CN112159777B

CN112159777B - Cyclopeptide antibiotic valinomycin high-yield strain and application thereof

Info

Publication number: CN112159777B
Application number: CN202011064961.0A
Authority: CN
Inventors: 陈小龙; 张栋; 陆跃乐; 朱林江; 吴楚天
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-11-09
Anticipated expiration: 2040-09-30
Also published as: CN112159777A

Abstract

The invention relates to a high-yield strain streptomycete IFE-v1m38 of cyclopeptide antibiotic Valinomycin, and application thereof in preparation of Valinomycin (Valinomycin) by microbial fermentation. The streptomycete IFE-v1m38(Streptomyces sp. IFE-v1m38) is preserved in the China general microbiological culture Collection center, and the address is as follows: west road No. 1 institute No. 3, north kyo, chaoyang district, preservation number CGMCC No.20046, preservation date: year 2020, month 06, and day 08. The invention provides a cyclopeptide antibiotic validamycin high-yield strain streptomycete IFE-v1m38, the quantity of the validamycin produced by the strain reaches 2279 mu g/m, which is far higher than that of a strain reported to produce the validamycin, and the strain is used for producing the validamycin to inhibit the half inhibitory concentration (MIC) of hypha growth of sclerotinia sclerotiorum by utilizing the strain₅₀) Is 0.056 mu g/mL, and provides a new way for the industrial production of the pesticide antifungal agent.

Description

Cyclopeptide antibiotic valinomycin high-yield strain and application thereof

(I) technical field

The invention relates to a high-yield strain of cyclopeptide antibiotic Valinomycin, namely Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m38), and application thereof in preparation of Valinomycin (Valinomycin) by microbial fermentation.

(II) background of the invention

The Sclerotinia sclerotiorum is a worldwide fungal natural disease caused by pathogenic fungi Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), has the characteristics of soil-borne diseases and air-borne diseases, is extremely wide in geographical distribution, and is epidemic in a plurality of countries in five continents. Sclerotinia sclerotiorum was first reported in 1915 by Shaw et al in India, and reports related to sclerotinia sclerotiorum disease in China were first published in 1932 by Zhufengmei. The sclerotinia rot of colza is the first disease of rape (sclerotinia rot of colza, downy mildew and virosis) in China, the morbidity is generally 10-30%, the disease can reach 80% when serious, the yield of diseased plants is generally reduced by 10-70%, and even the diseased plants are completely harvested when serious. The sclerotinia rot of rape is mainly concentrated in rape production areas such as the middle and lower reaches of Yangtze river in China, the coastal areas in south east China and the like, and the rape production in China is seriously influenced. Therefore, the research on sclerotinia rot of colza has been paid much attention. In the past 40 years, China mainly depends on chemical agents such as carbendazim, dimethachlon and the like to prevent and treat sclerotinia sclerotiorum, but the action mechanism of the bactericide is single, and the bactericide is continuously used for a long time, in a large area and in an excessive amount, so that the sclerotinia sclerotiorum is accelerated to generate resistance to the bactericide. At present, the reports of the drug resistance of sclerotinia sclerotiorum to carbendazim have appeared in Yangtze river basin of China, such as Anhui, Jiangsu, Zhejiang and other provinces. Therefore, a pesticide is required to be found to replace bactericides such as carbendazim and dimethachlon and be applied to the control of sclerotinia rot of rape.

Actinomycetes are gram-positive bacteria, which are facultative or obligate anaerobes. The bacterial colony of the actinomycetes is actinoid, has various varieties and wide distribution, and mainly exists in soil, air and water as hyphae or spores. Actinomycetes are a class of microorganisms of great practical value, and 22,000 known microbial secondary metabolites are currently found in microorganisms, 70% of which are derived from actinomycetes and most of which are antibiotics. The screening of biologically active substances from actinomycetes has been a focus of microbial antibiotic research.

Disclosure of the invention

The invention aims to provide a high-yield strain of cyclopeptide antibiotic validamycin, namely Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m38), and application thereof in preparation of validamycin (Valinomycin) by microbial fermentation.

The technical scheme adopted by the invention is as follows:

a high-yield strain of cyclopeptide antibiotic validamycin, namely Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m38), is deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the address is as follows: west road No. 1 institute No. 3, north kyo, chaoyang district, preservation number CGMCC No.20046, preservation date: year 2020, month 06, and day 08.

The strain is obtained by separating and screening soil, and the specific method comprises the following steps:

baking 1g of soil stored in a laboratory at 110 ℃ for 30min, adding the soil into a 50mL triangular flask containing 9mL of physiological saline and a proper amount of glass beads, shaking the soil in a shaking table at 28 ℃ and 200rpm for 30min, standing the soil at room temperature for 1h, taking supernatant, and diluting the supernatant to 10 times according to a gradient of 10 times^-2、 10^-3、10^-4、10^-5And 10^-6Coating the mixture on a coating containing 50-100 mu g/mL K₂CrO₄Culturing on the Gao's No. I solid culture medium at the constant temperature of 28 ℃ for 7 days; the inoculating needle picks up bacterial colony, streaks and separates until single bacterial colony actinomycete is obtained, inoculates the bacterial colony to a bacterial colony storage tube containing 12% glycerin, and stores in an ultra-low temperature refrigerator at-80 ℃. Screening actinomycetes which have an inhibition effect on the growth of sclerotinia sclerotiorum hyphae by using a plate confronting culture method by using sclerotinia sclerotiorum (S.sclerotiorum) which is a pathogenic fungus of sclerotinia sclerotiorum of rape as an indicator strain, then selecting 1 actinomycetes IFE-v1m38 with the best bacteriostatic activity, and carrying out physiological and biochemical identification and 16S rDNA identification on the actinomycetes.

The invention also relates to application of the streptomycete IFE-v1m38 in preparation of the sclerotinia sclerotiorum antibacterial agent through microbial fermentation. The strain fermentation culture has obvious inhibition effect on sclerotinia sclerotiorum hypha growth, and can be used for preparing sclerotinia sclerotiorum antibacterial agents.

Or more specifically, the streptomyces IFE-v1m38 is applied to the preparation of Valinomycin (Valinomycin) by microbial fermentation, and the structure of the Valinomycin is shown as the following formula:

the invention also relates to a method for preparing valinomycin by utilizing the streptomycete IFE-v1m38 microbial fermentation, which is characterized by comprising the following steps: inoculating Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m38) to a fermentation medium, regulating the pH value to be 7.0-7.4 by ammonia water under the conditions of 26-30 ℃, 150-500 rpm of rotation speed and 0-4.0 vvm of ventilation, carrying out fermentation culture for 48-192 h, obtaining fermentation liquor containing the valinomycin, and separating and purifying to obtain the valinomycin.

Specifically, the fermentation medium comprises the following components: 5-30.0 g/L of soybean cake powder, 5-20.0 g/L of soluble starch, 5-30 g/L of glucose, 1-10.0 g/L of tryptone, 1-10.0 g/L of yeast extract, 0.5-5.0 g/L of beef extract, 0.1-5.0 g/L of NaCl, and MgSO (MgSO) 2₄·7H₂O 0.1～1.5g/L， K₂HPO₄·3H₂O 0.1～1.5g/L，CaCO₃ 0.5～5.0g/L，FeSO₄·7H₂0.001-0.02 g/L of O, 0.1-3.0 per mill of defoaming agent, and sterilizing for 10-20 min at 121 ℃.

Preferably, the streptomycete IFE-v1m38(Streptomyces sp. IFE-v1m38) is inoculated to a seed culture medium for culture to obtain a seed solution, and the seed solution is inoculated to a fermentation culture medium for fermentation culture, wherein the seed culture medium comprises the following components: 5-20.0 g/L yeast extract, 1-15.0 g/L beef extract, 0.1-2.0 g/L NaCl and MgSO₄·7H₂O 0.1～2.0g/L，K₂HPO₄·3H₂O 0.1～2.0 g/L，FeSO₄·7H₂O 0.01～0.05g/L。

Inoculating Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m38) into a 500mL triangular flask containing 50-100 mL of liquid seed culture medium, and culturing at 28-30 ℃ and 200rpm for 24-72 h to obtain a fermented seed liquid; inoculating the strain liquid into a 5-L fermentation tank with the inoculation amount of 5-10%, regulating the pH value to 7.0-7.4 by ammonia water under the conditions of the temperature of 26-30 ℃, the rotating speed of 150-500 rpm and the ventilation volume of 0-4.0 vvm, and carrying out fermentation culture for 48-192 h to obtain the fermentation liquid containing the valinomycin.

Specifically, the separation and purification method comprises the following steps: adding 1-3 per mill of flocculant polyaluminium chloride into fermentation liquor containing valinomycin, carrying out suction filtration and solid-liquid separation by using a vacuum pump to obtain mycelium, repeatedly soaking and extracting the mycelium for 3-5 times by using pure methanol, concentrating an extract, repeatedly extracting the concentrated extract for 2-4 times by using ethyl acetate, washing the mycelium for 1-3 times by using saturated NaCl solution, standing and layering the mycelium by using a separating funnel, concentrating an organic phase by using a vacuum rotary evaporator, and dewatering the sample by using a vacuum drier; performing 200-300-mesh silica gel column chromatography, wherein the ratio of the sample loading amount to the silica gel dosage is 1.0: 30-60, 0-100% MeOH-CHCl₃Gradient elution, selecting active component for further separation and purification by preparative TLC, and developing agent is MeOH: CHCl₃20: 80; then, preparing active substances separated, purified and collected by a liquid phase by adopting Shimadzu LC-20AP, and carrying out column chromatography by using methanol: 1 per mill trifluoroacetic acid aqueous solution is 90:10 and is used as a mobile phase, the column temperature is 25 ℃, the detection wavelength is 210nm, and the flow rate is 2.0 mL/min; and concentrating the collected active components in a vacuum rotary evaporator and freeze-drying to obtain a white solid, namely the valinomycin. IR, UV, LC/MS, HRESIMS, TOF MS, of conjugated compounds,¹H-and ¹³C-NMR, HMQC, HMBC, DETP, COSY, NOESY and other data identify the structure of the compound, and the result shows that the compound is Valinomycin (Valinomycin).

The valinomycin can obviously inhibit the growth of sclerotinia sclerotiorum hyphae, and has good bacteriostatic effect on fungi such as phytophthora capsici, pyricularia oryzae and rhizoctonia solani by consulting documents, so that the valinomycin can be used as a potential pesticide antifungal agent.

The invention has the following beneficial effects: the invention provides a cyclopeptide antibiotic validamycin high-yield strain streptomycete IFE-v1m38, the quantity of the validamycin produced by the strain reaches 2279 mu g/m, which is far higher than that of a strain reported to produce the validamycin, and the strain is used for producing the validamycin to inhibit the half inhibitory concentration (MIC) of hypha growth of sclerotinia sclerotiorum by utilizing the strain₅₀) Is 0.056 mu g/mL, and provides a new way for the industrial production of the pesticide antifungal agent.

(IV) description of the drawings

FIG. 1 is a colony map of Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m 38);

FIG. 2 is a chart of methylene blue staining (10X 100) of Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m 38);

FIG. 3 is a 1H NMR spectrum of valinomycin;

FIG. 4 is a 13C NMR spectrum of valinomycin;

FIG. 5 is a 1H-1H COSY spectrum of valinomycin;

FIG. 6 is an HSQC spectrum of valinomycin;

FIG. 7 is an HMBC spectrum of valinomycin;

FIG. 8 is a diagram of the profile of valinomycin NOSEY;

FIG. 9 is a DEPT spectrum of valinomycin;

FIG. 10 is a graphic representation of the HRESIMS spectrum ([ M-Na ] +);

FIG. 11 is a TOF-MS/MS spectrum of valinomycin;

FIG. 12 is an IR spectrum of valinomycin;

FIG. 13 is a UV absorption spectrum of valinomycin.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: method for separating Streptomyces sp.IFE-v1m38 with resistance to sclerotinia sclerotiorum from soil

Baking and drying 1g of soil stored in a laboratory at 110 ℃ for 30min, adding the soil into a 250mL triangular flask containing 9mL of physiological saline and a proper amount of glass beads, shaking the soil in a shaking table at 28 ℃ and 200rpm for 30min, standing the soil at room temperature for 1h, taking supernatant, and diluting the supernatant to 10 times according to a gradient of 10 times^-2、10^-3、10^-4、10^-5And 10^-6Coating the mixture on a coating containing 50-100 mu g/mL K₂CrO₄Culturing on the Gao's No. I solid culture medium at the constant temperature of 28 ℃ for 7 days; the inoculating needle picks up bacterial colony, streaks and separates until single bacterial colony actinomycete is obtained, inoculates the bacterial colony to a bacterial colony storage tube containing 12% glycerin, and stores in an ultra-low temperature refrigerator at-80 ℃. Using sclerotinia sclerotiorum (S.sclerotiorum) as indicator strain, and culturing by plate confronting culture method in a culture chamber containing PThe two sides of a 9cm culture dish of a DA culture medium are firstly transferred with actinomycetes, after the culture is carried out for 96h at 28 ℃, a fungus cake with the diameter of 0.6cm is inoculated at the central position of a PDA culture medium, the phenomenon is observed after the culture is carried out for 48h at 23 ℃, actinomycetes which have the inhibition effect on the sclerotinia sclerotiorum hypha growth are screened, and then 1 actinomycetes which have the most obvious inhibition effect on the sclerotinia sclerotiorum hypha (a colony diagram is shown in figure 1, and a methylene blue staining diagram is shown in figure 2) are selected and named as IFE-v1m 38.

Example 2: strain classification identification-16S rDNA sequence determination

2.1 Actinomycete DNA extraction

The actinomycetes are subjected to streak culture on a 90mm glass culture dish containing a Gao's first solid culture medium, the actinomycetes grow for 7d in a constant temperature box at the temperature of 28 ℃, spores are picked by using an inoculating loop and transferred to CaCO containing 2g/L₃In 1mL of physiological saline, inducing seeds to germinate for 12-16h by a metal bath at 28 ℃, centrifuging at 12000rpm for 15min to obtain actinomycete spores, and extracting DNA by adopting an OMEGA bacterial DNA genome kit (model D3350-01) to be used as a PCR amplification template.

2.2PCR amplification

2.2.1 amplification primers

With reference to the literature reported by Weissburg et al, PCR amplification using universal primers 27F and 1541R (alternatively 1492R 5' TACGGYTACCTTGTTACGACTT)

Upstream primer (27F): 5 'AGAGAGTTTGATCCTGGCTCAG 3'

Downstream primer (1541R): 5 'AAGGAGGTGATCCAGCCGCA 3'

2.2.2 reaction System

PCR amplification reaction system

2.2.3 reaction sequence

2.2.4 identification of PCR products by gel electrophoresis

After the amplification is finished, 0.8% agarose H gel is prepared, a BIO-RAD gel electrophoresis apparatus is adopted, the operation is carried out for 30min under the condition of 110v voltage, and the molecular weight of the PCR product is detected by taking a DNA molecular weight standard Marker (100-5000 bp) as a reference.

2.3 sequence determination

And after determining a target product according to a gel electrophoresis result, transferring the amplified PCR product to Shanghai bioengineering company Limited for sequencing. After sequencing is completed, the obtained results are spliced by using DNAman 8, homologous sequences are compared by using a Blast tool of NCBI, and strains with high homology are selected for sequence comparison.

2.4 sequence results and analysis

Table 1: results of sequence alignment

Based on the results of the sequence comparison, it was named Streptomyces IFE-v1m38(Streptomyces sp. IFE-v1m 38).

Example 3: preparation of fermentation broth of Streptomyces sp.IFE-v1m38

Streptomyces sp.IFE-v1m38 was inoculated into a medium containing 50mL of liquid seed medium (yeast extract 10.0g/L, beef extract 5.0g/L, NaCl 2.0g/L, MgSO 2)₄·7H₂O 0.5g/L，K₂HPO₄·3H₂O0.5 g/L) was cultured at 28 ℃ for 24 hours at 200rpm in a 500mL Erlenmeyer flask to obtain a strain liquid. Inoculating the strain liquid into 3L fermentation medium (soybean cake powder 20.0g/L, soluble starch 10.0g/L (boiling water bath gelatinization), glucose 20g/L, tryptone 4.0g/L, yeast extract 5.0g/L, beef extract 3.0 g/L) with inoculation amount of 6%,NaCl 2.0g/L， MgSO₄·7H₂O 0.5g/L，K₂HPO₄·3H₂O 0.5g/L，CaCO₃ 3.0g/L，FeSO₄·7H₂o0.01 g/L, 1.5% antifoam) was added to the mixture, and the mixture was fermented and cultured for 168 hours at a temperature of 28 ℃, a rotation speed of a stirring paddle of 350rpm, an aeration rate of 3.0vvm, and a pH of 7.0 with ammonia water to obtain a fermentation broth.

Example 4: isolation, purification and characterization of Valinomycin (Valinomycin)

3 per mill of flocculant polyaluminum chloride is added into the streptomycete fermentation liquor obtained in the embodiment 3, solid-liquid separation is carried out by vacuum pump filtration, and mycelium is repeatedly soaked and extracted for 4 times by 2L of pure methanol. Concentrating the extract, repeatedly extracting with ethyl acetate for 3 times, washing with saturated NaCl solution for 2 times, standing in separating funnel for layering, concentrating organic phase with vacuum rotary evaporator, and removing water from the sample with vacuum drier. Performing 200-300-mesh silica gel column chromatography, wherein the ratio of the sample loading amount to the silica gel dosage is 1.0: 30-60, 0-100% MeOH-CHCl₃Gradient elution and further separation and purification of active fractions by preparative TLC in MeOH: CHCl₃20: 80; then, Shimadzu LC-20AP is adopted to prepare active substances which are separated, purified and collected by a liquid phase, and a column chromatography Agilent ZORBAX Eclipse XDB-C18 semi-preparative column (9.4 multiplied by 250mm,5Micron), methanol: water (1 ‰ trifluoroacetic acid, w/w) ═ 90:10 as mobile phase, column temperature 25 ℃, detection wavelength 210nm, flow rate 2.0 mL/min; the collected active fractions were concentrated and lyophilized on a vacuum rotary evaporator to give a white solid. In the separation and purification process, the pathogenic fungi sclerotinia sclerotiorum (S.sclerotiorum) of sclerotinia sclerotiorum of rape is taken as an indicator strain, and the active component is monitored by a bioassay method. IR, UV, LC/MS, HRESIMS, TOF MS, of conjugated compounds,¹H-and ¹³C-NMR, HMQC, HMBC, DETP, COSY, NOESY and the like (see figures 3-13), and the compound is cyclopeptide antibiotic Valinomycin (Valinomycin).

Table 2: valinomycin¹H and¹³C-NMR data (600MHz, DMSO-d6)

Example 5: HPLC (high performance liquid chromatography) for detecting the content of validamycin produced by Streptomyces sp.IFE-v1m38

20mg of valinomycin (commercially available) was dissolved in a 10mL volumetric flask and prepared as a standard sample with a gradient of 2, 1, 0.5, 0.25 mg/mL. 1mL of the fermentation liquid sample obtained in the embodiment 3 is shaken up, 9mL of methanol is added to be leached at 37 ℃ overnight, the mixture is centrifuged at 10000r/min for 10min, a 0.22 mu m micropore organic filter membrane is used for filtering supernatant, and the filtrate is stored at 4 ℃ for detection. The valinomycin is determined by an HPLC method, and the chromatographic conditions are as follows: c18 column (250 mm. times.4.6 mm), mobile phase V (methanol): v (water, 1 ‰ TFA) ═ 90:10, flow rate: 1mL/min, and the detection wavelength λ is 210 nm.

The content of the validamycin in the fermentation liquor is 2279 mug/mL through detection.

Example 6: inhibition of Validamycin on Sclerotinia sclerotiorum

A 16.0mg validamycin sample (white solid obtained in example 4) is added into a 10.0mL volumetric flask, dissolved in DMSO and subjected to constant volume to obtain a sample with the concentration of 1.6 mg/mL; then diluted with a DMSO gradient to 0.8mg/mL, 0.4mg/mL, 0.2mg/mL, 0.1mg/mL, 50.0. mu.g/mL, 25.0. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL. 0.1mL of the 9 solutions with different concentrations, 6.25. mu.g/mL-1.6 mg/mL, and 9.9mL of PDA were mixed well on a 90mm glass petri dish to prepare a plate. Under aseptic conditions, the 600mm sclerotinia sclerotiorum small fungus cakes are inoculated on PDA containing validamycin with different concentrations, 0.1mL DMSO and 9.9mL PDA are adopted in a blank control group to be uniformly mixed (v/v, 1% DMSO), and 3 parallel control groups are set in each group of experiments. Standing l h flat plate, placing in a constant temperature incubator at 23 ℃ for inverted culture for 48h, observing the transparency degree and edge uniformity degree of the antibacterial zone, measuring the diameter of the antibacterial zone by a cross method, recording the result and calculating the inhibition rate according to the following formula:

the diameter of the colony is the arithmetic mean of two perpendicular diameters-the diameter of the cake (600mm)

By using

The staticiscs 25.0 software performs regression analysis on the data to obtain the MIC of the valinomycin for inhibiting the hypha growth of sclerotinia sclerotiorum₅₀0.056 ± 0.004 μ g/mL. Therefore, the valinomycin can be used as a potential pesticide antibacterial agent to be applied to the prevention and treatment of sclerotinia rot of colza.

Sequence listing

<110> Zhejiang industrial university

<120> cyclopeptide antibiotic valinomycin high-producing strain and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

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<211> 1413

<212> DNA

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<400> 1

tcgccagtcc caccttcgac agctccctcc cacaaggggt tgggccaccg gcttcgggtg 60

ttaccgactt tcgtgacgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgca 120

gcaatgctga tctgcgatta ctagcaactc cgacttcatg gggtcgagtt gcagacccca 180

atccgaactg agaccggctt tttgagattc gctccgcctc gcggcatcgc agctcattgt 240

accggccatt gtagcacgtg tgcagcccaa gacataaggg gcatgatgac ttgacgtcgt 300

ccccaccttc ctccgagttg accccggcag tctcctgtga gtccccatca ccccgaaggg 360

catgctggca acacagaaca agggttgcgc tcgttgcggg acttaaccca acatctcacg 420

acacgagctg acgacagcca tgcaccacct gtataccgac cacaaggggg gcaccatctc 480

tgatgctttc cggtatatgt caagccttgg taaggttctt cgcgttgcgt cgaattaagc 540

cacatgctcc gctgcttgtg cgggcccccg tcaattcctt tgagttttag ccttgcggcc 600

gtactcccca ggcggggaac ttaatgcgtt agctgcggca ccgacgacgt ggaatgtcgc 660

caacacctag ttcccaacgt ttacggcgtg gactaccagg gtatctaatc ctgttcgctc 720

cccacgcttt cgctcctcag cgtcagtaat ggcccagaga tccgccttcg ccaccggtgt 780

tcctcctgat atctgcgcat ttcaccgcta caccaggaat tccgatctcc cctaccacac 840

tctagctagc ccgtatcgaa tgcagacccg gggttaagcc ccgggctttc acatccgacg 900

tgacaagccg cctacgagct ctttacgccc aataattccg gacaacgctt gcgccctacg 960

tattaccgcg gctgctggca cgtagttagc ccggcgcttc ttctgcaggt accgtcactt 1020

tcgcttcttc cctgctgaaa gaggtttaca acccgaaggc cgtcatccct cacgcggcgt 1080

cgctgcatca ggctttcgcc cattgtgcaa tattccccac tgctgcctcc cgtaggagtc 1140

tgggccgtgt ctcagtccca gtgtggccgg tcgccctctc aggccggcta cccgtcgtcg 1200

ccttggtagg ccattacccc accaacaagc tgataggccg cgggctcatc cttcaccgcc 1260

ggagctttta accccgtccc atgcgggaca gagtgttatc cggtattaga ccccgtttcc 1320

agggcttgtc ccagagtgaa gggcagattg cccacgtgtt actcacccgt tcgccactaa 1380

tccaccccga aaggcttcat cgttcgactg cat 1413

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<213> Unknown (Unknown)

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agagtttgat cctggctcag 20

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<213> Unknown (Unknown)

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aaggaggtga tccagccgca 20

Claims

1. Streptomyces IFE-v1m38 (a strain with high yield of cyclopeptide antibiotic valinomycinStreptomyces sp, IFE-v1m38), deposited in the general microbiological center of the chinese committee for culture collection, address: the number of the collection is CGMCC number 20046, and the collection date is as follows: year 2020, month 06, and day 08.

2. The use of the streptomyces IFE-v1m38 of claim 1 in the preparation of a sclerotinia sclerotiorum antimicrobial agent by microbial fermentation.

3. Use of the Streptomyces IFE-v1m38 according to claim 1 for the preparation of Valinomycin (Valinomycin) by microbial fermentation, said Valinomycin having the structure represented by the following formula:

。

4. a method for preparing valinomycin by utilizing the streptomyces IFE-v1m38 microorganism fermentation as described in claim 1, which is characterized in that the method comprises the following steps: streptomyces IFE-v1m38(Streptomyces sp, IFE-v1m38) is inoculated to a fermentation medium, the pH value is regulated to 7.0-7.4 by ammonia water under the conditions of the temperature of 26-30 ℃, the rotating speed of 150-500 rpm and the ventilation volume of 0-4.0 vvm, the fermentation culture is carried out for 48-192 h, and the validamycin-containing strain is obtainedThe valinomycin is obtained by separating and purifying the fermentation liquor of the valinomycin.

5. The method according to claim 4, characterized in that the fermentation medium consists of: 5-30.0 g/L of soybean cake powder, 5-20.0 g/L of soluble starch, 5-30 g/L of glucose, 1-10.0 g/L of tryptone, 1-10.0 g/L of yeast extract, 0.5-5.00 g/L of beef extract, 0.1-5.0 g/L of NaCl, and MgSO (MgSO) 2₄·7H₂O 0.1~1.5 g/L，K₂HPO₄·3H₂O 0.1~1.5 g/L，CaCO₃ 0.5~5.0 g/L，FeSO₄·7H₂0.001-0.02 g/L of O, 0.1-3.0 per mill of defoaming agent, and sterilizing for 10-20 min at 121 ℃.

6. The method according to claim 4, wherein said Streptomyces strain IFE-v1m38 (I)Streptomyces sp, IFE-v1m38) is firstly inoculated to a seed culture medium for culture to obtain a seed solution, and then the seed solution is inoculated to a fermentation culture medium for fermentation culture, wherein the seed culture medium comprises the following components: 5-20.0 g/L yeast extract, 1-15.0 g/L beef extract, 0.1-2.0 g/L NaCl and MgSO₄·7H₂O 0.1~2.0 g/L，K₂HPO₄·3H₂O 0.1~2.0 g/L， FeSO₄·7H₂O 0.001~0.02 g/L

7. The method according to claim 4, wherein the separation and purification method comprises the following steps: adding 1-3 per mill of flocculant polyaluminium chloride into fermentation liquor containing valinomycin, carrying out suction filtration and solid-liquid separation by using a vacuum pump to obtain mycelium, repeatedly soaking and extracting the mycelium for 3-5 times by using pure methanol, concentrating an extract, repeatedly extracting the concentrated extract for 2-4 times by using ethyl acetate, washing the mycelium for 1-3 times by using saturated NaCl solution, standing and layering the mycelium by using a separating funnel, concentrating an organic phase by using a vacuum rotary evaporator, and dewatering the sample by using a vacuum drier; performing 200-300-mesh silica gel column chromatography, wherein the ratio of the sample loading amount to the silica gel dosage is 1.0: 30-60, 0-100% MeOH-CHCl₃Gradient elution, selecting active component for further separation and purification by preparative TLC, and developing agent is MeOH: CHCl₃=20: 80; then preparing active substances separated, purified and collected by liquid phase by using Shimadzu LC-20APColumn chromatography, eluting with methanol: 1 per mill trifluoroacetic acid aqueous solution = 90:10 is a mobile phase, the column temperature is 25 ℃, the detection wavelength is 210nm, and the flow rate is 2.0 mL/min; and concentrating the collected active components in a vacuum rotary evaporator and freeze-drying to obtain a white solid, namely the valinomycin.