Method for improving fermentation yield of fidaxomicin

Abstract

The invention discloses a method for producing fidaxomicin through fermentation. From the perspective of strengthening fidaxomicin anabolic flow, a four-carbon branched chain precursor substance is added in the fermentation process and is converted into isobutyric acid through microbial utilization, and the fermentation unit of fidaxomicin is increased. The method is simple to operate, mild in condition, wide in raw material source and low in equipment requirement, the production cost is reduced, the fermentation yield is greatly increased, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of producing antibiotics by microbial fermentation, and in particular to the fermentation production of fidaxomicin. Background Art

[0002] Fidaxomicin is a new narrow-spectrum macrolide antibiotic developed by Optimer, with the trade name Dificid. It is mainly used to treat Clostridium difficile-associated diarrhea (CDAD) and was approved by the FDA on May 27, 2011. Compared with the current main therapeutic drugs metronidazole and vancomycin, fidaxomicin can significantly reduce the recurrence rate. The structural formula of fidaxomicin is as follows:

[0003]

[0004] Chemical Formula:C 52 H 74 Cl 2 O 18

[0005] Molecular Weight:1058.05

[0006] Fidaxomycin is a typical secondary metabolite produced by the fermentation of Dactylocystis citrinum. During the fermentation process, 0-48 hours, it is in the rapid growth stage of mycelial cells, and 48 hours to the end of the fermentation, the cells are in the product synthesis phase, and a large amount of Fidaxomycin products are synthesized. In the actual Fidaxomycin fermentation process, after the fermentation enters the synthesis phase, there are problems such as the decline of the anabolic flow of Fidaxomycin and the weakening of the intensity of cell respiration, and it is necessary to adopt other regulatory means to increase the metabolic intensity of the cell, and then strengthen the anabolic flow of Fidaxomycin. CN104561198A reports the addition of edible oil to the fermentation culture process, and the fermentation yield is increased by microbial utilization and conversion into a carbon source and precursor propionyl-CoA. In addition, there is no relevant report on this aspect of research. Summary of the invention

[0007] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a method for increasing the fermentation yield of fidaxomicin.

[0008] The present invention provides a method for producing fidaxomicin by fermentation, which is characterized in that a four-carbon branched chain precursor substance is added to the fermentation liquid during the fermentation process.

[0009] Furthermore, the fermentation process comprises:

[0010] 1) Cultivation of vegetative inoculum in seed medium

[0011] The seed culture medium preparation method:

[0012] Glucose 20g / L, soluble starch 30g / L, yeast extract 5g / L, peptone 5g / L, magnesium sulfate 2g / L, sodium chloride 2g / L, calcium carbonate 2g / L, PH7.0.

[0013] Prepare an appropriate amount of seed culture medium, sterilize at 120°C for 30 minutes, then cool to 27-29°C, inoculate 48h-old shake flask seeds at an inoculum rate of 0.1-0.3v / v% by volume of the seed culture medium, and then culture at 27-29°C for 28-36 hours to obtain vegetative inoculum.

[0014] 2) Inoculate the nutrient inoculum in the fermentation medium, add the four-carbon branched chain precursor, and continue fermentation to obtain fidaxomicin

[0015] The fermentation medium preparation method:

[0016] Glucose 20g / L, soluble starch 50g / L, yeast powder 10g / L, peptone 5g / L, soybean powder 20g / L, magnesium sulfate 2g / L, sodium chloride 2g / L, dipotassium hydrogen phosphate 1g / L, calcium carbonate 2g / L, PH7.0.

[0017] Prepare an appropriate amount of fermentation medium, sterilize at 120° C. for 30 min, then cool to 27-29° C., and inoculate the vegetative inoculum in the seed medium in step 1) at an inoculum volume ratio of 4-8 v / v% of the fermentation medium. During the fermentation process, the temperature is controlled at 27-29° C., the pressure is controlled at 0.04-0.06 MPa, the dissolved oxygen electrode is monitored online, and the dissolved oxygen is controlled to be above 30% by the rotation speed and ventilation.

[0018] The fermentation culture is continued, and the four-carbon branched chain precursor is added between 0 and 144 hours of fermentation; preferably, the four-carbon branched chain precursor is added when the dissolved oxygen in the fermentation liquid begins to rise from the lowest point, and the addition of the four-carbon branched chain precursor is stopped after 144 hours of fermentation; more preferably, the four-carbon branched chain precursor is added between 48 and 144 hours of fermentation. After 180 hours of fermentation, when the titer no longer increases, the fermentation is stopped and the product is detected.

[0019] Furthermore, the volume of the four-carbon branched chain precursor added is 0.1 to 4 v / v% of the volume of the fermentation broth;

[0020] Preferably, the volume of the four-carbon branched chain precursor added is 1 to 3 v / v% of the volume of the fermentation broth;

[0021] More preferably, the volume of the four-carbon branched chain precursor added is 1.5-2.5 v / v% of the volume of the fermentation broth.

[0022] Furthermore, in the fermentation method, the four-carbon branched chain precursor is added continuously, with the added amount controlled and added continuously and uninterruptedly; or the addition is intermittent, with the four-carbon branched chain precursor added 1 to 4 times per time unit.

[0023] Preferably, the four-carbon branched chain precursor is added continuously, with 0.02 to 0.4 v / v‰ of the volume of the fermentation liquid added per hour;

[0024] More preferably, the four-carbon branched chain precursor is added continuously, with 0.1 to 0.3 v / v‰ of the volume of the fermentation liquid added per hour.

[0025] Further, the four-carbon branched chain precursor is selected from any one or more of isobutyric acid, isobutyrate, isobutanol, esters derived from isobutyric acid, and esters derived from isobutanol;

[0026] Preferably, the four-carbon branched chain precursor is selected from any one or more of isobutyric acid, sodium isobutyrate, potassium isobutyrate, isobutanol, methyl isobutyrate, ethyl isobutyrate, propyl isobutyrate, isobutyl acetate, isobutyl propionate, and isobutyl oleate.

[0027] Furthermore, the fermentation broth is separated and purified to obtain high-purity Fidaxomicin.

[0028] In the present invention, the strain used for fermentation is Dactylosporangium aurantiacum, which can be obtained from the following institutions: Agricultural Culture Collection of China, strain collection number: ACCC40816.

[0029] The present invention aims at the deficiencies of the existing Fidaxomicin fermentation process, based on the microbial growth kinetics, from the perspective of strengthening the anabolic flow of Fidaxomicin, combined with the characteristics of Fidaxomicin biosynthesis, adds a four-carbon branched chain precursor substance in the fermentation process, and converts it into isobutyric acid through microbial utilization, thereby improving the production fermentation unit of Fidaxomicin, improving production efficiency, and reducing production costs. The present invention has the advantages of clear precursor guidance and simple process control, and can be applied to the industrial production of Fidaxomicin to significantly increase production and reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 HPLC chromatogram of Fidaxomycin detection with continuous addition of four-carbon branched chain precursor in the fermentation process of Example 1;

[0031] Figure 2This is the HPLC chromatogram of Fidaxomycin detected in Example 6 without adding a four-carbon branched chain precursor substance during the fermentation process. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not mean any limitation to the present invention. Dactylosporangium aurantiacum ACCC40816 was stored in a -80°C refrigerator freezing method. Shake flask culture: 1L of seed culture medium was prepared in a 2.5L seed tank, sterilized at 120°C for 30min, then cooled to 27°C, inoculated with ACCC40816 strains, and cultured in a shake flask at 30°C and a rotation speed of 200rpm.

[0033] Example 1 Continuous addition of four-carbon branched chain precursor during fermentation

[0034] Prepare 250 L of seed culture medium in a 500 L seed tank, sterilize at 120 °C for 30 min, then cool to 28 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.2% of the volume of the seed culture medium, and then culture at 28 °C for 28 h to obtain vegetative inoculum.

[0035] Prepare 3500L of fermentation medium in a 5000L fermenter, sterilize at 120°C for 30min, then cool to 27°C, and inoculate the nutrient inoculum in the above-mentioned seed medium at an inoculum volume ratio of 4v / v% of the fermentation medium. During the fermentation process, the temperature was controlled at 28°C, the pressure was controlled at 0.05Mpa, the ventilation was controlled at 0.5VVM, and the rotation speed was controlled at 100rpm to ensure that the dissolved oxygen was above 30%.

[0036] The fermentation culture was continued. After 72 hours, the dissolved oxygen content in the fermenter dropped to 32% and gradually began to rise. Methyl isobutyrate was continuously added to the fermenter at a flow rate of 900 mL / h, and the volume ratio of methyl isobutyrate to the fermentation liquid was maintained at 0.1‰ per hour. The fermentation was continued. After 144 hours of fermentation, the addition of methyl isobutyrate was stopped (the total added volume was 0.72% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased. HPLC detection showed that the fermentation unit in the fermentation liquid was 4190 mg / L.

[0037] Example 2 Intermittent addition of four-carbon branched chain precursor during fermentation

[0038] Prepare 100 L of seed culture medium in a 200 L seed tank, sterilize at 120 °C for 30 min, then cool to 28 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.2% of the volume of the seed culture medium, and then culture at 27 °C for 34 h to obtain vegetative inoculum.

[0039] Prepare 500 L of fermentation medium in a 1000 L fermenter, sterilize at 120 ° C for 30 min, then cool to 28 ° C, and inoculate the nutrient inoculum in the above seed medium at an inoculum volume ratio of 6 v / v%. During the fermentation process, the temperature is controlled at 28 ° C, the pressure is controlled at 0.05 MPa, the ventilation is controlled at 1.0 VVM, and the rotation speed is controlled at 200 rpm to ensure that the dissolved oxygen is above 30%.

[0040] The fermentation culture was continued. After 72 hours, the dissolved oxygen content in the fermenter dropped to 31% and gradually began to rise. Isobutyric acid was added to the fermenter at a rate of 8 hours per time. The volume of isobutyric acid added each time was 0.4% of the volume of the fermentation liquid. The fermentation was continued. The addition of isobutyric acid was stopped after 144 hours of fermentation (the total added volume was 3.6% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased. The product was obtained. According to HPLC detection, the fermentation unit in the fermentation liquid was 3890 mg / L.

[0041] Example 3: Intermittent addition of four-carbon branched chain precursors during fermentation

[0042] Prepare 300 L of seed culture medium in a 500 L seed tank, sterilize at 120 °C for 30 min, then cool to 27 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.1% of the volume of the seed culture medium, and then culture at 27 °C for 28 h to obtain vegetative inoculum.

[0043] Prepare 3000L fermentation medium in a 5000L fermenter, sterilize at 120℃ for 30min, then cool to 27℃, and inoculate the nutrient inoculum in the above seed medium at an inoculum volume ratio of 5v / v%. During the fermentation process, the temperature was controlled at 27℃, the pressure was controlled at 0.04Mpa, the ventilation was controlled at 0.5VVM, and the rotation speed was controlled at 100rpm to ensure that the dissolved oxygen was above 30%.

[0044] The fermentation culture was continued. After 48 hours, the dissolved oxygen content in the fermenter dropped to 33% and gradually began to rise. A mixed four-carbon branched-chain precursor substance of isobutanol:isobutyl acetate in a ratio of 1:1 was added to the fermenter at a rate of 8 hours / time. The volume of the mixed four-carbon branched-chain precursor substance added each time was 0.2% of the volume of the fermentation liquid. The fermentation was continued. After 136 hours of fermentation, the addition of the mixed four-carbon branched-chain precursor substance was stopped (the total added volume was 2.2% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased, and the product was obtained. According to HPLC detection, the fermentation unit in the fermentation liquid was 3930 mg / L.

[0045] Example 4 Continuous addition of four-carbon branched chain precursor during fermentation

[0046] Prepare 250 L of seed culture medium in a 500 L seed tank, sterilize at 120 °C for 30 min, then cool to 28 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.3% of the volume of the seed culture medium, and then culture at 28 °C for 32 h to obtain vegetative inoculum.

[0047] Prepare 600 L of fermentation medium in a 1500 L fermenter, sterilize at 120 ° C for 30 min, then cool to 29 ° C, and inoculate the nutrient inoculum in the above seed medium at an inoculum volume ratio of 7 v / v% of the fermentation medium. During the fermentation process, the temperature was controlled at 28 ° C, the pressure was controlled at 0.05 MPa, the ventilation was controlled at 1.2 VVM, and the speed was controlled at 200 rpm to ensure that the dissolved oxygen was above 30%.

[0048] The fermentation culture was continued. After 70 hours, the dissolved oxygen content in the fermenter dropped to 33%. A 10% by mass sodium isobutyrate solution was continuously added to the fermenter at a flow rate of 1500 mL / h. The sodium isobutyrate solution was added every hour at a volume ratio of 0.25‰ to the fermentation liquid, and the fermentation was continued. The addition was stopped after 144 hours of fermentation (the total added volume was 1.8% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased, and the product was obtained. HPLC detection showed that the fermentation unit in the fermentation liquid was 4170 mg / L.

[0049] Example 5 Continuous addition of four-carbon branched chain precursor during fermentation

[0050] Prepare 100 L of seed culture medium in a 200 L seed tank, sterilize at 120 ° C for 30 min, then cool to 27-29 ° C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.3% of the volume ratio of the seed culture medium, and then culture at 29 ° C for 36 h to obtain vegetative inoculum.

[0051] 1400 L of fermentation medium was prepared in a 2000 L fermenter, sterilized at 120 ° C for 30 min and then cooled to 29 ° C, and the vegetative inoculum in the above seed medium was inoculated at an inoculum volume ratio of 8 v / v% of the fermentation medium. During the fermentation process, the temperature was controlled at 29 ° C, the pressure was controlled at 0.06 MPa, the ventilation was controlled at 1.5 VVM, and the rotation speed was controlled at 250 rpm to ensure that the dissolved oxygen was above 30%.

[0052] The fermentation culture was continued. After 64 hours, the dissolved oxygen content in the fermenter dropped to 33% and gradually began to rise. Isobutyric acid was continuously added to the fermenter at a flow rate of 300 mL / h, and the ratio of isobutyric acid added per hour to the volume of the fermentation liquid was maintained at 0.3‰. The fermentation was continued. After 144 hours of fermentation, the addition of isobutyric acid was stopped (the total added volume was 2.4% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased, and the product was obtained. HPLC detection showed that the fermentation unit in the fermentation liquid was 3975 mg / L.

[0053] Example 6 No Four-Channel Branched Chain Precursor Added During Fermentation

[0054] Prepare 300 L of seed culture medium in a 500 L seed tank, sterilize at 120 °C for 30 min, then cool to 27 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.1% of the volume of the seed culture medium, and then culture at 28 °C for 28 h to obtain vegetative inoculum.

[0055] Prepare 3500L of fermentation medium in a 5000L fermenter, sterilize at 120°C for 30min, then cool to 27°C, and inoculate the nutrient inoculum in the above-mentioned seed medium at an inoculum volume ratio of 4v / v% of the fermentation medium. During the fermentation process, the temperature was controlled at 28°C, the pressure was controlled at 0.05Mpa, the ventilation was controlled at 0.5VVM, and the rotation speed was controlled at 100rpm to ensure that the dissolved oxygen was above 30%.

[0056] The fermentation culture was continued. After 180 hours of fermentation, the fermentation was stopped when the titer stopped increasing. HPLC detection showed that the fermentation unit in the fermentation broth was 2527 mg / L.

[0057] Example 7 Continuous addition of edible oil during fermentation

[0058] Prepare 250 L of seed culture medium in a 500 L seed tank, sterilize at 120 °C for 30 min, then cool to 28 °C, inoculate 48 h old shake flask seeds at an inoculation rate of 0.2% of the volume of the seed culture medium, and then culture at 28 °C for 28 h to obtain vegetative inoculum.

[0059] Prepare 3500L of fermentation medium in a 5000L fermenter, sterilize at 120°C for 30min, then cool to 27°C, and inoculate the nutrient inoculum in the above-mentioned seed medium at an inoculum volume ratio of 4v / v% of the fermentation medium. During the fermentation process, the temperature was controlled at 28°C, the pressure was controlled at 0.05Mpa, the ventilation was controlled at 0.5VVM, and the rotation speed was controlled at 100rpm to ensure that the dissolved oxygen was above 30%.

[0060] The fermentation culture was continued. After 72 hours, the dissolved oxygen content in the fermenter dropped to 32% and gradually began to recover. Soybean oil was continuously added to the fermenter at a flow rate of 900 ml / h, and the volume ratio of soybean oil added per hour to the fermentation liquid was maintained at 0.26‰. The fermentation was continued. The addition of soybean oil was stopped after 144 hours of fermentation (the total amount added was 1.9% of the volume of the fermentation liquid). After 180 hours of fermentation, the fermentation was stopped when the titer no longer increased. HPLC detection showed that the fermentation unit in the fermentation liquid was 2867 mg / L.

[0061] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A method for producing fidaxomicin by fermentation, It is characterized in that During the fermentation process, a four-carbon branched chain precursor is added to the fermentation broth.

2. The method according to claim 1, It is characterized in that The four-carbon branched chain precursor was added between 0 and 144 hours of fermentation; Preferably, when the dissolved oxygen in the fermentation liquid starts to rise from the lowest point, the addition of the four-carbon branched chain precursor substance begins, and the addition of the four-carbon branched chain precursor substance is stopped after 144 hours of fermentation; More preferably, the four-carbon branched chain precursor is added between 48 and 144 hours of fermentation.

3. The method according to claim 1, It is characterized in that The volume of the four-carbon branched chain precursor added is 0.1 to 4 v / v% of the fermentation broth volume; Preferably, the volume of the four-carbon branched chain precursor added is 1 to 3 v / v% of the volume of the fermentation broth; More preferably, the volume of the four-carbon branched chain precursor added is 1.5-2.5 v / v% of the volume of the fermentation broth.

4. The method according to claim 1, Features The addition method of the four-carbon branched chain precursor is continuous, the addition amount is controlled, and the addition is continuous and uninterrupted; or the addition method is intermittent, and the four-carbon branched chain precursor is added 1 to 4 times per time unit.

5. The method according to claim 4, It is characterized in that The addition method of the four-carbon branched chain precursor is continuous, and 0.02 to 0.4 v / v‰ of the volume of the fermentation liquid is added every hour; Preferably, 0.1 to 0.3 v / v‰ of the volume of the fermentation broth is added per hour.

6. The method according to claim 1, Features The four-carbon branched chain precursor is selected from any one or more of isobutyric acid, isobutyrate, isobutanol, esters derived from isobutyric acid, and esters derived from isobutanol; Preferably, the four-carbon branched chain precursor is selected from any one or more of isobutyric acid, sodium isobutyrate, potassium isobutyrate, isobutanol, methyl isobutyrate, ethyl isobutyrate, propyl isobutyrate, isobutyl acetate, isobutyl propionate, and isobutyl oleate.

7. The method according to claim 1, Features The fermentation process comprises: 1) cultivating a vegetative inoculum in a seed culture medium; 2) Inoculating the vegetative inoculum into the fermentation medium, adding the four-carbon branched chain precursor substance, and continuing the fermentation to obtain fidaxomicin.

8. The method according to claim 1 or 7, It is characterized in that The strain used in the fermentation is Dactylocystis aurantiaca.

9. The method according to claim 7, Features In step 2), the vegetative inoculum in the seed culture medium of step 1) is inoculated at an inoculum volume ratio of 4 to 8 v / v% of the fermentation culture medium.

10. The method according to claim 7, Features In step 2), the fermentation conditions are as follows: the temperature is 27-29° C., the pressure is 0.04-0.06 MPa, the ventilation is 0.5-1.5 VVM, and the rotation speed is 100-250 rpm.

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