WO2019233367A1 - Streptomyces strain and application thereof in production of staurosporine - Google Patents

Abstract

A streptomyces strain and an application thereof, and a method for preparing staurosporine by culturing the strain. The strain is named as streptomyces sp. HS-HY-153, and the preservation number thereof is CGMCC NO. 14806.

Description

A Streptomyces strain and application of staurosporin Technical field

The invention belongs to the technical field of microbial engineering, and particularly relates to a strain of Streptomyces and its application in preparing staurosporine.

Background technique

Staurosporine (the structural formula is shown in Formula 1), originally found in the fermentation product of Streptomyces staurosporeus (AM-2282) strain, has antibacterial, antifungal, blood pressure lowering, platelet aggregation, antitumor and neuroprotection, etc. A variety of biological activities, especially as effective inhibitors of protein kinase C, have great research and development value in the treatment of cancer. On April 28, 2017, the FDA approved its semi-synthetic drug Midostalin (Midostaurin (trade name: Rydapt, structural formula shown in Formula 2)) in combination with chemotherapy for newly diagnosed acute myeloid leukemia (acute myeloid leukemia, (AML) treatment of adult patients, suitable for patients with a specific gene mutation FLT3. At the same time, Rydapt was approved for use with the companion diagnostic reagent LeukoStrat CDx FLT3 Mutation Assay to detect FLT3 mutations in patients with AML. In addition to AML, Rydapt is also approved for the treatment of adult patients with certain types of rare hematological disorders, including aggressive systemic mastocytosis (ASM), systemic mast cell proliferation with hematological tumors Disease (SM-AHN) and mast cell leukemia (MCL).

There are many reported staurosporin-producing bacteria. US7608420B and EP0444503A disclose the use of Streptomyces hygroscopicus C39280-450-9 (ATCC 53730) to prepare staurosporine. The fermentation units are 392mg / L and 130mg / L; US4107297. The morphological, cultural, and physiological and biochemical characteristics of the astrosporin-producing strain Streptomyces sp. AM-2282 (Streptomyces staurosporeus nov.sp. NRRL11184) were disclosed; Gai Cuijuan et al. (Chinese Journal of Antibiotics, 2015, 40 (5) : 325-329) Mutagenic breeding of Streptomyces sp. YB-24, so that the fermentation unit for the production of staurosporine is 3.833 mg / L; Zhuang Yibin et al. (China Marine Medicine, 2011, 30 (2): 29 -33) Compound mutagenesis breeding with Streptomyces fradiae 007, Streptomyces arenae Z ₄ 007, and Streptomyces rubrolavendulae THW-12 as the starting strains, the fermentation unit is lower, 9.06ug / mL; Byung-Yong Kim et al. ( International Journal of Systematic and Evolutionary Microbiology, 2012, 62: 966-970) published Streptomyces staurosporininus sp.nov. Biochemistry, molecular biology identification, but did not publish its fermentation technology and fermentation unit. Pu Xiaoming et al. (Microbiology Bulletin, 2009, 36 (11): 1631-1637) optimized the fermentation process of marine actinomycetes H41-38 so that the fermentation unit of astrosporin reached 286.44ug / mL.

In summary, although there are many bacteriocin-producing bacteria in published patents and literatures, the strains are relatively primitive and the level of fermentation units is low, which is not suitable for industrialization. Therefore, the search for a new and efficient staurosporin-producing strain is still ongoing.

Summary of the Invention

One of the objectives of the present invention is to provide a new Streptomyces HS-HY-153, which has the ability to produce staurosporin, and its deposit number is CGMCC No. 14806.

The microbial strain of the streptomyces HS-HY-153 of the present invention was deposited on October 13, 2017 at the Common Microbial Center (CGMCC) of the China Microbial Strain Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China) ), Its deposit number is CGMCC No. 14806, it is classified as Streptomyces sp., And it is registered to prove its survival.

The object of the present invention is also to provide the application of a new streptomyces HS-HY-153 (CGMCC No. 14806) in preparing staurosporine or a pharmaceutical composition containing staurosporine.

The invention also provides a method for preparing staurosporin. The method includes the process of aerobic fermentation by using Streptomyces HS-HY-153 (CGMCC No. 14806) in a nutrient medium containing an assimitable carbon source and / or nitrogen source.

In a preferred embodiment, the assimitable carbon source is selected from the group consisting of corn starch, corn soluble starch, maltodextrin, sucrose, glucose, sorbitol, mannitol, glycerol, maltose, lactose, galactose, xylan, One of industrial molasses, soybean oil, methyl oleate, or any combination thereof.

In a preferred embodiment, the assimilated nitrogen source is selected from beef extract powder, beef extract, yeast extract, yeast extract, malt extract powder, yeast powder, peptone, gluten powder, wheat bran , Wheat germ flakes, cottonseed meal powder, cottonseed meal powder, peanut meal powder, soybean meal, wheat germ meal, soybean meal, fish meal, dried corn slurry meal, corn meal, urea, ammonium salt, nitrate, or one of the above substances random combination.

In a preferred embodiment, the nutrient medium further includes an inorganic salt selected from the group consisting of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, diammonium hydrogen phosphate, potassium chloride, and chloride. Sodium, magnesium chloride, cobalt chloride, sodium molybdate, trisodium citrate, calcium carbonate, calcium chloride, or any combination thereof.

In a preferred embodiment, the nutrient medium contains glucose 0-30.0g / L, glycerol 5.0-90.0g / L, peanut cake powder 5.0-60.0g / L, yeast powder 3.0-20.0g / L, and soybean cake Flour 0-40.0g / L, wheat germ flakes 0-30.0g / L, soybean flour 0-40.0g / L, magnesium sulfate 0-5.0g / L, calcium carbonate 1.0-5.0g / L, ferrous sulfate 0- 5.0 × 10 ^-2 g / L, zinc sulfate 0-2.0 × 10 ^-3 g / L, manganese sulfate 0-2.0 × 10 ^-4 g / L, cobalt chloride 0-5.0 × 10 ^-5 g / L, molybdenum Sodium 0-2.0 × 10 ^-4 g / L; preferably, the nutrient medium contains glucose 0-10.0g / L, glycerol 70.0.0-90.0g / L, peanut cake powder 40.0-60.0g / L, Yeast powder 5.0-20.0g / L, Soybean cake powder 0-40.0g / L, Wheat germ slices 0-20.0g / L, Soy flour 0-40.0g / L, Magnesium sulfate 0-1.0g / L, Calcium carbonate 2.0 -5.0g / L, ferrous sulfate 0-5.0 × 10 ^-2 g / L, zinc sulfate 0-5.0 × 10 ^-5 g / L, manganese sulfate 0-2.0 × 10 ^-4 g / L, cobalt chloride 0 -5.0 × 10 ^-5 g / L, sodium molybdate 0-2.0 × 10 ^-4 g / L.

In a preferred embodiment, the temperature of the aerobic fermentation is 20 ° C-30 ° C, preferably 24 ° C-28 ° C; the pH of the culture medium is 6.0-8.0, preferably 7.0-7.8; and the culture time is 24-300 hours, preferably 120-168 hours.

In a preferred embodiment, the streptomyces HS-HY-153 is inoculated into the nutrient medium through a seed liquid to perform the fermentation culture;

The seed solution is obtained by seed culture of Streptomyces HS-HY-153 in a seed medium; the conditions for the seed culture are: the temperature of the seed culture is 20 ° C-30 ° C, preferably 24 ° C-28 ° C The pH of the medium is 6.0-8.0, preferably 6.5-7.8; the culture time is 17-80 hours, preferably 19-72 hours.

In a preferred embodiment, the seed medium contains corn starch 2.0-40.0g / L, glucose 0-20.0g / L, maltodextrin 0-10.0g / L, glycerin 5.0-50.0g / L, peanut Cake powder 5.0-40.0g / L, yeast powder 5.0-20.0g / L, soybean cake powder 0-10.0g / L, calcium carbonate 1.0-4.0g / L.

The staurosporin of the present invention can be detected by HPLC under the following conditions:

Chromatographic column: C18 column (4.6mm × 250mm, 5μm);

Mobile phase: methanol: water: ammonia = 85: 15: 0.1 (v / v / v);

Injection volume: 20 μL;

Flow rate: 1.0mL / min;

Detection wavelength: 254nm;

The main biological characteristics of the Streptomyces HS-HY-153 (CGMCC No. 14806) of the present invention are: round colony shape, convex surface, colony diameter of about 5-7mm, smooth colony surface, grooves, and a slight depression in the middle The matrix mycelium is well developed, tightly combined with the culture medium, not easy to provoke, the color is colorless, beige or light yellow, the aerial mycelium is white, the spores are rich, the cyan is in the early stage, and the teal is turned into dark cyan, cyan grey, and agate gray in the later stage No soluble pigments.

The strain HS-HY-153 of the present invention has high production capacity; and its ability to produce staurosporin has been greatly improved compared with other strains in the prior art, which is beneficial to the realization of industrial production.

Preservation Instructions

Strain name: Streptomyces

Latin name: Streptomyces sp.

Strain number: HS-HY-153

Preservation institution: General Microbial Center of China Microbial Collection Management Committee

Depository organization abbreviation: CGMCC

Address: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing

Deposit Date: October 13, 2017

Depository Center Registration Number: CGMCC No. 14806

Brief description of the drawings:

Fig. 1 is a micrograph (400 ×) of strain HS-HY-153 (CGMCC No. 14806) on ISP2 medium.

Figure 2 is a colony characteristic diagram of strain HS-HY-153 (CGMCC No. 14806) on ISP2 medium.

Figure 3 is a colony characteristic diagram of strain HS-HY-153 (CGMCC No. 14806) on ISP1, ISP3, ISP4, ISP5, calcium malate, Gao No. 1, nutrient agar medium.

Detailed ways:

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials, reagents, etc. used in the following examples are ordinary commercially available products and can be purchased on the market.

The actinomycete DNA extraction kit used was a product of Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

The SanPrep column PCR product purification kit used for the purification and recovery of PCR products is a product of Biotech Bioengineering (Shanghai) Co., Ltd.

Yeast extraction powder (YEAST EXTRACT) is a product of OXOID Company, article number LP0021.

Malt Extract is a product of BD Co., Ltd., product number 218630.

Corn starch is a product of Weifang Shengtai Pharmaceutical Co., Ltd.

Peanut cake powder is a product of Jinan Huilong Biotechnology Co., Ltd.

Wheat germ flakes are products of Haining Dawei Food Co., Ltd.

The staurosporine standard was made by Zhejiang Hisun Pharmaceutical Co., Ltd., and its chromatographic purity was 99%.

The HPLC method used in the following examples to detect staurosporine in the fermentation broth uses the staurosporine standard as a control. The HPLC detection conditions are as follows:

Chromatographic column: C18 column (4.6mm × 250mm, 5μm);

Mobile phase: methanol: water: ammonia = 85: 15: 0.1 (v / v / v);

Injection volume: 20 μL;

Flow rate: 1.0mL / min;

Detection wavelength: 254nm.

The following further describes the present invention in combination with specific examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1: Strain source

The Streptomyces HS-HY-153 (CGMCC No. 14806) of the present invention is obtained by isolating the original strain from a soil sample in Tiantai Mountain, Zhejiang Province, and then breeding it through mutagenesis.

After the original strain was cultured in an ISP2 slant medium at 28 ° C. for 7-10 days, the mycelium was scraped off with an inoculating shovel under aseptic conditions to prepare a bacterial suspension for NTG (nitrosoguanidine) mutagenesis.

2 mg of NTG crystals were weighed and dissolved in 2 mL of sterile Tris buffer (pH 6.0). Pipette 1mL NTG solution into a 1mL bacterial suspension, and shake with a rotary or reciprocating shaker at 28 ° C for 1 hour. The remaining specific steps are as follows:

(1) Mycelium culture

Solid medium formula: ISP2. After sterilizing at 121 ° C for 20 minutes, cool to 50-60 ° C and pour the plate. The mutagenized bacteria suspension was appropriately diluted, and 0.2 mL of the bacterial solution was pipetted onto the ISP2 plate. The non-mutagenic bacteria suspension was also appropriately diluted and coated on the ISP2 plate as a control. Box culture, colony culture for 7 days, mycelium is mature.

(2) Preparation of seed medium and seed liquid culture

Seed medium formula: yeast powder 7.5g / L, glucose 8.0g / L, corn starch 10.0g / L, glycerin 20.0g / L, peanut cake powder 20.0g / L, calcium carbonate 2.0g / L, the balance is tap water , Adjust the pH to 7.2-7.4 with 6mol / L NaOH solution; the volume of the triangle shake bottle is 25mL / 250mL, and sterilize at 121 ℃ for 20min. Each seed shake flask was inoculated with about 0.5 colonies, and cultured at 28 ± 1 ° C and 250 rpm for 24 hours to obtain seed liquid.

(3) Preparation of fermentation medium and culture of fermentation broth

Formulation of fermentation medium: glycerin 50.0g / L, glucose 5.0g / L, peanut cake powder 20.0g / L, yeast powder 5.0g / L, calcium carbonate 3.0g / L, the balance is tap water, using 6mol / L NaOH solution Adjust the pH to 7.4-7.6; the volume of the triangle shake bottle is 20mL / 250mL, and sterilize at 121 ° C for 20min. The seed liquid is inserted into the fermentation medium with an inoculation amount of 5-10% (volume percentage), the cultivation temperature is 28 ± 1 ° C., the rotation speed is 250 rpm, and the cultivation is performed for 120 hours.

800 single colonies after NTG mutagenesis were selected and subjected to shake flask fermentation. The astrosporin content in the fermentation broth was detected by HPLC method. The strain with the highest astrosporin production was selected as Streptomyces HS-HY-153. (CGMCC No. 14806).

Example 2: The morphology, culture characteristics and physiological and biochemical characteristics of Streptomyces HS-HY-153 (CGMCC No. 14806).

Refer to the relevant contents in the "Streptomyces Identification Manual", "Classification and Identification of Actinomycetes", "Common Bacterial System Identification Manual" and other books for experiments: the color judgment refers to the colors in the RAL K7 color card for comparison.

1. Morphological characteristics of the strain: Strain HS-HY-153 (CGMCC No. 14806) was inoculated in ISP2 medium for insert culture. After 3-5 days of cultivation at 28 ° C, coverslips were taken on slides, and optically Observe at 400 × magnification under a microscope. The results are shown in Figure 1.

2. Strain culture characteristics: After strain HS-HY-153 (CGMCC No. 14806) was cultured on ISP2 medium at 28 ° C for 7-10 days, the colonies were round in shape and the surface was convex, and the diameter of the colonies was about 5-7mm. The surface of the colony is smooth, with grooves, with a slight depression in the middle, and the matrix hyphae is developed. It is tightly combined with the culture medium and difficult to provoke. The color is pale yellow, the aerial hyphae is white, the spore production is rich, the early stage is blue, and the late stage is dark Cyan, cyan gray, no soluble pigment, the results are shown in Figure 2.

For other culture characteristics, ISP1, ISP3, ISP4, ISP5, calcium malate, Gao No.1, and nutrient agar were used in 7 kinds of medium. After culturing at 28 ° C for 7-10 days, observe the colonies, mycelia, spores, and pigments. The situation occurs, and the results are shown in Table 1 and Figure 3.

Table 1 Culture characteristics of strain HS-HY-153 (CGMCC No. 14806) on 7 media

3. Physiological and biochemical characteristics test: Results Table 2-Table 7.

a) Utilization of carbon source: ISP9 is used as the basic medium, and the final concentration of each carbon source is 1.0% (1.0g / 100mL), see Table 2.

b) Utilization of inorganic nitrogen source: ISP9 was used as the basic medium, and the concentrations of potassium nitrate and ammonium sulfate were both 0.1% (0.1g / 100mL), as shown in Table 2.

c) Degradation test and NaCl tolerance test uses GYEA (pH 6.8) as the basic medium. The concentration of various degradation products and the results of the degradation test are shown in Table 3; the results of the NaCl tolerance test are shown in Table 7.

d) Catalase test, pH test and temperature test all use ISP2 medium. The catalase test results are shown in Table 4, the pH test results are shown in Table 5, and the temperature test results are shown in Table 6.

e) M.R, V-P and other experiments adopt the method of "Common Bacterial System Identification Manual". The results are shown in Table 4.

f) Except for temperature experiments, all were cultured at 28 ° C for 7-10 days.

Table 2 Utilization of carbon and nitrogen sources for strain HS-HY-153 (CGMCC No. 14806)

Carbon source	growing situation	Carbon source	growing situation	Inorganic nitrogen source	growing situation
D-glucose	4	Salicin	2	Ammonium sulfate	+
D-raffinose	2	D-lactose	2	Potassium nitrate	-
D-xylose	2	Galactose	3
D-sorbitol	2	Inositol	2
L-arabinose	2	Mannitol	3
glycerin	2	Glycine	2
maltose	2	Xylan	4
D-fructose	2	Inulin	2
D-sucrose	2	D	2

Table 3 Degradation test results of strain HS-HY-153 (CGMCC No. 14806)

Degradation	Degradation concentration	result	Degradation	Degradation concentration	result
Adenine	0.5%	2,-	Casein	1.0%	4,-
Guanine	0.5%	4,-	Tyrosine	1.0%	4, +
Xanthine	0.4%	4,-	Tween-40	1.0%	2,-
Xylan	0.4%	4,-	Tween-60	1.0%	2,-
Hypoxanthine	0.4%	4, +	Tween-80	1.0%	2,-

Table 4 Main physiological and biochemical characteristics of strain HS-HY-153 (CGMCC No. 14806)

Pilot projects	result	Pilot projects	result	Pilot projects	result
Gelatin liquefaction	+	Milk mash	-	Cellulose utilization	-
Starch hydrolysis	+	Nitrate reduction	-	Catalase	+
Milk coagulation	-	Hydrogen sulfide production	-
V-P experiment	-	M.R experiment	-

Table 5 pH test for growth of strain HS-HY-153 (CGMCC No. 14806)

pH	3.5	4.0	4.5	5.0	5.5	6.0	6.5	7.0	7.5
growing situation	0	0	0	0	4	4	4	4	4

Table 6 Temperature test for the growth of strain HS-HY-153 (CGMCC No. 14806)

Temperature (℃)	7	14	28	37	45
growing situation	2	4	4	0	0

Table 7 Tolerance of strain HS-HY-153 (CGMCC No. 14806) to NaCl

NaCl concentration (%)	1.0	4.0	7.0	10.0
Strain growth	4	3	0	0

* Note: In Table 2-7: 0, no growth; 1, weak growth; 2, can grow with a small amount of spores; 3, good growth with a large number of spores; 4, the best growth, with rich spores; +, Positive;-, negative.

Example 3: Identification of bacteria

1. 16S rDNA sequence analysis of Streptomyces HS-HY-153 (CGMCC No. 14806)

Follow the instructions in the "Molecular Cloning Experiment Guide" book for experiments. Mycelia were collected, and total DNA was extracted using an actinomycete DNA extraction kit. 16S rDNA sequence amplification was performed using the universal primers 27F (SEQ ID NO: 1) / 1495R (SEQ ID NO: 2). The amplification system and PCR reaction program are shown in Table 8. The PCR product was detected using a 0.8% agarose gel. Electrophoresis, purification and recovery of PCR products using SanPrep column PCR product purification kit, the purified PCR products were directly sent to Nanjing Kingsray Biotechnology Co., Ltd. for sequence determination.

Table 8 PCR amplification system and reaction procedures

The sequence of 16SrDNA detected by strain HS-HY-153 (CGMCC No. 14806) was verified, and then homologous sequence BLAST comparison was performed with sequences of related species and genera in GenBank database to determine the taxonomic status of the strain.

The 16S rDNA sequence (SEQ ID NO: 3) measured by strain HS-HY-153 (CGMCC No. 14806) was submitted to NCBI and related sequences in GenBank were compared for BLAST. Highly sensitive model strains).

Table 9 Homology of strain HS-HY-153 (CGMCC No. 14806) and typical model strain

Taxon Name	GenBank No.	Similarity (%)
Streptomyces xanthophaeus NRRL B-5414 (T)	JOTT01000080	99.45

Streptomyces cirratus NRRL B-3250 (T)	AY999794	99.45
Streptomyces spororaveus LMG 20313 (T)	AJ781370	99.45
Streptomyces nojiriensis LMG 20094 (T)	AJ781355	99.45
Streptomyces vinaceus NBRC 13425 (T)	AB184394	99.44
Streptomyces sporoverrucosus NBRC 15458 (T)	AB184684	99.44
Streptomyces goshikiensis NBRC 12868 (T)	AB184204	99.44
Streptomyces colombiensis NRRL B-1990 (T)	DQ026646	99.38
Streptomyces lavendulae subsp.lavendulae NRRL B-2774 (T)	JOEW01000098	99.38
Streptomyces avidinii NBRC 13429 (T)	AB184395	99.17

The 16S rDNA region of strain HS-HY-153 (CGMCC No. 14806) was sequenced and compared with the sequences of related species and genus in the GenBank database by homologous sequence BLAST. It was found to be related to Streptomyces xanthophaeus and tendril Streptomyces cirratus, Streptomyces spororaveus, Streptomyces nojiriensis all have a homology of 99.45%. At the same time, the strain HS-HY-153 (CGMCC No. 14806) has apparent characteristics. It was found that the strain and Streptomyces sp. Classification-related parameters were very close. Therefore, the strain HS-HY-153 (CGMCC No. 14806) was identified as a Streptomyces sp. Strain.

2. The comparison between the present Streptomyces HS-HY-153 (CGMCC No. 14806) and other staurosporin-producing bacteria is as follows:

US7608420B and EP0444503A disclose the fermentation units of Streptomyces hygroscopicus C39280-450-9 (ATCC 53730) for producing staurosporine are 392 mg / L and 130 mg / L, respectively, and the fermentation of HS-HY-153 (CGMCC No. 14806) of the present invention The unit is as high as 1540mg / L.

US4107297 discloses the morphological, cultural, and physiological and biochemical characteristics of Streptomyces sp. AM-2282 (later named Streptomyces staurosporeus nov.sp. NRRL11184). The bacteria HS-HY-153 (CGMCC No. 14806) and Streptomyces of the present invention The cultural characteristics of sp.AM-2282 are shown in Table 10, and the physiological and biochemical characteristics of Streptomyces sp.AM-2282 are shown in Table 11. It can be seen from Tables 10 and 11 that the bacteria HS-HY-153 (CGMCC No. 14806) and Streptomyces sp. AM-2282 of the present invention have physiological and biochemical characteristics such as culture characteristics, milk coagulation, milk mash, cellulose utilization All are different.

Table 10 Comparison of the cultural characteristics of strain HS-HY-153 (CGMCC No. 14806) and strain Streptomyces sp. AM-2282

Table 11 Comparison of physiological and biochemical characteristics of strain HS-HY-153 (CGMCC No. 14806) and strain Streptomyces sp. AM-2282

Note: In Table 11, 0, no growth; 1, weak growth; 2, can grow, with a small number of spores; 3, good growth, with a large number of spores; 4, the best growth, with rich spores; +, positive;-, Negative; w, suspected / weak positive.

Zhuang Yibin et al. (China Marine Drugs, 2011, 30 (2): 29-33) reported that astrosporin was improved by selective mutagenesis and breeding of strains Streptomyces fradiae 007, Streptomyces arenae Z ₄ 007 and Streptomyces rubrolavendulae THW-12 Fermentation unit. As a result, a high-yield strain was obtained from the strain Streptomyces fradiae 007. The fermentation unit was increased from the original 1.02 mg / L to 9.06 mg / L. The fermentation unit of the present invention HS-HY-153 (CGMCC No. 14806) Up to 1540mg / L.

Gai Cuijuan et al. (Chinese Journal of Antibiotics, 2015, 40 (5): 325-329) After high-yield strains of Streptomyces sp. YB-24 were subjected to combined mutagenesis with ultraviolet and lithium chloride, the metabolism The capacity is 3.833 mg / L, and the HS-HY-153 (CGMCC No. 14806) of the present invention produces a maximum of 1540 mg / L of staurosporin fermentation unit.

Byung-Yong Kim et al. (International Journal of Systematic and Evolutionary Microbiology, 2012, 62: 966-970) disclose the astrosporin-producing bacteria Streptomyces staurosporininus sp.nov. Culture characteristics and physiological and biochemical characteristics on ISP4 Table 12 compares the culture characteristics and physiological and biochemical characteristics of the inventive bacteria HS-HY-153 (CGMCC No. 14806) and Streptomyces staurosporininus sp. Nov. On ISP4 medium. It can be seen from Table 12 that the bacterium HS-HY-153 (CGMCC No. 14806) and Streptomyces staurosporininus sp.nov. Have different culture characteristics on the ISP4 medium, such as nitrate reduction, hydrogen sulfide production, and casein degradation. There are differences in physiological and biochemical characteristics, such as xylan degradation, fructose utilization.

Table 12 Comparison of culture characteristics and physiological and biochemical characteristics of strain HS-HY-153 (CGMCC No. 14806) and strain Streptomyces staurosporininus sp.nov.

Note: In Table 12, 0, no growth; 1, weak growth; 2, can grow, with a small number of spores; 3, good growth, with a large number of spores; 4, the best growth, with rich spores; +, positive;-, negative.

The marine actinomycete H41-38 reported by Pu Xiaoming et al. (Microbiology Bulletin, 2009, 36 (11): 1631-1637), after further adjusting the fermentation conditions, the fermentation unit for the production of staurosporine reached 286.44ug / mL, while the HS-HY-153 (CGMCC No. 14806) produced by the present invention has a maximum fermentation unit of 1540 mg / L. In addition, Ma Chunxiu disclosed the physiological and biochemical characteristics of H4138 (same as H41-38) and the use of carbon and nitrogen sources in "Identification, Mutagenesis and Toxin Mixing Insecticide Study of Streptomyces H4138". -HY-153 (CGMCC No. 14806) for the cultural characteristics of strain H4138 is shown in Table 13, and the physiological and biochemical characteristics of strain H4138 are shown in Table 14; as can be seen from Tables 13 and 14, HS-HY of the present invention -153 (CGMCC No. 14806) and strain H4138 are different in physiological and biochemical characteristics such as culture characteristics, hydrogen sulfide production, milk coagulation, milk mash, and cellulose utilization.

Table 13 Comparison of the cultural characteristics of strain HS-HY-153 (CGMCC No. 14806) and strain H4138

Table 14 Comparison of physiological and biochemical characteristics of strain HS-HY-153 (CGMCC No. 14806) and strain H4138

Note: In Table 14, 0, no growth; 1, weak growth; 2, can grow with a small number of spores; 3, good growth, with a large number of spores; 4, the best growth, with rich spores; +, positive;-, negative.

Based on the morphology, culture characteristics, physiological and biochemical characteristics and 16S rDNA sequence identification results of the aforementioned Streptomyces HS-HY-153 (CGMCC No. 14806) of the present invention, it can be known that the strain HS-HY-153 (CGMCC No. 14806) of the present invention ) Belongs to Streptomyces sp. Strain, and is different from other known staurosporin-producing bacteria, so the streptomyces HS-HY-153 (CGMCC No. 14806) of the present invention is a brand new astrospore Streptozotocin-producing bacteria.

Example 4: Preparation of a staurosporin-containing fermentation broth

(1) Preparation of slanted medium and spore culture:

Slant medium formula: yeast extraction powder 4.0g / L, malt extract 10.0g / L, glucose 4.0g / L, agar 20.0g / L, the balance is purified water, and the pH is adjusted with 6mol / L NaOH solution 7.2-7.4; 250mL eggplant-shaped bottle, filled with 60mL, sterilized at 121 ° C for 20min, cooled to about 55 ° C, and placed in a 37 ° C incubator for 1-2 days after cooling and solidification, inoculated with Streptomyces HS -HY-153 (CGMCC No. 14806) spores or mycelia to oblique plane, after culturing at 28 ± 1 ℃ for 7-10 days, the spores mature.

(2) Preparation of seed medium and seed liquid culture:

Seed medium formula: yeast powder 7.5g / L, glucose 8.0g / L, corn starch 10.0g / L, glycerin 20.0g / L, peanut cake powder 20.0g / L, calcium carbonate 2.0g / L, the balance is tap water Use a 6mol / L NaOH solution to adjust the pH to 7.2-7.4; 250mL triangular shake flask with a capacity of 25mL and sterilize at 121 ° C for 20min. The spores obtained in step (1) were inoculated into the seed medium at 10 ⁷ -10 ⁸ cfu / mL, and cultured at 28 ± 1 ° C and shaking at 250 rpm for 22-24 hours. At this time, the pH of the culture solution was 6.8-7.2, and the concentration of mycelium 8-10% (volume percentage) to obtain a seed liquid.

(3) Preparation of fermentation medium and culture of fermentation broth:

Formulation of fermentation medium: glycerin 50.0g / L, glucose 5.0g / L, peanut cake powder 20.0g / L, yeast powder 5.0g / L, calcium carbonate 3.0g / L, the balance is tap water, using 6mol / L NaOH solution Adjust the pH to 7.4-7.6; 250mL triangular shake flask with a volume of 20mL and sterilize at 121 ℃ for 20min. The seed liquid obtained in step (2) is inserted with an inoculation amount of 5-10% (volume percentage). The culture was shaken at 28 ± 1 ° C and 250 rpm for 168 hours to obtain a fermentation broth. Using the staurosporin standard as a control, the astrosporin content in the fermentation broth was measured by HPLC method, and it was found to be 1310 μg / mL.

Example 5: Preparation of a staurosporin-containing fermentation broth

(1) Preparation of slanted medium and spore culture:

The specific steps are the same as step (1) in Example 4.

(2) Preparation of seed medium and seed liquid culture

The specific steps are the same as step (2) in Example 4.

(3) Preparation of fermentation medium and culture of fermentation broth:

Fermentation medium formula: glycerol 70.0g / L, glucose 5.0g / L, peanut cake powder 40.0g / L, yeast powder 5.0g / L, magnesium sulfate 1.0g / L, calcium carbonate 3.0g / L, the balance is tap water Use a 6mol / L NaOH solution to adjust the pH to 7.4-7.6; a 250mL triangular shake flask with a volume of 20mL and sterilize at 121 ° C for 20min. The seed liquid obtained in step (2) is inserted with an inoculation amount of 5-10% (volume percentage). The culture was shaken at 28 ± 1 ° C and 250 rpm for 168 hours to obtain a fermentation broth. Using the staurosporine standard as a control, the astrosporin content in the fermentation broth was measured by HPLC method, and it was found to be 1380 μg / mL.

Example 6: Preparation of a staurosporin-containing fermentation broth

(1) Preparation of slanted medium and spore culture:

The specific steps are the same as step (1) in Example 4.

(2) Preparation of seed medium and seed liquid culture

The specific steps are the same as step (2) in Example 4.

(3) Preparation of fermentation medium and culture of fermentation broth:

Fermentation medium formula: glycerol 80.0g / L, peanut cake powder 40.0g / L, yeast powder 5.0g / L, wheat germ flakes 20.0g / L, magnesium sulfate 1.0g / L, calcium carbonate 3.0g / L, subsulfate Iron 5.0 × 10 ^-2 g / L, zinc sulfate 2.0 × 10 ^-3 g / L, the balance is tap water, adjust the pH to 7.4-7.6 with 6mol / L NaOH solution; 250mL triangle shake flask, 20mL, Sterilize at 121 ° C for 20min. The seed liquid obtained in step (2) is inserted with an inoculation amount of 5-10% (volume percentage). The culture was shaken at 28 ± 1 ° C and 250 rpm for 168 hours to obtain a fermentation broth. Using the staurosporine standard as a control, the astrosporin content in the fermentation broth was measured by HPLC method, and it was found to be 1480 μg / mL.

Example 7: Preparation of a staurosporin-containing fermentation broth

(1) Preparation of slanted medium and spore culture:

The specific steps are the same as step (1) in Example 4.

(2) Preparation of seed medium and seed liquid culture

The specific steps are the same as step (2) in Example 4.

(3) Preparation of fermentation medium and culture of fermentation broth:

Formulation of fermentation medium: glycerin 90.0g / L, glucose 10.0g / L, peanut cake powder 50.0g / L, yeast powder 10.0g / L, wheat germ flakes 10.0g / L, magnesium sulfate 1.0g / L, calcium carbonate 4.0 g / L, ferrous sulfate 5.0 × 10 ^-2 g / L, zinc sulfate 5.0 × 10 ^-5 g / L, the balance is tap water, adjust the pH to 7.4-7.6 with 6mol / L NaOH solution; 250mL triangle shake 20ml bottle, sterilize at 121 ℃ for 20min. The seed liquid obtained in step (2) is inserted with an inoculation amount of 5-10% (volume percentage). The culture was shaken at 28 ± 1 ° C and 250 rpm for 168 hours to obtain a fermentation broth. Using the staurosporin standard as a control, the astrosporin content in the fermentation broth was measured by HPLC method, and it was found to be 1505 μg / mL.

Example 8: Preparation of a staurosporin-containing fermentation broth

(1) Preparation of slanted medium and spore culture:

The specific steps are the same as step (1) in Example 4.

(2) Preparation of primary seed medium and primary seed liquid culture

The specific steps are the same as step (2) in Example 4.

(3) Preparation of seed tank seed liquid:

The formula of the seed liquid medium in the seed tank is the same as the seed medium in step (2) in Example 4;

Put 10L of seed medium into a 15L seed tank, sterilize it with steam, and sterilize it at 121 ° C for 20min. After cooling to 28 ° C, insert 200mL of the first-stage seed solution prepared in step (2). The stirring speed was 200 rpm, the aeration was 1.0 vvm, and the culture was performed at 28 ± 1 ° C. for 24 hours. At this time, the seed solution had a pH of 7.0-7.4 and a hyphae concentration of 12-15% (volume percentage) to obtain a seed tank seed solution.

(4) Preparation of fermentation liquid of fermentation tank:

The formulation of the fermentation medium is the same as the fermentation medium in step (3) in Example 6;

The fermentation tank has a volume of 50L and a feed volume of 30L. It is sterilized with steam at 121 ° C for 20min. After cooling to 28 ° C, it is connected to 3L of the seed tank seed solution prepared in step (3). The stirring rotation speed is 200-400 rpm (the rotation speed is gradually increased from 200 rpm to 320 rpm in the first 3 days), the aeration volume is 0.8-1.0 vvm, and the culture is performed at 28 ° C. for 168 hours to obtain a fermentation tank fermentation broth. Using the staurosporine standard as a control, the astrosporin content in the fermentation broth was measured by HPLC method to be 1540 μg / mL.

Example 9: Isolation, purification and structural identification of staurosporine

30 L of the fermentation broth obtained in Example 8 was centrifuged (4000 rpm, 15 min) to obtain mycelia, and the mycelia was extracted twice with acetone to obtain an acetone extract. The acetone extract was concentrated to dryness in vacuo and extracted with ethyl acetate to obtain an extract containing staurosporine. After the sample was extracted with silica gel, the sample was loaded on a silica gel column and eluted with a chloroform / methanol gradient of 99: 1, 98: 2, 97: 3, 96: 4 (V: V). The eluted fractions were collected in stages and detected by TLC. (Chloroform / methanol = 9: 1 (V: V)), the fractions containing chromatographic purity greater than 90% were combined and concentrated to dryness in vacuo to obtain a crude product. The crude astrosporin was recrystallized using chloroform / methanol = 1: 1 (V: V) to obtain 18.4 g of pure product.

The obtained pure product was determined by MS analysis (ESI-MS m / z 467 [M + H] ⁺ ) to have a molecular weight of 466. The ¹ H NMR (400 MHz, CDCl ₃ ) data was as follows: δ9.42 (d, J = 7.7 Hz, H-4), 7.91 (d, J = 8.4 Hz, H-11), 7.85 (d, J = 7.7 Hz, H-8), 7.43 (t, J = 7.7 Hz, H-2), 7.38 (dd, J = 8.4, 7.7 Hz, H-10), 7.35 (t, J = 7.7 Hz, H-3), 7.31 (t, J = 7.7 Hz, H-9), 7.27 (d, J = 7.7 Hz, H-1), 6.54 (br s, H-6), 6.53 (br d, J = 5.1 Hz, H-6 '), 5.03 (d, J = 15.5 Hz, H-7a), 4.95 (d , J = 15.5 Hz, H-7b), 3.86 (d, J = 3.3 Hz, H-3 '), 3.38 (s, H-3'-OMe), 3.32 (m, H-4'), 2.70 ( dd, J = 14.7, 3.1Hz, H-5'a), 2.33 (m, H-5'b), 2.33 (s, H-2'-Me), 1.54 (s, H-4'-NMe) These data and references 45 (2): 195-198), and its structure was determined to be staurosporin.

The fermentation broth obtained in Example 4-7 was processed according to the same isolation, purification, and structure identification steps as described above, and structural data consistent with the above were obtained, confirming that the target component in the fermentation broth was staurosporine. At the same time, the target peak collected by HPLC in Examples 4-8 was identified by the above structure, and it was also confirmed that the target was staurosporin.

Claims (10)

1. A Streptomyces sp. HS-HY-153, the deposit number is CGMCC No. 14806.
2. The use of streptomyces HS-HY-153 according to claim 1 in the preparation of staurosporine or a pharmaceutical composition containing staurosporin.
3. A method for preparing staurosporin comprises a process of aerobic fermentation of the streptomyces HS-HY-153 according to claim 1 in a nutrient medium containing an assimitable carbon source and / or nitrogen source.
4. The method according to claim 3, wherein the assimitable carbon source is selected from the group consisting of corn starch, corn soluble starch, maltodextrin, sucrose, glucose, sorbitol, mannitol, glycerol, maltose, lactose, hemi One of lactose, xylan, industrial molasses, soybean oil, methyl oleate, or any combination thereof.
5. The method according to claim 3, wherein the assimilationable nitrogen source is selected from the group consisting of beef extract powder, beef extract, yeast extract, yeast extract, malt extract powder, yeast powder, peptone, Gluten, wheat bran, wheat germ flakes, cottonseed meal, cottonseed powder, peanut meal, soybean meal, wheat germ meal, soybean meal, fish meal, corn starch dried meal, corn meal, urea, ammonium salt, nitrate One or any combination of the above.
6. The method according to any one of claims 3-5, wherein the nutrient medium further comprises an inorganic salt selected from the group consisting of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, Diammonium hydrogen phosphate, potassium chloride, sodium chloride, magnesium chloride, cobalt chloride, sodium molybdate, trisodium citrate, calcium carbonate, calcium chloride, or any combination thereof.
7. The method according to any one of claims 3-6, wherein the nutrient medium contains glucose 0-30.0g / L, glycerin 5.0-90.0g / L, peanut cake powder 5.0-60.0g / L, Yeast powder 3.0-20.0g / L, soybean meal powder 0-40.0g / L, wheat germ flakes 0-30.0g / L, soybean powder 0-40.0g / L, magnesium sulfate 0-5.0g / L, calcium carbonate 1.0 -5.0g / L, ferrous sulfate 0-5.0 × 10 ^-2 g / L, zinc sulfate 0-2.0 × 10 ^-3 g / L, manganese sulfate 0-2.0 × 10 ^-4 g / L, cobalt chloride 0 -5.0 × 10 ^-5 g / L, sodium molybdate 0-2.0 × 10 ^-4 g / L.
8. The method according to any one of claims 3 to 7, characterized in that the temperature of the aerobic fermentation is 20 ° C-30 ° C, preferably 24 ° C-28 ° C; the pH of the culture medium is 6.0-8.0, preferably 7.0- 7.8; culture time is 24-300 hours, preferably 120-168 hours.
9. The method according to any one of claims 3 to 8, characterized in that: the streptomyces HS-HY-153 is inoculated into the nutrient medium through a seed liquid to perform the fermentation culture;

   The seed liquid is obtained by seed-culturing the streptomyces HS-HY-153 according to claim 1 in a seed medium; the conditions for the seed culture are: the temperature of the seed culture is 20 ° C-30 ° C, It is preferably 24 ° C-28 ° C; the pH of the medium is 6.0-8.0, preferably 6.5-7.8; and the culture time is 17-80 hours, preferably 19-72 hours.
10. The method according to claim 9, characterized in that the seed medium contains corn starch 2.0-40.0g / L, glucose 0-20.0g / L, maltodextrin 0-10.0g / L, and glycerol 5.0- 50.0g / L, peanut cake powder 5.0-40.0g / L, yeast powder 5.0-20.0g / L, soybean cake powder 0-10.0g / L, calcium carbonate 1.0-4.0g / L.

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