Glycopeptide antibiotic with decapeptide skeleton and application thereof

Abstract

The invention relates to the technical field of biology, in particular to glycopeptide antibiotic with a decapeptide skeleton and application of the glycopeptide antibiotic. A brand new GPA biosynthetic gene cluster aqu is excavated, brand new glycopeptide antibiotics Aquilumycin and Aquilumycin M are synthesized in a heterologous host streptomyces coelicolor M1154, technical support is provided for preparation of the compounds, a strain and a separation and purification method are provided for large-scale fermentation production of Aquilumycin and Aquilumycin M in the future, candidate molecules are provided for research and development of new antibiotic drugs, and the new GPA biosynthetic gene cluster aqu can be used for preparing the new glycopeptide antibiotics Aquilumycin and Aquilumycin M. Important application prospects and economic values are realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a glycopeptide antibiotic with a decapeptide skeleton and applications thereof. Background Art

[0002] Glycopeptide antibiotics (GPA) are first-line drugs used clinically to treat Gram-positive resistant pathogens, such as methicillin-resistant Staphylococcus aureus infections. The first-generation GPAs commonly used clinically are vancomycin and teicoplanin. However, with the widespread use of GPA, GPA-resistant pathogens are constantly being detected clinically. The pathogen resistance report published by the US Centers for Disease Control and Prevention in 2019 pointed out that the detection rate of vancomycin-resistant enterococci in the United States is as high as 30%. Faced with the severe threat of GPA-resistant pathogens, researchers used chemical semi-synthetic technology to derive from the structural skeleton of natural glycopeptide antibiotics and developed the second-generation semi-synthetic glycopeptide antibiotics telavancin, oritavancin and dalbavancin. [1] , which can effectively inhibit the infection of vancomycin-resistant pathogens. Currently, the World Health Organization has listed antimicrobial resistance as one of the top ten global public health threats facing humanity. It is estimated that by 2050, antibiotic-resistant infections will claim up to 10 million lives each year and cause approximately $100 trillion in losses to the global economy. [2] The research and development of antibiotics with new structures and new mechanisms of action is of great significance for alleviating the crisis of antimicrobial resistance and protecting human life and health. With the advancement of science and technology, the innovation capacity of the pharmaceutical industry has been continuously improved. The application level of new antibiotics in my country has also increased significantly in recent years. At present, China has become the world's second largest antimicrobial drug market. [3] .

[0003] GPA has long been a hot topic in the research and development of new antibiotics. As an important class of clinical drugs, GPA plays an irreplaceable role in the treatment of infections such as methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Although the second-generation semi-synthetic glycopeptide antibiotics can effectively control infections caused by drug-resistant pathogens, with their application, corresponding drug-resistant pathogens will inevitably appear. With the continuous increase in the level of GPA resistance, the development of a new generation of highly effective GPA has become particularly urgent. At present, GPA can be divided into 5 subtypes according to the characteristics of its chemical structure: Type IV [4], usually composed of a heptapeptide or nonapeptide backbone, whose peptide backbone is highly modified by glycosylation, acylation and methylation to synthesize mature GPA. However, V-type GPA generally lacks post-glycosidic modifications. V-type GPA initially only included Complestatin and Kistamicin. With the rapid development of genome sequencing and bioinformatics, Wright's research group was the first to discover a new GPA with a nonapeptide backbone, Corbomycin. [5] Subsequently, the Wright team developed a new GPA synthetic biology discovery system (Glycopeptide antibiotic heterologous expression system, GPAHex). Using this platform, they discovered new V-type GPA GP6738, Rimomycin and Misaugamycin from three strains of Streptomyces, Streptomyces sp. WAC06738, Streptomyces sp. WAC 06783 and Streptomyces sp. WAC 00631. [6-7] Subsequent studies on the antibacterial mechanism showed that V-type GPA inhibits the activity of cell autolysins by binding to bacterial cell wall peptidoglycan, thereby inhibiting the degradation of the cell wall and, in turn, inhibiting bacterial division and proliferation. This unique antibacterial mechanism of V-type GPA enables it to exhibit broad-spectrum antibacterial activity against a variety of clinically resistant Gram-positive pathogens, making it the focus of clinical research and development of new antibiotics with new mechanisms. [8] .

[0004] References:

[0005] 1.Blaskovich MAT,Hansford KA,Butler MS,Jia Z,Mark AE,CooperMA.Developments in Glycopeptide Antibiotics.ACS Infect Dis.2018May 11;4(5):715-735.doi:10.1021 / acsinfecdis.7b00258.Epub 2018Feb 19. PMID: 29363950; PMCID: PMC5952257.

[0006] 2. Strathdee SA, Davies SC, Marcelin JR. Confronting antimicrobial resistance beyond the COVID-19 pandemic and the 2020US election. Lancet.

[0007] 3. Wu Ying. Global antibiotic market overview analysis[J]. Foreign Medical Antibiotics, 2012, 33(3):119-124.

[0008] 4. Nicolaou, KCBoddy, CNC S. et al. "Chemistry, biology, and medicine of the glycopeptide antibiotics," Angewandte Chemie International Edition, 1999, 38, 2096-2152.

[0009] 5. Koteva, K. Xu, M. Wang, W. et al. "Synthetic biology facilitates semisynthetic development of type V glycopeptide antibiotics targeting vancomycin-resistant Enterococcus," Journal of Medicinal Chemistry, 2023, 66, 9006-9022.

[0010] 6. Xu, M. Wang, W. Waglechner, N. et al. "GPAHex-A synthetic biology platform for Type IV–V glycopeptide antibiotic production and discovery," Nature Communications, 2020, 11, 5232.

[0011] 7. Xu, M. Wang, W. Waglechner, N. et al. "Phylogeny-informed syntheticbiology reveals unprecedented structural novelty in type V glycopeptideantibiotics," ACS Central Science, 2022, 8, 615-626.

[0012] 8. Culp, E. J. Waglechner, N. Wang, W. et al. "Evolution-guided discovery ofantibiotics that inhibit peptidoglycan remodelling," Nature, 2020, 578, 582-587. Summary of the Invention

[0013] This study used known glycopeptide antibiotic P450 enzyme sequences as templates to perform a BlastP search in NCBI. The retrieved gene sequences were downloaded and used antiSMASH to predict gene clusters involved in glycopeptide antibiotic synthesis in metabolic pathways. This led to the discovery of a novel glycopeptide antibiotic (GPA) biosynthetic gene cluster within the gene sequence of the Streptomyces aquilus JCM 33584 strain. Through direct cloning of the biosynthetic gene cluster and subsequent heterologous expression and fermentation experiments in Streptomyces, we confirmed that this GPA biosynthetic gene cluster, derived from Streptomyces aquilus JCM 33584, can be heterologously expressed in Streptomyces coelicolor M1154 to synthesize a novel decapeptide backbone glycopeptide antibiotic, aquilumycin. This study provides a new method for rapidly and massively synthesizing this class of compounds using commonly used microbial platforms such as S. coelicolor. Furthermore, a high-yield strain capable of heterologously expressing the novel glycopeptide antibiotic aquilumycin was developed, laying the foundation for further research and application of this class of compounds.

[0014] Therefore, the present invention discovered that the gene sequence of Streptomyces aquilus JCM 33584 carries a novel glycopeptide antibiotic (GPA) biosynthetic gene cluster. Through direct cloning of the biosynthetic gene cluster and heterologous expression and fermentation experiments in Streptomyces, we confirmed that this GPA biosynthetic gene cluster from Streptomyces aquilus JCM 33584 can be heterologously expressed in Streptomyces coelicolor M1154 to synthesize a novel decapeptide backbone glycopeptide antibiotic, aquilumycin. This provides a new method for the rapid and large-scale synthesis of such compounds using commonly used microbial platforms such as S. coelicolor. Furthermore, a high-yield strain capable of heterologously expressing the novel glycopeptide antibiotic aquilumycin was developed, laying the foundation for further research and application of this class of compounds.

[0015] Therefore, the present invention provides a glycopeptide antibiotic with a decapeptide backbone, which is the glycopeptide compound Aquilumycin or Aquilumycin M.

[0016] The structural formula of the compound Aquilumycin is as follows:

[0017]

[0018] The structural formula of Aquilumycin M is as follows:

[0019]

[0020] The present invention further provides the use of the decapeptide backbone glycopeptide antibiotic in the preparation of antibacterial products.

[0021] Specifically, the antibacterial activity refers to antifungal and antibacterial activity, specifically antifungal activity against the following bacteria: Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Mycobacterium smegmatis, Saccharomyces cerevisiae, Candida albicans or drug-resistant Staphylococcus aureus.

[0022] The present invention also provides a GPA biosynthetic gene cluster aqu, whose nucleotide sequence is shown in Sequence 1, which includes the following genes in order from the 5' end to the 3' end: aquR1 gene, aquR2 gene, aquR3 gene, aquA gene, aquB gene, aquC gene, aquD gene, aquE gene, aquF gene, aquG gene, aquH gene, aquI gene, aquT gene, aquR4 gene, aquR5 gene, hpgT gene, dgpA gene, dgpB gene, dgpC gene, dgpD gene, and aquJ gene;

[0023] Specifically, the nucleotide sequence of the aquR1 gene is shown in SEQ ID NO: 1.

[0024] The present invention also provides a recombinant vector containing the GPA biosynthesis gene cluster aqu.

[0025] Specifically, the target gene cluster was directionally cloned into the vector pCGW using the yeast transformation recombination cloning system.

[0026] The present invention provides a recombinant strain containing the GPA biosynthetic gene cluster aqu or the recombinant vector; specifically, the recombinant vector is electrotransferred into an Escherichia coli strain, and then the recombinant vector is transferred into Streptomyces coelicolor through triparental conjugation to obtain a recombinant strain.

[0027] The present invention also provides a method for preparing the compound Aquilumycin and / or Aquilumycin M, which comprises the following steps: fermenting and culturing the recombinant strain to produce the compound Aquilumycin and / or Aquilumycin M.

[0028] Furthermore, the method further comprises the step of isolating pure compound Aquilumycin and / or Aquilumycin M.

[0029] Optionally, the separation and purification method is separation and purification by extraction, molecular sieve chromatography, reverse phase medium pressure preparative chromatography and reverse phase high performance preparative chromatography system.

[0030] Compared with the existing technology, the present invention has the following beneficial effects: the present invention has discovered a new GPA biosynthetic gene cluster aqu, and synthesized new glycopeptide antibiotics Aquilumycin and Aquilumycin M in the heterologous host Streptomyces coelicolor M1154, providing technical support for the preparation of such compounds, providing strains and separation and purification methods for the future large-scale fermentation production of Aquilumycin and Aquilumycin M, and providing candidate molecules for the research and development of new antibiotic drugs, which has important application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the map of the plasmid pGP33584;

[0032] Figure 2 is the liquid phase diagram of the compound Aquilumycin;

[0033] Figure 3 is the liquid phase diagram of compound Aquilumycin M;

[0034] Figure 4 is the chemical structural formula of the compound Aquilumycin;

[0035] Figure 5 is the secondary mass spectrum of compound Aquilumycin;

[0036] Figure 6 is the chemical structural formula of the compound Aquilumycin M;

[0037] Figure 7 is the secondary mass spectrum of compound Aquilumycin M;

[0038] Figure 8 The NMR of compound Aquilumycin 1 H spectrum;

[0039] Figure 9 The NMR of compound Aquilumycin 13 C spectrum;

[0040] Figure 10 The NMR of compound Aquilumycin M 1 H spectrum;

[0041] Figure 11 The NMR of the compound Aquilumycin M 13 C spectrum. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0044] Example 1: Cloning of the biosynthetic gene cluster aqu and construction of its recombinant plasmid.

[0045] The present invention discovered a GPA biosynthetic gene cluster in Streptomyces aquilus JCM33584 by searching the NCBI database and cloned its genomic DNA. The specific implementation scheme is as follows:

[0046] 1) Add 50 mL of TSBY medium (containing 0.4%-0.5% glycine) to a 250 mL shake flask, add 2 mL of seed solution from a fresh tube, and incubate at 30°C, 250 rpm, and shake for 48 h.

[0047] 2) Collect the culture medium into a 50 mL centrifuge tube and centrifuge at 5,000 rpm for 10 minutes at room temperature to collect the cells;

[0048] 3) Weigh 0.5 g of fresh cells into a 2 mL centrifuge tube and wash twice with 1 mL of ddH2O.

[0049] 4) Transfer the cells to a 50 mL centrifuge tube and resuspend in 5 mL of SET solution (75 mM NaCl, 25 mM EDTA, 20 mM Tris-Cl, pH 7.5).

[0050] 5) Add lysozyme to a final concentration of 1 mg / mL and incubate at 37°C for 10 min;

[0051] 6) Add 1 / 10 volume of 10% SDS solution, slowly invert to mix, then add proteinase K to a final concentration of 0.5 mg / mL, slowly invert to mix, and incubate in a 55°C water bath for 2 h;

[0052] 7) After the incubation period, add 1 / 3 volume of 5M NaCl and 1 volume of CHCl3 and mix gently at room temperature until the solution becomes a uniform milky white.

[0053] 8) Centrifuge at 7,000 rpm, 4°C for 15 min. Transfer the upper layer to a new 50 mL centrifuge tube, avoiding aspirating the middle protein layer.

[0054] 9) Add 1 volume of CHCl3 and mix gently at room temperature until the solution becomes a uniform milky white;

[0055] 10) Centrifuge at 7,000 rpm, 4°C for 15 min. Transfer the upper layer to a new 50 mL centrifuge tube, avoiding aspirating the middle protein layer.

[0056] 11) Add 1 volume of pre-cooled isopropanol and mix slowly until flocs are formed, which is genomic DNA.

[0057] 12) Slowly pour the isopropyl alcohol along the wall and add 5 mL of 70% ethanol to wash twice;

[0058] 13) Pour out the 70% ethanol and place it on a clean bench to dry;

[0059] 14) Add 500 μL TE to dissolve genomic DNA;

[0060] 15) After the DNA is fully dissolved, add 1% RNase A and incubate at 37°C for 1 hour to degrade RNA.

[0061] 16) Add proteinase K to a final concentration of 0.5 mg / mL and incubate at 55°C for 1 h;

[0062] 17) Add an equal volume of CHCl3 for extraction and gently mix the two phases;

[0063] 18) Centrifuge at 13,300 rpm at 4°C for 10 min. Transfer the upper layer to a new 2 mL centrifuge tube and add 1 / 10 volume of 3M NaOAc (pH 5.2) to a final concentration of 0.3 M. Invert to mix thoroughly, then add 0.7-1 volume of pre-chilled isopropanol and mix slowly until flocs form, which is the DNA.

[0064] 19) Slowly pour the isopropyl alcohol along the wall and add 1 mL of 70% ethanol to wash twice;

[0065] 20) Pour out the 70% ethanol and place it on a clean bench to dry;

[0066] 21) Add 200 μL TE to dissolve genomic DNA;

[0067] After linearization of the obtained genomic DNA and the vector plasmid pCGW, a plasmid for the GPA biosynthetic gene cluster was constructed using the yeast transformation associated recombination (TAR) system (Zhang JJ, Yamanaka K, Tang X, Moore BS. Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination. Methods Enzymol. 2019; 621: 87-110. doi: 10.1016 / bs.mie.2019.02.026. Epub 2019 Mar 21. PMID: 31128791; PMCID: PMC6555405.). The specific implementation method is as follows:

[0068] 1) Inoculate a single clone of the yeast strain Sacchromyces cerevisiae VL6-48N into 3 mL of YPD liquid medium (supplemented with adenine to 100 μg / mL) and incubate at 30°C with shaking overnight.

[0069] 2) Transfer 2 mL of overnight culture to a new 100 mL YPD liquid medium (add Adenine to 100 μg / mL, 500 mL Erlenmeyer flask) and culture at 30°C with shaking until OD 600 =0.7~1.0;

[0070] 3) Place the Erlenmeyer flask on ice for 10 minutes, then transfer to a 50 mL centrifuge tube and centrifuge at 1800 g, 4°C for 3 minutes;

[0071] 4) Discard the supernatant, resuspend the cells in 50 mL of pre-chilled sterile water (4°C) by low-speed vortexing, and centrifuge at 1800 g for 3 min at 4°C;

[0072] 5) Discard the supernatant and resuspend the cells in 50 mL of pre-chilled 1 M sorbitol (4°C) by low-speed vortexing. Place the 50 mL centrifuge tube containing the yeast cell suspension on ice at 4°C overnight to stabilize the yeast cell osmotic pressure.

[0073] 6) Gently invert 50 mL of yeast cell suspension that has been ice-bathed at 4°C overnight to mix thoroughly, then centrifuge at 1800 g for 3 min at 4°C.

[0074] 7) Discard the supernatant and add 20 mL of SPE solution (0.01 M HEPES, pH 7.5; 25 mM EDTA; Sorbitol 18.2 g / 100 mL). Resuspend the cells by vortexing at low speed. Add 40 μL of β-mercaptoethanol and mix by inversion. Add 80 μL of zymolyase-20T and mix by inversion.

[0075] 8) Incubate in a 30°C incubator for 40 min;

[0076] 9) Measure the OD of the cell suspension 600 To check the protoplast level: Take two 200 μL aliquots of cells. Add one to 800 μL 2% SDS and the other to 800 μL 1M sorbitol. Mix by pipetting gently. Measure the OD value of the two suspensions. 600 The optical density values ​​at the two sites were measured and compared. When the difference between them was 10-20 times (about 90-95% of the cells were converted into protoplasts), the protoplasts had the best effect, and more than 75% could be used for subsequent experiments.

[0077] 10) Add pre-cooled 1 M sorbitol (4°C) to 50 mL along the tube wall, gently invert to mix, and centrifuge at 600 g, 4°C for 10 min.

[0078] 11) Slowly pour out the supernatant and suck out the remaining liquid with the tip of a pipette;

[0079] 12) Gently add 5 mL of pre-chilled 1 M sorbitol (4°C) along the wall to resuspend the cells. Mix thoroughly by slowly pipetting with a cut pipette tip to avoid creating bubbles that could damage the protoplasts.

[0080] 13) Slowly add pre-cooled 1 M sorbitol (4°C) along the wall to make up to 50 mL and invert to mix;

[0081] 14) Centrifuge at 600g, 4°C for 10 min to collect the cells;

[0082] 15) Slowly pour out the supernatant and remove any remaining liquid with a pipette tip;

[0083] 16) Add 2 mL of STC solution (0.01 M Tris-HCl, pH 7.5; 10 mM CaCl2; Sorbitol 18.2 g / 100 mL) and mix thoroughly by slowly pipetting with a sheared pipette tip to avoid creating bubbles that could damage the protoplasts.

[0084] 17) Let stand at room temperature for 10 minutes;

[0085] 18) Meanwhile, prepare a DNA solution in a 2 mL centrifuge tube containing 0.5 μg of linearized vector and 4 μg of genomic DNA;

[0086] 19) Using a pipette tip with a cut tip, slowly add 200 μL of yeast protoplasts to the DNA solution along the wall. Gently pipette to mix, avoiding bubbles.

[0087] 20) The protoplast / DNA mixture was allowed to stand at room temperature for 10 minutes;

[0088] 21) Add 800 μL of 20% PEG8000 solution and invert the tube 10 times to mix;

[0089] 22) Let stand at room temperature for 20 minutes;

[0090] 23) Centrifuge at 700 g, 4°C for 10 min to collect protoplasts.

[0091] 24) Aspirate the supernatant with a pipette and add 800 μL of SOS solution (6.6 mM CaCl2, 0.25 g yeast extract / 100 ml, 1 g peptone, 18.2 g Sorbitol / 100 ml). Resuspend the suspension by slowly pipetting with a cut pipette tip to avoid creating bubbles.

[0092] 25) Incubate the resuspended cells in a 30°C incubator for 30-40 minutes;

[0093] 26) Add the cell suspension to 8 mL of melted SD-Trp top screening agar (1 M sorbitol; 2% glucose; 2% agar powder; autoclave, cool, then add 50 mL of 10× Yeast Nitrogen Base and 5 mL of 100× adenine) (maintained at 60°C in a water bath) and mix by slowly pipetting with a cut pipette tip.

[0094] 27) Immediately pour the cell suspension onto a SD-Trp bottom screening agar plate (1 M sorbitol; 2% glucose; 1.5% agar powder autoclaved and cooled, followed by the addition of 50 mL 10× Yeast Nitrogen Base and 5 mL 100× adenine).

[0095] 28) After the SD-Trp agar plate solidifies, wrap it in tin foil and place it in a 30°C incubator in the dark for 3-5 days.

[0096] 29) Screen the positive yeast clones by PCR and extract the plasmids.

[0097] The nucleotide sequence of the GPA biosynthetic gene cluster of Streptomyces aquilus JCM 33584 is shown in SEQ ID NO: 1, which includes the following genes from the 5' end to the 3' end: aquR1 at positions 59312-9965, aquR2 at positions 58534-9190, aquR3 at positions 57210-8532, aquA at positions 47722-6988, aquB at positions 19189-41607, aquC at positions 18012-19181, aquD at positions 17701-18006, aquE at positions 17051-17638, aquF at positions 15753-17054, and aquF at positions 11347-1136. The gene sequences and functional annotations are shown in Table 1.

[0098] Table 1 shows the GPA biosynthetic gene cluster and its detailed functional annotation of Streptomyces aquilus JCM 33584.

[0099]

[0100] The GPA biosynthetic gene cluster was cloned into the plasmid vector pCGW to obtain plasmid pGP33584 ( Figure 1 ).

[0101] Example 2: Obtaining a high-yield recombinant strain containing the recombinant vector

[0102] Conjugative transfer is a mechanism by which genetic material (usually plasmid DNA) is transferred between bacteria and other single-celled organisms through direct contact. Plasmid pGP33584 was electroporated into the strain Escherichiacoli ET12567. Using E. coli ET12567 / pR9406 as a helper strain, the plasmid was transferred to Streptomyces coelicolor M1154 via triparental conjugative transfer, resulting in the generation of the recombinant strain S. coelicolor M1154 / pGP33584.

[0103] The specific implementation methods are as follows:

[0104] 1) Take 1 mL each of overnight cultures of E. coli ET12567 / pR9406 and E. coli ET12567 / pGP33584, wash twice with LB medium, and resuspend in 100 μL of LB medium;

[0105] 2) Take 20 μL of Treptomyces coelicolor M1154 spore suspension, discard the supernatant, resuspend in 100 μL of 2×YT medium, and incubate at 50°C.

[0106] After heat shock for 10 min, cool to room temperature;

[0107] 3) The above solutions were mixed evenly, plated, and cultured at 30°C to obtain the S. coelicolor M1154 / pGP33584 recombinant strain.

[0108] Example 3. Preparation, separation, purification and product characterization of fermentation products

[0109] The compounds synthesized by fermentation of the recombinant strain S. coelicolor M1154 / pGP33584 will be isolated and purified by extraction, molecular sieve chromatography, reversed-phase medium-pressure preparative chromatography, and reversed-phase high-performance preparative chromatography systems, and their chemical structures will be elucidated using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

[0110] The specific steps are as follows:

[0111] 1) Inoculate the seed solution at a 2% inoculum into SAM medium (15 g glucose; 15 g soy peptone; 5 g NaCl; 1 g yeast extract; 1 g CaCO3 (added last after pH adjustment); 2.5 mL glycerol; pH 6.8 (adjusted with NaOH)). After 7 days of fermentation, centrifuge at 7,000 rpm for 10 min, discard the supernatant, and transfer the cells to a 50 mL centrifuge tube.

[0112] 2) Add an equal volume of 20% methanol in water (containing 0.05% acetic acid) to each tube of mycelial pellet and vortex to mix thoroughly. Centrifuge at 4,000 rpm for 10 minutes and discard the supernatant.

[0113] 3) Wash the mycelia again with an equal volume of 20% methanol in water (containing 0.05% acetic acid), vortex to mix, centrifuge at 4,000 rpm for 10 min, and discard the supernatant.

[0114] 4) Add an equal volume of DMSO (1% ammonia) to extract the compound, vortex to mix, and sonicate for 10 minutes. Centrifuge at 4,000 rpm for 10 minutes. Pipette the supernatant into a new 50 mL centrifuge tube.

[0115] 5) Repeat step 4 and transfer the supernatant to a new 50 mL centrifuge tube;

[0116] 6) Take 100 μL of the supernatant obtained in steps 3 and 4, add 1 μL of acetic acid to adjust the pH, centrifuge at 13,000 rpm for 10 min, and transfer the supernatant to a liquid chromatography vial for liquid chromatography analysis;

[0117] Liquid phase analysis conditions:

[0118] Chromatographic column: Symmetry Shield RP8 Column, 3.5 μm, 4.6 mm × 150 mm (Waters); detector: PDA; sample volume: 10 μL

[0119] Mobile phase A: 0.1% TFA ultrapure water; Mobile phase B: 0.1% TFA acetonitrile;

[0120] Gradient program: t = 0 min, 5% mobile phase B, t = 20 min, 95% mobile phase B, t = 25 min, 95% mobile phase B, t = 26 min, 5% mobile phase B, t = 30 min, 5% mobile phase B

[0121] 7) Repeat the extraction step until the liquid chromatography test shows no compound in the extract, and then combine the extracts according to the purity of the compound indicated by the liquid chromatography results (a total of 5 extractions, 50 mL / extraction, a total of 250 mL);

[0122] 8) completely freeze-drying the extract;

[0123] 9) Dissolve each lyophilized sample in 25 mL of DMSO (ultrasonicate for 30 min until the solution is clear) and centrifuge at 12,000 rpm for 20 min;

[0124] 10) Methanol was added dropwise to the DMSO solution in step 9 to slowly precipitate, and the mixture was centrifuged at 12,000 rpm for 20 min. This was repeated three times until no more precipitate was formed after the addition of methanol.

[0125] 11) Combine the supernatants from step 10 and perform rotary evaporation to remove the methanol, then freeze-dry the sample overnight in a freeze dryer;

[0126] 12) Extract the precipitates from step 10 and step 11 three times with equal volumes of DMSO, and freeze-dry the combined extracts overnight;

[0127] 13) Take 100 μL of the sample from steps 11 and 12, centrifuge at 13,000 rpm for 10 min, and transfer the supernatant to a liquid chromatography vial for liquid chromatography analysis;

[0128] 14) The lyophilized sample from step 12 was fully resuspended in 5 mL of DMSO and an equal volume of 50% acetonitrile aqueous solution (1% ammonia solution) was added;

[0129] 15) Centrifuge at 8,000 rpm for 10 min, and apply the supernatant to an LH20 molecular sieve column (40 × 610 mm) for separation.

[0130] 16) Elute with 50% acetonitrile in water (1% ammonia) as the mobile phase, collecting fractions in 10 mL / tube;

[0131] 17) Take 100 μL of the fraction obtained in step 16, add 1 μL of acetic acid to adjust the pH, centrifuge at 13,000 rpm for 10 min, and transfer the supernatant to a liquid chromatography vial for liquid chromatography analysis;

[0132] 18) Based on the purity of the compound indicated by the liquid chromatography results, the fractions from step 16 were combined, concentrated, and lyophilized on a freeze dryer;

[0133] 19) Dissolve the lyophilized sample in 2 mL of DMSO and purify it on a medium-pressure preparative column;

[0134] 20) Medium pressure preparation conditions:

[0135] Column: Airs Science Flash C8, 20-35 μm, 40g; detector: PDA; flow rate: 30mL / min

[0136] Mobile phase A: 0.1% Formic acid in ultrapure water; Mobile phase B: 0.1% Formic acid in acetonitrile;

[0137] Gradient program: t = 0 min, 5% mobile phase B, t = 20 min, 95% mobile phase B, t = 25 min, 95% mobile phase B, t = 26 min, 5% mobile phase B, t = 30 min, 5% mobile phase B

[0138] 21) Transfer 100 μL of the eluate obtained in step 20 to a liquid chromatography vial for liquid chromatography analysis;

[0139] 22) Based on the liquid phase results, the collected solutions were combined and freeze-dried;

[0140] 23) Sample weighing;

[0141] Aquilumycin and aquilumycin M were obtained through preparative liquid phase purification with retention times of 9 minutes and 10 minutes, respectively. High-resolution primary and secondary mass spectra of aquilumycin and aquilumycin M were acquired using a Sciex TripleTOF 6600. NMR spectra were acquired on a Bruker AVANCE III 600 MHz NMR spectrometer using samples dissolved in DMSO-d6.

[0142] Figure 2 and Figure 3 These are the liquid phase diagrams of compounds Aquilumycin and Aquilumycin M, respectively. Figure 4 and Figure 6 These are the chemical structural formulas of compounds Aquilumycin and Aquilumycin M, respectively. Figure 5 and Figure 7 They are the secondary mass spectra of compounds Aquilumycin and Aquilumycin M respectively; Figure 8 and Figure 10 are the NMR spectra of compounds Aquilumycin and Aquilumycin M, respectively. 1 H spectrum; Figure 9 and Figure 11 The NMR spectra of compounds Aquilumycin and Aquilumycin M are 13 C spectrum.

[0143] Example 4: Application of Aquilumycin and Aquilumycin M

[0144] Aquilumycin and Aquilumycin M were tested for their antibacterial activity, focusing on Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Mycobacterium smegmatis, Saccharomyces cerevisiae, Candida albicans, and drug-resistant Staphylococcus aureus. The minimum inhibitory concentration (MIC) was used to measure the antibacterial activity of the target compounds against different strains.

[0145] The specific steps are as follows:

[0146] 1) Activate the strain by plate streak method;

[0147] 2) Pick a single colony from the above sample and place it in 4 mL of LB medium. Incubate the culture in a shaking incubator at 37°C overnight.

[0148] 3) Dilute the bacterial solution 100 times with physiological saline and measure the OD 600 Numeric value;

[0149] 4) According to the OD in step 3 600 Dilute the bacterial solution in step 3 with the test medium to a final concentration of OD 600 =0.0004-0.0005

[0150] 5) Use a dispenser to add the bacterial solution to a 96-well plate: add 198 μL of bacterial solution to each well in the first column (the amount can be adjusted based on the amount of test compound added, for a total volume of 200 μL), add 100 μL of bacterial solution to each well in columns 2 to 11, and add 100 μL of culture medium to each well in column 12 (column 11 is a positive control for bacterial growth, and column 12 is a negative control for culture medium);

[0151] 6) Add 2 μL of compound to each well in the first column and mix thoroughly by pipetting (avoid introducing bubbles during pipetting).

[0152] 7) Use a dispenser to draw 100 μL of sample from the first column and add it to the second column, pipette and mix, then draw 100 μL from the second column and add it to the third column, pipette and mix, and so on, until 100 μL is drawn from the ninth column and added to the tenth column (gradient dilution), and finally draw 100 μL from the tenth column and discard.

[0153] 8) Place the 96-well plate in an incubator and incubate for 16-20 hours, then read the corresponding MIC value of the compound.

[0154] Experiments have shown that Aquilumycin has antibacterial activity against Bacillus subtilis and Staphylococcus aureus, while Aquilumycin M has antibacterial activity against Enterococcus faecalis and Mycobacterium smegmatis. Specific antibacterial activity data are shown in Table 2.

[0155] Table 2. MIC antibacterial activity results

[0156]

[0157] The nucleotide sequence of SEQ ID NO: 1 is as follows:

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Claims

1. A glycopeptide antibiotic with a decapeptide backbone, characterized in that: It is the glycopeptide compound Aquilumycin or Aquilumycin M.

2. Use of the decapeptide backbone glycopeptide antibiotic according to claim 1 in the preparation of antibacterial products.

3. The use according to claim 2, characterized in that The antibacterial agent refers to antifungal and antibacterial agents, specifically antifungal agents against the following bacteria: Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Mycobacterium smegmatis, Saccharomyces cerevisiae, Candida albicans or drug-resistant Staphylococcus aureus.

4. A GPA biosynthetic gene cluster aqu, characterized in that The genes included in order from the 5' end to the 3' end are: aquR1 gene, aquR2 gene, aquR3 gene, aquA gene, aquB gene, aquC gene, aquD gene, aquE gene, aquF gene, aquG gene, aquH gene, aquI gene, aquT gene, aquR4 gene, aquR5 gene, hpgT gene, dgpA gene, dgpB gene, dgpC gene, dgpD gene, and aquJ gene; Specifically, the nucleotide sequence of the aquR1 gene is as shown in SEQ ID NO: 59312-9965, the nucleotide sequence of the aquR2 gene is as shown in SEQ ID NO: 58534-9190, the nucleotide sequence of the aquR3 gene is as shown in SEQ ID NO: 57210-8532, the nucleotide sequence of the aquA gene is as shown in SEQ ID NO: 47722-6988, the nucleotide sequence of the aquB gene is as shown in SEQ ID NO: 19189-41607, and the nucleotide sequence of the aquR4 gene is as shown in SEQ ID NO: 1 The nucleotide sequence of the uC gene is shown in SEQ ID NO: 18012-19181, the nucleotide sequence of the aquD gene is shown in SEQ ID NO: 17701-18006, the nucleotide sequence of the aquE gene is shown in SEQ ID NO: 17051-17638, the nucleotide sequence of the aquF gene is shown in SEQ ID NO: 15753-17054, the nucleotide sequence of the aquG gene is shown in SEQ ID NO: 15034-15756, the nucleotide sequence of the aquH gene is shown in SEQ ID NO: 1 The nucleotide sequence of the aquI gene is shown in SEQ ID NO: 13778-15037, the nucleotide sequence of the aquI gene is shown in SEQ ID NO: 13253-13477, the nucleotide sequence of the aquT gene is shown in SEQ ID NO: 11077-13212, the nucleotide sequence of the aquR4 gene is shown in SEQ ID NO: 9762-10769, the nucleotide sequence of the aquR5 gene is shown in SEQ ID NO: 6682-9447, the nucleotide sequence of the hpgT gene is shown in SEQ ID NO: 1 Positions 5221-6561 as shown in SEQ ID NO: 1, the nucleotide sequence of the dgpA gene is as shown in SEQ ID NO: 1, positions 4009-5151, the nucleotide sequence of the dgpB gene is as shown in SEQ ID NO: 1, positions 2031-3329, the nucleotide sequence of the dgpD gene is as shown in SEQ ID NO: 1, positions 1222-2034, and the nucleotide sequence of the aquJ gene is as shown in SEQ ID NO: 1; More preferably, the nucleotide sequence is as shown in SEQ ID NO: 1 or a degenerate sequence thereof.

5. A recombinant vector containing the GPA biosynthetic gene cluster aqu according to claim 4.

6. The recombinant vector according to claim 5, wherein The target gene cluster was cloned into the vector pCGW using the yeast transformation recombination cloning system.

7. A recombinant strain containing the GPA biosynthetic gene cluster aqu according to claim 4 or the recombinant vector according to claim 5; specifically, the recombinant vector according to claim 5 or 6 is electroporated into an Escherichia coli strain, and the recombinant vector is transferred into Streptomyces coelicolor by triparental conjugation to obtain the recombinant strain.

8. A method for preparing the compound Aquilumycin and / or Aquilumycin M, characterized in that: The method comprises the following steps: fermenting and culturing the recombinant strain as claimed in claim 7 to produce the compound Aquilumycin and / or Aquilumycin M.

9. The method according to claim 8, wherein The method also includes the step of isolating pure compound Aquilumycin and / or Aquilumycin M.

10. The method according to claim 8, wherein The separation and purification method is to separate and purify through extraction, molecular sieve chromatography, reverse phase medium pressure preparative chromatography and reverse phase high performance preparative chromatography system.

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