A strain of Streptomyces, its metabolites and their applications

Abstract

This invention provides a Streptomyces strain, its metabolites, and their applications. Specifically, it relates to a Streptomyces scabrisporus that has the ability to produce stetamycin, stetamycin B, shilomycin, lepromycin A, lepromycin B, and stetamycin aglycone. The Streptomyces strain of this invention is the same strain as the Streptomyces strain deposited on January 18, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, and confirmed to be viable on January 25, 2022, with accession number CCTCC NO:M 2022087. The strain according to this invention can provide metabolites that are effective as antibiotics, offering new possibilities for green pesticides.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a microorganism with biopesticide potential, its metabolites, and their applications. Background Technology

[0002] Soil microorganisms are diverse and interact with each other. Soil-derived Streptomyces have developed complex and unique metabolic pathways. In their later stages of growth, Streptomyces can produce various secondary metabolites with practical applications, including polyketides, alkaloids, glycopeptides, polyenes, lactones, and lactams. Qu Jia et al. isolated a dark blue Streptomyces caeruleatus from walnut rhizosphere soil and found that the extract from its fermentation broth had antagonistic effects against walnut black spot disease. Niu Hongjie et al. isolated a small Streptomyces from cucumber rhizosphere soil, which showed good biological activity against cucumber pathogens. Qiang Wei et al. screened seven actinomycetes from ginseng rhizosphere soil that exhibited high antagonistic activity against a single ginseng pathogen.

[0003] However, these microorganisms are still insufficient to meet the demand for developing novel bioactive natural products. Summary of the Invention

[0004] To address the aforementioned problems, the inventors of this invention screened soil biological resources and discovered a new Streptomyces strain capable of inhibiting bacteria and fungi, named HBERC-53204, which has the potential for direct application or modification for use in related fields.

[0005] The Streptomyces HBERC-53204 strain, identified molecularly as Streptomyces cabrispora HBERC-53204, was isolated by the inventor from the rhizosphere soil of bamboo forests in Zhongpo National Forest Park, Huaihua City, Hunan Province on March 11, 2013. The strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China (Wuhan University), with accession number CCTCC NO: M2022087, on January 18, 2022. It was confirmed to be viable on January 25, 2022.

[0006] Six compounds were isolated from the fermentation extract of strain HBERC-53204, exhibiting antifungal, antibacterial, and antiviral activities. After isolation and preparation, pure compounds were obtained. NMR analysis revealed these compounds to be: stefmycin, stefmycin B, stefmycin aglycone, hitachimycin, leptomycin A, and leptomycin B. Laboratory bioactivity assays confirmed that these compounds possessed a variety of biological activities. This completes the present invention.

[0007] According to one aspect of the invention, a Streptomyces scabrisporus is provided, which has the ability to produce stetamycin, stetamycin B, shilomycin, lepromycin A, lepromycin B, and stetamycin aglycone.

[0008] The Streptomyces strain of the present invention may be the same strain as the Streptomyces strain deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University on January 18, 2022, and detected as viable on January 25, 2022, with accession number CCTCC NO:M 2022087.

[0009] According to another aspect of the present invention, metabolites produced by the aforementioned Streptomyces are provided, preferably including stetamycin, stetamycin B, shilomycin, lepromycin A, lepromycin B, and stetamycin aglycone, more preferably including stetamycin B.

[0010] These metabolites have been shown to be effective as antibiotics and anticancer agents. Therefore, the present invention provides a new way of producing the aforementioned substances.

[0011] According to another aspect of the present invention, a method for producing the aforementioned Streptomyces or the aforementioned metabolites is provided, wherein the Streptomyces is cultured by fermentation, and optionally the culture medium includes: glucose, yeast powder, and MgSO4.

[0012] Preferably, the culture medium formula is: glucose 36.22 g / L, peptone 8 g / L, yeast powder 8.51 g / L, acid-hydrolyzed casein 1.5 g / L, MgSO4 0.68 g / L, KNO3 1 g / L, and the preferred fermentation time is 120 h.

[0013] The culture medium formulation and fermentation time can maximize the potency of stetamycin B.

[0014] The aforementioned production method also includes the following steps:

[0015] (1) After the fermentation broth is freeze-dried, it is extracted and precipitated with a solvent to obtain stethomycin B. The preferred extraction solvent is ethyl acetate.

[0016] (2) The content of stimycin B was further increased by repeatedly using the dissolution-centrifugation precipitation method;

[0017] (3) The precipitate obtained in step (2) is placed in a container and dissolved in a solvent, and allowed to stand to crystallize. The solvent is preferably any one of methanol, ethanol, or acetonitrile.

[0018] Once enough crystals have formed in step (3), carefully pour off the solvent and let it evaporate. It is preferable to use clean air at 40-45°C to dry it or to accelerate the evaporation of the solvent by depressurization. Bright red stetamycin B crystals will then be visible.

[0019] The above method allows for the simple preparation of high-purity stethromycin B crystals.

[0020] According to another aspect of the invention, metabolites produced by Streptomyces HBERC-53204 are provided for use as chemical intermediates, said metabolites preferably stetamicin aglycone or stetamicin B.

[0021] stetamycin B and stetamycin aglycone have multiple chemically active sites and molecular weights of 588 and 398, respectively, and can be used as intermediates in chemical synthesis.

[0022] According to another aspect of the present invention, the use of metabolites produced by Streptomyces HBERC-53204 for the control of crop diseases is provided.

[0023] The crop diseases mentioned include potato scab, rice blast, rice sheath blight, rice false smut, cotton wilt, or cotton verticillium wilt.

[0024] The metabolites produced by Streptomyces HBERC-53204 are intended for use in the manufacture of veterinary drugs for diseases caused by Erysipelothrix rhusiopathiae, Streptococcus suis, and Staphylococcus aureus.

[0025] According to another aspect of the invention, the use of stetam or stetam B for the prevention and control of potato scab is provided, preferably said potato scab is caused by Streptomyces galeris, more preferably said stetam or stetam B is derived from Streptomyces brucellosis strain HBERC-53204.

[0026] According to another aspect of the present invention, a pharmaceutical composition for the prevention and control of plant and animal diseases is provided, comprising a metabolite of Streptomyces HBERC-53204, said metabolite comprising any one of stetamycin, stetamycin B, shilomycin, lepromycin A, lepromycin B, and stetamycin aglycone; or containing stetamycin or stetamycin B or a derivative thereof.

[0027] Stetamycin and stetamycin B have anti-Gram-positive bacterial activity. For example, the MIC (minimum inhibitory concentration) against Staphylococcus aureus, Erysipelothrix rhusiopathiae, Streptococcus suis, and Streptococcus galactiae (the scabies of the potato) are 6.25 μg / mL, 1.56 μg / mL, 1.56 μg / mL, and 6.25 μg / mL, respectively. At a drug concentration of 5 μg / mL, stetamycin B showed an inhibition rate of 53.65% against porcine pseudorabies virus.

[0028] Rilibromycin exhibits good activity against both bacteria and fungi. For example, its MICs against Bacillus subtilis, Micrococcus occulta, and rice blast fungus are 0.4 μg / mL, 0.4 μg / mL, and 3.1 μg / mL, respectively.

[0029] Leptomycin A and Leptomycin B have antifungal activity, with MICs of 3.12 μg / mL and 6.3 μg / mL against rice sheath blight and rice blast fungus, respectively.

[0030] To date, there are no reports of using stetamicin B for the prevention and treatment of potato scab, swine erysipelas, swine streptococcus, or swine pseudorabies virus. In particular, there are no reports in the literature of using stetamicin B for the treatment of *Streptomyces galeris*, a pathogen specific to potato scab in my country. Attached Figure Description

[0031] Figure 1-1 Colony morphology of strain HBERC-53204 on ISP-2 solid medium;

[0032] Figure 1-2 The mycelial morphology of strain HBERC-53204 after 3 days of growth in ISP-2 liquid medium;

[0033] Figure 2 Phylogenetic tree of strain HBERC-53204 based on the 16S rRNA gene;

[0034] Figure 3 This is a state diagram of the pure compound stetamycin B;

[0035] Figure 4 It is a crystal of the compound stetamycin B;

[0036] Figure 5 This study describes the inhibitory effect of stetam B on Streptomyces galactiae (the pathogen causing potato scab). Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the embodiments described are only some representative embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0038] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be used in this invention.

[0039] (I) Streptomyces HBERC-53204 of the present invention

[0040] 1. Source and preservation unit of the strain

[0041] The Streptomyces HBERC-53204 involved in this invention was isolated by the inventor from the rhizosphere soil of bamboo forests collected from Zhongpo National Forest Park in Huaihua City, Hunan Province, and has completely independent intellectual property rights.

[0042] The strain was deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University). The accession number is CCTCC NO: M 2022087. The deposit date was January 18, 2022, and the strain was found to be viable on January 25, 2022.

[0043] 2. Morphological characteristics of the strain

[0044] The colony morphology of HBERC-53204 on ISP-2 solid medium is as follows: Figure 1-1 As shown.

[0045] HBERC-53204 mycelial morphology after 3 days of growth in ISP-2 liquid medium is as follows: Figure 1-2 As shown.

[0046] 3. Sequencing and analysis of the 16S rRNA gene of the strain

[0047] The 16SDNA gene sequence of a pair of strains, HBERC-53204, was analyzed and identified. This strain has 100% similarity to Streptomyces scabrisporus and is therefore identified as Streptomyces scabrisporus.

[0048] Figure 2 This is a Neighbor-Joining phylogenetic tree constructed based on the 16S rRNA or (16S rDNA) gene sequence alignment results, with the outer branch of Nonomuraea deserti KC310 (MG770639) as the phylogenetic tree.

[0049] 4. Fermentation of the strain

[0050] A wide range of culture media can be used, such as glucose, maltose, dextrin, soluble starch, starch syrup, corn starch, soybean peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, tryptone, beef extract, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, and magnesium sulfate. When using corn starch or potato starch, it must be hydrolyzed with α-amylase before use.

[0051] When fermenting in a fermenter, defoamers such as polyether and silicone need to be added to control foam.

[0052] For laboratory cultivation, commercially available raw materials such as glucose, malt extract, soybean peptone, and yeast extract can be used, and formulated according to formulas such as Gao's No. 1 and ISP-2, or you can prepare your own.

[0053] The culture apparatus can use common equipment such as glass bottles, Erlenmeyer flasks, and shakers, and the culture can be carried out using common shake flask culture methods. The liquid culture obtained from shake flask culture can be used for observation, preservation, or as a strain for large-scale culture.

[0054] Fermentation can also be carried out in fermentation tanks. Depending on the actual needs, fermentation tanks ranging from 10L to 200,000L can be used. The seed volume should be determined based on the fermentation volume, and the inoculum amount (the ratio of seed volume to fermentation volume) should be controlled at 1%-10%. When the fermentation volume is greater than 5000L, three-stage fermentation is recommended. The volume of ingredients in the fermentation tank should be controlled at 60%-70% of the tank's volume.

[0055] When the inventors investigated the effects of culture medium composition on the fermentation of the strain, they found that glucose, yeast extract, and MgSO4 significantly affected the yield of the strain.

[0056] Furthermore, through further optimization and analysis of the culture medium, the optimal medium formulation was determined to be: glucose 36.22 g / L, peptone 8 g / L, yeast extract 8.51 g / L, acid-hydrolyzed casein 1.5 g / L, MgSO4 0.68 g / L, and KNO3 1 g / L. Using this medium formulation, the target product yield increased by 17 times compared to the previous formulation.

[0057] In addition, the inventors' fermentation verification results show that the optimal fermentation time for the aforementioned formula is 120 hours, and the optimal stetamycin B (SMB) potency can be achieved after 120 hours of cultivation.

[0058] 5. Methods for preserving bacterial strains

[0059] The strains can be preserved using common preservation methods, such as in ISP-2 slant culture, glycerol tubes, or lyophilized tubes, at -20 to -80°C.

[0060] 6. Metabolites of the strain

[0061] Six compounds were isolated from the fermentation extract of strain HBERC-53204, exhibiting antifungal, antibacterial, and antiviral activities. After isolation and preparation, pure compounds were obtained, and NMR analysis identified these compounds as: stetamycin, stetamycin B, stetamycin aglycone, shilomycin, lepromycin A, and lepromycin B.

[0062] Indoor bioactivity assays revealed that the above compounds exhibited different bioactivities.

[0063] Stetamycin and stetamycin B have anti-Gram-positive bacterial activity. For example, the MIC (minimum inhibitory concentration) against Staphylococcus aureus, Erysipelothrix rhusiopathiae, Streptococcus suis, and Streptococcus galactiae (the scabies of the potato) are 6.25, 1.56, 1.56, and 6.25 μg / mL, respectively. At a drug concentration of 5 μg / mL, stetamycin B showed an inhibition rate of 53.65% against porcine pseudorabies virus.

[0064] Rilibromycin exhibits good activity against both bacteria and fungi. For example, its MICs against Bacillus subtilis, Micrococcus occulta, and Bacillus oryzae are 0.4 μg / mL, 0.4 μg / mL, and 3.1 μg / mL, respectively.

[0065] Leptomycin A and Leptomycin B have antifungal activity, with MICs of 3.12 μg / mL and 6.3 μg / mL against rice sheath blight and rice blast fungus, respectively.

[0066] stetamycin B and stetamycin aglycone have multiple chemically active sites and molecular weights of 588 and 398, respectively, and can be used as intermediates in chemical synthesis.

[0067] The present invention will be described below through specific embodiments.

[0068] Example 1: Preservation of Streptomyces HBERC-53204

[0069] Streptomyces HBERC-53204 can be preserved in ISP-2 slant medium or glycerol tubes and then cryopreserved at low temperature, following the method described below.

[0070] ISP-2 slant culture medium formula: 4.0 g glucose, 10.0 g malt extract powder, 4.0 g yeast extract powder, 15-20 g agar powder. Prepare slant culture in 15-18 mm glass test tubes, and fill each tube with 4-10 mL of culture medium.

[0071] Once the culture medium is prepared, sterilize it at 118-125℃ for 20-35 minutes. After cooling, remove it and place it on a slant (the length of the slant should not exceed 2 / 3 of the test tube length). After solidification, inoculate the bacterial culture according to aseptic operation requirements and incubate at 25-30℃ for 7-14 days.

[0072] Glycerin tube preparation: Prepare glycerin by mixing pure water with glycerin, maintaining a glycerin concentration between 20-40%. Use 2mL plastic cryovials, filling each tube with 1.0-1.5mL of glycerin. Sterilize at 118-125℃ for 20-35 minutes. Transfer spores or mycelia into the cryovials, mix well, and store at -20 to -80℃.

[0073] Freeze-dried tube preservation: Prepare 20%-40% skim milk, wash off the bacterial growth on the slant, mix well, transfer to sterilized freeze-drying tubes, freeze-dry in a freeze dryer, seal, and store at -20 to -80℃.

[0074] Example 2: Preparation of Streptomyces HBERC-53204

[0075] (1) Seed liquid preparation:

[0076] Take an HBERC-53204 slant test tube filled with spores, and use an inoculation spatula to pick up a 1cm square culture and put it into 100mL of ISP-2 liquid medium. Incubate at 150-200r / min and 28-32℃ for 3-4 days.

[0077] HBERC-53204 mycelial morphology after 3 days of growth in ISP-2 liquid medium is as follows: Figure 1-2 As shown.

[0078] (2) Fermentation by strain:

[0079] The seed culture was transferred to the fermentation medium at a ratio of 1:10 (seed culture / fermentation medium volume) and cultured at 150 rpm and 28°C for 4 days. The fermentation medium formula used was: glucose 25-36 g / L, peptone 8-10 g / L, yeast extract 8.5-12 g / L, acid-hydrolyzed casein 1-1.5 g / L, MgSO4 0.68 g / L, KNO3 1 g / L, which yielded a large quantity of Streptomyces HBERC-53204.

[0080] As mentioned above, the inventors demonstrated through experiments that the optimal culture medium formulation suitable for the strain of this invention is: glucose 36.22 g / L, peptone 8 g / L, yeast extract 8.51 g / L, acid-hydrolyzed casein 1.5 g / L, MgSO4 0.68 g / L, and KNO3 1 g / L. In this fermentation medium formulation, after 4 days of shake-flask fermentation, the SMB titer reached 477.26 mg / L, representing a 1773.08% increase in the yield of the target compound compared to the previous formulation.

[0081] The optimized shake-flask fermentation results were validated in a 20L fermenter. The optimal fermentation time was 120 h, and the SMB titer reached 214.48 mg / L. This optimized culture medium formulation significantly improved the yield of the target compound SMB.

[0082] Based on the optimized formula, the fermentation level of SMB was significantly improved. Combined with the inherent physicochemical properties of SMB, gram-level preparation of pure product could be easily achieved.

[0083] Example 3: Metabolites of Streptomyces HBERC-53204

[0084] The Streptomyces HBERC-53204 fermentation broth obtained in Example 2 was aliquoted into lyophilization cups and lyophilized in a vacuum freeze dryer according to the standard lyophilization procedure. The lyophilized sample was moistened with a small amount of 50% methanol-water mixture, then ethyl acetate (W / V = 1:10) was added, and the mixture was extracted by shaking at 150 rpm for 30 min. After centrifugation, the ester phase was collected and concentrated under reduced pressure to obtain the crude extract. The crude extract contained various metabolites, with stetamycin B accounting for the largest proportion.

[0085] After dissolving the above sample, six compounds were obtained by chromatography or preparative HPLC, and each compound was purified. Mass spectrometry and NMR analysis revealed these compounds to be stetamycin, stetamycin B, stetamycin aglycone, shilomycin, lepromycin A, and lepromycin B. Their structural formulas are as follows:

[0086]

[0087]

[0088] Example 4: Application of Metabolites

[0089] As mentioned above, the above compounds were found to have different biological activities according to in vitro bioactivity assays.

[0090] Stetamycin and stetamycin B have anti-Gram-positive bacterial activity. For example, the MIC (minimum inhibitory concentration) against Staphylococcus aureus, Erysipelothrix rhusiopathiae, Streptococcus suis, and Streptococcus galactiae (the scabies of the potato) are 6.25 μg / mL, 1.56 μg / mL, 1.56 μg / mL, and 6.25 μg / mL, respectively. At a drug concentration of 5 μg / mL, stetamycin B showed an inhibition rate of 53.65% against porcine pseudorabies virus.

[0091] Rilibromycin exhibits good activity against both bacteria and fungi. For example, its MICs against Bacillus subtilis, Micrococcus occulta, and rice blast fungus are 0.4 μg / mL, 0.4 μg / mL, and 3.1 μg / mL, respectively.

[0092] Leptomycin A and Leptomycin B have antifungal activity, with MICs of 3.12 μg / mL and 6.3 μg / mL against rice sheath blight and rice blast fungus, respectively.

[0093] stetamycin B and stetamycin aglycone have multiple chemically active sites and molecular weights of 588 and 398, respectively, and can be used as intermediates in chemical synthesis (see Example 6).

[0094] Since agricultural fertilizers and formulations generally have relatively simple requirements, the fermentation broth obtained in Example 2 can be directly concentrated and dried to produce liquid, powder, granule, or tablet forms for the prevention and control of crop diseases. For example, to reduce costs, the fermentation broth can be applied directly to farmland or soil; alternatively, the fermentation broth can be concentrated to a solid content of 20-30%, and 0.1-0.2% benzoic acid or potassium sorbate can be added to produce a liquid product for application via spraying.

[0095] From the perspective of convenient application and stable quality, the fermentation broth can also be concentrated at low temperature and then spray-dried or freeze-dried to directly obtain the freeze-dried strain powder.

[0096] Other excipients or small amounts of other fertilizers that do not affect the activity of the bacterial strain (such as about 5-10%) can also be added to make compound granules. To improve dosage accuracy, fillers and adjuvants can also be added to compress them into tablets. Adjuvants can also be added to make veterinary drugs for administration to the corresponding animals.

[0097] Example 5: Preparation of stetamycin B

[0098] Of the six compounds obtained in Example 3, stetamycin B had a high content in the sample and could be prepared in large quantities as a pure product by crystallization.

[0099] HBERC-53204 fermentation broth was obtained using the two-stage fermentation method described in Example 2;

[0100] (1) After the fermentation broth was freeze-dried, crude stethomimycin B was obtained by ethyl acetate extraction and precipitation method.

[0101] (2) Repeatedly using the dissolution-centrifugation precipitation method, the content of stetamycin B was increased to over 90%;

[0102] (3) Dissolve the stetamycin B sample in methanol, transfer it to an Erlenmeyer flask, seal the mouth of the flask with a semi-permeable membrane, and place it in a fume hood to allow the stetamycin B to crystallize.

[0103] (4) When enough crystals appear in the Erlenmeyer flask, carefully pour off the methanol, then place the Erlenmeyer flask on a nitrogen blower and dry it with clean air at 40-45℃. Bright red stethomycin B crystals will be visible.

[0104] (5) Collect the above crystals into a clean vial to obtain stetamycin B crystals with a purity of over 95%. The state of stetamycin B crystals under a microscope is as follows: Figure 4 As shown.

[0105] This crystal can be used as a biochemical reagent, an intermediate in organic chemical synthesis, and also for the prevention and control of bacterial diseases in plants and animals.

[0106] When a large quantity of the compound is required, the procedure described above can be scaled up appropriately. Furthermore, Example 4 illustrates the use of an Erlenmeyer flask as the container and ethyl acetate as the extraction solvent; those skilled in the art will understand that other containers or suitable solvents are also applicable to this invention.

[0107] Example 6: Transformation of stetamycin B

[0108] The aglycone of stetamicin has multiple active sites, such as -OCH3, -C=O, and -OH in its structural formula, all of which exhibit high reactivity and can undergo various chemical reactions to form derivatives. Therefore, stetamicin B or its aglycone can be used as a chemical starting material to obtain other compounds.

[0109]

[0110] For example, weigh 20 mg of pure stetamycin B and put it into a 200 mL flask. Add formaldehyde as a catalyst and 10 mg of piperidine. React under stirring and heating conditions. After 2 hours of treatment, a piperidine substituent can be added to stetamycin B.

[0111] Chemically synthesized drugs are widely used in pesticides, pharmaceuticals, and biochemicals. Chemical synthesis is generally suitable for synthesizing compounds with smaller molecular weights, such as less than 300 Da; larger molecules are difficult to synthesize. The compounds stetamycin B and stetamycin aglycone produced by Streptomyces HBERC-53204 are characterized by high yield and abundant chemical modification sites, making them suitable as intermediates in chemical synthesis and for synthesizing related products.

[0112]

[0113] Example 7: Application of stetamycin B

[0114] The pure stetamicin B obtained in Example 5 can be formulated into a product by adding solvents and dispersants for disease control, such as potato scab. The pure stetamicin B compound, with the addition of adjuvants, can be used to control animal diseases, such as swine erysipelas and swine streptococcal disease.

[0115] One of the main pathogens causing potato scab in my country is *Streptomyces galliculitis*. Currently, the primary approach to controlling potato scab is prevention; there are no effective treatments or control methods.

[0116] The inventors of this invention unexpectedly discovered that the metabolite stetamycin B produced by the strain of this invention has a good inhibitory effect on Streptomyces galactiae (see [link]). Figure 5 ).

[0117] The strain of this invention produces stetamycin B with a MIC (minimum inhibitory concentration) of 6.25 μg / mL against Streptomyces galactiae, the causal agent of potato scab.

[0118] Currently, stetamicin B is generally used in anticancer applications, and there are no reports of its use in agricultural pesticides. The strain and culture method of this invention enable the large-scale production of stetamicin B and other antibiotics, providing a possibility for solving agricultural diseases.

[0119] As described above, the metabolites of *Streptomyces* of the present invention possess antibacterial, antifungal, and antiviral activities, and therefore can be developed as green pesticides to control crop diseases, especially providing a possible solution for currently difficult-to-control crop diseases such as potato scab. Alternatively, they can be developed as veterinary drugs to provide new solutions for the control of livestock diseases. Furthermore, the metabolites are provided and can be purified using simple methods, making them very useful as precursors for chemical drugs or as raw materials for chemical drugs.

[0120] While specific embodiments of the present invention have been described and illustrated, it will be apparent to those skilled in the art that various other changes and modifications can be made, and combinations of embodiments can be made, without departing from the general scope of this disclosure. Therefore, all changes, modifications, and combinations that do not depart from the technical spirit of the present invention are within the scope of this invention.

[0121] For example, regarding the conversion of stetamycin B, only the reaction with piperidine is cited as an example. Those skilled in the art will understand that, based on well-known chemical reaction principles and conditions, it is possible to react with other reagents as needed to generate other compounds.

[0122] Furthermore, examples of pathogens that can be inhibited or killed by the metabolites of the strains of the present invention include potato scab, rice blast, rice sheath blight, rice false smut, cotton wilt, or cotton verticillium wilt. However, those skilled in the art will understand that this method can also be applied to other diseases known to respond to stetamycin, stetamycin B, shilomycin, lepromycin A, lepromycin B, and stetamycin aglycone.

[0123] Furthermore, although the specification exemplifies the application of the strain-containing metabolites in agriculture, those skilled in the art will understand that the purified metabolites can also be used for human pharmaceutical purposes.

[0124] Furthermore, although the specification exemplifies the use of liquid fermentation of strain HBERC-20716 to produce live bacterial preparations, those skilled in the art will understand that live bacterial preparations can also be produced via solid-state fermentation. However, liquid fermentation is a more efficient and cost-effective method.

[0125] The examples of strain fermentation illustrate small-scale fermentation, and the purification of stetamycin B illustrates gram-scale preparation. However, those skilled in the art can scale up the process as needed without departing from the principles of the invention. Appropriate adjustments can also be made to the instruments, reagents, and apparatus.

Claims

1. A strain of Streptomyces roughi ( Streptomyces scabrisporus ), characterized in that, It was preserved in China Center for Type Culture Collection (CCTCC) on January 18, 2022, and the preservation number is CCTCC NO: M 2022087.

2. The culture method of Streptomyces roughensia of claim 1, characterized by, The Streptomyces scabrisporus is cultured by fermentation.

3. The culturing method according to claim 2, wherein, The culture medium used in fermentation includes glucose, yeast powder and MgSO4.

4. The culturing method according to claim 3, wherein, The formula of the culture medium used in fermentation is: glucose 36.22 g / L, peptone 8 g / L, yeast powder 8.51 g / L, acid hydrolysis casein 1.5 g / L, MgSO4 0.68 g / L and KNO3 1 g / L.

5. The culturing method according to claim 4, wherein, The fermentation time is 120 h.

6. A method for producing streptimidate B, the method comprising: the step of culturing Streptomyces scabrisporus by the method of any one of claims 2-5; and the step of extracting streptimidate B.

7. The method of claim 6, wherein, The step of extracting streptimidate B comprises: (1) after freeze-drying the fermentation broth, extracting, precipitating with a solvent to obtain streptimidate B; (2) repeatedly using the method of dissolving-centrifugal precipitation to further improve the content of streptimidate B; (3) placing the precipitate obtained in step (2) in a container and dissolving it in a solvent, and standing to crystallize; (4) when enough crystals appear in step (3), carefully pour off the solvent and evaporate the solvent.

8. The method of claim 7, wherein, The extraction solvent in step (1) is ethyl acetate.

9. The method of claim 7, wherein, The crystallization solvent in step (3) is any one of methanol, ethanol and acetonitrile.

10. The method of claim 7, wherein, In step (4), the solvent is blown dry with clean air or vacuum pumping to accelerate solvent evaporation.

11. Use of the Streptomyces scabrisporus of claim 1 for producing streptimidate aglycone, streptimidate B, streptimidate, hillimycin, leptomycin A and leptomycin B.

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