Antioxidant active polypeptide catdehas, preparation method thereof and application of antioxidant active polypeptide catdehas in preparation of antioxidant drugs

Abstract

The invention relates to antioxidant active polypeptide catdehas, a preparation method thereof and application of the antioxidant active polypeptide catdehas in preparation of antioxidant drugs. And the antioxidant active polypeptide catdehas is selected from the following compounds 1-3. The compound provided by the invention is a non-standard amino acid derivative modified by 2, 3-dihydroxybenzoyl and obtained from streptomycete by means of gene cloning and heterologous expression, and in an antioxidant experiment, the compound shows remarkable DPPH free radical scavenging capacity, and can be used for preparing an antioxidant drug. Therefore, the compound has wide application value in various fields as a novel antioxidant. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a compound with antioxidant activity, its preparation method and antioxidant use; more specifically, it relates to a class of non-ribosomal peptide compounds modified with 2,3-dihydroxybenzoyl group obtained by in vitro cloning and expression of a Streptomyces biosynthetic gene cluster, a preparation method of such compounds and their application in antioxidant. Background Art

[0002] Oxidative damage is the damage of cells and tissues caused by reactive oxygen species (ROS). ROS include superoxide, hydrogen peroxide and hydroxyl radicals, etc., which are produced during normal metabolism, but can cause cell damage when in excess. Oxidative damage is closely related to a variety of diseases and the aging process. For example, oxidative stress is considered an important pathogenic factor for cardiovascular diseases, cancer, diabetes and neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). In addition, oxidative damage is also related to skin aging, decreased immune function and enhanced inflammatory response. Therefore, developing compounds with antioxidant activity is of great significance for the prevention and treatment of these diseases.

[0003] Streptomyces is a treasure trove of natural active ingredients. However, it has become increasingly difficult to discover new active substances from Streptomyces by traditional methods. Heterologous expression refers to introducing foreign genes into host cells to express and produce corresponding proteins or metabolites in the host cells. This technology has played an important role in the discovery and production of novel bioactive natural products. Through heterologous expression, bioactive natural products can be efficiently produced in microorganisms, plants or animal cells, overcoming the problems of limited sources and difficult extraction of natural products.

[0004] DPPH (1,1-diphenyl-2-picrylhydrazyl) is widely used in antioxidant experiments. DPPH has a stable free radical structure. In antioxidant experiments, DPPH free radicals will react with antioxidants, and the color changes from purple to yellow, so that the activity of antioxidants can be evaluated by measuring the color change. Therefore, the DPPH experiment is an effective means for screening and discovering antioxidants. Summary of the Invention

[0005] Technical Objectives

[0006] One of the objectives of the present invention is to provide a class of non-ribosomal peptide compounds with antioxidant activity.

[0007] Another objective of the present invention is to provide a biosynthetic gene cluster of non-ribosomal peptide compounds.

[0008] Another objective of the present invention is to provide a preparation method of the above compounds.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising the above compound.

[0010] Another object of the present invention is to provide the relevant applications of the above compound in industries with antioxidant requirements.

[0011] Technical content

[0012] On the one hand, the present invention provides a class of compounds with antioxidant activity (also referred to as non-ribosomal peptide compounds catedehas herein), and the compounds are selected from the following Compounds 1-3:

[0013]

[0014] On the other hand, the present invention provides a biosynthetic gene cluster of non-ribosomal peptide compound catedehas, and the biosynthetic gene cluster comprises the elements shown in the following table:

[0015]

[0016]

[0017] In some embodiments, the biosynthetic gene cluster comprises the elements shown in the following table:

[0018]

[0019]

[0020] In some embodiments, the biosynthetic gene cluster comprises the bases at positions 294146-341799 of the nucleotide sequence of NCBI Reference Sequence: CP023702.1.

[0021] The biosynthetic gene cluster comprises 35 coding genes and 47,654 bases. It includes non-ribosomal peptide synthetase coding genes, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase coding genes, regulatory genes, iron transport protein genes, and multifunctional (2,3-dihydroxybenzoyl) adenylate synthetase coding genes.

[0022] On yet another aspect, the present invention provides a vector comprising the catedehas biosynthetic gene cluster of the present invention.

[0023] In some embodiments, the vector is a recombinant plasmid obtained by introducing the catedehas biosynthetic gene cluster of the present invention into pBE45 or pBE44 plasmid.

[0024] On the other hand, the present invention provides a host bacterium comprising the above vector.

[0025] On yet another aspect, the present invention provides a method for preparing the above compound, the method comprising the following steps:

[0026] 1) In vitro clone the biosynthetic gene cluster of the above non-ribosomal peptide compound catedehas and prepare a recombinant strain for its heterologous expression;

[0027] 2) Ferment the recombinant strain for heterologous expression in step 1), after fermentation, centrifuge to separate the mycelium and the fermentation broth, and extract the mycelium and the fermentation broth with methanol and ethyl acetate respectively to obtain extract A and extract B;

[0028] 3) Mix the extract A and the extract B in step 2) to obtain a total extract, and separate and purify the total extract by normal phase, reverse phase column chromatography and preparative liquid chromatography to obtain compound 1, compound 2 and compound 3.

[0029] In a specific embodiment, step 1) is carried out through the following processes:

[0030] 1-1) Extract the genomic DNA of Streptomyces S. nitrosporeus ATCC 12769;

[0031] 1-2) Synthesize the guide RNA (gRNA) at both ends of the cda gene cluster and the pBE45 and pBE44 backbone DNAs (receivers). Among them, the gRNA molecule is obtained by in vitro transcription of a dsDNA template with a size of 60 bp formed by annealing two ssDNA oligonucleotides. The dsDNA template consists of a T7 promoter sequence and a gRNA sequence. The primers are shown in the table. The pBE45 and pBE44 receivers respectively contain 39 bp homologous fragments at both ends of the gene cluster. The amplification primer sequences are shown in the following table:

[0032]

[0033]

[0034] 1-3) Digest to obtain the cda gene cluster by FnCas12a;

[0035] 1-4) In vitro assemble the biosynthetic gene cluster cda recombinant plasmid and transform it into E. coli pBE14 Cre competent cells, and verify the recombinant plasmid by in vitro PCR; and

[0036] (1-5) The recombinant plasmid was transferred into the donor bacterium E. coli WM6026, and Streptomyces albus J1074 was used as the recipient bacterium for conjugation transfer to obtain the engineered bacterium S. lividans TK24::cda.

[0037] In a specific embodiment, in step 2), the fermentation medium used was MYM liquid medium, containing 10 g / L malt extract broth, 4 g / L yeast extract, and 4 g / L maltose monohydrate.

[0038] In a specific embodiment, in step 3), the normal-phase silica gel used was of 100-200 mesh, and petroleum ether / ethyl acetate was used as the eluent for gradient elution, with the volume ratios of petroleum ether / ethyl acetate being 9:1, 8:2, 7:3, 5:5, pure ethyl acetate, and pure methanol for elution.

[0039] In a specific embodiment, in step 3), the packing material used for reverse-phase chromatography was ODS-18, the packing material for semi-preparative high-performance liquid chromatography column was ODS-18, and the eluent was an acetonitrile-water system with a volume ratio of 3:7 to 7:3.

[0040] On the other hand, the present invention also provides an engineered bacterium S. lividans TK24::cda constructed according to step 1) of the above method.

[0041] On the other hand, the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of the above compound and optionally a pharmaceutically acceptable carrier.

[0042] On the other hand, the present invention provides the use of the above compound as an antioxidant in the preparation of drugs, cosmetics, health products or foods.

[0043] In a specific embodiment, the drug can be used to treat cardiovascular diseases, diabetes, nervous system diseases (Alzheimer's disease and Parkinson's disease, Down syndrome), mental diseases (depression, schizophrenia, bipolar disorder), kidney diseases, and lung diseases (chronic obstructive pulmonary disease, lung cancer) by inhibiting the reactive oxygen species (ROS) pathway.

[0044] In a specific embodiment, the compound exerts an antioxidant effect by scavenging free radicals.

[0045] Technical effects

[0046] The compounds involved in the present invention (named catedehas herein, also known as non-ribosomal peptide compound catedehas) are a class of non-canonical amino acid derivatives with 2,3-dihydroxybenzoyl modification obtained from Streptomyces by means of gene cloning and heterologous expression. In antioxidant experiments, they exhibit significant DPPH radical scavenging ability, and thus have broad application value as novel antioxidants in various fields. Brief Description of the Drawings

[0047] Figure 1 : Colony PCR verification of the recombinant plasmid of the catedehas biosynthetic gene cluster cda.

[0048] Figure 2 : Restriction enzyme digestion verification of the recombinant plasmid of the catedehas biosynthetic gene cluster cda.

[0049] Figure 3 : HPLC results of the heterologous expression of the catedehas biosynthetic gene cluster cda. In the figure, 1, 2, and 3 correspond to Compounds 1 - 3.

[0050] Figure 4 : Structures of Compounds 1 - 3.

[0051] Figure 5 : Antioxidant activities of Compounds 1 - 3. *** indicates p < 0.001 vs. the positive drug ascorbic acid.

[0052] Figure 6 : For Compound 1 1 1H NMR spectrum.

[0053] Figure 7 : For Compound 1 13 13C NMR spectrum.

[0054] Figure 8 : For Compound 1 1 1H- 1 1H COSY spectrum.

[0055] Figure 9 : HSQC spectrum of Compound 1.

[0056] Figure 10 : HMBC spectrum of Compound 1.

[0057] Figure 11 : For Compound 2 1 1H NMR spectrum.

[0058] Figure 12 : For Compound 2 13 13C NMR spectrum.

[0059] Figure 13 : The 1 H- 1 H COSY spectrum of Compound 2.

[0060] Figure 14 : The HSQC spectrum of Compound 2.

[0061] Figure 15 : The HMBC spectrum of Compound 2.

[0062] Figure 16 : The NOESY spectrum of Compound 2.

[0063] Figure 17 : The 1 H NMR spectrum of Compound 3.

[0064] Figure 18 : The 13 C NMR spectrum of Compound 3.

[0065] Figure 19 : The 1 H- 1 H COSY spectrum of Compound 3.

[0066] Figure 20 : The HSQC spectrum of Compound 3.

[0067] Figure 21 : The HMBC spectrum of Compound 3.

[0068] Figure 22 : The NOESY spectrum of Compound 3. Detailed implementation manners

[0069] In the following, the implementation schemes of the present invention will be described in detail in combination with the implementation manners. However, the following implementation manners and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0070] Term

[0071] In this application, the term "catedehas biosynthetic gene cluster" can be abbreviated as: "cda" or "cda gene cluster".

[0072] Materials, reagents and methods

[0073] The strains and plasmids used in this study are shown in Table 1 below. The strains required for the cloning system were kindly provided by Professor Zhao Huimin, and the Streptomyces strains were purchased from the China General Microbiological Culture Collection Center. Chemical reagents and other molecular biology reagents were purchased from standard commercial sources and used according to the manufacturer's recommendations. DPPH was purchased from Shanghai Macklin Biochemical Co., Ltd., and the positive control ascorbic acid was purchased from Shanghai Aladdin Biochemical Reagent Co., Ltd.

[0074] 1H, 13C, and 2D NMR spectra were recorded at 298 K on an Avance NEO 600 MHz nuclear magnetic resonance spectrometer (Bruker) equipped with a cryoprobe.

[0075] FID measurements of each compound were performed using standard COSY, HSQC, HMBC, and NOESY experiments with solvent suppression, with a 1H spectral width of 11,905 Hz. The FIDs of the COSY, HSQC, and HMBC spectra were recorded with 1024, 512, and 2048 data points, respectively, and the integration of the NOESY spectrum with 1024 and 256 data points. The mixing times used for COSY, HSQC, HMBC, and NOESY were 100 - 150 ms, 50 - 100 ms, 200 - 250 ms, and 100 - 150 ms, respectively.

[0076] High-resolution electrospray ionization mass spectrometry (HRESIMS) data were determined using an Agilent 6230 LC-TOF MS mass spectrometer and a Sciex ZenoTOF 7600 system. The experimental conditions included a source temperature of 480 °C, ion source gas 1 at 50 psi, ion source gas 2 at 45 psi, positive polarity, a spray voltage of 5500 V, and a total scan time of 0.278 s.

[0077] Analytical HPLC was performed on an Agilent 1260 Infinity series instrument equipped with a DAD detector, using an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). The solvent system consisted of solvent A (water containing 0.1% trifluoroacetic acid) and B (acetonitrile containing 0.1% trifluoroacetic acid). The elution process was run with the following program: 5% B to 50% B (linear gradient, 0 - 15 min), 50% B to 80% B (linear gradient, 15 - 20 min), 80% B to 100% B (20 - 21 min), 100% B (isocratic elution, 21 - 24 min), 100% B to 5% B (24 - 25 min), 5% B (isocratic elution, 25 - 30 min); the flow rate was 1 ml / min. -1 。

[0078] Table 1

[0079]

[0080]

[0081] Example 1 Cloning of the Catedehas Biosynthetic Gene Cluster and Construction of Recombinant Plasmids

[0082] 1. Mining of the Catedehas Biosynthetic Gene Cluster

[0083] Through bioinformatics analysis of the genomic sequence of Streptomyces nitrosporeus ATCC 12769 (GenBank accession number: GCA_000715845.1), the inventors located the biosynthetic gene cluster cda of Catedehas, and its encoding genes are shown in Table 2, including non-ribosomal peptide synthetase (NRPS) encoding gene cassettes, multiple post-modification genes, regulatory genes, transporter-encoding genes, etc. The nucleotide sequence of the biosynthetic gene cluster cda of non-ribosomal peptide compounds is the nucleotide sequence from position 294146 to 341799 of the nucleotide sequence of NCBI Reference Sequence: CP023702.1.

[0084] Table 2

[0085]

[0086]

[0087]

[0088]

[0089] 2. Cloning of the biosynthetic gene cluster of Catedehas non-ribosomal peptide compounds

[0090] 1) Extraction of genomic DNA (gDNA) of Streptomyces nitrosporeus ATCC 12769

[0091] The spore suspension of S. nitrosporeus ATCC 12769 was inoculated into 50 mL of MYG liquid medium (10 g / L malt extract broth, 4 g / L yeast extract, 4 g / L glucose) at a ratio of 0.1%, and cultured in a shaking flask at 28 °C until the late exponential growth phase. Subsequently, the cells were collected by centrifugation at 4000 rpm for 10 min. The cell pellet was resuspended in 12 mL of resuspension buffer (50 mM Tris-HCl, pH 8.0, 25 mM EDTA). Then, 20 mg of lysozyme and 0.5 mg of RNase A were added, and after thorough mixing, the mixture was incubated at 37 °C for 2 hours. Then, 5 mg of proteinase K was added and incubated at 37 °C for 1 hour. Next, 1.2 mL of 10% SDS was added and incubated at 50 °C for 2 hours. After that, 4.8 mL of 5 mol / L NaCl was added, and the mixture was mixed well and left at room temperature for 20 minutes. Finally, gDNA was recovered by phenol-chloroform extraction.

[0092] 2) Synthesis of guide RNA (gRNA) at both ends of the cda gene cluster and pBE45 and pBE44 backbone DNA (receivers).

[0093] The gRNA molecule was obtained by in vitro transcription using a T7 High Yield Transcription Kit (Thermo Scientific) with a 60 bp dsDNA template formed by annealing two ssDNA oligonucleotides. The dsDNA template consists of a T7 promoter sequence and a gRNA sequence. The primer sequences are shown in Table 2.

[0094] pBE45 and pBE44 receivers were amplified using the pBE45 and pBE44 plasmids (https: / / www.addgene.org / 174654 / ) as templates with Phanta Max Super-Fidelity DNA Polymerase (Vazyme). The reaction system and PCR amplification program were operated according to the product instructions. pBE45 and pBE44 receivers each contain 39 bp homologous fragments at both ends of the gene cluster. The amplification primer sequences are shown in Table 3.

[0095] Table 3

[0096]

[0097]

[0098] 3) FnCas12a-mediated targeted cleavage of the gene cluster

[0099] After mixing the reaction system shown in Table 3, incubate at 37 °C for 2 h, then incubate at 65 °C for 30 min. Then add 3 μL of 10 mg / mL RNase A, mix well and incubate at 37 °C for 30 min. Finally, add 3 μL of 20 mg / mL proteinase K, mix well and incubate at 50 °C for 30 min. Transfer the reaction solution to a Phase Lock Gel (TIANGEN) centrifuge tube, and extract the cda gene cluster digested by FnCas12a using the phenol-chloroform method.

[0100] The reaction system is shown in Table 4 below:

[0101] Table 4

[0102]

[0103] 3. In vitro assembly and transformation of the recombinant plasmid of the Catedehas biosynthetic gene cluster cda

[0104] The reaction system is shown in Table 5 below:

[0105] Table 5

[0106]

[0107] The above reaction system was first incubated at 65 °C for 10 min without adding T4 DNA polymerase. After cooling to room temperature, T4 enzyme was added, and then incubated at 25 °C for 1 h, 75 °C for 20 min, and 50 °C for 30 min. Then 1 μL of 1 mM NAD + , 0.4 μL of 10 mM dNTPs, 3 U of T4 DNA polymerase and 1 μL of E. coli DNA ligase were added, and incubated at 37 °C for 1 h and 75 °C for 20 min. The reaction solution was dropped onto an MF-Millipore TM filter membrane above distilled water for dialysis for 30 min.

[0108] 10 μL of the dialyzed reaction solution was added to 70 μL of prepared E. coli pBE14 Cre competent cells (Enghiad B, Huang C, Guo F, Jiang G, Wang B, Tabatabaei SK, Martin TA, Zhao H. 2021. Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination. Nat Commun. 12(1):1171.). Transient electroporation was performed using a Gene Pulser XCell Electroporation system (Bio-Rad) (1 mm cuvette, 1250 V, 100 Ω, 25 μF). Then it was added to 1 mL of LB liquid medium containing 5 mM MgCl2, and shaken at 37 °C for 75 min. Finally, the cells were collected and evenly spread on an LB solid medium (50 μg / mL apramycin, 0.2 mM IPTG, 40 mg / L X-Gal).

[0109] 4. In vitro verification of the recombinant plasmid of the Catedehas biosynthetic gene cluster cda

[0110] Multiple white single colonies were picked for PCR identification. The verified band length was 722 bp. The results were as Figure 1 shown. The clone corresponding to the correct target band was transferred to 5 mL of LB liquid medium and shaken at 37 °C for 15 h for plasmid extraction (Hipure BAC DNA Mini Kit, Magen). The plasmid was digested with restriction enzymes BstBⅠ and MfeⅠ. The results were as Figure 2As shown, the bands after enzymatic digestion are all correct, indicating the successful construction of the recombinant plasmid of the catedehas biosynthetic gene cluster cda.

[0111] Example 2 Heterologous expression of the recombinant plasmid of the catedehas biosynthetic gene cluster cda

[0112] The recombinant plasmid constructed in Example 1 was transferred into E. coli WM6026 (containing 40 μg / mL 2,6-diaminopimelic acid) as the donor bacterium, and Streptomyces albus J1074 was used as the recipient bacterium for conjugation transfer. The conjugants were grown on SFM solid medium (15 g / L mannitol, 15 g / L soybean powder, 2 g / L NaCl, 15 g / L CaCO3, 20 g / L agar, pH 7.0 - 7.4). After about 3 days of growth, spores of about 4 mm 2 in size were taken into 20 μL DMSO, shaken at 100 °C and 2000 rpm for 20 min, centrifuged at 20000 g for 30 s, and 2 μL of the supernatant was taken into a 25 μL system for Colony PCR identification. The correct conjugants were harvested with 40% glycerol spores, inoculated at 1‰ into 50 mL of MYM liquid medium (10 g / L malt extract broth, 4 g / L yeast extract, 4 g / L maltose monohydrate), and shaken at 28 °C for 5 days to obtain the engineered strain S. lividans TK24::cda.

[0113] The fermentation broth was centrifuged, and the supernatant was mixed with ethyl acetate at a ratio of 1:1 for extraction. The ethyl acetate phase was concentrated under reduced pressure and the crude extract was dissolved in methanol. After analysis by high performance liquid chromatography (HPLC) and comparison with the control group S. lividans TK24 (without the cda gene cluster), three new absorption peaks corresponding to compounds 1, 2, and 3 were found, as Figure 3 shown, indicating the successful heterologous expression of the recombinant plasmid of the Catedehas biosynthetic gene cluster cda.

[0114] Example 3 Preparation of Compounds 1, 2, and 3

[0115] 1. Isolation and purification of monomeric compounds

[0116] 1) The engineered strain S. lividans TK24::cda in Example 2 was inoculated into MYM liquid medium (10 g / L malt extract broth, 4 g / L yeast extract, 4 g / L maltose monohydrate) at 0.1%, and shaken at 28 °C for 5 - 7 days.

[0117] 2) Separate the fermentation broth and mycelium of the fermentation culture. The mycelium is extracted with acetone three times. The remaining aqueous mixture after recovering acetone from the extract is then extracted with ethyl acetate. The ethyl acetate phase is concentrated under reduced pressure to obtain extract A; the fermentation broth is extracted with ethyl acetate at a volume ratio of 1:1 (v / v). The ethyl acetate phase is concentrated under reduced pressure to obtain extract B.

[0118] 3) Mix extract A and extract B. The mixed extracts A and B are mixed with silica gel of 100 - 200 mesh for sample loading, packed into a column by dry method, and gradient elution is carried out using petroleum ether / ethyl acetate as the eluent (volume ratios 9:1, 8:2, 7:3, 5:5), pure ethyl acetate, and pure methanol for elution. TLC is used for detection, and the same fractions are collected to obtain Fr.1 - Fr.6 in sequence.

[0119] 4) The fraction Fr.5 eluted with ethyl acetate is separated by repeated gel column chromatography (Sephadex-LH20, methanol), preparative thin layer chromatography (PTLC), and semi-preparative high performance liquid chromatography (semi-HPLC); compounds 1 - 3 are obtained by purification through high performance liquid chromatography (HPLC).

[0120] The solvent system used in HPLC includes solvent A (water added with 0.1% trifluoroacetic acid) and B (acetonitrile added with 0.1% trifluoroacetic acid). The following professional program is run during the elution process: 5% B to 50% B (linear gradient, 0 - 15 min), 50% B to 80% B (linear gradient, 15 - 20 min), 80% B to 100% B (20 - 21 min), 100% B (isocratic elution, 21 - 24 min), 100% B to 5% B (24 - 25 min), 5% B (static elution, 25 - 30 min), and the flow rate is 1 ml min -1 。

[0121] 2. Structure identification of monomeric compounds

[0122] The structures of the new compounds 1 - 3 were identified by 1D NMR, 2D NMR, MS, IR, optical rotation, etc. ( Figure 4 ) and named catedehas A (1), catedehas B (2), catedehas C (3). The specific physicochemical data are as follows:

[0123] Compound 1: 1 H NMR (600 MHz, DMSO-d6) δ H6.97 (1H, d, J = 7.0 Hz, H-4), 6.76 (1H, dd, J = 7.0, 7.9 Hz, H-5), 7.39 (1H, d, J = 7.9 Hz, H-6), 5.79 (2H, s, 8-CH2), 6.54 (2H, s, 8-CH2), 10.67 (1H, s, 9-NH). 13 13C NMR (150 MHz, DMSO-d6) δ C 119.1 (C-1), 145.3 (C-2), 146.0 (C-3), 118.6 (C-4), 119.2 (C-5), 120.3 (C-6), 164.4 (C-7), 107.8 (C-8), 133.1 (C-9), 165.0 (C-10).

[0124] Compound 2: 1 1H NMR (600 MHz, CD3OD) δ H 6.96 (1H, dd, J = 1.3, 7.7 Hz, H-4), 6.76 (1H, dd, J = 7.7, 8.1 Hz, H-5), 7.38 (1H, dd, J = 1.3, 8.1 Hz, H-6), 5.96 (2H, s, 8-CH2), 6.53 (2H, s, 8-CH2), 6.95 (1H, dd, J = 1.2, 7.8 Hz, H-4’), 6.74 (1H, dd, J = 7.8, 8.1 Hz, H-5’), 7.32 (1H, dd, J = 1.2, 8.1 Hz, H-6’), 4.67 (2H, dd, J = 6.0, 11.3 Hz, 8’-CH2), 4.77 (2H, dd, J = 4.2, 11.3 Hz, 8’-CH2), 5.08 (1H, dd, J = 4.2, 6.0 Hz, H-9’), 3.78 (1H, s, H-11’). 13 13C NMR (150 MHz, CD3OD) δ C 118.9 (C-1), 147.8 (C-2), 147.1 (C-3), 119.8 (C-4), 120.3 (C-5), 121.0 (C-6), 167.9 (C-7), 111.5 (C-8), 133.4 (C-9), 164.9 (C-10), 116.9 (C-1’), 149.6 (C-2’), 147.2 (C-3’), 120.0 (C-4’), 119.9 (C-5’), 119.7 (C-6’), 170.8 (C-7’), 65.6 (C-8’), 53.2 (C-9’), 171.1 (C-10’), 53.3 (C-11’).

[0125] Compound 3:1 1H NMR (600 MHz, CD3OD) δ H 6.96 (1H, dd, J = 1.5, 7.9 Hz, H-4), 6.76 (1H, dd, J = 7.9, 8.1 Hz, H-5), 7.36 (1H, dd, J = 1.5, 8.1 Hz, H-6), 5.92 (2H, s, 8-CH2), 6.48 (2H, s, 8-CH2), 6.94 (1H, dd, J = 1.4, 7.8 Hz, H-4’), 6.72 (1H, dd, J = 7.8, 8.1 Hz, H-5’), 7.30 (1H, dd, J = 1.4, 8.1 Hz, H-6’), 4.68 (2H, dd, J = 5.9, 11.4 Hz, 8’-CH2), 4.75 (2H, dd, J = 4.1, 11.4 Hz, 8’-CH2), 5.05 (1H, dd, J = 4.1, 5.9 Hz, H-9’), 6.91 (1H, dd, J = 1.5, 7.8 Hz, H-4”), 6.70 (1H, dd, J = 7.8, 8.1 Hz, H-5”), 7.27 (1H, dd, J = 1.5, 8.1 Hz, H-6”), 4.55 (2H, dd, J = 5.8, 11.4 Hz, 8”-CH2), 4.77 (2H, dd, J = 4.1, 11.4 Hz, 8”-CH2), 5.02 (1H, dd, J = 4.1, 5.8 Hz, H-9”), 3.71 (1H, s, H-11”). 13 13C NMR (150 MHz, CD3OD) δ 118.9 (C-1), 147.9 (C-2), 147.1 (C-3), 119.8 (C-4), 120.3 (C-5), 121.0 (C-6), 167.9 (C-7), 111.6 (C-8), 133.3 (C-9), 164.9 (C-10), 116.9 (C-1'), 149.5 (C-2’), 147.2 (C-3’), 120.2 (C-4’), 119.9 (C-5’), 119.8 (C-6’), 170.9 (C-7’), 65.6 (C-8’), 53.3 (C-9’), 170.2 (C-10’), 116.7 (C-1”), 149.6 (C-2”), 147.2 (C-3”), 120.0 (C-4”), 119.9 (C-5”), 119.6 (C-6”), 170.8 (C-7”), 65.5 (C-8”), 53.2 (C-9”), 171.0 (C-10”), 53.2 (C-11”).

[0126] Determination of the antioxidant activities of compounds catedehas A - C (1–3) in Test Example 1

[0127] 1. Experimental principle

[0128] DPPH (1,1-diphenyl-2-picrylhydrazyl) is a very stable free radical. Its methanol solution is purple and has a maximum absorption at a wavelength of 518 nm. After adding an antioxidant, DPPH will capture the free single electron of the antioxidant. After pairing with it, its color fades, and the absorption at 518 nm disappears. The degree of fading is quantitatively related to the number of electrons received. Therefore, the antioxidant capacity is quantitatively determined by colorimetry (spectrophotometer).

[0129] 2. Experimental procedures

[0130] 1) Add samples or the positive drug ascorbic acid with concentration gradients (6.25 - 200 μM) dissolved in 50 μL of DMSO to a 96-well plate, and then immediately add 150 μL of DPPH methanol solution (0.2 mM, freshly prepared), and pipette to mix well.

[0131] 2) Place the above 96-well plate in the dark for 30 minutes, and measure the absorbance value A at 518 nm using a microplate reader.

[0132] 3) Add 150 μL of DPPH methanol solution (0.2 mM, freshly prepared) to 50 μL of blank DMSO as a negative control, and measure its absorbance value as A0; if the sample has color, then add 150 μL of methanol to 50 μL of the DMSO solution of the corresponding concentration of the sample, and record its absorbance as A blank ; The calculation method for the scavenging rate of each sample against DPPH free radicals is as follows:

[0133] Scavenging rate = (A0 - (A - A blank )) / A0 × 100%

[0134] 3. Test results

[0135] The IC of the scavenging rate of compounds catedehas A–C (1–3) against DPPH free radicals was collected and calculated through the above experiments 50 (repeated 3 times, with errors expressed as ±SD). The results are shown in Table 6, and the concentrations required to scavenge 50% of the free radicals are 27.52, 12.51, and 8.32 μM respectively.

[0136] Table 6

[0137]

[0138]

[0139] Statistical analysis using GraPhadprism10 software found that the IC of compounds 1–3 50Significantly less than that of the positive drug ascorbic acid (93.63 μM, p < 0.001), as Figure 5 shown. This indicates that compounds 1–3 have significant antioxidant activity and have important value for development into antioxidant drugs and related preparations.

[0140] The description presented in the above exemplary embodiments is only for the purpose of illustrating the technical solutions of the present invention and is not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, many changes and variations are possible for those of ordinary skill in the art according to the above teachings. The selection of the exemplary embodiments and the description thereof are for the purpose of explaining the specific principles of the present invention and its practical applications, so that other technical personnel in the art can easily understand, implement and utilize various exemplary embodiments of the present invention and their various alternative forms and modifications. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

Claims

1. A class of non-ribosomal peptide compounds, which are selected from the following compounds 1-3:

2. A biosynthetic gene cluster of the non-ribosomal peptide compound according to claim 1, the biosynthetic gene cluster comprising the elements shown in the following table:

3. The biosynthetic gene cluster according to claim 2, the biosynthetic gene cluster comprising or consisting of the elements shown in the following table:

4. The biosynthetic gene cluster according to claim 2 or 3, the biosynthetic gene cluster comprising the nucleotides at positions 294146-341799 of the nucleotide sequence of NCBI Reference Sequence: CP023702.

1.

5. A vector, which comprises the biosynthetic gene cluster according to any one of claims 2-4.

6. The vector according to claim 5, which is a recombinant plasmid obtained by introducing the biosynthetic gene cluster into the pBE45 or pBE44 plasmid.

7. A host bacterium, which comprises the vector according to any one of claims 4-6.

8. A method for preparing the non-ribosomal peptide compound according to claim 1, the method comprising the following steps: 1) In vitro clone the biosynthetic gene cluster according to any one of claims 2-4 and prepare a recombinant strain for its heterologous expression; 2) Ferment the recombinant strain for heterologous expression in step 1), after fermentation, centrifuge to separate the mycelium and the fermentation broth, and extract the mycelium and the fermentation broth with methanol and ethyl acetate respectively to obtain extract A and extract B; 3) Mix the extract A and the extract B in step 2) to obtain a total extract, and separate and purify the total extract by normal phase, reverse phase column chromatography and preparative liquid chromatography to obtain compound 1, compound 2 and compound 3.

9. The method according to claim 8, wherein Step 1) is carried out through the following processes: 1-1) Extract the genomic DNA of Streptomyces S. nitrosporeus ATCC 12769; 1-2) Synthesize the guide RNA (gRNA) at both ends of the biosynthetic gene cluster (cda gene cluster) and the pBE45 and pBE44 backbone DNAs (receivers), wherein the gRNA molecule is obtained by in vitro transcription of a dsDNA template with a size of 60 bp formed by annealing two ssDNA oligonucleotides, the dsDNA template consists of a T7 promoter sequence and a gRNA sequence, the primers are shown in the following table, the pBE45 and pBE44 receivers respectively contain 39 bp homologous fragments at both ends of the gene cluster, and the amplification primer sequences are shown in the following table: 1-3) Digest to obtain the cda gene cluster by FnCas12a; 1-4) In vitro assemble the recombinant plasmid of the biosynthetic gene cluster cda and transform it into E. coli pBE14 Cre competent cells, and verify the recombinant plasmid by in vitro PCR; and 1-5) Transfer the recombinant plasmid into the donor bacterium E. coli WM6026, and perform conjugation transfer using the model Streptomyces S. albus J1074 as the recipient bacterium to obtain the engineered bacterium S. lividans TK24::cda.

10. An engineered bacterium S. lividans TK24::cda constructed according to step 1) of claim 9.

11. A pharmaceutical composition comprising a therapeutically effective amount of the non-ribosomal peptide compound as claimed in claim 1, and optionally a pharmaceutically acceptable carrier.

12. Use of the non-ribosomal peptide compound as claimed in claim 1 as an antioxidant in the preparation of a drug, a cosmetic, a health product or a food, preferably, the drug is used for treating cardiovascular diseases, diabetes, neurological diseases, mental diseases, kidney diseases or lung diseases by inhibiting the reactive oxygen species pathway.

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