CN112409372A

CN112409372A - Rubicin analogue, preparation method and application thereof

Info

Publication number: CN112409372A
Application number: CN202011223059.9A
Authority: CN
Inventors: 段燕文; 朱湘成; 易理伟
Original assignee: Changsha Cihang Pharmaceutical Institute Co ltd; Changsha Tianci Biomedicine Technology Co ltd; Hayao Cihang Pharmaceutical Co ltd
Current assignee: Changsha Cihang Pharmaceutical Institute Co ltd; Changsha Tianci Biomedicine Technology Co ltd; Hayao Cihang Pharmaceutical Co ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-02-26
Anticipated expiration: 2040-11-05
Also published as: CN112409372B

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a novel rubicin analogue, and a preparation method and application thereof. The invention provides four new structural rubicin analogues, the structural formula of which is shown as the formula (I):

Description

Rubicin analogue, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a novel rubicin analogue, a preparation method and application thereof.

Background

Tumors, as a serious disease endangering human health, have become the first killer of human in the new century, and are the biggest public health threat all over the world. Therefore, the research and development of anti-cancer drugs are always the key points of urgent needs of social development and civilian life. According to the statistical analysis of the tumor tissue specimens detected at present, the positive rate of the telomerase activity of malignant tumor tissues reaches 85% -95%, and the detection rate of the telomerase activity of benign tumors and normal tissues is only about 4%. Telomeres and telomerase are used as new 'targets' for the research of human antitumor drugs, and the development of telomerase inhibitors also becomes a hot spot for the development of antitumor drugs.

Rubigins (rubins, Rubs) natural products have a5, 6 spiroketal core backbone. Of the 20 reported rubicin analogs, the naphthoquinone structure can be divided into two types: one is naphthoquinone with a quinone ring linked to a5, 6 spiroketal, such as β -rubicin; the other is the benzene ring of naphthoquinone linked to a5, 6 spiroketal, such as gamma-rubicin. The vast majority of the rubicin natural products are gamma-rubicin type skeletons, and only have 3 beta-rubicin type structures. The zearalenone natural product has wide biological activity, such as antibacterial activity, cytotoxicity, reverse transcriptase inhibiting activity and the like, and especially, part of compounds show good inhibiting activity on human telomerase. Of the 20 zearalenone natural products reported, 5 showed good inhibitory activity against telomerase, with β -rubicin having a unique naphthoquinone structure being the best. The natural products of the type have poor solubility in most solvents, and the development of the rubicin natural products as a new generation of antitumor drugs taking telomerase as a target is greatly limited. Therefore, the discovery of new structures of rubicin is of particular importance.

Disclosure of Invention

The invention provides four new structural rubicin analogues and a preparation method and application thereof.

The technical scheme of the invention is as follows:

a rubicin analog having a structural formula as shown in formula (I):

the invention also provides a preparation method of the rubicin analogue, which is prepared by fermenting Streptomyces sp.CB00271, genetically engineered strain Streptomyces sp.ZX02 or genetically engineered strain Streptomyces sp.ZX06, wherein the genetically engineered strain Streptomyces sp.ZX02 is a genetically engineered strain obtained by knocking out orf9 gene in the genome of Streptomyces sp.CB00271, and the genetically engineered strain Streptomyces sp.ZX06 is a genetically engineered strain obtained by knocking out orf4 gene in the genome of Streptomyces sp.CB00271; the nucleotide sequence of the orf9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the orf4 gene is shown as SEQ ID NO. 2.

Preferably, in the above rubicin analog, the compound 3 is prepared by fermenting Streptomyces sp.cb00271, which comprises the following steps:

s1, fermentation: inoculating Streptomyces sp.CB00271 spores into a culture medium containing 50mL of Tryptic Soy Broth (TSB) for culturing for 1-2 days, then inoculating the Streptomyces sp.CB00271 spores into an mR2A culture medium according to the inoculation amount of 4%, continuously culturing for 7 days, adjusting the pH of the fermentation liquid to 10-11, continuing to react for 12 hours, then adjusting the pH of the fermentation liquid after the reaction to 3-4, separating out red precipitates, centrifuging, collecting the precipitates and thalli, performing ultrasonic extraction on the precipitates and the thalli by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;

s2, separation: dissolving the crude extract, passing through silica gel chromatographic column for the first time, and eluting to obtain four components (Fr.1-Fr.4); subjecting Fr.3 to silica gel chromatography for the second time to obtain 5 components (Fr.3.1-Fr.3.5); fr.3.2 was separated by semi-preparative HPLC to give compound 3.

Preferably, the pH of the fermentation broth is adjusted to 10-11 with saturated NaOH solution.

Preferably, the pH of the fermentation broth is adjusted to 11 with saturated NaOH solution.

Through experimental exploration, the supernatant product in the mR2A culture medium after 7 days of fermentation only contains the compound 1 and a small amount of 3, and the compound 3 is gradually increased along with the increase of the fermentation days, because the compound 1 can be completely converted into the compound 3 in 12 hours under the alkaline condition.

The structural formula of all compounds in the invention is shown as formula (II):

preferably, the mixed solvent in S1 is a mixed solvent of dichloromethane and methanol, and the dichloromethane and methanol are mixed at a ratio of 1:1 in the mixed solvent.

The mixed solvent has better solubility to the precipitation than other pure solvents or mixed solvents with other proportions.

Preferably, the solvent in which the crude extract is dissolved is dichloromethane.

Preferably, the mesh number of the first silica gel column in the S2 is 300-400 meshes.

Preferably, the elution in S2 is gradient elution, which is performed sequentially by using mixed solutions of dichloromethane and methanol at a mass ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90: 10.

According to different components eluted by different gradients, mixed solvents with 10 proportions are sequentially eluted, impurities are washed off to the maximum extent, and target compounds are enriched.

Preferably, the mesh number of the second silica gel column chromatography in the S2 is 200 meshes.

The mesh number of the two times of silica gel chromatographic columns is different, the first time is rough division, and the second time is fine division.

Preferably, the mR2A medium consists of: 0.5g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, 0.5g/L acid hydrolyzed casein, 0.3g/L sodium pyruvate、0.025g/L MgSO₄·7H₂O、0.5g/L K₂HPO₄， pH 7.0。

Preferably, in the rubicin analog, the compound 4 is prepared by fermentation of a genetically engineered strain Streptomyces sp.zx02, and the specific process comprises the following steps:

A. fermentation: inoculating a genetically engineered strain Streptomyces sp.ZX02 to an R2A plate containing thiostrepton for 7-9 days, inoculating spores into a TSB culture medium for culturing for 1-2 days, then inoculating the spores into an mR2A culture medium according to the inoculation amount of 4%, continuously culturing for 7 days, adjusting the pH value of a fermentation liquid to 3-4, separating out a reddish brown precipitate, centrifugally collecting the precipitate and thalli, performing ultrasonic extraction by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;

B. separation: dissolving the crude extract, passing through silica gel chromatographic column, and eluting to obtain five components (Fr.1-Fr.5); combining Fr.2 and Fr.3, and purifying with silica gel column chromatography to obtain 5 components (Fr.2.1-Fr.2.5); further isolation of fr.2.4, using semi-preparative HPLC, finally isolated to give compound 4.

Preferably, Fr.2.4 is further isolated using semi-preparative HPLC, while isolating Compound 5.

The retention time of Compound 4 was 12.002min, its M/z 511.0875[ M + H ]]Determining its molecular formula as C by HRESIMS₂₅H₁₈O₁₂The retention time of Compound 5 was 14.331min, which is (M/z 495.0923[ M + H ]](+) HRESIMS determining its molecular formula as C₂₅H₁₈O₁₁. Based on molecular weight and characteristic UV absorption, compound 5 was identified as 7, 8-dideoxygriseorrodin C, while compound 4 had one more hydroxyl group than compound 5, and further analysis by C and H spectra and comparison with previous compounds confirmed that compound 4 has a structure as shown in FIG. 1, which is a novel rubicin analog.

Preferably, in the above rubicin analog,

compounds

8 and 11 are prepared by fermentation of genetically engineered strain Streptomyces sp.zx06, and the specific process is as follows:

C. fermentation: inoculating a genetically engineered strain Streptomyces sp.ZX06 to an R2A plate containing thiostrepton for 7-9 days, inoculating spores into a TSB culture medium for culturing for 1-2 days, then inoculating the spores into an mR2A culture medium according to the inoculation amount of 4%, continuously culturing for 7 days, adjusting the pH value of a fermentation liquid to 3-4, separating out a reddish brown precipitate, centrifugally collecting the precipitate and thalli, performing ultrasonic extraction by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;

D. separation: dissolving the crude extract, passing through silica gel chromatographic column, and eluting to obtain 8 components (Fr.1-Fr.8); combining Fr.2 and Fr.3, and purifying with silica gel column chromatography to obtain 5 components (Fr.2.1-Fr.2.5); fr.2.2 and fr.2.3 are combined for further separation, using semi-preparative HPLC, to give compound 10 and compound 11 by final separation; fr.4 and fr.5 are combined for further separation, using semi-preparative HPLC, to give compound 8 and compound 9.

The invention also provides application of the

compounds

3, 4, 8 and 11 in preparing antitumor drugs.

The invention uses mR2A culture medium to ferment original strain Streptomyces sp.CB00271, and analyzes the secondary metabolite by UPLC-MS. Analysis by the UPLC program showed that multiple rubicin analogs could be produced in the original strain. By HRLCMS analysis, peak 3 with retention time of 13.342min and characteristic UV absorption values (lambda max 238nm,309nm and 508nm) and M/z 523.0876[ M + H ] of]+, peak 6 at a retention time of 14.051min and having characteristic UV absorbance values (. lamda.max 236nm,316nm and 496nm), which is (M/z 555.31[ M + H ]](+) at a retention time of 14.431min and having peaks 9 of characteristic UV absorbance values (. lamda.max 231nm,312nm and 512nm), which are (M/z 553.0982[ M + H ]]⁺) Peak 1 with a retention time of 15.590min and characteristic UV absorption values (. lamda.max 239nm,313nm and 513nm), which is (M/z 537.1035[ M + H ]](+) at a retention time of 15.98min and having a peak 7 of characteristic UV absorbance (. lamda.max 239nm,313nm and 513nm), which is (M/z 509.0714[ M + H)](+) at a retention time of 16.976min and having a peak 2 of characteristic UV absorption values (. lamda.max 230nm,312nm and 504nm), which is (M/z 523.0792[ M + H ]]⁺). According to the molecular weight and the characteristic ultraviolet absorption, the compound 1 is determined to be beta-rubicin, the compound 2 is determined to be gamma-rubicin, the compound 6 is heliomycin, the compound 7 is determined to be rubicin CA1, and the compound 10 is determined to be 3' -hydroxy-beta-rubicin. Compound 3, which has the same molecular weight as compound 2 and differs from compound 1 by 1 methylene group, may be a novel rubicin derivative. By sampling different fermentation days for HPLC analysis, as shown in fig. 4, it can be seen that on day 2 of fermentation, the supernatant product is only compound 1 and a small amount of compound 2, compound 3 gradually increases with the number of fermentation days, and we also observe that the pH of the fermentation broth gradually increases. When we collected the fermentation broth for 7 days and centrifuged at high speed, the supernatant was taken, the pH of the supernatant was adjusted to 11 with 1M NaOH, the supernatant was allowed to stand at room temperature for 12 hours, samples were taken at different time points for HPLC detection, and the results are shown in FIG. 5. under alkaline conditions, compound 1 could be completely converted into compound 3 within 12 hours, and then, in combination with the molecular weight analysis between the two, it can be presumed that compound 1 could be deprived of a methylene group to form compound 3 under alkaline conditions. In order to determine the structural formula of the other compound 3, 8L scale fermentation and subsequent in vitro conversion are adopted to enrich the compound 3, and a chloroform methanol system is utilized to carry out silica gel column separation and HPLC-UV preparation to obtain a pure product of the compound 3; by nuclear magnetic resonance analysis and in vitro transformation analysis, we determined that the structure of compound 3 is shown in fig. 1, and that compound 3 is beta-rubicin acid, a novel rubicin analog.

The original strain Streptomyces sp.CB00271 is added with 20mM Mg²⁺The MS plate of (2) can grow well and produce more spores. When the sensitivity of the strain to different antibiotics was tested, the strain was found to be supplemented with 20mM Mg²⁺And no growth on MS containing 50. mu.g/mL of apramycin or 20. mu.g/mL of thiostrepton, the strain grew well on a medium containing 40. mu.g/mL of nalidixic acid; coli S17-1 nalidixic acid is extremely sensitive, so that the screening marker of apramycin and thiostrepton can be used for screening mutant strains. The escherichia coli and Streptomyces species joint transfer is carried out by using the shuttle plasmid pSET152 of the escherichia coli and Streptomyces, the jointed Streptomyces and the Streptomyces species are covered by the medicine of the apramycin and nalidixic acid, and more spliceosomes can grow on a flat plate after about 4 days, which shows that Streptomyces sp.CB00271 has better joint transferMobility. The establishment of the genetic operation system lays a solid foundation for the genetic engineering transformation of the strain.

The biosynthetic gene cluster of the rubicin in the CB00271 is determined by carrying out whole gene sequencing and secondary metabolite biosynthetic gene cluster analysis on an original strain Streptomyces sp.CB00271 and subsequent gene knockout experiments. By comparative analysis with the reported rubicin gene cluster, we found that the rubicin gene cluster in CB00271 differs from the known gene cluster in comparison to the reported rubicin gene cluster rub^DSM2012 (GenBank accession no.AF293355),rub^CB00271The gene cluster is provided with a plurality of genes, wherein the genes comprise an anthraquinone oxidoreductase gene orf 9; compared with the reported griseoviridin gene cluster grh (GenBank accession No. AF509565), rub^CB00271There are several unique modifying enzyme genes in the gene cluster, including an ester cyclase gene orf4, the details of which are shown in table 1. In the later period, two special genes orf9 and orf4 are knocked out to obtain 2 engineering strains Streptomyces sp.ZX02 and Streptomyces sp.ZX06 capable of directionally producing the rubicin analogue. The strain was fermented with mR2A medium and analyzed for secondary metabolites by UPLC-MS. Analysis by the UPLC program showed that two

rubicin analogs

4 and 5 could be produced in a targeted manner in Streptomyces sp. By HRLCMS analysis, peak 4 with retention time of 12.002min and characteristic UV absorption values (lambda max 239nm,313nm and 510nm) and M/z 511.0875[ M + H [ (. sup.]⁺ Peak 5, which has a retention time of 14.331min and characteristic UV absorption values (. lamda.max 232nm,315nm and 502nm), (M/z 495.0923[ M + H ]]⁺). Based on molecular weight and characteristic uv absorption, compound 5 was identified as 7, 8-dideoxygriseorrodin C. In order to determine the structural formula of the other compound, a 6L scale fermentation is adopted, and a chloroform methanol system is utilized to carry out silica gel column separation and HPLC-UV preparation to obtain a pure product of the compound 4; by NMR analysis, we determined compound 4 to be a novel rubicin analog (FIG. 1).

ZX06 mutants of Streptomyces sp.ZX06 were analyzed using the same UPLC procedure as described aboveIt was shown that this mutant produced 4 rubicin analogues 8/9/10/11, which, after further HRLCMS analysis, had a retention time of 12.869min and peaks 8 with characteristic UV absorbances (. lamda.max 231nm,308nm and 516nm), M/z 539.0822[ M + H ] of]⁺ Peak 9 with a retention time of 13.979min and characteristic UV absorption values (. lamda.max 231nm,312nm and 495nm), which is (M/z 525.0663[ M + H ]]⁺) Peak 10 at a retention time of 14.431min and having characteristic UV absorption values (. lamda.max 231nm,312nm and 512nm), which is (M/z 553.0982[ M + H ]]⁺) Peak 11 at a retention time of 15.421min and with characteristic UV absorption values (. lamda.max 230nm,312nm and 494nm), which is (M/z 539.0822[ M + H ]]⁺). There is a difference of one methylene group between

compounds

8 and 10 and also between

compounds

9 and 11. During fermentation, four analogues were found to be linked, with ZX06 fermented for 2 days, the product was only compounds 10 and 11, with increasing days of fermentation and increasing pH of the broth, with increasing

compounds

8 and 9, as shown in figure 2. When we collected 2 days of fermentation broth and centrifuged at high speed, the supernatant was taken, the pH of the supernatant was adjusted to 11 with 1M NaOH and left at room temperature for 24 hours, it was observed that compounds 10 and 11 could be completely converted to

compounds

8 and 9 in 24 hours under alkaline conditions as shown in FIG. 3, and in combination with molecular weight analysis between 4, it was speculated that

compounds

10 and 11 were deprived of a methylene group under alkaline conditions to form

compounds

8 and 9, respectively. Based on molecular weight and characteristic uv absorption, compound 9 was identified as rubicin CA2 and compound 10 was identified as 3' -hydroxy- β -rubicin, where

compounds

8 and 11 should be new. In order to determine the structures of the compounds corresponding to the two peaks, 8L scale fermentation treatment, silica gel column separation and HPLC-UV preparation are carried out by a chloroform methanol system to obtain pure products of the

compounds

8 and 11 respectively; nuclear magnetic resonance analysis and in vitro transformation analysis confirmed that the structures of

compounds

8 and 11 are shown in fig. 1, compound 8 is 3 '-hydroxy- β -rubicin acid, compound 11 is 3' -hydroxy- γ -rubicin, both of which are novel rubicin analogs.

The invention relates to a gene orf9 (the nucleotide sequence of which is shown as SEQ ID NO. 1) and an ester cyclase gene orf4 (the nucleotide sequence of which is shown as SEQ ID NO. 2) in a rubicin biosynthesis gene cluster, wherein anthraquinone oxidoreductase is responsible for modification after oxidation, and the engineering strains for directionally producing rubicin analogs can be obtained after the genes are respectively replaced and mutated separately. In the biosynthetic gene cluster for rubicin, orf9 encodes a polypeptide of 325 amino acids, which is defined as anthraquinone oxidoreductase. orf4 encodes a polypeptide of 288 amino acids, defined as an ester cyclase. To determine the specific role of the proteins Orf9 and Orf4 encoded by the Orf9 and Orf4 genes in the biosynthesis of rubicin, in-frame substitutions were made with a thiostrepton-resistant DNA fragment, respectively, to inactivate Orf9 (fig. 6) and Orf4 (fig. 7). Transferring the knockout vector into an E.coli S17-1 strain, performing interspecies conjoint transfer with a wild type CB00271 strain to obtain some conjugants, selecting a ZX02 strain with correct resistance phenotype (thiostrepton tolerance and apramycin sensitivity) and genotype, fermenting the ZX06 strain and the wild type CB00271 strain under the same condition, and taking the wild type strain as a positive control. Analysis of the fermentation broth extract of mutant strain ZX02 by HPLC revealed that the mutant produced 2 rubicin analogues (FIG. 8) and the mutant strain ZX06 produced 4 rubicin analogues (FIG. 8). These data demonstrate that Orf9 and Orf4 proteins have an important role in the biosynthesis of rubicin.

The nucleotide sequence or partial nucleotide sequence provided by the invention can obtain genes similar to orf9 and orf4 from other organisms by using a Polymerase Chain Reaction (PCR) method or a method comprising DNA at positions 1 to 978 of the sequence shown in SEQ ID NO.1 or DNA at positions 1 to 867 of the sequence shown in SEQ ID NO.2 of the invention as a probe by Southern hybridization and the like.

The nucleotide sequences or at least part of the nucleotide sequences provided by the present invention may be modified or mutated in vitro and in vivo, including insertions, substitutions or deletions, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutations, re-ligation of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical agents, etc.

The cloned gene comprising the nucleotide sequence provided by the invention or at least part of the nucleotide sequence can be expressed in an exogenous host by a suitable expression system to obtain the corresponding enzyme or other higher bioactive substances or yields. These exogenous hosts include E.coli, Streptomyces, Micromonospora, Pseudomonas, Bacillus, yeast, plants, animals, and the like.

Polypeptides comprising the amino acid sequences or at least partial sequences provided herein may have biological activity, even new biological activity, after removal or substitution of certain amino acids, or increased yield or optimized protein kinetics or other properties sought to be achieved.

The invention provides a way for targeted use in genetically engineered microorganisms, comprising DNA fragments or genes that can be used to construct mutants that are targeted for production of rubicin or a derivative thereof.

The invention also provides application of anthraquinone oxidoreductase Orf9 in the biosynthesis gene cluster of the rubicin in oriented production of the rubicin analogue.

The discovery of the biosynthesis gene cluster of the natural product and the development of gene knockout technology provide possibility and means for the genetic engineering modification of the strain. Many researches have been carried out to successfully complete structural modification and transformation of more natural products with complex structures or directional accumulation of certain target components by a biosynthesis means, construct a series of derivatives or high-yield mutant strains with equivalent or stronger activity, provide good cases for genetic engineering transformation of streptomycete, and bring new hopes for solving the treatment of increasingly serious tumor diseases.

The novel rubicin analogue is discovered through fermentation of the original strain or the rubicin is structurally modified or reformed through a gene knockout means to obtain more rubicin structural analogues, thereby accelerating the early clinical researches such as anticancer activity analysis and action mechanism of the natural products and the application and development of novel antitumor drugs.

The invention also provides the application of the nucleotide sequence shown as SEQ ID NO.2 in preparing the compound rubicin analogue.

The application is that four compounds in the rubicin analogue can be directionally produced after the nucleotide sequence shown as SEQ ID NO.2 is deleted.

In a word, the post-modification gene and protein information related to the biosynthesis of the rubicin provided by the invention can help people to understand the biosynthesis mechanism of anthraquinone natural products, and provides possibility for obtaining engineering strains by further utilizing a genetic modification mode. The gene and the protein thereof provided by the invention can also be used for searching and discovering compounds or genes and proteins which can be used for medicine, industry or agriculture.

The Streptomyces is Streptomyces sp.CB00271, which is preserved in China center for type culture Collection in 6.2.2020, and is addressed to Wuhan university school with eight paths 299 in Wuchang district, Wuhan city, Hubei province, with the preservation number of CCTCC M2020176.

Has the advantages that:

the

compounds

3 and 8 prepared by the invention have better tumor inhibition activity and can be used for preparing antitumor drugs. Particularly, the activity of the compound 3 is far better than that of the similar compound 1 and is also far better than that of a positive anti-tumor western medicine control group, so that the application of the compound in future anti-tumor medicines is proved to have very good prospects. Provides a greater possibility for accelerating the anticancer activity analysis and the clinical early research of action mechanism and the like of the natural products and the application and development of new antitumor drugs.

Drawings

FIG. 1 shows the chemical structural formula of rubicin.

FIG. 2 is a fermentation time HPLC analysis of the double crossover mutant ZX 06.

FIG. 3 shows the reaction HPLC analysis of the supernatant product of the second day of fermentation of the double crossover mutant ZX06 under alkaline conditions.

FIG. 4 is a wild type CB00271 fermentation time HPLC analysis.

FIG. 5 is a reaction HPLC analysis of wild type CB00271 fermentation day 7 supernatant products under alkaline conditions.

FIG. 6 shows the construction and PCR validation of the double-crossover mutant ZX02, wherein 1 is wild type, 2 is the double-crossover mutant ZX02, and M is Marker.

FIG. 7 shows the construction and PCR validation of the double-crossover mutant ZX06, wherein 1 is wild type, 2 is the double-crossover mutant ZX02, and M is Marker.

FIG. 8 is a detailed embodiment of the double crossover mutant ZX02/ZX06 and wild type post-fermentation HPLC analysis.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. All the following percentages refer to mass percentages. The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1 culture of Streptomyces sp.CB00271 and establishment of genetic manipulation System and Gene Cluster analysis

1.1 solid culture of CB 00271:

streptomyces sp.CB00271 is obtained by separating from Yunnan forest soil, the strain is preserved on the slant of R2A culture medium, and the composition of R2A culture medium is as follows: 0.5g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, 0.5g/L acid hydrolyzed casein, 0.3g/L sodium pyruvate, 0.025g/L MgSO₄·7H₂O、0.5g/L K₂HPO₄And 20 g/L agar, pH7.0, sterilized for use.

1.2 establishment of Streptomyces sp.CB00271 genetic operation System

We investigated the growth and sporulation of Streptomyces sp.CB00271 on various media and found it to contain 20mM Mg²⁺The MS medium of (3) produced spores well. Meanwhile, the sensitivity of Streptomyces sp.cb00271 to various antibacterial agents was studied, and the antibacterial agents tested included: sensitivity to Apramycin (Apramycin, Am), nalidixic acid, Thiostrepton (Tsr), etc., Streptomyces sp.CB00271 was found to be sensitive to Apramycin (50. mu.g/mL) and Thiostrepton (20. mu.g/mL). Nalidixic acid (40 μ g/mL) is not toxic to Streptomyces sp.CB00271. Coli S17-1 strain was very sensitive to nalidixic acid (40. mu.g/mL). This work demonstrates that we can introduce sulfur filaments during gene replacement mutationsThe bacterin resistance gene can be used for screening and obtaining a conjugator by using thiostrepton and nalidixic acid during conjugal transfer. The feasibility of this genetic manipulation was demonstrated by conjugative transfer of S17-1 E.coli containing the integrative plasmid pSET152 to Streptomyces sp.CB00271, covering the conjugation plate with apramycin and nalidixic acid, and finding that many zygotes appeared in about 4 days. And a good foundation is laid for the development of subsequent experiments.

1.3 Gene Cluster analysis of CB00271

The biosynthetic gene cluster of the rubicin in the CB00271 is determined by carrying out whole gene sequencing and secondary metabolite biosynthetic gene cluster analysis on an original strain Streptomyces sp.CB00271 and subsequent gene knockout experiments. By comparative analysis with the reported rubicin gene cluster, we found that the rubicin gene cluster in CB00271 differs from the known gene cluster in comparison to the reported rubicin gene cluster rub^DSM2012 (GenBank accession no.AF293355),rub^CB00271The gene cluster is provided with a plurality of genes, wherein the genes comprise an anthraquinone oxidoreductase gene orf 9; compared with the reported griseoviridin gene cluster grh (GenBank accession No. AF509565), rub^CB00271There are several unique modifying enzyme genes in the gene cluster, including an ester cyclase gene orf4, the details of which are shown in table 1.

TABLE 1 comparison of analysis of the CB00271 rubicin biosynthetic gene cluster

Example 2Streptomyces sp.CB00271 fermentation analysis and subsequent large-scale fermentation and separation, purification and structural identification of rubicin analogue

CB00271 produces a number of rubicin analogs with retention times of 13.342min and peak 3 with characteristic UV absorbance values (. lamda.max 238nm,309nm and 508nm), M/z 523.0876[ M + H ] of]+, molecular formula is C₂₆H₁₈O₁₂(ii) a Has a retention time of 14.051min and a characteristic UV absorption value (lambda max 236nm, 316)nm and 496nm), M/z 555.31[ M + H ]]+, molecular formula is C₂₆H₁₈O₁₄(ii) a At a retention time of 14.431min and having peaks 10 of characteristic UV absorption values (. lamda.max 231nm,312nm and 512nm), which are (M/z 553.0982[ M + H ]]⁺) Molecular formula is C₂₇H₂₀O₁₃(ii) a At a retention time of 15.590min and having a peak 1 of characteristic UV absorption values (. lamda.max 239nm,313nm and 513nm), which is (M/z 537.1035[ M + H ]](+) and the molecular formula is C₂₇H₂₀O₁₂(ii) a At a retention time of 16.076min and having a peak 2 of characteristic UV absorption values (. lamda.max 230nm,312nm and 504nm), which is (M/z 523.0792[ M + H ]](+) and the molecular formula is C₂₆H₁₈O₁₂. Based on the molecular weight and the characteristic uv absorption and retention time, compound 1 was determined to be β -rubicin, compound 2 was determined to be γ -rubicin, compound 6 was a heliomycin, compound 10 was 3' -hydroxy- β -rubicin, compound 3 had the same molecular weight as compound 2, differing by 1 methylene from compound 1, and possibly a new rubicin derivative. By sampling different fermentation days for HPLC analysis, as shown in fig. 6, it can be seen that at day 2 of fermentation, the supernatant product is only compound 1 and a small amount of compound 2, compound 3 gradually increases with the number of fermentation days, and we also observe that the pH of the fermentation broth gradually increases. When we collected the fermentation broth for 7 days and centrifuged at high speed, the supernatant was taken, the pH of the supernatant was adjusted to 11 with 1M NaOH, the supernatant was allowed to stand at room temperature for 12 hours, samples were taken at different time points for HPLC detection, and the results are shown in FIG. 7. under alkaline conditions, compound 1 could be completely converted into compound 3 within 12 hours, and then, in combination with the molecular weight analysis between the two, it can be presumed that compound 1 could be deprived of a methylene group to form compound 3 under alkaline conditions.

To identify the structure of compound 3, we performed a large-scale fermentation of 8L followed by in vitro transformation to enrich compound 3 as follows: a part of the CB00271 spores was inoculated into a medium containing 50mL of TSB and subjected to shake culture at 30 ℃ for 1 to 2 days, and inoculated into mR2A medium in an inoculum size of 4%, followed by further shake culture at 30 ℃ at 220rpm for 7 days. After fermenting CB00271 for 7 days, the pH of the fermentation broth was adjusted to 11 with saturated NaOH solution and left to mix on the shaker overnight. Then, 1M hydrochloric acid is used for adjusting the pH value of the fermentation liquor after reaction to 3-4, red precipitate is separated out, high-speed centrifugation is carried out, and the supernatant is discarded to collect the precipitate and thalli. And dissolving and extracting the precipitate and the thallus collected by centrifugation for multiple times by using a mixed solvent of dichloromethane and methanol respectively until an extracting solution has no color, combining soluble parts after centrifugation, and concentrating under vacuum to obtain a crude extract. The crude extract was dissolved in a small amount of methylene chloride and subjected to 300-mesh 400-mesh silica gel column chromatography with gradient elution with methylene chloride and methanol to give four fractions (Fr.1-Fr.4). Fr.3 was further purified by 200 mesh silica gel column chromatography in order to obtain 5 fractions (Fr.3.1-Fr.3.3). Fr.3.2 was further isolated using semi-preparative HPLC to afford compound 3. Further by analysis of the C and H spectra and comparison with previous compounds, it was confirmed that compound 3, having the structure shown in fig. 1, is a novel rubicin analog that can be produced by hydrolysis of β -rubicin under basic conditions, thus designating compound 3 as β -rubicin acid.

Example 3 construction of orf9 and orf4 Gene knockout plasmids in Streptomyces sp.CB00271

3.1 construction of orf9 knockout plasmid in Streptomyces sp.CB00271

2 pairs of primers are respectively designed by taking S sp.CB00271 total DNA as a template

pD9F：5′-AATGTGAACACGCCTCCTCGACCCGCAG-3′(SEQ ID NO.3)；

pD 9R: 5'-GCGGCCGCGGATCCTCTAGAGGCAAGACCACCTTGTCGCG-3' (SEQ ID NO. 4); and

pD10F：5′-ACGACGGCCAGTGCCAAGCTTGGCGAAGACCGAGATGC-3′(SEQ ID NO.5)；

pD 10R: 5'-CAACCGATAAAAGGCGCACGAGCTGATG-3' (SEQ ID NO. 6); the method is used for PCR amplification of left and right homologous arms, specific products with the length of 1.5kb are purified and recovered, and a pair of primers are designed:

p9TF：5′-CGTGCGCCTTTTATCGGTTGGCCGCGAG-3′(SEQ ID NO.7)；

p9 TR: 5'-CGAGGAGGCGTGTTCACATTCGAACGGTCTCTG-3' (SEQ ID NO. 8); is used for amplifying thiostrepton resistance gene cassettes, and specific products with the length of 0.9kb are purified and recovered. The construction method was performed with reference to (TSV-S1) Trelief SoSoo Cloning Kit operating manual, using a strategy for seamless Cloning. The left and right homologous arms, the resistance gene and a HindIII and XbaI enzyme-digested linearized pOJ260 vector are seamlessly connected according to the concentration of 3:3:3:1, a link product is transformed into competent E.coli DH5 alpha, a single clone is selected for colony verification, a positive clone is extracted to obtain a plasmid for enzyme digestion verification and sequencing verification, and the correctly sequenced plasmid is orf9/10 gene knockout plasmid pYLW003 for subsequent gene knockout.

3.2 construction of orf4 knockout plasmid in Streptomyces sp.CB00271

2 pairs of primers are respectively designed by taking S sp.CB00271 total DNA as a template:

pD4UPF：5′-AATGTGAACAGGCGTCGTGGAACAGC-3′(SEQ ID NO.9)；

pD4 UPR: 5'-GCGGCCGCGGATCCTCTAGAGGATGTCGCGGCCCGA-3' (SEQ ID NO. 10); and

pD4DNF：5′-ACGACGGCCAGTGCCAAGCTTGGACTGGCCCATGGTGAC-3′(SEQ ID NO.11)；

pD4DNR (5'-GCCAACCGATAAAAGGTCTCCGAGCACTGG-3' (SEQ ID NO. 12)) was used for PCR amplification of the left and right homology arms, and specific products of 1.3kb in length were purified and recovered, while a pair of primers was designed:

p4TF：5′-CTCGGAGACCTTTTATCGGTTGGCCGCGAG-3′(SEQ ID NO.13)；

p4 TR: 5'-CACGACGCCTGTTCACATTCGAACGGTCTCTG-3' (SEQ ID NO. 14); is used for amplifying thiostrepton resistance gene cassettes, and specific products with the length of 0.9kb are purified and recovered. The construction method was performed with reference to (TSV-S1) Trelief SoSoo Cloning Kit operating manual, using a strategy for seamless Cloning. The left and right homologous arms, the resistance gene and a pOJ260 vector linearized by enzyme digestion with HindIII and XbaI are seamlessly connected according to the concentration of 3:3:3:1, a link product is transformed into competent E.coli DH5 alpha, a single clone is selected for colony verification, a positive clone is extracted to obtain a plasmid for enzyme digestion verification and sequencing verification, and the plasmid with correct sequencing is orf9 gene knockout plasmid pYLW010 for subsequent gene knockout.

Example 4 construction of orf9 Gene deletion mutant Strain Streptomyces sp.ZX02

The constructed knock-out plasmid pYLW003 is transferred into E.coli S17-1 to obtain E.coli S17-1 which is transferred with the knock-out plasmid pYLW003 and is named as E.coli S17-1/pJu2014/pYLW003, and the E.coli S17-1 is jointed and transferred with a wild strain. The bonding transfer process is as follows: freezing and storing in S sp.CB00271 spore 2 tube (about 10 deg.C) of-80 deg.C refrigerator⁹Spores) were washed 2 times with LB, resuspended in 1mL of LB medium, heat-shocked in a water bath at 50 ℃ for 10min, and then allowed to stand to room temperature to serve as a conjugative transfer recipient. Transforming the constructed recombinant plasmid into E.coli S17-1, selecting a single clone from an LB plate containing two antibiotics (Am and Km) to inoculate to 3mL of LB culture medium (containing the corresponding antibiotics), carrying out shaking culture at 37 ℃ and 230rpm for 12-15h, sucking 500 mu l of bacterial liquid to inoculate to 50mL of LB culture medium (containing the corresponding antibiotics), and carrying out shaking culture at 37 ℃ and 230rpm for 4-8 h until OD is reached₆₀₀To 0.3-0.4. And pouring the bacterial liquid into a 50mL sterilized centrifuge tube, and centrifuging at 4000rpm for 10min to collect thalli. The cells were washed twice with 25mL of LB medium and resuspended in 2mL of LB medium as a conjugative transfer donor. And (3) carrying out heat shock on the S sp.CB00271 spores and E.coli S17-1 (containing recombinant plasmids) bacterial liquid according to the proportion of 1:1 volume of the suspension was mixed well, centrifuged to remove a portion of the supernatant, and resuspended and pipetted in M S medium (containing 20mM MgCl. RTM₂) And simultaneously making a negative control and a positive control. M S medium used as positive and negative controls was coated with only 20. mu. l S sp. CB00271 spores. Culturing M S culture medium coated with bacterial liquid in a constant temperature incubator at 30 ℃ for 18-20h, washing out surface escherichia coli by using sterile water, covering 50mg/L Am (or 20mg/L Tsr) and 40mg/L nalidixic acid on a joint transfer culture medium and a negative control, covering 40 mu g/mL nalidixic acid on a positive control, and placing on an ultra-clean bench for purging for 2-3h until the surface is dried. The culture medium is placed in an incubator at 30 ℃ for inverted culture, and white zygote single colonies grow out after 4-7 days. Double crossover mutant strain ZX02 passes through its resistance phenotype to antibiotics (Am)^S&Tsr^R)And genotype, wherein the PCR verification of orf9 gene deletion utilizes primers:

pYZ9F：5′-CCCGCGCTTCCATCAGC-3′(SEQ ID NO.15)；

pYZ 9R: 5'-ACGTCCTGCGGGTCGAG-3' (SEQ ID NO. 16); PCR amplification reaction was performed to confirm that S sp.CB00271 in which orf9 gene was deletion-mutated was obtained and named as a double crossover knockout strain Streptomyces sp.ZX02.

The construction process of orf4 gene deletion mutant strain is the same as Streptomyces sp.ZX02, and the verification primer:

pYZ4F：5′-TCATGGCGTGGGCAGTCC-3′(SEQ ID NO.17)；

pYZ 4R: 5'-CCGGAACGGAGGGACTC-3' (SEQ ID NO.18), thereby obtaining the orf4 gene deletion mutant S sp.CB00271 which was named as a double crossover knockout strain Streptomyces sp.ZX06.

Example 5 fermentation of wild-type strains with double-crossover mutant strains Streptomyces sp.ZX02 and Streptomyces sp.ZX06 and HPLC analysis thereof

The wild type strain and the double-crossover mutant strain are cultured for 7-9 days at 30 ℃ on an R2A solid medium plate, wherein the plate of the double-crossover mutant strain simultaneously contains the thiostrepton with the final concentration of 20 mug/mL; selecting a proper amount of spores, inoculating the spores into a triangular flask filled with 50mL of TSB culture medium, and culturing for 1-2 days on a shaking table at the temperature of 30 ℃ and the rotating speed of 220 rpm; inoculating the strain in a fermentation medium mR2A with an inoculation amount of 4%, wherein the fermentation medium mR2A comprises the following components: 5 parts of soluble starch, 5 parts of glucose, 2.5 parts of peptone, 2.5 parts of yeast powder, 0.5 part of acid hydrolyzed casein, 0.3 part of sodium pyruvate, and 0.25 part of MgSO 2₄·7H₂O, 0.5 part of K₂HPO₄pH 7.0. The culture was carried out for 7 days at 30 ℃ on a shaker at a rotation speed of 220 rpm. Adjusting the pH value of 50mL of fermentation liquor to 3-4 by using 1M HCl, uniformly mixing, precipitating a reddish brown precipitate, centrifuging the fermentation liquor, collecting the precipitate and thalli, performing ultrasonic extraction on the centrifuged precipitate and thalli for multiple times by using a mixed solvent of dichloromethane and methanol until the extract is colorless, combining the extracts to obtain a mixed solution, concentrating and drying, then re-metering the volume to 50mL, centrifuging at 12000rpm for 5min, and taking the supernatant for HPLC analysis. HPLC analytical conditions and procedure were as follows: the mobile phase A is 99.9 percent of deionized water and 0.1 percent of formic acid; the mobile phase B is 100% acetonitrile, the flow rate is 1.0 mL/min, the ultraviolet detector wavelength is 310nm, and the linear gradient analysis program is as follows: 0-1 min, 95% a; 1-18 minutes, 95% a to 5% a; 18-20 minutes, 5% a; 20-20.5 minutes, 5% a to 95% a; 20.5-25 min, 95% A. The results of the HPLC analysis are shown in FIGS. 4 and 5. From the figure, it can be seen that the ZX02 mutant produced only 2 rubicin analogs (FIG. 4); ZX06 produced 4 rubicin analogs (fig. 5).

Example 6 Mass fermentation of Streptomyces sp.ZX02 mutant strains and isolation, purification and structural identification of rubicin analogs

Firstly, the mutant strain ZX02 was inoculated onto an R2A plate containing 20. mu.g/mL S.thiostrepton, after the spores grew well for 7 to 9 days, a part of the spores was inoculated into a medium containing 50mL of TSB and shake-cultured at 30 ℃ for 1 to 2 days, and then inoculated into an mR2A medium in an inoculum size of 4%, and the medium was further shake-cultured at 30 ℃ at 220rpm for 7 days for a total of 6L. And then, adjusting the pH value of the fermentation liquor to 3-4 by using 1M HCl, uniformly mixing, separating out a reddish brown precipitate, centrifuging the fermentation liquor, collecting the precipitate and thalli, performing ultrasonic extraction on the centrifugal precipitate and the thalli for multiple times by using a mixed solvent of dichloromethane and methanol until the extract is colorless, combining the extracts, mixing the obtained mixed liquor, mixing the mixed liquor, and enriching and separating the target compound. The crude extract was dissolved in a small amount of methylene chloride and subjected to 300-mesh 400-mesh silica gel column chromatography with gradient elution with methylene chloride and methanol to give five fractions (Fr.1-Fr.5). Fr.2 and Fr.3 were combined and further purified by 200 mesh silica gel column chromatography in order to give 5 fractions (Fr.2.1-Fr.2.5). Further isolation of fr.2.4, using semi-preparative HPLC, final isolation gave 4 as well as 5. The retention time of Compound 4 was 12.002min, its M/z 511.0875[ M + H ]]⁺HRESIMS determined molecular formula C₂₅H₁₈O₁₂The retention time of Compound 5 was 14.331min, which is (M/z 495.0923[ M + H ]]⁺) HRESIMS determined molecular formula C₂₅H₁₈O₁₁. Based on molecular weight and characteristic UV absorption, compound 5 was identified as 7, 8-dideoxygriseorrodin C, while compound 4 had one more hydroxyl group than compound 5, and further analysis by C and H spectra and comparison with previous compounds confirmed that compound 4 has a structure as shown in FIG. 1, which is a novel rubicin analog.

Example 7 fermentation analysis and subsequent large-scale fermentation of Streptomyces sp.ZX06 mutant strains and separation, purification and structural identification of rubicin analogs

ZX06 produced 4 rubicin analogs, which, upon further HRLCMS analysis, had retention times at 12.869min and peaks 8 with characteristic UV absorbance values (. lamda.max 231nm,308nm and 516nm), with M/z 539.0822[ M + H ] of]⁺Molecular formula is C₂₆H₁₈O₁₃(ii) a At a retention time of 13.979min and having peaks 9 of characteristic UV absorbance values (. lamda.max 231nm,312nm and 495nm), of M/z 525.0663[ M + H ]]⁺Molecular formula is C₂₅H₁₆O₁₃(ii) a At a retention time of 14.431min and having peaks 10 of characteristic UV absorption values (. lamda.max 231nm,312nm and 512nm), M/z 553.0982[ M + H ]]⁺Molecular formula is C₂₇H₂₀O₁₃(ii) a At a retention time of 15.421min and having peaks 11 of characteristic UV absorption values (. lamda.max 230nm,312nm and 494nm), M/z 539.0822[ M + H ]]⁺Molecular formula is C₂₆H₁₈O₁₃. Based on molecular weight and characteristic uv absorption, compound 9 was identified as rubromycin CA2 and compound 10 was identified as 3' -hydroxy- β -rubicin; there is a difference of one methylene group between compound 8 and compound 10, and a difference of one methylene group between compound 9 and compound 11. During the fermentation process, the four analogues were found to have a certain relation, as shown in figure 2, when ZX06 was fermented for 2 days, the product was only compound 10 and compound 11, and as the fermentation days increased, and the pH of the fermentation broth became higher, compound 8 and compound 9 gradually increased. When we collected 2 days of fermentation broth and centrifuged at high speed, the supernatant was taken, the pH of the supernatant was adjusted to 11 with 1M NaOH, the supernatant was left at room temperature for 24 hours, samples were taken at different time points for HPLC detection, and the results are shown in FIG. 3. under alkaline conditions, compound 10 and compound 11 could be completely converted into compound 8 and compound 9 within 24 hours, and then combined with molecular weight analysis between 4, compound 10 and compound 11 could be presumed to remove one methylene group under alkaline conditions to form compound 8 and compound 9, respectively.

The fermentation method and extraction method of the ZX06 mutant strain are the same as the treatment method of the ZX04 mutant strain. The isolation procedure for compounds in the ZX06 mutant was as follows: the crude extract was dissolved in a small amount of methylene chloride and subjected to 300-mesh 400-mesh silica gel column chromatography with gradient elution with methylene chloride and methanol to obtain 8 fractions (Fr.1-Fr.8). Fr.2 and Fr.3 were combined and further purified by 200 mesh silica gel column chromatography in order to give 5 fractions (Fr.2.1-Fr.2.5). Fr.2.2 and fr.2.3 are combined for further separation, using semi-preparative HPLC, to give compound 11 and compound 10 by final separation; fr.4 and fr.5 are combined for further separation, using semi-preparative HPLC, to give compound 9 and compound 8. Through C spectrum and H spectrum analysis and the relationship of mutual conversion between the compound and the four, the compound 9 can be determined to be the rubicin CA2, and the compound 10 is the 3' -hydroxy-beta-rubicin; wherein the structures of compound 8 and compound 11 are shown in figure 2, compound 8 is 3 '-hydroxy-beta-rubicin acid, and compound 11 is 3' -hydroxy-gamma-rubicin, which are novel rubicin analogs.

TABLE 2 Nuclear magnetic data for Compound 3/4/11

The structural formulas of the compounds 1 to 11 are shown in figure 1 respectively.

EXAMPLE 8 antitumor assay of Compound 1/2/3/4/8/9/10/11

Tumor cell strains used in the experiment were HeLa (human cervical cancer cells), A549 (human small cell lung cancer cells) and Caco-2 (human colon adenocarcinoma cells).

The experimental method used the conventional CCK-8 method. Collecting cells in logarithmic growth phase, adjusting cell suspension concentration to make cell density to be measured 2 × 10³～5×10³Cells/well (cell density depends on cell type), cultured in 96-well plates, 100. mu.L per well, placed at 37 ℃ and 5% CO₂After incubation in the incubator of (1) for 24h, the medium was aspirated off using a calandria, and then a gradient of different concentrations (100. mu.M, 50. mu.M, 25. mu.M, 10. mu.M, 1. mu.M, 0.1. mu.M, 0.01. mu.M) of drug formulated with the medium was added, each concentration being in 3 duplicate wells, and incubated for 72 h. The medium was aspirated off, 100 μ L of medium solution containing 10% CCK-8 was added to each well, incubated for 1-2h until the medium color turned orange, the plate was removed and the absorbance OD value of each well was measured at 450nm in an elisa detector. The experiment required the setting of a zero-setting well (blank medium) and a control well (medium containing the same cells without drug) and a positive control well (bleomycin), with PBS added to the edge wells. The cell survival rate [% ] is (the OD value of the drug addition group-the OD value of the blank group)/(the OD value of the control group-the OD value of the blank group) × 100%. Taking the average value of 3 multiple wells in each group, drawing a cell growth inhibition curve, and calculating IC₅₀The value is obtained. The results are shown in Table 3.

TABLE 3 antitumor Activity test analysis results of Compound 1/2/3/4/8/9/10/11

As can be seen from Table 2, the

compounds

3 and 8 have better tumor inhibition activity and can be used for preparing antitumor drugs. Particularly, the activity of the compound 3 is far better than that of the similar compound 1 and is also far better than that of a positive anti-tumor western medicine control group, so that the application of the compound in future anti-tumor medicines is proved to have very good prospects.

SEQUENCE LISTING

<110> Changsha Tianci biological medicine science and technology Limited

<120> rubicin analogue, preparation method and application thereof

<130> 1

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 978

<212> DNA

<213> Artificial Synthesis

<400> 1

atgcgtatcg tccgccacca cgcgttcggg gcccccgacg tcctgcgggt cgaggaggcg 60

ccccgccccg aacccggtgc gggccaggtc ctgatccgga ccgaggccgt cggtgtgaac 120

ttcgccgaat gccagcgccg tcagggcatc ccggtcggcg gccccgccac cctgccgggc 180

tcgccgggcg gcgatgtcgc cggagtcgtc gaggcgctcg gcgagggggt gagcgaggtg 240

agcgtcggcg accgcgtcgt caccggcgca cccttcgacg cctacgccga atacgtcgtc 300

gcgcgggccg actggctctt tccggtcccg gagtccatcg acgcggccca ggccacgtcg 360

atgccgatcc ccgcgcagac ggcgtaccac gtcatcgcga cggcggcccg gctcgccagg 420

ggtgaatcga tgctgatcac ggcagcggcc ggtggtatcg ggcacctgct cgtccaactg 480

ggcaaggcgc tcggcgccgg ccgggtgatc gctgccgcgg gcagcgcggc caagctggac 540

ttcgtccgcg ggctgggcgc cgacgtcgcg atcaactaca ccgacgagga ctgggacgag 600

caggtcaggg cggccaccga cggccgtggc gccgatgtgg tgctggagac cgtgggcggc 660

gacatcctgc ggcggtcggt cggtctgatc gccccgttcg ggcggatggt ggtctacggc 720

acggccggag gggaactgcc tgccgtggag atcgcggaca tcttcgacaa ccggacggtg 780

atgggcttca gcatgtgggg cgtgatggcc catcgcccgc aggagatggc ggacggcgcc 840

aaagccctgc tcgacctggt gggatccggc cgggtgagcc cggtgatcca cgcccggatc 900

ccgctcgacc gggccggtgc ggcgcacgcc ctgatggagg cccggtccca gctcggccgg 960

gtggtgctgg tgccctga 978

<210> 2

<211> 867

<212> DNA

<213> Artificial Synthesis

<400> 2

atgacgatcc cgatccccgc ggagagcagg gcgcgcaacg ccgggctggt ccgcggtctg 60

tacgacgtgc tttaccggcg gcaccggccg gacgaggccg ggcggctgtt ccacgacgcc 120

catgtcgacc acgacccgcg ggtggggccc ggcccctccg ggccgttgcg gcactgggag 180

tcgatgcgcg cggcctttcc cgggctgcgc gcccgtatcg gccgtctggt cgcggacgag 240

gaccgggtca tgctgttcgc ggcctggacc ggacggactc cggccggtga cccgttcgag 300

ctgcacacgg ccgagttgta ccggatcgcg gagggccgga tcgccgagca ctggaatgtc 360

accgaccggg acgtgctcgc cgggcatgtc ccggacgtgc cggacgcgct cgtggggccg 420

gagccggccg tgctgcacgg tctccacagc gaggacgagc gggccaacgc ggcggtggtg 480

ctcgacgcgt accgggtggt gttcagcgag caccggctgg atctggccga ccgctactac 540

caccgggact accggcacca caacgcgcgt acggaccggg tgcccgacgg cctggaggcg 600

ttcaccgcgt tcttcgccga caacctggcc gccttccccg atctggccgt caccgtcgag 660

cacgtggtcg ccgccgggga ccgggtgatg gtcttcgtca cctggaccgg aacgctcacc 720

ggcgcctgga gcggtggtgg cccgtccggg ctgccgttgg tgatgcgcac ctgcgaccac 780

ttccggctgg aggacgggaa ggtctccgag cactgggagg tcgtcgacta tctgccggtc 840

cggcgtgccg gactgcccac gccatga 867

<210> 3

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 3

aatgtgaaca cgcctcctcg acccgcag 28

<210> 4

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 4

gcggccgcgg atcctctaga ggcaagacca ccttgtcgcg 40

<210> 5

<211> 38

<212> DNA

<213> Artificial Synthesis

<400> 5

acgacggcca gtgccaagct tggcgaagac cgagatgc 38

<210> 6

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 6

caaccgataa aaggcgcacg agctgatg 28

<210> 7

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 7

cgtgcgcctt ttatcggttg gccgcgag 28

<210> 8

<211> 33

<212> DNA

<213> Artificial Synthesis

<400> 8

cgaggaggcg tgttcacatt cgaacggtct ctg 33

<210> 9

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 9

aatgtgaaca ggcgtcgtgg aacagc 26

<210> 10

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 10

gcggccgcgg atcctctaga ggatgtcgcg gcccga 36

<210> 11

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 11

acgacggcca gtgccaagct tggactggcc catggtgac 39

<210> 12

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 12

gccaaccgat aaaaggtctc cgagcactgg 30

<210> 13

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 13

ctcggagacc ttttatcggt tggccgcgag 30

<210> 14

<211> 32

<212> DNA

<213> Artificial Synthesis

<400> 14

cacgacgcct gttcacattc gaacggtctc tg 32

<210> 15

<211> 17

<212> DNA

<213> Artificial Synthesis

<400> 15

cccgcgcttc catcagc 17

<210> 16

<211> 17

<212> DNA

<213> Artificial Synthesis

<400> 16

acgtcctgcg ggtcgag 17

<210> 17

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 17

tcatggcgtg ggcagtcc 18

<210> 18

<211> 17

<212> DNA

<213> Artificial Synthesis

<400> 18

ccggaacgga gggactc 17

Claims

1. A rubicin analog, characterized in that it has the structural formula shown in formula (I):

2. a process for producing a rubicin analog as claimed in claim 1, wherein said process is carried out by fermentation using Streptomyces sp.CB00271, genetically engineered sp.ZX02 or genetically engineered Streptomyces sp.ZX06, wherein said genetically engineered sp.ZX02 is obtained by knocking out the orf9 gene in the genome of Streptomyces sp.CB00271, and wherein said genetically engineered sp.ZX06 is obtained by knocking out the orf4 gene in the genome of Streptomyces sp.CB00271; the nucleotide sequence of the orf9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the orf4 gene is shown as SEQ ID NO. 2.

3. The process of claim 2, wherein the compound 3 of the rubicin analog of claim 1 is prepared by fermentation with Streptomyces sp.cb00271, which is a Streptomyces sp.cb00271 strain prepared by:

s1, fermentation: inoculating Streptomyces sp.CB00271 spores into a Trypticase Soy Broth (TSB) for culturing for 1-2 days, then inoculating the Streptomyces sp.CB00271 spores into an mR2A culture medium according to the inoculation amount of 4 percent for continuous culturing for 7 days, adjusting the pH of a fermentation liquid to 10-11, continuing to react for 12 hours, then adjusting the pH of the fermentation liquid after the reaction to 3-4, separating out a red precipitate, centrifugally collecting the precipitate and thalli, ultrasonically extracting the precipitate and the thalli by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;

4. The method according to claim 3, wherein the pH of the fermentation broth is adjusted to 10 to 11 with a saturated NaOH solution.

5. The method according to claim 3, wherein the mixed solvent in S1 is a mixed solvent of dichloromethane and methanol, and the dichloromethane and methanol are mixed at a ratio of 1: 1.

6. The preparation method according to claim 3, wherein the elution in S2 is gradient elution, and the gradient elution is sequentially performed by using mixed solutions of dichloromethane and methanol in a volume ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9 and 90: 10.

7. The method according to claim 3, wherein the mR2A medium consists of: 0.5g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, 0.5g/L acid hydrolyzed casein0.3g/L sodium pyruvate, 0.025g/L MgSO₄·7H₂O、0.5g/L K₂HPO₄，pH 7.0。

8. The process according to claim 2, wherein the rubicin analog of claim 1, wherein compound 4 is prepared by fermentation of a genetically engineered strain Streptomyces sp.zx02 by the process of:

B. separation: dissolving the crude extract, passing through silica gel chromatographic column, and eluting to obtain five components (Fr.1-Fr.5); combining Fr.2 and Fr.3, and purifying with silica gel column chromatography to obtain 5 components (Fr.2.1-Fr.2.5); further isolation of fr.2.4, using semi-preparative HPLC, finally isolated to give compounds 4 and 5.

9. The process according to claim 2, wherein the rubicin analog of claim 1, compound 8 and compound 11 are prepared by fermentation of a genetically engineered strain Streptomyces sp.zx06 by:

C. fermentation: inoculating a genetically engineered strain Streptomyces sp.ZX02 to an R2A plate containing thiostrepton for 7-9 days, inoculating spores into a TSB culture medium for culturing for 1-2 days, then inoculating the spores into an mR2A culture medium according to the inoculation amount of 4%, continuously culturing for 7 days, adjusting the pH value of a fermentation liquid to 3-4, separating out a reddish brown precipitate, centrifugally collecting the precipitate and thalli, performing ultrasonic extraction by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;

D. separation: dissolving the crude extract, passing through silica gel chromatographic column, and eluting to obtain 8 components (Fr.1-Fr.8); combining Fr.2 and Fr.3, and purifying with silica gel column chromatography to obtain 5 components (Fr.2.1-Fr.2.5); fr.2.2 and fr.2.3 are combined for further separation using semi-preparative HPLC to give compounds 10 and 11; fr.4 and fr.5 are combined for further separation, using semi-preparative HPLC, to give compounds 8 and 9.

10. Use of a rubicin analog according to claim 1 or prepared by the process of any one of claims 2-9 in the preparation of an anti-tumor medicament.