CN114058516B - Penicillium fungus N29 capable of producing brefeldin A from ocean source and application thereof - Google Patents

Abstract

The invention discloses a marine medicinal mangrove plant acanthus trifoliatus source brefeldin A penicillium fungus N29, which is classified, identified and named as penicilliumPenicilliumsp.N29 with the preservation number of CGMCC No. 17193. The extract content in the strain fermentation liquor can reach 560 mg/L, wherein the relative percentage content of the brefeldin A is 90%, the yield is 504 mg/L, and a novel production path is provided for obtaining the brefeldin A medicine source. The SRB method proves that the fermentation broth extract of the penicillium fungus N29 has inhibition effect on liver cancer, leukemia, breast cancer, colon adenocarcinoma, prostate cancer, lung cancer, cervical cancer and pancreatic cancer cells, and the inhibition rate can reach 94.53 percent.

Description

Penicillium fungus N29 capable of producing brefeldin A from ocean source and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a marine medicinal mangrove plant acanthus trifoliatus source penicillium fungus N29, which has the characteristic of stably producing brefeldin A in high yield, and further relates to application of the penicillium fungus N29 in a medicament for treating tumors.

Background

Brefeldin A (English name: brefeldin A, abbreviated BFA) is a class of macrolide fungal metabolites. From Singleton et al in 1958Penicillium decumbens(Singleton, V.L).et al. Nature1958, 181, 1072-1073) by Weber et al by single crystal, CD, asymmetric synthesis determined its absolute configuration (Weber, h.p).et al. Helv. Chim. Acta,1971, 54, 2763-2766). The structural formula of BFA is as follows:

。

BFA has a wide range of biological activities including antiviral, antifungal, anti-nematode, antimitotic, and antitumor etc. (Tamura, G.et al. J. Antibiot. 1968, 21, 160−161; Takatsuki, A. et al. J. Antibiot. 1969, 22, 442−445；Betina, V. et al. Folia Microbiol. 1962, 7,353-357；Harri, E. et al. Helv. Chim. Acta,1963, 46, 1235-1243；Bacikova,´D, et al. Naturwissenschaften 1964, 51, 445；Betina, V. et al. Bull. Soc. Chim. Biol. 1966, 48, 194−198；Betina, V, et al. Naturwissenschaften,1962, 49, 241；Betina, V., et al. Neoplasma,1969, 16, 23-32). Compared with the currently used chemotherapeutic drugs, the BFA has novel structure, and the perfect annular conformation and functional group distribution and perfect wedging of the hydrophobic residue binding site of the Sec7 domain of the targeting protein are key to play the action mode (Cherfils, J).et alNature, 2003, 426, 525-530). A series of researches find that BFA can inhibit the transport of protein from an endoplasmic reticulum to a Golgi complex by inducing the decomposition of the Golgi apparatus, and the BFA has become an important molecular tool for biologists to study the signal transduction pathway of mammalian cells because the BFA influences the protein transport and processing process. In addition, the American tumor institute (NCI) tested BFA for in vitro antiproliferative activity of 60 tumor cell lines, GI ₅₀ The mean value (MGM) was 40 nM (Cushman, M.et al. J. Med. Chem.2006, 49, 3897-3905) shows a certain selectivity (GI) for melanoma cells and prostate cancer cells ₅₀ The value was about 20 nM) (Grever, m.r.et al. Cancer J. Sci. Am. 1996, 2, 52-58). Akira et al use of low doses of BFA in combination increased the effect of gemcitabine on the MIA PaCa-2 cell line of human pancreatic cancer cells (Togawa, A).et al. Pancreas, 2003, 27, 220-224.). Furthermore, huang et al found that BFA in combination with docetaxel had a greater effect on both prostate cancer PC-3 cell growth inhibition and apoptosis induction than drug alone (patent No. CN 105456255A). HealdIn the above, the ability of BFA to induce tumor cell differentiation and apoptosis (independent of p53 apoptosis pathway) has wide application prospect as a chemotherapeutic agent for tumor treatment.

With the widespread and deep research of BFA, especially the BFA has great market demands as candidate antitumor drugs, medicine and pesticide intermediates and important molecular tool reagents. Therefore, the demand for the amount of the compound is becoming more urgent, and particularly, tests with more consuming compounds, such as structural modification, in vivo drug generation in animals, drug effect test, toxicological test, and the like, are being conducted. The BFA society demand is large, the total amount of the global annual BFA consumption reaches the kilogram level according to the conservation estimation, and the BFA society demand is mostly used for scientific research experiments, and the recovery rate and the utilization rate are low. BFA is commercially expensive, reaching 1 mg/RMB 114 (Selleck China, selleck Chemicals).

In order to solve the problems of medicine source shortage and ecological damage caused by microorganism resource shortage and other factors, people are also trying to produce BFA by other methods, such as chemical synthesis method, plant extraction and the like, and the main characteristics are as follows:

(1) The chemical total synthesis BFA method has been successful, but the synthetic route is complex and the whole process is very expensive, and the yield is not high (mg level is not broken through), which is not an ideal approach (Furstner, a).et al. Angew. Chem. Int. Ed. Engl. 2015, 54, 3978−3982.) 。

(2) Chinese angelicaAngelica sinensisThe BFA is the only reported plant source, the chemical composition of the extract is complex, the BFA content is extremely low, the separation difficulty is extremely high, and the method is not an ideal path (Han, L).et al. Chinese Traditional and Herbal Drugs, 2011, 42, 1900−1904.) 。

Isolation of endophytic fungi that produce BFA or the like has long been a means of effectively solving the problem of BFA resources. Searching endophytic fungi capable of producing BFA, and producing BFA in large quantity by a microbial fermentation method is expected to improve the current situation that the BFA is expensive and is in short supply. Compared with chemical synthesis, the plant is a scarce source, and the like, and the fermentation scale can be controlled manually due to the short fermentation period (10-15 days) of fungi, so that the production capacity of the plant is certain to be superior to that of the plant. Therefore, screening and development of high-yield strains or optimization of fermentation conditions of target strains becomes a key for obtaining BFA with high yield. The guarantee of BFA raw materials becomes a key factor for the success of the medicine in market. Thus, searching for new resources of BFA and even new microbial resources is the focus of current research.

According to the prior literature report, we find that the genus Mycosporidium is [ ]Cladosporium) Genus PhomaPhoma) Paecilomyces genusPaecilomyces) Genus PhytophthoraPhyllosticta) Penicillium genusPenicillium) Penicillium plantEupenicillium) Aspergillus genusAspergillus) Strains of the genus Alternaria (Alternaria) and the like are all capable of fermentation to produce BFA. However, the yield of BFA produced by microbial fermentation reported at present is low, secondary metabolites of the microorganism are complex, byproducts are more, separation difficulty is high, and the increasing commercial production requirements are difficult to meet. U.S. patent No. 3896002, issued to Howard et al, disclosesPhyllosticta medicaginisThe yield of the fermented BFA is 300 mg/L. McCloud et al P.baenaE. brefeldianum The fermentation production of BFA by ATCC 58665 was studied with yield 169 mg/L. Zhao Yufen et al report PenicilliumPenicilliumThe BFA produced by the SHZK-15 after optimizing the fermentation condition is 151.6 mg/L. Chinese patent CN 101445784a by the inventors Zheng Yuguo et al discloses a variation of eupenicillium breve obtained by mutagenesis using low energy ion implantation techniquesEupenicillium brefeldianumZJB 08702 is capable of fermentation to produce BFA up to 943.8 mg/L. Further optimizing the fermentation conditions of the P.breuifimbriae variant ZJB 08702 in 2012, wang Yajun, etc., finally results in a yield of BFA of 1304.7 mg/L.

2013, oin et alPanax ginsengEndophytic plant fungiPenicillium janthinellumBFA is obtained by separating from potato glucose fermentation broth of Yuan-27, specifically, 1 g crude extract is extracted from 60L fermentation broth, the BFA yield is only 180 mg, namely 3 mg/L, the content of the total metabolite is 18%, and the yield is extremely low (Qin, L.P).et al. Appl. Microbiol. Biotechnol.2013, 97, 7617−7625.)。

2016-2018, ma et al pairPenicillium janthinellumThe metabolic status of the rice solid medium of DT-F29 was studied and 12 compounds, including 9 BFA natural product analogues and 2 pencilids, were isolated therefrom. The metabolism is complex and various, the analogues are more, and the separation difficulty is extremely high (Ma, Z, J).et al. Nat. Prod. Res.2016, 30, 2311−2315; Ma, Z. J. et al. Nat. Prod. Res. 2018, 32, 282−286.)。

In 2019, zhang et al have grown from a strain of soil-borne Penicillium fungusPenicillium janthinellumThe ethanol and ethyl acetate extracts of the rice solid culture medium are separated to obtain 12 Brefeldin compounds including BFA, and the metabolism is complex. Specifically, BFA 2.5 mg is separated from the 25 g crude extract, the yield is extremely low, and the separation difficulty is extremely high (Zhang Y.H).et al. Bioorg. Chem.2019, 86, 176−182)。

Disclosure of Invention

The invention aims to provide a new choice for producing BFA by microbial fermentation.

The invention aims to provide a mangrove acanthus that comes from south China sea medicinal mangrove acanthusAcanthus ilicifoliusEndophytic fungi penicillium of (a)Penicillium sp.N29 has the characteristic of stably and efficiently producing the brefeldin A, and the strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation date of 2019 and 03 month of 20 days and the preservation number of CGMCC No. 17193.

The application of the endophytic fungi in preparing the medicine for treating tumors proves that the penicillium N29 fermentation broth extract has obvious inhibition effect on liver cancer, leukemia, breast cancer, colon adenocarcinoma, prostate cancer, lung cancer, cervical cancer, pancreatic cancer and gastric cancer cells, and the inhibition rate can reach 94.53 percent.

The invention relates to an application of penicillium N29 in preparation of brefeldin A.

The method for preparing the brefeldin A is liquid fermentation.

The liquid fermentation of the invention is as follows: taking out from-80deg.C refrigerator and storingPenicillium sp.A freezing tube of N29, the strain is inoculated on a PDA flat plate, and the strain is activated for 3 to 5 days in a biochemical incubator at the temperature of 28 ℃; on an ultra-clean bench, inoculating the strain into a 500mL conical flask containing 250mL liquid fermentation medium by using an inoculating loop, and placing the inoculated conical flask in a shaking table at 120-200 rpm and 26-30 ℃ for culturing for 10-15 days.

The liquid fermentation medium comprises 200.0-g/L of potato, 20.0-40.0-g/L of carbon source and 5.0-15.0-g/L of sea salt; wherein the carbon source is at least one of glucose, sucrose, maltose or soluble starch.

The liquid fermentation culture medium comprises 200-g/L of potato, 20.0-g/L of glucose and 10-g/L of sea salt, wherein the pH value of the culture medium is 7.

The fermentation speed according to the invention is preferably 120rpm.

The fermentation temperature according to the invention is preferably 28 ℃.

The fermentation time according to the invention is preferably 15 days.

The method for separating and extracting the high-purity brefeldin A from the fermentation broth comprises the following steps: filtering the fermentation liquor by using four layers of gauze, performing solid-liquid separation to obtain a supernatant, performing solid-phase extraction by using HP20 macroporous adsorption resin, and eluting by using 50-70% ethanol to obtain a crude extract of the brefeldin A with the content of more than 90%; concentrating under reduced pressure, and further treating with CH ₃ OH-CH ₂ Cl ₂ Recrystallizing to obtain the high-purity brefeldin A.

The extract content in the strain fermentation liquor can reach 560 mg/L, wherein the relative percentage content of the brefeldin A is 90%, the yield is 504 mg/L, and a novel production path is provided for obtaining the brefeldin A medicine source.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) The invention provides a mangrove-derived endophytic fungus penicilliumPenicillium sp. N29 is capable of stably producing high levels of BFA by fermentation. Most importantly, the metabolite is single, the relative percentage of BFA is 90%, the yield is 504 mg/L, and the high yield is ensuredOn the premise of the rate, the separation difficulty of the single component of the BFA is greatly reduced. The method is far superior to the existing similar strains (Qin, L.P) in terms of yield and separation difficulty.et al. Appl. Microbiol. Biotechnol. 2013, 97, 7617−7625; Ma, Z. J. et al. Nat. Prod. Res.2016, 30, 2311−2315; Ma, Z. J. et al. Nat. Prod. Res. 2018, 32, 282−286; Zhang Y. H. et al. Bioorg. Chem.2019, 86, 176-182). Thus, there is a large difference between individuals of the same genus of microorganism.

(2) The method of combining macroporous resin solid phase extraction with recrystallization is adopted, so that the complex workload of liquid phase extraction is reduced, the separation means is simplified, the sample loss caused by the traditional technology such as positive and negative phases, gel column chromatography, HPLC and the like is avoided, the separation time is greatly shortened, the separation purity is improved, an ethanol-water elution system is adopted, the solvent is friendly, the recycling is realized, and the production cost is reduced.

(3) The SRB method is adopted to test the inhibition effect of the penicillium fungus N29 on various types of cancer cells, and the inhibition activity is obvious, and the inhibition rate can reach 94.53%.

(4) The method for producing the brefeldin A by fermenting the mangrove endophyte can be used for producing the brefeldin A in a short time and on a large scale without being limited by resources, environment, conditions, equipment and the like.

Drawings

Figure 1 shows the medicinal mangrove acanthus trifoliatusAcanthus ilicifoliusPictures of the samples.

FIG. 2 shows PenicilliumPenicillium sp.Photographs of N29 growth on PDA plates and shaker fermentation photographs.

FIG. 3 shows Penicillium plantPenicillium sp. The genetic relationship system of N29 evolves the tree.

FIG. 4 shows the chemical formula and the X-ray single crystal diffraction structure of Brefeldin A.

FIG. 5 shows Penicillium plantPenicillium sp.And (3) a high performance liquid chromatography analysis chart of the N29 fermentation liquor extract, wherein the labeling point is BFA.

Detailed Description

The invention will be further illustrated with reference to specific examples.

EXAMPLE 1 medicinal Acanthopanax trifoliatusAcanthus ilicifoliusIsolation and purification of coanda fungi

1. Acanthopanax trifoliatus (Bunge.) kuntzeAcanthus ilicifoliusPretreatment of samples

Acanthopanax trifoliatus is collected from medicinal mangrove in south China seaAcanthus ilicifoliusThe root, stem, leaf, mud and the former three are washed by sterile water, the absorbent paper is sucked dry, then the absorbent paper is soaked in 75 percent alcohol for 30 s, the absorbent paper is washed by sterile artificial seawater for three times, and the root, stem and She Jian are formed into 1 cm tissue for standby.

2. Acanthopanax trifoliatus (Bunge.) kuntzeAcanthus ilicifoliusIsolation of Coepiphyte

Tissue section method: under aseptic environment, taking pretreated root, stem and leaf materials, and placing the materials on a PDA culture medium plate; homogenizing: in a sterile environment, mud is diluted into three concentrations with sterile artificial seawater, respectively: 10 ^-1 、10 ^-2 、10 ^-3 (final four-concentration homogenates were obtained), 100. Mu.L of each was inoculated into PDA medium plates, and the plates were spread with a spreader. Culturing the flat plate in a constant temperature incubator at 28 ℃; PDA medium: potato 200g, glucose 20g, sea salt 30 g, water 1L, agar 20 g/L.

3. Acanthopanax trifoliatus (Bunge.) kuntzeAcanthus ilicifoliusPurification of coanda fungi

After about 2 days of culture, it was observed whether or not a single colony grew, and the grown single colony was transferred to a newly configured PDA medium plate, and the isolation and purification were repeated in this manner until a pure strain was obtained.

4. Identification of the Costump fungus Penicillium N29

The fungus was identified by ITS sequencing, and the use of the upstream primer ITS1 (5'-CTT GGT CAT TTA GAG GAA GTA A-3') and the downstream primer ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') was selected and used for most fungi.

The specific operation steps of molecular identification are as follows:

(1) Extraction of genomic DNA: picking hypha of single colony in 50 mu L of Lysis buffer (lysate) by using a gun head; thermal denaturation at 80℃for 15 min, centrifugation at 8000 rpm for 1 min, and taking 3. Mu.L of supernatant as PCR template.

(2) And (3) PCR amplification: add dd H sequentially into sterile 200 μl centrifuge tubes ₂ O34.5. Mu.L; 10 XPCR Buffer 5. Mu.L; dNTP 5. Mu.L; primer ITS 11. Mu.L; primer ITS4 1. Mu.L; ex Taq enzyme 0.5. Mu.L; 3 μl of the extracted fungal template; and then the PCR tube is tightly covered, centrifuged at a low speed to 3000 rpm for 1 min, and then put into a PCR instrument, and parameters of the PCR instrument are set: initial denaturation: 94 ℃ for 5 min; cycling 30 times: denaturation: denaturation at 94 ℃,30 s: 57 ℃,30 s, extension: 72 ℃,50 s; extension: 72 ℃ for 10 min. I.e., start the PCR amplification. Sealing the amplified centrifuge tube, and sending to Qingdao qing Ke catalpa, biological technology Co.

(3) BLAST comparison is carried out on ITS sequences fed back by sequencing of Qingdao Optimax Biotechnology limited company on NCBI to obtain similar strain with homology of more than 99% of the fungus, and the genus of the fungus is determined asPenicillium sp.。

5. Preservation of bacterial species

The glycerol and PDA culture medium (without agar) are uniformly mixed in a volume ratio of 1:4, placed in freezing pipes of 1 mL, mycelia of fungi growing vigorously are picked into each freezing pipe by an inoculating loop, and placed in an ultralow temperature refrigerator at-80 ℃ for preservation.

EXAMPLE 2 construction of the Penicillium fungus N29 phylogenetic tree

Downloading 10 strains of sequences closest to the fungus homology from NCBI, adjusting the sequences to FASTA format, comparing the sequences by using Bioeidt software, manually correcting the sequences while removing fragments with uneven ends, finally performing molecular biological analysis in MEGA5.05 by adopting an adjacent method (N-J method) and a maximum reduction method, establishing a phylogenetic tree (figure 3), and confirming the fungus to be penicilliumPenicillium sp.。

EXAMPLE 3 Penicillium fungus N29 fermentation

Taking out from-80deg.C refrigerator and storingPenicillium sp.N29 freezing tube, inoculating strain on PDA plate, activating strain in biochemical incubator at 28deg.C for 3-5 daysThe method comprises the steps of carrying out a first treatment on the surface of the On a super clean bench, the strain was inoculated with an inoculating loop into 500mL conical flasks containing 250mL of PDB medium, and the inoculated conical flasks were placed on a shaking table at 120rpm and 28℃for 15 days. PDB medium: potato 200g, glucose 20g, sea salt 10g, water 1L.

EXAMPLE 4 high Performance liquid chromatography of Penicillium fungus N29 fermentation broth extract

(1) Crude extract treatment: filtering 2 bottles of fermentation liquor with gauze, extracting the bacterial liquor with equal amount of ethyl acetate for 3 times, combining ethyl ester phases, concentrating under reduced pressure to obtain a crude extract, dissolving a proper amount of the crude extract with acetonitrile, absorbing supernatant fluid, and performing treatment on the crude extract by 0.22μAnd filtering with m-aperture filter membrane to remove impurities, and reserving the sample.

(2) The chromatographic conditions are as follows: chromatography column, ODS (C18) column 4.6X100 mm,5 nm; column temperature, normal temperature; mobile phase, methanol-water system; HPLC analysis conditions: gradient elution is carried out on 20-50% methanol in 0-10 min, 50-100% methanol in 10-60 min, and isocratic elution is carried out on 100% methanol in 60-65 min; flow rate, 2.0 mL/min; sample injection amount of 50 mu L; ultraviolet detection wavelength, 230 nm;

(3) The fingerprint analysis chart (figure 4) of the crude extract is measured, the labeling peak is consistent with Rt (retention time) of the BFA standard substance, and is 35.5-36.5 min, and the relative percentage content is about 90% calculated by peak area.

EXAMPLE 5 evaluation of Penicillium by SRB methodPenicillium sp.Cytotoxic Activity of crude N29 fermentation broth extract

Detection principle:

SRB is a water-soluble protein dye that binds basic amino acids of biological macromolecules, and the amount of protein bound in cells reflects the total protein and thus the number of cells. The OD value at 540 nm has a good linear relationship with the number of living cells.

Cell culture and test compound preparation

Human lung cancer cell A549, human breast cancer cell MCF-7, human colon cancer cell HCT116, human pancreas cancer cell BXPC-3, human prostate cancer cell PC-3, human liver cancer cell HepG2, human cervical cancer cell Hela, human peripheral blood leukemia T cell Jurkat, human gastric cancer cell MKN-45 are arrangedF-12K, RPMI-1640,McCoy's 5A,DMEM medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100U/mL penicillin and 100 g/mL streptomycin at 37deg.C, 5% CO ₂ Is cultured in a cell culture incubator. The liquid is changed once every two days, and after 80% of cells are fused, pancreatin digestion and passage are carried out, so that the cells are kept in a good logarithmic growth phase. All samples to be tested were dissolved in DMSO and sterilized by 0.22 μm filtration.

The detection method comprises the following steps:

a549, MCF-7, HCT, BXPC-3, PC-3, hepG2, hela, jurkat, MKN-45 cells in logarithmic growth phase were seeded in 96-well plates at 4000, 5000, 5000, 5000, 5000, 4000, 4000, 5000, 4000/well (180 μl/well), and after culturing 24 h, crude extract 50 of sample N29 to be tested was addedμg/mL, 4 duplicate wells per sample. The amount of DMSO used in the solvent control group was 0.1% of the maximum dose used in the test group. After drug action 72 h, cells were fixed by adding 50% (m/v) ice-cold trichloroacetic acid (TCA) to each well, after SRB staining, 150. Mu.L/well Tris solution was added and OD at 540 nm was measured on a microplate reader.

The inhibition rate of tumor cell growth was calculated according to the following formula:

inhibition = [ (OD) ₅₄₀ Control well-OD ₅₄₀ Drug administration well)/OD ₅₄₀ Control wells]×100％

TABLE 1 inhibition of Penicillium fungus N29 fermentation broth extract on different types of tumor cells

And (3) analyzing the initial screening cytotoxicity activity data, wherein the inhibition rate of the penicillium fungus N29 fermentation broth extract on A549, MCF-7, HCT116, BXPC-3, PC-3, hepG2 and Hela, jurkat, MKN-45 cells is between 77.23 and 94.53 percent, which is equivalent to the activity of positive control doxorubicin and has strong inhibition activity.

EXAMPLE 6 isolation and purification of BFA

Filtering the fermentation broth with four layers of gauze, separating solid and liquid to obtain supernatant, and fixing with HP20 macroporous adsorbent resinPhase extraction, eluting with 50-70% ethanol to obtain crude extract of the brefeldin A with the content of more than 90%; concentrating under reduced pressure, and further treating with CH ₃ OH-CH ₂ Cl ₂ Recrystallizing to obtain the high-purity brefeldin A. The compound is determined to be BFA by comparing literature data through nuclear magnetic hydrogen spectrum and carbon spectrum. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.34 (1H, dd, J=15.5, 3.0 Hz), 5.75 – 5.60 (2H, overlapped), 5.20 (1H, dd, J=15.2, 9.6 Hz), 5.10 (1H, s), 4.76 – 4.64 (1H, m), 4.48 (1H, s), 4.08 – 4.00 (1H, m), 3.92 (1H, d, J=9.2 Hz), 2.36 – 2.26 (1H, m), 2.02 – 1.87 (2H, overlapped), 1.87 – 1.60 (6H, overlapped), 1.54 – 1.41 (1H, m), 1.34 – 1.25 (1H, m), 1.18 (3H, d, J=6.3 Hz), 0.80 – 0.69 (1H, m); ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 165.7, 154.4, 137.1, 129.2, 116.3, 74.3, 70.9, 70.5, 51.7, 43.3, 43.1, 40.9, 33.4, 31.5, 26.5, 20.7; ESIMS m/z 281.17 [M + H] ⁺ .

Sequence listing

<110> university of ocean in China

<120> Penicillium fungus N29 producing brefeldin A from marine source and application thereof

<130> 20200721

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 583

<212> DNA

<213> Penicillium (Penicillium sp.)

<400> 2

cttccgtaag gtgaacctgc ggaaggatca ttaccgagtg agggccctct gggtccaacc 60

tcccacccgt gtttatctta cctagttgct tcggcgggcc cgccgtcagg ccgccggggg 120

gcacccgccc ccgggcccgc gcccgccgaa gccccccctg aacgctgtct gaagattgca 180

gtctgagcga ttagctaaat cagttaaaac tttcaacaac ggatctcttg gttccggcat 240

cgatgaagaa cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc 300

gagtctttga acgcacattg cgccccctgg tattccgggg ggcatgcctg tccgagcgtc 360

attgctgccc tcaagcacgg cttgtgtgtt gggcccccgc cccccggctc ccggggggcg 420

ggcccgaaag gcagcggcgg caccgcgtcc ggtcctcgag cgtatggggc ttcgtcaccc 480

gctctgtagg cccggccggc gcccgccggc gacccccctc aatctttctc aggttgacct 540

cggatcaggt agggataccc gctgaactta agcatatcaa taa 583

Claims (11)

1. The endophytic fungus of the marine medicinal mangrove acanthus trifoliatus source for producing brefeldin A is characterized in that the endophytic fungus is Penicillium sp.N29, and the preservation number is CGMCC No. 17193.

2. Use of the penicillium fungus N29 as claimed in claim 1 for the preparation of brefeldin a.

3. Use according to claim 2, characterized in that the process for preparing brefeldin a is liquid fermentation.

4. Use according to claim 3, wherein the liquid fermentation is: taking out the freezing tube stored with Penicilliumsp.N29 from the ultralow temperature refrigerator at-80 ℃, inoculating the strain on a PDA flat plate, and activating the strain in a biochemical incubator at 28 ℃ for 3-5 days; on an ultra-clean bench, the strain is inoculated into a 500mL conical flask containing 250mL liquid fermentation medium by an inoculating loop, and the inoculated conical flask is placed in a shaking table at 120-200 rpm and at 26-30 ℃ for culturing for 10-15 days.

5. The application of the liquid fermentation medium according to claim 4, wherein the liquid fermentation medium comprises 200g/L of potato, 20-40 g/L of carbon source and 5-15 g/L of sea salt; wherein the carbon source is at least one of glucose, sucrose, maltose or soluble starch.

6. The use according to claim 5, wherein the liquid fermentation medium comprises the following components: 200g/L of potato, 20g/L of glucose, 10g/L of sea salt and pH of a culture medium of 7.

7. Use according to any one of claims 4 to 6, characterized in that: the fermentation speed is 120rpm.

8. Use according to any one of claims 4 to 6, characterized in that: the fermentation temperature is 28 ℃.

9. Use according to any one of claims 4 to 6, characterized in that: the fermentation time is 15 days.

10. The use according to claim 3, wherein the method for separating and extracting high purity brefeldin a from fermentation broth comprises the following steps: filtering the fermentation liquor by four layers of gauze, carrying out solid-liquid separation to obtain supernatant, adopting HP20 macroporous adsorption resin for solid-phase extraction, and eluting by 50-70% ethanol to obtain crude extract of the brefeldin A with the content of more than 90%; concentrating under reduced pressure, and further treating with CH ₃ OH-CH ₂ Cl ₂ Recrystallizing to obtain the high-purity brefeldin A.

11. The use of the endophytic fungus of claim 1 in preparing a medicament for treating tumors, wherein the tumors are liver cancer, leukemia, breast cancer, colon adenocarcinoma, prostate cancer, lung cancer, cervical cancer, pancreatic cancer and gastric cancer.