lobophorins N1-N3, their preparation methods, and applications

Abstract

This invention discloses the antibiotics lobophorin E and lobophorins N1-N3, their preparation methods, and applications. The invention constructed an engineered strain, *Streptomyces sp. SCSIO 01127 / ΔlobP1*, from which the known compound lobophorin E (1) and the novel compounds lobophorins N1-N3 (2-4) can be obtained in large quantities from the fermentation culture of this strain. Lobophorin E possesses antitumor activity, providing a candidate compound for the development of new antitumor drugs; lobophorins N1-N3 possess antibacterial activity and can be used to prepare antibacterial drugs.

Description

Technical fields:

[0001] This invention belongs to the field of industrial microbiology, specifically relating to the novel antibiotics lobophorins N1-N3 (2-4) and the known antibiotic lobophorin E (1), as well as their preparation methods and applications. Background technology:

[0002] Lobophorins are spirocyclic acetoacetate lactone antibiotics with various biological activities, including antibacterial, antitumor, and anti-inflammatory effects. Lobophorin E is a known member of the lobophorins family and possesses antibacterial activity. Summary of the Invention:

[0003] The first object of the present invention is to provide three new spirocyclic antibiotics lobophorins N1-N3 (2-4) and a method for obtaining large quantities of the known compound lobophorin E (1).

[0004] Lobophorins N1-N3 are novel lobophorins family compounds. In this study, we knocked out the P450 hydroxylase gene lobP1 in the lobophorins biosynthesis gene cluster to obtain sufficient amounts of the known compound lobophorin E and three new lobophorins family compounds lobophorins N1-N3. We then investigated the antibacterial and antitumor activities of these compounds.

[0005] The present invention comprises three novel spirocyclic antibiotics lobophorins N1-N3 (2-4) and a known compound lobophorin E (1), the structures of which are shown in formula (I):

[0006]

[0007] The second objective of this invention is to provide a method for constructing the engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1, which involves knocking out the P450 hydroxylase gene lobP1 in the lobophorin biosynthesis gene cluster of strain Streptomyces sp.SCSIO 01127 to obtain the genetically engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1.

[0008] A third objective of this invention is to provide a method for preparing the antibiotics lobophorins N1-N3 and lobophorin E, characterized in that the compounds lobophorins N1-N3 and lobophorin E are prepared and isolated from the fermentation culture of the engineered strain Streptomyces sp.SCSIO01127 / ΔlobP1.

[0009] Preferably, the specific steps are as follows:

[0010] a. Preparation of fermentation culture of engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1: The fermentation broth and mycelium of the fermentation culture were separated. The fermentation broth was adsorbed onto macroporous resin XAD-16, then eluted with acetone, concentrated under reduced pressure to recover acetone, and the remaining aqueous phase was extracted with butanone and concentrated to dryness under reduced pressure to obtain extract A. The mycelium was first extracted with acetone, concentrated under reduced pressure to recover acetone, and the remaining aqueous phase was extracted with butanone and concentrated to dryness under reduced pressure to obtain extract B. Extracts A and B were combined to obtain crude extract, thus obtaining the crude extract of engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1.

[0011] The crude extract of the engineered strain Streptomyces sp. SCSIO 01127 / ΔlobP1 was separated by normal-phase silica gel column chromatography. The fraction was eluted with chloroform / methanol at volume ratios of 1:0, 4:1, 2:1, and 0:1. The fraction Fr.2 at a chloroform / methanol volume ratio of 4:1 was collected. Fr.2 was then separated by Sephdex LH-20 gel column chromatography with isocratic elution using chloroform / methanol at a volume ratio of 1:1. The eluted fraction was purified to yield the compounds lobophorin N1, lobophorin N2, lobophorin N3, and lobophorin E.

[0012] The fermentation culture of the engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1 was prepared by directly inoculating the activated engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1 into the fermentation medium and culturing at 28°C, 200 rpm, and shaking for 120 h. The fermentation medium was formulated as follows: per liter, it contained: 3g soybean flour, 3g yeast extract, 10g trehalose, 1g L-proline, 3g beef extract, 6g glycerol, 0.5g FeSO4·7H2O, 0.5g MgSO4·7H2O, 0.3g K2HPO4, 2g CaCO3, 30g sea salt, with the remainder being water, and a pH of 7.2-7.4.

[0013] A fourth object of the present invention is to provide the use of any of the compounds of the above-mentioned spirocyclic antibiotics lobophorins N1-N3 in the preparation of antibacterial drugs.

[0014] The antibacterial drug is preferably an antibacterial drug against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, or MRSA.

[0015] A fifth object of the present invention is to provide the use of any of the above-mentioned spirocyclic antibiotics lobophorin N1, lobophorin N3 and lobophorin E in the preparation of antitumor drugs.

[0016] The antitumor drugs are preferably drugs for treating neuroma, liver cancer, breast cancer, and / or non-small cell lung cancer.

[0017] This invention constructs an engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1, and isolates lobophorins N1-N3 and lobophorin E with antibacterial and antitumor activities from its fermentation culture, which can serve as lead compounds for antibacterial and antitumor compounds.

[0018] The wild-type strain Streptomyces sp. SCSIO 01127 of this invention was deposited on May 19, 2011, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2011178. It is disclosed in patent number ZL201110149558.2, entitled "Antibiotics Lobophorin E and F and their preparation methods and their application in the preparation of antibacterial and antitumor drugs". Attached image description:

[0019] Figure 1 This is a diagram of the lobophorin biosynthesis gene cluster in Streptomyces sp.SCSIO 01127;

[0020] Figure 2 This describes the construction process of the engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1;

[0021] Figure 3 The images show the HPLC analysis results of the fermentation broth of the engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1 and the wild-type strain, as well as the structures of compounds 1-4.

[0022] High-performance liquid chromatography (HPLC) conditions: Column: Phenomex Kinetex C18, 250 × 4.6 mm, 5 μm; Mobile phase: Phase A: 10% (v / v) acetonitrile + 0.1% (v / v) formic acid, solvent: water; Mobile phase B: 90% (v / v) acetonitrile, solvent: water; Injection program: 0-20 min, mobile phase ratio A / B (v / v): 95:5-0:100; 20-30 min, mobile phase ratio A / B (v / v): 0:100; 30-31 min, mobile phase ratio A / B (v / v): 0:100-95:5; 31-35 min, mobile phase ratio A / B (v / v): 95:5; Detection wavelength: 265 nm; Flow rate: 1 mL / min. -1 , where 1 represents compound 1, 2 represents compound 2, 3 represents compound 3, and 4 represents compound 4.

[0023] Figure 4 This is the HRESIMS spectrum of compound 2;

[0024] Figure 5 It is compound 2. 1 H-NMR spectrum;

[0025] Figure 6 It is compound 2. 13 C-NMR spectrum;

[0026] Figure 7 This is the DEPT135 spectrum of compound 2;

[0027] Figure 8 This is the COSY spectrum of compound 2;

[0028] Figure 9 This is the HSQC spectrum of compound 2;

[0029] Figure 10 This is the HMBC spectrum of compound 2;

[0030] Figure 11 This is the HRESIMS spectrum of compound 3;

[0031] Figure 12 It is compound 3. 1 H-NMR spectrum;

[0032] Figure 13 It is compound 3. 13 C-NMR spectrum;

[0033] Figure 14 This is the DEPT135 spectrum of compound 3;

[0034] Figure 15 This is the COSY spectrum of compound 3;

[0035] Figure 16 This is the HSQC spectrum of compound 3;

[0036] Figure 17 This is the HMBC spectrum of compound 3;

[0037] Figure 18 This is the HRESIMS spectrum of compound 4;

[0038] Figure 19 It is compound 4. 1 H-NMR spectrum;

[0039] Figure 20 It is compound 4. 13 C-NMR spectrum;

[0040] Figure 21 This is the DEPT135 spectrum of compound 4;

[0041] Figure 22 This is the COSY spectrum of compound 4;

[0042] Figure 23 This is the HSQC spectrum of compound 4;

[0043] Figure 24 This is the HMBC spectrum of compound 4;

[0044] Figure 25 This is the NOESY spectrum of compound 4. Detailed implementation method:

[0045] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0046] Construction and fermentation detection of the lobP1 knockout strain Streptomyces sp. SCSIO 01127 / ΔlobP1 mutant.

[0047] The lobophorins biosynthesis gene cluster in Streptomyces sp. SCSIO 01127 was analyzed using PCR-targeting. Figure 1Knockout of the P450 hydroxylase gene lobP1 in Streptomyces sp.SCSIO01127 yielded the mutant strain Streptomyces sp.SCSIO01127 / ΔlobP1. Comparison with the fermentation assay of the wild-type Streptomyces sp.SCSIO01127 showed that the mutant strain Streptomyces sp.SCSIO01127 / ΔlobP1 produced a large amount of the known compound 1, and also produced new compounds 2-4. Figure 3 ), via HRESIMS, 1 H, 13 Data analysis of C, DEPT135, HSQC, HMBC, and COSY confirmed that these three new compounds, lobophorin N1(2)( Figure 4-10 lobophorin N2(3)( Figure 11-17 ) and lobophorin N3(4)( Figure 18-25 The structure of ).

[0048] This invention successfully obtained several new lobophorin structural analogs lobophorins N1-N3 (2-4) through gene knockout, and obtained sufficient quantities of the known compound lobophorin E (1) for antitumor activity testing.

[0049] The following are further embodiments, which are intended to help understand the present invention and are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] Example 1: Construction of engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1

[0051] Its construction process is as follows Figure 2 As shown, the specific steps are as follows:

[0052] lobophorin biosynthetic gene clusters such as Figure 1As shown, the lobP1 gene encoding the P450 hydroxylase gene, genbank accession number: AGI99476.1 in pCSG517 (Li,S.;Xiao,J.;Zhu,Y.;Zhang,G.;Yang,C.;Zhang,H.;Ma,L.;Zhang,C.,Dissecting glycosylation steps in lobophorin biosynthesis implies an iterative glycosyltransferase. Organicletters 2013,15(6),1374-1377.) was replaced by the resistance cassette aac(3)IV and oriT to obtain the lobT1-deficient mutant plasmid pCSG713. The specific PCR-targeting method is as follows: (1) Transform plasmid pCSG517 into E. coli BW25113 / pIJ790 to obtain E. coli BW25113 / pIJ790 / pCSG517, induce λ / red engineering system expression with 10 mmol / L L-arabinose, and prepare it into chemically competent cells for use. (2) Plasmid pIJ773 was digested with restriction enzymes EcoRI and HindIII. A DNA fragment containing the transfer origin and apopramine resistance gene, approximately 1.4 kb, was recovered and used as a PCR template. A 1.4 kb PCR product was amplified using primers lobP1-tarF / R (Table 1). The 50 μL PCR reaction mixture consisted of: 2 μL high-fidelity DNA polymerase FastPfu, 5 μL 10×Buffer, 0.5 mmol / L dNTPs, 2.5 μL DMSO, 0.5 μmol / L primers, approximately 1 ng DNA template, and water to a final volume of 50 μL. The PCR conditions were: pre-denaturation at 94℃ for 5 min; amplification cycles of 94℃ denaturation for 45 s, 58℃ annealing for 45 s, and 72℃ extension for 90 s, for 30 cycles; and a final extension at 72℃ for 10 min. The 1.4 kb PCR product was recovered, purified, and ready for use. (3) Transform the PCR product into the competent cells prepared in step (1) to induce recombination, plate it on LB selection plates (containing 50 μg / mL apopramycin), and incubate overnight at 37°C. Pick positive single clones from the plates and verify them using the verification primers lobP1-TF / TR (Table 1). Name the plasmid that has been verified by PCR as pCSG713. (4) Transform the constructed plasmid pCSG713 into E. coli ET12567 / pUZ8002 to construct E. coli ET12567 / pUZ8002 / pCSG713, which will serve as the donor bacteria for conjugation transfer.

[0053] The recipient bacterium Streptomyces sp. SCSIO 01127, which underwent conjugation transfer, was streaked at 38°. # (4g yeast extract, 4g glucose, 5g malt extract, 500μL multivitamins, 30g sea salt, 15-20g agar powder, 1000mL water, pH 7.2-7.4.) Plate the culture and incubate at 28℃ for 5-7 days. Collect the spores with sterile cotton swabs in TSB medium and vortex to disperse them. Filter to separate the mycelium and spores. Suspend the spores in 2mL of TSB medium, heat-shock at 50℃ for 10min, and then germinate at 28℃ for 2h, serving as the recipient bacteria for conjugation transfer.

[0054] Donor strain E. coli ET12567 / pUZ8002 / pCSG713 was grown to OD in 50 mL of LB liquid medium containing 50 μg / mL apopramycin, 50 μg / mL kanamycin, and 50 μg / mL chloramphenicol at 37°C. 600 The value was approximately 0.8. The bacterial cells were collected by centrifugation (4000 rpm, 10 min), washed three times with LB broth, and resuspended in 400 μL of LB medium as donor bacteria for conjugation transfer. 400 μL of the recipient bacteria and 100 μL of the donor bacteria were mixed thoroughly and spread onto ISP4 solid medium without any antibiotics. After drying, the mixture was incubated at 28°C for 18-20 h. The plates were then removed and covered with water containing antibiotics (final antibiotic concentrations of 50 μg / mL apopramycin and 100 μg / mL trimethoprim). After drying, the plates were placed in a 28°C incubator and incubated for 5-7 days before observation. After colonies grew on the conjugation transfer plate, they were transferred to an ISP4 plate containing 50 μg / mL apopramycin and 100 μg / mL trimethoprim using a sterile toothpick. After incubation at 28°C for 3 days, genomic DNA was extracted from each mutant strain. Single or double crossover mutants were screened by PCR using the detection primers lobP1-TF / TR (Table 1). The mutant strains were streaked onto antibiotic-free ISP4 plates. After spores grew, the spores were diluted and spread onto antibiotic-free ISP4 plates. The grown single clones were spotted onto ISP4 plates containing Kan resistance and ISP4 plates containing Apr resistance, respectively. PCR was used to verify clones that grew on Apr plates but not on Kan plates. Finally, a double crossover mutant strain was obtained and named Streptomyces sp.SCSIO01127 / ΔlobP1. The lobP1 gene was knocked out and inactivated in this mutant strain.

[0055] Table 1. Primers used in this invention

[0056]

[0057] Example 2: Fermentation and product preparation of engineered strain Streptomyces sp. SCSIO 01127 / ΔlobP1

[0058] 1. Scale-up fermentation culture:

[0059] The fermentation medium consists of: 3g soybean flour, 3g yeast extract, 10g trehalose, 1g L-proline, 3g beef extract, 6g glycerol, 0.5g FeSO4·7H2O, 0.5g MgSO4·7H2O, 0.3g K2HPO4, 2g CaCO3, and 30g sea salt. These are added to 1000mL of water, the pH is adjusted to 7.2-7.4, and the mixture is then sterilized.

[0060] After activation, the engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1 spores were directly inoculated into 50 mL of fermentation medium (250 mL Erlenmeyer flask). A total of 14 L of fermentation medium was prepared, and the culture was carried out at 28 °C and 200 rpm for 120 h to obtain the fermentation culture of engineered strain Streptomyces sp.SCSIO 01127 / ΔlobP1.

[0061] 2. Extraction of fermentation broth:

[0062] The fermentation culture was first centrifuged (3500 rpm, 8 min) to obtain the supernatant (fermentation broth) and mycelium. The fermentation broth was subjected to solid-phase extraction with macroporous resin XAD-16, followed by elution of the macroporous resin three times with acetone. After recovering the acetone from the eluent, the remaining aqueous mixture was extracted three times with butanone. The butanone layer was concentrated by distillation to obtain the supernatant extract - extract A. The mycelium was extracted three times with 2 L of acetone at room temperature for 3 hours each time. The extracts were combined, and after recovering the acetone under reduced pressure, the remaining aqueous mixture was extracted with 6 L of butanone. The butanone layer was distilled under reduced pressure to obtain the mycelial extract - extract B.

[0063] 3. Separation of compounds:

[0064] Extracts A and B obtained from the fermentation culture of the engineered strain Streptomyces sp. SCSIO 01127 / ΔlobP1 were combined and separated by normal-phase silica gel column chromatography. The fractions were sequentially eluted with chloroform / methanol gradients (volume ratios of 1:0, 4:1, 2:1, and 0:1) to obtain four fractions (Fr.1-Fr.4). Fraction Fr.2 was analyzed by Sephdex chromatography. Separation was performed using an LH-20 gel column (120cm × 3cm), eluted isocratically with chloroform / methanol 1:1, with one 15mL vial collected. Based on TLC analysis (developing solvent: chloroform / methanol 10:1, v / v), fractions with the same Rf value were combined to obtain four subfractions Fr.1.1–Fr.1.4 (Fr.1.1 was combined from vials 1–5; Fr.1.2 from vials 6–19; Fr.1.3 from vials 20–29; Fr.1.4 from vials 30–39). Fraction Fr.1.2 was then separated under medium pressure by reversed-phase chromatography (packing material: YMC*GEL ODS-A-HG; 12nm, 50μm; mobile phase: A phase: water with 0.1% formic acid; B phase: 100% acetonitrile; elution program: 0%). B-85%, 0-60 min; 85% B-100% B, 60-80 min; 100% B, 80-100 min; flow rate 20 mL / min. -1 Nine components (Fr.1.2.1-Fr.1.2.9) were obtained by detection at a wavelength of 265 nm.

[0065] Fr.1.2.2 (fractions eluted from 55 to 70 min) were purified by semi-preparative HPLC (column parameters: Phenomenex Kinetex C18, 250 × 4.6 mm, 5 μm; mobile phase: phase A was water with 0.1% formic acid, phase B was 90% acetonitrile / water, eluted isocratically at 90% of phase B, flow rate was 2.5 mL / min). -1 The detection wavelength was 265 nm. Lobophorin N3(4) (Rt = 23.7 min), lobophorin N2(3) (Rt = 24.0 min), and lobophorin N1(2) (Rt = 24.8 min) were obtained. The fraction eluted from Fr. 1.2.8 (80-90 min) was purified by semi-preparative HPLC (column parameters: Phenomenex Kinetex C18, 250 × 4.6 mm, 5 μm; mobile phase: phase A was water with 0.1% formic acid, phase B was 90% acetonitrile / water, 95% of phase B was eluted isocratically, flow rate was 2.5 mL·min). -1 The compound lobophorin E(1) was obtained by detection at a wavelength of 265 nm (Rt = 27.0 min).

[0066] 4. Identification of compounds:

[0067] The structures of compounds 2-4 were determined by HRESIMS, 1 H, 13 C, DEPT135, HSQC, HMBC, and COSY were used for identification. The NMR data are assigned in Table 2, and the spectrum of compound lobophorin N1(2) is shown in [Table 2]. Figure 4-10 The spectrum of lobophorin N2(3) is shown in [reference needed]. Figure 11-17 The spectrum of lobophorin N3(4) is shown in [reference needed]. Figure 18-25 .

[0068] Therefore, the structural formula of the new compound 2-4 is determined as follows:

[0069]

[0070] Table 2. NMR (700MHz) data assignments for compounds 2-4

[0071]

[0072]

[0073] Example 3: Determination of the antibacterial activity of compounds 1-4

[0074] The inhibitory activities of compounds 1-4 against four indicator bacteria—Bacillus subtilis 1064, Micrococcus luteus SCSIO ML01, Staphylococcus aureus ATCC 29213, and MRSA shhs-A1 (clinical sample)—were determined using the microdilution method. The four indicator bacteria were cultured on a shaker at 37°C and 200 rpm for 16 h, diluted with sterile medium to an OD value (600 nm) of 0.04-0.06, and then further diluted 10-fold into 96-well plates. After adding the sample, the plates were uniformly diluted to a final concentration of 64-0.125 μg / mL. -1 Each concentration was tested in triplicate; the cells were incubated at 37°C for 18 hours, and the absorbance of each well was measured using a microplate reader. The minimum inhibitory concentration (MIC) of each compound was calculated, with the inhibition rate (%) = (1 - (sample A - sample A background) / (negative control A - blank control A)) × 100%. An inhibition rate > 80% was considered the MIC value. The results are shown in Table 3.

[0075] Table 3. Antibacterial activity (MIC, μg / mL) of compounds 1-4

[0076]

[0077] Example 4: Determination of the antitumor activity of compounds 1, 2 and 4

[0078] The inhibitory activities of compounds 1, 2, and 4 on four tumor cell lines (SF-268, HepG2, MCF-7, and A549) were determined using the SRB method. The four tumor cell lines were cultured in RPMI medium, with 180 μL of culture (concentration 3 × 10⁻⁶) added. 4 Add 10 μL of cells per mL to a 96-well plate and incubate at 37°C with 5% CO2 for 18 h; add 20 μL of the test sample (final concentrations of 1, 10 and 100 μM, solvent DMSO) to the corresponding wells of the 96-well plate, using DMSO as a negative control, perform 3 replicates for each concentration, and continue incubation for 72 h; add 50 μL of 50% trichloroacetic acid and mix, then add 0.4% SRB (dissolved in 1% acetic acid) and let stand for 30 min; remove the supernatant, dissolve the dye-bound protein in 200 μL of 10 mM Tris buffer, measure the OD value (570 nm) of each well using a microplate reader, and calculate the corresponding inhibition rate; use doxorubicin as a positive control. The results are shown in Table 4, indicating that lobophorin E(1) has an IC50 inhibitory effect on 4 indicator cell lines. 50 The corresponding IC50 was calculated using the non-linear curve-fitting method in SigmaPlot 14.0 software, ranging from 3.11 to 12.07 μM. Its anti-hepatocellular carcinoma activity was comparable to that of the positive control doxorubicin.

[0079] Table 4. Cytotoxic activities of compounds 1, 2 and 4

[0080]

Claims

1. An engineered strain Streptomyces sp. SCSIO 01127 / ∆ lobP1 Its characteristics are, strain Streptomyces The P450 hydroxylase gene in the lobophorin biosynthesis gene cluster of sp. SCSIO 01127 lobP1 Genetically engineered strains were obtained after knockout. Streptomyces sp. SCSIO 01127 / ∆ lobP1 The aforementioned Streptomyces sp. SCSIO 01127, with accession number CCTCC NO: M 2011178, describes the P450 hydroxylase gene. lobP1 Its genbank login number is: AGI99476.

1.

2. A method for preparing lobophorins N1-N3 and the antibiotic lobophorin E, characterized in that, Compounds lobophorin E and lobophorins N1-N3 are derived from the engineered strains described in claim 1. Streptomyces sp.SCSIO 01127 / ∆ lobP1 It was prepared and isolated from fermentation culture; The compounds lobophorins N1-N3 have the structure shown in formula (I): Formula (I).

3. The preparation method according to claim 2, characterized in that, The specific steps are as follows: a. Preparation of engineered strains Streptomyces sp. SCSIO 01127 / ∆ lobP1 The fermentation culture was prepared by separating the fermentation broth and mycelium. The fermentation broth was adsorbed onto a macroporous resin, then eluted with acetone, and the acetone was recovered by vacuum concentration. The remaining aqueous phase was extracted with butanone and then concentrated to dryness under vacuum to obtain extract A. The mycelium was first extracted with acetone, and the acetone was recovered by vacuum concentration. The remaining aqueous phase was extracted with butanone and then concentrated to dryness under vacuum to obtain extract B. Extracts A and B were combined to obtain the crude extract, thereby obtaining the engineered strain. Streptomyces sp. SCSIO 01127 / ∆ lobP1 Crude extract; engineered strains Streptomyces sp. SCSIO 01127 / ∆ lobP1 The crude extract was separated by normal-phase silica gel column chromatography, eluted with chloroform / methanol at volume ratios of 1:0, 4:1, 2:1 and 0:

1. The fraction Fr.2 eluted with chloroform / methanol at a volume ratio of 4:1 was collected and then separated by Sephdex LH-20 gel column chromatography with isocratic elution of chloroform / methanol at a volume ratio of 1:

1. The eluted fractions containing the target components were combined and purified semi-preparatively to obtain compounds lobophorin E, lobophorin N1, lobophorin N2 and lobophorin N3.

4. The preparation method according to claim 3, characterized in that, The engineered strains Streptomyces sp.SCSIO 01127 / ∆ lobP1 The fermentation culture is made by activating engineered strains. Streptomyces sp. SCSIO01127 / ∆ lobP1 The culture was directly inoculated into the fermentation medium and cultured at 28 °C and 200 rpm for 120 h with shaking to obtain the fermentation culture. The fermentation medium was formulated as follows: per liter of medium, the following ingredients were added: 3 g soybean flour, 3 g yeast extract, 10 g trehalose, 1 g L-proline, 3 g beef extract, 6 g glycerol, 0.5 g FeSO4·7H2O, 0.5 g MgSO4·7H2O, 0.3 g K2HPO4, 2 g CaCO3, 30 g sea salt, with the balance being water, and the pH was 7.2-7.4.

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