CN110218244B

CN110218244B - Compound ilamycin F and application thereof

Info

Publication number: CN110218244B
Application number: CN201910335521.5A
Authority: CN
Inventors: 马俊英; 鞠建华; 黄洪波; 张春燕; 贾艳玺; 李岩
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2016-10-09
Filing date: 2016-10-09
Publication date: 2021-04-09
Anticipated expiration: 2036-10-09
Also published as: CN106497827A; CN110218244A; CN106497827B

Abstract

The invention discloses a genetic engineering strain for directionally producing antitubercular active and antitumor active compounds and application thereof. A genetic engineering strain is obtained by knocking out an ialL gene or an ilaR gene in a genome of Streptomyces atratus SCSIO ZH16, wherein the nucleotide sequence of the ialL gene is shown as SEQ ID No.1, and the nucleotide sequence of the ialR gene is shown as SEQ ID No. 2. The genetically engineered strain can produce the compounds 1, 2, 3, 4 and 5 with antitubercular activity and antitumor activity, and shows great value in the development of antitubercular drugs. Therefore, the successful construction of the genetic engineering strain for directionally producing the compound with the anti-tuberculosis and anti-tumor activity can accelerate the industrialization process of the compound and promote the development of marine drugs in China.

Description

Compound ilamycin F and application thereof

The divisional application is 2016 patent application, 10-09 application number 201610885104.4, and is named as a genetic engineering strain for directionally producing antituberculous active and antitumor active compounds and application thereof.

Technical Field

The invention belongs to the field of microbial genetic engineering and metabolic engineering, and particularly relates to a genetic engineering strain for directionally producing an antitubercular active and antitumor active compound cyclopeptide antibiotic ilamycins and application thereof.

Background art:

tuberculosis is a serious and worldwide epidemic infectious disease with the leading position in deaths caused by infectious diseases. Chemotherapy is still the mainstay of treatment for tuberculosis in adults at present. However, due to the unique pathogenic mechanism and invasion characteristics of mycobacterium tuberculosis, the drugs capable of effectively treating mycobacterium tuberculosis are still limited, the use period of the drugs is long, and meanwhile, the drugs are accompanied by very serious toxic and side effects, the compliance of patients is poor or the drugs cannot be completely taken according to the treatment course, so that the emergence and the rapid propagation of multi-drug-resistant bacteria or pan-drug resistance are caused. In addition, the concurrence of tuberculosis and HIV makes the prevention and control of tuberculosis in humans more troublesome and difficult. There is a pressing need for new antitubercular drugs with new mechanism of action and fewer toxic side effects and which do not cross-react with other drugs.

In addition, with the growing population, deterioration of the ecological environment and changes in lifestyle, cancer has become the first killer to harm human health. 1270 ten thousand cancer patients exist in 2008 globally, and by 2030, the global cancer cases may increase by 75%, and the number of cancer patients is estimated to reach 2220 ten thousand. Moreover, chemotherapy drugs are still the main means of cancer resistance at present. However, the toxic and side effects and the rapid generation of drug resistance of the existing drugs are urgent, and anti-tumor drugs with novel structures, unique action mechanisms and less toxic and side effects are urgently needed.

The marine microorganisms (especially actinomycetes) have evolved unique enzyme systems adapting to extreme environments due to the unique living environments of the marine microorganisms, have the potential of generating compounds with novel structures and unique action mechanisms, and become new resources for drug development. Some strains can produce multiple secondary metabolites simultaneously, which is an advantage for compound diversity, but becomes a disadvantage for the development of targeted drugs of interest. Because the drug development company wants to specifically obtain the compound with specific activity, the cost of drug development can be saved, the efficiency of drug development can be improved, and the continuous and rapid supply of the target compound can be ensured.

The discovery of biosynthetic gene clusters of antibiotics and the development of gene knockout technologies provide possibilities and means for genetic engineering of strains producing active secondary metabolites. Many researches have been carried out to successfully complete structural modification and reconstruction of more natural products with complex structures or directional accumulation of certain target components through biosynthesis and combined biosynthesis technologies, construct a series of derivatives with equivalent or stronger activity or high-yield mutant strains, provide a useful methodology reference sample for genetic engineering reconstruction of streptomycete, and bring new hopes for solving the increasingly troublesome drug resistance problem of pathogenic microorganisms and treating tumor diseases.

Ilamycins are cyclic peptide antibiotics with novel structures and complex components. The structure of the cyclopeptide compound is shown as a formula (I). In vitro activity analysis shows that part of the components have obvious inhibitory activity on mycobacterium tuberculosis.

The invention content is as follows:

the first purpose of the invention is to provide a genetic engineering strain capable of directionally producing antitubercular and antitumor compounds.

The genetic engineering strain for directionally producing the antituberculous and antitumor compound is constructed by the following method, and is obtained by knocking out an ialL gene or an ilaR gene in the genome of Streptomyces atratus SCSIO ZH16, wherein the nucleotide sequence of the ialL gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ialR gene is shown as SEQ ID NO. 2.

The second purpose of the invention is to provide the application of the genetic engineering strain in which the ialL gene in the genome of Streptomyces atratus SCSIO ZH16 is knocked out in the preparation of the compound 1 and/or the compound 2;

the structural formulas of the

compounds

1 and 2 are respectively shown as 1 and 2 in the formula (I);

the third purpose of the invention is to provide the application of the genetic engineering strain in which the ialR gene in the genome of the Streptomyces atratus SCSIO ZH16 is knocked out in the preparation of the

compounds

1, 3, 4 and/or 5;

the structural formulas of the

compounds

1, 3, 4 and 5 are respectively shown as 1, 3, 4 and 5 in the formula (II);

a compound 5 represented by the formula (III):

the application of the compound 5 shown as the formula (III) in the preparation of antituberculosis drugs or antitumor drugs.

The antituberculous drug is preferably an anti-M.semagamit or M.Tuberluccosis drug.

An antituberculous drug characterized by containing a compound 5 represented by the formula (III) as an active ingredient.

The original strain Streptomyces atratus SCSIO ZH16 is a marine strain Streptomyces which is added with 20mM of Mg in ISP2 with the content of 3 percent²⁺The ISP2 plate of (1) was able to grow well and produce more spores. When the sensitivity of the strain to different antibiotics is tested, the strain is added with 20mM Mg²⁺And ISP2 containing 50. mu.g/mL of apramycin or 10. mu.g/mL of thiostrepton, and the strain does not grow onThe strain grew well on a medium containing 50. mu.g/mL trimethoprim; coli ET12567/pUZ8002 trimethoprim is extremely sensitive, so that we can use apramycin and trimethoprim as screening markers for mutant screening. Thiostrepton and trimethoprim can be used as screening marks of anaplerotic mutant strains during gene anaplerosis. 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 medicines of the apramycin and trimethoprim, and after about 4 days, more splicers grow on a plate, which shows that the Streptomyces atratus SCSIO ZH16 has better joint transfer performance. The establishment of the genetic operation system lays a solid foundation for the genetic engineering transformation of the strain.

The original strain Streptomyces atratus SCSIO ZH16 is a marine Streptomyces, and 2 engineering strains delta ilaL and delta ilaR capable of directionally producing anti-tubercular activity ilamycins are obtained by knocking out genes ilaL and ilaR of 2P 450 family cytochrome oxidase in a biosynthesis gene cluster of secondary metabolites. The strain was fermented with Am2ab medium, the fermentation broth was extracted with butanone, and the secondary metabolites were analyzed by HPLC-DAD-UV. Analysis by HPLC program (to be supplemented) showed that two ilamycins homologues could be produced in a targeted manner in the mutant Δ ilaL. HRLCMS analysis shows that the retention time is 24.25min and the retention time has a characteristic ultraviolet absorption value (lambda)_max230nm,285nm and 354nm), M/z 1012.5880[ M + H ]]⁺and m/z1034.5702[M+Na]⁺Has a retention time of 23.01min and a characteristic ultraviolet absorption value (lambda)_max230nm,285nm and 354nm), which is (M/z1028.5824[ M + H)]⁺and m/z 1050.5654[M+Na]⁺). In order to determine the structural formulas of the two compounds, 8L scale fermentation is adopted, and a chloroform methanol system and a petroleum ether ethyl acetate system are utilized to carry out silica gel column separation and HPLC-UV preparation to obtain a pure product of the compound 2; by NMR analysis and associated experience with the separation of these two compounds in conjunction with our previous two patents, we determined that Compound 1 was neutralizedCompound 2 is the antibiotics ilamycin B1 and ilamycin B, respectively₂(FIG. 1). Analysis of the mutant strain of Δ ilaR using the same HPLC procedure as described above showed that the mutant strain produced 4 ilamycins homologues which, after further HRLCMS analysis, exhibited a characteristic UV absorbance (. lamda.) at a retention time of 24.25min_max230nm,285nm and 354nm), its M/z 1012.5880[ M + H ]]⁺and m/z 1034.5430[M+Na]⁺The compound should be ilamycin B₁(ii) a At a retention time of 23.6min, its M/z 1026.5669[ M + H [)]⁺and m/z 1048.5512[M+Na]⁺The retention time is 23.0min, its M/z 1026.5645[ M + H [)]⁺and m/z 1048.5564[M+Na]⁺The retention time is 21.8min, and the corresponding M/z 1042.5616[ M + H ] of the compound]⁺and m/z 1064.5428[M+Na]⁺. The latter three compounds should be new. In order to determine the structures of the compounds corresponding to the three peaks, the applicant adopts 8L scale fermentation treatment, and silica gel column separation and HPLC-UV preparation are carried out on a perchloro-chloroform system and a petroleum ether ethyl acetate system to respectively obtain pure products of the compounds 3-5; the structure of compound 3-5 is shown in FIG. 1, and is antibiotic ilamycin E1 by NMR analysis₁、ilamycin E₂And ilamycin F (FIG. 1), wherein ilamycin E2 and ilamycin F are novel compounds.

The invention relates to genes ilaL (the nucleotide sequence of which is shown in SEQ ID NO. 1) and ilaR (the nucleotide sequence of which is shown in SEQ ID NO. 2) of p450 cytochrome oxidase in an ilamycin biosynthesis gene cluster, which are responsible for modification after oxidation, and engineering strains for directionally producing ilamycins homologues can be obtained after the genes ilaL (the nucleotide sequence of which is shown in SEQ ID NO. 1) and ilaR (the nucleotide sequence of which is shown in SEQ ID NO. 2) are respectively replaced and mutated independently. In the biosynthetic gene cluster of ilamycins, there are two genes for p450 cytochrome oxidase designated ilaL and ilaR, respectively, which encode a polypeptide of 402 amino acids and a polypeptide of 399 amino acids, respectively. To determine the specific role of the ilaL and ilaR genes encoding the proteins ilaL and ilaR in the biosynthesis of the antibiotic ilamycins, in-frame substitutions were made with a DNA fragment resistant to apramycin to inactivate ilaL (fig. 2) and ilaR (fig. 3), respectively. The mutant cosmids were transferred to methylation-deficient e.coli ET12567/pUZ8002 strains, and were interspecific conjoint transferred with wild-type s.atraus SCSIO ZH16 strains to obtain several conjugants, 3 resistant phenotypes (apra-tolerant, kanamycin-sensitive) and correct genotypes Δ ilaL, Δ ilaR and wild-type s.atraus SCSIO ZH16 strains were selected respectively and fermented under the same conditions, and the wild-type strains served as positive controls. Analysis of the mutant strain Δ ilaL by HPLC for butanone extraction in fermentation broth showed that the three mutants consistently produced 2 ilamycins homologues (fig. 4) and the Δ ilaR mutant consistently produced 4 ilamycins homologues (fig. 5). These data demonstrate that the IlaL and IlaR proteins play an important role in the biosynthesis of ilamycins.

The nucleotide sequence or partial nucleotide sequence provided by the invention can obtain genes similar to ilaL and ilaR from other organisms by using a Polymerase Chain Reaction (PCR) method or a DNA (deoxyribonucleic acid) containing the 1 st to 1209 th sites of the sequence shown by the SEQ ID NO.1 or the SEQ ID NO.2 as a probe by a Southern hybridization method 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.

The amino acid sequences provided by the invention can be used for separating the required protein and can be used for preparing antibodies.

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 the production of ilamycins or derivatives thereof.

The invention also provides application of p450 cytochrome oxidase IlaR in the ilamycins biosynthesis gene cluster in directional production of ilamycins homologues.

The invention also provides application of the nucleotide sequence shown as SEQ ID NO.2 in preparation of the ilaamycins homolog of the compound.

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

In conclusion, the post-modified gene and protein information related to the biosynthesis of ilamycins provided by the invention can help people to understand the biosynthesis mechanism of the cyclic depsipeptides 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 starting strain Streptomyces atrophaeus SCSIO ZH16 of the present invention has been stored in the common microorganism center of the china committee for culture collection of microorganisms (CGMCC) 3, 10 days 2016, with the address: the postcode 100101 of institute for microbiology, china academy of sciences, west road No.1, north chen, chaoyang, beijing, the strain preservation number is: CGMCC No. 12198.

Description of the drawings:

FIG. 1 is the chemical structural formula of ilamycins.

FIG. 2 shows the construction and PCR validation of double-crossover mutants of. DELTA.ialL 1,. DELTA.ialL 2 and. DELTA.ialL 3, where WT was a wild-type strain and M was Marker.

FIG. 3 is a diagram showing the construction and PCR confirmation of a.DELTA.ilaR double-crossover mutant strain, in which. DELTA.ialR 1,. DELTA.ialR 2 and. DELTA.ialR 3 are all the.DELTA.ialR double-crossover mutant strains, WT is a wild-type strain, and M is Marker.

FIG. 4 shows the HPLC analysis results of fermentation of both Δ ialL1, Δ ialL2 and Δ ialL3, which are Δ ialL double-crossover mutants, and wild-type strain WT, respectively.

FIG. 5 shows the HPLC analysis results of the respective fermentations of Δ ilaR double-crossover and wild-type strains, in which Δ ialR1, Δ ialR2 and Δ ialR3 are all Δ ialR double-crossover mutants and WT is a wild-type strain.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

culture of Streptomyces atratus SCSIO ZH16 and establishment of genetic manipulation System

Solid culture of s.atraus SCSIO Zh 16:

1.1 marine streptomyces S. atraus SCSIO Zh16 is separated from deep sea sediment in south sea, the strain is preserved on the inclined plane of ISP-2 culture medium, and the ISP-2 culture medium comprises the following components: 4g of yeast extract powder, 4g of glucose, 10g of malt extract powder, 30g of crude sea salt, 20g of agar powder, 1000mL of water, pH7.2-7.4 and sterilizing for later use.

1.2 establishment of the Streptomyces atratus SCSIO ZH16 genetic operating System

We studied the growth and sporulation of Streptomyces atratus SCSIO ZH16 on different media and found it to contain 20nM of Mg²⁺The production of spores was good in the ISP2 medium. Meanwhile, the sensitivity of Streptomyces atraus SCSIO ZH16 to various antibacterial drugs including: sensitivity to Apramycin (Apramycin, Apm), ampicillin, kanamycin, chloramphenicol, trimethoprim (Tmp), thiostrepton, etc., S.atraus SCSIO ZH16 was found to be sensitive to Apramycin (50. mu.g/mL) and thiostrepton (10. mu.g/mL) and resistant to ampicillin (100. mu.g/mL), chloramphenicol (25. mu.g/mL) and kanamycin (100. mu.g/mL). Trimethoprim (50. mu.g/mL) is not toxic to S.atraus SCSIO ZH 16. Coli ET12567/pUZ8002 strain is very sensitive to trimethoprim (50. mu.g/mL). This work demonstrates that one can introduce the apramycin resistance gene during gene substitution mutation and select for zygotes using apramycin and trimethoprim during conjugal transfer. The feasibility of this genetic manipulation was demonstrated by conjugative transfer of E.coli containing ET12567/pUZ8002 of the integrative plasmid pSET152 to S.atraus SCSIO ZH16, by overlaying the conjugation plates with apramycin and trimethoprim, and by finding more zygotes to appear around 4 days. And a good foundation is laid for the development of subsequent experiments.

Construction of a genomic library of S.atraus SCSIO ZH16 and obtaining of a cosmid containing ilaL and ilaR.

2.1 construction of genomic library of S.atraus SCSIO ZH16

The genomic library was constructed by reference to SuperCos1 Cosmid Vector Kit and Gigapck III XL packing Extract operating manual. Culturing Streptomyces atratus SCSIO ZH16, centrifuging after 1.5 days, collecting thalli, extracting genome DNA according to a mature method, taking a proper amount of high-purity DNA, carrying out enzyme digestion treatment with different enzyme digestion times and different Sau3AI enzyme concentrations, carrying out multitubular enzyme digestion of the same system after determining the optimal reaction time and enzyme reaction concentration, and then combining enzyme digestion products for dephosphorylation treatment; meanwhile, the SuperCos1 vector is treated by XbaI restriction endonuclease, and then dephosphorylation treatment is carried out after treatment; then, the SuperCos1 vector was digested with BamHI, purified and recovered by phenol chloroform extraction, ligated with genomic DNA digested with Sau3AI and dephosphorylated, packaged in vitro and infected. The infected strain is coated on a resistant plate containing kanamycin, 1920 clones are randomly picked and stored in a 96-well plate, and the construction of a genome library of the strain is completed.

2.2 screening and obtaining of Cosmid201E containing genes ilaL and ilaR.

Sequencing of the complete genome map of S.atratus SCSIO ZH16 was carried out by Shanghai Linn Bio Inc. According to the sequence provided by Linn bioinformatics, Inc., combined with on-line bioinformatics software and structural characteristics of ilamycins, we obtained a biosynthetic gene cluster of ilamycins, in which both the ilaL gene (whose nucleotide sequence is shown in SEQ ID NO. 1) and the IlaR gene (whose nucleotide sequence is shown in SEQ ID NO. 2) in the gene cluster encoded cytochrome p450 oxidase. To investigate the function of ilaL and IlaR genes in the biosynthesis of ilamycins, we performed gene knock-outs separately. According to the requirement of PCR-targeting operation, in order to obtain cosmids capable of knocking out ilaL and IlaR, two pairs of primers, SAF (5'-CCAGCCACCTCTTCGTAGC-3'), SAR (5'-CGGTCCGGTCGATCTTTCC-3') and SBF (5'-CGGAGCCAAGCAGGTCGTC-3') and SBR (5'-GAGGTCAGGGAAGCGGTTCAG-3'), are designed on the upstream of the ilaL gene sequence and the downstream of the ilaR gene sequence. The two pairs of primers are respectively used for screening positive clones from the constructed genome library by using a PCR technology. The PCR procedure was as follows: 5min at 95 ℃; 45s at 95 ℃, 45s at 58 ℃, 60s at 72 ℃ and 30 cycles; 10min at 72 ℃. A plurality of positive clones were selected, and by end sequencing, a clone (named Cosmid201E) corresponding to the (1, E) position in No. 20 96-well plate was selected for further knock-out of ilaL and ilaR genes.

Construction of the mutant Strain Δ ilaL with deletion of ilaL Gene

The in-frame inactivation of ilaL gene adopts lambda-RED mediated PCR-Targeting technology. Cosmid201E containing the ilaL gene was transformed into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/12H 8. PCR amplification reaction of the aac (3) IV-oriT resistant fragment with Apr resistance was carried out using the pIJ773 plasmid fragment digested with EcoRI and HindIII as a template and a knock-out primer having 39bp homology arms with the ilaL gene, respectively (ilaLdelF:5'-CCCGAGGTGTTGCACAATCCGATTTCCGCCTATGGGCAGattccggggatccgtcgacc-3', ilaLdelR:5'-GAGGTCAGGGAAGCGGTTCAGCAGTGCGCCGAGCGCCACtgtaggctggagctgcttc-3' capital letter part represents homology with the ilaL gene). The amplified aac (3) IV-oriT resistant fragment is electrically transformed into E.coli BW25113/pIJ790/12H8 competent cells, and the recombinant cosmid after modification is obtained through the homologous recombination mediated by lambda-RED and is named as p 201E-delta ilaL. The correctness of the recombinant cosmid was confirmed by performing an amplification reaction using a PCR-verified primer for the ilaL gene (IDilaLF:5'-TCCTGTGCGGTGCTGCTGAT-3', IDilaLR: 5'-GCGATTCTGCGGGCTGCTTT-3'). Subsequently, the recombinant p201E- Δ ilaL was transferred to e.coli ET12567/pUZ8002 to obtain e.coli ET12567/pUZ8002, named e.coli ET12567/pUZ8002/pJu2014, into which the recombinant p201E- Δ ilaL was transferred, and the e.coli ET12567/pUZ8002/pJu was transferred by conjugation to a wild-type strain.

The process of bond transfer is as follows: culturing spores of wild S.atraus SCSIO ZH16 in TSB culture solution at 28 deg.C under shaking for 6-10 hr to make the spores germinate; coli ET12567/pUZ8002/pJu2014 was cultured in LB medium supplemented with kanamycin (Kan, final concentration 50. mu.g/mL), ampicillin (Amp, final concentration 100. mu.g/mL), chloramphenicol (Cml, final concentration 25. mu.g/mL) and adriamycin (Apm, final concentration 50. mu.g/mL) to an optical density OD of 0.6, cells were collected by centrifugation, washed twice with sterile LB medium liquid, and then mixed with germinated wild-type spores, and the mixture was plated on a plate supplemented with 20mM MgSO 2₄And 3% of sea salt₄On a solid culture medium; after 23 hours of growth at 28 ℃, each solid medium plate was coated with 800 μ L of sterile water containing 30 μ L of trimethoprim (Tmp,50mg/mL) and 30 μ L of apramycin (Apm,50mg/mL) added with antibiotic drugs of trimethoprim and apramycin; allowing it to grow in an incubator at 30 ℃ for 4-5 days until zygotes appear; double crossover mutant strain Δ ilaL passes through its resistance phenotype to antibiotics (Kan)^S&Apm^R) And the genotype, wherein the confirmation of the ilaL gene deletion by PCR amplification reaction using primers (IDilaLF, IDilaLR) was confirmed, and S.atratus SCSIO ZH16, which was the ilaL gene deletion mutant, was obtained and named as a double crossover mutant strain Δ ilaL.

The construction process of the ilaR gene deletion mutant strain is the same as that of delta ilaL, but the gene deletion primer of the ilaR is delilarF:5'-TATTCCAAGGACGACGGAAAAGCACTCCAGGACTGGTTCattccggggatccgtcgacc-3', delilaRR:5'-CAGCACCGGGAACCGTCGCAGCAGCGCGGAGAGTGCGATtgtaggctggagctgcttc-3' capital letter part which represents the part homologous with the ilaR gene, and the verification primer IDilarBF:5'-CGTTAGCAGCGGAGTCGGAATC-3', IDilaRR: 5'-GGTGGATGCGGTGAGGAGGAA-3'. This gave S.atratus SCSIO ZH16 which was deletion mutant for the ilaR gene, which was designated as double crossover mutant strain Δ ilaR.

4. Fermentation and HPLC analysis of wild type strains and double-crossover mutant strains delta ialL and delta ilaR

Both the wild type strain and the double crossover mutant strain contained 20mM MG²⁺ISP (A-S)₂(M-ISP₄) Culturing on solid culture medium plate at 30 deg.C for 7-9 days, wherein the double-crossover mutant plate simultaneously contains apramycin with final concentration of 30 μ g/mL; selecting appropriate amount of spore, inoculating into a container containing 50ml of soybean powder 2ab (Am2ab formula: 5g/L of soybean powder, 5g/L of soluble starch, 20g/L of glucose, 2g/L of peptone, 2g/L of yeast extract powder, MgSO4.7H₂O0.5g/L，KH₂PO₄0.5g/L，NaCl4g/L，CaCO₃2g/L, sea salt 30g/L, pH7.2-7.4, by dissolving the above components in water according to their contents, sterilizing, and culturing for 8-9 days on a shaker at 30 deg.C and 200 rpm). Extracting the fermentation liquor with butanone 2 times the volume of the fermentation liquor, evaporating the extracted organic phase liquid by a rotary evaporator to dryness, dissolving the organic phase liquid in 1mL of methanol, centrifuging the organic phase liquid at 12000rpm for 5min, and taking the supernatant for HPLC analysis. The composition of the mobile phase used for HPLC analysis was as follows: phase A, 15% acetonitrile, 85% water and 0.1% glacial acetic acid; phase B, 15% water + 85% acetonitrile + 0.1% glacial acetic acid. The HPLC procedure was: gradient elution is carried out on the phase B by 10-100 percent for 0-20 min; eluting with 100% B phase constant gradient for 20.1-25.0 min; 25.1-30.0min, 10% B phase elution. UV detection wavelength (230nm, 285nm and 352nm) flow rate of 1 min/mL. The results of the HPLC analysis are shown in FIGS. 4 and 5. From the figure it can be seen that the Δ ial mutant produces only 2 ilamycins homologues (fig. 4); Δ ilaar produced only 4 ilamycins homologs (fig. 5) and the retention time between each peak was much different, greatly facilitating isolation of the compounds.

5. Large-scale fermentation of delta ialL and delta ilaR mutant strains, separation, purification and structural identification of ilamcylins homologs

5.1, first, the mutant strain delta ialL is inoculated on an ISP2 plate containing 30 mu g/mL of apramycin and 3% of sea salt, after the spores grow well for 7-9 days, a part of the spores are inoculated into a culture medium containing 50mLAm2ab and are subjected to shake culture at 30 ℃ for 60 hours, and the spores are transferred to Am3 (soybean meal 5g/L, bacteriological peptone 15g/L, soluble starch 15g/L, glycerol 15g/L, CaCO) according to the ratio of 1:10₃2g/L, sea salt 30g/L, pH7.2-7.4, the preparation method comprises dissolving the above components in water according to their content, adjusting pH, sterilizing for use) in culture medium, placing in shaking table at 30 deg.C of rotation speed of 200rpm, and continuously culturing for 7-9 days for 8L total fermentation. And then, centrifugally separating mycelium and supernatant by using a large-scale centrifuge, extracting the supernatant for 3 times by using isovolumetric butanone, extracting the mycelium for 3 times by using acetone, distilling the upper organic phase at 40 ℃ under reduced pressure to obtain an extract, combining the mycelium extract and the supernatant extract according to the HPLC analysis result of the mycelium extract and the supernatant extract, mixing the samples, and enriching and separating the target compound.

And combining the mycelium extract and the supernatant extract, separating by silica gel (100-200 meshes) column chromatography, gradient eluting by chloroform-methanol (volume ratio of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20 and 50:50), and concentrating under reduced pressure to obtain corresponding fractions AFr.1-AFr.8 in sequence. By HPLC analysis, fractions AFr.1 (chloroform-methanol elution fraction with volume ratio of 100: 0) to AFr3 (chloroform-methanol elution fraction with volume ratio of 96: 4) are combined, separated by silica gel (100-mesh) column chromatography, subjected to gradient elution by a petroleum ether-ethyl acetate-methanol system through a B column (volume ratio of 100:0:0, 8:2:0, 6:4:0, 4:6:0, 2:8:0, 0:100:0,0:95:5 and C: M (ethyl acetate-methanol) ═ 9:1), and subjected to concentration under reduced pressure to obtain fractions BFr.1-BFr.8 in sequence. Bfr.5-bfr.8 fractions (bfr.5, bfr.6, bfr.7, bfr.8 are petroleum ether-ethyl acetate-methanol volume ratios 2:8:0, 0:100:0,0:95:5 and C: M ═ 9:1 fractions) were combined and separated by semi-preparative HPLC (preparation system was water (a): acetonitrile (B) system, detection wavelength was 270nm and 354nm, procedure was 0min 30% a, 0.1-18min 30% -0% a, 18.1-23min 100% B, 23.1-25min 0% -30% a, 25.1-30min 30% a, preparation column specification was 5 μ M × 21.2mm × 250mm, invaval C18, flow rate 10mL/min) to finally give compound 2(232mg) and compound 1(89 mg). Retention time of compound 1 is R_t＝24.25；m/z1012.5880[M+H]⁺；m/z1034.5430[M+Na]⁺HRESIMS determined molecular formula C₅₄H₇₇N₉O₁₀Retention time of Compound 2 is R_t＝23.01，m/z1028.5824[M+H]⁺；m/z1050.5054[M+Na]⁺HRESIMS determined molecular formula C₅₄H₇₇N₉O₁₁. The two compounds are determined to be known compounds, i.e. ilamycinB, by C-spectrum and H-spectrum analysis and comparison with the previous compounds₁And ilamycinB₂. The structures are shown in1 and 2 in figure 1.

5.2、

The mutant Δ ilaR produces 4 ilamycins homologues which, after further HRLCMS analysis, have a retention time of 24.25min and a characteristic UV absorbance (. lamda.) (λ. RTM.)_max230nm,285nm and 354nm), M/z 1012.5880[ M + H ]]⁺andm/z 1034.5430[M+Na]⁺The compound is ilamycin B₁(ii) a At a retention time of 23.6min, its M/z 1026.5669[ M + H [)]⁺and m/z 1048.5512[M+Na]⁺The molecular formula is C₅₄H₇₅N₉O₁₁The compound is ilamycin E₁(ii) a At a retention time of 23.0min, its M/z 1026.5645[ M + H [)]⁺and m/z 1048.5564[M+Na]⁺The molecular formula is C₅₄H₇₅N₉O₁₁The compound is ilamycin E₂(ii) a At a retention time of 21.8min, the corresponding M/z 1042.5616[ M + H ] of this compound]⁺and m/z 1064.5428[M+Na]⁺The molecular formula is C₅₄H₇₅N₉O₁₂The compound is ilamycin F. The latter three compounds should be new. The fermentation and extraction methods for the Δ ilaR mutant strains were the same as for the Δ ilaL mutant strains. The isolation procedure for compounds in the Δ ilaR mutant was as follows:

firstly, the mutant strain delta ilaR was inoculated on an ISP2 plate containing 30. mu.g/mL of apramycin and 3% of sea salt, after the spores grew well for 7-9 days, a part of the spores was inoculated into a culture medium containing 50mL of Am2ab and subjected to shake culture at 30 ℃ for 60 hours, and the spores were transferred to Am3 (soybean meal 5g/L, bacteriological peptone 15g/L, soluble starch 15g/L, glycerol 15g/L, CaCO) at a volume ratio of 1:10₃2g/L, 30g/L sea salt, pH 7.2-7.4), placing in a shaking table at 30 ℃ with the rotation speed of 200rpm for continuous culture for 7-9 days, and fermenting 8L. Then, the mycelium was centrifuged by a large centrifuge and the supernatant was collectedExtracting the supernatant with butanone 3 times, extracting mycelium with acetone 3 times, distilling the upper organic phase at 40 deg.C under reduced pressure to obtain extract, mixing the crude extracts, and concentrating and separating the target compounds.

And combining the mycelium extract and the supernatant extract, separating by silica gel (100-200 meshes) column chromatography, gradient eluting by chloroform-methanol (volume ratio of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20 and 50:50), and concentrating under reduced pressure to obtain corresponding fractions AFr.1-AFr.8 in sequence. By HPLC analysis, fractions AFr.1 (chloroform-methanol elution fraction with volume ratio of 100: 0) to AFr3 (chloroform-methanol elution fraction with volume ratio of 96: 4) are combined, separated by silica gel (100-mesh) column chromatography, subjected to gradient elution by a petroleum ether-ethyl acetate-methanol system through a B column (volume ratio of 100:0:0, 8:2:0, 6:4:0, 4:6:0, 2:8:0, 0:100:0,0:95:5 and C: M (ethyl acetate-methanol) ═ 9:1), and subjected to concentration under reduced pressure to obtain fractions BFr.1-BFr.8 in sequence. Bfr.5 was stirred by HPLC analysis, gradient eluted through a C column (100: 0:0, 8:2:0, 6:4:0, 4:6:0, 2:8:0, 0:100:0,0:95:5 and C: M (ethyl acetate-methanol) ═ 9:1 by volume ratio) in a petroleum ether-ethyl acetate-chloroform-methanol system, received in a vial, and analyzed by TLC detection showing that cfr.6-cfr.19 contained different target products, and further combined and prepared by preparative HPLC based on the results of TLC. Similarly, BFr.7 containing target component is mixed and passed through D column, and is eluted by using solution and system identical to C column, and HPLC analysis shows that DFr.6-DFr.8 contain compound 4-5; DFr.5, DFr.6 and BFr.8 were combined and subjected to E column chromatography using chloroform methanol system (volume ratio 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20) to obtain 7 fractions, EFr.1 to EFr.7, which were analyzed by HPLC to show that EFr.3 to EFr.6 contained the target compound 5, and then the three fractions were combined and subjected to semi-preparative HPLC to prepare them. Using a system (water (A): acetonitrile (B) as a system for preparation) with detection wavelengths of 270nm and 354nm, a procedure of 0min 30% A, 0.1-18min 30% -0% A, 18.1-23min 100% B, 23.1-25min 0% -30% A, 25.1-30min 30% A, a column specification of 5. mu.M.times.21.2 mm.times.250 mm, innovalC18, a flow rate of 10mL/min) was prepared to obtain compound 5(234mg, retention time of 21.8min), and a part of compound 5 was subjected to 1-and 2-dimensional NMR analysis, which revealed that compound 5 was a novel compound. Fractions cfr.6-cfr.19 were prepared using the same semi-preparative procedure described above to give compounds 1 (retention time 24.25min), 3 (retention time 23.6min) and 4 (retention time 23min), and the chemical structures of compounds 3-5 were determined by 1 and 2 dimensional NMR analysis, as shown in figure 1. The nuclear magnetic data for

compounds

3, 4 and 5 are shown in table 1.

Table 1, nuclear magnetic data for compounds 3-5.

The structural formulas of the compounds 1-5 are shown as 1, 2, 3, 4 and 5 in figure 1 respectively.

6. Antituberculous activity assay and antitumor assay of Compounds 1-5

6.1 test of Compounds 1-5 vs. M.smegmatis mc by Broth double dilution method²155 and m. tuberculosis H37R_VThe antibacterial activity of (1). Wherein for M.smegmatis mc²155 the procedure for the antimicrobial activity test is briefly described as follows: 1) m. smegmatis mc²155, reviving for 36 hours, and diluting 1000 times to be used as bacterial liquid to be detected; 2) dissolving the compounds 1-5 by DMSO to prepare a stock solution with the concentration of 3200 mu g/mL; 3) diluting the drug stock solution with 7H9 broth (0.2% glycerol, 0.05% Tween80) at a certain ratio, and diluting to a final concentration of 256-0.125 μ g/mL, wherein the volume of the drug broth per well is 100 μ L; 4) 100. mu.L of diluted bacterial suspension was added to each well of a 96-well plate by means of a line gunSo that the final concentration of each hole in each row is 128-0.0625 mug/mL in sequence; diluted broth and blank broth were used as positive and negative controls, respectively, with 3 replicates per compound. 5) The 96-well plate was incubated at 37 ℃ in an incubator, and the results were observed and recorded for about 24 hours.

Double dilution method for determining the ratio of 1-5 to M.tuberculosis H37R_VThe procedure for the antibacterial activity test of (1) is briefly described as follows: 1) inoculating 2ml of self-luminescence M.tubericulosis H37Rv (UAlRa) frozen at-80 ℃ into an Erlenmeyer flask containing 50ml of 7H9 (containing 0.1% twin80) culture medium, and culturing until the OD value reaches between 0.3 and 1.0; 2) preparing a compound 1-5 into a mother solution of 10mg/mL by using DMSO, diluting each compound to 5120-2.5 mu g/mL according to a certain proportion, taking RIF (10ug/mL, 1ug/mL) as a positive control and DMSO as a negative control, adding the corresponding compound into a 96-well enzyme label plate by taking 5 mu L of each well, and setting each compound to be three times; 3) diluting the bacterial liquid stock solution, and taking diluted bacterial liquid with a luminous value of 3000-5000/200 mu L as bacterial liquid for detection; 4) adding 195. mu.L of diluted bacteria solution into each well of a 96-well enzyme label plate by using a calandria gun to ensure that the final concentration of each compound is 128-0.0625. mu.g/mL in sequence, culturing in a 37-degree incubator, and detecting the luminescence value at 0-7 d. The results of the antituberculosis activity tests are shown in table 2. The results show that the compounds 2-5 have remarkable antitubercular activity and show great economic value and superior market prospect in the field of antitubercular drug development.

Table 2, antitubercular activity of compounds 1-5.

6.2 testing the growth inhibition effect of the compounds 1-5 on tumor cells.

The normal cell lines used in the experiment were LO2 and Hum V12; the tumor cell strains are HeLa (human cervical cancer cells), HepG2 (human hepatoma cells), A549 (human small cell lung cancer cells), CNE-2 (human nasopharyngeal carcinoma cells) and MCF-7 (human breast cancer cells).

The experimental procedure used a conventional tetramethylazoazolium salt colorimetric Method (MTT). Experimental design blank control group, positive control group, treatment group 1, treatment group 2 and treatment group 3. Compound 1, compound 2, compound 3, compound 4, compound 5 and doxorubicin were formulated in DMSO at different concentrations, respectively. The 7 cells LO2, HumV12, HeLa, HepG2, A549, CNE-2 and MCF-7 were cultured in 1640 medium (containing 10% calf serum) in 5% CO₂Cultured in an incubator of (1), and then diluted to a concentration of 2X 10³Cells were seeded in 96-well plates at 100. mu.L/100. mu.L cell suspension, and incubated in 37 ℃ incubator for 12 h. Blank control was added with 50 μ L DMSO, positive control with doxorubicin at different concentrations, treatment 1 with compound 1 at different concentrations, treatment 2 with compound 2 at different concentrations, and treatment 3 with compound 3 at different concentrations, 3 wells per drug concentration. Exposure to ophilobolin O for 48h, addition of 50. mu.L MTT solution (1mg/mL in PBS), incubation at 37 ℃ for 4h, addition of 200. mu.L DMSO, and measurement of absorbance at 550nm using a microplate reader. The cell survival rate and half maximal Inhibitory Concentration (IC) of

compounds

1, 2, 3, 4 and 5 after tumor cell treatment were calculated according to the formula₅₀) The results are shown in Table 3.

Table 3 antitumor activity assay results of compounds 1-5.

As can be seen from Table 3, compounds 2, 3 and 4 have better tumor inhibition activity and can be used for preparing antitumor drugs.

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> Compound ilamycin F and application thereof

<160>2

<210>1

<211>1209

<212>DNA

<213> streptomyce atratus SCSIO ZH16

<400> 1

gtgctcaaca gcgagaatgc ccggaacgac atccctgaga tcgccatgat cgaccccgag 60

gtgttgcaca atccgatttc cgcctatggg caggccaggg aacggtcccc catagtgcgg 120

ctcatggcac ccggttacgg ctcgatatgg gcggtcaccc gccacggcga cgcgaaggcc 180

atgctcggcg attcgcgcct cgccatgacc gccgacagct ggaatcagcg cataccggat 240

gacatcaagc cgtacatgct gacgatgtcg cacatggacg gggccgacca cggccggctg 300

cgcaagctcg ccgtgcgggc cttcaccgcc cgccgcgtcg ccgcccgccg cgacatgatc 360

ctgaagagca ccgaacggct gctcgacgcc ctgcccggcc atgccgagca gggcaccgtc 420

gacctgctgg aacacttcgc ccggccgctg cccaccttcg tggtcggcga cgtgatgggc 480

atccccgagg cggaccggcc cctgtggcgc gcctacgtcg cccggatggg ctccggccag 540

acgtacgacg acgccctgcg cgacgccgtc gccggagcca agcaggtcgt cgcgctgcgc 600

cgcgaggagc ccggcgacga cctcgtctcc gacctcctcc aagcgcaggc cgaggccgcc 660

gaccggatca ccgacaccga gctggtcacc atggtgtggc acaccgtctt cgcaggccag 720

gacaaccttg cgaacttcat cgccaactcc gtggtggccc tgctctcaca tcccgagcag 780

ctcgccgccc tgcgcgccga cccgggcctg atgccccggg ccgtcgagga gctgatgcgc 840

tggcggcccc cgctgttgct gaccgccatc cgctacgccc tggaggactt cgagctgtgc 900

ggcgcgccgg tgcgccgcgg ggactccgtg gtggcggtga tcgcctcggc caaccgtgac 960

ccgcgggtct tcgccgaccc ggacacactc gacatcaccc ggcctgccgg caccgcggtc 1020

cagttcggct tcggccacgg cccgcactac tgcatcggcg catcactggc cgccgtcgag 1080

gcggaggtgg cgctcggcgc actgctgaac cgcttccctg acctcgcgct ggccgtcccg 1140

ccggaggagg tgccgcggct gcccaacccc ggcagctggc acctgaccgc gctcccggtc 1200

accctctga 1209

<210>2

<211>1200

<212>DNA

<213> streptomyce atratus SCSIO ZH16

<400> 2

gtggaaaagt cgacgccgcc cccgccctat tccaaggacg acggaaaagc actccaggac 60

tggttccgcg tcatgcggga cgagcggccc gtccaccagg atcccgcgac cggcgcctgg 120

atggttctcc ggtacgccga cgtcgtcgcg gccagcctgg accacgccac gtactcctcg 180

gagctgtggc gcgcgtaccc cgccgagtgg ggaaagggcg acgcctgggg cgagggcagg 240

ctcaccgaga tggacccgcc gaaacaccgg ctgctgcggg ccgtgatcgg caaggcgttc 300

accaaccgga cggtcgccgg cctcgcaccc gagatcgagg ccaccgtgga acgcctgttg 360

gacgccgcgg acggtcgcac ggagatcgac gtggcccgcg acctggccga cccgctgccg 420

gtcatggtga tcgccgaact gatcggactt cccttcgagg accgcgagtt gctgcgcggc 480

tgggcggacc ggcttctctc cttcgaggtc ggcgacctgg ccggcgagga cctcgtcaag 540

gcgatcgacg cggccggcgc ggaactgctc gcctaccttc gcgagcacta ccggctgcga 600

cgcgccgccc cgaaggacga cctcttcagc aggctggtga cggcggaggt ggaaggcgaa 660

cggctcaccg aggaaacggt ggtcaacctc ggcaagctgc tgctcatcgg cggacatgcc 720

accaccgcct gctcactggc cagtctcgtc ttcgaactgc tgcgccaccc cgatgcgctg 780

accgccgtac ggcaggacgc cgacctgata ccggccgcca tcgaggaatc ggtccgctac 840

cggcccgccg tggtgaacag cctgcggctg accacacgtg agaccgagct cggcgacgtc 900

accatccccg ccggacagtt cgtgtcgctc tccgcgctct cggccaacca cgacgagcgg 960

cagttcgacg cccccgaacg cttcgacatc caccggcagc acaaccagca cgtcggcttc 1020

ggccagggcg tccactactg cctgggcgca ccactggccc gtacggagat ccggatcgca 1080

ctctccgcgc tgctgcgacg gttcccggtg ctggaacgga ccgacggccc cctccagtac 1140

taccgcaacc cgagcatcgc cggactcagg tccttcccgg tggccgtcgg ccgaggctga 1200

Claims

1. A compound 5 represented by the formula (III):

2. use of compound 5 according to claim 1, as represented by formula (iii), in the manufacture of an anti-tubercular drug, said anti-tubercular drug being an anti-m.semagamit or m.tuberlucosis drug.

3. An antituberculous drug characterized by containing a compound 5 represented by the formula (III) as an active ingredient, wherein the antituberculous drug is an anti-M.semagamit or M.Tuberluccosis drug.