CN114854603B - Strain for high-yield echinocandin B and application thereof - Google Patents

Strain for high-yield echinocandin B and application thereof Download PDF

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CN114854603B
CN114854603B CN202210496764.9A CN202210496764A CN114854603B CN 114854603 B CN114854603 B CN 114854603B CN 202210496764 A CN202210496764 A CN 202210496764A CN 114854603 B CN114854603 B CN 114854603B
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吕雪峰
刘永娟
黄雪年
王贝贝
周宇
谷猛
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Qingdao High Energy Hecheng Biotechnology Co ltd
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The invention provides a strain for high-yield echinocandin B, which is aspergillus nidulans (Aspergillus nidulans) Gen-9 and is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.40074, the preservation date is 2022, 1 month and 29 days, and the address is: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355; the invention also provides application of the strain in production of echinocandin B.

Description

Strain for high-yield echinocandin B and application thereof
Technical Field
The invention relates to the technical field of microorganism breeding, in particular to an echinocandin B high-yield strain obtained by dimethyl sulfoxide-mediated ribosome engineering screening, and also relates to application of the strain.
Background
The echinocandin medicine is a novel antifungal medicine, and the synthesis of fungal cell walls is interfered by inhibiting the activity of beta-1, 3-glucan synthase, and mammalian cells have no cell walls, so that the medicine has small toxic and side effects, high safety and antibacterial activity on candida, aspergillus and partial azole-resistant fungi. Echinocandins that have been approved for marketing include caspofungin (marketed in the united states in 2004), micafungin (marketed in japan in 2005), and anidulafungin (marketed in the united states in 2006), all of which have similar non-ribosomal hexacyclic peptide-like parent core structures, differing only in the side chain groups, amino acid attachment sequences, and post-modification groups. The caspofungin and the micafungin are marketed in China, and the anidulafungin has low yield and high market price, so that the application and popularization in clinic are affected to a certain extent, and the micafungin has a wide market prospect.
The industrial production of anidulafungin includes three processes, namely, the precursor compound echinocandin B is synthesized by Aspergillus nidulans fermentation, then fatty chains are hydrolyzed by actinoplanes utahensis fermentation, and finally, anidulafungin is obtained by adding an artificially synthesized side chain through a chemical method. Wherein, aspergillus nidulans is the first step of industrial production and is also the key core of the whole process. High performance fermentation strains are key factors for controlling production cost and are technical thresholds for limiting the admission of the industry. At present, research on improving the fermentation level of echinocandin B by scholars at home and abroad mainly aims at mutation breeding, fermentation optimization, regulation and control and metabolic engineering. However, further systematic analysis has found that core pathways have limited targets for rational modification, and that the randomness of mutagenic breeding is advantageous when the overall fermentation performance of the strain is improved, especially for relatively complex multicellular microorganisms, so that most actinomycetes and filamentous fungal strains used in current industrial production are obtained by mutagenic breeding. The lack of an effective microorganism breeding method limits the further substantial increase in the production of echinocandin B, and in order to meet the requirements of industrial production, the fermentation level of the echinocandin B producing strain needs to be rapidly increased by suitable genetic breeding means. The invention obtains a high-yield echinocandin B strain through dimethyl sulfoxide (DMSO) -mediated ribosome engineering technology, fermentation optimization and extraction treatment optimization.
Disclosure of Invention
The invention provides a strain for high-yield echinocandin B, which is aspergillus nidulans (Aspergillus nidulans) Gen-9 and is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.40074, the preservation date is 2022, 1 month and 29 days, and the address is: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355.
On the other hand, the invention also provides a microbial inoculum, which comprises the strain.
In one embodiment, the microbial agent is a liquid formulation or a solid formulation.
In another aspect, the present invention also provides a method for fermenting the above strain, comprising the step of fermenting the above strain with a medium.
In one embodiment, the fermentation temperature is 20 ℃ to 40 ℃, preferably 25 ℃; the fermentation time is 2d-20d, for example, 10d-15d.
In one embodiment, the medium comprises mannitol, peanut oil, glycerol, soybean meal, peptone, feSO 4 ·7H 2 O，K 2 HPO 4 ，MgSO 4 ·7H 2 O，MnSO 4 ·H 2 O，CuSO 4 ·5H 2 O，CaCl 2 。
On the other hand, the invention also provides application of the strain or the microbial inoculum in producing echinocandin B.
In another aspect, the present invention also provides a process for preparing echinocandin B, comprising the step of fermenting the strain described above.
Further, the method for preparing echinocandin B further comprises a step of isolating/purifying the echinocandin B.
In another aspect, the present invention also provides a method of preparing anidulafungin, the method comprising the steps of:
(1) Preparing echinocandin B by using the strain disclosed by the invention;
(2) And (3) preparing anidulafungin by using the echinocandin B obtained in the step (1).
In one embodiment, the above step (2) may be implemented by: the fatty chain is hydrolyzed by the fermentation of echinocandin B through actinoplanes utahensis, and finally the anidulafungin is obtained by adding a side chain through a chemical method.
Further, the above method for preparing anidulafungin further comprises a step of isolating/purifying anidulafungin.
The invention discloses a breeding method for screening echinocandin B high-yield strains by using a DMSO (dimethyl sulfoxide) -mediated ribosome engineering technology. Specifically, the method comprises the steps of screening DMSO to destroy proper concentration of cell walls, screening effective antibiotics through DMSO mediation, obtaining a strain Gen-9 for producing echinocandin B in high yield on a proper gentamicin resistance plate, and optimizing through fermentation and extraction treatment, wherein the strain for producing the echinocandin B in high yield is 111.4% higher than that of a control strain. The invention has obvious effect, can be used for screening excellent strains of echinocandin B, improves the existing screening method, accelerates the breeding speed, and improves the titer of fermentation liquor and the fermentation yield.
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The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 determination of optimal DMSO concentration.
Figure 2 dmso mediated screening for potent antibiotics.
FIG. 3 shows the fermentation yields of different fermentation strains, echinocandin B, with the position of the horizontal line in the figure being the fermentation yield of the wild type control strain.
FIG. 4 shows the fermentation yield of the wild-type control strain at the horizontal line position, after optimizing the fermentation conditions, by mutating the production of echinocandin B.
FIG. 5 shows the fermentation yield of wild-type control strain at the horizontal line position, after optimizing the extraction conditions.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the invention is not limited to the examples. The materials, reagents, instruments and methods used in the examples below, without any particular description, are conventional in the art and are commercially available.
PDA solid medium: 39g/L potato medium PDA dry powder (BD company product, catalog number: 633840), balance deionized water, and autoclaved at 115℃for 30min, and cooled to about 60℃to prepare a flat plate.
Seed culture medium: 5-30 g/L cottonseed cake powder, 5-20 g/L glucose, 5-10 g/L glycerol and the balance deionized water, regulating the pH to 5.0-7.0 by NaOH, and sterilizing at 121 ℃ for 20min under high pressure.
Fermentation medium: 60-150 g/L mannitol, 15-50 g/L peanut oil, 5-20 g/L glycerol, 5-20 g/L soybean cake powder, 5-20 g/L peptone, 0.01-0.07 g/L FeSO 4 ·7H 2 O，5～10g/L K 2 HPO 4 ，0.2～1g/L MgSO 4 ·7H 2 O，0.1～0.8g/L MnSO 4 ·H 2 O，0.3～0.8g/L CuSO 4 ·5H 2 O，0.1～0.5g/L CaCl 2 Deionized water for the rest, natural pH, and autoclaving at 121deg.C for 20min.
EXAMPLE 1 determination of optimal DMSO concentration
Taking a wild strain ATCC58397 (AN 01) as AN original strain, firstly taking out a frozen glycerol tube from a refrigerator at the temperature of minus 80 ℃, coating a PDA plate after melting, inversely culturing for 6-8 days at the temperature of 25 ℃, and waiting for spores of the strain to be in a mature state. 2-3 mL of sterile physiological saline is taken to be placed in a PDA flat plate, a sterile small writing brush is used for brushing spores, the washed spores are filtered by a 300-500 mesh filter cloth, spore suspension is collected and washed for 2-3 times by the sterile physiological saline, and the spores are counted by a spore counter and diluted to 10 4 ～10 6 CFU/mL spore suspension.
Respectively taking 800 mu L, 900 mu L, 1mL, 1.1mL and 1.2mL of spore suspension liquid into a sterile 5mL centrifuge tube, respectively adding 1.2mL, 1.1mL, 1mL, 900 mu L and 800 mu L of DMSO with purity of 99.9% into the centrifuge tube, replacing a control experiment with sterile water, uniformly mixing, then placing the mixture into a refrigerator with the temperature of 4 ℃ for 24-48 h, then taking 100-200 mu L of coated PDA flat plate, culturing for 5-8 d at the temperature of 25 ℃, and determining the optimal concentration of the DMSO. The results are shown in FIG. 1, in which strain growth was gradually inhibited with increasing DMSO concentration compared to control, and both 50% and 55% DMSO could be used as antibiotic-mediated concentrations for ribosome engineering screening, but the optimum concentration was 50%.
EXAMPLE 2 DMSO-mediated screening of potent antibiotics
Taking out a frozen glycerol tube from a refrigerator at the temperature of minus 80 ℃, diluting, coating a PDA plate, and inversely culturing for 6-8 d at the temperature of 25 ℃ to enable spores of the strain to be in a mature state. 2-3 mL of sterile physiological saline is taken to be placed in a PDA flat plate, a sterile small writing brush is used for brushing spores, the washed spores are filtered by a 300-500 mesh filter cloth, spore suspension is collected and washed for 2-3 times by the sterile physiological saline, and the spores are counted by a spore counter and diluted to 10 4 ～10 6 CFU/mL spore suspension.
1mL of spore suspension is respectively taken in 3 sterile 5mL centrifuge tubes, 1mL of DMSO with purity of 99.9% is respectively added into the centrifuge tubes, 2mg, 4mg and 10mg of gentamicin are respectively weighed in the centrifuge tubes, sterile water is used for replacing control experiments, the mixture is uniformly mixed and then placed in a refrigerator at 4 ℃ for 24-48 hours, 150 mu L of coated PDA plates are then taken, and the mixture is cultured for 3-5 days at 25 ℃. As a result, as shown in FIG. 2, only gentamicin at 1mg/mL grew on the plate, but gentamicin at 2mg/mL after 8d of incubation, and also grew on the plate, compared to the control.
EXAMPLE 3 fermentation verification of gentamicin resistant Strain
10 single colonies growing in gentamicin resistant plates with the concentration of 1mg/mL and 2mg/mL are picked on PDA solid plates and cultured for 9-12 d at 25 ℃. 1-2 mL of sterile physiological saline is taken to be placed in a PDA flat plate, a sterile small writing brush is used for brushing spores, the washed spores are filtered by 300-500 meshes of filter cloth, spore suspension is collected and counted by a spore counter, and 10 is taken 7 The CFU/mL spore suspension was inoculated into 50mL of seed medium at 25℃and 220rpm, followed by shaking for 2d. Taking 5-50 mL of fermentation medium from the above-mentioned cultured seed solution, 25 deg.C, 220rpm, shake culturing for 10-12 d, every strainThe plants were arranged in 3 replicates. 1mL of each bottle of fermentation broth is taken, an equal volume of methanol is added, vortex shaking extraction is carried out for 1h, and the supernatant is taken after centrifugation. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous formic acid, mobile phase B:0.05% (volume ratio) acetonitrile formate solution, flow rate is 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 25min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 40% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 40% to 60% by volume of the mobile phase for 5-15min, and the mobile phase B is linearly increased from 60% to 100% by volume of the mobile phase for 15-20 min. As a result, as shown in FIG. 3, the yield of echinocandin B was 4 strains higher than that of the starting strain AN01, the highest strain was G-9, and the yield of echinocandin B was 731.0mg/L.
Example 4 fermentation verification of highly-produced echinocandin B strains after fermentation optimization
The above 10 resistant strains and the control strain AN01 were re-inoculated on PDA solid plates and cultured at 25℃for 9-12 d. 1-2 mL of sterile physiological saline is put in a PDA flat plate, a sterile small writing brush is used for brushing spores, the washed spores are filtered by a 300-500-mesh filter cloth, spore suspension is collected and counted by a spore counter, and the diameter of the spores is increased (10) 8 CFU/mL) spore suspension was inoculated in 50mL of seed medium, 25 ℃,220rpm, shaking for 2d. Taking 5-50 mL of fermentation medium from the seed liquid, fermenting and culturing for 13d at 25 ℃ and 220rpm, wherein each strain is arranged in 3 parallels, and the humidity of a shaking table needs to be controlled to avoid evaporation in the whole fermentation process. 1mL of each bottle of fermentation broth is taken, an equal volume of methanol is added, vortex shaking extraction is carried out for 1h, and the supernatant is taken after centrifugation. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous formic acid, mobile phase B:0.05% (volume ratio) acetonitrile formate solution, flow rate is 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 25min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 40% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 40% to 60% by volume of the mobile phase for 5-15min, and the mobile phase B is linearly increased from 60% to 100% by volume of the mobile phase for 15-20 min. As a result, as shown in FIG. 4, the yield of echinocandin B at the end of fermentation was 9 strains higher than that of the starting strain AN01, only one strain was lower than that of the control strain, the positive mutation rate was very high, and the highest yield of echinocandin B was 1070.0mg/L.
Example 5 fermentation verification of highly productive echinocandin B Strain after optimization of extraction treatment
The above 9 mutant strains and the control strain AN01 were re-inoculated on PDA solid plates and cultured at 25℃for 9-12 d. 1-2 mL of sterile physiological saline is taken to be placed in a PDA flat plate, a sterile small writing brush is used for brushing spores, the washed spores are filtered by 300-500 meshes of filter cloth, spore suspension is collected and counted by a spore counter, and 10 8 CFU/mL spore suspension was inoculated in 50mL of seed medium at 25℃and 220rpm, followed by shaking for 2d. Taking 5-50 mL of fermentation medium from the seed liquid, shaking the culture medium at 25 ℃ and 220rpm for 13d, setting 3 strains in parallel for each strain, and controlling the humidity of a shaking table to avoid evaporation in the whole fermentation process. 1mL of each bottle of fermentation liquor is taken, methanol with the volume of 5-10 times is added, vortex vibration extraction is carried out for 1h, and the supernatant is obtained after centrifugation. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous formic acid, mobile phase B:0.05% (volume ratio) acetonitrile formate solution, flow rate is 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 25min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 40% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 40% to 60% by volume of the mobile phase for 5-15min, and the mobile phase B is linearly increased from 60% to 100% by volume of the mobile phase for 15-20 min. As shown in FIG. 5, the yield of 9 mutant bacteria echinocandin B is higher than that of the original strain by 111.4% compared with the original strain AN01 through optimizing the fermentation condition and the extraction treatment condition, and the highest strain is 1257.6 mg/L. We name Aspergillus nidulans (Aspergillus nidulans) Gen-9, which is preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC No.40074 and the preservation date of 2022, 1 month and 29 days, address: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355, the yield of echinocandin B produced by the strain through fermentation is 111.4 percent higher than that of the original strain.
While the invention has been described in detail with respect to the general description and specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The strain for producing echinocandin B is aspergillus nidulansAspergillus nidulans) Gen-9, the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.40074, the preservation date is 2022, 1 month and 29 days, and the address is: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355.
2. A microbial agent comprising the strain of claim 1.
3. The microbial agent of claim 2, wherein the microbial agent is a solid or liquid formulation.
4. Use of the strain of claim 1 or the microbial agent of claim 2 for the production of echinocandin B.
5. A method of fermenting the strain of claim 1, comprising the step of fermenting the strain with a medium.
6. A method of preparing echinocandin B, said method comprising the step of fermenting the strain of claim 1.
7. A method of preparing anidulafungin, the method comprising the steps of:
(1) Fermenting the strain of claim 1 to produce echinocandin B;
(2) And (3) preparing anidulafungin by using the echinocandin B obtained in the step (1).
8. The method according to any one of claims 5 to 7, wherein the fermentation temperature is 20 ℃ to 40 ℃.
9. The method of any one of claims 5-7, wherein the fermentation is for a period of time ranging from 2d to 20 d.
10. The method according to any one of claims 5 to 7, wherein the components of the fermentation medium comprise mannitol, peanut oil, glycerol, soybean meal, peptone, feSO 4 ，K 2 HPO 4 ，MgSO 4 ，MnSO 4 ，CuSO 4 ，CaCl 2 。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103289901A (en) * 2012-02-22 2013-09-11 上海来益生物药物研究开发中心有限责任公司 Echinocandin B high-producing strain and application thereof
CN103724403A (en) * 2012-10-12 2014-04-16 重庆乾泰生物医药有限公司 Separation purification method and use of echinocandin B
WO2016056022A2 (en) * 2014-10-07 2016-04-14 Alaparthi Lakshmi Prasad Intermediates and processes to prepare anidulafungin
CN107779402A (en) * 2016-08-27 2018-03-09 鲁南制药集团股份有限公司 A kind of screening technique of ECB production bacterium superior strain

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103289901A (en) * 2012-02-22 2013-09-11 上海来益生物药物研究开发中心有限责任公司 Echinocandin B high-producing strain and application thereof
CN103724403A (en) * 2012-10-12 2014-04-16 重庆乾泰生物医药有限公司 Separation purification method and use of echinocandin B
WO2016056022A2 (en) * 2014-10-07 2016-04-14 Alaparthi Lakshmi Prasad Intermediates and processes to prepare anidulafungin
CN107779402A (en) * 2016-08-27 2018-03-09 鲁南制药集团股份有限公司 A kind of screening technique of ECB production bacterium superior strain

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