Myxococcus flavus for producing epothilone as well as screening method and application of myxococcus flavus

Abstract

The invention discloses myxococcus flavus with high yield of epothilone, the name of the myxococcus flavus is Myxococcus flavus ZE9N-R22, the myxococcus flavus belongs to the technical field of biology, the strain is preserved in China Center for Type Culture Collection (CCTCC) on September 27, 2024, and the preservation number of the strain is CCTCC M 20242100. The invention also discloses a screening method of the ZE9N-R22 strain and an application of the ZE9N-R22 strain in preparation of epothilone. Myxococcus flavus dispersedly grows in a liquid culture medium, can be subjected to high-density fermentation in a fermentation tank and is stable in strain fermentation performance, the epothilone yield is as high as 93.4 mg / L, main products are epothilone B and epothilone A, no other by-products exist, the content of high-activity epothilone B is increased, the separation and purification difficulty of subsequent products is greatly reduced, and the method is suitable for industrial production. The strain is an epothilone production strain with great research and development values.

Description

Technical Field

[0001] The invention relates to a strain of Myxococcus xanthus, in particular to a strain of Myxococcus xanthus producing epothilone and a screening method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Epothilone is a 16-membered macrolide antibiotic produced by myxobacteria, which has the activity of stabilizing microtubules and inhibiting the proliferation of tumor cells. Epothilone has a similar anti-tumor mechanism to paclitaxel, but due to its smaller molecular weight and better water solubility, it has good inhibitory activity against paclitaxel-resistant tumor cells, and can be produced by microbial fermentation. It is considered to be a newer alternative to paclitaxel (Goodin et al., 2004). At present, there are two epothilone anti-tumor drugs, isapilone (Peterson et al., 2005) and eutilide (Xu et al., 2021), which are marketed abroad and domestically, respectively, for the clinical treatment of advanced breast cancer.

[0003] The original producer of epothilone is the myxobacterium Sorangium cellulosum. The fermentation yield of this strain is low, it grows in clusters in liquid culture medium, and the growth generation time is as long as 16 hours, which seriously affects the genetic modification and yield optimization of the strain. Sorangium cellulosum produces a variety of epothilone structural analogs, including epothilone A, B, C, D, etc., among which the main products are epothilone A and epothilone B. Epothilone B has the highest anti-tumor activity and is also the raw material for the production of isapilone. However, the proportion of epothilone B produced by fermentation of Sorangium cellulosum is low, and the ratio of epothilone B to epothilone A is usually 1:2, which increases the difficulty and cost of separating the target product. After the biosynthetic gene cluster of epothilone was reported, it was heterologously expressed in hosts such as Streptomyces, Escherichia coli, Myxococcus xanthus, Pseudomonas putida, and Burkholderia. Among them, Myxococcus xanthus, as a close host of the original producing bacteria, showed the highest initial heterologous expression yield, and thus has great application potential (Yue et al., 2023). For example, the epothilone gene cluster from S. cellulosum So0157-2 was introduced into Myxococcus xanthus, and the resulting strain M. xanthus ZE9 had an epothilone yield of up to 1.2 mg / L, which was much higher than the heterologous expression yield of other hosts (Zhu et al., 2015). Most of the studies on heterologous expression of epothilone in Myxococcus xanthus have optimized the epothilone yield by optimizing fermentation conditions and regulating transcription levels. However, the current epothilone yield is still far lower than that of the original bacteria and cannot meet the needs of industrial production.

[0004] Ribosome engineering is a breeding technique that uses antibiotics targeting ribosomes or RNA polymerases to screen resistant mutants and activate the synthesis of secondary metabolism in strains (Hosaka et al., 2009). This technology is widely used in actinomycetes and effectively activates the synthesis of new secondary metabolites or increases the yield of known natural products (Zhu et al., 2019). After searching the literature and patents, this technology has never been used in myxobacteria. Whether ribosome engineering is suitable for optimizing the yield of epothilone in Myxococcus xanthus has not been reported. Summary of the invention

[0005] In view of the above-mentioned problems in the prior art, the object of the present invention is to provide a strain of Myxococcus xanthus that produces epothilone and a screening method and application thereof.

[0006] As the first aspect of the present invention, a strain of Myxococcus xanthus producing epothilone is provided, named Myxococcus xanthus ZE9N-R22, which was deposited in the China Center for Type Culture Collection (CCTCC) on September 27, 2024, with a deposit number of CCTCC M 20242100, and the address is Wuhan University, Wuhan, China.

[0007] The epothilone-producing Myxococcus xanthus of the present invention is Myxococcus xanthus ZE9N-R22 obtained by screening the epothilone heterologous expression strain Myxococcus xanthus ZE9 with neomycin and rifampicin.

[0008] As a second aspect of the present invention, a method for screening the Myxococcus xanthus ZE9N-R22 strain is also disclosed, comprising the following steps:

[0009] S1, Myxococcus aureus ZE9 was cultured in CYE medium, transferred to a culture plate, and antibiotics acting on ribosomes or RNA polymerase were added.

[0010] The antibiotic is selected from tetracycline, paromomycin, neomycin, streptomycin, rifampicin, gentamicin and spectinomycin;

[0011] Furthermore, tetracycline, paromomycin, neomycin, and rifampicin with a minimum inhibitory concentration of less than 256 μg / ml were selected for screening of resistant mutant strains;

[0012] S2, single antibiotic screening, antibiotics were selected from tetracycline, paromomycin, neomycin, and rifampicin;

[0013] The concentrations were tetracycline 8 μg / ml, paromomycin 100 μg / ml, neomycin 64 μg / ml, and rifampicin 2 μg / ml;

[0014] S3, double-resistant mutant strain, continue to screen for double-resistant mutant strains resistant to rifampicin; rifampicin concentration is 2 μg / ml;

[0015] S4, screen out the dual-resistant strain with the highest epothilone production.

[0016] As a third aspect of the present invention, it provides the use of the Myxococcus xanthus ZE9N-R22 strain in the preparation of epothilone.

[0017] Specifically, ZE9N-R22 was transferred to a shake flask of CYE medium to culture seed liquid, and the seed liquid was transferred to a fermenter of CMO medium. After fermentation, the resin was collected by filtering with a filter, and methanol was added for extraction to obtain a crude extract of epothilone.

[0018] CYE medium composition: 10g / L casein peptone, 5g / L yeast extract, 4mM magnesium sulfate·7H2O 10mM MOPS, pH 7.6. Add 1.5% agar powder to the solid medium and sterilize with high pressure steam at 115℃ for 30min;

[0019] CMO medium composition: On the basis of CYE medium, XAD 16 macroporous adsorption resin was added at a ratio of 2%, and sterilized by high-pressure steam at 115°C for 30 minutes; 7 mL / L of filtered and sterilized methyl oleate was added before use.

[0020] Further, the seed liquid was transferred to a fermentation tank filled with CMO culture medium at a ratio of 30%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The yellow myxococcus provided by the present invention grows dispersedly in a liquid culture medium, can be fermented at a high density in a fermenter, has stable fermentation performance, and has an epothilone yield of up to 93.4 mg / L. The main products are epothilone B and epothilone A, without other by-products, and the content of highly active epothilone B is increased, which greatly reduces the difficulty of separation and purification of subsequent products. The strain is an epothilone production strain with great research and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1.Growth and fermentation status of Myxococcus xanthus ZE9N-R22 strain. A, electron microscope observation photo of ZE9N-R22; B, colony morphology of ZE9N-R22 on CYE plate; C, growth status of ZE9N-R22 in CYE liquid medium; D, fermentation status and biomass statistics of ZE9N-R22 in fermenter.

[0025] Figure 2 .Identification diagram of epothilone produced by fermentation of Myxococcus xanthus ZE9N-R22. A, mass spectrometry identification of epothilone A; B, mass spectrometry identification of epothilone B.

[0026] Figure 3 .Statistical chart of epothilone production produced by fermentation of Myxococcus xanthus ZE9N-R22. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] In the embodiment of the present invention, the screening method of the ZE9N-R22 strain provided is:

[0029] Myxococcus xanthus ZE9 was cultured overnight in CYE medium, and the OD 600The value was diluted to 0.005 and transferred to a 96-well culture plate at 150uL / well. Antibiotics acting on ribosomes or RNA polymerase were added respectively, including but not limited to tetracycline, paromomycin, neomycin, streptomycin, rifampicin, gentamicin and spectinomycin. The final concentrations of antibiotics were 256, 128, 64, 32, 16, 8, 4, 2 and 1μg / mL, respectively. After culturing at 30℃ for 36h, the minimum inhibitory concentration of antibiotics was observed, and tetracycline, paromomycin, neomycin and rifampicin with a minimum inhibitory concentration of less than 256μg / ml were selected for screening of resistant mutant strains. The overnight culture seed solution of ZE9 strain was diluted to OD 0.1, 1 ml of the bacterial solution was mixed with 2.5 mL of soft agar (agar powder content of 0.3%), and plated on screening plates containing tetracycline (8 μg / ml), paromomycin (100 μg / ml), neomycin (64 μg / ml) and rifampicin (2 μg / ml), respectively, and single resistant colonies were picked after culturing at 30°C for 7 days. Eight tetracycline-resistant strains were obtained by single antibiotic screening, named ZE9-T1 to ZE9-T8; 28 paromomycin-resistant strains were obtained, named ZE9-P1 to ZE9-P28; 37 neomycin-resistant strains were obtained, named ZE9-N1 to ZE9-N37; 19 rifampicin-resistant strains were obtained, named ZE9-R1 to ZE9-R1. All the above monoclonal antibody mutants were transferred to a shake flask containing 50mL CYE medium, and the seed solution was cultured overnight at 30℃200rpm. At a ratio of 2%, the shake flask containing 50mL CMO medium was transferred, and fermented at 30℃200rpm for 7 days. The resin was collected by filtration, and extracted with 3mL methanol overnight. The extract was filtered through a 0.22μm pore size filter membrane and analyzed for epothilone production by high performance liquid chromatography-mass spectrometry. The mobile phase for liquid phase analysis was 60% methanol and 40% water, the flow rate was 1ml / min, the sample volume was 20μL each time, and the detection wavelength was 249nm. According to the peak area A of the characteristic ultraviolet absorption peak of epothilone at 249nm, the formula Y(mg / L)=(A×1.1×10 -7 -3×10 -5 )×60 to quantify the epothilone production. After testing, the neomycin-resistant strain ZE9-N18 had the highest epothilone production, reaching 34.5 mg / L. Using the ZE9-N18 strain, the same method was used to continue screening for dual-resistant mutant strains resistant to rifampicin (2 μg / ml), and a total of 63 dual-resistant strains were obtained, named ZE9N-R1 to ZE9N-R63. The epothilone production of 63 dual-resistant strains was analyzed by shake flask fermentation, and the dual-resistant strain ZE9N-R22 with the highest production was screened out, with an epothilone production of 54.8 mg / L.

[0030] The epothilone-producing Myxococcus xanthus provided in the embodiment of the present invention is obtained by screening the epothilone heterologous expression strain Myxococcus xanthus ZE9 using neomycin (64 μg / ml) and rifampicin (2 μg / ml) to obtain Myxococcus xanthus ZE9N-R22 (CCTCC M 20242100). The biological characteristics of the strain are: the vegetative cells are short rod-shaped ( Figure 1 -A), the colonies on the plate expanded and grew bright yellow ( Figure 1 -B). The strains were grown in shake flasks containing CYE medium ( Figure 1 -C), high-density fermentation can be carried out in the fermenter, with the maximum cell dry weight reaching 24g / L ( Figure 1 -D). The strain was fermented in a shake flask using CMO medium for 7 days, and the main product was epothilone A ( Figure 2 A) and epothilone B ( Figure 2 B), and the content of epothilone B increased, and the ratio of epothilone B to epothilone A increased to 1.1:1 ( Figure 3 ). The strain was fermented in a 10L fermenter in batch mode for 20 days, and the total epothilone production reached 93.4 mg / L.

[0031] CYE medium composition: 10g / L casein peptone, 5g / L yeast extract, 4mM magnesium sulfate·7H2O 10mM MOPS, pH 7.6. Add 1.5% agar powder to the solid medium. Sterilize with high pressure steam at 115℃ for 30min.

[0032] CMO medium composition: XAD 16 macroporous adsorption resin was added to CYE medium at a ratio of 2%, and sterilized by high pressure steam at 115°C for 30 minutes. 7 mL / L of filtered and sterilized methyl oleate was added before use.

[0033] The embodiment of the present invention also provides the use of Myxococcus xanthus ZE9N-R22 in the preparation of epothilone:

[0034] ZE9N-R22 is preferably subjected to continuous fed-batch fermentation using the following conditions: ZE9N-R22 is transferred to a shake flask containing 50 mL of CYE medium, and the seed solution is cultured overnight at 30°C. The seed solution is preferably transferred to a 10 L fermenter containing 5 L of CMO medium at a ratio of 30%, and the pH is set to 7.4, and the dissolved oxygen is 50%. Feeding is started after 5 days of fermentation, and the feeding is performed at a ratio of 6 ml / L of methyl oleate, 4 g / L of casein peptone, and 2 g / L of yeast extract. The feeding is performed once every 48 hours, for a total of 5 times. After 20 days of fermentation, the resin is collected by filtration using a filter, and 300 mL of methanol is added for overnight extraction to obtain a crude epothilone extract.

[0035] The following conditions are preferred for separation and quantification of epothilone compounds:

[0036] The methanol extract was filtered using a 0.22 μm pore size filter membrane and analyzed by high performance liquid mass spectrometry using a C18 chromatographic column. The following analysis conditions were preferably used: the mobile phase was 60% methanol and 40% water, the flow rate was 1 ml / min, the sample load was 20 μL each time, and the detection wavelength was 249 nm. Epothilone A and Epothilone B peaked at 13.5 min and 16.6 min, respectively. According to the peak area A of the characteristic ultraviolet absorption peak of Epothilone at 249 nm, the formula Y (mg / L) = (A × 1.1 × 10 -7 -3×10 -5 )×60 was used to quantify the epothilone production.

[0037] Example 1. Construction of the Epothilone Heterologous Expression Strain ZE9

[0038] Plasmid p15A-TP-CF carries the epothilone biosynthetic gene cluster from Sorangium cellulosum So0157-2 (Accession: EU414841.2). The plasmid was introduced into Myxococcus xanthus DZ2 (strain accession number ATCC25232) by electroporation. The gene cluster was integrated into different sites of the DZ2 host by transposition to obtain an epothilone heterologous expression strain (Zhu et al., 2015). The specific operation process is as follows:

[0039] (1) Scrape the yellow Myxococcus DZ2 cultured on the CYE plate for 2-3 days and transfer it to 50mL CYE liquid medium. Cultivate it at 30℃ and 200rpm for 24h. The OD600 is about 1.0. Take 5mL of fresh bacterial liquid, centrifuge it at 4℃ and 8000rpm for 5min, wash it three times with pre-cooled double distilled water, and finally resuspend it with 50μL double distilled water. Dispense it into 1.5mL EP tubes at 100μL / tube for later use.

[0040] (2) Take 100 μg of p15A-TP-CF plasmid and add it to the EP tube. After mixing by pipetting, transfer it to the eppendorf electroporation cup for electroporation. The electroporation parameters are voltage 1250 V, resistance 400 Ω, and capacitance 25 μF.

[0041] (3) After the electroporation, 500 μL of fresh CYE medium was quickly added, and the culture was transferred to a 10 mL centrifuge tube containing 3 mL of CYE medium after aspiration, and cultured at 30°C, 200 rpm for 4 h. The recovered culture was mixed with CYE soft agar medium containing 0.3% agar powder, and plated on CYE plates containing apramycin (30 μg / mL) and chloramphenicol (10 μg / mL), and cultured at 30°C for 7 days. The 14 mutant strains obtained were screened and named ZE1-ZE14.

[0042] (3) ZE1-ZE14 strains were transferred to fresh CYE plates. After culturing for 2-3 days, they were transferred to shake flasks containing 50 mL of CYE medium and cultured overnight at 30°C and 200 rpm. The seed liquid was transferred to shake flasks containing 50 mL of CYE medium at a ratio of 2% and fermented at 30°C and 200 rpm for 7 days.

[0043] (4) Collect the resin by filtering through a filter, add 3 mL of methanol and extract overnight, filter the extract through a 0.22 μm filter membrane, and analyze the epothilone fermentation yield by high performance liquid chromatography mass spectrometry. The sample volume is 20 μL, the mobile phase is 60% methanol-40% water, the flow rate is 1 mL / min, and the detection wavelength is 249 nm. According to the peak area A of the characteristic ultraviolet absorption peak of epothilone, the formula Y (mg / L) = (A × 1.1 × 10 -7 -3×10 -5 ) × 60 to quantify the epothilone production. After testing, ZE9 had the highest epothilone production, reaching 1.2 mg / L, and this strain was used for subsequent ribosome engineering screening of strains.

[0044] Example 2. Screening of ribosomal engineered antibiotics from Myxococcus xanthus ZE9

[0045] Myxobacteria that can grow dispersedly in liquid culture medium can use this method to screen antibiotics that can be used for ribosome engineering. This example uses ZE9 as an example to explain the antibiotic screening method in detail. The specific operation is as follows:

[0046] (1) ZE9 strain was cultured on CYE plates for 2-3 days, then transferred to a shake flask containing 50 mL CYE medium and cultured at 30°C, 200 rpm for 24 h. The OD value of the bacterial solution was measured using a spectrophotometer. 600 value and diluted to OD using CTT medium 600 The concentration of 0.005 was transferred to a 96-well culture plate at a rate of 150 μL / well.

[0047] (2) Select antibiotics whose targets are ribosomes or RNA polymerase, including but not limited to tetracycline, paromomycin, neomycin, streptomycin, rifampicin, gentamicin and spectinomycin, and add the antibiotic solution to a 96-well culture plate in a gradient dilution to a final concentration of 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg / mL, respectively.

[0048] (3) The culture plate was placed in a 30°C incubator for 36 h, and the bacterial growth was observed. The minimum concentration that could completely inhibit the growth of the bacterial solution was determined as the minimum inhibitory concentration of the antibiotic to ZE9. Antibiotics with a minimum inhibitory concentration lower than 256 μg / mL were selected for screening resistant strains.

[0049] Example 3. Screening of ribosomal engineered antibiotics in Myxococcus xanthus DK1622

[0050] Myxococcus xanthus DK1622 (DSM 16526) is a model strain of myxobacteria, and the strain grows dispersedly in CTT liquid culture medium. The method of the present invention can be used to screen antibiotics for ribosome engineering of Myxococcus xanthus DK1622, and the specific operation is as follows:

[0051] (1) DK1622 strain was cultured on CTT plates for 2-3 days, transferred to a shake flask containing 50 mL CYE medium, and cultured at 30°C, 200 rpm for 24 h. The OD value of the bacterial solution was measured using a spectrophotometer. 600 value and diluted to OD using CTT medium 600 The concentration of 0.005 was transferred to a 96-well culture plate at a rate of 150 μL / well.

[0052] (2) Select antibiotics whose targets are ribosomes or RNA polymerase, including but not limited to tetracycline, paromomycin, neomycin, streptomycin, rifampicin, gentamicin and spectinomycin, and add the antibiotic solution to a 96-well culture plate in a gradient dilution to a final concentration of 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg / mL, respectively.

[0053] (3) The culture plate was placed in a 30°C incubator for 36 h, and the bacterial growth was observed. The minimum concentration that could completely inhibit the growth of the bacterial solution was determined as the minimum inhibitory concentration of the antibiotic to ZE9. Antibiotics with a minimum inhibitory concentration lower than 256 μg / mL were selected for screening resistant strains.

[0054] Example 4. Screening of Epothilone High-Producing Bacteria ZE9N-R22

[0055] ZE9 obtained ZE9N-R22 with high epothilone production after two rounds of antibiotic screening, but the construction of high-yield strains is not limited to two rounds of screening. For different strains, two or more rounds of screening using two or more antibiotics can construct multi-resistant mutants and select high-yield strains from them. The construction method of ZE9N-R22 is as follows:

[0056] (1) The ZE9 strain was cultured on a CYE plate for 2-3 days, then transferred to a shake flask containing 50 mL of CTT medium and cultured at 30°C, 200 rpm for 24 h.

[0057] (2) Take 1 mL of bacterial solution and mix it with CYE soft agar (agar powder content is 0.3%), and evenly spread it on a CYE screening plate containing antibiotics (Rif, 2 μg / mL; Neo, 150 μg / mL; Par, 200 μg / mL; Tet, 10 μg / mL). After the soft agar solidifies, incubate the plate at 30°C for 7 days.

[0058] (3) Pick the resistant colonies grown on the screening plate, transfer them to a new plate containing the same antibiotic, culture them at 30°C for 2-3 days, and confirm the resistance again. The resistant mutants screened using rifampicin, neomycin, tetracycline and paromomycin were named ZE9-R, ZE9-N, ZE9-T and ZE9-P, respectively. Each mutant contains multiple mutant clones, and the different clones are numbered in sequence according to the name of the bacteria.

[0059] (4) All clones of the four monoclonal antibody mutants were transferred to shake flasks containing 50 mL of CYE medium and cultured at 30°C and 200 rpm for 24 h. 2OD of bacterial solution was transferred to a shake flask containing 50 mL of CMO medium and fermented at 30°C and 200 rpm for 7 days. The resin was collected by filtration using a 100-mesh filter, transferred to a 10 mL centrifuge tube, and 3 mL of methanol was added for overnight extraction. After the extract was filtered using a 0.22 μm filter membrane, the epothilone fermentation yield was analyzed by a high-performance liquid chromatography-mass spectrometry detector. The neomycin-tolerant strain ZE9-N18 had the highest epothilone yield in shake flask fermentation, reaching 34.7 mg / L.

[0060] (5) After the ZE9-N18 strain was cultured on the plate for 2-3 days, it was transferred to a shake flask containing 50 mL of CYE medium and cultured at 30°C and 200 rpm for 24 h. 1 mL of the bacterial solution was mixed with CYE soft agar and evenly plated on a screening plate containing Rif antibiotic (2 μg / mL). After the soft agar solidified, the plate was incubated at 30°C for 7 days.

[0061] (6) The resistant bacteria growing on the Rif plate were named ZE9N-R, and different clones were numbered in sequence. All clones were transferred to a dual-resistance plate containing neomycin and rifampicin, cultured at 30°C for 2-3 days, and the drug resistance was confirmed again.

[0062] (7) All clones of ZR9N-R were transferred to a shake flask containing 50 mL of CYE medium and cultured at 30°C and 200 rpm for 24 h. 2 OD of bacterial culture was transferred to a shake flask containing 50 mL of CMO medium. After fermentation at 30°C and 200 rpm for 7 days, the resin was collected and the product was extracted with 3 mL of methanol. The extract was filtered with a 0.22 μm filter membrane and the epothilone fermentation yield was analyzed by high performance liquid chromatography-mass spectrometry. The sample volume was 20 μL, the mobile phase was 60% methanol-40% water, the flow rate was 1 mL / min, and the detection wavelength was 249 nm. According to the peak area A of the characteristic ultraviolet absorption peak of epothilone, the formula Y (mg / L) = (A×1.1×10 -7 -3×10 -5 ) × 60 to quantify the epothilone production. The experimental results showed that the total epothilone production of the 22nd dual-resistance clone ZE9N-R22 was the highest, reaching 54.8 mg / L.

[0063] Example 5. Application of ZE9N-R22 in the preparation of epothilone

[0064] (1) Seed liquid culture: ZE9N-R22 was cultured on CYE plates for 2-3 days, transferred to a shake flask containing 50 mL of CYE medium, and cultured at 30°C, 200 rpm for 24 h. The overnight culture was transferred to a shake flask containing 50 mL of CYE medium at a ratio of 2%, and cultured at 30°C, 200 rpm for 24 h to prepare the fermentation seed liquid.

[0065] (2) Continuous feeding fermentation: The seed liquid was inoculated into a 10L fermenter containing 5L CMO medium at a ratio of 30%. The fermenter automatically monitored the dissolved oxygen, temperature, pH value and stirring speed of the fermentation liquid. The pH was set to 7.4 and the dissolved oxygen was set to 50%. Feeding began after 5 days of fermentation. Each time, the feed was made at a ratio of 6mL / L methyl oleate, 4g / L casein peptone and 2g / L yeast extract. Feeding was performed once every 48h for 5 consecutive times.

[0066] (3) Detection of bacterial fermentation biomass: The total fermentation time was 480 h. During the fermentation process, samples were taken every 48 h from 72 h onwards to detect bacterial biomass. When sampling, 80 mL of bacterial cells were taken each time and divided into two tubes. The cells were collected by centrifugation at 8000 rpm for 5 min. One tube was resuspended in an equal volume of CYE medium and the OD was measured. 600 The cell dry weight was determined after drying another tube at 55°C.

[0067] Epothilone product detection: After the fermentation is completed, the resin is collected by filtration using a filter and 300 mL of methanol is added for overnight extraction. The extract is filtered using a 0.22 μm filter membrane and analyzed by high performance liquid chromatography-mass spectrometry. The mobile phase is 60% methanol-40% water, the flow rate is 1 mL / min, the sample volume is 20 μL each time, and the detection wavelength is 249 nm. Mass spectrometry detection analysis conditions for epothilone molecules: ESI is connected to LC, the vertebral foramen voltage is 30 V, the nitrogen flow rate is 1 mL / min, the positive ion mode, and the m / z scanning range is 100-1000. The molecular ion peak of epothilone A [M+H] + 494m / Z( Figure 2 A), [M+H] of epothilone B + 508m / Z( Figure 2 B). According to the peak area A of the characteristic ultraviolet absorption peak of epothilone, the formula Y (mg / L) = (A × 1.1 × 10 -7 -3×10 -5 )×60 to quantify the epothilone production. The production of epothilone A and epothilone B were 44.8 mg / L and 48.6 mg / L, respectively, and the total production was 93.4 mg / L ( Figure 3 ).

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Myxococcus xanthus that produces epothilone, characterized in that: The strain is named Myxococcusxanthus ZE9N-R22, and was deposited in the China Center for Type Culture Collection on September 27, 2024. Its deposit number is: CCTCC M 20242100, and its address is Wuhan University, Wuhan, China.

2. The epothilone-producing Myxococcus xanthus according to claim 1, characterized in that: Starting from the epothilone heterologous expression strain Myxococcus xanthus ZE9, neomycin and rifampicin were used to screen and obtain Myxococcus xanthus ZE9N-R22.

3. The method for screening the Myxococcus xanthus ZE9N-R22 strain according to claim 1, characterized in that: The following steps are included: S1, Myxococcus xanthus ZE9 was cultured in CYE medium, transferred to culture plates, and antibiotics acting on ribosomes or RNA polymerase were added; S2, single antibiotic screening, antibiotics were selected from tetracycline, paromomycin, neomycin, streptomycin, rifampicin, gentamicin, and spectinomycin; S3, double-resistant mutant strains, continue to screen for double-resistant mutant strains resistant to rifampicin; S4, screen out the dual-resistant strain with the highest epothilone production.

4. The method for screening Myxococcus xanthus ZE9N-R22 strain according to claim 3, characterized in that: In step S1, the antibiotic acting on ribosome or RNA polymerase is selected from tetracycline, paromomycin, neomycin and rifampicin.

5. The method for screening Myxococcus xanthus ZE9N-R22 strain according to claim 3, characterized in that: In step S1, tetracycline, paromomycin, neomycin and rifampicin with a minimum inhibitory concentration of less than 256 μg / ml are selected for screening of resistant mutant strains.

6. The method for screening Myxococcus xanthus ZE9N-R22 strain according to claim 3, characterized in that: In step S2, the antibiotic concentrations are 8 μg / ml of tetracycline, 100 μg / ml of paromomycin, 64 μg / ml of neomycin and 2 μg / ml of rifampicin.

7. The method for screening Myxococcus xanthus ZE9N-R22 strain according to claim 3, characterized in that: In step S3, the concentration of rifampicin is 2 μg / ml.

8. Use of the epothilone-producing Myxococcus xanthus according to claim 1 in the preparation of epothilone.

9. The use according to claim 8, characterized in that: Myxococcus xanthus was transferred to a shake flask of CYE medium to culture seed liquid, and the seed liquid was transferred to a fermenter of CMO medium. After fermentation, the resin was collected by filtration using a filter, and methanol was added for extraction to obtain a crude extract of epothilone; CYE medium composition: 10 g / L casein peptone, 5 g / L yeast extract, 4 mM magnesium sulfate·7H2O 10 mM MOPS, pH 7.6; 1.5% agar powder was added to the solid medium and sterilized by high pressure steam at 115°C for 30 min; CMO medium composition: On the basis of CYE medium, XAD 16 macroporous adsorption resin was added at a ratio of 2%, and sterilized by high-pressure steam at 115°C for 30 minutes; 7 mL / L of filtered and sterilized methyl oleate was added before use.

10. The use according to claim 9, characterized in that: The seed liquid was transferred to a fermentation tank containing CMO culture medium at a ratio of 30%.

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