Use of a group of peptide compounds with antiviral activity, omicine b group compounds

Abstract

The present application relates to a group of peptide compounds with antiviral activity, the structure of the peptide compound is shown in formula (1): the virus is a respiratory virus, especially influenza virus or coronavirus. The present application also relates to a gene cluster for biosynthesis of the peptide compound by microorganisms, the gene cluster is Streptomyces sp. CPCC 200451 genome chromosome 1:7,822,964-7,875,615, with a full length of 52.6 kb.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and more specifically, relates to the application of a group of peptide compounds, specifically Omixin B compounds, which have antiviral activity. Background Technology

[0002] Highly contagious viral infections pose a serious threat to human health. Among them, severe acute respiratory syndrome (SARS) caused by coronaviruses, which has been prevalent since the beginning of the new century, is notorious for its strong infectivity and high mortality rate. Meanwhile, influenza caused by influenza viruses and avian influenza, which circulate globally each year, have become a significant public health concern due to their wide reach and high mortality rates. These highly contagious viral diseases urgently require safe and effective broad-spectrum antiviral drugs, especially anti-coronavirus drugs, for the effective treatment of the novel coronavirus.

[0003] Recently, whole-genome sequencing data of the 2019-nCoV coronavirus, published in the *New England Journal of Medicine* on January 24, 2019, and *The Lancet* on January 29, 2019, by the Chinese Center for Disease Control and Prevention (CCDC) and other institutions, showed that it shares only 79% homology with the SARS coronavirus, classifying it as a novel coronavirus distinct from SARS. The 2019-nCoV coronavirus genome encodes 29,844 bases in protein-coding regions, encoding 12 proteins: 1ab, S, 3, E, M, 7, 8, 9, 10b, N, 13, and 14. Among these, 1ab encodes a non-structural protein precursor polyprotein composed of 7,096 amino acids, S encodes the spike protein, and it also contains other envelope proteins such as E, M, and N proteins. Generally speaking, the 7096 amino acid non-structural precursor polyprotein encoded by 1ab is cleaved by the virus-encoded proteases 3CLpro and PLpro to form 16 non-structural proteins (NSPs), most of which are involved in the formation of the viral replication complex [1,2]. According to the research on drug targets related to SARS coronavirus and Middle East Respiratory Syndrome coronavirus MERS, the homology comparison study of the 2019-nCoV coronavirus genome shows that the key drug targets that can be utilized by 2019-nCoV are the interaction between the spike protein and angiotensin-converting enzyme 2 (ACE2) on the human cell membrane, i.e., the entry mechanism, the RNA-dependent RNA polymerase RdRp, and the cysteine ​​proteases 3CLpro and PLpro responsible for hydrolyzing the 7096 amino acid polyprotein into functional proteins [3].

[0004] Influenza virus belongs to the genus Influenza A of the family Orthomyxoviridae. Avian influenza A virus particles are pleomorphic, with spherical particles having a diameter of 80-120 nm and an envelope. The genome is a segmented single-stranded negative-sense RNA. Based on the different antigenicity of its outer membrane hemagglutinin (H) and neuraminidase (N) proteins, it can currently be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9) [4]. Among them, the hemagglutinin (HA) on the surface of influenza virus exists in the form of precursor protein HA0 before being cleaved into HA1 and HA2 by intracellular proteases in the host cell. Therefore, host cell proteases are crucial for viral infection [5].

[0005] Respiratory-associated viruses such as coronaviruses, influenza viruses, and parainfluenza viruses require host cell proteases to cleave and activate viral proteins before they can enter and replicate in respiratory epithelial cells [6]. Therefore, inhibitors of these host cell-encoded proteases may have broad-spectrum antiviral activity against these respiratory-associated viruses. More importantly, antiviral drugs targeting host cell proteases can also effectively prevent viral escape and mutation.

[0006] Microbial-derived natural products are the main source of new anti-infective antibiotics. According to statistics, among the existing 25,000 microbial secondary metabolites with certain biological activities, about 10% of microbial metabolites have antiviral activity. For example, the anti-syncytial virus drug ribavirin comes from the microbial secondary metabolite ribavirin. Broad-spectrum antiviral antibiotics such as spongiurin, vidarabine, fosfomycin, and formycin also come from microbial natural products or modifications of microbial natural products [7].

[0007] Finding new drug lead compounds from natural products has always been a research hotspot. Compared with secondary metabolites of plants and animals, microbial secondary metabolites have the characteristics of resource sustainability and no damage to the ecological environment, and therefore have greater development and utilization value.

[0008] As early as the 1960s, researchers at the Institute of Pharmaceutical Biotechnology isolated and screened a Streptomyces sp. CPCC 200451 strain with good antiviral activity from soil samples collected in southern my country. They then used classic screening methods and ion-exchange resin column chromatography to obtain its active component from the fermentation broth of CPCC 200451. This component was tested in clinical trials as an antiviral drug against human influenza virus, with excellent results in reducing high fever when administered as nasal drops of the extract solution. The study also found that this active component exhibited high sensitivity to various viruses, such as influenza virus, coronavirus, and Newcastle disease virus. However, due to limitations in experimental conditions and isolation methods at the time, and the use of harsh conditions such as strong acids during the isolation process, a stable and precise pharmacologically accurate component of Streptomyces sp. CPCC 200451 could not be obtained, and the structure of the antiviral active ingredient could not be determined.

[0009] With the rapid development of whole-genome DNA sequencing technology for microorganisms, more and more microbial genomes have been sequenced and information sharing has been achieved. In addition, a series of cutting-edge technologies such as bioinformatics and molecular biology have been widely applied in the field of genome research, which has greatly accelerated the process of researchers mining microbial gene resources [8]. Studies have found that the biosynthetic genes of microbial secondary metabolites are often arranged in clusters and have a high degree of conservation. Through bioinformatics analysis of microbial genomes, the discovery and analysis of related secondary metabolite gene clusters can infer the structure and physicochemical properties of the products, and can also guide the separation and purification of target compounds [9]. The rise of bioinformatics has not only provided a new opportunity for the development of microbial drugs, but also played a very important role in the discovery of new microbial secondary metabolites, and has provided new research ideas for us to solve the problem of identifying antiviral active compounds produced by CPCC200451.

[0010] With the arrival of the "post-genomic era", omics technologies such as transcriptomics and metabolomics based on high-throughput sequencing have emerged and been widely used

[10] . Transcriptomics is the collection of all transcripts produced by organisms in a certain functional state. At present, the research object of prokaryotic transcriptome sequencing is mainly mRNA. By comparing the transcriptome of microorganisms under different fermentation conditions, the changes in gene expression profiles can be obtained, thereby finding the biosynthetic gene information that leads to expression differences [11,12]. Combining genome sequencing, bioinformatics and other analysis techniques can help locate the biosynthetic gene cluster of target metabolites. Metabonomics refers to the dynamic whole of endogenous metabolites in organisms. Since microorganisms can produce different secondary metabolites under different fermentation conditions, and the diversity of metabolites is attributed to the diversity of biosynthetic genes, the combined application of genomics, transcriptomics and metabolomics analysis can not only detect the differences in metabolites from the phenomenon, but also explain the reasons for the changes in metabolites at the gene level

[13] . Therefore, by analyzing the changes in the transcriptome and metabolome of microorganisms under active fermentation conditions and comparing them with gene expression and metabolites under inactive fermentation conditions—that is, activity-oriented comparative transcriptomics and comparative metabolomics analysis—we can help us find activity-related biosynthetic gene clusters and metabolites, and guide the isolation, purification, and structural analysis of target compounds.

[0011] To identify the effective antiviral components in Streptomyces sp. CPCC 200451, we started with the whole genome information of Streptomyces sp. CPCC 200451. Through genomic bioinformatics analysis, we identified secondary metabolism-related gene clusters. Using activity-guided comparative transcriptomics analysis, we pinpointed the biosynthetic gene clusters of active substances in CPCC 200451. Combining molecular biology techniques, we performed genetic manipulations such as knockout and overexpression of key genes in the target gene clusters to determine the biosynthetic gene clusters containing the active components of CPCC 200451. Furthermore, through activity-guided comparative metabolomics analysis, we obtained the structural characteristics of the active substances to aid in the isolation and structural confirmation of the target products. By comprehensively utilizing bioinformatics and chemical separation techniques, we isolated the active substance monomers, ultimately identifying the pharmacodynamic components responsible for the antiviral activity of Streptomyces sp. CPCC 200451. Currently, we have discovered that some of the antiviral compounds produced by Streptomyces sp. CPCC200451 are known protease inhibitors, such as antipain and chymostatin. This indicates that the omexine series of compounds can target proteases in both viruses and host cells, which may explain their antiviral activity against a variety of respiratory-associated viruses, especially coronaviruses and influenza viruses. [References]

[0012] [1]Na Zhu, Dingyu Zhang, Wenling Wang, et al., A Novel Coronavirus from Patients with Pneumonia in China, The New England Journal of Medicine, January 24, 2020, DOI: 10.1056 / NEJMoa2001017.

[0013] [2]Nanshan Chen, Min Zhou, Xuan Dong, et al., Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study, The Lancet, January 30, 2020, DOI: 10.1016 / S0140-6736(20)30211-7.

[0014] [3]Guangdi Li&Erik De Clercq, Therapeutic options for the 2019 novelcoronavirus(2019-nCoV), Nature, 10th February, 2020, doi:10.1038 / d41573-020-00016-0.

[0015] [4]Bernadeta Dadonaite, Brad Gilbertson, et al.The structure of theinfluenza A virus genome,Nature Microbiology|VOL 4|NOVEMBER 2019|1781–1789

[0016] [5] Chen Wei, Wang Lili, Research progress on anti-influenza virus drug targets [J], International Journal of Pharmaceutical Research, 2013, 40(1):1-7

[0017] [6] Manon Laporte, Lieve Naesens, et al., Airway proteases: an emerging drug target for influenza and other respiratory virus infections. Current Opinion in Virology, 2017, 24, 16-24.

[0018] [7]Naoki Takizawa and Manabu Yamasaki, Current landscape and future prospects of antiviral drugs derived from microbial products, The Journal of Antibiotics (2018) 71, 45–52.

[0019] [8] Chen Ming. Bioinformatics in the post-genomic era [J]. Bioinformatics, 2004, 21(2):29-34.

[0020] [9]Kim ES J.Midostaurin:First Global Approval[J].Drugs,2017,77(11):1-9.

[0021]

[10] Lockhart D J,Winzeler E A.Genomics,gene expression and DNA arrays[J].Nature,2000,405(6788):827-836.

[0022]

[11] Velculescu V E,Zhang L,Zhou W,et al.Characterization of the yeasttranscriptome[J].Cell,1997,88(2):243-251.

[0023]

[12] Wang Z,Gerstein M,Snyder M.RNA-Seq:a revolutionary tool fortranscriptomics[J].Nature Reviews Genetics,2009,10(1):57-63.

[0024]

[13] Tang J.Microbial metabolomics[J].Current Genomics,2011,12(6):391-403.

[0025]

[14] Benson D A,Cavanaugh M,Clark K,et al.GenBank[J].Nucleic AcidsResearch,2017,45(D1):D37-d42.

[0026]

[15] Finn R D,Coggill P,Eberhardt R Y,et al.The Pfam protein familiesdatabase:towards a more sustainable future[J].Nucleic Acids Research,2016,44(D1):D279-285.

[0027]

[16] Eddy S R.Accelerated Profile HMM Searches[J].PLoS ComputationalBiology,2011,7(10):e1002195.

[0028]

[17] Wang Shiyuan. Research progress on the mechanism of action and application of nonribosomal peptide synthases (NRPSs) [J]. Acta Microbiologica Sinica, 2007, 47(4):734-737.

[0029]

[18] Suda H, Aoyagi T, Hamada M, et al. Antipain, a new protease inhibitor isolated from actinomycetes[J]. Journal of Antibiotics (Tokyo), 1972, 25(4): 263-266.

[0030]

[19] Umezawa H, Aoyagi T, Morishima H, et al. Chymostatin, a newchymotrypsin inhibitor produced by actinomycetes[J]. Journal of Antibiotics (Tokyo), 1970, 23(8): 425-427.

[0031]

[20] Umezawa H, Aoyagi T, Okura A, et al. Letter: Elastatinal, a newelastase inhibitor produced by actinomycetes [J]. Journal of Antibiotics (Tokyo), 1973, 26 (12): 787-789.

[0032]

[21] Maxson T, Tietz JI, Hudson GA, et al. Targeting Reactive Carbonyls for Identifying Natural Products and Their Biosynthetic Origins[J]. Journal of the American Chemical Society, 2016, 138(46): 15157-15166.

[0033]

[22] Imker HJ,Walsh CT,Wuest W M.SylC catalyzes ureido-bondformation during biosynthesis of the proteasome inhibitor syringolin A[J].Journal of the American Chemical Society,2009,131(51):18263-18265.

[0034]

[23] Chen Y, Mcclure RA, Zheng Y, et al. Proteomics guided discovery offlavopeptins: anti-proliferative aldehydes synthesized by a reductase domain-containing non-ribosomal peptide synthetase[J]. Journal of the American Chemical Society, 2013, 135(28): 10449-10456.

[0035]

[24] Yan N.Structural Biology of the Major Facilitator SuperfamilyTransporters[J].Annual Review of Biophysics,2015,44:257-283. Summary of the Invention

[0036] This invention first relates to the application of a group of peptide compounds (Omicin) in the preparation of antiviral drugs, wherein the structure of the peptide compounds is shown in formula (1):

[0037]

[0038] The various substituents that can be selected for R1 to R5 are shown in the table below:

[0039]

[0040]

[0041] Preferably, the virus is a respiratory virus;

[0042] The most preferred virus is an influenza virus or a coronavirus.

[0043] This invention also relates to a method for preparing the aforementioned peptide compound (Ommicin), the method comprising the following steps:

[0044] (1) Fermentation of Streptomyces sp. CPCC 200451, and collection of supernatant after centrifugation of fermentation broth (4000 rpm / min, 15 min);

[0045] (2) Active components were collected by HPLC using a cascaded reversed-phase C18 chromatography column with macroporous adsorption resins.

[0046] (3) The active component obtained in step (2) is separated by semi-preparative RP-HPLC to obtain the peptide compound Omicin.

[0047] The fermentation described in step (1) is as follows:

[0048] The inoculum was transferred to A3 fermentation medium at a rate of 10%, and cultured at 28℃ and 200 rpm for 3–10 days. The fermentation broth was then collected.

[0049] The A3 fermentation medium contains the following components (in g / L): glycerol 20, dextrin 20, peptone 10, yeast extract 5, ammonium sulfate 2, calcium carbonate 2; pH 7.2-7.4.

[0050] The step parameters for the macroporous adsorption resin cascade reversed-phase HPLC method in step (2) are as follows:

[0051] HP20 macroporous adsorption resin and C18 reversed-phase HPLC column were used.

[0052] The separation steps are as follows:

[0053] 1) After the supernatant is adsorbed by the macroporous adsorption resin Diaion HP20, it is washed with two column volumes of deionized water.

[0054] 2) Use ethanol-water gradient elution (elute with 20%, 50% and 100% ethanol in sequence), elute with each gradient until the eluent is colorless or the color remains unchanged, collect the eluent of the 50% ethanol gradient, concentrate it under reduced pressure and use it for later use.

[0055] 3) After concentrating the 50% ethanol gradient eluent obtained in step 2), perform C18 column chromatography with acetonitrile-water gradient elution (10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 80%, and 100% acetonitrile), and collect 15% to 80% of the gradient eluent. Preferably, collect 15% to 20% of the acetonitrile-water eluent.

[0056] The semi-preparative RP-HPLC parameters and methods described in step (3) are as follows: Column: SHISEIDO Capcell-Pak PFP 5μm, 10×250mm; Mobile phase:

[0057] Omekaxin A series compounds: 25% ACN / H2O containing 0.1% TFA;

[0058] Omekaxin B series compounds: 20% ACN / H2O containing 0.1% HCOOH;

[0059] Omekaxin C-series compounds: 40% ACN / H2O containing 0.1% TFA;

[0060] Flow rate: 1.5 mL / min.

[0061] This invention also relates to a new group of peptide compounds (Omicoxin), namely Omicoxin A1, A2, A6, B1, B2, B3, B5, B6, C1, C2, and C6, with the general structural formula shown in formula (1). The substituents of R1 to R4 of each compound are shown in the table below:

[0062]

[0063]

[0064] This invention also relates to gene clusters for the microbial biosynthesis of said peptide compounds.

[0065] The gene cluster mentioned is chromosome 1:7,822,964-7,875,615 of the genome of Streptomyces sp. CPCC 200451, with a full length of 52.6kb;

[0066] Preferably, the gene cluster is gene 7092-7102 (chromosome 1:7,841,516-7,857,514), with a total length of 15.99kb.

[0067] Among them, genes 7094 and 7098 are key biosynthetic genes of omega-3, and the amino acid sequences of their encoded proteins are shown in SEQ ID NO.1 and 2, respectively. Gene 7102 is a positive regulatory gene of the omega-3 biosynthetic gene cluster, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.3.

[0068] The present invention also relates to a regulatory protein 7102 that enhances the expression level of the omegaxin gene cluster, the amino acid sequence of which is shown in SEQ ID NO.3, and its expression level is directly proportional to the content of omegaxin in the fermentation broth of Streptomyces sp. CPCC 200451.

[0069] This invention also relates to key synthetic genes for the microbial biosynthesis of the peptide compound (Omicin), wherein the synthetic genes are genes 7094 and 7098, and the amino acid sequences of the proteins they encode are shown in SEQ ID NO.1 and 2, respectively.

[0070] The present invention also relates to the use of the aforementioned gene cluster or regulatory gene in the preparation of the peptide compound (Omicin) of formula (1) by microbial fermentation. Attached Figure Description

[0071] Figure 1 Transcriptome alignment results of Streptomyces CPCC 200451 under different fermentation conditions

[0072] Figure 2 Comparison results of Cluster 27 of Streptomyces CPCC 200451 under different active fermentation conditions

[0073] Figure 3 Comparison results of Cluster 28 of Streptomyces CPCC 200451 under different active fermentation conditions

[0074] Figure 4 Comparison results of Cluster 36 of Streptomyces CPCC 200451 under different active fermentation conditions

[0075] Figure 5 Detection of Cluster 27 gene expression level by real-time quantitative RT-PCR

[0076] Figure 6 Detection of Cluster 36 gene expression levels by real-time quantitative RT-PCR

[0077] Figure 7 A1, A3 and A3-Fe 3+ Cluster 36 alignment results of Streptomyces CPCC 200451 in culture medium

[0078] Figure 8 Quantitative real-time RT-PCR detection of A1, A3, and A3-Fe 3+ - Gene expression of Streptomyces CPCC 200451 strain Cluster 36

[0079] Figure 9Comparison of Cluster 36 and Deimino-antipain biosynthetic gene cluster

[0080] Figure 10 Deimino-antipain biosynthetic gene cluster and related natural products

[19]

[0081] Figure 11 Electrophoresis image of plasmid pOJ7094LR after restriction enzyme digestion.

[0082] Figure 12 Electrophoresis image of plasmid pOJ7098LR after restriction enzyme digestion.

[0083] Figure 13 Schematic diagram of PCR screening for single / double crossover mutants

[0084] Figure 14 PCR screening of 7094 gene double crossover mutant strains

[0085] Figure 15 PCR screening of 7098 gene double crossover mutants

[0086] Figure 16 RT-qPCR verification of the gene within Cluster 36 in *Streptomyces* strain 7094-KO and wild-type strain CPCC 200451

[0087] Figure 17 RT-qPCR verification of the gene within Cluster 36 in *Streptomyces* strain 7098-KO and wild-type strain CPCC 200451

[0088] Figure 18 Comparison results of Streptomyces CPCC 200451 and Cluster 36 in strains overexpressing the 7102 gene in A1 medium.

[0089] Figure 19 RT-qPCR of Streptomyces CPCC 200451 and Cluster 36 of strain overexpressing the 7102 gene in A1 medium

[0090] Figure 20 Separation process and technical route of active compounds

[0091] Figure 21 Chemical structures of Omicsynin A class compounds (Omicsynin A1, Omicsynin A2)

[0092] Figure 22 Omega-Sin A1 1 H(22A), 13C(22B), DEPT(22C), 1 H- 1 Spectra of H COSY (22D), HSQC (22E), HMBC (22F), NOESY (22G) and HRMS (22H) analysis data

[0093] Figure 23 Omega-Syne A2 1 H(23A), 13 C(23B), DEPT(23C), 1 H- 1 Spectra of H COSY (23D), HSQC (23E), HMBC (23F), NOESY (23G) and HRMS (23H) analysis data graph

[0094] Figure 24 HRMS analysis data chart of OmegaXin A6

[0095] Figure 25 HRMS analysis data chart of Omega-1 B1

[0096] Figure 26 HRMS analysis data chart of Omega-Syne B2

[0097] Figure 27 HRMS analysis data chart of Omega-3 B3

[0098] Figure 28 HRMS analysis data chart of Omega-B5

[0099] Figure 29 HRMS analysis data chart of Omega-B6

[0100] Figure 30 HRMS analysis data chart of Omegacin C1

[0101] Figure 31 HRMS analysis data chart of Omegacin C2

[0102] Figure 32 HRMS analysis data chart of Omega C6 Detailed Implementation

[0103] Materials and Methods

[0104] Strains:

[0105] Streptomyces sp. CPCC 200451, China Pharmaceutical Culture Collection, accession number CPCC 200451;

[0106] Escherichia coli DH5α;

[0107] Escherichia coli ET12567 / pUZ8002;

[0108] plasmid:

[0109] pSET152, an integrated vector for Streptomyces;

[0110] pOJ260, Streptomyces gene knockout plasmid;

[0111] Primers:

[0112]

[0113]

[0114]

[0115]

[0116] Fermentation medium:

[0117] A1 medium (g / L): glucose 5, malt extract 10, cottonseed meal 10, soluble starch 20, yeast extract 5, dipotassium hydrogen phosphate 0.5, ammonium sulfate 5, calcium carbonate 3, sodium chloride 1, pH 7.2-7.4.

[0118] A2 medium (g / L): glucose 5, yeast extract 5, peptone 5, beef extract 5, corn steep liquor 4, soybean meal 10, calcium carbonate 4, cobalt chloride 0.02, soluble starch 20, pH 7.2-7.4.

[0119] A3 medium (g / L): glycerol 20, dextrin 20, peptone 10, yeast extract 5, ammonium sulfate 2, calcium carbonate 2, pH 7.2-7.4.

[0120] A4 medium (g / L): soluble starch 30, soybean meal 15, sodium thiosulfate 20 μ, ferrous sulfate 0.5, dipotassium hydrogen phosphate 0.5, potassium chloride 0.3, pH 7.2-7.4.

[0121] B1 glucose-asparagine medium (g / L): glucose 10, asparagine 0.5, dipotassium hydrogen phosphate 0.5, pH 7.2-7.4.

[0122] B2 Synthesis Medium No. 5 (g / L): Potassium nitrate 1, Sodium chloride 0.5, Dipotassium hydrogen phosphate 0.5, Ferrous sulfate 0.01, Magnesium sulfate 0.5, Soluble starch 20, pH 7.0.

[0123] B3 Sodium propionate medium (g / L): Sodium propionate 2, ammonium nitrate 0.1, potassium chloride 0.1, magnesium sulfate 0.05, ferrous sulfate 0.05, pH 7.2.

[0124] B4 medium (g / L): sodium succinate 0.9, ammonium dihydrogen phosphate 0.5, magnesium sulfate 0.1, ferrous sulfate 0.01, pH 7.2.

[0125] B5 Waksman medium (g / L): ammonium sulfate 0.2, dipotassium hydrogen phosphate 3, magnesium sulfate 0.5, calcium chloride 0.126, pH 7.2.

[0126] B6 TWYE medium (g / L): yeast extract 0.25, dipotassium hydrogen phosphate 0.5, pH 7.2.

[0127] B7 Kröss Synthesis No. 1 Medium (g / L): Dipotassium hydrogen phosphate 1, Magnesium carbonate 0.3, Sodium chloride 0.2, Potassium nitrate 1, Ferrous sulfate 0.01, Calcium carbonate 0.5, Glucose 20, pH 7.0.

[0128] B8 Czapek's medium (g / L): sucrose 30, potassium nitrate 2, dipotassium hydrogen phosphate 1, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate 0.01, pH 7.2-7.4.

[0129] B9 ISP7 medium (g / L): tyrosine 0.5, glycerol 15, asparagine 1, dipotassium hydrogen phosphate 0.5, magnesium sulfate 0.5, sodium chloride 0.5, ferrous sulfate 0.01, pH 7.2-7.4.

[0130] B10 medium (g / L): soluble starch 2, ferrous sulfate 0.01, magnesium sulfate 0.5, potassium nitrate 1, sodium chloride 0.4, dipotassium hydrogen phosphate 0.5, pH 7.2.

[0131] Other culture media are standard culture media commonly used in this field.

[0132] Other commonly used reagents are domestically produced analytical grade or chromatographic grade.

[0133] Example 1: Culture and sequencing of Streptomyces CPCC 200451

[0134] 1. Cultivation and preservation of Streptomyces sp. CPCC 200451

[0135] The mycelium of CPCC 200451 was cultured in YMG or TSB liquid medium at 28°C and 200 rpm for 36-72 h. When solid culture of Streptomyces CPCC 200451 was performed, YMG solid medium was used and cultured in an incubator at 28°C for 5-7 days. MS medium was used for sporulation solid medium.

[0136] All bacterial strains used in the experiment were preserved using the low-temperature glycerol preservation method, and were frozen at -20℃ or -80℃.

[0137] 2. Fermentation culture of Streptomyces sp. CPCC 200451

[0138] CPCC 200451 was inoculated onto the surface of YMG solid medium and cultured at 28°C for 7 days. It was then transferred to a 500mL shake flask containing 100mL YMG liquid medium and cultured at 28°C and 200rpm for 48 hours. After that, 10% of the inoculum was transferred to 100mL / 500mL fermentation medium and cultured at 28°C and 200rpm for another 5 days. The fermentation broth was then collected.

[0139] TSB medium (g / L): peptone 2, sodium chloride 5, glucose 2.5, dipotassium hydrogen phosphate 2.5.

[0140] YMG medium (g / L): glucose 10, malt extract 10, yeast extract 10, agar 15, pH 7.0.

[0141] MS medium (g / L): mannitol 20, soybean flour 20, agar 20, prepared with tap water. Add magnesium chloride to a final concentration of 10 mM before use.

[0142] Other culture media:

[0143] YS medium (g / L): yeast extract 2, soluble starch 10, agar 15, pH 7.2-7.4.

[0144] PYG medium (g / L): peptone 3, yeast extract 5, glycerol 10, agar 15, pH 7.2-7.4.

[0145] ISP4 medium (g / L): soluble starch 10, dipotassium hydrogen phosphate 1, sodium chloride 1, ammonium sulfate 2, magnesium sulfate 1, calcium carbonate 2, agar 15, pH 7.0-7.4.

[0146] 3. CPCC 200451 whole genome sequencing

[0147] To identify the effective antiviral components in Streptomyces sp. CPCC 200451 and to reveal its biosynthetic gene clusters and biosynthetic mechanisms, we first performed whole-genome DNA sequencing on Streptomyces sp. CPCC 200451 provided by the China Pharmaceutical Microbiology Culture Collection Center.

[0148] This high-throughput sequencing was performed by the Beijing Genomics Institute. The entire genome DNA of CPCC200451 was sequenced using the novel third-generation sequencing platform PacBio RSII, combined with the second-generation sequencing platform Illumina Hiseq 4000, and the genome was assembled to obtain a fine map. The genome of CPCC 200451 is a linear chromosome of 8,918,347 bp in length, with a (G+C)mol percentage of 73.6%. No free plasmids were found outside the chromosome.

[0149] The CPCC 200451 genome contains 316 tandem repeat sequences, totaling 151,923 bp in length, accounting for 1.7% of the total genome length. Gene annotation analysis revealed 8,151 protein-coding genes. Alignment with an rRNA library and the rRNAmmer software identified 11 rRNA operons. The tRNAscan-SE software further predicted 73 tRNA-coding genes.

[0150] Example 2: Analysis and identification of the biosynthetic gene cluster of secondary metabolites of anti-influenza virus in CPCC 200451

[0151] Transcriptional regulation is one of the most important regulatory mechanisms in prokaryotes. This embodiment compares and analyzes the differences in transcriptional levels across the entire genome of Streptomyces sp. CPCC200451 in fermentation broth samples with different antiviral activities (high activity, low activity, and no activity), thereby preliminarily identifying the biosynthetic gene cluster containing the effective antiviral component of CPCC 200451.

[0152] 1. Screening of fermentation conditions

[0153] To obtain fermentation broth samples with different anti-influenza virus activities, we first screened the fermentation conditions of CPCC 200451, tried 14 fermentation media and 4 fermentation time points, and determined the anti-influenza virus activity of the fermentation broth.

[0154] (1) Screening of fermentation media

[0155] Spore suspensions of Streptomyces CPCC 200451 were spread on the surface of YMG solid medium and cultured in a constant temperature incubator at 28°C for 7 days. After the same size cells were scooped out with an inoculation spatula, the cells were dispersed and inoculated into 14 different fermentation media and cultured in a shaker at 28°C and 200 rpm.

[0156] (2) Selection of fermentation time

[0157] Fermentation broth samples from different culture media were collected at 3, 5, 7 and 10 days. After centrifugation, the supernatant was collected to determine its anti-influenza virus activity.

[0158] 2. Anti-influenza virus activity assay

[0159] The determination of anti-influenza virus activity was completed by the virology laboratory of this institute, using influenza virus strains A / FM / 1 / 47 (H1N1) and A / Hanfang / 359 / 95 (H3N2).

[0160] Test method:

[0161] (1) Canine kidney cells MDCK were seeded in 96-well culture plates and cultured at 37°C with 5% CO2.

[0162] (2) Infected with influenza virus 24 hours later, the virus solution was discarded after 2 hours of adsorption, and maintenance solution containing sample and positive control drug was added. Cell control wells and virus control wells were set up and cultured.

[0163] (3) The degree of cytopathic effect in each group was observed based on the degree of cytopathic effect in the virus control group, and the median lethal concentration (TC) of different samples for cells was calculated using the Reed-Muench method. 50 ), and the half-maximal inhibitory concentration (IC50) for the virus. 50 ), and calculate the selection index (SI = TC) 50 / IC 50 ).

[0164] Activity test results showed that A3 culture medium was derived from the fermentation broth sample of Streptomyces sp. CPCC 200451. Influenza virus A / Hanfang / 359 / 95(H3N2) Highest activity The fermentation medium A1 and A2 were the most effective, with the fermentation broth sample at 5 days showing significantly higher values ​​than the other three time points (Table 1). Furthermore, based on the growth status of the strains and the results of anti-influenza virus activity assays, fermentation medium B7 was selected as an inactive fermentation medium to serve as a negative control for subsequent comparative transcriptome analysis, thus helping to narrow down the screening range of target biosynthetic gene clusters.

[0165] Further assay results showed that the fermentation broth of Streptomyces sp. CPCC 200451 from A3 medium not only had good activity against influenza virus A / Hanfang / 359 / 95 (H3N2), but also showed some activity against influenza virus A / FM / 1 / 47 (H1N1) (Table 2).

[0166] Table 1. Determination of anti-influenza virus activity of CPCC 200451 fermentation broth

[0167]

[0168] Table 2. Determination of antiviral activity of CPCC 200451 fermentation broth

[0169]

[0170] 3. Transcriptome sequencing analysis and RT-qPCR validation

[0171] Based on the results of the anti-influenza virus activity assay, A3 was selected as the high-activity fermentation medium and B7 as the inactive fermentation medium to ferment CPCC 200451. The bacterial cells were collected in the early stage of fermentation, total RNA was extracted, and transcriptome sequencing (RNA-seq) and data analysis were performed.

[0172] (1) Preparation of RNA samples

[0173] Based on the results of the anti-influenza virus activity assay of the fermentation broth, A3 and B7 were selected as high-activity and inactive fermentation media for Streptomyces CPCC200451, respectively. Cells were collected at 12h, 24h and 48h in the early stage of fermentation. Total RNA of Streptomyces CPCC200451 was extracted using the modified TRIzol method. The sample names were A3-24, A3-48, A3-72, B7-24, B7-48 and B7-72, respectively.

[0174] After testing, the extraction quality of the six RNA samples was good, with no obvious genomic DNA contamination or severe degradation, which basically met the requirements of high-throughput sequencing.

[0175] (2) RNA-Seq transcriptome sequencing and data analysis

[0176] The transcriptomes were constructed using the six RNA samples described above by Beijing BGI Genomics Co., Ltd., and high-throughput sequencing was performed using the BGISEQ-500 next-generation sequencing platform. A visualization of the transcriptomes is shown below. Figure 1As shown. Based on the secondary metabolite biosynthesis gene cluster information predicted by antiSMASH, we identified three biosynthesis gene clusters that showed significant differences between A3 and B7: Cluster 27, Cluster 28, and Cluster 36.

[0177] The first differentially expressed gene cluster is Cluster 27, which is a biosynthetic gene cluster of NRPS-type siderophores. The transcriptome visualization is shown below. Figure 2 As shown in the figure, Cluster 27 exhibits significantly higher expression in the A3 high-activity fermentation medium compared to the B7 low-activity fermentation medium.

[0178] Cluster 28 is also a biosynthetic gene cluster of siderophores, as shown in the transcriptome visualization. Figure 3 The results showed that the transcription of Cluster 28 differed significantly under high-activity and low-activity fermentation conditions, and it was also significantly highly expressed in A3 fermentation medium.

[0179] The third gene cluster exhibiting significant differences at the transcriptional level is Cluster 36, an NRPS-type biosynthetic gene cluster that shows significantly high expression in A3 high-activity fermentation medium. Transcriptome visualization is shown below. Figure 4 As shown.

[0180] (2) RT-qPCR validation of transcriptome data analysis results

[0181] The transcriptional levels of these gene clusters were verified using quantitative real-time RT-qPCR.

[0182] Since both Cluster 27 and Cluster 28 are siderophore biosynthetic gene clusters, Cluster 27 will be used as an example for this study. In this experiment, three siderophore biosynthesis-related genes from Cluster 27 were selected: C27_5814 (dhb), C27_5819 (NRPS), and C27_5821 (transporter). RT-qPCR assays were performed, and the results are as follows: Figure 5 As shown, Cluster 27 in Streptomyces sp. CPCC 200451 was significantly highly expressed under A3 high-activity fermentation conditions, meaning that the RT-qPCR results were consistent with the transcriptome analysis results, indicating that the transcriptome sequencing data were reliable.

[0183] Three functional genes from the Cluster 36 biosynthetic gene cluster, namely C36_7094 (NRPS), C36_7097 (NRPS), and C36_7098 (NRPS), were selected. The results are as follows: Figure 6 As shown, the RT-qPCR validation results are consistent with the transcriptome sequencing results.

[0184] Example 3: Bioinformatics and Functional Analysis of Three Key Gene Clusters

[0185] antiSMASH prediction indicated that clusters 27 and 28 are biosynthetic gene clusters of siderophores. Bioinformatics analysis showed that both clusters 27 and 28 contain multiple binding sites for iron repressor proteins, suggesting that their expression is regulated by iron ion concentration, i.e., high expression in oligoferric media and no expression in iron-rich media.

[0186] Therefore, we added 0.05% iron ions, namely A3-Fe, to the A3 high-activity fermentation medium. 3+ Fermentation medium. Streptomyces sp. CPCC 200451 was fermented using both A3 and A3-Fe. 3+ The culture medium was used for fermentation under the same conditions. Cells were collected 48 hours into the fermentation process to extract RNA for RNA-Seq sequencing and data analysis, as well as RT-qPCR validation. Results showed that, compared to A3 medium, A3-Fe... 3+ Streptomyces sp. CPCC 200451, Cluster 27 and Cluster 28 collected under fermentation conditions were no longer expressed.

[0187] However, the results of the anti-influenza virus activity assay showed that the fermentation broth of Streptomyces sp. CPCC200451, even after the addition of iron ions, still maintained high activity. Therefore, Prove that these two siderophore gene clusters (Cluster 27 and The synthetic product of Cluster 28 is not Streptomyces CPCC. 200451 Main anti-influenza virus active substances .

[0188] After excluding the first two gene clusters with differential transcriptional expression, we turned our attention to the third gene cluster showing significant differential expression—Cluster 36. We compared Streptomyces sp. CPCC 200451 in A3 and A3-Fe... 3+ Transcriptome data of Cluster 36 under fermentation conditions were analyzed, with A1 fermentation medium as a control. The analysis revealed that the biosynthetic gene cluster Cluster 36 in the genome is located in A3-Fe... 3+ It is still in a state of high expression in the culture medium. Figure 7) Validation was performed using RT-qPCR, and the results showed that Cluster 36 was present in A3 and A3-Fe. 3+ Expression in the culture medium was significantly higher than in the A1 culture medium, consistent with the results compared with the transcriptome. Figure 8 Based on the results of the anti-influenza virus activity assay after the addition of excess iron ions, Speculation on Cluster 36 This may be the biosynthetic gene cluster containing the antiviral active ingredient of streptomyces sp. CPCC 200451. .

[0189] Cluster 36 is located in chromosome 1:7,822,964-7,875,615 of the Streptomyces sp. CPCC 200451 genome, with a total length of 52.6 kb. Bioinformatics analysis predicts that this gene cluster contains 50 open reading frames, and the core region of the gene cluster is gene 7092-7102 (chromosome 1:7,841,516-7,857,514).

[0190] Using GenBank database [14-16] The BLASTP function was used to perform homology analysis on the amino acid sequences of the proteins encoded by the 50 open reading frames contained in Cluster 36. The results showed that Cluster 36 belongs to the NRPS class of biosynthetic gene clusters. NRPS (Nonribosomal peptide synthetases) play a key role in the synthesis of nonribosomal peptides. They are multifunctional protein complexes composed of multiple independent modules tandemly arranged in a specific spatial order. They can specifically recognize, activate, and transport specific amino acid substrates, and condense them in a certain order to form peptide chains, synthesizing and releasing nonribosomal peptides. Each module in an NRPS contains at least three core domains, including an adenylate domain (A domain), a peptidyl carrier protein domain (PCP domain), and a condensation domain (C domain).

[17] The final component of NRPS contains a special domain located downstream of the synthase peptide chain, called the thioesterase domain (TE domain), which is responsible for releasing the peptide chain from the NRPS module. In addition, it may include other specific domains, such as epimerization (E domain) and methyltransferase (M domain), to modify the substrate amino acids accordingly.

[0191] According to antiSMASH prediction, Cluster 36 and Deimino-antipain's biosynthetic gene cluster share the highest similarity at 66%. These similar genes are located in the core region of Cluster 36. The protein sequences encoded by these similar genes in Cluster 36 and Deimino-antipain were compared using the BLASTP tool, and the results are as follows: Figure 9 As shown. This family of protease inhibitors has existed for over 40 years and typically features relatively low molecular weight, hydrophobicity, the presence of C-terminal aldehydes, and internal urea bonds. Their high structural similarity suggests a shared biosynthetic pathway, thus leading to the evolution of related biosynthetic gene clusters. [18-20] .

[0192] In 2016, Maxson et al. used probes to detect and isolate Deimino-antipain from Streptomyces albulus NRRL B-3066, and analyzed its biosynthetic gene clusters.

[21] ,like Figure 10 As shown, NRPS is composed of genes anpC-G, where the A domains of genes anpD, anpE, and anpF are responsible for assembling Arg, Phe, and Val, respectively. However, the fourth A domain responsible for assembling Arg (or Cit) is speculated to be the Phe loading module (anpE), which also loads Arg / Cit, similar to the biosynthesis of syringolin or the Arg-specific module (anpD) performing its function twice in a discontinuous manner.

[22] Another scenario is that anpD installs Cit in a specific, discontinuous manner (or Arg is subsequently replaced by Cit), followed by Arg. The anpC gene contains only the C domain, while anpG contains both the PCP and C domains, as well as a NAD reduction (R) domain that may be responsible for release (instead of the conventional thioesterase), ultimately producing a C-terminal aldehyde product.

[23] In addition to the NRPS gene, the anpA gene may encode a hydrolase that could act before or after Arg assembly, playing a role in Cit formation. anpB belongs to the MFS transporter superfamily.

[24] The product encoded by anpH is a regulatory histidine kinase. Subsequently, Maxson et al. used heterologous expression to demonstrate that the assembly of Cit in Deimino-antipain requires the function of genes outside this biosynthetic gene cluster.

[0193] Furthermore, literature reports that the biosynthetic gene clusters responsible for the synthesis of these peptide aldehyde compounds, anpB-G genes, maintain a consistent direction and sequence. Based on the presence and arrangement of the gene anpI encoding acyl-CoA dehydrogenase, they can be roughly divided into three categories: the first category consists of biosynthetic gene clusters that do not contain the anpI gene, such as Deimino-antipain; the second category consists of gene clusters where the anpI gene is located between anpD and anpE, and this arrangement constitutes the vast majority of gene clusters; only a few gene clusters have the anpI gene located after the anpG gene, which is the third type of anp-class biosynthetic gene cluster.

[21] Therefore, Cluster 36 in Streptomyces sp. CPCC 200451 belongs to the second type of anp gene cluster, which is relatively common. The difference is that it also has an additional gene encoding SDR reductase, the function of which needs further research and confirmation.

[0194] In summary, we can infer that the coding products of Cluster 36 in Streptomyces sp. CPCC 200451 are diverse and may be structurally similar to compounds such as Deimino-antipain, chymostatin, elastatinal, and MAPI. To further confirm whether Cluster 36 is the biosynthetic gene cluster containing the anti-influenza virus active component of Streptomyces CPCC 200451, we constructed a genetic manipulation system for knockout and overexpression of this strain.

[0195] Example 4: Verification of the function of Cluster 36 gene cluster in Streptomyces CPCC 200451 using gene knockout method

[0196] CPCC 200451 spores reached their optimal morphology and quantity after culturing in MS medium for 96-120 hours; 120 hours was selected as the spore collection time for CPCC200451. Furthermore, since CPCC 200451 is sensitive to apramycin, apramycin was chosen as a selection marker for CPCC 200451, and aztreonam was selected as an inhibitor of Escherichia coli in the conjugation transfer experiment.

[0197] Establishment of the CPCC 200451 Knockout Genetic Operating System

[0198] Two NRPS-type functional genes within Cluster 36, namely gene 7094 (Chromosome 1:7,844,718-7,847,825) and gene 7098 (Chromosome 1:7,850,958-7,852,772), were selected to construct a knockout genetic operating system.

[0199] Primers were designed to amplify two fragments (the forearm and hindarm) containing the upstream and downstream of the target gene, respectively. These fragments were then ligated to the multiple cloning site of the suicide plasmid pOJ260, and the recombinant plasmid was introduced into Streptomyces sp. CPCC 200451 via conjugation transfer. Single-crossover strains were screened using apopramine resistance markers. After successful identification, the strains were passaged approximately five times on MS solid medium without apopramine. Double-crossover mutant strains that lost apopramine resistance were then screened by copying, and the results were verified by PCR. Finally, a blocking strain lacking the target gene was obtained. The specific steps are roughly as follows.

[0200] (1) Construction of blocking plasmids

[0201] Using CPCC 200451 genomic DNA as a template, two pairs of primers were designed approximately 2000 bp to the left and right sides of genes 7094 and 7098, respectively. PCR was used to amplify the left and right homologous arms for double crossover. The lengths of the two arms of gene 7094 were 2129 bp and 2215 bp, respectively; the lengths of the two arms of gene 7098 were 2056 bp and 2173 bp, respectively. HindIII and EcoRI restriction sites were introduced at both ends of the left arm, and EcoRI and HindIII restriction sites were introduced at both ends of the right arm, respectively.

[0202] The pOJ260 suicide plasmid was selected for constructing the blocking strain. First, the left and right homologous arms obtained from PCR amplification were ligated into a T vector and transformed into E. coli competent cells. The recombinant plasmid was extracted and sequenced for verification. The correctly sequenced recombinant plasmid was digested with EcoRI and HindIII. Simultaneously, plasmid pOJ260 was digested with HindIII, and the large vector DNA fragment was recovered and ligated into the digested left and right homologous arms. The ligation product was transformed into E. coli DH5α competent cells. Positive transformants were screened using the apopramine resistance marker on plasmid pOJ260, and verified by plasmid extraction and restriction enzyme digestion. Figure 11 and Figure 12 Thus, the correct recombinant plasmids were obtained and named pOJ7094LR and pOJ7098LR, respectively.

[0203] Lanes 1-3, pOJ7094LR / HindIII; lanes 4-6, pOJ7094LR / EcoRI; lanes 7-9, pOJ7094LR / PstI; lanes 10-12, pOJ7094LR / KpnI.

[0204] Lanes 1-3, pOJ7098LR / HindIII; lanes 4-6, pOJ7098LR / EcoRI; lanes 7-9, pOJ7098LR / PstI; lanes 10-12, pOJ7098LR / NcoI.

[0205] (2) Screening of single crossover mutants

[0206] Recombinant plasmids pOJ7094LR and pOJ7098LR were transformed into E. coli ET12567 / pUZ8002 competent cells, and then introduced into CPCC 200451 via conjugative transfer. Apopramycin and aztreonam were used for resistance selection. After 3-5 days of antibiotic coverage, conjugates resistant to apopramycin grew on the plates. Single colonies were picked and imprinted onto plates containing apopramycin; this strain is a possible single crossover mutant.

[0207] Total genomic DNA was extracted from the strain, and single-crossover mutants were identified using PCR. Three pairs of primers (P1P2, P3P4, and P5P6) were designed to amplify fragments of the left homologous arm and its flanking regions, the right homologous arm and its flanking regions, and the target gene, respectively. Figure 13 If it is a left-single-crossover mutant, when using primer P1P2 for PCR, the left homologous arm of the product fragment, approximately 2kb in size, can be amplified. Conversely, when primer P3P4 amplifies the right homologous arm of the product fragment, approximately 2kb in size, it is a right-single-crossover strain.

[0208] (3) Screening of double crossover mutants

[0209] After PCR verification, the single-crossover strain was cultured on MS plates without apopramine for approximately 5 generations. Strains lacking apopramine resistance were then screened by photocopying, and double-crossover mutants were identified using PCR. When using primer P3 (located on the right edge of the left homologous arm) and primer P2 (located on the left edge of the right arm) for PCR verification, only a small target band was amplified, not a large fragment equivalent to the knocked-out gene. Primer P5P6 failed to amplify the internal gene fragment, while primer P1P4 amplified approximately 4kb of the ligation product between the left and right homologous arms. This 4kb PCR product was sequenced for verification. If the sequence correctly identified the left and right homologous arms, the strain was confirmed to be a double-crossover mutant.

[0210] Twelve single-crossover conjugates of gene 7094 were obtained. One left-crossover and one right-crossover mutant were selected. After five generations of subculture on MS plates without apopramycin resistance, spores were collected, diluted, and plated. Two suspected double-crossover mutants (named 7094-KO-10 and 7094-KO-33) were screened from the right-crossover mutant and verified by PCR. Figure 14 As shown.

[0211] Only one conjugate of the 7098 gene was obtained by screening on a plate containing apopramine resistance, and this was verified by PCR. Similarly, after five generations of subculturing on MS plates without apopramine, we screened for double-crossover mutants (named 7098-KO-37 and 7098-KO-47) that had lost apopramine, and identified them by PCR. Figure 15 As shown.

[0212] (4) Validation of the blocking strain by RT-qPCR

[0213] The blocking strains 7094-KO and 7098-KO obtained from the above screening were combined with the wild-type strain CPCC 200451 and simultaneously treated with A3-Fe containing excess iron ions. 3+ The culture medium was used for fermentation under identical conditions. After the initial 48 hours of fermentation, bacterial cells were collected and RNA extracted. This RNA was reverse transcribed into cDNA, and the relevant genes of Cluster 36 were verified by qRT-PCR. Figure 16 and 17 The results showed that the target gene was successfully knocked out; it was also found that when gene 7094 was knocked out, gene 7098 was no longer expressed, and the expression of genes 7097 and 7099 was also affected; while after gene 7098 was knocked out, the expression of gene 7094 was not affected.

[0214] (5) Construction of complement strains

[0215] Using genomic DNA from wild-type Streptomyces sp. CPCC 200451 as a template, primers 7094_F (containing NdeI restriction site) and 7094_R (containing XbaI restriction site), and primers 7098_F (containing NdeI restriction site) and 7098_R (containing BamHI restriction site) were designed. The 7094 and 7098 genes were amplified using PCR and cloned into the corresponding restriction sites of the pSET152 plasmid (containing a strong erythromycin promoter and a phage ΦC31 integration site, and a selection marker for apopramycin resistance). Genetic complementation recombinant plasmids for the 7094 and 7098 genes were constructed. After verification by restriction enzyme digestion and sequencing, the complement plasmids pL-7094 and pL-7098 were obtained.

[0216] The verified complement plasmids were introduced into the blocking strains 7094-KO and 7098-KO via conjugation transfer. Apopramine resistance in plasmid pSET152 was used as a selection marker. Three conjugates were selected for each gene, and PCR verification was performed using three pairs of primers for apopramine resistance, pSET152 integration site, and complement gene. The results proved that the genetically complementary strains 7094-KOC and 7098-KOC were successfully constructed.

[0217] (6) Validation of each strain by RT-qPCR

[0218] The blocking strains 7094-KO and 7098-KO obtained from the above screening were used to reintroduce strains 7094-KOC, 7098-KOC, and wild-type Streptomyces sp. CPCC 200451, while simultaneously using A3-Fe... 3+ Fermentation medium was used, and fermentation was carried out under the same conditions. At the initial 48 hours of fermentation, bacterial cells were collected and RNA was extracted. After reverse transcription into cDNA, the 7094 and 7098 genes were verified by RT-qPCR. The results showed that the 7094 gene was successfully reintroduced into the knockout strain 7094-KO; and the 7098 gene was successfully reintroduced into the blocked strain 7098-KO.

[0219] (7) Determination of the anti-influenza virus activity of blocking and complement strains

[0220] The anti-influenza virus activity of fermentation broths from blocking strains 7094-KO and 7098-KO, replenished strains 7094-KOC and 7098-KOC, and wild-type CPCC 200451 was determined. The results showed that... Knock out structural genes 7094 or 7098 of this gene cluster. After gene sequencing, the anti-influenza virus activity of Streptomyces CPCC200451 disappeared. Replacing the 7094 gene does not restore its ability to fight influenza viruses, but... The strains that had the 7098 gene restored exhibited good anti-influenza virus activity (Table 3).Therefore, it is demonstrated that the expression of Cluster 36 is closely related to the anti-influenza virus activity of Streptomyces sp. CPCC 200451.

[0221] Table 3. Results of anti-influenza virus activity assays of fermentation broths from CPCC 200451 wild-type strain, blocking strain, and complement strain.

[0222]

[0223] Example 5: Establishment of the CPCC 200451 overexpression genetic operating system

[0224] To further confirm that Cluster 36 is the biosynthetic gene cluster containing the anti-influenza virus active component of Streptomyces sp. CPCC 200451, we selected five regulatory genes within this cluster and constructed overexpression plasmids based on plasmid pSET152. These plasmids were then introduced into Streptomyces sp. CPCC 200451 for overexpression. The recombinant strain was fermented under the same conditions using A1 fermentation medium. By detecting and comparing changes in anti-influenza virus activity, we identified the regulatory genes and their regulatory roles in the expression of the anti-influenza active component in Cluster 36.

[0225] 1. Construction of regulatory gene overexpression plasmids

[0226] First, using the genomic DNA of Streptomyces sp. CPCC 200451 as a template, five expression regulatory genes 7081, 7082, 7083, 7089 and 7102 in Cluster 36 were selected, and primers were designed for each gene (primer sequences are detailed in the materials). The DNA fragments of these five regulatory genes were amplified using PCR technology.

[0227] The integrated plasmid pSET152 was digested with NdeI and BamHI. Since the regulatory genes 7081 and 7089 contain BamHI sites, NdeI and XbaI restriction sites were introduced at both ends of them, and the plasmid pSET152 was digested with NdeI and XbaI.

[0228] NdeI and BamHI restriction sites were introduced at both ends of the 7082, 7083 and 7102 regulatory genes.

[0229] First, the PCR product is ligated into the pEASY-T vector. After sequencing to verify the sequence is correct, the regulatory gene is digested with the corresponding restriction enzyme sites and the product is recovered. Then, it is ligated into the pSET152 vector, which has been digested with the same enzymes, to obtain the recombinant plasmid.

[0230] 2. Import Streptomyces sp. CPCC 200451 via electroconversion.

[0231] The recombinant plasmids were introduced into the wild-type Streptomyces sp. CPCC 200451 strain via electroporation. Apopramycin resistance was used for screening to obtain overexpressing recombinant strains. Simultaneously, the empty vector pSET152 was introduced into the wild-type Streptomyces sp. CPCC 200451 strain as a control. The PCR verification primers were pSET152 and attB-Streptomyces. If the recombinant plasmid was correctly integrated into the genome of Streptomyces sp. CPCC 200451, PCR could amplify a 1.6kb target band. After PCR verification, three overexpressing strains were obtained from regulatory genes 7081, 7082, 7083 and 7102, and two recombinant strains were obtained from regulatory gene 7089. They were named 200451 / pL-7081, 200451 / pL-7082, 200451 / pL-7083, 200451 / pL-7089 and 200451 / pL-7102, respectively.

[0232] 3. Determination of the anti-influenza virus activity of the overexpression strains

[0233] To further investigate the effect of upregulation of regulatory genes within Cluster 36 on the anti-influenza virus activity of Streptomyces sp. CPCC 200451, we selected three fermentation media (A1, A3, and B7) and fermented strains overexpressing the above-mentioned regulatory genes under the same conditions. Fermentation broth samples were then collected for determination of anti-influenza virus activity (Table 4). The results showed that overexpression of regulatory gene 7102 increased the anti-influenza virus activity of fermentation broth samples from A1 and A3 media to some extent. Overexpression of the other four regulatory genes did not significantly change the antiviral activity of the fermentation broth. However, the fermentation product using B7 medium did not show a significant increase in anti-influenza virus activity, which is likely because B7 is an oligotrophic medium, which limits the growth of Streptomyces to some extent, thus preventing the synthesis of abundant secondary metabolites.

[0234] Table 4. Determination of anti-influenza virus activity of overexpressing strains.

[0235]

[0236]

[0237] 4. Transcriptome analysis of overexpression strains

[0238] Given that overexpression of the regulatory gene 7102 resulted in a change in the anti-influenza virus activity of fermentation broth samples in A1 medium from a lower level to a higher level, we further investigated the differences in transcriptional levels. We simultaneously fermented wild-type Streptomyces sp. CPCC 200451 and strains overexpressing the regulatory gene 7102 in A1 fermentation medium, collecting cells at the initial 48 hours of fermentation to extract RNA for transcriptome sequencing (RNA-Seq) and data analysis. The transcriptional status of Cluster 36 was visualized using visualization tools. Figure 18 The results showed that... Regulatory genes After overexpression of 7102, the genes in the core region of Cluster 36 (Chromosome 1:7,841,516-7,858,166) showed significant upexpression. Adjust .

[0239] 5. Validation of overexpression strains by RT-qPCR

[0240] To verify the reliability of the transcriptome data, we also reverse transcribed the extracted RNA samples into cDNA for quantitative RT-qPCR validation. The results are as follows: Figure 19 As shown, in bacterial cells collected using A1 fermentation medium, compared to the wild-type Streptomyces sp. CPCC 200451, overexpression of the regulatory gene 7102 resulted in a 2-8 fold upregulation of genes in the Cluster 36 core region. This change is consistent with the transcriptome results. Therefore, we infer that the regulatory gene 7102 regulates the expression of genes in the Cluster 36 core region; and that upregulation of gene 7102 leads to an increase in the anti-influenza virus activity of Streptomyces sp. CPCC 200451, indicating that the regulatory gene 7102 plays a positive regulatory role.

[0241] In summary, this study selected two functional genes and constructed a knockout genetic operating system. RT-qPCR validation results showed that the two functional genes were successfully knocked out. Fermentation and anti-influenza virus activity assays were performed on the knockout strain and the wild-type Streptomyces sp. CPCC 200451 strain. The results showed that the anti-influenza virus activity of Streptomyces sp. CPCC 200451 disappeared after knocking out the structural genes of this gene cluster. Five regulatory genes were selected, and an overexpression strain of the target gene cluster was successfully constructed. Transcriptome analysis and RT-qPCR validation results showed that overexpression of the regulatory gene 7102 caused upregulation of gene expression in the core region of this gene cluster. Therefore, Cluster 36 was ultimately identified as the gene cluster containing the anti-influenza virus active substances of Streptomyces sp. CPCC 200451.

[0242] Example 6: Chemical separation and purification of antiviral active ingredients

[0243] Previous experimental results have shown that adding excess iron ions to A3 high-activity medium can stop the expression of the siderophore biosynthetic gene cluster, while the target gene cluster Cluster 36 can be expressed normally. Therefore, this study uses A3-Fe 3+ Large-scale fermentation of wild-type Streptomyces sp. CPCC 200451 was performed on the culture medium. The supernatant was collected by centrifugation. Simultaneously, the blocking strains 7094-KO and 7098-KO (without anti-influenza virus activity) from Cluster 36 (containing genes 7094 and 7098) were used as negative controls. Fermentation was carried out under the same conditions, and the supernatant of the fermentation broth was collected and treated in the same way as the active fermentation broth. Based on the combined results of activity assays and HPLC analysis, the components with anti-influenza virus activity were identified, and the samples were prepared and purified by HPLC. The separation process and technical route of the active compounds are as follows: Figure 20 As shown.

[0244] A total of 14 L of the supernatant from the fermentation broth of wild-type Streptomyces sp. CPCC 200451 was adsorbed onto HP20 macroporous adsorption resin, and the flow-through sample was collected. The sample was then washed with two column volumes of deionized water, and the wash water sample was collected. A gradient elution was performed using ethanol-water (20%, 50%, and 100% ethanol sequentially), eluting at each gradient until the eluent was colorless or unchanged. The eluents from each gradient were collected, concentrated under reduced pressure, and then lyophilized for later use. The antiviral activity of the above fermentation broth, flow-through, wash water, and 20%, 50%, and 100% ethanol eluents was determined. The results showed that… The antiviral active ingredients are mainly concentrated in the 50% ethanol eluent fraction. In addition, the 100% ethanol eluent also has certain activity (Table 5).

[0245] Table 5 Results of anti-influenza virus activity assay in fermented samples

[0246]

[0247]

[0248] The 50% ethanol fraction (200451-50E) exhibiting the best anti-influenza virus activity was further analyzed by reversed-phase C18 (ODS-A-HG) open column chromatography with gradient elution of acetonitrile-water (10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 80%, and 100% acetonitrile). HPLC analysis was performed using an Agilent-C18-Aq column (5 μm, 4.6 × 150 mm) with acetonitrile and water (containing 0.1% TFA) as the mobile phase. The analytical conditions were 0–30 min (0–30% acetonitrile) and 30–60 min (30–100% acetonitrile). Based on the HPLC analysis results of the main components in each fraction, 10 fractions (AJ) were obtained, denoted as 50E-C18-A to J, and their anti-influenza virus activity was tested (Table 6). The results showed that all components of 50E-C18-C to I possessed certain anti-influenza virus activity, among which, The antiviral activity of the 50E-C18-E and 50E-C18-F components was significant. The value was higher than that of other components. .

[0249] Table 6. Determination of anti-influenza virus activity of each component of 50E-C18

[0250]

[0251]

[0252] Example 7: Isolation, purification, and structural identification of secondary metabolites

[0253] The active component 50E-C18-G was directly prepared by RP-HPLC (SHISEIDO Capcell-Pak PFP 5μm, 10×250mm, 25% ACN / H2O containing 0.1% TFA, 1.5mL / min) to obtain a group of compounds. Named Omekaxin A (Omicsynin A) .

[0254] The active components 50E-C18-E and 50E-C18-F were directly prepared by RP-HPLC (SHISEIDO Capcell-Pak PFP 5μm, 10×250mm, 20% ACN / H2O containing 0.1% HCOOH, 1.5mL / min) to obtain a group of compounds. name Omicsynin B .

[0255] The active component 200451-100E was directly prepared by RP-HPLC (SHISEIDO Capcell-Pak PFP 5μm, 10×250mm, 40% ACN / H2O containing 0.1% TFA, 1.5mL / min) to obtain a group of compounds. Named Omekaxin C (OmicsyninC) .

[0256] Comprehensive application of modern spectroscopic techniques, including HRESIMS, 1 H-NMR, 13 C-NMR, DEPT 1 H- 1 The chemical structures of the collected compounds were identified and resolved using HCOSY, HSQC, HMBC, and NOESY. The chemical structures of each compound were deduced using fragment ion characteristics provided by HRESIMS / MS, as shown in Table 7 below.

[0257] Table 7. Structural Identification of Omekaxin AC

[0258]

[0259]

[0260]

[0261] Among them, the chemical structures of the new Omicsynin A class compounds (Omicsynin A1, Omicsynin A2) are as follows: Figure 21 As shown in Table 8, the NMR data are as follows. 1 H-NMR, 13 C-NMR, DEPT 1 H- 1 H COSY, HSQC, HMBC, NOESY spectra and HRMS analysis data are as follows: Figure 22 , Figure 23 As shown.

[0262] HRMS analysis data for compounds A6, B1, B2, B3, B5, B6, C1, C2, and C6 are as follows: Figures 24-32 As shown

[0263] Table 8. NMR data (600 MHz, DMSO-d6) of compounds Omicsynin A1 and Omicsynin A2

[0264]

[0265]

[0266] Example 8: Determination of the inhibitory activity of Omicsynin-like compounds against coronaviruses

[0267] 1. CPE method for antiviral efficacy testing:

[0268] (1) The experiment was conducted in passaged hepatocytes Huh7.5, with 1 × 10⁶ cells per cell line. 4 Inoculate 100 cells / well into a 96-well plate and incubate overnight;

[0269] (2) Cells were infected with 100 TCID50 coronavirus solution. The test drug was diluted with culture medium and tested under two administration regimens: administration at the same time as infection and administration 2 hours after infection. The test drug was tested by three-fold dilution of 8 doses of sample. The positive control drug ribavirin injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd. and diluted to the required concentration before use.

[0270] (3) Two parallel wells were set for each dose. The results were observed when the lesions of the virus control group reached the CPE evaluation standard 4+. The results were recorded and the half-maximal inhibitory concentration (IC50) of the drug against the virus was calculated using the Reed-Muench method (the formula is as follows). The selection index (SI = TC50 / IC50) was also calculated.

[0271]

[0272] Where: A = drug concentration with cumulative inhibition rate < 50%, B = inhibition rate with cumulative inhibition rate > 50%, C = inhibition rate with cumulative inhibition rate < 50%, D = log dilution factor.

[0273] CPE evaluation criteria: The percentage of cell death is marked as 4+ (75%–100% cell death), 3+ (50%–75% cell death), 2+ (25%–50% cell death), 1+ (0–25% cell death), and 0+ (all cells survive).

[0274] Repeat the experiment at least twice and provide representative results.

[0275] 2. Efficacy against HCoV-229E strain

[0276] (1) Simultaneous administration during infection: In Huh7.5 cells, the inhibitory effect of 0h administration on HCoV-229E strain was determined by CPE method, and the activity of ribavirin (RBV) was determined at the same time.

[0277]

[0278]

[0279] Sample Description:

[0280] RBV, ribavirin injection, was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., batch number 31712252, specification 100mg / ml;

[0281] Antipain (#37682-72-7, 5mg) was purchased from Shanghai Yifei Biotechnology Co., Ltd., and is equivalent to the monomeric compound Omicsynin B4 isolated from the fermentation sample of Streptomyces CPCC200451 as described in this invention.

[0282] Chymostatin (#9076-44-2, 5 mg) was purchased from Sigma-Aldrich. This reagent contains three compounds, Chymostatin A, B, and C, which are equivalent to the compounds Omicsynin C3 and C5 described in this invention.

[0283] The test samples 7094-KO-20E, 7094-KO-50E, and 7094-KO-100E were 20% ethanol, 50% ethanol, and 100% ethanol eluents obtained by macroporous adsorption resin column chromatography of the fermentation broth of the blocking strain 7094-KO (a mutant strain that knocks out the 7094 gene in Cluster36).

[0284] Test samples 200451-20E, 200451-50E, and 200451-100E are 20% ethanol, 50% ethanol, and 100% ethanol eluents obtained by macroporous adsorption resin column chromatography of the fermentation broth of wild-type Streptomyces CPCC 200451.

[0285] ODS 24 and ODS 26 are the 24th and 26th fractions collected from sample 200451-50E after reverse-phase C18 column chromatography and elution with 30% acetonitrile-water. They both belong to the 50E-C18-G component described in Example 6 of this invention. Fraction 24 mainly contains compounds Omicsynin A3 and A4, and fraction 26 mainly contains compounds Omicsynin A1 and A2.

[0286] Test samples 200451-A3 and 200451-A3Fe were respectively tested using Streptomyces CPCC 200451 A3 and A3-Fe. 3+ (0.05% Fe) 3+ The solid sample obtained by freeze-drying the fermentation supernatant obtained from the fermentation medium.

[0287] (2) Administration 2 hours after infection: In Huh7.5 cells, the inhibitory effect of administration 2 hours after infection on HCoV-229E strain was determined by CPE method, and the activity of ribavirin (RBV) was determined at the same time.

[0288]

[0289] Sample description: Same as above

[0290] The results show that the fermentation broth of Streptomyces CPCC 200451 containing omexixin and the omexixin-like compounds purified from the fermentation broth also have a good inhibitory effect on coronaviruses.

[0291] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A group of Omicin B6-like compounds, the structure of the Omicin B6-like compounds is shown in formula (1) : wherein The optional various substituents of R1-R5 are shown in the following table: 2.A preparation method of the Omicin B6-like compounds in claim 1, The method comprises the following steps, (1) collecting the supernatant after centrifugation of the fermentation broth of Streptomyces sp. CPCC 200451 at 4000 rpm for 15 min; (2) collecting the active components by using macroporous adsorption resin cascade reverse phase C18 chromatographic column HPLC method; (3) separating the active components obtained in step (2) by semi-preparative RP-HPLC to obtain the peptide compounds; The fermentation broth in step (1) is: After inoculation in A3 fermentation medium at an inoculation amount of 10%, the fermentation broth is collected after culturing at 28℃, 200 rpm for 3-12 d; The content of each component in the A3 fermentation medium is: glycerol 20 g / L, dextrin 20 g / L, peptone 10 g / L, yeast powder 5 g / L, ammonium sulfate 2 g / L, calcium carbonate 2 g / L; pH 7.2-7.4; The step parameters of the macroporous adsorption resin cascade reverse phase HPLC method in step (2) are: HP20 macroporous adsorption resin and C18 reverse phase HPLC chromatographic column are used, The separation steps are: 1) after adsorption of the supernatant by macroporous adsorption resin HP20, two column volumes of deionized water are used for washing; 2) gradient elution is performed by using ethanol-water, and the eluate of 50% ethanol gradient is collected after each gradient elution until the effluent is colorless or the color does not change, and the eluate is concentrated under reduced pressure for standby; 3) after concentration of the eluate of 50% ethanol gradient obtained in step 2), the eluate is loaded into a C18 column and eluted by gradient elution of acetonitrile-water, and the eluate of 15%-80% gradient is collected; The semi-preparative RP-HPLC parameters and method in step (3) are: chromatographic column: SHISEIDO Capcell-Pak PFP 5μm, 10×250mm; the mobile phase is acetonitrile and water containing 0.1% HCOOH, respectively: 20% ACN / 0.1% HCOOH H2O to collect Omicin B series compounds, Flow rate: 1.5 mL / min.

3. The Omicin B6-like compounds in claim 1 are used for preparing an antiviral drug, and the virus is an influenza virus.

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