7H20, 0.2g / L LiCl2, 0.2g / L COCl2, 0.3g / L MnSO4), and the kanamycin concentration is 50μg / mL.
[0053] (3) Enzyme production stage: After 6 hours of fermentation, the OD600 of the fermentation broth was measured to be 20, the temperature of the fermentation broth was lowered to 26°C, 0.3 mM IPTG inducer was added, and the fermentation was continued for 22 hours, during which the dissolved oxygen was maintained at 25-55%; the feed rate was 20 g / L / H. After the fermentation was completed, OD600 was obtained to be 126, and the cell dry weight was 56 g / L.
[0054] The feed was: 80% glycerol.
[0055] (4) Enzymatic conversion of cyclic diguanylate: After the fermentation culture is completed, the fermentation liquid is diluted to a state containing 5% bacterial cells, and then the fermentation tank temperature is raised to 90°C, kept warm for 15 minutes, and then cooled to 70°C. At this time, 10mM magnesium chloride, 50mM pH7.5 phosphate buffer and 3g / L substrate GTP are added, and the mixture is fully mixed. The pH value is adjusted to 6.0-7.5, and the temperature is controlled at 70°C to start the conversion reaction, which ends in 100 minutes. HPLC liquid phase detection is the same as in Example 2.
[0056] After adding the flocculant, the reaction solution was filtered through a plate and frame filter, and the content of cyclic diguanylic acid in the reaction solution was 2780ug / ml.
Claims
1. A method for preparing cyclic diguanylate, characterized in that: The fermentation liquid of the recombinant bacteria expressing diguanylate cyclase is prepared into a reaction system, and cyclic diguanylate is generated by a one-step reaction.
2. The preparation method according to claim 1, characterized in that: The recombinant bacteria express the diguanylate cyclase shown in SEQ ID NO:
2.
3. The preparation method according to claim 2, characterized in that: The recombinant bacteria are obtained by transforming Escherichia coli BL21 (DE3) with a pET28a plasmid carrying the gene sequence shown in SEQ ID NO:
1.
4. The preparation method according to any one of claims 1 to 3, characterized in that: After the fermentation of the fermentation liquid of the recombinant bacteria is completed, the temperature is raised to 85-95° C. and kept warm for 10-15 minutes before adding other components of the reaction system.
5. The preparation method according to claim 4, characterized in that: After the fermentation of the fermentation liquid of the recombinant bacteria is completed, the temperature is raised to 90° C. and kept warm for 10 to 15 minutes before adding other components of the reaction system.
6. The preparation method according to claim 1, characterized in that: The reaction system also contains magnesium chloride, buffer salt and catalytic substrate, the pH of the reaction system is 6.0-7.5, and the reaction temperature is 70-75°C; after the reaction is finished, the reaction is terminated by boiling.
7. The preparation method according to claim 6, characterized in that: The concentration of magnesium chloride is 10-20 mM; the buffer salt is 50 mM phosphate, pH 7.5; the catalytic substrate is guanosine triphosphate, the concentration is 2-4 g / L; the reaction time is 90-120 minutes.
8. The preparation method according to claim 7, characterized in that: After the reaction is completed, a flocculant is added to the reaction system and then filtered through a plate and frame filter press to obtain a clear liquid containing cyclic diguanylate.
9. The preparation method according to any one of claims 1 to 3, characterized in that: The fermentation of the recombinant bacteria is divided into a rapid propagation stage and an induction recombinant protein expression stage: the fermentation temperature in the rapid propagation stage is 33-37°C and the dissolved oxygen content is 25-75%; the fermentation temperature in the induction recombinant protein expression stage is 22-28°C and the dissolved oxygen content is 35-55%.
10. The preparation method according to claim 9, characterized in that: The fermentation medium comprises: 10 g / L glycerol, 12 g / L peptone, 24 g / L yeast extract, 12.5 g / L K2HPO4·12H20, 3.0 g / L KH2PO3, 0.2 g / L MgSO4·7H20, 3-5 ml / L of trace elements, and a kanamycin concentration of 50 μg / mL; wherein the trace elements comprise: 6 g / L FeCl3, 0.58 g / L ZnSO4·7H20, 0.2 g / L LiCl2, 0.2 g / L COCl2, and 0.3 g / L MnSO4.
11. The preparation method according to claim 10, characterized in that: During the stage of inducing recombinant protein expression, 80% glycerol was added at a rate of 20 to 30 g / L / H.