CN114540212A - Streptomyces and method for producing lepomycin B by fermenting streptomyces - Google Patents
Streptomyces and method for producing lepomycin B by fermenting streptomyces Download PDFInfo
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- CN114540212A CN114540212A CN202011350157.9A CN202011350157A CN114540212A CN 114540212 A CN114540212 A CN 114540212A CN 202011350157 A CN202011350157 A CN 202011350157A CN 114540212 A CN114540212 A CN 114540212A
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- streptomyces
- hdcc00025
- lepomycin
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Abstract
The invention discloses Streptomyces (Streptomyces sp.) HDCC00025 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.20886 and the preservation date of 2020 and 10 months as well as 14 days. The Streptomyces sp is a novel lepomycin B producing strain, the production capacity is high, the lepomycin B producing capacity is greatly improved compared with other strains in the prior art, the titer of the lepomycin B can be maintained at more than 1200mg/L, and the industrial production is favorably realized.
Description
Technical Field
The invention relates to the technical field of industrial microbial fermentation, in particular to streptomyces and a method for producing lepomycin B by fermentation of the streptomyces.
Background
Leptomycin B (CAS number: 87081-35-4) is an unsaturated branched chain fatty acid with molecular formula C33H48O6Molecular weight is 540.73, and chemical structural formula is shown in figure 4.
Leptomycin B was originally found in fermentation broth of Streptomyces sp.ATS1287 (Hamamoto T, Gunji S, Tsuji H, Beppu T.Leptomycins A and B, new anti-microbial antibodies.I.Taxomon of the production and transformation, purification and characterization.J. Antibiot (Tokyo) 1983; 36(6):639-645 doi: 10.7164/anti-microbial.36.639), identified as an unsaturated branched-chain fatty acid with antifungal activity.
The lepomycin B serving as a commonly used nuclear transport inhibitor can penetrate cells and inhibit protein transport from cell nucleus to cytoplasm, and the mechanism of inhibiting nuclear transport is that the lepomycin B can be directly combined with CRM1 (Chromosome main amino protein 1, a nuclear export receptor participating in active cell transport) to inhibit the interaction of CRM1 and a nuclear localization signal of a target export protein, so that the combination of CRM1 and a protein with a nuclear signal is inhibited, and finally the inhibition of nuclear transport mediated by CRM1 is caused.
In recent years, with the progress of research, the compound is found to have remarkable antitumor activity, so that the lepomycin B also becomes a potential targeting drug in the aspect of resisting malignant tumors. Lorenzo Brunetti et al published in the journal of Blood 2017 showed that lepomycin B induces Leukemia cell differentiation in patients with Acute Myelogenous Leukemia (AML) subtype M2 by inhibiting nucleation of mutant NPM1 protein (Brunetti, Lorenzo et al, autologous Leukemia with Mutated NPM1 Is dependenton the cytoplastic Localization of NPM1c, [ J ] Blood,2017,877.). An article published in the journal of Chinese pathophysiology by Navy et al in 2018 shows that by inhibiting the nuclear transport of RUNX3 protein, LMB can obviously reduce the activity of breast cancer cells and inhibit the proliferation of the breast cancer cells (Navy, Liuhui. RUNX3 protein expression and localization research in different molecular subtype breast cancer cell lines [ J ]. Chinese pathophysiology journal, 2018,34(09): 1603-1609.).
In the prior art, Hamamoto T et al reported that a certain amount of the lepomycin B can be obtained by culturing Streptomyces sp. ATS1287 and separating and purifying, but the highest titer of the lepomycin B in the fermentation broth is about 40mg/L in the mycelium, the fermentation level is low, the strain is relatively original and is not suitable for industrial application (Hamamoto T, Gunji S, Tsuji H, Beppu T. Leptomycins A and B, new antibacterial and fermentation. I. Taxomy of the production and fermentation, purification and fermentation. J antibacterial (Tokyo) 1983; 36(6) 639-doi: 10.7164/antibacterial.36.639). Therefore, the search for a new strain which can efficiently ferment to obtain the leprosy B is of great significance.
Disclosure of Invention
One of the purposes of the present invention is to provide Streptomyces (Streptomyces sp.) HDCC00025, which is deposited in China general microbiological culture Collection center (CGMCC) at 10-14.2020 with the following addresses: no. 3 of Xilu No. 1 of Beijing, Chaoyang, Beijing, the institute of microbiology, national academy of sciences, with the collection number of CGMCC NO.20886, and registered in a book to prove survival.
The invention also aims to provide application of Streptomyces sp HDCC00025(CGMCC NO.20886) in preparing the lepomycin B or the pharmaceutical composition containing the lepomycin B.
The invention also provides a preparation method of the lepomycin B, which comprises the step of carrying out aerobic fermentation by adopting Streptomyces sp HDCC00025(CGMCC NO.20886) in a nutrient medium containing assimilable carbon sources and/or nitrogen sources.
In a preferred embodiment, the assimilable carbon source is selected from one of corn starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or a combination thereof, preferably one of corn starch, maltodextrin, glucose, sucrose, soybean oil or a combination of any of them.
In a preferred embodiment, the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salt or a combination of the above substances, preferably one of soybean cake powder, soybean lecithin, yeast extract powder and yeast powder or a combination of any several of the above substances.
In a preferred embodiment, the nutrient medium further comprises an inorganic salt selected from one of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, or a combination thereof, preferably calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride.
In a preferred embodiment, the nutrient medium contains 10-50g/L of corn starch, 10-50g/L of maltodextrin, 5-20g/L of soybean oil, 1-15g/L of yeast extract powder, 5-20g/L of soybean lecithin, 5-20g/L of soybean cake powder, 5-20g/L of yeast extract, 1-10g/L of magnesium sulfate, 2-8g/L of dipotassium phosphate and 0-50g/L of calcium carbonate.
In a preferred embodiment, the temperature of the aerobic fermentation is 20-35 ℃, preferably 23-30 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24-240 hours, preferably 72-168 hours; the oxygen throughput is from 0.1 to 2.0vvm, preferably from 0.5 to 2.0 vvm.
In a preferred embodiment, said Streptomyces sp HDCC00025(CGMCC No.20886) is subjected to said fermentative culture by seed liquid inoculation into said nutrient medium; the seed solution is obtained by seed culture of Streptomyces sp HDCC00025(CGMCC NO.20886) in a seed culture medium; the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24 to 80 hours, preferably 24 to 60 hours.
In a preferred embodiment, the seed culture medium contains 5-20g/L of glucose, 5-20g/L of corn starch, 5-20g/L of soybean cake powder, 1-10g/L of yeast extract powder, 1-20g/L of calcium carbonate, 1-10g/L of magnesium sulfate and 1-10g/L of dipotassium phosphate.
The inventive lepomycin B is detected by HPLC under the following conditions:
a chromatographic column: agilent poroshell 120EC-C18 (4.6X 50mm, 2.7 μm)
Column temperature: 30 ℃;
sample introduction volume: 2 mu L of the solution;
flow rate: 0.5 mL/min;
detection wavelength: 225 nm;
analysis time: 10 min;
mobile phase:
mobile phase A: 0.1% (volume percent) phosphoric acid aqueous solution;
mobile phase B: 0.1% (volume percent) acetonitrile phosphate solution;
| time (min) | 0 | 7.5 | 8.5 | 8.6 | 10 |
| Mobile phase A (%) | 38 | 10 | 10 | 38 | 38 |
| Mobile phase B (%) | 62 | 90 | 90 | 62 | 62 |
The Streptomyces sp HDCC00025(CGMCC NO.20886) has the following main biological characteristics: the colony is round in shape, the surface is convex, the diameter of the colony is about 7-15 mm, the surface of the colony is grooved, the middle is slightly concave, the substrate hyphae are developed, the combination with the culture medium is tight, the colony is not easy to pick up, the color is beige or faint yellow, the aerial hyphae are white, the spore production is rich, the early stage is faint yellow, the later stage is light cyan and gray, and no soluble pigment exists.
The strain (Streptomyces sp.) HDCC00025(CGMCC NO.20886) is a completely novel lepomycin B producing strain, has high production capacity, has the capability of producing lepomycin B greatly improved compared with other strains in the prior art, has the titer of lepomycin B maintained above 1200mg/L, and is beneficial to realizing industrial production.
Drawings
FIG. 1 is a microscopic image (400X) of strain HDCC00025(CGMCC NO.20886) on ISP2 medium.
FIG. 2 is a characteristic diagram of the colony of the strain HDCC00025(CGMCC NO.20886) on ISP2 medium.
FIG. 3 shows the spectrum of the lepomycin B detected by HPLC after the separation and extraction of thalli after the fermentation culture of the bacterial strain HDCC00025(CGMCC NO. 20886).
FIG. 4 shows the chemical structural formula of lepomycin B (Leptomycin B).
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The materials, reagents and the like used in the following examples are all common commercially available products and are commercially available unless otherwise specified.
Wherein the actinomycete DNA extraction kit is purchased from Beijing Sanbo Polygala tenuifolia biotechnology, LLC;
purification and recovery of PCR products the SanPrep column type PCR product purification kit used was purchased from Biotechnology engineering (Shanghai) Ltd.
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope.
Example 1: the source of the strain
Streptomyces sp HDCC00025(CGMCC NO.20886) is separated from soil of a certain hillside of Moganshan mountain in Hu of Zhejiang province of China.
Cross sampling is carried out on soil in Moganshan area, 5 sampling points are randomly selected, 10g of soil sample is taken at each point, the soil samples are put into a conical flask, 10g of sample is taken after uniform mixing, the sample is added into the conical flask filled with 90mL of sterile water (a magnetic stirrer is arranged in the conical flask), vortex stirring is carried out for 30 minutes, the mixture is fully and uniformly mixed to prepare suspension, namely 10-1And (4) bacterial suspension. Mixing the suspension with sterile water according to a volume ratio of 1: 9 to 10-2，10-3，10-4，10-5And (3) taking 0.1mL of bacterial suspension with different dilution times, coating the bacterial suspension on an ISP2 culture medium plate, lightly coating the bacterial suspension on the surface of the culture medium by using a sterile coating rod, standing the bacterial suspension for 30 minutes at room temperature, and then placing the bacterial suspension in a constant-temperature incubator at 28 ℃. And observing and recording the color, transparency, surface and edge morphology of the colony after the colony grows out. Finally, 1000 strains are picked and inoculated in an ISP2 culture medium to prepare a slant, and fermentation verification is carried out. The slant culture of thallus with inoculating loop is taken, inoculated to seed culture containing 20mLAfter shake culture at 28 ℃ for 1 day in a 250mL conical flask containing the medium, 1mL is sucked and transplanted into a 250mL conical flask containing 20mL of fermentation medium, after shake culture at 28 ℃ for 3 days, the content of the hapomycin B in the obtained fermentation liquid is detected by HPLC, and the most highly productive strain, namely Streptomyces sp HDCC00025(CGMCC NO.20886) is selected.
Seed culture medium formula (g/L): 10g/L of glucose, 10g/L of starch, 10g/L of soybean cake powder, 5g/L of yeast extract powder, 15g/L of calcium carbonate, 1.5g/L of magnesium sulfate and 1g/L of monopotassium phosphate, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0 +/-0.1.
Fermentation medium formula (g/L): 40g/L of corn starch, 20g/L of maltodextrin, 5g/L of yeast extract powder, 10g/L of yeast extract and 30g/L of calcium carbonate, and adding water to a constant volume of 1000mL and a pH value of 7.0 +/-0.1.
Example 2: the morphology, the cultural characteristics and the physiological and biochemical characteristics of Streptomyces sp HDCC00025(CGMCC NO. 20886).
Experiments are carried out according to relevant contents in books such as Streptomyces appraisal handbook, Classification and appraisal of actinomycetes, common bacteria system appraisal handbook and the like: the color was determined by reference to the color in the RAL K7 color chart.
1. Morphological characteristics of the strains: bacterial strain HDCC00025(CGMCC NO.20886) is inoculated in ISP2 culture medium for insert cultivation, after cultivation for 3-5 days at 28 ℃, a cover glass is taken out and put in a slide glass, and the observation is carried out under an optical microscope at 400 times, and the result is shown in figure 1.
2. The strain culture characteristics are as follows: after the bacterial strain HDCC00025(CGMCC NO.20886) is cultured for 7-10 days at 28 ℃ on an ISP2 culture medium, the colony is oval in shape, has radial stripes and raised surface, the diameter of the colony is about 7-15 mm, the surface of the colony is provided with furrows, the middle part of the colony is slightly concave, the substrate hyphae are developed, the colony is tightly combined with the culture medium, is not easy to pick up, is beige in color, is white in aerial hyphae, is rich in spore, is light yellow at the early stage, is changed into light gray at the later stage, does not contain soluble pigment, and the result is shown in figure 2.
And for other culture characteristics, 7 culture media of ISP1, ISP3, ISP4, ISP5, calcium malate, Gao's I and nutrient agar are adopted, and after the culture media are cultured for 7-10 days at the temperature of 28 ℃, the generation conditions of colonies, hyphae, spores and pigments are observed, and the results are shown in Table 1.
TABLE 1 culture characteristics of the strain HDCC00025(CGMCC NO.20886) on 7 media
3. Physiological and biochemical characteristic test: the results are shown in tables 2 to 7.
a) Utilization of carbon source: using ISP9 as the basal medium, the final concentration of each carbon source was 1.0%, as shown in Table 2.
b) Utilization of inorganic nitrogen source: using ISP9 as the basal medium, the potassium nitrate and ammonium sulfate concentrations were 0.1% each, as shown in Table 2.
c) The basic culture medium adopted in the degradation test and the NaCl tolerance test is GYEA (pH6.8), and the concentration of various degradation products and the degradation test results are shown in Table 3; the results of the NaCl tolerance experiments are shown in Table 7.
d) The catalase test, the pH test and the temperature test all use the ISP2 culture medium. The results of the catalase test are shown in Table 4, the pH test in Table 5, and the temperature test in Table 6.
e) M.R, V-P, etc., the results are shown in Table 4, using the method of "handbook of identifying common bacteria systems".
f) Except for the temperature experiment, the culture is carried out for 7-10 days at 28 ℃.
TABLE 2 utilization of the carbon and nitrogen sources of Strain HDCC00025
| Carbon source | Growth conditions | Carbon source | Growth conditions | Inorganic nitrogen source | Growth conditions |
| D- |
4 | |
2 | Ammonium sulfate | + |
| D-raffinose | 1 | D- |
4 | Potassium nitrate | - |
| D- |
1 | Galactose | 2 | ||
| D- |
3 | Inositol | 1 | ||
| L- |
2 | |
3 | ||
| |
3 | |
1 | ||
| |
4 | Xylan | 2 | ||
| D- |
3 | Inulin powder | 3 | ||
| D- |
3 | |
3 |
TABLE 3 degradation test results of Strain HDCC00025
| Degradation product | Concentration of degradants | Results | Degradation product | Concentration of degradants | Results |
| Adenine | 0.5％ | 3，+ | |
1％ | 4，+ |
| Guanine and its preparing process | 0.5％ | 4，- | |
1％ | 3，- |
| Xanthine | 0.4％ | 3，- | Tween-40 | 1％ | 4，- |
| Xylan | 0.4％ | 3，- | Tween-60 | 1％ | 3，- |
| Hypoxanthine | 0.4％ | 4，+ | Tween-80 | 1％ | 3，- |
TABLE 4 major physio-biochemical characteristics of Strain HDCC00025
TABLE 5 pH test for growth of Strain HDCC00025
TABLE 6 temperature test for growth of Strain HDCC00025
| Temperature (. degree.C.) | 7 | 14 | 28 | 37 | 45 |
| |
1 | 2 | 4 | 2 | 0 |
TABLE 7 tolerance of strain HDCC00025 to NaCl
| Concentration of |
1％ | 4％ | 7％ | 10％ |
| Growth of the |
3 | 1 | 0 | 0 |
Remarking: in tables 2 to 7, 0: no growth occurs; 1: the growth is very weak; 2: can grow, has a small amount of spores; 3: the growth is good, and a large number of spores exist; 4: the growth is best, and spores are abundant; +: positive; -: and (4) negativity.
Example 3 species identification
1. 16S rDNA sequence analysis of streptomycete HDCC00025(CGMCC NO.20886)
Experiments were performed with reference to the book "molecular cloning, laboratory Manual". Mycelia were collected, and then total DNA was extracted using an actinomycete DNA extraction kit. The 16SrDNA sequence amplification is carried out by adopting universal primers 27F (27F: 5'-A G A G T T T G A T C C T G G C T C A G-3')/1495R (1492R: 5'-TACGGCTACCTTGTTACGACTT-3'), an amplification system and a PCR reaction program are shown in Table 8, 0.8% agarose gel electrophoresis is adopted for PCR product detection, a SanPrep column type PCR purification product kit is adopted for PCR product purification and recovery, and the purified PCR product is directly sent to Nanjing Kingsry biotechnology and Co., Ltd for sequence determination.
TABLE 8 PCR amplification System and reaction procedure
The 16S rDNA sequence measured by the strain HDCC00025(CGMCC NO.20886) is compared with the homologous sequence BLAST of the related species and genus sequence in the GenBank database after being corrected so as to determine the classification status of the strain.
The 16S rDNA sequence (SEQ ID NO:3) determined by strain HDCC00025(CGMCC NO.20886) was subjected to BLAST comparison with related sequences in GenBank, and the results are shown in Table 9 (only the more homologous model strains are listed in the table).
TABLE 9 homology of HDCC00025(CGMCC NO.20886) and typical model strains
Sequencing a rDNA region of the strain HDCC 0002516S, carrying out BLAST comparison on homologous sequences of related species and genera in a GenBank database, finding that the strain is 99.72% or more of the same homology with Streptomyces scabrispora strain and Streptomyces sp, and carrying out an apparent characteristic test on the strain HDCC00025 to find that the strain is very close to related parameters of Streptomyces sp classification, so that the strain HDCC00025 is identified as a Streptomyces sp strain.
2. The streptomyces HDCC00025 of the invention was compared with other lepomycin B producing bacteria as follows:
hamamoto T et al reported Streptomyces sp. ats1287, which showed colony morphology and spore color in various media described in the literature, that are significantly different from Streptomyces HDCC00025 of the present invention.
By combining the morphological, culture, physiological and biochemical characteristics and 16S rDNA sequence identification results of the Streptomyces HDCC00025, the strain HDCC00025 belongs to Streptomyces sp strains and is different from other known lepomycin B producing bacteria, so that the Streptomyces HDCC00025 is a novel lepomycin B producing strain.
EXAMPLE 4 preparation of Leptomycin B fermentation broth
(1) Preparing and culturing slant spores:
slant culture medium formula (g/L): 4.0g/L of yeast extract powder, 10.0g/L of malt extract, 4.0g/L of glucose, 20.0g/L of agar, 7.2-7.4 of pH before digestion, 30 x 200mm of test tube, 15mL of loading capacity, sterilizing at 121 ℃ for 20min, cooling to 55-60 ℃ and swinging an inclined plane, inoculating a ring of spores or mycelia to the inclined plane after cooling and solidification, and culturing at 28 +/-1 ℃ for 7-10 days to ensure that the spores are mature.
(2) Preparing and culturing a seed solution:
seed culture medium formula (g/L): 5g/L of glucose, 5g/L of corn starch, 20g/L of soybean cake powder, 10g/L of yeast extract powder, 1g/L of calcium carbonate, 1g/L of magnesium sulfate and 1g/L of dipotassium phosphate. pH 7.0 before digestion; a250 mL triangular shake flask with the specification is filled with 50mL and sterilized at 121 ℃ for 20 min. Inoculation 107～108cfu/mL into seed medium, 28 + -1 deg.C, 250rpm shaking culture for 24 hours, at which time the culture solution pH is 6.8-7.2, mycelium concentration is 8% (volume percent).
(3) Preparing and culturing a fermentation medium:
fermentation medium formula (g/L):
10g/L of corn starch, 10g/L of maltodextrin, 5g/L of soybean oil, 15g/L of yeast extract powder, 5g/L of soybean lecithin, 5g/L of soybean cake powder, 5g/L of yeast extract, 1g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate and 0g/L of calcium carbonate. pH 5.0 before digestion. A250 mL triangular shake flask with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 5% (by volume). The cells were cultured at 28. + -. 1 ℃ for 120 hours with shaking at 250 rpm.
The content of the hapramycin B in the fermentation liquor is detected by an HPLC method and is 1174 ug/mL.
EXAMPLE 5 preparation of Leptomycin B fermentation broth
(1) The formulation of the slant medium and the culture conditions were the same as those in (1) of example 4
(2) The formulation of the seed culture medium and the culture conditions were the same as those in (2) of example 4
(3) Preparing and culturing a fermentation medium:
20g/L of corn starch, 20g/L of maltodextrin, 10g/L of soybean oil, 10g/L of yeast extract powder, 10g/L of soybean lecithin, 10g/L of soybean cake powder, 10g/L of yeast extract, 5g/L of magnesium sulfate, 5g/L of dipotassium hydrogen phosphate and 20g/L of calcium carbonate. pH 6.0 before digestion. A250 mL triangular shake flask with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 8% (by volume). The culture was carried out at 23. + -. 1 ℃ for 72 hours with shaking at 250 rpm.
The content of the hapramycin B in the fermentation liquor is detected by an HPLC method and is 986 ug/mL.
EXAMPLE 6 preparation of Leptomycin B fermentation broth
(1) The formulation of the slant culture medium and the culture conditions were the same as those in step (1) of example 4; the formulation and culture conditions of the primary seed medium were the same as those in step (2) of example 4.
(2) Preparing seed liquid in a seeding tank:
the formula of the seed liquid culture medium in the seeding tank is the same as the seed culture medium in the step (2) in the example 4;
putting 10L of seed culture medium into a 15L seed tank, sterilizing by steam at 121 ℃ for 20min, cooling to 28 ℃, and inoculating 200mL of first-stage shake flask seed liquid. The mixture was stirred at 200rpm, and the aeration rate was 1.0 vvm, and the mixture was incubated at 28. + -. 1 ℃ for 24 hours, at which time the pH of the seed solution was 7.4 and the hypha concentration was 15% (by volume).
(3) Preparing fermentation broth of a fermentation tank:
formula of fermentation medium
50g/L of corn starch, 50g/L of maltodextrin, 20g/L of soybean oil, 15g/L of yeast extract powder, 20g/L of soybean lecithin, 20g/L of soybean cake powder, 20g/L of yeast extract, 10g/L of magnesium sulfate, 8g/L of dipotassium hydrogen phosphate and 50g/L of calcium carbonate. pH 7.0 before digestion.
The volume of the fermentation tank is 50L, the feeding volume is 30L, the fermentation tank is sterilized by steam, the temperature is 121 ℃, the fermentation tank is 20min, and 3L seed solution in the seed tank is inoculated after the fermentation tank is cooled to 28 ℃. The stirring speed was 300-600rpm (the speed was gradually increased from 300 rpm to 600rpm in the first 3 days), the aeration rate was 2.0vvm, and the culture was carried out at 30 ℃ for 168 hours.
The content of the lepomycin B in the fermentation liquor is 1218ug/mL through HPLC detection.
Sequence listing
<110> Zhejiang a kind of jade Biotech Co., Ltd
<120> streptomycete and method for producing lepomycin B by fermenting streptomycete
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcactggcgc tgcttaccat gcagtcgaac gctgaagccc ttcggggtgg atgagtggcg 60
aacgggtgag taacacgtgg gcaacctgcc ctgcactctg ggataacttc gggaaaccgg 120
agctaatacc ggataatatc ttccaccgca tggtgggggg ttgaaagttc cggcggtgca 180
ggatgggccc gcggcctatc agcttgttgg tggggtagtg gcctaccaag gcgacgacgg 240
gtagccggcc tgagagggcg accggccaca ctgggactga gacacggccc agactcctac 300
gggaggcagc agtggggaat attgcgcaat gggcgaaagc ctgacgcagc gacgccgcgt 360
gagggatgac ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg aaagtgacgg 420
tacctgcaga agaagcaccg gctaactacg tgccagcagc cgcggtaata cgtagggtgc 480
gagcgttgtc cggaattatt gggcgtaaag agctcgtagg cggcctgtcg cgtcggatgt 540
gaaaactcgg ggcttaaccc cgagcctgca ttcgatacgg gcaggctaga gttcggcagg 600
ggagactgga attcctggtg tagcggtgaa atgcgcagat atcaggagga acaccggtgg 660
cgaaggcggg tctctgggcc gatactgacg ctgaggagcg aaagcgtggg gagcgaacag 720
gattagatac cctggtagtc cacgccgtaa acgttgggaa ctaggtgtgg gcgacattcc 780
acgtcgtccg tgccgcagct aacgcattaa gttccccgcc tggggagtac ggccgcaagg 840
ctaaaactca aaggaattga cgggggcccg cacaagaggc ggagcatgtg gcttaattcg 900
acgcaacgcg aagaacctta ccaaggcttg acatacaccg gaaacatctg gagacaggtg 960
ccccttttgg tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1020
gggttaagtc ccgcaacgag cgcaaccctc gttctgtgtt gccagcatgc ctttcggggt 1080
gatggggact cacaggagac tgccggggtc aactcggagg aaggtgggga cgacgtcaag 1140
tcatcatgcc ccttatgtct tgggctgcac acgtgctaca atggccggta caatgagctg 1200
cgataccgca aggtggagcg aatctcaaaa agccggtctc agttcggatc ggggtctgca 1260
actcgacccc gtgaagtcgg agtctctagt aatcgcagat cagcattgct gcggtgaata 1320
cgttcccggg ccttgtacac accgcccgtc acgtcacgaa agtcggtaac acccgaagcc 1380
ggtggcccaa ctcgcaagag ggggagccgt cgaagggtac agccc 1425
Claims (14)
1. Streptomyces sp HDCC00025 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.20886 and the preservation date of 2020 and 10 months and 14 days.
2. Use of Streptomyces sp HDCC00025 according to claim 1 for the preparation of lepomycin B or of a pharmaceutical composition containing lepomycin B.
3. A process for the preparation of lepomycin B by Streptomyces according to claim 1, wherein: the method comprises aerobic fermentation of Streptomyces sp HDCC00025 in a nutrient medium containing assimilable carbon and/or nitrogen sources to obtain lepomycin B.
4. The method of claim 3, wherein: the assimilable carbon source is selected from one of corn starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, mannitol or a combination thereof.
5. The method of claim 3, wherein: the assimilable nitrogen source is selected from one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea and ammonium salt or the combination of any more of the substances;
6. the method of claim 4, wherein; the assimilable carbon source is selected from one or the combination of any of corn starch, maltodextrin, glucose, sucrose and soybean oil.
7. The method of claim 5, wherein: the assimilable nitrogen source is selected from one or the combination of any several of soybean cake powder, soybean lecithin, yeast extract powder and yeast powder.
8. The method of claim 3, wherein: the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from one of trisodium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate or the combination of any of the above substances.
9. The method of claim 8, wherein: the inorganic salt is selected from calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate or potassium chloride or a combination of any of the substances.
10. The method of claim 3, wherein: the nutrient medium contains 10-50g/L of corn starch, 10-50g/L of maltodextrin, 5-20g/L of soybean oil, 1-15g/L of yeast extract powder, 5-20g/L of soybean lecithin, 5-20g/L of soybean cake powder, 5-20g/L of yeast extract, 1-10g/L of magnesium sulfate, 2-8g/L of dipotassium phosphate and 0-50g/L of calcium carbonate.
11. The method of claim 3, wherein: the temperature of the aerobic fermentation is 20-35 ℃, and the preferential temperature is 23-30 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24-240 hours, preferably 72-168 hours; the oxygen throughput is from 0.1 to 2.0vvm, preferably from 0.5 to 2.0 vvm.
12. The method of any one of claims 3-11, wherein: the Streptomyces (Streptomyces sp.) HDCC00025 is inoculated into the nutrient medium through seed liquid to carry out fermentation culture;
wherein the seed solution is obtained by seed culture of the Streptomyces sp HDCC00025 in a seed culture medium.
13. The method of claim 12, wherein: the seed culture medium contains 5-20g/L of glucose, 5-20g/L of corn starch, 5-20g/L of soybean cake powder, 1-10g/L of yeast extract powder, 1-20g/L of calcium carbonate, 1-10g/L of magnesium sulfate and 1-10g/L of dipotassium phosphate.
14. The method of claim 13, wherein: the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, and the optimal temperature is 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the culture time is 24 to 80 hours, preferably 24 to 60 hours.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08239379A (en) * | 1995-03-03 | 1996-09-17 | Kirin Brewery Co Ltd | Kr 2827 derivative as new substance, its production and use |
| CN1964718A (en) * | 2004-06-03 | 2007-05-16 | 高山生物科学股份有限公司 | Leptomycin compounds |
| CN101732308A (en) * | 2008-11-17 | 2010-06-16 | 中国人民解放军军事医学科学院毒物药物研究所 | New application of leptomycin B, pharmaceutical compositions and products thereof |
| WO2010127645A2 (en) * | 2009-05-04 | 2010-11-11 | Institute Of Microbiology As Cr, V. V. I. | The method of biotechnological preparation of lincomycin derivatives and its using |
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2020
- 2020-11-26 CN CN202011350157.9A patent/CN114540212B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08239379A (en) * | 1995-03-03 | 1996-09-17 | Kirin Brewery Co Ltd | Kr 2827 derivative as new substance, its production and use |
| CN1964718A (en) * | 2004-06-03 | 2007-05-16 | 高山生物科学股份有限公司 | Leptomycin compounds |
| CN101732308A (en) * | 2008-11-17 | 2010-06-16 | 中国人民解放军军事医学科学院毒物药物研究所 | New application of leptomycin B, pharmaceutical compositions and products thereof |
| WO2010127645A2 (en) * | 2009-05-04 | 2010-11-11 | Institute Of Microbiology As Cr, V. V. I. | The method of biotechnological preparation of lincomycin derivatives and its using |
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