Streptomycete of staurosporine and application thereof in preparation of staurosporine compound

Abstract

The invention relates to streptomyces staurosporininus and an application of the streptomyces staurosporininus in preparation of a staurosporininus compound, and the collection number of the streptomyces staurosporininus is CGMCC (China General Microbiological Culture Collection Center) No. 27633. The strain can effectively improve the titer of staurosporine compounds in fermentation liquor.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to a staurosporine Streptomyces and its application in preparing a staurosporine compound. Background Art

[0002] Acute myeloid leukemia (AML) is a blood malignancy characterized by the abnormal proliferation of hematopoietic stem cells in the bone marrow, which interferes with the production of normal hematopoietic cells. AML develops in the bone marrow, leading to an increase in the number of abnormal white blood cells in the blood and bone marrow, and typically progresses rapidly. Consequently, AML is one of the most aggressive and difficult-to-treat blood cancers, with very limited treatment options. In 2017, over 20,000 patients were diagnosed with the disease in the United States, and up to one-third of these patients harbored a FLT3 gene mutation. The protein encoded by the FLT3 gene is a cell surface receptor that plays a critical role in the proliferation of specific blood cells. Mutations in this gene can lead to rapid disease progression in leukemia patients. Consequently, compared with other AML patients, patients with FLT3 mutations have a higher relapse rate and lower survival. However, over the past two to three decades, leukemia treatment strategies have remained relatively unchanged, placing patients with FLT3 mutations at significant risk.

[0003] Staurosporine was originally discovered in the fermentation products of Streptomyces staurosporeus (AM-2282) strain. It has multiple biological activities such as antibacterial, antifungal, antihypertensive, platelet aggregation, anti-tumor and neuroprotective activities. In particular, as an effective inhibitor of protein kinase C, it is of great research and development value in the treatment of cancer.

[0004] On April 28, 2017, the FDA approved Novartis's semi-synthetic staurosporine drug, midostaurin (trade name Rydapt), for use in combination with chemotherapy for the treatment of newly diagnosed adult patients with acute myeloid leukemia (AML) who harbor a specific FLT3 mutation. Rydapt was also approved for use with the companion diagnostic LeukoStrat CDx FLT3Mutation Assay to detect FLT3 mutations in AML patients. For this indication, midostaurin is the first new drug approved in nearly 25 years and the first targeted therapy used in combination with chemotherapy for the treatment of acute myeloid leukemia. In addition to AML, Rydapt is also approved for the treatment of adult patients with certain rare hematologic disorders, including aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematologic neoplasms (SM-AHN), and mast cell leukemia (MCL).

[0005] Midostaurin is semi-synthesized using staurosporine as a precursor. The chemical semi-synthesis requires only one step, resulting in a simple synthesis process and high yield. Therefore, staurosporine is a key intermediate of midostaurin, and its production process directly impacts the production cost and quality of midostaurin. However, the current yield of staurosporine from wild-type fungi is extremely low, and strain preservation, genetic improvement, and fermentation processes present technical challenges. While a few domestic companies are working on strain selection, fermentation optimization, and small- and pilot-scale process development, their fermentation capabilities are still far from commercialization. A major contributing factor to this problem is the poor fermentation activity and stability of existing fermentation strains. Therefore, identifying strains with strong fermentation activity is crucial for the fermentation production of staurosporine compounds. Summary of the Invention

[0006] Based on this, the present application provides a Streptomyces for preparing staurosporine, which has a deposit number of CGMCC No. 27633, to solve the problem of low fermentation titer of staurosporine compounds in the prior art, and further provides a method for producing staurosporine by fermentation using the Streptomyces.

[0007] The technical solution is as follows:

[0008] Streptomyces staurosporininus, its deposit number is CGMCC No.27633.

[0009] The invention relates to an application of the staurosporine Streptomyces in the preparation of staurosporine.

[0010] A method for preparing staurosporine comprises the steps of fermenting the staurosporine Streptomyces in a fermentation medium; wherein the fermentation medium contains a carbon source, a nitrogen source and an inorganic salt.

[0011] In one embodiment, the step of fermenting the Streptomyces staurosporinus in a fermentation medium includes inoculating the seed liquid of the Streptomyces staurosporinus into the fermentation medium at an inoculum rate of 1% to 20% for fermentation culture.

[0012] In one embodiment, the fermentation medium satisfies one or more of the following conditions:

[0013] The carbon source includes one or more of glycerol, soluble sediment and glucose;

[0014] The nitrogen source includes one or more of peptone, yeast powder and soybean cake powder; and

[0015] The inorganic salt includes one or more of K2HPO4, NaCl, MgSO4·7H2O and CaCO3.

[0016] In one embodiment, the fermentation medium contains the following components in mass percentage: 6% to 8% glycerol, 0.5% to 1.5% peptone, 0.5% to 2% yeast powder, 1% to 2.5% soybean cake powder, 0.05% to 0.15% K2HPO4, 0.5% to 1.5% NaCl, 0.05% to 0.15% MgSO4·7H2O, and 0.6% to 0.8% CaCO3.

[0017] In one embodiment, the fermentation medium further contains 0.05% to 1.0% by weight of a precursor, wherein the precursor includes one or more of vitamin B1, niacin, isonicotinic acid, nicotinic acid tyramine, tyrosine, and tryptophan.

[0018] In one embodiment, the rotation speed is controlled at 200 rpm to 500 rpm, the ventilation volume is controlled at 0.08 vvm to 2.0 vvm, and the fermentation culture is carried out at 26° C. to 30° C.

[0019] In one embodiment, the step of preparing the seed solution of Streptomyces staurosporinus comprises inoculating the Streptomyces staurosporinus into a seed culture medium for seed solution culture.

[0020] In one embodiment, the seed culture medium comprises the following components in mass percentage: 1.0% to 2.0% glycerol, 1.0% to 2.0% soluble starch, 1.0% to 2.0% peptone, 1.0% to 2.0% yeast powder and 0.2% to 0.4% calcium carbonate.

[0021] In one embodiment, before preparing the seed solution of Streptomyces staurosporinus, the process further includes inoculating Streptomyces staurosporinus into a slant culture medium for slant culture and preservation.

[0022] In one embodiment, the slant culture medium comprises the following components in mass percentage: 0.4% to 0.6% glucose, 1.0% to 1.2% malt extract, 0.4% to 0.6% yeast extract, and 1.8% to 2.0% agar.

[0023] Compared with traditional technologies, this application has the following beneficial effects:

[0024] This application presents a novel Streptomyces staurosporin strain, obtained through strain mutagenesis and screening, that is highly productive of the staurosporin compound. This strain is capable of effectively increasing the potency of the staurosporin compound in the fermentation broth. In fermentation experiments, this strain demonstrated high potency and low impurity content in the staurosporin produced by fermentation with this strain, greatly facilitating the extraction and purification of staurosporin and meeting industrial requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of this application easier to understand, the following further describes this application in detail based on the specific embodiments of this application and in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 is the evolutionary tree of strain ST-327;

[0027] Figure 2 is a microscopic image of strain ST-327;

[0028] Figure 3 is the colony morphology characteristic diagram of strain ST-327;

[0029] Figure 4 This is a blast comparison analysis of the staurosporine secondary biosynthesis gene cluster of strain ST-327. DETAILED DESCRIPTION

[0030] To make the above-mentioned objects, features, and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below. The following description sets forth many specific details to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0032] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] One embodiment of the present application provides a new Streptomyces, which is classified and named Streptomyces taurosporininus (Streptomyces staurosporinus, FIM18-0327, referred to as ST-327), and has been deposited in the General Microbiology Center of the China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 27633 and a deposit date of June 14, 2023.

[0034] The strain ST-327 is a Gram-positive bacterium with yellow-white, wavy-edged colonies and slender, branched hyphae. The ST-327 strain produces high-titer staurosporine with low impurity content.

[0035] One embodiment of the present application provides the use of Streptomyces staurosporin in preparing a crude staurosporin product or staurosporin.

[0036] In one specific example, the main component of the crude staurosporine product is staurosporine.

[0037] One embodiment of the present application provides a method for preparing staurosporine, which comprises the steps of fermenting Streptomyces staurosporinus ST-327 in a fermentation medium.

[0038] In one embodiment, the fermentation medium contains an available carbon source, an available nitrogen source, and inorganic salts.

[0039] In a specific example, the seed liquid of Streptomyces staurosporinus ST-327 is inoculated into the fermentation medium at an inoculum rate of 1% to 20% for fermentation culture.

[0040] In this application, the inoculation volume refers to the volume ratio of the seed solution transferred in to the culture solution after inoculation.

[0041] In a specific example, the available carbon source includes one or more of glycerol, soluble starch and glucose.

[0042] In one specific example, the available carbon source is glycerol.

[0043] In a specific example, the available nitrogen source includes one or more of peptone, yeast powder and soybean cake powder.

[0044] In a specific example, the available nitrogen sources are peptone, yeast powder and soybean cake powder.

[0045] In a specific example, the inorganic salt includes one or more of K2HPO4, NaCl, MgSO4·7H2O, and CaCO3.

[0046] In a specific example, the inorganic salts are K2HPO4, NaCl, MgSO4 and CaCO3.

[0047] In a specific example, the fermentation medium includes the following components in mass percentage: 6% to 8% glycerol, 0.5% to 1.5% peptone, 0.5% to 2% yeast powder, 1% to 2.5% soybean cake powder, 0.05% to 0.15% K2HPO4, 0.5% to 1.5% NaCl, 0.05% to 0.15% MgSO4·7H2O, and 0.6% to 0.8% CaCO3.

[0048] In one specific example, the pH of the fermentation medium is 7.0-7.5.

[0049] Optionally, the mass percentage of glycerol in the fermentation medium may be 6%, 6.5%, 7%, 7.5%, 8%, or a range consisting of any two values.

[0050] Optionally, the mass percentage of peptone in the fermentation medium can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.5%, or a range consisting of any two values.

[0051] Optionally, the mass percentage of yeast powder in the fermentation medium may be 0.5%, 1.0%, 1.5%, 2.0%, or a range consisting of any two values.

[0052] Optionally, the mass percentage of soybean cake powder in the fermentation medium can be 1%, 1.5%, 2.0%, 2.5%, or a range consisting of any two values.

[0053] Optionally, the mass percentage of K2HPO4 in the fermentation medium can be 0.05%, 0.1%, 0.15%, or a range consisting of any two values.

[0054] Optionally, the mass percentage of NaCl in the fermentation medium can be 0.5%, 1.0%, 1.5%, or a range consisting of any two values.

[0055] Optionally, the mass percentage of MgSO4·7H2O in the fermentation medium can be 0.05%, 0.1%, 0.15%, or a range consisting of any two values.

[0056] Optionally, the mass percentage of CaCO3 in the fermentation medium can be 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, or a range consisting of any two values.

[0057] In one specific example, the fermentation medium further includes 0.05% to 1.0% by weight of a precursor, wherein the precursor includes one or more of vitamin B1, niacin, isonicotinic acid, nicotinic acid tyramine, tyrosine, and tryptophan. The addition of the precursor to the fermentation medium increases the titer of staurosporine, reaching approximately 1450 mg / L.

[0058] Optionally, the mass percentage of the precursor in the fermentation medium can be 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, or a range consisting of any two values.

[0059] In a specific example, the fermentation culture conditions include: controlling the rotation speed to 200 rpm to 500 rpm, the ventilation volume to 0.08 vvm to 2.0 vvm, performing the fermentation culture at 26° C. to 30° C., and the culture time to 6 days to 10 days.

[0060] In a specific example, the step of preparing the seed liquid of Streptomyces staurosporinus ST-327 includes inoculating Streptomyces staurosporinus into a seed culture medium for seed liquid culture.

[0061] In a specific example, the seed culture medium includes the following components in mass percentage: 1.0% to 2.0% glycerol, 1.0% to 2.0% soluble starch, 1.0% to 2.0% peptone, 1.0% to 2.0% yeast powder and 0.2% to 0.4% calcium carbonate, with a pH value of 7.0 to 7.5.

[0062] The culture conditions of the seed liquid culture include: culture at 28°C to 32°C and shaking at 200rpm to 250rpm for 15h to 30h.

[0063] In a specific example, before preparing the seed solution of Streptomyces staurosporinus, the method further includes inoculating Streptomyces staurosporinus into a slant culture medium for slant culture and preservation.

[0064] In a specific example, the slant culture medium includes the following components in mass percentage: 0.4% to 0.6% glucose, 1.0% to 1.2% malt extract, 0.4% to 0.6% yeast extract and 1.8% to 2.0% agar, with a pH value of 7.0 to 7.5.

[0065] In a specific example, the slant culture conditions include constant temperature culture at 26° C. to 30° C. for 12 to 15 days.

[0066] In a specific example, the storage is performed at 2°C to 6°C.

[0067] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0068] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0069] Example 1 Strain Source

[0070] Between 2017 and 2018, the Screening Laboratory of the Fujian Institute of Microbiology isolated 1,501 actinomycete strains from samples collected from mountainous areas, farmlands, tidal flats, and the ocean in Fuzhou, Nanping, Zhangzhou, and Xiamen. These isolates were then compared, and strains with similar color, appearance, and shape were eliminated, resulting in a preliminary screening of 1,124 actinomycete strains. Fermentation extracts were screened for antimicrobial activity against Aspergillus niger and Candida albicans, yielding 87 active strains. Further screening against Bacillus subtilis and Escherichia coli yielded eight suspected staurosporine strains. Further secondary fermentation in shake flasks and liquid chromatography confirmed the presence of one of these strains, whose metabolites exhibited a similar peak time to the main peak of a staurosporine standard and whose UV absorption peaks were essentially identical to those of the standard. Following natural isolation, purification, fermentation, and post-extraction, the compound staurosporine was obtained, which was confirmed to be homologous to staurosporine (ST) by mass spectrometry and nuclear magnetic resonance. However, the titer of staurosporine produced by fermentation of the original strain was low, so this strain was used as the starting strain for strain mutagenesis in this example.

[0071] Preparation of spore suspension: Inoculate the starting strain onto an ISP2 slant and culture at 28°C for about 12 days. Take the cultured slant and add 15 mL of sterile saline. Gently scrape it with an inoculating spatula and pour it into a sterile shake flask with glass beads and shake it at 150 rpm for 15 minutes. Then filter the mycelium through sterile double-layer lens paper, leaving the spore suspension for later use.

[0072] ARTP (atmospheric pressure room temperature plasma) mutagenesis: The ARTP mutagenesis breeding instrument was set to 100 W, argon gas source, 10 L / min gas flow, 2 mm irradiation distance between the plasma emission source and the sample, and 60 s irradiation time. 10 μl of the prepared spore suspension was evenly spread on a sterile metal slide, which was then placed on the sample stage of the ARTP instrument. Plasma irradiation was performed according to the procedure. The mutagenized sample was appropriately diluted and spread on a separate plate. After incubation in a 28°C constant temperature incubator for 12 days, it was used to calculate the mutagenesis lethality and strain screening.

[0073] Screening for high-yield strains: Spread the ARTP-induced bacterial suspension onto dual-resistance plates containing 0.5 U streptomycin and 1.0% tryptophan. Incubate at 28°C for 10-15 days. Pick out single colonies and perform shake flask fermentation screening. Strains with a fermentation titer increase of 20% or more are selected for further rescreening.

[0074] After multiple rounds of mutagenesis screening, strains 69# and 108# were obtained, demonstrating stable fermentation titers and relatively high relative titers, approximately eightfold higher than the starting strain. Strain 69# reached a titer of 528 mg / L, while strain 108# reached a titer of 530 mg / L. The two strains exhibited distinct characteristics when cultured on slant medium. Strain 69# exhibited grayish-black mycelia with abundant, continuous spores that formed an off-white, powdery appearance. Strain 108# exhibited grayish-black mycelia with fewer, scattered spores that formed a pale yellowish-white, powdery appearance that darkened to an oily appearance upon absorption. Based on these considerations, both strains 69# and 108# were selected as starting strains for subsequent genetic improvement and culture medium optimization. Multiple passage experiments revealed that strain 69# exhibited relatively abundant spores and demonstrated high shake flask fermentation stability. Strain 108# exhibited slightly fewer spores and a slightly higher titer, but this varied significantly between shake flasks. After multiple natural separation and passage stability experiments, the results showed that the mutant strain 69-4-30# had good genetic stability, so it was further preserved and named ST-327.

[0075] Example 2 Morphological and cultural characteristics of Streptomyces ST-327

[0076] After identification, the Streptomyces ST-327 described in this example was positive by Gram staining. After growing on medium such as Gao's synthetic agar No. 1 and ISP1 for 10 days, the color and morphology of its colonies were observed: the surface of the colonies was raised, yellow-white, and had wavy edges ( Figure 2 ); hyphae are lightly stained, slender and branched ( Figure 3 ).

[0077] ISP1, ISP2, ISP3, ISP4 and other series of culture media were used respectively. After culturing at 28°C for 21 days, the color and pigment of the mycelium were observed. The results are shown in Table 1 below.

[0078] Table 1 ST-327 culture characteristics

[0079]

[0080] Note: +++: good; ++: medium; +: poor.

[0081] Physiological and biochemical characterization of the strain was performed according to standard methods for Streptomyces classification. Strain ST-327 was highly efficient in utilizing 18 carbon sources, but was unable to utilize or had a weak ability to utilize the other five, as shown in Table 2. The strain was sensitive to pH, NaCl, and culture temperature. Growth was optimal at pH 4-5, 1%-3% NaCl, and 30°C. It also tested positive for gelatin liquefaction, but negative for starch hydrolysis, cellulose hydrolysis, melanin production, and hydrogen sulfide production.

[0082] Table 2 Carbon source utilization of strain ST-327

[0083]

[0084]

[0085] Note: “+” indicates growth promotion and “-” indicates growth inhibition

[0086] 16S rDNA sequence analysis was performed on the preserved strain: genomic DNA was extracted from fresh cells using the lysozyme method, and 16S rDNA was amplified using universal primers. The PCR product was detected and purified, then directly sequenced using the Taq Deoxy Terminator Cyele ​​Sequencing Kit. Electrophoresis and sequencing were performed automatically using an Applied Biosystems DNA Sequencer (model 377). The measured 16S rDNA sequence was proofread and spliced, and then compared with sequences of related species in the GenBank database using BLAST. The 16S rDNA phylogenetic tree of strain ST-327 is shown in the attached figure. Figure 1 .

[0087] Based on the comprehensive analysis of the morphological characteristics, cultural characteristics, physiological and biochemical characteristics and 16SrDNA sequence of the strain, it was proved that the strain ST-327 screened in this application was identified as Streptomyces staurosporininus (staurosporininus). The above-mentioned Streptomyces ST-327 is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, referred to as CGMCC). Its registration number is CGMCC No. 27633, and the preservation date is June 14, 2023. This strain is a high-producing strain of staurosporin.

[0088] The whole genome of Streptomyces ST-327 was sequenced, and the secondary metabolite biosynthesis gene cluster was analyzed using the online software antiSMASH. The staurosporine biosynthesis gene cluster was located and submitted to NCBI blast for comparison. The comparison results are shown in Figure 4 The comparison results show that the similarity between this strain and the currently published staurosporine biosynthesis gene cluster is less than 90%, which indicates that this strain is different from the currently published staurosporine-producing bacteria and has different industrialization potential.

[0089] Example 3 Fermentation culture

[0090] Prepare ISP2 solid slant medium: 0.4% glucose, 1.0% malt extract, 0.4% yeast extract, 1.8% agar, and distilled water, adjusting the pH to 7.5. Inoculate the ISP2 slant with Streptomyces staurosporinus ST-327 (Deposit Number CGMCC No. 27633), culture at 28°C for 12-15 days, and store at 4°C.

[0091] Preparation of shake flask seed liquid culture medium: 2% glycerol, 1.0% soluble starch, 1.0% peptone, 1.0% soybean cake powder, 0.2% CaCO3, tap water, adjusted to pH 7.5.

[0092] Shake flask seed liquid culture: Place 80 ml of the above seed culture medium in a 500 mL Erlenmeyer flask, sterilize under high pressure at 121°C for 30 min, inoculate with the slant spore suspension after cooling, and culture in a shaker at 250 rpm and 28-32°C for 30 h to prepare the seed liquid.

[0093] 600 mL of shake flask seed liquid was prepared according to the above formula, and then inoculated into a 20 L seed tank (actual liquid volume 12 L) at an inoculum size of 1.0%. Seed tank culture was carried out, and the temperature was controlled at 28°C, the rotation speed was 200-500 rpm, the tank pressure was 0.03-0.05 MPa, the ventilation volume was 1:1.5 vvm, and the culture was carried out for 30 hours to obtain the fermentation tank seed liquid.

[0094] Fermentation tank culture medium: 6.0% glycerol, 1.5% peptone, 1.0% yeast powder, 1.5% soybean cake powder, 0.3% tryptophan, 0.05% K2HPO4, 0.5% NaCl, 0.05% MgSO4·7H2O, 0.6% calcium carbonate, prepared with tap water, adjusted to pH 7.5.

[0095] The seed liquid cultured in the above seed tank was inoculated into a 100 L fermenter (actual liquid volume 75 L) at an inoculum size of 1.0%. The fermentation speed was controlled at 200-500 rpm and the ventilation volume was controlled at 0.08-2.0 vvm. The culture was carried out for 6 days. 2 ml of the fermentation liquid was collected and added to 10 mL of methanol, mixed and shaken for 30 min, and centrifuged at 10,000 rpm / min for 10 min. The supernatant was then filtered through a 0.22 μm filter membrane and sampled. The staurosporine yield was 986 mg / L as determined by HPLC.

[0096] Example 4 Shake flask fermentation

[0097] ISP2 solid slant was prepared according to the method in Example 3 for strain slant storage.

[0098] The shake flask seed liquid culture medium was prepared according to the method in Example 3, and the seed liquid culture was carried out to obtain the seed liquid.

[0099] The shake flask fermentation medium of this example was prepared with the following ingredients: 6.0% glycerol, 1.5% peptone, 1.0% yeast powder, 1.5% soybean meal, 0.05% K2HPO4, 0.5% NaCl, 0.05% MgSO4·7H2O, 0.6% calcium carbonate, and tap water, with the pH adjusted to 7.5.

[0100] Shake flask fermentation: 30 mL of the above fermentation medium was placed in a 250 mL Erlenmeyer flask and autoclaved at 121°C for 30 min. After cooling, the cultured seed solution was inoculated at a 2.0% inoculum and cultured in a shaker at 250 rpm and 28-32°C for 7 days. The fermentation broth was collected and analyzed by HPLC. The titer of the target product, staurosporine, in the fermentation broth was 560 mg / L.

[0101] Example 5 Shake flask fermentation

[0102] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0103] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.6% tryptophan, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.6% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 3 to produce a fermentation broth.

[0104] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 1450 mg / L.

[0105] Example 6 Shake flask fermentation

[0106] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0107] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 1.0% tryptophan, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, 0.6% calcium carbonate, and tap water, adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0108] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 1208 mg / L.

[0109] Example 7 Shake flask fermentation

[0110] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0111] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.3% niacin, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.6% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0112] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 785 mg / L.

[0113] Example 8 Shake flask fermentation

[0114] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0115] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.3% isonicotinic acid, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.6% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0116] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 658 mg / L.

[0117] Example 9 Shake flask fermentation

[0118] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 5.

[0119] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.3% nicotinic acid tyramine, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.6% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 3 to produce a fermentation broth.

[0120] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 860 mg / L.

[0121] Example 10 Shake flask fermentation

[0122] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0123] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.1% vitamin B1, 0.05% K2HPO4, 0.5% NaCl, 0.05% MgSO4·7H2O, 0.6% calcium carbonate, and tap water, adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0124] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 610 mg / L.

[0125] Example 11 Shake flask fermentation

[0126] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0127] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 1.0% yeast extract, 1.5% soybean meal, 0.3% tyrosine, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.6% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0128] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 680 mg / L.

[0129] Example 12 Shake flask fermentation

[0130] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0131] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 0.5% peptone, 1.0% yeast extract, 2.5% soybean meal, 0.3% tryptophan, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, 0.6% calcium carbonate, and tap water, adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0132] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 980 mg / L.

[0133] Example 13 Shake flask fermentation

[0134] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0135] The shake flask fermentation medium of this example consisted of 6.0% glycerol, 1.5% peptone, 0.5% yeast extract, 2.5% soybean meal, 0.3% tyrosine, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, 0.6% calcium carbonate, and tap water, adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0136] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 880 mg / L.

[0137] Example 14 Shake flask fermentation

[0138] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0139] The shake flask fermentation medium of this example consisted of 8.0% glycerol, 1.0% peptone, 1.0% yeast extract, 1.0% soybean meal, 0.3% tyrosine, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, and 0.8% calcium carbonate, prepared in tap water and adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0140] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 780 mg / L.

[0141] Example 15 Shake flask fermentation

[0142] The culture medium selection and culture conditions in the strain preservation and seed solution preparation steps in this example are the same as those in Example 3.

[0143] The shake flask fermentation medium of this example consisted of 8.0% glycerol, 1.0% peptone, 2.0% yeast extract, 2.0% soybean meal, 0.3% tyrosine, 0.05% K₂HPO₄, 0.5% NaCl, 0.05% MgSO₄·7H₂O, 0.8% calcium carbonate, and tap water, adjusted to pH 7.5. Shake flask fermentation was carried out according to the method of Example 5 to produce a fermentation broth.

[0144] After the fermentation was completed, the fermentation broth was collected and subjected to HPLC detection, and the titer of the target product staurosporine in the fermentation broth was measured to be 1280 mg / L.

[0145] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0146] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims, and the specification may be used to interpret the content of the claims.

Claims

1. Streptomyces staurosporininus, characterized in that Its deposit number is CGMCC No.27633.

2. Use of the staurosporine Streptomyces according to claim 1 in the preparation of staurosporine.

3. A method for preparing staurosporine, characterized in that: The preparation method comprises the steps of: fermenting the Streptomyces staurosporinus described in claim 1 in a fermentation medium; Wherein, the fermentation medium contains a carbon source, a nitrogen source and an inorganic salt.

4. The preparation method according to claim 3, characterized in that The step of using the staurosporine Streptomyces to ferment in a fermentation medium comprises inoculating the seed liquid of the staurosporine Streptomyces into the fermentation medium at an inoculation rate of 1% to 20% for fermentation.

5. The preparation method according to claim 3, characterized in that The fermentation medium satisfies one or more of the following conditions: The carbon source includes one or more of glycerol, soluble sediment and glucose; The nitrogen source includes one or more of peptone, yeast powder and soybean cake powder; and The inorganic salt includes one or more of K2HPO4, NaCl, MgSO4·7H2O and CaCO3.

6. The preparation method according to claim 5, characterized in that The fermentation medium contains the following components in percentage by mass: 6% to 8% glycerol, 0.5% to 1.5% peptone, 0.5% to 2% yeast powder, 1% to 2.5% soybean cake powder, 0.05% to 0.15% K2HPO4, 0.5% to 1.5% NaCl, 0.05% to 0.15% MgSO4·7H2O and 0.6% to 0.8% CaCO3.

7. The preparation method according to any one of claims 3 to 6, characterized in that: The fermentation medium further contains 0.05% to 1.0% by mass of precursors, wherein the precursors include one or more of vitamin B1, nicotinic acid, isonicotinic acid, nicotinic acid tyramine, tyrosine and tryptophan.

8. The preparation method according to any one of claims 3 to 6, characterized in that The rotation speed is controlled to be 200 rpm to 500 rpm, the ventilation volume is 0.08 vvm to 2.0 vvm, and the fermentation is carried out at 26° C. to 30° C.

9. The preparation method according to any one of claims 4 to 6, characterized in that: The step of preparing the seed liquid of the staurosporine Streptomyces comprises inoculating the staurosporine Streptomyces into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components in percentage by mass: 1.0% to 2.0% glycerol, 1.0% to 2.0% soluble starch, 1.0% to 2.0% peptone, 1.0% to 2.0% yeast powder and 0.2% to 0.4% calcium carbonate.

10. The preparation method according to claim 9, characterized in that Before preparing the seed solution of the staurosporine Streptomyces, the method further includes inoculating the staurosporine Streptomyces into a slant culture medium for slant culture and preserving the slant culture medium; The slant culture medium comprises the following components in percentage by mass: 0.4% to 0.6% of glucose, 1.0% to 1.2% of malt extract, 0.4% to 0.6% of yeast extract and 1.8% to 2.0% of agar.

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