CN114854603A - 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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CN114854603A
CN114854603A CN202210496764.9A CN202210496764A CN114854603A CN 114854603 A CN114854603 A CN 114854603A CN 202210496764 A CN202210496764 A CN 202210496764A CN 114854603 A CN114854603 A CN 114854603A
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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 of echinocandin B, which is 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 years, 1 month, 29 days, the address: the microbial research institute of the national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing, and Beijing: 010-; the invention also provides application of the strain in producing echinocandin B.

Description

Strain for high-yield echinocandin B and application thereof
Technical Field
The invention relates to the technical field of microbial breeding, in particular to an echinocandin B high-yield strain obtained by screening through ribosome engineering mediated by dimethyl sulfoxide, and also relates to application of the strain.
Background
Echinocandin medicine is a novel antifungal medicine, which interferes the synthesis of fungal cell walls by inhibiting the activity of beta-1, 3-glucan synthetase, and mammalian cells have no cell walls, so the medicine has small toxic and side effects and high safety, and has antibacterial activity on candida, aspergillus and part of fungi resistant to azoles. Echinocandin class of drugs that have been approved for marketing include caspofungin (marketed in the us 2004), micafungin (marketed in japan 2005) and anidulafungin (marketed in the us 2006), all of which have a similar non-ribosomal six-membered cyclic peptide parent nucleus structure differing only in side chain groups, amino acid linkage order and post-modification groups. Wherein caspofungin and micafungin are on the market at home, and anidulafungin has a certain influence on application and popularization in clinic due to low yield and high market price, so that the method has a wide market prospect.
The industrial production of anidulafungin includes three processes, firstly, the precursor compound echinocandin B is synthesized by fermentation of Aspergillus nidulans, then the fatty chain is hydrolyzed by fermentation of Actinoplanes utahensis, and finally, the anidulafungin is obtained by adding an artificially synthesized side chain by a chemical method. Wherein, the fermentation and synthesis of the anidulafungin precursor echinocandin B by 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 in controlling production costs and are also technical thresholds limiting the industry's admission. At present, researchers at home and abroad mainly focus on mutation breeding, fermentation optimization and regulation and metabolic engineering for improving the fermentation level of echinocandin B. However, further systematic analysis shows that the core approach has limited target sites for rational modification, and compared with rational modification, the random nature of mutation breeding can exert advantages in improving the comprehensive fermentation performance of strains, especially for relatively complex multi-cell microorganisms, so that most of actinomycetes and filamentous fungi strains used in industrial production are obtained by mutation breeding. The lack of an effective microbial breeding method limits further and great improvement of the yield of echinocandin B, and in order to meet the requirement of industrial production, the fermentation level of an echinocandin B production strain needs to be rapidly improved by a proper genetic breeding means. The echinocandin B high-yield strain is obtained by a dimethyl sulfoxide (DMSO) -mediated ribosome engineering technology, fermentation optimization and extraction treatment optimization.
Disclosure of Invention
The invention provides a strain for high yield of echinocandin B, which is 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 years, 1 month, 29 days, the address: the microbial research institute of the national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing, and Beijing: 010-64807355.
On the other hand, the invention also provides a microbial inoculum which comprises the bacterial strain.
In one embodiment, the microbial inoculum 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 using a medium.
In one embodiment, the temperature of the fermentation is 20 ℃ to 40 ℃, preferably, 25 ℃; the fermentation time is 2d-20d, for example, 10d-15 d.
In one embodiment of the method of the present invention,the culture medium comprises mannitol, peanut oil, glycerol, soybean cake powder, peptone, and 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 method for preparing echinocandin B, which comprises the step of fermenting the above-mentioned strain.
Further, the method for preparing echinocandin B further comprises a step of isolating/purifying the echinocandin B.
In another aspect, the present invention provides a method for preparing anidulafungin, said method comprising the steps of:
(1) preparing echinocandin B by using the strain provided by the invention;
(2) preparing anidulafungin by using echinocandin B obtained in the step (1).
In one embodiment, the step (2) may be implemented by: the echinocandin B is fermented and hydrolyzed by actinoplanes utahensis to remove a fatty chain, and finally the anidulafungin is obtained by adding a side chain through a chemical method.
Further, the above-mentioned 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 applying a DMSO-mediated ribosome engineering technology. The method specifically comprises the steps of screening the appropriate concentration of DMSO for damaging cell walls, screening effective antibiotics through mediation of DMSO, obtaining a bacterial strain Gen-9 with high echinocandin B yield on an appropriate gentamycin resistance plate, and improving the high-yield bacterial strain by 111.4% compared with a control bacterial strain through fermentation optimization and extraction treatment optimization. The method has obvious effect, can be used for screening the echinocandin B excellent strains, improves the existing screening method, accelerates the breeding speed, and improves the titer and fermentation yield of fermentation liquor.
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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 embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 determination of optimal DMSO concentration.
Figure 2 screen for DMSO-mediated potent antibiotics.
FIG. 3 shows the yield of echinocandin B from different fermentation strains, the position of the horizontal line in the figure is the fermentation yield of the wild-type control strain.
FIG. 4 shows the yield of echinocandin B of the mutagenized strain after optimization of the fermentation conditions, the position of the horizontal line in the figure is the fermentation yield of the wild-type control strain.
FIG. 5 shows the yield of echinocandin B of the mutagenized strain after optimization of the extraction treatment conditions, and the position of the horizontal line in the figure is the fermentation yield of the wild-type control strain.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples. The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are conventional in the art and are commercially available.
PDA solid medium: 39g/L of potato/potato culture medium PDA dry powder (product of BD company, catalog No. 633840), and the balance of deionized water, autoclaving at 115 deg.C for 30min, cooling to about 60 deg.C, and making into plate.
Seed culture medium: 5-30 g/L cottonseed cake powder, 5-20 g/L glucose, 5-10 g/L glycerol and the balance of deionized water, adjusting the pH value to 5.0-7.0 by using NaOH, and performing autoclaving at 121 ℃ for 20 min.
Fermentation medium: 60-150 g/L mannitol, 15-50 g/L peanut oil, 5-20 g/L glycerin, 5-20 g/L soybean cake powder, 5-20 g/L peptone and 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 And the balance of deionized water, and sterilizing at 121 ℃ for 20min under high pressure.
Example 1 determination of optimal DMSO concentration
Taking a wild strain ATCC58397(AN01) as AN initial strain, firstly taking a frozen glycerin tube out of a refrigerator at the temperature of-80 ℃, coating a PDA flat plate after melting, and carrying out inverted culture at the temperature of 25 ℃ for 6-8 days until the spores of the strain are in a mature state. 2-3 mL of sterile normal saline is taken to be put into a PDA flat plate, spores are scrubbed by a sterile small brush pen, the washed spores are filtered by 300-500-mesh filter cloth, spore suspension is collected and washed for 2-3 times by the sterile normal saline, a spore counter counts and dilutes the spores to 10 4 ～10 6 CFU/mL spore suspension.
Respectively putting 800 mu L, 900 mu L, 1mL, 1.1mL and 1.2mL of spore suspension into a sterile 5mL centrifuge tube, respectively adding 1.2mL, 1.1mL, 1mL, 900 mu L and 800 mu L of DMSO with the purity of 99.9% into the centrifuge tube, replacing sterile water for a control experiment, uniformly mixing, putting into a refrigerator at 4 ℃ for treatment for 24-48 h, then taking 100-200 mu L of coated PDA plate, culturing at 25 ℃ for 5-8 d, and determining the optimal concentration of DMSO. As shown in FIG. 1, the growth of the strain was gradually inhibited as the DMSO concentration increased compared to the control, and 45%, 50% and 55% DMSO were used as antibiotic-mediated concentrations for ribosome engineering screening, but the optimum concentration was 50%.
Example 2 DMSO-mediated screening for potent antibiotics
Taking out the frozen glycerin pipe from a refrigerator at the temperature of minus 80 ℃, coating a PDA flat plate after dilution, and carrying out inverted culture at the temperature of 25 ℃ for 6-8 days to enable the strain spores to be in a mature state. 2-3 mL of sterile normal saline is taken to be put into a PDA flat plate, spores are scrubbed by a sterile small brush pen, the washed spores are filtered by 300-500-mesh filter cloth, spore suspension is collected and washed for 2-3 times by the sterile normal saline, a spore counter counts and dilutes the spores to 10 4 ～10 6 CFU/mL spore suspension.
Respectively taking 1mL of spore suspension liquid into 3 sterile 5mL centrifuge tubes, respectively adding 1mL of DMSO with the purity of 99.9% into the centrifuge tubes, respectively weighing 2mg, 4mg and 10mg of gentamicin into the centrifuge tubes, respectively replacing sterile water for a control experiment, uniformly mixing, placing in a refrigerator at 4 ℃ for treating for 24-48 h, then taking 150 mu L of PDA coated plate, and culturing at 25 ℃ for 3-5 d. As shown in FIG. 2, only the concentration of gentamicin was 1mg/mL, and a single colony grew on the plate, but the concentration of gentamicin was 2mg/mL after 8 days of culture, and a single colony grew on the plate, as compared to the control.
Example 3 fermentation validation of gentamicin resistant strains
And (3) selecting 10 single colonies growing in gentamicin resistant plates with the concentrations of 1mg/mL and 2mg/mL on a PDA solid plate, and culturing for 9-12 days at 25 ℃. 1-2 mL of sterile physiological saline is taken to be placed in a PDA flat plate, spores are scrubbed by a sterile small brush pen, the washed spores are filtered by 300-500-mesh filter cloth, spore suspension is collected and counted by a spore counter, 10 parts of sterile physiological saline are taken 7 CFU/mL spore suspension was inoculated in 50mL seed medium, incubated at 25 deg.C, 220rpm, and shaken for 2 days. Taking 5-50 mL of fermentation medium from the cultured seed solution, carrying out shake culture at 25 ℃ and 220rpm for 10-12 d, and arranging 3 strains in parallel. Taking 1mL of fermentation liquor from each bottle, adding methanol with the same volume, extracting for 1h by vortex oscillation, centrifuging, and taking supernatant. 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 an Agilent C-18 reverse phase column 883975-902 (4.6X 150mm, 5 μm); the mobile phase is A: 0.05% (by volume) aqueous formic acid solution, mobile phase B: 0.05% (volume ratio) formic acid acetonitrile solution, the flow rate is 1mL/min, the ultraviolet detection wavelength: 210nm, 30 ℃ and a total elution time of 25 min. Gradient elution conditions: and (3) the time is 0-5min, the volume of the mobile phase B is linearly increased from 5% to 40% in the mobile phase B, the time is 5-15min, the volume of the mobile phase B is linearly increased from 40% to 60% in the mobile phase B, the time is 15-20min, and the volume of the mobile phase B is linearly increased from 60% to 100%. As shown in FIG. 3, compared with the original strain AN01, the yield of echinocandin B is 4 strains higher than that of the original strain, the highest strain is G-9, and the yield of echinocandin B is 731.0 mg/L.
Example 4 fermentation validation of echinocandin B high producing strains after fermentation optimization
The above 10 resistant strains and a control strain AN0 were added1, re-inoculating on a PDA solid plate, and culturing at 25 ℃ for 9-12 days. 1-2 mL of sterile physiological saline is taken to be put into a PDA flat plate, spores are scrubbed by a sterile small brush pen, the washed spores are filtered by 300-500 meshes of filter cloth, a spore suspension is collected and counted by a spore counter, and the number of the spores is increased (10) 8 CFU/mL) spore suspension was inoculated in 50mL seed medium at 25 deg.C, 220rpm, and shake-cultured for 2 d. Taking 5-50 mL of fermentation medium from the cultured seed solution, carrying out fermentation culture at 25 ℃ and 220rpm for 13d, setting 3 strains in parallel, and controlling the humidity of a shaking table to avoid evaporation in the whole fermentation process. Taking 1mL of fermentation liquor from each bottle, adding methanol with the same volume, extracting for 1h by vortex oscillation, centrifuging, and taking supernatant. 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 an Agilent C-18 reverse phase column 883975-902 (4.6X 150mm, 5 μm); the mobile phase is A: 0.05% (by volume) aqueous formic acid solution, mobile phase B: 0.05% (volume ratio) formic acid acetonitrile solution, the flow rate is 1mL/min, the ultraviolet detection wavelength: 210nm, 30 ℃ and a total elution time of 25 min. Gradient elution conditions: and (3) the volume of the mobile phase B is linearly increased from 5% to 40% in 0-5min and is increased from 5% to 15min, the volume of the mobile phase B is linearly increased from 40% to 60% in 15-20min, and the volume of the mobile phase B is linearly increased from 60% to 100%. As shown in FIG. 4, compared with the starting strain AN01, the yield of echinocandin B after fermentation is 9 strains, only one strain is lower than that of the control strain, the positive mutation rate is high, and the yield of the highest echinocandin B strain is 1070.0 mg/L.
Example 5 fermentation validation of echinocandin B high producing strains after optimization of extraction treatment
The 9 mutant strains and the control strain AN01 are re-inoculated on a PDA solid plate and cultured for 9-12 days at 25 ℃. 1-2 mL of sterile physiological saline is taken to be placed in a PDA flat plate, spores are scrubbed by a sterile small brush pen, the washed spores are filtered by 300-500-mesh filter cloth, spore suspension is collected and counted by a spore counter, and the count is 10 8 The CFU/mL spore suspension was inoculated into 50mL seed medium at 25 deg.C, 220rpm, and shake cultured for 2 days. Culturing the above5-50 mL of fermentation medium is taken as seed culture liquid, shaking culture is carried out at 25 ℃ and 220rpm for 13d, each strain is arranged in 3 parallel, and the humidity of the shaking table needs to be controlled in order to avoid evaporation in the whole fermentation process. Taking 1mL of fermentation liquor from each bottle, adding methanol with the volume 5-10 times of that of the fermentation liquor, performing vortex oscillation extraction for 1 hour, centrifuging, and taking supernatant. 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 an Agilent C-18 reverse phase column 883975-902 (4.6X 150mm, 5 μm); the mobile phase is A: 0.05% (by volume) aqueous formic acid solution, mobile phase B: 0.05% (volume ratio) formic acid acetonitrile solution, the flow rate is 1mL/min, the ultraviolet detection wavelength: 210nm, 30 ℃ and a total elution time of 25 min. Gradient elution conditions: and (3) the volume of the mobile phase B is linearly increased from 5% to 40% in 0-5min and is increased from 5% to 15min, the volume of the mobile phase B is linearly increased from 40% to 60% in 15-20min, and the volume of the mobile phase B is linearly increased from 60% to 100%. The result is shown in figure 5, through the optimization of fermentation conditions and extraction treatment conditions, compared with the original strain AN01, the yield of 9 mutant bacteria echinocandin B is higher than that of the original strain, the highest one is 1257.6mg/L, and the yield is increased by 111.4% compared with that of the original strain. We named it as 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 years, 1 month and 29 days, address: the microbial research institute of the national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing, and Beijing: 010-64807355, the yield of echinocandin B produced by the strain fermentation is increased by 111.4 percent compared with the original strain.
The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A strain for high yield of echinocandin B is Aspergillus nidulans (Aspergillus nidulans) Gen-9, is preserved in China general microbiological culture Collection center (CGMCC), has a preservation number of CGMCC No.40074, a preservation date of 2022 years, 1 month and 29 days, and has an address: the microbial research institute of the national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing, and Beijing: 010-64807355.
2. A microbial preparation comprising the strain of claim 1.
3. The microbial inoculum according to claim 2, which 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 culture medium.
6. A method for preparing echinocandin B, comprising the step of fermenting the strain of claim 1.
7. A process for the preparation of anidulafungin, said process comprising the steps of:
(1) preparing echinocandin B by fermentation using the strain of claim 1;
(2) preparing anidulafungin by using echinocandin B obtained in the step (1).
8. The method according to any one of claims 5 to 7, wherein the temperature of the fermentation is 20 ℃ to 40 ℃.
9. The method according to any one of claims 5 to 7, wherein the fermentation time is 2d to 20 d.
10. The method of any one of claims 5 to 7, wherein the fermentation medium comprises 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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