Application of a strain of aspergillus and a cyclic depsipeptide compound thereof

Abstract

The application discloses an Aspergillus strain and application of a cyclic depsipeptide compound of the Aspergillus strain, and belongs to the technical field of microorganisms.The strain is Aspergillus sp BTBU20212048, and the preservation number is CGMCC NO.40328.The application further discloses application of the Aspergillus strain, and a cyclic depsipeptide compound is obtained through fermentation, extraction, separation and chromatography.The cyclic depsipeptide compound is stable in structure, has inhibiting effect on Candida albicans, and has a minimum inhibiting concentration of 200 μg / mL; and when the compound is used in combination with rapamycin, the minimum inhibiting concentrations of the two can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to an application of an Aspergillus strain and a cyclic depsipeptide compound thereof. BACKGROUND

[0002] Microorganisms are one of the most abundant biological resources on earth, and are the second largest group in the world of life, next to insects, and play an important role in the synthesis and decomposition of organic matter in geochemical cycles. Microorganisms widely exist in our daily life and play a huge role in food, medicine, agriculture, environmental protection and other fields.

[0003] In the field of medicine, microorganisms play an irreplaceable role and provide great help for human health. For example, paclitaxel produced by Taxus chinensis endophytic fungi has significant anticancer effect; more than 50% of lichen species contain lichenin and usnic acid and other specific antibacterial and anticancer active substances. Penicillin, as a metabolic product in the growth process of fungi, can inhibit a variety of harmful bacteria and is non-toxic to humans, so that human life has a lifesaving drug, and people have also begun to explore the use of genetic methods to continuously improve the culture medium and fermentation conditions, so that microorganisms can produce penicillin in large quantities according to the synthesis route required by people. Microorganisms have made important contributions to human health and drug development.

[0004] However, with the increase of drug resistance of various pathogenic bacteria, the effect of many antibiotics is weakening or even disappearing, and improving antibiotics and finding new antibiotics have become the key problems of scientists, and finding new antibiotics through microbial fermentation is a research meaningful way. SUMMARY

[0005] In order to solve the above problems, the present application provides an application of an Aspergillus strain and a cyclic depsipeptide compound thereof. The Aspergillus strain BTBU20212048 provided by the present application can metabolically produce a cyclic depsipeptide compound, which has stable structure and has inhibitory effect on Candida albicans, and can reduce the minimum inhibitory concentration of both when used in combination with rapamycin.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides an Aspergillus strain (Aspergillus sp) BTBU20212048 strain, and the preservation number of the strain is CGMCC NO.40328.

[0008] The present application further provides a cyclic depsipeptide compound, which has the structural formula shown in formula I:

[0009]

[0010] The cyclic depsipeptide compound is a metabolite of the BTBU20212048 strain.

[0011] The preparation method of the cyclic depsipeptide compound is characterized in that it comprises the following steps:

[0012] (1) inoculating the BTBU20212048 strain into a rice medium and culturing for 10-30 days to obtain a fermentation product;

[0013] (2) sequentially extracting and extracting the fermentation product to obtain a crude extract;

[0014] (3) performing normal phase silica gel column separation on the crude extract, sequentially performing gradient elution using a n-hexane-dichloromethane system and a dichloromethane-methanol system, and obtaining A-L fractions in the order of component outflow;

[0015] (4) performing dextran gel column separation on the G fraction, performing elution using a dichloromethane-methanol system, and obtaining G1-G7 fractions through thin layer chromatography analysis of the eluate;

[0016] (5) performing high performance liquid chromatography separation on the G1 fraction to obtain the cyclic depsipeptide compound.

[0017] Preferably, the extraction agent in step (2) is a mixture of ethyl acetate and methanol.

[0018] Preferably, the extraction agent in step (2) is a mixture of ethyl acetate and water.

[0019] Preferably, the strain is activated and expanded in culture before being inoculated into the rice medium in step (1).

[0020] Preferably, the culture medium for activation is a MEA solid medium containing 30.0 g of malt extract powder, 3.0 g of soybean peptone, and 15.0 g of agar per liter, with the rest being water, and the activation culture temperature is 28°C for 7 days.

[0021] Preferably, the culture medium for expansion is a PDB medium containing 5 g of potato extract powder and 20 g of glucose per liter, with the rest being water, and the expansion culture temperature is 28°C for 3 days at a speed of 180 rpm.

[0022] Preferably, the volume ratio of ethyl acetate to methanol in the mixture of ethyl acetate and methanol in step (2) is 4:1.

[0023] Preferably, the volume ratio of ethyl acetate to water in the mixture of ethyl acetate and water in step (2) is 1:1.

[0024] Preferably, the volume ratio of n-hexane and dichloromethane in the n-hexane-dichloromethane system in step (3) is 50:50, 30:70, 10:90, 3:97, 1:99, 0:100, respectively.

[0025] Preferably, the volume ratio of dichloromethane and methanol in the dichloromethane-methanol system in step (3) is 99:1, 98:2, 97:3, 95:5, 90:10, 80:20, respectively.

[0026] Preferably, the volume ratio of dichloromethane and methanol in the dichloromethane-methanol system in step (4) is (1-2):1.

[0027] Preferably, the chromatographic column for high-phase liquid chromatography separation in step (5) is C 18 The reverse phase chromatographic column, and the mobile phase is acetonitrile-water system, and the elution is gradient elution.

[0028] Preferably, the gradient elution program for high-performance liquid chromatography separation in step (5) is:

[0029] 0-15min: the volume percentage of acetonitrile in the mobile phase is increased from 50% to 75% at a constant rate;

[0030] 15-15.5min: the volume percentage of acetonitrile in the mobile phase is increased from 75% to 100% at a constant rate.

[0031] The application also provides the use of the above-mentioned cyclic thioester peptide compound or the cyclic thioester peptide compound prepared by the above-mentioned preparation method in the field of antibiosis.

[0032] Compared with the prior art, the application has the following beneficial effects:

[0033] The application provides an Aspergillus strain BTBU20212048 strain, and the preservation number of the strain is CGMCC NO.40328. The Aspergillus strain BTBU20212048 strain provided by the application can metabolically produce a cyclic thioester peptide compound, the cyclic thioester peptide compound is stable in structure, has an inhibitory effect on Candida albicans, and has a minimum inhibitory concentration of 200 mu g / mL. When the cyclic thioester peptide compound is combined with rapamycin, the minimum inhibitory concentration of the two on Candida albicans can be reduced, at this time, the concentration of rapamycin in the drug combination is 0.0625 mu g / mL, and the concentration of the compound is 12.5 mu g / mL.

[0034] Depository: Aspergillus sp. BTBU20212048 strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on September 28, 2022, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 40328. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A phylogenetic tree of Aspergillus sp. BTBU20212048 strain;

[0036] Figure 2 A high-performance liquid chromatogram of the cyclic thioester peptide compound prepared in Example 2;

[0037] Figure 3 A high-resolution mass spectrum of the cyclic thioester peptide compound prepared in Example 2;

[0038] Figure 4 A nuclear magnetic resonance hydrogen spectrum of the cyclic thioester peptide compound prepared in Example 2;

[0039] Figure 5 A nuclear magnetic resonance carbon spectrum of the cyclic thioester peptide compound prepared in Example 2. DETAILED DESCRIPTION

[0040] The present application provides an Aspergillus sp. BTBU20212048 strain, and the deposit number of the strain is CGMCC NO. 40328.

[0041] Unless otherwise specified, the present application does not have special requirements for the preparation raw materials, and commercially available goods known to those skilled in the art can be used. The technical solutions of the present application are further described below through the drawings and examples.

[0042] Example 1

[0043] I. Isolation of the strain

[0044] 1. Preparation of malt extract agar (MEA) medium:

[0045] The malt extract agar (MEA) medium includes malt extract 30.0 g, soybean peptone 5.0 g, agar 15.0 g, and pH 6.0, in 1 L;

[0046] The medium was prepared according to the above components, and then the prepared medium was sterilized, cooled to a temperature of about 40 DEG C, and chloramphenicol and streptomycin were added to a final concentration of 0.2 mg / mL (chloramphenicol and streptomycin were prepared in advance, filtered with a 0.22 mu m microporous filter, and stored at -20 DEG C), mixed, and poured into a plate to obtain the MEA medium.

[0047] 2. Isolation method: 10 g of Nanhui beach mud (Shanghai) was weighed, added with 100 ml of sterile water, soaked for 10 min, shaken to disperse, and a suspension was obtained. 200 mu L of the suspension was diluted 10 times and 100 times, respectively, and then coated on the isolation medium MEA, and inverted and cultured at 28 DEG C. After the second day of culture, the fungal colony growth on the plate was observed, and the fungal colony was picked out in time. Then, the fungal colony was observed once a day, and new fungal colonies were picked out in time.

[0048] II. Identification of the strain

[0049] After the strain was cultured on the MEA plate medium for 7 days, the color of the colony was black, the colony characteristics were observed, the colony was round and diffuse, the texture was loose, and the colony was villous. The connection between the colony and the medium was not tight, the mycelium was easy to pick, the middle mycelium was gray-black, and further identification was performed by combining the ITS DNA sequence.

[0050] In the present application, the nucleotide sequence of the ITS gene of the Aspergillus sp BTBU20212048 strain is shown in SEQ ID NO: 1:

[0051] CGATGTGACTGCGGAGGTCATTACCGAGTGCTGGGTCCTTCGGGGCCCAACCTCCCACCCGTGCTTACCGTACCCTGTTGCTTCGGCGGGCCCGCCTTCGGGCGGCCCGGGGCCTGCCCCCGGGACCGCGCCCGCCGGAGACCCCAATGGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGATACCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTCCCCTCCAGCCCCGCTGGTTGTTGGGCCGCGCCCCCCCGGGGGCGGGCCTCGAGAGAAACGGCGGCACCGTCCGGTCCTCGAGCGTATGGGGCTCTGTCACCCGCTCTATGGGCCCGGCCGGGGCTTGCCTCGACCCCCAATCTTCTCAGATTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCATAAAGGCGGGAGGAATTTTTTTTTTT.

[0052] About 1 cm2of the 7-day-old culture was cut with a inoculation needle 2 The bacterial cells were placed in a centrifuge tube, 800 μL of DNA extraction reagent was added, a steel ball was added (to facilitate cell disruption), the centrifuge tube was symmetrically placed in a SCIENTZ-48 high-throughput tissue grinder, the bacterial cells were disrupted, the genomic DNA of the strain to be tested was obtained, and PCR amplification was performed.

[0053] The ITS sequence of the ribosomal rDNA of the strain was amplified using the fungal universal primer ITS4 as shown in SEQ ID NO: 2: 5'-TCCTCCGCTTATTGATATGC-3' and ITS5 as shown in SEQ ID NO: 3: 5'-GGAAGTAAAAGTCGTAACAAGG-3'.

[0054] PCR amplification system (50 μL): 10×Buffer 5 μL, 2.5 mM dNTP 5 μL, 25 mM MgCl2 5 μL, template DNA 5 μL, primers ITS4 and ITS5 0.5 μL each, Taq DNA polymerase 0.5 μL, ddH2O 34.5 μL. Reaction program: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 1 min, 55℃ annealing for 4 min, 72℃ extension for 1.5 min, for a total of 37 cycles, followed by a 72℃ final extension for 10 min, and finally storage at 25℃.

[0055] The PCR amplification products were sequenced (Beijing Ruiboxingke Biotechnology Co., Ltd.), and the sequenced sequence is shown in the sequence listing SEQ ID NO: 1.

[0056] Phylogenetic analysis was performed on the ITS gene sequence obtained from the sequencing and the nucleotide sequence of the reference strain. The results of the phylogenetic analysis are shown below. Figure 1 ,from Figure 1 It can be seen that the ITS sequence of strain BTBU20212048 is most similar to the ITS gene sequence of the type strain Aspergillus assiutensis AUMC 5748, with a similarity of 98.76%.

[0057] Example 2

[0058] The Aspergillus sp. BTBU20212048 strain, frozen at -80℃, was removed from the cryovial. After thawing on ice, 50 μL of the bacterial suspension was evenly spread onto MEA solid medium. After incubation at 28℃ for 7 days, the morphology of the strain was photographed and recorded. A 1 cm sample was taken from the medium. 2 Large and small colonies were inoculated into PDB medium (50 mL culture base in a 250 mL Erlenmeyer flask) and cultured at 180 rpm and 28°C for 3 days. Then, 5 mL of seed culture was inoculated into sterilized rice medium (1 L Erlenmeyer flask containing 150 g rice and 120 mL water) and cultured statically at 28°C for about 30 days. The fermentation yield was 3.0 kg.

[0059] After fermentation, the culture medium was soaked in a mixture of ethyl acetate and methanol (volume ratio of 4:1) for 5 hours, then sonicated for 20 minutes. The culture medium and bacterial cells were removed by filtration. The extraction was performed three times, and the extracts were combined. The extracts were then concentrated to dryness and mixed with ethyl acetate and water (volume ratio of 1:1) for extraction. The extraction was performed three times, and the ethyl acetate phases obtained from the extraction were combined and concentrated to dryness to obtain the crude extract.

[0060] The crude extract was dissolved in dichloromethane, mixed with silica gel with a particle size of 100-200 mesh, and then air-dried.

[0061] Then, the volume ratio (V) is used sequentially. 正己烷 V 二氯甲烷 Hexane-dichloromethane systems with volume ratios of 50:50, 30:70, 10:90, 3:97, 1:99, and 0:100, respectively, and volume ratios (V...) 二氯甲烷 V 甲醇 The dichloromethane-methanol systems were eluted in ratios of 99:1, 98:2, 97:3, 95:5, 90:10, and 80:20, and the AL fraction was obtained according to the elution order of the components.

[0062] The G fraction was separated by Sephadex LH-20 gel column chromatography using a dichloromethane-methanol system with a volume ratio of 2:1. The resulting eluent was analyzed by thin-layer chromatography to obtain fractions G1-G9.

[0063] The obtained G1 fraction was separated by high-performance liquid chromatography (HPLC) to obtain the cyclic ester peptide compound. The HPLC column used for separation was an Agilent Eclipse XDB-C18 reversed-phase column, and the preferred gradient elution program was: 0-15 min: the volume percentage of acetonitrile in the mobile phase increased uniformly from 50% to 75%; 15-15.5 min: the volume percentage of acetonitrile in the mobile phase increased uniformly from 75% to 100%.

[0064] The purity of the prepared cyclic ester peptide compound was determined, and the results are shown in the figure. Figure 2 ,from Figure 2 It can be seen that the purity of the cyclic ester peptide compound prepared by this invention is greater than 98%.

[0065] The structures of the prepared cyclic ester peptide compounds were identified using mass spectrometry and nuclear magnetic resonance (NMR). NMR was performed using a Bruker AVANCE DRX-500 NMR spectrometer; mass spectrometry was performed using a Waters G2-XS mass spectrometer. The results are shown below. Figures 3-5 , Figure 3 The high-resolution mass spectrum of the cyclic ester peptide compound prepared in Example 2 is shown below. Figure 3 It can be seen that the quasi-molecular ion peak [M+Na] in the positive ion mode is... + The m / z is 749.3856, and the molecular formula is C. 37 H 54 N6O9. Figure 4 The above is the 1H NMR spectrum of the cyclic ester peptide compound prepared in Example 2. Figure 5 The image shows the carbon NMR spectrum of the cyclic ester peptide compound prepared in Example 2. Figures 3-5 The data shown is in Table 1.

[0066] Table 1 1 H and 13C NMR data (500 MHz)

[0067]

[0068]

[0069] The present application tests the synergistic antibacterial activity of the cyclic lactone peptide compound prepared in Example 2, which is divided into a compound group (dissolved by DMSO solution), a positive control (rapamycin) and a negative control (DMSO solution). The test is carried out by microdilution method, and rapamycin (dissolved by DMSO solution) is configured to 0.4 mg / mL, and the compound is configured to 4 mg / mL.

[0070] A single bacterium of Candida albicans with a diameter of about 1 mm is added to 1 mL of 1640 culture medium. After counting under a microscope, the bacterial concentration is adjusted to 1.0 x 10 4 cfu / mL.

[0071] This experiment uses a positive drug plate (rapamycin) and a compound plate.

[0072] First, 49 μL of bacterial solution is added to A1-A10 wells, and then 1 μL of 0.4 mg / mL rapamycin is added, and two-fold gradient dilution is carried out to H1. At this time, the concentration of A row is 8 μg / mL, and the concentration of H row is 0.0625 μg / mL. The specific rapamycin corresponding well dilution concentration is shown in Table 1.

[0073] Table 1 Positive drug plate (rapamycin corresponding well dilution concentration)

[0074]

[0075] In A2-H2 wells, 45 μL of bacterial solution is added, and then 5 μL of 4 mg / mL compound is added, and two-fold gradient dilution is carried out to A10; in A11 column wells, 45 μL of bacterial solution is added, and then 5 μL of 4 mg / mL compound is added, and two-fold gradient dilution is carried out to H11. At this time, the compound concentration in column A2 is 400 μg / mL, the compound concentration in column A10 is 1.5625 μg / mL, the compound concentration in column A11 is 400 μg / mL, the compound concentration in column B11 is 200 μg / mL, the compound concentration in column C11 is 100 μg / mL, the compound concentration in column D11 is 50 μg / mL, the compound concentration in column E11 is 25 μg / mL, the compound concentration in column F11 is 12.5 μg / mL, the compound concentration in column G11 is 6.25 μg / mL, and the compound concentration in column H11 is 3.125 μg / mL. The specific compound corresponding well dilution concentration is shown in Table 2.

[0076] Table 2 compound plate (compound corresponding hole dilution concentration)

[0077]

[0078] The above compound plate is combined with each hole in the drug plate, 95 μL of bacteria solution and 5 μL of DMSO solution are added in the A12 column as a negative control. The 96-well plate is placed in a 28°C incubator for 16-18 h, and the growth state of the fungus is observed.

[0079] Test results: the minimum inhibitory concentration of positive control rapamycin is 0.5 μg / mL, and the minimum inhibitory concentration of the compound is 200 μg / mL. The combination of the compound and rapamycin can reduce the minimum inhibitory concentration of Candida albicans, and the minimum inhibitory concentration of rapamycin in the combination is 0.0625 μg / mL, and the minimum inhibitory concentration of the compound is 12.5 μg / mL.

[0080] The above-described embodiments are only to describe the preferred modes of the present application, and not to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An Aspergillus sp. BTBU20212048 strain, with a preservation number of CGMCC NO. 40328.

2. Use of the Aspergillus strain according to claim 1 for the preparation of a cyclic depsipeptide compound, characterized in that, The cyclic ester peptide compound has a structural formula shown in formula I: Formula I.

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