Preparation of macrolide compound pa-46101s c-e and use thereof

Abstract

Disclosed in the present invention are the preparation of a macrolide compound PA-46101s C-E and the use thereof. In the present invention, Streptomyces sp. SCSIO 40066 is mutated to obtain mutant strains Streptomyces sp. SCSIO 40066 / ΔpasO1, Streptomyces sp. SCSIO 40066 / ΔpasO4 and Streptomyces sp. SCSIO 40066 / ΔpasO1O4; and three new spiro acetoacetate compounds PA-46101s C-E having an antibacterial effect are separated from fermentation cultures of the mutant strains.

Description

Preparation and application of macrolide compound PA-46101s CE Technical field:

[0001] The present invention belongs to the field of industrial microorganisms, and in particular relates to a new antibiotic compound PA-46101s CE (3-5) and a preparation method thereof. Background technology:

[0002] PA-46101s are a class of spiroacetoacetic acid lactone polyketides. This family of compounds is structurally diverse and exhibits diverse activities, including anti-inflammatory, antibacterial, and anti-tumor activities. It is well known that the development of unmodified natural products into druggable forms is challenging due to various factors. Advances in molecular biology and gene editing technologies have made it possible to genetically engineer natural product biosyntheses to produce structurally and bioactively optimized natural products, offering advantages such as high specificity, high catalytic efficiency, and environmental friendliness.

[0003] Summary of the invention:

[0004] The first object of the present invention is to provide three new spirocyclic acetoacetic acid lactone compounds PA-46101s CE (3-5).

[0005] The three new PA-46101s macrolide compounds PA-46101s CE (3-5) of the present invention have structures as shown in (I):

[0006] The second object of the present invention is to provide the use of compound PA-46101s CE in the preparation of antibacterial drugs.

[0007] The antibacterial drug is a drug against Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0008] The third object of the present invention is to provide a P450 oxidase encoding gene knockout mutant strain Streptomyces sp.SCSIO 40066 / ΔpasO1, which is characterized in that the P450 oxidase encoding gene pasO1 in the pas gene cluster is knocked out, and the nucleotide sequence of the gene pasO1 is shown in SEQ ID NO.1.

[0009] The fourth object of the present invention is to provide a P450 oxidase encoding gene knockout mutant strain Streptomyces sp.SCSIO 40066 / ΔpasO4, which is characterized in that the P450 oxidase encoding gene pasO4 in the pas gene cluster is knocked out, and the nucleotide sequence of the gene pasO4 is shown in SEQ ID NO.2.

[0010] A fifth object of the present invention is to provide a P450 oxidase encoding gene knockout mutant strain, Streptomyces sp.SCSIO 40066 / ΔpasO1O4, characterized in that the P450 oxidase encoding gene pasO4 in the pas gene cluster is first deleted in-frame, and then the P450 oxidase encoding gene pasO1 in the gene cluster is deleted in-frame, the nucleotide sequence of the gene pasO1 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene pasO4 is shown in SEQ ID NO.2.

[0011] The sixth object of the present invention is to provide a method for preparing PA-46101s CE (3-5), characterized in that PA-46101C (3) is isolated from the metabolites of the strain Streptomyces sp. SCSIO 40066 / ΔpasO1; PA-46101D (4) is isolated from the metabolites of the strain Streptomyces sp. SCSIO 40066 / ΔpasO4; and PA-46101E (5) is isolated from the metabolites of the strain Streptomyces sp. SCSIO 40066 / ΔpasO1O4.

[0012] The specific steps are:

[0013] Fermentation cultures of Streptomyces sp. SCSIO 40066 / ΔpasO1, Streptomyces sp. SCSIO 40066 / ΔpasO4, and Streptomyces sp. SCSIO 40066 / ΔpasO1O4 were prepared respectively, and the fermentation broth and mycelia of the fermentation cultures were separated by centrifugation; the fermentation broth was adsorbed on a macroporous resin, eluted with acetone, concentrated under reduced pressure to recover the acetone, the remaining aqueous phase was extracted with butanone, and then concentrated under reduced pressure to dryness to obtain extract A; the mycelia were first extracted with acetone, concentrated under reduced pressure to recover the acetone, the remaining aqueous phase was extracted with butanone, and then concentrated under reduced pressure to dryness to obtain extract B; and extracts A and B were combined to obtain a crude fermentation extract;

[0014] a. The crude fermentation extract of Streptomyces sp.SCSIO 40066 / ΔpasO1 was separated by normal phase silica gel column chromatography using gradient elution with chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1, v / v, 500 mL, to obtain five fractions, Frs.A1-A5. Thin layer chromatography (TLC) and HPLC high-performance liquid chromatography determined that the target compound was concentrated in Frs.A2. The extract was then separated by gel chromatography on Sephadex LH-20 using isocratic elution with chloroform / methanol = 1 / 1, v / v, with each 15 mL fraction collected into a vial. A total of approximately 30 vials were collected. After HPLC analysis, 8-12 vials containing the target compound were combined to obtain subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative high-performance liquid chromatography using the following column parameters: Phenomenex Kinetex C18, 250×4.6mm, 5μm; preparation conditions are acetonitrile / water, 70 / 30, v / v, 2.5mL / min, to obtain subfraction Frs.A2-1-1, t R =17.8min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC. The preparation conditions were chloroform / methanol, 10 / 1, v / v, R f =0.45, to obtain compound PA-46101C;

[0015] b. The crude extract of Streptomyces sp. SCSIO 40066 / ΔpasO4 was separated by normal phase silica gel column chromatography using a gradient elution solvent of chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1, v / v, in 500 mL. Five fractions, Frs.A1-A5, were sequentially separated. Thin layer chromatography and HPLC high-performance liquid chromatography determined that the target compound was concentrated in Frs.A2. The extract was then separated by gel chromatography on Sephadex LH-20 using an isocratic elution solvent of chloroform / methanol = 1 / 1, v / v. 15 mL of the extract was collected into one vial, and approximately 30 vials were collected. After HPLC analysis, 16-20 vials containing the target compound were combined to obtain subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative high-performance liquid chromatography using the following column parameters: Phenomenex Kinetex C18, 250×4.6mm, 5μm; preparation conditions are acetonitrile / water, 75 / 25, v / v, 2.5mL / min, to obtain subfraction Frs.A2-1-1, t R = 22.3min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC. The preparation conditions were chloroform / methanol / formic acid, 10 / 1 / 0.011, v / v, R f=0.60, to obtain compound PA-46101D;

[0016] c. A crude extract of Streptomyces sp. SCSIO 40066 / ΔpasO1O4 was separated by normal-phase silica gel column chromatography using a gradient elution solvent of chloroform / methanol (1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1, v / v). Five fractions, Frs.A1-A5, were sequentially separated. TLC and HPLC high-performance liquid chromatography confirmed that the target compound was concentrated in Frs.A2. The extract was then separated by isocratic elution using a chloroform / methanol (1 / 1, v / v) solvent on a Sephadex LH-20 column. 15 mL of the extract was collected in a vial, and approximately 30 vials were collected. After HPLC analysis, vials 18-22 containing the target compound were combined to obtain subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative HPLC. The column parameters were: Phenomenex Kinetex C18, 250×4.6 mm, 5 μm; the preparation conditions were acetonitrile / water, 95 / 5, v / v, 2.5 mL / min, and the subfractions Frs.A2-1-1, t R = 13.2 min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC. The preparation conditions were chloroform / methanol, 10 / 1, v / v, R f =0.55, obtaining compound PA-46101E.

[0017] The fermentation cultures of the mutant strains Streptomyces sp. SCSIO 40066 / ΔpasO1, Streptomyces sp. SCSIO 40066 / ΔpasO4, and Streptomyces sp. SCSIO 40066 / ΔpasO1O4 are prepared by the following method: the activated strains are inoculated into conical flasks filled with A1 medium, cultured at 28°C and 200 rpm to obtain seed liquid; the seed liquid is then inoculated into a conical flask filled with A1 medium at a 10% inoculum volume, and cultured with shaking at 28°C and 200 rpm to obtain a fermentation culture. The A1 medium contains 10g of fish meal, 10g of glycerol, 0.2g of KBr, 3g of CaCO3, 30g of sea salt, and 1000ml of water per liter, with a pH of 7.2-7.4.

[0018] The invention mutates Streptomyces sp. SCSIO 40066 to obtain mutant strains Streptomyces sp. SCSIO 40066 / ΔpasO1, Streptomyces sp. SCSIO 40066 / ΔpasO4, and Streptomyces sp. SCSIO 40066 / ΔpasO1O4, and isolates three new spiroacetoacetic acid lactone compounds PA-46101s CE with antibacterial activity from fermentation cultures of the mutant strains.

[0019] Streptomyces sp. SCSIO 40066 is disclosed in NCBI (Streptomyces sp. strain SCSIO 40066 16S ribosomal RNA gene, partial sequence-Nucleotide-NCBI (nih.gov), GenBank: PP037986.1). The applicant also holds this strain and guarantees that it will be available to the public for 20 years from the date of application. Description of the drawings:

[0020] FIG1 is a diagram of the PA-46101 biosynthetic gene cluster in Streptomyces sp. SCSIO 40066;

[0021] Figure 2 is a schematic diagram of pasO1 knockout;

[0022] Figure 3 is a schematic diagram of pasO4 knockout;

[0023] Figure 4 is a schematic diagram of pasO1O4 knockout;

[0024] FIG5 is a high performance liquid chromatogram of a fermentation extract of a gene knockout mutant;

[0025] High-performance liquid chromatography (HPLC) conditions: chromatographic column: Phenomenex C18, 150×4.6 mm, 5 μm; mobile phases: phase A and phase B; mobile phase A: 10% (volume fraction) acetonitrile + 0.1% (volume fraction) formic acid, solvent: water; mobile phase B: 90% (volume fraction) acetonitrile, solvent: water; injection program: 0-20 min, mobile phase ratio: phase A / phase B (volume ratio): 95:5-5:95, 20-21 min, mobile phase ratio: phase A / phase B (volume ratio): 5:95, 21-25 min, mobile phase ratio: phase A / phase B (volume ratio): 5:95-95:5, 25-30 min, mobile phase ratio: phase A / phase B (volume ratio): 95:5; detection wavelength: 236 nm; flow rate: 1 ml min -1 .

[0026] Figure 6 is the HRESIMS spectrum of compound 3

[0027] Figure 7 is the 1 H-NMR spectrum

[0028] Figure 8 is the 13 C-NMR spectrum

[0029] Figure 9 is the DEPT135 spectrum of compound 3

[0030] Figure 10 is the COSY spectrum of compound 3

[0031] Figure 11 is the HSQC spectrum of compound 3

[0032] Figure 12 is the HMBC spectrum of compound 3

[0033] Figure 13 is the HRESIMS spectrum of compound 4

[0034] Figure 14 is a graph showing the structure of compound 4. 1 H-NMR spectrum

[0035] Figure 15 is a graph showing the structure of compound 4. 13 C-NMR spectrum

[0036] Figure 16 is the DEPT135 spectrum of compound 4

[0037] Figure 17 is the COSY spectrum of compound 4

[0038] Figure 18 is the HSQC spectrum of compound 4

[0039] Figure 19 is the HMBC spectrum of compound 4

[0040] Figure 20 is the HRESIMS spectrum of compound 5

[0041] Figure 21 is a graph showing the structure of compound 5. 1 H-NMR spectrum

[0042] Figure 22 is a graph showing the structure of compound 5. 13 C-NMR spectrum

[0043] Figure 23 is the DEPT135 spectrum of compound 5

[0044] Figure 24 is the COSY spectrum of compound 5

[0045] Figure 25 is the HSQC spectrum of compound 5

[0046] Figure 26 is the HMBC spectrum of compound 5 Specific implementation method:

[0047] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0048] 1. Construction of the gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1 (Figure 2)

[0049] The P450 oxidase gene pasO1 in the PA-46101 biosynthetic gene cluster (Figure 1) of Streptomyces sp. SCSIO 40066 was knocked out by PCR-targeting to generate the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1. Compared with the fermentation assay of wild-type Streptomyces sp. SCSIO 40066, the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1 produced a new compound 3 (Figure 5). 1 H. 13 C, DEPT135, HSQC, HMBC and COSY data analysis confirmed the structure of the new compound PA-46101C.

[0050] 2. Construction of the gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO4 (Figure 3)

[0051] The P450 oxidase gene pasO4 in the PA-46101 biosynthetic gene cluster (Figure 1) of Streptomyces sp. SCSIO 40066 was knocked out by PCR-targeting to generate the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO4. Compared with the fermentation assay of wild-type Streptomyces sp. SCSIO 40066, the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO4 produced a new compound 4 (Figure 5). 1 H. 13 C, DEPT135, HSQC, HMBC and COSY data analysis confirmed the structure of the new compound PA-46101D.

[0052] 3. Construction of the gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1O4 (Figure 4)

[0053] The P450 oxidase gene pasO4 in the PA-46101 biosynthetic gene cluster (Figure 1) of Streptomyces sp. SCSIO 40066 was knocked out by the thermosensitive plasmid pKC1139-mediated in-frame deletion method to obtain the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO4-inframe. Then, the P450 oxidase gene pasO1 in the PA-46101 biosynthetic gene cluster (Figure 1) in Streptomyces sp. SCSIO 40066 / ΔpasO4-inframe was knocked out by the thermosensitive plasmid pKC1139-mediated in-frame deletion method to obtain the mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1O4. Compared with the fermentation detection chart of wild-type Streptomyces sp.SCSIO 40066, the mutant strain Streptomyces sp.SCSIO 40066 / ΔpasO1O4 produced a new compound 5 (Figure 5), which was analyzed by HRESIMS, 1 H. 13 C, DEPT135, HSQC, HMBC and COSY data analysis confirmed the structure of the new compound PA-46101E.

[0054] Table 1. Primers used in the present invention

[0055] Example 1: Construction of gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1

[0056] Using a λRED-mediated PCR-targeted gene knockout method, the Apr resistance gene in the pIJ773 plasmid was used as a conjugate screening tag, and the target gene pasO1 was inactivated by insertional mutagenesis. The conjugative transfer process is as follows: Streptomyces sp. SCSIO 40066 was streaked onto plates in SFM medium (30 g soybean powder, 30 g mannitol, 30 g sea salt, 20 g agar powder, 1000 mL water, pH 7.0; the preparation method is to mix all ingredients, adjust the pH, and sterilize them for later use) for 5-7 days. Spores were collected with a sterile cotton swab in TSB medium and vortexed to disperse the spores. Mycelium and spores were separated by filtration, and the spores were suspended in 5 mL of TSB medium, heat-shocked at 50°C for 10 minutes, and then germinated at 28°C for 2 hours to serve as the recipient bacteria for conjugative transfer. Donor bacteria E. coli BW25113 / pIJ790 / pCGS6003 in 50 mL containing 50 μg mL-1 The cells were grown in LB liquid medium with apramycin at 37°C until the OD 600 The value is about 0.8. The shuttle plasmid containing E. coli ET12567 / pUB307 contains 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were grown in LB liquid medium containing chloramphenicol at 37°C until the OD 600 The value is about 0.8. Collect the two bacteria by centrifugation (4000 rpm, 10 min), wash the bacteria three times with LB, and suspend them in 300 μL LB medium to serve as the donor bacteria for conjugation transfer. Take 400 μL of the above recipient bacteria and 100 μL of the donor bacteria, mix them evenly, spread them on ISP4 solid medium without any antibiotics, blow dry, and incubate at 28°C for 18-20 hours. Then remove the plate and cover it with water containing antibiotics. The final concentration of antibiotics is 50 μg mL -1 Apramycin and 100 μg mL -1 After drying, place the trimethoprim in a 28°C incubator and observe after culturing for 5-7 days. When colonies grow on the conjugation transfer plate, use a sterile toothpick to transfer them to a plate containing 50 μg mL -1 Apramycin and 100 μg mL -1 After culturing on trimethoprim-containing SFM plates at 28°C for three days, genomic DNA was extracted from each mutant. PCR verification of the genomic DNA using the validation primers PA-O1TF / TR (using the wild-type strain as a negative control) yielded a DNA fragment approximately 1.9 kb in size (the PCR product for the wild-type strain control was 1.7 kb). After passage and antibiotic control, a double crossover conjugate was obtained, resulting in the single gene knockout mutant SCSIO 40066 / ΔpasO1.

[0057] Example 2: Construction of gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO4

[0058] The λRED-mediated PCR targeting gene knockout method was used, and the Apr resistance gene in the pIJ773 plasmid was used as a conjugate screening tag. The target gene pasO4 was inactivated by insertion mutation. The conjugation transfer process is described as follows: Streptomyces sp. SCSIO 40066 was streaked and cultured on SFM culture plates for 5-7 days. The grown spores were collected in TSB culture medium with a sterile cotton swab and vortexed to disperse the spores. The mycelium and spores were separated by filtration, and the spores were suspended in 5 mL of TSB culture medium, heat-shocked at 50°C for 10 minutes, and then germinated at 28°C for 2 hours as the recipient bacteria for conjugation transfer. The donor bacteria E. coli BW25113 / pIJ790 / pCGS6004 were added to 50 mL containing 50 μg mL -1 The cells were grown in LB liquid medium with apramycin at 37°C until the OD 600 The value is about 0.8. The shuttle plasmid containing E. coli ET12567 / pUB307 contains 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were grown in LB liquid medium containing chloramphenicol at 37°C until the OD 600 The value is about 0.8. Collect the two bacteria by centrifugation (4000 rpm, 10 min), wash the bacteria three times with LB, and suspend them in 300 μL LB medium to serve as the donor bacteria for conjugation transfer. Take 400 μL of the above recipient bacteria and 100 μL of the donor bacteria, mix them evenly, spread them on ISP4 solid medium without any antibiotics, blow dry, and incubate at 28°C for 18-20 hours. Then remove the plate and cover it with water containing antibiotics. The final concentration of antibiotics is 50 μg mL -1 Apramycin and 100 μg mL -1 After drying, place the trimethoprim in a 28°C incubator and observe after culturing for 5-7 days. When colonies grow on the conjugation transfer plate, use a sterile toothpick to transfer them to a plate containing 50 μg mL -1 Apramycin and 100 μg mL -1 After culturing on trimethoprim-containing SFM plates at 28°C for three days, genomic DNA was extracted from each mutant. PCR verification of the genomic DNA using the validation primers PA-O4TF / TR (using the wild-type strain as a negative control) yielded a DNA fragment approximately 2.6 kb in size (the PCR product for the wild-type strain control was 1.7 kb). After passage and antibiotic control, a double crossover conjugate was obtained, resulting in the single gene knockout mutant SCSIO 40066 / ΔpasO4.

[0059] Example 3: Construction of gene knockout mutant strain Streptomyces sp. SCSIO 40066 / ΔpasO1O4

[0060] The homologous recombination-mediated gene in-frame deletion method was used, and the Apr resistance gene was used as a conjugate screening tag. The target gene pasO4 was inactivated by insertion mutation. The conjugation transfer process is described as follows: Streptomyces sp. SCSIO 40066 was streaked and cultured on SFM culture plates for 5-7 days. The grown spores were collected in TSB culture medium with a sterile cotton swab and vortexed to disperse the spores. The mycelium and spores were separated by filtration, and the spores were suspended in 5 mL of TSB culture medium, heat-shocked at 50°C for 10 minutes, and then germinated at 28°C for 2 hours to serve as the recipient bacteria for conjugation transfer. The donor bacteria E. coli DH5α / pCSG6006 was diluted in 50 mL containing 50 μg mL -1 The cells were grown in LB liquid medium with apramycin at 37°C until the OD 600 The value is about 0.8. The shuttle plasmid containing E. coli ET12567 / pUB307 contains 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were grown in LB liquid medium containing chloramphenicol at 37°C until the OD 600 The value is about 0.8. Collect the two bacteria by centrifugation (4000 rpm, 10 min), wash the bacteria three times with LB, and suspend them in 300 μL LB medium to serve as the donor bacteria for conjugation transfer. Take 400 μL of the above recipient bacteria and 100 μL of the donor bacteria, mix them evenly, spread them on ISP4 solid medium without any antibiotics, blow dry, and incubate at 28°C for 18-20 hours. Then remove the plate and cover it with water containing antibiotics. The final concentration of antibiotics is 50 μg mL -1 Apramycin and 100 μg mL -1 After drying, place the trimethoprim in a 28°C incubator and observe after culturing for 5-7 days. When colonies grow on the conjugation transfer plate, use a sterile toothpick to transfer them to a plate containing 50 μg mL -1 Apramycin and 100 μg mL -1After culturing on trimethoprim-containing SFM plates at 28°C for three days, genomic DNA was extracted from each mutant. PCR verification of the genomic DNA using the validation primers PA-O4TF / TR (using the wild-type strain as a negative control) yielded a DNA fragment approximately 1.1 kb in size (the PCR product for the wild-type control was 1.7 kb). Subculture at 37°C eliminated the vector fragment containing the resistance gene, and single colonies were selected for antibiotic spot testing, resulting in the single-gene in-frame deletion mutant SCSIO 40066 / ΔpasO4-inframe, which did not grow on Apr-resistant plates but grew on non-resistant plates.

[0061] The gene in-frame deletion method mediated by homologous recombination was used, and the Apr resistance gene was used as a conjugate screening tag. The target gene pasO1 was inactivated by insertion mutation. The conjugation transfer process is described as follows: Actinomycetes SCSIO 40066 / ΔpasO4-inframe were streaked and cultured on SFM culture plates for 5-7 days. The grown spores were collected in TSB culture medium with a sterile cotton swab and vortexed to disperse the spores. The mycelium and spores were separated by filtration, and the spores were suspended in 5 mL of TSB culture medium, heat-shocked at 50°C for 10 minutes, and then germinated at 28°C for 2 hours to serve as the recipient bacteria for conjugation transfer. The donor bacteria E. coli DH5α / pCSG6007 was added to 50 mL containing 50 μg mL -1 The cells were grown in LB liquid medium with apramycin at 37°C until the OD 600 The value is about 0.8. The shuttle plasmid containing E. coli ET12567 / pUB307 contains 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were grown in LB liquid medium containing chloramphenicol at 37°C until the OD 600 The value is about 0.8. Collect the two bacteria by centrifugation (4000 rpm, 10 min), wash the bacteria three times with LB, and suspend them in 300 μL LB medium to serve as the donor bacteria for conjugation transfer. Take 400 μL of the above recipient bacteria and 100 μL of the donor bacteria, mix them evenly, spread them on ISP4 solid medium without any antibiotics, blow dry, and incubate at 28°C for 18-20 hours. Then remove the plate and cover it with water containing antibiotics. The final concentration of antibiotics is 50 μg mL -1 Apramycin and 100 μg mL -1 After drying, place the trimethoprim in a 28°C incubator and observe after culturing for 5-7 days. When colonies grow on the conjugation transfer plate, use a sterile toothpick to transfer them to a plate containing 50 μg mL -1 Apramycin and 100 μg mL -1After culturing on trimethoprim-containing SFM plates at 28°C for three days, genomic DNA was extracted from each mutant. PCR verification of the genomic DNA using the validation primers PA-O1TF / TR (using the wild-type strain as a negative control) yielded a DNA fragment approximately 0.9 kb in size (the PCR product for the wild-type control was 1.7 kb). Subculture at 37°C eliminated the vector fragment containing the resistance gene, and single colonies were selected for antibiotic spot testing, resulting in the single-gene in-frame deletion mutant SCSIO 40066 / ΔpasO1O4, which did not grow on Apr-resistant plates but grew on non-resistant plates.

[0062] Example 4: Fermentation and preparation of PA-46101s compounds

[0063] 1. Scale-up fermentation culture

[0064] The seed culture medium and fermentation medium used for the fermentation of mutant strains Streptomyces sp.SCSIO 40066 / ΔpasO1, Streptomyces sp.SCSIO 40066 / ΔpasO4, and Streptomyces sp.SCSIO 40066 / ΔpasO1O4 were A1 (fish meal 10 g, glycerol 10 g, KBr 0.2 g, CaCO3 3 g, sea salt 30 g, water 1000 mL, pH 7.2-7.4, the preparation method of which is to mix all the ingredients evenly, adjust the pH value and sterilize for use).

[0065] After all mutant strains were activated, the spores were inoculated into seed culture medium and cultured at 28°C, 200 rpm for 48 h to prepare seed liquid; the seed liquid was inoculated into fermentation medium (14 L) at a volume ratio of 10%, cultured at 28°C, 200 rpm for 120 h to prepare fermentation cultures of each mutant strain.

[0066] 2. Extraction of fermentation broth:

[0067] The fermentation culture was first centrifuged (3500 rpm, 8 minutes) to obtain a supernatant (fermentation broth) and mycelia. The fermentation broth was adsorbed on a macroporous resin XAD16, eluted with acetone, and concentrated under reduced pressure to recover the acetone. The remaining aqueous phase was extracted with butanone and then concentrated to dryness under reduced pressure to obtain extract A. The mycelia were first extracted with acetone, concentrated under reduced pressure to recover the 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 a crude fermentation extract.

[0068] 3. Separation of compounds:

[0069] a. A crude fermentation extract of Streptomyces sp. SCSIO 40066 / ΔpasO1 was separated by normal-phase silica gel column chromatography using a gradient elution solvent of chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1 (v / v) in 500 mL portions, yielding five fractions, Frs.A1-A5. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) confirmed that the target compound was concentrated in Frs.A2 (the fraction eluted with chloroform / methanol = 10 / 1). Separation was performed by gel chromatography on Sephadex LH-20 (isocratic elution with chloroform / methanol = 1 / 1 (v / v)). Approximately 30 vials were collected in 15 mL increments. After HPLC analysis, 8-12 vials containing the target compound were combined to yield subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative HPLC (column parameters: Phenomenex Kinetex C18, 250×4.6 mm, 5 μm; preparation conditions: acetonitrile / water, 70 / 30, v / v, 2.5 mL / min) to obtain subfraction Frs.A2-1-1 (t R =17.8min) Finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC (preparation conditions: chloroform / methanol, 10 / 1, v / v, R f =0.45), yielding compound PA-46101C (3).

[0070] b. A crude extract of Streptomyces sp. SCSIO 40066 / ΔpasO4 was separated by normal-phase silica gel column chromatography using a gradient elution system of chloroform / methanol (1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1, v / v) in 500 mL portions to yield five fractions, Frs.A1-A5. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) confirmed that the target compound was concentrated in Frs.A2 (the fraction eluted with chloroform / methanol (10 / 1). Separation was performed by gel chromatography on Sephadex LH-20 (isocratic elution with chloroform / methanol (1 / 1, v / v)). Approximately 30 vials were collected in 15 mL increments. After HPLC analysis, 16-20 vials containing the target compound were combined to yield subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative HPLC (column parameters: Phenomenex Kinetex C18, 250×4.6 mm, 5 μm; preparation conditions: acetonitrile / water, 75 / 25, v / v, 2.5 mL / min) to obtain subfraction Frs.A2-1-1 (t R=22.3min) Finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC (preparation conditions: chloroform / methanol / formic acid, 10 / 1 / 0.011, v / v, R f =0.60) to obtain compound PA-46101D (4).

[0071] c. A crude extract of Streptomyces sp. SCSIO 40066 / ΔpasO1O4 was separated by normal-phase silica gel column chromatography using a gradient elution solvent of chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1, and 0 / 1 (v / v) in 500 mL portions, yielding five fractions, Frs.A1-A5. TLC and HPLC analysis confirmed that the target compound was concentrated in Frs.A2 (the fraction eluted with chloroform / methanol = 10 / 1). Separation was performed by gel chromatography on Sephadex LH-20 (isocratic elution with chloroform / methanol = 1 / 1 (v / v)). Approximately 30 vials were collected in 15 mL increments. After HPLC analysis, vials 18-22 containing the target compound were combined to yield subfraction Frs.A2-1. The target compound was further separated and purified by semi-preparative HPLC (column parameters: Phenomenex Kinetex C18, 250×4.6 mm, 5 μm; preparation conditions: acetonitrile / water, 95 / 5, v / v, 2.5 mL / min) to obtain subfraction Frs.A2-1-1 (t R =13.2min). Finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC (preparation conditions: chloroform / methanol, 10 / 1, v / v, R f =0.55), yielding compound PA-46101E (5).

[0072] 4. Identification of compounds:

[0073] Figure 6 is the HRESIMS spectrum of compound 3, and Figure 7 is the 1 H-NMR spectrum, Figure 8 is compound 3 13 C-NMR spectrum, Figure 9 is the DEPT135 spectrum of compound 3, Figure 10 is the COSY spectrum of compound 3, Figure 11 is the HSQC spectrum of compound 3, Figure 12 is the HMBC spectrum of compound 3, Figure 13 is the HRESIMS spectrum of compound 4, and Figure 14 is the 1 H-NMR spectrum, Figure 15 is compound 4 13C-NMR spectrum, Figure 16 is the DEPT135 spectrum of compound 4, Figure 17 is the COSY spectrum of compound 4, Figure 18 is the HSQC spectrum of compound 4, Figure 19 is the HMBC spectrum of compound 4, Figure 20 is the HRESIMS spectrum of compound 5, and Figure 21 is the 1 H-NMR spectrum, Figure 22 is compound 5 13 C-NMR spectrum, Figure 23 is the DEPT135 spectrum of compound 5, Figure 24 is the COSY spectrum of compound 5, Figure 25 is the HSQC spectrum of compound 5, and Figure 26 is the HMBC spectrum of compound 5. The structure of PA-46101s CE (3-5) is shown in Figure 24. 1 H. 13 C, DEPT135, HSQC, HMBC and COSY analysis confirmed its structural formula as follows:

[0074] Example 5: Construction of plasmid pCSG6003 for gene knockout

[0075] Based on the DNA sequence (SEQ ID NO.1) of the gene pasO1, the protein translation sequence, and the PCR targeting knockout sequence, a pair of long-fragment knockout primers (about 60 bp) were designed. The sequence of the 39 bp uppercase portion of the primer at the 5' end matched the gene, while the sequence of the lowercase portion at the 3' end matched the sequences on both sides of the DNA fragment containing the transfer initiation sequence oriT and the Apr resistance gene aac(3)IV in pIJ773. The plasmid pIJ773 was treated with restriction endonucleases EcoRI and HindIII, and a 1.4 kb DNA fragment containing the transfer origin oriT and the Apr resistance gene aac(3)IV was recovered. As a PCR template, a 1.4 kb fragment was amplified using gene knockout primers (pasO1-tarF1 and pasO1-tarR1), and the fragment was recovered by gel PCR for use. Prepare E. coli BW25113 / pIJ790 chemically competent cells (the plasmid pIJ790 in this strain is a temperature-sensitive plasmid that only contains Crm resistance). Chemically transform the positive clones from the cosmid library plasmid pCSG6001, containing the target gene, into the E. coli BW25113 / pIJ790 competent cells. Verify that the resulting colonies are positive using screening primers (the strain culture temperature is controlled at 28-30°C throughout to ensure that the plasmid pIJ790 is not lost; positive strains are resistant to Crm and Kan). Recombinant strains are then prepared as chemically competent cells. Note that when expanding the culture (50 mL) of this strain, add 500 μL of L-arabinose solution (1 mol / L) and 250 μL of MgCl2 solution (2 mol / L) to LB liquid medium and maintain the culture temperature at 28-30°C to induce accumulation of the homologous recombination element λRED. The purified PCR product (1.4 kb in size) was introduced into competent cells by chemical transformation, and the homologous recombination element λRED in the host cells induced the aac(3)IV and oriT replacement resistance fragment containing the homologous arms to target the target gene pasO1. The strain was cultured overnight on LB solid culture plates (containing AMP, Kan, and Apr resistance) in a shaker at 37°C. A single colony was picked from the plate and transferred to 5 mL LB liquid culture medium and cultured overnight in a shaker at 37°C. The plasmid was extracted for verification and sequencing. The partial fragment of the cytochrome P450 monooxygenase encoding gene pasO1 on the cosmid plasmid in this recombinant strain was replaced with a gene containing the aac(3)IV and oriT fragments, and was named E. coli BW25113 / pIJ790 / pCGS6003.

[0076] Example 6: Construction of plasmid pCSG6004 for gene knockout

[0077] Based on the DNA sequence (SEQ ID NO.2) of the gene pasO4, the protein translation sequence, and the PCR targeting knockout sequence, a pair of long-fragment knockout primers (about 60 bp) were designed. The sequence of the 39 bp uppercase portion of the primer at the 5' end matched the gene, while the sequence of the lowercase portion at the 3' end matched the sequences on both sides of the DNA fragment containing the transfer initiation sequence oriT and the Apr resistance gene aac(3)IV in pIJ773. The plasmid pIJ773 was treated with restriction endonucleases EcoRI and HindIII, and a 1.4 kb DNA fragment containing the transfer origin oriT and the Apr resistance gene aac(3)IV was recovered. As a PCR template, the gene knockout primers (pasO4-tarF1 and pasO4-tarR1) were used to amplify a 1.4 kb fragment, which was then recovered by gel filtration for later use. Prepare E. coli BW25113 / pIJ790 chemically competent cells (the plasmid pIJ790 in this strain is a temperature-sensitive plasmid that only contains Crm resistance). Chemically transform the positive clone containing the target gene from the cosmid library plasmid pCSG6002 into the E. coli BW25113 / pIJ790 competent cells. Verify that the resulting colony is positive using screening primers (the strain culture temperature is controlled at 28-30°C throughout to ensure that the plasmid pIJ790 is not lost; positive strains are resistant to Crm and Kan). This recombinant strain is then prepared for chemical transformation. Note that when expanding the culture (50 mL) of this strain, add 500 μL of L-arabinose solution (1 mol / L) and 250 μL of MgCl2 solution (2 mol / L) to LB liquid medium and maintain the culture temperature at 28-30°C to induce accumulation of the homologous recombination element λRED. The purified PCR product (1.4 kb in size) was introduced into competent cells by chemical transformation, and the homologous recombination element λRED in the host cell induced the aac(3)IV and oriT replacement resistance fragment containing the homologous arms to target the target gene pasO1. The strain was cultured overnight on LB solid culture plates (containing AMP, Kan and Apr resistance) in a shaker at 37°C. A single colony was picked from the plate and transferred to 5 mL LB liquid culture medium and cultured overnight in a shaker at 37°C. The plasmid was extracted for verification and sequencing. The partial fragment of the cytochrome P450 monooxygenase encoding gene pasO4 on the cosmid plasmid in this recombinant strain was replaced with a gene containing the aac(3)IV and oriT fragments, and was named E. coli BW25113 / pIJ790 / pCGS6004, resulting in the strain E. coli DH5α / pCSG6004.

[0078] Example 7: Construction of plasmid pCSG6006 for gene knockout

[0079] Based on the DNA sequence (SEQ ID NO. 2) and protein translation sequence of the pasO4 gene, as well as the in-frame deletion gene knockout sequence, upstream and downstream homology arm amplification primers (approximately 40 bp) were designed. Using genomic DNA from Streptomyces SCSIO 40066 as a template, primers pasO4-UHA-F / pasO4-UHA-R and pasO4-DHA-F / pasO4-DHA-R were used to amplify the upstream and downstream homology arms of the pasO4 gene, designated ArmA and ArmB, respectively. The fragments were purified and recovered according to the instructions of the gel recovery kit. The temperature-sensitive shuttle plasmid pKC1139 was double-digested with EcoRI and HindIII, and the fragments were purified and recovered according to the instructions of the gel recovery kit. The linearized plasmid and upstream and downstream homology arms were assembled using the Gibson Assembly method to generate a plasmid for in-frame gene deletion, named pCSG6006. The plasmid was transformed into E. coli DH5α, and approximately 50 monoclonal colonies were selected for PCR verification. Five to ten monoclonal colonies were inoculated into 5 mL of LB liquid medium (containing an Apr-resistant strain) and cultured overnight at 37°C in a shaker at 200 rpm to generate the strain E. coli DH5α / pCSG6006.

[0080] Example 8: Construction of plasmid pCSG6007 for gene knockout

[0081] Based on the DNA and protein sequences of the pasO1 gene and the in-frame deletion gene knockout sequence, primers (approximately 40 bp) were designed to amplify the upstream and downstream homology arms. Using genomic DNA from Streptomyces SCSIO 40066 as a template, primers pasO1-UHA-F / pasO1-UHA-R and pasO1-DHA-F / pasO1-DHA-R were used to amplify the upstream and downstream homology arms of the pasO4 gene, designated ArmA1 and ArmB1, respectively. The fragments were purified and recovered according to the gel extraction kit instructions. The temperature-sensitive shuttle plasmid pKC1139 was double-digested with EcoRI and HindIII, and the fragments were purified and recovered according to the gel extraction kit instructions. The linearized plasmid and upstream and downstream homology arms were assembled using the Gibson Assembly method to generate a plasmid for in-frame gene deletion, named pCSG6007. The plasmid was transformed into E. coli DH5α, and approximately 50 monoclonal colonies were selected for PCR verification. Five to ten monoclonal colonies were inoculated into 5 mL of LB liquid medium (containing an Apr-resistant strain) and cultured overnight at 37°C in a shaker at 200 rpm to generate the strain E. coli DH5α / pCSG6007.

[0082] Example 9: Construction and screening of genomic cosmid library plasmids pCSG6001 and pCSG6002

[0083] A cosmid library of strain Streptomyces sp. SCSIO 40066 was constructed according to the instructions of the library packaging protein kit MaxPlaxTM Lambda Packaging Extracts (purchased from Epicentre, USA). The prepared genomic cosmid library template was screened using the designed library screening primers pasO1-cosmid-F1 / R1 and pasO4-cosmid-F1 / R1, and the target clones were located and inoculated into 5 mL of LB liquid culture medium (containing Kan resistance) and cultured overnight in a constant temperature shaker at 37°C (200 rpm) to obtain plasmids pCSG6001 and pCSG6002.

[0084] Example 10: Determination of antibacterial activity of compound PA-46101s

[0085] Compound PA-46101s was tested for inhibitory activity against Micrococcus luteus ML01, Bacillus subtilis 1064, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus MRSA, Vibrio alginolyticus 13214, Escherichia coli 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, and Acinetobacter baumannii ATCC 19606. The minimum inhibitory concentration (MIC) of the isolated compounds was determined by the broth microdilution method. The experimental method was based on the literature [Tan B, Chen S, Zhang Q, et al. Heterologous expression leads to discovery of diversified lobophorin analogues and a flexible glycosyltransferase[J]. Organic Letters, 2020, 22(3): 1062-1066.], with TMP, vancomycin, and AMP as controls. The results showed that PA-46101C-E had different degrees of inhibitory effects on Micrococcus luteus ML01, Bacillus subtilis 1064, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and methicillin-resistant Staphylococcus aureus MRSA, ranging from 0.125-64 μg mL -1The growth inhibition concentrations of PA-46101C against Micrococcus luteus ML01, Bacillus subtilis 1064, Bacillus siamese BS01, and Enterococcus faecalis ATCC 29212 were 2, 32, 32, and 64 μg mL -1 The growth inhibition concentrations of A-46101D against Micrococcus luteus ML01, Bacillus subtilis 1064, Bacillus siamea BS01, Enterococcus faecalis ATCC 29212, and methicillin-resistant Staphylococcus aureus MRSA were 2, 2, 2, 4, and 4 μg mL -1 The growth inhibition concentrations of A-46101E against Micrococcus luteus ML01, Bacillus subtilis 1064, Bacillus siamese BS01, and Staphylococcus aureus ATCC 29213 were 2, 32, 4, and 32 μg mL -1 Through bioengineering or chemical modification, it is expected to be developed into a new drug against Gram-positive bacteria.

[0086] Example 11: Enzyme activity inhibition assay of compound PA-46101s

[0087] The inhibitory activity of compound PA-46101s against tyrosinase, acetylcholinesterase, glucosidase and porcine pancreatic elastase was determined by in vitro assays to determine the half inhibitory concentration (IC50) of the isolated compound. 50 ), experimental methods refer to [Yang CF, Huang CS, Fang CY, et al. Inactivation of Flavoenzyme-Encoding Gene flsO in Fluostatin Biosynthesis Leads to Diversified Angucyclinone Derivatives[J]. Journal of Organic Chemistry, 2021, 86(16): 11019-11028.], kojic acid, huperzine potassium A, acarbose and sivelestat sodium were used as controls. The results showed that PA-46101C had a significant inhibitory effect on tyrosinase, IC 50 The value was 141.1±11.0μM (positive control was 195.1±4.25μM), which showed a moderate inhibitory effect on acetylcholinesterase, IC 50 The value was 11.03±0.17μM (positive control was 0.058±0.01μM), and it is expected to be developed into a new enzyme inhibitor drug through bioengineering or chemical modification.

[0088] PasO1 gene sequence (SEQ ID NO. 1)

[0089] PasO4 gene sequence (SEQ ID NO.2)

Claims

1. Compound PA-46101 C, D or E, whose structure is shown in formula (I):

2. Use of the compound PA-46101 C, D or E according to claim 1 in the preparation of antibacterial drugs.

3. The use according to claim 2, characterized in that: The antibacterial drug is a drug against Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

4. A method for preparing the compound PA-46101 C, D or E according to claim 1, characterized in that: PA-46101 C was isolated from the metabolites of strain Streptomyces sp.SCSIO 40066 / ΔpasO1; PA-46101 D was isolated from the metabolites of strain Streptomyces sp.SCSIO 40066 / ΔpasO4; PA-46101 E was isolated from the metabolites of strain Streptomyces sp.SCSIO 40066 / ΔpasO1O4; The strain Streptomyces sp.SCSIO 40066 / ΔpasO1 is obtained by knocking out the P450 oxidase encoding gene pasO1 in the pas gene cluster of Streptomyces sp.SCSIO 40066, and the nucleotide sequence of the gene pasO1 is shown in SEQ ID NO.1; The strain Streptomyces sp.SCSIO 40066 / ΔpasO4 is obtained by knocking out the P450 oxidase encoding gene pasO4 in the pas gene cluster of Streptomyces sp.SCSIO 40066, and the nucleotide sequence of the gene pasO4 is shown in SEQ ID NO.2; The strain Streptomyces sp.SCSIO 40066 / ΔpasO1O4 is obtained by firstly deleting the P450 oxidase encoding gene pasO4 in the pas gene cluster of Streptomyces sp.SCSIO 40066 in the same frame, and then deleting the P450 oxidase encoding gene pasO1 in the gene cluster in the same frame.

5. The preparation method according to claim 4, characterized in that: The specific steps are: Fermentation cultures of strains Streptomyces sp.SCSIO 40066 / ΔpasO1, Streptomyces sp.SCSIO 40066 / ΔpasO4 and Streptomyces sp.SCSIO 40066 / ΔpasO1O4 were prepared respectively, and the fermentation broth and mycelium of the fermentation culture were separated by centrifugation; the fermentation broth was adsorbed by macroporous resin, then eluted with acetone, concentrated under reduced pressure to recover acetone, the remaining aqueous phase was extracted with butanone, and then concentrated under reduced pressure to dryness to obtain extract A; the mycelium was firstly extracted with acetone, concentrated under reduced pressure to recover acetone, the remaining aqueous phase was extracted with butanone, and then concentrated under reduced pressure to dryness to obtain extract B, and the extract A was combined and B to obtain a crude fermentation extract; a. The fermentation crude extract of strain Streptomyces sp.SCSIO 40066 / ΔpasO1 was separated by normal phase silica gel column chromatography, gradient elution, elution solvent was chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1 and 0 / 1, v / v, 500 mL, and five fractions Frs.A1-A5 were separated in sequence. Thin layer chromatography analysis and HPLC high performance liquid chromatography detection determined that the target compound was concentrated in Frs.A2, and separated by gel chromatography column chromatography Sephadex LH-20, isocratic elution, elution solvent was chloroform / methanol = 1 / 1, v / v, each 15 mL was collected into one bottle, a total of 30 bottles were collected, and 8-12 bottles containing the target compound were combined to obtain subfraction Frs.A2-1 after HPLC detection, and the target compound was further separated and purified by semi-preparative high performance liquid chromatography, and the column parameters were: Phenomenex Kinetex C18, 250×4.6mm, 5μm; preparation conditions: acetonitrile / water, 70 / 30, v / v, 2.5mL / min, subfraction Frs.A2-1-1 was obtained, t R = 17.8min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC. The preparation conditions were chloroform / methanol, 10 / 1, v / v, R f =0.45, to obtain compound PA-46101 C; b. The crude extract of strain Streptomyces sp.SCSIO 40066 / ΔpasO4 was separated by normal phase silica gel column chromatography, gradient elution, elution solvent was chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1 and 0 / 1, v / v, 500 mL, and five fractions Frs.A1-A5 were separated in sequence. Thin layer chromatography analysis and HPLC high performance liquid chromatography detection determined that the target compound was concentrated in Frs.A2, and gel chromatography column chromatography Sephadex LH-20 was separated, isocratic elution, elution solvent was chloroform / methanol = 1 / 1, v / v, each 15 mL was collected into one bottle, a total of 30 bottles were collected, and 16-20 bottles containing the target compound were combined by HPLC detection to obtain subfraction Frs.A2-1, and the target compound was further separated and purified by semi-preparative high performance liquid chromatography, and the column parameters were: Phenomenex Kinetex C18, 250×4.6mm, 5μm; preparation conditions: acetonitrile / water, 75 / 25, v / v, 2.5mL / min, subfraction Frs.A2-1-1 was obtained, t R = 22.3min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC method, and the preparation conditions were chloroform / methanol / formic acid, 10 / 1 / 0.011, v / v, R f =0.60, to obtain compound PA-46101 D; c. The crude extract of strain Streptomyces sp.SCSIO 40066 / ΔpasO1O4 was separated by normal phase silica gel column chromatography, gradient elution, elution solvent was chloroform / methanol = 1 / 0, 10 / 1, 5 / 1, 1 / 1 and 0 / 1, v / v, 500 mL, and five fractions Frs.A1-A5 were separated in sequence. The target compound was determined to be concentrated in Frs.A2 by TLC and HPLC high-performance liquid chromatography. It was separated by gel chromatography column chromatography on Sephadex LH-20, isocratic elution, elution solvent was chloroform / methanol = 1 / 1, v / v, each 15 mL was collected into one bottle, and a total of 30 bottles were collected. After HPLC detection, 18-22 bottles containing the target compound were combined to obtain subfraction Frs.A2-1, and the target compound was further separated and purified by semi-preparative high-performance liquid chromatography. The column parameters were: Phenomenex Kinetex C18, 250×4.6mm, 5μm; preparation conditions: acetonitrile / water, 95 / 5, v / v, 2.5mL / min, subfraction Frs.A2-1-1 was obtained, t R = 13.2min, and finally, Frs.A2-1-1 was further separated and purified by semi-preparative TLC method. The preparation conditions were chloroform / methanol, 10 / 1, v / v, R f =0.55, and compound PA-46101 E was obtained.

6. The preparation method according to claim 5, characterized in that: The fermentation cultures of the strains Streptomyces sp.SCSIO 40066 / ΔpasO1, Streptomyces sp.SCSIO 40066 / ΔpasO4 and Streptomyces sp.SCSIO 40066 / ΔpasO1O4 are prepared by the following method: the activated strains are inoculated into conical flasks filled with A1 culture medium respectively, and cultured at 28°C and 200rpm to obtain seed liquid; the seed liquid is then inoculated into a conical flask filled with A1 culture medium at an inoculation rate of 10%, and cultured at 28°C and 200rpm with shaking to obtain fermentation culture, wherein the A1 culture medium contains 10g of fish meal, 10g of glycerol, 0.2g of KBr, 3g of CaCO3, 30g of sea salt, 1000mL of water, and a pH of 7.2-7.4 per liter.

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