Antibiotic compound as well as preparation method and application thereof

Abstract

The invention discloses an antibiotic compound as well as a preparation method and application thereof, and the antibiotic compound disclosed by the invention comprises antibiotic dihydrotecamycin (dhTDM) and antibiotic tecamycin (TDM). The chemical structural formulas of the dhTDM and the antibiotic ticamycin TDM are shown as a formula (I): # imgabs0 #. The antibiotic dhTDM and the antibiotic ticamycin TDM with unique tetracyclic systems (decahydronaphthalene ring, seven-membered oxygen heterocyclic ring and acetoacetic acid lactone) are separated and identified from marine actinomycetes CGMCC4.1698 for the first time, and a lead molecule with a novel structure is provided for research and development of a novel antibacterial preparation. Not only is the blank of a tetracyclic polyketone antibiotic biosynthesis mechanism filled, but also a powerful candidate molecule and an innovative technical path are provided for solving the problem of antibiotic drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an antibiotic compound, a preparation method and application thereof. Background Art

[0002] Many natural products contain decahydronaphthalene ring building blocks, the cyclohexene building blocks of which are constructed via the Diels-Alder (DA) reaction. Reported DA enzymes include LovB in the lipid-lowering drug lovastatin, PyrE3 in the biosynthesis of pyrroindomycin, and Sol5 in solanapyrone. In recent years, teams both domestically and internationally have reported numerous enzymatic DA reactions, characterized the corresponding DA enzymes, and revealed their catalytic mechanisms. Based on this understanding of the catalytic mechanisms, enzyme engineering can be used to manipulate the regio- and stereoselectivity of the enzymes, enabling the generation of single-configuration DA products. This has important applications in the precise and efficient synthesis of DA products. Using novel DA enzymes as probes will help uncover more drug-derived molecules.

[0003] Early studies suggested that tetrodecamycin originates from the polyketide synthesis pathway, with the decahydronaphthalene ring structure likely constructed by a DA reaction. However, the biosynthetic process remained largely unresolved. Whole-genome sequencing, bioinformatics analysis, and in vivo gene knockout identified the biosynthetic gene cluster. Protein expression and purification, combined with in vitro biochemical experiments, further elucidated the biosynthetic process, revealing that an FAD-dependent 4+2 cyclase constructs the core trans-decahydronaphthalene ring, a P450 enzyme constructs the seven-membered oxygen heterocycle and hydroxylates at the C-13 position, and NADH / NADPH reduces the tetronate double bond. This study refines the biosynthetic process of dihydrotetrodecamycin, further enhancing our understanding of tetronate-containing natural products and providing a probe for bacterial gene mining, thereby facilitating the discovery of lead active molecules.

[0004] At present, an antibiotic compound, a preparation method and application thereof have not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide an antibiotic compound and a preparation method and application thereof.

[0006] The purpose of this invention is to address the current challenge of antibiotic resistance, discover new active molecules, and analyze their biosynthetic pathways, thereby facilitating the efficient production of large quantities of antibiotics through chemoenzymatic and synthetic biology approaches. The biosynthetic gene clusters, biosynthetic pathways, and enzymes involved in the synthesis of TDM and dhTDM are protected.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions: In a first aspect, the present application provides an antibiotic compound.

[0008] In a second aspect, the present application provides a method for preparing an antibiotic compound.

[0009] In a third aspect, the present application provides an application of an antibiotic dihydroticamycin compound dhTDM and an antibiotic ticamycin TDM in the preparation of an antibacterial preparation.

[0010] In a fourth aspect, the present application provides a gene cluster related to the biosynthesis of the antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM.

[0011] In a fifth aspect, the present application provides an application of a gene cluster in catalyzing the biosynthesis of dhTDM and the antibiotic ticamycin TDM.

[0012] In a first aspect, the present application provides an antibiotic compound, which includes the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM. The chemical structures of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are shown in formula (I):

[0013]

[0014] A second aspect of the present application provides a method for preparing an antibiotic compound, comprising the following steps:

[0015] (1) The strain CGMCC4.1698 was cultured in solid ISP3 medium; the strain CGMCC4.1698 was purchased from the China General Microbiological Culture Collection (CGMCC).

[0016] (2) cutting the culture obtained in step (1) into pieces, placing them in a culture medium, and culturing them at 30° C. for 5-7 days;

[0017] (3) extracting the culture medium obtained in step (2) with ethyl acetate and methanol organic solvents, and concentrating to obtain a crude extract F1;

[0018] (4) The extracted ethyl acetate was combined and concentrated using a rotary evaporator to obtain a crude paste. After the crude paste was weighed, 2 times the weight of the reverse phase filler was weighed and mixed with the sample. After the sample was completely dried, it was ground and gradient eluted using a methanol-water system from 10% methanol to 100% methanol for a total of 12 column volumes. The eluted fraction was analyzed by analytical HPLC and further purified by gel column chromatography. The fraction purified by gel column chromatography was analyzed again;

[0019] (5) The components obtained in step (4) were separated and purified by preparative HPLC to obtain antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM.

[0020] Furthermore, in step (1), the solid ISP3 medium was prepared by boiling 20 g of oats in boiling water for 20 minutes, filtering out the oat grains to obtain oat juice; then weighing 0.2 g of KNO3, 0.2 g of MgSO4·7H2O, 0.5 g of K2HPO4·3H2O, 20g agar, are mixed with oat juice and diluted with water to 1L; the culture conditions for SP3 plate fermentation are 30°C for 3-4 days; in step (2), the preparation method of F medium is as follows: 20g sucrose, 10g glucose, 0.1g casein hydrolysate casminoacids, 5g yeast extract, 5g 3-(N-morpholino)propanesulfonic acid MOPS, 100μL trace element solution, 0.25g K2SO4, 1g MgCl2·6H2O, and the volume is diluted to 1L with distilled water; the preparation method of trace element solution is as follows: 0.04g ZnCl2, 0.2g FeCl3·6H2O, 0.01g CuCl2·2H2O, 0.01g MnCl2·4H2O, 0.01g Na2B4O7·10H2O, 0.01g (NH4)6Mo7O 24 0.01 g of 4H2O was dissolved in 100 ml of ultrapure water and used as mother liquor. In step (3), the organic solvents were ethyl acetate and methanol.

[0021] Furthermore,

[0022]

[0023] (a) A polyketide chain is formed under the catalysis of polyketide synthases TedS1, TedS2, TedS3, and TedS4, and then tetronate synthesis is carried out to obtain ticamycin-6;

[0024] (b) Ticamycin-6 undergoes a 4+2 cycloaddition reaction catalyzed by the FAD-dependent 4+2 cyclase TedJ to construct the core skeleton of the molecule, the trans-decalin ring, to give ticamycin-7;

[0025] (c) Ticamycin-7 forms a seven-membered oxygen heterocycle catalyzed by the P450 enzyme ScTedH and undergoes hydroxylation at position 13 to produce 13-deoxyticamycin and ticamycin;

[0026] (d) Ticamycin is reacted with NADH / NADPH to reduce its double bond and generate the final product dhTDM.

[0027] Furthermore, the key enzymes include polyketide synthases TedS1, TedS2, TedS3, and TedS4; FAD-dependent 4+2 cyclase TedJ; and P450 enzyme ScTedH. The enzymes are used to catalyze the core skeleton construction and functional group modification of dhTDM and the antibiotic ticamycin TDM.

[0028] The third aspect of the present application provides a use of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM in the preparation of an antibacterial agent.

[0029] Furthermore, the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are used to prepare lead molecules for antibacterial preparations.

[0030] In a fourth aspect, the present application provides a gene cluster related to the biosynthesis of the antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM, wherein the gene cluster comprises the nucleotide sequences shown in SEQ ID NOs: 1-6.

[0031] Further, the gene cluster contains the following genes:

[0032] Genes encoding polyketide synthase TedS1, polyketide synthase TedS2, polyketide synthase TedS3, and polyketide synthase TedS4;

[0033] The gene encoding the FAD-dependent 4+2 cyclase TedJ;

[0034] the gene encoding the P450 enzyme TedH;

[0035] In a fifth aspect, the present application provides an application of a gene cluster in catalyzing the biosynthesis of dhTDM and the antibiotic ticamycin TDM.

[0036] Beneficial effects: This invention fills the gap in the biosynthesis of tetracyclic polyketide antibiotics for the first time, and also provides a potent candidate molecule and innovative technical path for solving the problem of antibiotic resistance, which has significant academic value and application prospects.

[0037] Compared with the existing technologies, the present invention has the following advantages: (1) Through whole genome sequencing, gene knockout and in vitro biochemical experiments, the present invention fully reveals the biosynthetic pathway of dhTDM and clarifies the catalytic functions of key enzymes such as polyketide synthase (TedS1, TedS2, TedS3, TedS4), FAD-dependent 4+2 cyclase (TedJ), P450 hydroxylase (ScTedH). Among them, the cycloaddition reaction catalyzed by TedJ constructs the core skeleton of the trans-decalin ring for the first time, providing a new paradigm for the study of the ring system construction mechanism of natural products.

[0038] (2) The TDM of the present invention has a significant inhibitory effect on Gram-negative bacteria Photobacterium damselae ssp. Piscicida (MIC1.56-6.25μg / mL) and Gram-positive bacteria (such as MRSA, Staphylococcus aureus, etc.). Its activity is better than that of many existing antibiotics, especially showing important application potential in the field of drug-resistant bacteria prevention and control.

[0039] (3) The key enzymes identified in this invention (such as TedJ and ScTedH) lay the foundation for the chemoenzymatic synthesis of TDM compounds. The catalytic efficiency and selectivity can be optimized through enzyme engineering to achieve efficient and targeted synthesis, thereby reducing the cost and complexity of traditional chemical synthesis.

[0040] (4) The protection of the CGMCC4.1698 biosynthetic gene cluster by the present invention provides core biological resources for subsequent gene mining, combinatorial biosynthesis and drug development, and facilitates the high-throughput screening and modification of new antibiotics.

[0041] (5) The exo-methylene group (Michael acceptor) in the TDM molecule of the present invention provides an active site for structural modification, and different functional groups can be introduced through chemical modification to optimize the pharmacokinetic properties and antibacterial spectrum, thereby accelerating the clinical transformation of the lead compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0043] Figure 1 Figure 1 is a diagram of the gene cluster and biosynthesis pathway of dhTDM of the present invention.

[0044] Figure 2 This is a diagram of protein expression and purification involved in the biosynthesis process of the present invention.

[0045] Figure 3 is the hydrogen spectrum of dihydroticamycin of the present invention;

[0046] Figure 4 is the carbon spectrum of dihydroticamycin of the present invention;

[0047] Figure 5 is the DEPT spectrum of dihydroticamycin of the present invention;

[0048] Figure 6 is the COSY correlation spectrum of dihydroticamycin of the present invention;

[0049] Figure 7 is the HSQC spectrum of dihydroticamycin of the present invention;

[0050] Figure 8 The HMBC spectrum of the dihydroticamycin of the present invention;

[0051] Figure 9 is the NOESY correlation spectrum of dihydroticamycin of the present invention;

[0052] Figure 10 This is a high-resolution mass spectrum of the dihydroticamycin of the present invention. DETAILED DESCRIPTION

[0053] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0054] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0055] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0056] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0057] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0058] In a first aspect, an embodiment of the present application provides an antibiotic compound, comprising the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM, wherein the chemical structures of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are shown in formula (I):

[0059]

[0060]

[0061] A second aspect of the present invention provides a method for preparing an antibiotic compound, comprising the following steps:

[0062] (1) The strain CGMCC4.1698 was cultured in solid ISP3 medium; the strain CGMCC4.1698 was purchased from the China General Microbiological Culture Collection (CGMCC).

[0063] (2) cutting the culture obtained in step (1) into pieces, placing them in a culture medium, and culturing them at 30° C. for 5-7 days;

[0064] (3) extracting the culture medium obtained in step (2) with an organic solvent such as ethyl acetate and concentrating the extract to obtain a crude extract F1;

[0065] (4) The extracted ethyl acetate was combined and concentrated using a rotary evaporator to obtain a crude paste. After the crude paste was weighed, 2 times the weight of the reverse phase filler was weighed and mixed with the sample. After the sample was completely dried, it was ground and gradient eluted using a methanol-water system from 10% methanol to 100% methanol for a total of 12 column volumes. The eluted fractions were analyzed by analytical HPLC and combined as needed, or further purified by gel column chromatography. The fractions purified by gel column chromatography were analyzed again;

[0066] (5) The components obtained in step (4) were separated and purified by preparative HPLC to obtain antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM.

[0067] In some embodiments, in step (1), the solid ISP3 medium is prepared by boiling 20 g of oats in boiling water for 20 minutes, filtering out the oat particles to obtain oat juice; then weighing 0.2 g of KNO3, 0.2 g of MgSO4·7H2O, 0.5gK2HPO4·3H2O, 20g agar, mixed with oat juice, and diluted to 1L with water; SP3 plate fermentation culture conditions are 30°C for 3-4 days; in step (2), the preparation method of F culture medium is as follows: 20g sucrose, 10g glucose, 0.1g casein hydrolyzate casminoacids, 5g yeast extract, 5g 3-(N-morpholino)propanesulfonic acid MOPS, 100 microliters of trace element solution, 0.25g K2SO4, 1g MgCl2·6H2O, and diluted to 1L with distilled water; the preparation method of trace element solution is as follows: 0.04g ZnCl2, 0.2g FeCl3·6H2O, 0.01g CuCl2·2H2O, 0.01g MnCl2·4H2O, 7·10H2O 0.01g,(NH4)6Mo7O 24 0.01 g of 4H2O was dissolved in 100 ml of ultrapure water and used as mother liquor. In step (3), the organic solvents were ethyl acetate and methanol.

[0068]

[0069] (a) A polyketide chain is formed under the catalysis of polyketide synthases TedS1, TedS2, TedS3, and TedS4, and then tetronate synthesis is carried out to obtain ticamycin-6;

[0070] (b) Ticamycin-6 undergoes a 4+2 cycloaddition reaction catalyzed by the FAD-dependent 4+2 cyclase TedJ to construct the core skeleton of the molecule, the trans-decalin ring, to give ticamycin-7;

[0071] (c) Ticamycin-7 forms a seven-membered oxygen heterocycle catalyzed by the P450 enzyme ScTedH and undergoes hydroxylation at position 13 to produce 13-deoxyticamycin and ticamycin;

[0072] (d) Ticamycin is reacted with NADH / NADPH to reduce its double bond and generate the final product dhTDM.

[0073] In some embodiments, key enzymes include polyketide synthases TedS1, TedS2, TedS3, and TedS4; FAD-dependent 4+2 cyclase TedJ; and P450 enzyme ScTedH. These enzymes are used to catalyze the core skeleton construction and functional group modification of dhTDM and the antibiotic ticamycin TDM.

[0074] A third aspect of the embodiments of the present application provides a use of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM in the preparation of an antibacterial agent.

[0075] In some embodiments, the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are used to prepare lead molecules for antibacterial agents.

[0076] In a fourth aspect, an embodiment of the present application provides a gene cluster related to the biosynthesis of the antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM, wherein the gene cluster comprises the nucleotide sequences shown in SEQ ID NOs: 1-6.

[0077] In some embodiments, the gene cluster comprises the following genes:

[0078] Genes encoding polyketide synthase TedS1, polyketide synthase TedS2, polyketide synthase TedS3, and polyketide synthase TedS4;

[0079] The gene encoding the FAD-dependent 4+2 cyclase TedJ;

[0080] the gene encoding the P450 enzyme TedH;

[0081] In a fifth aspect, the embodiments of the present application provide an application of a gene cluster in catalyzing the biosynthesis of dhTDM and the antibiotic ticamycin TDM.

[0082] Example 1

[0083] An antibiotic compound of the present invention includes the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM. The chemical structures of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are shown in formula (I):

[0084]

[0085]

[0086] Example 2

[0087] A method for preparing an antibiotic compound of the present invention comprises the following steps:

[0088] (1) The strain CGMCC4.1698 was cultured in solid ISP3 medium; the strain CGMCC4.1698 was purchased from the China General Microbiological Culture Collection (CGMCC). The solid ISP3 medium was prepared by boiling 20 g of oats in boiling water for 20 minutes, filtering out the oat grains to obtain oat juice; then weighing 0.2 g of KNO3, 0.2 g of MgSO4·7H2O, 0.5 g of K2HPO4·3H2O, and 20 g of agar, mixing with the oat juice, and adding water to make the volume 1 L; the SP3 plate fermentation culture was cultured at 30°C for 3-4 days;

[0089] (2) The culture obtained in step (1) was cut into pieces, placed in a culture medium, and cultured at 30°C for 5-7 days; the preparation method of F culture medium was as follows: 20g sucrose, 10g glucose, 0.1g casein hydrolyzate casmino acids, 5g yeast extract, 5g 3-(N-morpholino)propanesulfonic acid MOPS, 100μL trace element solution, 0.25g K2SO4, 1g MgCl2·6H2O, and the volume was adjusted to 1L with distilled water; the preparation method of trace element solution was as follows: 0.04g ZnCl2, 0.2g FeCl3·6H2O, 0.01g CuCl2·2H2O, 0.01g MnCl2·4H2O, 0.01g Na2B4O7·10H2O, 0.01g (NH4)6Mo7O 24 Dissolve 0.01 g of 4H2O in 100 ml of ultrapure water and use as the mother solution.

[0090] (3) extracting the culture medium obtained in step (2) with an organic solvent such as ethyl acetate and concentrating the extract to obtain a crude extract F1; the organic solvent is ethyl acetate;

[0091] (4) The extracted ethyl acetate was combined and concentrated using a rotary evaporator to obtain a crude paste. After the crude paste was weighed, 2 times the weight of the reverse phase filler was weighed and mixed with the sample. After the sample was completely dried, it was ground and gradient eluted using a methanol-water system from 10% methanol to 100% methanol for a total of 12 column volumes. The eluted fractions were analyzed by analytical HPLC and combined as needed, or further purified by gel column chromatography. The fractions purified by gel column chromatography were analyzed again;

[0092] (5) The components obtained in step (4) were separated and purified by preparative HPLC to obtain antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM.

[0093]

[0094] (a) A polyketide chain is formed under the catalysis of polyketide synthases TedS1, TedS2, TedS3, and TedS4, and then tetronate synthesis is carried out to obtain ticamycin-6;

[0095] (b) Ticamycin-6 undergoes a 4+2 cycloaddition reaction catalyzed by the FAD-dependent 4+2 cyclase TedJ to construct the core skeleton of the molecule, the trans-decalin ring, to give ticamycin-7;

[0096] (c) Ticamycin-7 forms a seven-membered oxygen heterocycle catalyzed by the P450 enzyme ScTedH and undergoes hydroxylation at position 13 to produce 13-deoxyticamycin and ticamycin;

[0097] (d) Ticamycin is reacted with NADH / NADPH to reduce its double bond and generate the final product dhTDM.

[0098] In some embodiments, the key enzymes include polyketide synthases TedS1, TedS2, TedS3, and TedS4; FAD-dependent [4+2] cyclase TedJ; and P450 enzyme ScTedH. The enzymes are used to catalyze the core skeleton construction and functional group modification of dhTDM and the antibiotic ticamycin TDM.

[0099] Example 3

[0100] The difference between Example 3 and Example 2 is that in step (3), the culture medium obtained in step (2) is extracted with an organic solvent such as ethyl acetate and concentrated to obtain a crude extract F1; the organic solvent is methanol.

[0101] Example 4

[0102] The invention discloses an application of an antibiotic dihydroticamycin dhTDM and an antibiotic ticamycin TDM in the preparation of an antibacterial agent. The antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are used to prepare a lead molecule of an antibacterial preparation.

[0103] Example 5

[0104] The present invention discloses a gene cluster related to the biosynthesis of the antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM, wherein the gene cluster comprises the nucleotide sequences shown in SEQ ID NOs: 1-4.

[0105] The following genes are included in the gene cluster:

[0106] Genes encoding polyketide synthase TedS1, polyketide synthase TedS2, polyketide synthase TedS3, and polyketide synthase TedS4;

[0107] The gene encoding the FAD-dependent 4+2 cyclase TedJ;

[0108] the gene encoding the P450 enzyme TedH;

[0109] Example 6

[0110] The invention discloses an application of a gene cluster in catalyzing the biosynthesis of dhTDM and antibiotic ticamycin TDM.

[0111] Example 7

[0112] As 3 to Figure 10 The following are the nuclear magnetic resonance data and mass spectrometry data spectra of dihydroticarcin dhTDM and ticarcin TDM of the present invention. Table 1 and Table 2 are the signal attributions of the nuclear magnetic resonance (NMR) spectra of the present invention.

[0113]

[0114] Table 1 Attribution of NMR signals of compound 1

[0115]

[0116]

[0117] Table 2 Attribution of NMR signals of compound 2

[0118]

[0119] Figure 2 This is a diagram of protein expression and purification involved in the biosynthesis process of the present invention.

[0120] The present invention discloses an antibiotic dihydroticamycin compound, which includes dihydroticamycin (dhTDM) and ticamycin (TDM). The chemical structures of dhTDM and the antibiotic ticamycin TDM are shown in formula (I):

[0121]

[0122]

[0123] Example 7

[0124] The difference between Example 7 and Example 2 is that the inventors isolated and identified dihydroticamycin (dhTDM) and ticamycin (TDM) from an actinomycete CGMCC4.1698 of marine origin.

[0125] Both TDM and dhTDM possess a unique tetracyclic ring system consisting of a decahydronaphthalene ring, a seven-membered heterocyclic ring containing an oxygen atom, and a tetronate. A key feature of TDM is the presence of an exo-methylene group within the tetronate unit. This allows for Michael addition reactions, allowing the introduction of different substituents at this position to influence its biological activity. In contrast, the dhTDM structure contains a methyl group at the same position. TDM exhibits inhibitory activity against the Gram-negative bacterium Photobacterium damselae ssp. Piscicida (MIC values ​​1.56-6.25 μg / mL), Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Micrococcus luteus, and Bacillus subtilis (MIC values ​​6.25-12.5 μg / mL).

[0126] Extraction and separation of dhTDM and antibiotic ticamycin TDM

[0127] The crude extract obtained from fermentation was weighed, and then twice the weight of reverse-phase filler was weighed and mixed. After the sample was completely dried, it was ground and gradient eluted using a methanol-water system, from 10% methanol to 100% methanol for a total of 12 column volumes. The eluted fractions were analyzed by analytical HPLC, combined as needed, or further purified by gel column chromatography. The fractions purified by gel column chromatography were analyzed again. The fractions containing the target compound were concentrated under reduced pressure, dissolved in methanol, centrifuged, and the supernatant was aspirated. The purified compound was finally isolated and purified by semi-preparative HPLC. The compound dhTDM was isolated by semi-preparative HPLC (40% acetonitrile-water, 2.5 mL / min). TDM was isolated by semi-preparative HPLC (40% acetonitrile-water, 2.5 mL / min).

[0128] Principle of synthesis during biosynthesis:

[0129] like Figure 1 As shown, Figure 1 The gene cluster and biosynthetic pathway of dhTDM of the present invention are shown. The biosynthetic pathways of the antibiotic compounds dihydroticarcin dhTDM and ticarcin TDM are analyzed.

[0130] The biosynthesis process of an antibiotic dihydroticamycin compound invented includes the following steps:

[0131]

[0132] (a) A polyketide chain is formed under the catalysis of polyketide synthases TedS1, TedS2, TedS3, and TedS4, and then tetronate synthesis is carried out to obtain ticamycin-6;

[0133] (b) Ticamycin-6 undergoes a 4+2 cycloaddition reaction catalyzed by the FAD-dependent 4+2 cyclase TedJ to construct the core skeleton of the molecule, the trans-decalin ring, to give ticamycin-7;

[0134] (c) Ticamycin-7 forms a seven-membered oxygen heterocycle under the catalysis of the P450 enzyme ScTedH and undergoes hydroxylation at the C-13 position to produce 13-deoxyticamycin and ticamycin;

[0135] (d) Ticamycin undergoes double bond reduction via the action of NADH / NADPH, producing the final product, dhTDM, dihydroticamycin. Key enzymes involved include polyketide synthases TedS1, TedS2, TedS3, and TedS4; the FAD-dependent 4+2 cyclase TedJ; and the P450 enzyme ScTedH. These enzymes catalyze the core backbone construction and functional group modification of dhTDM and the antibiotic ticamycin TDM.

[0136] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. An antibiotic compound, characterized in that: The antibiotic compounds include the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM. The chemical structural formulas of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are shown in formula (I):

2. The method for preparing the antibiotic compound according to claim 1, characterized in that The steps include: (1) Cultivate strain CGMCC4.1698 on solid ISP3 medium; (2) Cut the culture obtained in step (1) into pieces, inoculate them into F medium, and culture them at 30°C for 5-7 days; (3) extracting the culture medium obtained in step (2) with an organic solvent and concentrating the extract to obtain a crude extract F1; (4) The extracted ethyl acetate was combined and concentrated using a rotary evaporator to obtain a crude paste. After the crude paste was weighed, 2 times the weight of the reverse phase filler was weighed and mixed with the sample. After the sample was completely dried, it was ground and gradient eluted using a methanol-water system from 10% methanol to 100% methanol for a total of 12 column volumes. The eluted fraction was analyzed by analytical HPLC and further purified by gel column chromatography. The fraction purified by gel column chromatography was analyzed again; (5) The components obtained in step (4) were separated and purified by preparative HPLC to obtain antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM.

3. The method for preparing an antibiotic compound according to claim 2, wherein: In step (1), the preparation method of solid ISP3 culture medium is as follows: 20g of oats are boiled in boiling water for 20 minutes, and oat particles are filtered out to obtain oat juice; then 0.2g of KNO3, 0.2g of MgSO4·7H2O, 0.5g of K2HPO4·3H2O, and 20g of agar are weighed, mixed with the oat juice, and the volume is adjusted to 1L with water; the culture conditions of SP3 plate fermentation culture are 30°C for 3-4 days; in step (2), the preparation method of F culture medium is as follows: 20g of sucrose, 10g of glucose, 0.1g of casein hydrolyzate casmino acids, 5g of yeast extract, 5g of 3-(N-morpholino)propanesulfonic acid MOPS, 100 microliters of trace element solution, 0.25g of K2SO4, and 1g of MgCl2·6H2O are added, and the volume is adjusted to 1L with distilled water; the preparation method of trace element solution is as follows: 0.04g of ZnCl2, FeCl3·6H2O 0.2g, CuCl2·2H2O 0.01g, MnCl2·4H2O0.01g, Na2B4O7·10H2O 0.01g, (NH4)6Mo7O 24 0.01 g of 4H2O was dissolved in 100 ml of ultrapure water and used as mother liquor. In step (3), the organic solvents were ethyl acetate and methanol.

4. The method for preparing an antibiotic compound according to claim 2, wherein: (a) A polyketide chain is formed under the catalysis of polyketide synthases TedS1, TedS2, TedS3, and TedS4, and then tetronate synthesis is carried out to obtain ticamycin-6; (b) Ticamycin-6 undergoes a 4+2 cycloaddition reaction catalyzed by the FAD-dependent 4+2 cyclase TedJ to construct the core skeleton of the molecule, the trans-decalin ring, to give ticamycin-7; (c) Ticamycin-7 forms a seven-membered oxygen heterocycle catalyzed by the P450 enzyme ScTedH and undergoes hydroxylation at position 13 to produce 13-deoxyticamycin and ticamycin TDM; (d) Ticamycin is reacted with NADH / NADPH to reduce the double bond and generate the final product dhTDM.

5. The method for preparing an antibiotic compound according to claim 4, wherein: The key enzymes include polyketide synthases TedS1, TedS2, TedS3, and TedS4; FAD-dependent 4+2 cyclase TedJ; and P450 enzyme ScTedH. The enzymes are used to catalyze the core skeleton construction and functional group modification of dhTDM and the antibiotic ticamycin TDM.

6. Use of the antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM according to any one of claims 1 to 4 in the preparation of an antibacterial agent.

7. The use according to claim 6, characterized in that: The antibiotic dihydroticamycin dhTDM and the antibiotic ticamycin TDM are used to prepare lead molecules for antibacterial preparations.

8. A gene cluster related to the biosynthesis of the antibiotic compounds dihydroticamycin dhTDM and ticamycin TDM according to claim 1, characterized in that: The gene cluster comprises the nucleotide sequences shown in SEQ ID NOs: 1-6.

9. The gene cluster according to claim 8, characterized in that: The gene cluster contains the following genes: Genes encoding polyketide synthase TedS1, polyketide synthase TedS2, polyketide synthase TedS3, and polyketide synthase TedS4; The gene encoding the FAD-dependent 4+2 cyclase TedJ; The gene encoding the P450 enzyme TedH.

10. Use of the gene cluster according to claim 8 in catalyzing the biosynthesis of dhTDM and the antibiotic ticamycin TDM.

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