CN106434702A - Biosynthetic gene cluster of paquete amide and application thereof - Google Patents
Biosynthetic gene cluster of paquete amide and application thereof Download PDFInfo
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- CN106434702A CN106434702A CN201610807614.XA CN201610807614A CN106434702A CN 106434702 A CN106434702 A CN 106434702A CN 201610807614 A CN201610807614 A CN 201610807614A CN 106434702 A CN106434702 A CN 106434702A
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- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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- C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
- C12Y103/05005—15-Cis-phytoene desaturase (1.3.5.5)
Abstract
The invention discloses a biosynthetic gene cluster of paquete amide and an application thereof. The biosynthetic gene cluster of paquete amide comes from (Streptomyces pactum)SCSIO 02999, and the cluster comprises five genes including heterozygous polyketone/ nonribosomal peptide synthetases genes ptmA, FAD dependent redox enzyme genes ptmB1, phytoene dehydrogenase genes ptmB2, cyclase genes ptmC and hydroxylase genes ptmD. According to the biosynthetic gene cluster of paquete amide and the application thereof, information in genes and proteins related to biosynthesis of the paquete amide provides theoretical foundations and materials for conducting genetic modification on the biosynthesis of multi-ring tetramate macrocylic lactam family. By conducting genetic modifications on the biological synthetic genes, 6 new structures antineoplastic paquete amide compounds pactamide A-F are obtained, and thus effective compound entities are provided for research and development of antineoplastic drugs.
Description
Technical field
The invention belongs to microbiological genetic engineering field and in particular to a kind of Parker spy acid amides biological synthesis gene cluster and
Its application.
Background technology
The synthetic biology technology flourishing in recent years, the silence biosynthesis contained for activation Deep-Sea Microorganisms
Gene cluster, discovery new antitumoral lead compound bring new opportunity.Illustrating biosynthesis pathway, the understanding of nature
On the basis of Combinatorial biosynthesis mechanism, the biosynthesis gene using the raw element of Combinatorial biosynthesis technical antagonism is struck in vivo
The operation such as remove and recombinate, and is not only able to produce the Structures of Natural Products analog of " non-natural ", but also can improve natural product
Natural products required for the yield of thing, or orientation accumulation, the discovery for natural products and drug development provide compound structure
With diverse biological activities.
So far, about living things catalysis preparation Parker's spy's acyl such as the biological synthesis gene cluster of Parker's spy's acid amides, cyclase
There is not been reported both at home and abroad for amine and promoter engineering acquisition antitumor Parker spy's acid amides and the like etc..
Content of the invention
First purpose of the present invention is to provide a kind of biosynthesis base of the spy's acid amides of the Parker in Deep-Sea Microorganisms
Because of cluster.
The present invention, carries out biological letter to streptomycete (Streptomyces pactum) SCSIO 02999 whole genome sequence
Breath credit analysis, obtains the biological synthesis gene cluster of Parker's spy's acid amides.With synthetic biologies such as promoter engineering and heterogenous expressions
Technology, the biological synthesis gene cluster of Parker's spy's acid amides of successful activation silence, obtain 6 new Parker's spy's amide-type chemical combination
Thing, provides chemical entities for screening anti-tumor medicine.
The biological synthesis gene cluster of Parker's spy's acid amides of the present invention derives from South China Sea sediments streptomycete (Streptomyces
Pactum) SCSIO 02999 it is characterised in that the nucleotide sequence such as SEQ ID NO.1 of this biological synthesis gene cluster
Shown in the base sequence of 14290-29055 position, including 5 genes, specially：
(1) it is responsible for the gene of the polyketone-ornithine-polyketone skeleton synthesis of Parker's spy's acid amides, i.e. ptmA, positioned at nucleotides sequence
At row SEQ the 15332-24676 base of ID NO.1, length is 9345 base-pairs, the polyketone/non-ribosomal of encoding hybrid
Poly- peptide synthetase, 3114 amino acid；
(2) it is responsible for another gene of Parker's spy's amide backbone synthetic modification, i.e. ptmB1, positioned at nucleotide sequence SEQ
At the 24663-26348 base of ID NO.1, length is 1686 base-pairs, the oxidoreducing enzyme that coding FAD relies on, 561
Amino acid；
(3) it is responsible for the gene ptmB2 of Parker's spy's amide backbone synthetic modification, positioned at nucleotide sequence SEQ ID NO.1 the
At 26360-28003 base, length is 1644 base-pairs, encodes phytoene dehydrogenase, 547 amino acid；
(4) it is responsible for the gene ptmC of Parker's spy's amide backbone cyclisation, positioned at nucleotide sequence SEQ ID NO.1 28000-
At 29055 bases, 1056 base-pairs of length, encode cyclase, 351 amino acid；
(5) it is responsible for the gene ptmD that Parker's spy's acid amides hydroxylating is modified, positioned at nucleotide sequence SEQ ID NO.1 the
At 14290-15195 base, length is 906 base-pairs, encoding hydroxylase, 301 amino acid.
Shown in SEQ ID NO.1, the complementary series of 14290-29055 base sequence of sequence can be mutual according to DNA base
Mend principle to obtain at any time, and SEQ ID 14290-29055 base sequence of NO.1 or partial nucleotide sequence can be by poly-
Polymerase chain reacts (PCR) or is obtained with suitable digestion with restriction enzyme corresponding DNA or DNA recombinant technique.The present invention carries
Supply to build the recombinant DNA including at least DNA fragmentation in the 14290-29055 position of sequence shown in part SEQ ID NO.1 to carry
Body approach.
Present invention also offers initiative Parker's spy's acid amides biosynthesis gene is blocked or other genes such as heterogenous expression change
The approach made, at least one of gene comprises DNA fragmentation in the 14290-29055 position of sequence shown in SEQ ID NO.1.
Nucleotide sequence provided by the present invention or partial nucleotide sequence, can be miscellaneous using PCR sonde method or Southern
The technology such as friendship, obtain the biological synthesis gene cluster homologous gene of Parker's spy's acid amides from the screening of other biological body weight.
Nucleotide sequence or partial nucleotide sequence that the present invention provides, can be used for streptomycete (Streptomyces
Pactum) more Library plasmid are positioned in SCSIO 02999 genomic library.These Library plasmid are at least included in the present invention
Partial sequence, also comprise adjacent domain in streptomycete (Streptomyces pactum) SCSIO 02999 genome and do not clone
DNA.
Comprise nucleotide sequence provided by the present invention or the DNA fragmentation of at least part of nucleotide sequence, can be internal
Outer mutation etc. is modified, including insertion, displacement, disappearance, error-prone PCR, mutation site-specific, different sequence weight
Group, orthogenesis etc..
Comprise the clone of nucleotide sequence provided by the present invention or partial nucleotide sequence, can be by suitable expression system
The system corresponding enzyme of Expression product or raising bioactive compound yield in foreign host.These foreign host include large intestine bar
Bacterium, streptomycete, pseudomonad, bacillus, yeast and animals and plants etc..
The amino acid sequence that the present invention provides can be used for separating desirable proteins and can be used for Antibody preparation.
Comprise the polypeptide of amino acid sequence provided by the present invention or partial sequence, removing or may substitute some
Still have the even new biologically active of biologically active after amino acid, or improve yield or optimize protein dynamics feature or its
He is devoted to the property obtaining.
Comprise nucleotide sequence provided by the present invention or the gene of partial nucleotide sequence or gene cluster can be heterologous
Express and disclose their functions in host metabolism in host.
Comprise nucleotide sequence provided by the present invention or the gene of partial nucleotide sequence or gene cluster can be by DNA weight
Group technique construction recombinant vector, to obtain new bio route of synthesis it is also possible to by insertion, displacement, disappearance or inactivate, and then
Obtain other novel compounds based on biosynthesis pathway.
Second object of the present invention be provide Parker spy acid amides biological synthesis gene cluster preparation Parker spy acid amides or
Application in its analog, the particularly application in preparing the arbitrary compound of pactamide A-F,
Third object of the present invention be provide a kind of cyclase gene ptmC it is characterised in that its nucleotide sequence such as
Shown in the base sequence of 28000-29055 position of SEQ ID NO.1.
The present invention also provides a kind of cyclase PtmC by above-mentioned cyclase gene ptmC coding.Cyclase PtmC can be with
NADPH becomes pactamide A for co-factor catalysis pactamide C-shaped.Therefore, the present invention also provides cyclase PtmC in catalysis
Compound pactamide C-shaped becomes the application in compound pactamide A,
The present invention also provide Parker spy acid amides the promoter engineered mutant of biological synthesis gene cluster and its with cyclase base
Because ptmC deletion mutation, oxide-reductase gene ptmB1 deletion mutation or Phytoene dehydrogenase gene ptmB2 disappearance is prominent
The genetic engineering bacterium becoming；Described oxide-reductase gene ptmB1, the 24663- of its nucleotide sequence such as SEQ ID NO.1
Shown in the base sequence of 26348；Described Phytoene dehydrogenase gene ptmB2, its nucleotide sequence such as SEQ ID
Shown in the base sequence of 26360-28003 position of NO.1.
The present invention also provides application in preparation Parker's spy's acid amides or its analog for the described genetic engineering bacterium.
Preferably, the genetic engineering bacterium of the promoter engineered mutant of biological synthesis gene cluster of described Parker's spy's acid amides is
Streptomyces pactum SCSIO 02999PTMp1.
In a word, all genes including Parker's spy's acid amides biosynthesis correlation provided by the present invention and Protein Information, side
Help it is appreciated that the key mechanism such as reduced form cyclisation of polycyclic tetramate Macrocyclic lactams family natural products, for further
Genetic modification provides material and theoretical foundation.Gene provided by the present invention and protein can be used to find and discovery can be used for
The polycyclic tetramate Macrocyclic lactams family of medicine, health or agricultural, gene or albumen.
The present inventor is found through experiments, and the present invention newly separates the compound pactamide A F of identification to human cancer cell
There is cytotoxic activity (table including breast cancer MCF-7, human liver cancer HepG2, human lung cancer H460 and human glioma cell SF-268
8), wherein pactamide A includes breast cancer MCF-7, human lung cancer H460 cell, human liver cancer HepG2 and people god to human cancer cell
IC through glioma cell SF-26850It is 0.26 μM, 0.24 μM, 0.37 μM and 0.51 μM respectively.
Therefore, fourth object of the present invention is to provide new compound pactamide A, B, C, D, E and F,
The present invention also provides compound pactamide A, B, C, D, E or F application in preparing antineoplastic.
Described antineoplastic is preferably anti-breast cancer, the medicine of human lung cancer, human liver cancer or human glioma.
5th purpose of the present invention is to provide a kind of antineoplastic it is characterised in that comprising effective dose
Pactamide A, B, C, D, E or F are as active ingredient.
Described antineoplastic is preferably anti-breast cancer, the medicine of human lung cancer, human liver cancer or human glioma.
The present invention from streptomycete (Streptomyces pactum) SCSIO 02999 separate obtain 6 new have anti-swollen
The Macrocyclic lactams Alkaloid of tumor activity, it can be used for preparing antineoplastic, is expected by bioengineering or chemical modification
It is developed into anti-cancer agent.
In the present invention Parker spy acid amides produce strain streptomycete (Streptomyces pactum) SCSIO 02999 in
September is preserved in the micro- life in the Chinese Academy of Sciences of Chinese Academy of Science Nanhai Ocean Research Institute of Guangzhou, Guangdong Province, China city ocean on the 8th within 2012
Thing research center (RNAM Center for Marine Microbiology, CAS), deposit number is SCSIO 02999.Should
The bacterial strain of collection is for sale, and therefore those skilled in the art can be from September in 2012 8 days afterwards from this preservation
The heart buys this bacterial strain.
Streptomycete (Streptomyces pactum) SCSIO 02999 of the present invention is also disclosed in September in 2012 on the 8th
In non-patent literature (webpage), its network address is：http://www.scsio.ac.cn/xwzx/tpxw/201209/ t20120908_3640561.html(open with title " streptomycete Streptomyces sp.SCSIO 02999 "), this bacterial classification
Applicant also holds, and ensures to provide to the public in 20 years from the applying date.
Brief description
Fig. 1 is representative PTM class compound and the chemical constitution of Parker spy amide compound pactamide A-F.
Fig. 2 be in Streptomyces pactum SCSIO 02999 Parker spy acid amides biological synthesis gene cluster and its
Functional analysis；Wherein, A is the biological synthesis gene cluster of Parker spy acid amides in Streptomyces pactum SCSIO 02999
Structural representation；B is the HPLC analysis chart of different strains butanone crude extract in the fermentation medium：(i)：The same frame of xiaP gene
The S.pactum SCSIO 02999XM47i of disappearance, (ii)：The mutant strain S.pactum activating in situ before ptmA gene loci
SCSIO 02999PTMp1, (iii)：TK64/pCSG2801, (iv)：TK64/pSET152, (v)：TK64/pCSG2804, (vi)
TK64/pCSG2805, (vii) TK64/pCSG2809, (viii) TK64/pCSG2811, (ix) TK64/pCSG2814, " # " generation
The still unidentified PTM class compound of table, in figure Arabic numerals represent Parker's spy's amide compound, and wherein 11-16 distinguishes successively
The compound that formula 11-16 in Fig. 1 that represents represents is pactamide A-F；C be enzyme PtmA, PtmB1, PtmB2, PtmC and
PtmD participates in the possible function analysis of synthesis Parker's spy's acid amides.
Fig. 3 is the conceptual design in ptmA gene front target to insertion ermE*p promoter；Wherein, A is that design of primers is used for
ErmE*p promoter is inserted before ptmA gene；B is that the biological synthesis gene cluster of activation Parker's spy's acid amides can be used for pattern lead-changing penicillium
The clay pCSG2804 of streptomycete TK64 heterogenous expression builds flow chart；C is primer amplification aadA, ermE*p promoter, aadA-
The PCR electrophoretogram of ermE*P DNA fragmentation product；D is to be proved to be successful aadA-ermE*P DNA fragmentation in ptmA gene front target
PCR electrophoretogram to insertion.
Fig. 4 is that same frame lacks ptmC gene and in ptmA gene front target to the conceptual design inserting ermE*p promoter；Its
In, A be complete Parker's spy's acid amides synthetic gene cluster promoter is engineered and the clay pCSG2809 of the same frame disappearance of ptmC
Build flow chart；B be proved to be successful the same frame disappearance of ptmC and by aadA-ermE*P DNA fragmentation ptmA gene front target to
Insert the PCR electrophoretogram successfully building clay pCSG2809.
Fig. 5 is the expression and purification SDS-PAGE testing result of recombinant protein PtmC, and wherein, M is albumen maker, and PtmC is
Cyclase PtmC.
Fig. 6 is that pactamide C (compound 13) generates pactamide A (compound 11) under enzyme PtmC catalysis
HPLC analysis chart；(i) reaction system：Compound 13, PtmC and NADPH；(ii) reaction system：Compound 13, PtmC and NADH；
(iii) compound 11 standard items；(iv) control systems：Compound 13, heat inactivation PtmC, NADPH；(V) control systems：Compound
13, PtmC, lack NADPH.
Fig. 7 is the high-resolution electrospray ionization mass spectrometry of pactamide A (compound 11).
Fig. 8 is the hydrogen spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Fig. 9 is the carbon spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 10 is the hsqc spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 11 is the HMBC spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 12 is the COSY spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 13 is the NOESY spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 14 is the NOESY spectrum of pactamide A (compound 11), solvent C DCl3/CD3OD(9:1).
Figure 15 is that pactamide A-E spectrogram compares；A is that pactamide A-E key NOESY is related；B is
The ECD spectrum of pactamide A, B, D, E；C is the ECD spectrum of lysobacteramide B and HSAF.
Figure 16 is the high-resolution electrospray ionization mass spectrometry of pactamide B (compound 12).
Figure 17 is the DMSO-d of pactamide B (compound 12)6Hydrogen is composed.
Figure 18 is the DMSO-d of pactamide B (compound 12)6Carbon is composed.
Figure 19 is the DMSO-d of pactamide B (compound 12)6Hsqc spectrum.
Figure 20 is the DMSO-d of pactamide B (compound 12)6HMBC composes.
Figure 21 is the DMSO-d of pactamide B (compound 12)6COSY composes.
Figure 22 is the DMSO-d of pactamide B (compound 12)6NOESY composes.
Figure 23 is the high-resolution electrospray ionization mass spectrometry of pactamide C (compound 13).
Figure 24 is the DMSO-d of pactamide C (compound 13)6Hydrogen is composed.
Figure 25 is the DMSO-d of pactamide C (compound 13)6Carbon is composed.
Figure 26 is the DMSO-d of pactamide C (compound 13)6Hsqc spectrum.
Figure 27 is the DMSO-d of pactamide C (compound 13)6HMBC composes.
Figure 28 is the DMSO-d of pactamide C (compound 13)6COSY composes.
Figure 29 is the DMSO-d of pactamide C (compound 13)6NOESY composes.
Figure 30 is the DMSO-d of pactamide C (compound 13)6NOESY composes.
Figure 31 is the high-resolution electrospray ionization mass spectrometry of pactamide D (compound 14).
Figure 32 is the DMSO-d of pactamide D (compound 14)6Hydrogen is composed.
Figure 33 is the DMSO-d of pactamide D (compound 14)6Carbon is composed.
Figure 34 is the DMSO-d of pactamide D (compound 14)6Hsqc spectrum.
Figure 35 is the DMSO-d of pactamide D (compound 14)6HMBC composes.
Figure 36 is the DMSO-d of pactamide D (compound 14)6COSY composes.
Figure 37 is the DMSO-d of pactamide D (compound 14)6NOESY composes.
Figure 38 is the high-resolution electrospray ionization mass spectrometry of pactamide E (compound 15).
Figure 39 is the DMSO-d of pactamide E (compound 15)6Hydrogen is composed.
Figure 40 is the DMSO-d of pactamide E (compound 15)6Carbon is composed.
Figure 41 is the DMSO-d of pactamide E (compound 15)6COSY composes.
Figure 42 is the DMSO-d of pactamide E (compound 15)6Hsqc spectrum.
Figure 43 is the DMSO-d of pactamide E (compound 15)6HMBC composes.
Figure 44 is the DMSO-d of pactamide E (compound 15)6NOESY composes.
Figure 45 is the high-resolution electrospray ionization mass spectrometry of pactamide F (compound 16).
Figure 46 is the DMSO-d of pactamide F (compound 16)6Hydrogen is composed.
Figure 47 is the DMSO-d of pactamide F (compound 16)6Carbon is composed.
Figure 48 is the DMSO-d of pactamide F (compound 16)6Hsqc spectrum.
Figure 49 is the DMSO-d of pactamide F (compound 16)6HMBC composes.
Figure 50 is the DMSO-d of pactamide F (compound 16)6COSY composes.
Figure 51 is the DMSO-d of pactamide F (compound 16)6NOESY composes.
Specific embodiment
The following is and the present invention is further illustrated, rather than limitation of the present invention.
1. the biological synthesis gene cluster of clonal analysis Parker spy acid amides
Parker spy's acid amides (Fig. 1) belongs to polycyclic tetramate Macrocyclic lactams family (PTM) natural products, by heterozygosis
The catalysis of polyketone/non-ribosomal poly- peptide synthetase produces.To streptomycete (Streptomyces pactum) the full base of SCSIO 02999
Because organizing sequence analysis, find there is PTM biological synthesis gene cluster.To streptomycete (Streptomyces pactum) SCSIO
02999 genomic library about 2000 is cloned into performing PCR screening, obtains and contains complete PTM biological synthesis gene cluster positive colony
pCSG2404.
2. each gene in vivo functionality analysis in the biological synthesis gene cluster of Parker spy acid amides
The present invention is studied to the biosynthesis mechanism of Parker's spy's acid amides.Sequencing knot to positive colony pCSG2404
Really, with bioinformatic analysis, show that it comprises 18 ORF, predict ptmA, ptmB1, ptmB2, ptmC and ptmD totally 5
Gene is responsible for the biosynthesis of Parker's spy's acid amides and rear modification (table 1 and Fig. 2A).
The gene of biological synthesis gene cluster of table 1 Parker spy's acid amides and its function
A amino acid number；Homologous protein GenBank accession number is comprised in b bracket；C uniformity/similitude (identity/
similarity).
3. the promoter of Parker spy acid amides biological synthesis gene cluster is engineered：
XiaP gene in wild type streptomycete (Streptomyces pactum) SCSIO 02999 is responsible for tall building mycin
(xiamycin) the biosynthetic key gene of skeleton, by PCR targeting strategy (Gust B, et al.2003,
Proc.Natl.Acad.Sci.100:1541 1546.) after, this gene being lacked with frame, the mutant strain S.pactum of acquisition
SCSIO 02999XM47i loses the ability producing tall building mycin.Wild type streptomycete (Streptomyces pactum) SCSIO
02999 and mutant strain S.pactum SCSIO 02999XM47i (table 3) traditional zymotic culture be all not detected by Parker spy acid amides
The generation (Fig. 2 B) of class compound.Speculate Parker's spy's acyl that streptomycete (Streptomyces pactum) SCSIO 02999 exists
Amine biosynthesis cluster is in silence state, and it is engineered that the present invention has carried out promoter to Parker's spy's acid amides biosynthesis cluster, from
And activate Parker's spy's acid amides biological synthesis gene cluster or heterogenous expression Parker spy's acid amides biological synthesis gene cluster in situ.
With pPWW50 carrier as template, PCR amplification obtains 0.3kb ermE*p promoter, and (the primer is：ermE-p1-
F/ermE-p1-R, is shown in Table 2)；With pIJ778 as template, amplification obtains 1.4kb spectinomycin resistance gene aadA (the primer
For：P1-aadA-F/p-aadA-R, is shown in Table 2)；Connected using bridge-type round pcr, be configured to the aadA-ermE*P of 1.7kb
DNA fragmentation.According to PCR targeting strategy, the aadA-ermE*P DNA fragmentation of 1.7kb passes through homologous recombination (the primer
For：P1-aadA-F/ermE-p1-R, is shown in Table 2, Fig. 3 B, C), (use primer ermE- before the ptmA gene being inserted into pCSG2404
P1-det F/ermE-p1-det R verifies, is shown in Table 2, Fig. 3 B, D), build and obtain recombinant cosmid pCSG2802.DNA fragmentation aac
(3) IV-oriT-Int φ C31 comes from plasmid pSET152A ' B (Zhang, Y et al.2013.Org.Lett.15,3254-
3257), for engaging transfer with muta lead mycillin Streptomyces lividans TK64.Plasmid pSET152A ' B is made
Use BamHI/EcoRI double digestion, DNA fragmentation aac (3) the IV-oriT-Int φ C31 of acquisition passes through PCR further
Targeting technology, replaces the kalamycin resistance gene of pCSG2802, builds and obtains clay pCSG2804 (Fig. 3 B).
Before ptmA gene in pCSG2804, by PCR-targeting technology, insert strong promoter ermE*p, in addition kanamycins
Resistant gene by aac (3) IV-oriT-Int φ C31 substitute, thus can in TK64 heterologous integration (Fig. 3).Clay pCSG2805
Construction strategy similar with pCSG2804, (the primer is by homologous recombination for the aadA-ermE*P DNA fragmentation of 1.7kb：
P2-aadA-F/ermE-p2-R, is shown in Table 2), (with primer ermE-p2-det F/ before being inserted into the ptmD gene of pCSG2404
ErmE-p2-det R verifies, is shown in Table 2), build and obtain recombinant cosmid pCSG2803；PCSG2803 kalamycin resistance further
Gene is substituted by aac (3) IV-oriT-Int φ C31, thus the clay pCSG2805 obtaining can heterologous integration in TK64.
The same frame disappearance of 4.ptm gene
As shown in Figure 4 A, pCSG2801 is without promoter engineering, only using PCR-targeting technology, using DNA fragmentation
Aac (3) IV-oriT-Int φ C31 replaces the kalamycin resistance gene of pCSG2404, thus can heterologous integration in TK64.
As shown in Figure 4 A, the ptmC gene of clay pCSG2801, by PCR-targeting technology, is come from plasmid
(the primer is aadA (the spectinomycin resistance gene)+oriT aliquot replacement of pIJ778：PtmC-dis-F/ptmC-dis-R,
It is shown in Table 2), obtain clay pCSG2806.To clay pCSG2806, carry out 42 DEG C of induction FLP restructuring expression of enzymes, remove aadA+
OriT box, completes ptmC and deletes (with primer ptmC-det F/ptmC-det R checking, being shown in Table 2, Fig. 4 B) with frame, obtain clay
pCSG2808.
According to PCR targeting strategy, by homologous recombination, the aadA-ermE*P DNA fragmentation of 1.7kb is integrated into
(with primer ermE-p1-det F/ermE-p1-det R checking, it is shown in Table 2, Fig. 4 B) before the ptmA gene of pCSG2808, complete handkerchief
The promoter of cut acid amides synthetic gene cluster is engineered and the same frame disappearance of ptmC, builds and obtains recombinant cosmid pCSG2809
(Fig. 4).
Clay pCSG2811 and pCSG2814, respectively by similar with the construction strategy of pCSG2809, achieves respectively
The same frame of ptmB1 or ptmB2 is deleted and the promoter of Parker's spy's acid amides synthetic gene cluster is engineered.
5. engineered Parker's spy's acid amides biological synthesis gene cluster activation or the heterogenous expression in situ of promoter
Using the genetic operating system of streptomycete (Streptomyces pactum) SCSIO 02999, to Parker's spy's acid amides
Biological synthesis gene cluster carries out activating in situ, structure promoter engineered mutant strain S.pactum SCSIO 02999PTMp1 (
Insert strong promoter ermE*p before the ptmA gene of S.pactum SCSIO 02999XM47i engineered mutant strain and build
Arrive, table 2-3, the primer and method with build strong promoter ermE*p in pCSG2802 insert identical before ptmA gene).
S.pactum SCSIO 02999PTMp1 is fermented and HPLC analysis, pactamide A-F can be produced
(Fig. 2 B).
Clay pCSG2801, pCSG2804, pCSG2805, pCSG2809, pCSG2811, pCSG2814 and comparison empty carrier
PSET152, is shifted by joint respectively, imports heterogenous expression host muta lead mycillin TK64 (Fig. 2 B).
As shown in Figure 2 B, the muta lead mycillin TK64 transformant of pCSG2801, Parker's spy's acid amides biological synthesis gene cluster
Un-activation, is not detected by polycyclic tetramate Macrocyclic lactams family natural products.The muta lead mycillin TK64 of pCSG2804
Transformant, successful expression pactamide A-E.The muta lead mycillin TK64 transformant of pCSG2809 (Δ ptmC), principal product
For pactamide C (compound 13).The muta lead mycillin TK64 transformant of pCSG2811 (Δ ptmB1), principal product is
Pactamide E (compound 15).The muta lead mycillin TK64 transformant of pCSG2814 (Δ ptmB2), principal product is chemical combination
Thing 10.
Mutant strain is fermented and metabolite HPLC analysis, further speculate ptmA, ptmB1, ptmB2, ptmC and
5 genes such as ptmD are responsible for the biosynthesis (Fig. 2 C) of Parker's spy's acid amides.
Primer used in table 2 present invention
Bacterial strain and plasmid used in table 3 present invention
6. the training of Parker spy acid amides biological synthesis gene cluster original position activated mutant strain S.pactum SCSIO 02999PTMp1
Hair care ferment and
The separation of Parker's spy's amide compound
The product spore culture medium of S.pactum SCSIO 02999PTMp1 is using the 38# culture medium adding 3% sea salt.
S.pactum SCSIO 02999PTMp1 single bacterium colony is inoculated in R5 culture medium (the R5 training of interpolation mass fraction 3% sea salt of 50ml
Foster base contains：Sucrose 103g/L, potassium sulfate 0.25g/L, magnesium chloride 10.12g/L, glucose 10g/L, casein hydrolysate
0.1g/L, dusty yeast 5.0g/L, dipotassium hydrogen phosphate 0.5g/L, micro- 0.1mL/L, pH 7.2-7.4), 200rpm, 28 DEG C
Culture 5-7 days.According to 1:10 ratio carries out seed switching, cultivates 7 days for 20 DEG C, equipped with 200ml culture medium in the shaking flask of 1L, altogether
Fermentation 20L culture medium.The 20L zymotic fluid of S.pactum SCSIO 02999PTMp1 uses equal-volume butanone to extract 3 times, rotation
It is evaporated, obtain 7g crude extract.Crude extract is dissolved in volume ratio 1:After 1 chloroform methanol, mix with appropriate silica gel and mix sample, make
With normal-phase silica gel column chromatography (Flash silica post 14.5 × 2.5cm, 40g), using chloroform/methanol mixed solution, according to volume ratio
100/0th, 95/5,90/10 and 80/20, carry out gradient elution respectively, obtain 4 components (Fr1-Fr4).(applied sample amount) 500mg's
Fr2 component (cut of chloroform/methanol 95/5v/v wash-out), using sephadex lh-20 (pillar parameter 120 × 3.5cm
I.d.), volume ratio 5:5 chloroform/methanol is eluted, and elutes 1000ml, obtains component Fr2B-Fr2D.Merge 200-
300ml eluent, rotation is evaporated acquisition Fr2B (100mg), and this component is using configuration C-18 reversed-phase column (250 × 10.0mm
I.d., 5 μm) preparative high performance liquid chromatography (acetonitrile：Water 20:80, flow velocity 2.5ml/min), isolate and purify further, collect
Retention time is the stream part of the 12nd minute, and rotation is evaporated and prepares pactamide B (26mg)；Collect the 15th point of retention time
The stream part of clock, rotation is evaporated and prepares pactamide E (2.5mg).300-400ml eluent is merged, rotation is evaporated
Obtain component Fr2C (200mg), using the preparative efficient liquid phase of configuration C-18 reversed-phase column (250 × 10.0mm i.d., 5 μm)
Chromatogram (acetonitrile：Water 60:40, flow velocity 2.5ml/min), isolate and purify further, collect the retention time stream part of the 14th minute, rotation
Turn to be evaporated and prepare pactamide D (3.2mg)；Collect the stream part that retention time is the 23rd minute, rotation is evaporated preparation and obtains
Obtain pactamide A (30mg).400-500ml eluent is merged, rotation is evaporated acquisition Fr2D component, high by preparative
Effect liquid phase chromatogram (250 × 10.0mm i.d., 5 μm；Acetonitrile：Water 50:50, flow velocity 2.5ml/min), collect 17 points of retention time
The stream part of clock, rotation is evaporated, and purifies and obtains pactamide F (4.7mg).
7. the heterogenous expression mutant strain fermentation of Parker spy acid amides biological synthesis gene cluster and dividing of Parker's spy's amide compound
From
Clay pCSG2801, pCSG2804, pCSG2805, pCSG2809, pCSG2811, pCSG2814, comparison empty carrier
The muta lead mycillin TK64 transformant of pSET152 uses the R5 culture medium of 50ml, cultivates 5-7 days for 28 DEG C.The change of pCSG2809
Lead green grass or young crops streptomycete TK64 transformant, using R5 culture medium fermentation 10L, butanone extracts 3 times, obtains 4.7g crude extract.By crude extract
It is dissolved in volume ratio 1:After 1 chloroform methanol, mix with appropriate silica gel and mix sample, using normal-phase silica gel column chromatography (Flash silica
Post 14.5 × 2.5cm, 40g), using chloroform/methanol mixed solvent, gradient elution (100:0-0:100, v/v), 4 groups are obtained
Divide (Fr1-Fr4).Collecting chloroform/methanol is 95:The stream part eluting when 5 is Fr2.The Fr2 component of 165mg (applied sample amount), uses
Sephadex lh-20 (pillar parameter 120 × 3.5cm i.d.), volume ratio 5:5 chloroform/methanol are eluted, wash-out
1000ml, obtains component Fr2B-Fr2D respectively.200-300ml eluent is merged, rotation is evaporated acquisition Fr2B (65mg).
Fr2B component, further using the preparative high performance liquid chromatography of C-18 reverse phase silica gel post (250 × 10.0mm i.d., 5 μm)
(acetonitrile：Water 60:40, flow velocity 2.5ml/min), collect the stream part of retention time 18min, purify and obtain compound
pactamide C(25mg).
8. compound structure identification：
Parker's spy's acid amides A (pactamide A) structure elucidation (table 4-5, Fig. 7-14,15)：
The molecular formula of pactamide A is defined as by C according to HRESIMS29H40N2O4(m/z 481.3098[M+H]+,
calcd 481.3066).The nuclear magnetic data of pactamide A is much like with the nuclear magnetic data of lysobacteramide B, different
Place be that pactamide A lacks OH in C-14 position, lack methyl in N-22 position.The relative configuration of pactamide A is by idol
Close constant (Z Δ2,3J2,311.5Hz；EΔ17,18J17,1815.0Hz) related with NOESY belonging to.Related, the H- according to NOESY
5/H-11/H-13 is attributed to the same side, and H-6/H-8/H-10/H-12/H-16/H3-31 is attributed to opposite side.By comparing
The CD spectrum of pactamide A and lysobacteramide B, the absolute configuration of pactamide A is defined as 5R, 6S, 8S,
10S, 11R, 12R, 13S, 16R, 23S.
Parker spy's acid amides B (pactamide B) structure elucidation (table 4-5, Figure 15,16-22)：
According to HRESIMS (m/z 493.2707 [M+H]+, calcd 493.2708) and will be true for the molecular formula of pactamide B
It is set to C29H38N2O5.The H NMR spectroscopy of pactamide B is similar to pactamide A's, compares discovery pactamide B and lacks one
Individual bimodal methyl,1In H NMR, the methylene hydrogen of many a pair mutual couplings is thus it is speculated that methyl is substituted.From H3- 30 arrive δC
136.6 HMBC correlation and two increased sp2The quaternary carbon of hydridization illustrates that the singly-bound between C10/C11 is changed into double bond.From δH(3.05,
4.05) determine CH to the HMBC of C-10/C-11/C-123- 31 are substituted, and the chemical shift of binding molecule formula and methylene pushes away
Break and replaced by OH.The relative configuration of pactamide B is by coupling constant (Z Δ2,3J2,311.5Hz；EΔ17,18J17,18
15.5Hz) related with NOESY belonging to.Related according to NOESY, H-5/H-13 is attributed to the same side, H-6/H-8/H-12/H-16
It is attributed to opposite side.By comparing the CD spectrum of pactamide B and lysobacteramide B, pactamide B is absolute
Configuration is defined as 5R, 6S, 8R, 12S, 13S, 16R, 23S.
Parker spy's acid amides C (pactamide C) structure elucidation (table 4-5, Figure 15,23-30)：
According to HRESIMS (m/z 479.2901 [M+H]+, calcd 479.2904) and will be true for the molecular formula of pactamide C
It is set to C29H38N2O4.The H NMR spectroscopy of pactamide C is similar to pactamide A's, is pushed away by carefully comparing the H NMR spectroscopy of the two
Survey pactamide C and lack a hexatomic ring, this supposition is confirmed by the NMR data ownership of pactamide C.
The relative configuration of pactamide C is by coupling constant (E Δ2,3J2,315.0Hz；EΔ4,5J4,514.5Hz；EΔ15,16J15, 1614.5Hz；EΔ17,18J17,1815.0Hz) related with NOESY belonging to.Related according to NOESY, H-11/H-13 is attributed to same
Side, H-6/H-8/H-10/H-12/H3-31 is attributed to opposite side.By comparing pactamide C and lysobacteramide
The CD spectrum of B, the absolute configuration of pactamide C is defined as 6S, 8S, 10S, 11R, 12R, 13S, 23S.
Parker spy's acid amides D (pactamide D) structure elucidation (table 6-7, Figure 15,31-37)：
According to HRESIMS (m/z 517.2665 [M+H]+, calcd 517.2678) and will be true for the molecular formula of pactamide D
It is set to C29H38N2O5.The H NMR spectroscopy of pactamide D is similar to pactamide B's, and unlike pactamide B,
The many sp of pactamide D2Methine (the δ of hydridizationH5.24br s,δC127.8, C-9), a few sp2The season of hydridization
Carbon.From H3- 30 and δH5.24 arriving δC144.0 HMBC related description C9/C10 in pactamide D is double bond.H-8/H-
Noesy correlation between 9, H-9/H-30, H-9/H-7 further demonstrate that above-mentioned ownership.The relative configuration of pactamide D by
Coupling constant (Z Δ2,3J2,311.0Hz；EΔ17,18J17,1814.5Hz) related with NOESY belonging to.It is related according to NOESY,
H-5/H-11/H-13 is attributed to the same side, and H-6/H-8/H-12/H-16 is attributed to opposite side.By compare pactamide D with
The CD spectrum of lysobacteramide B, the absolute configuration of pactamide D is defined as 5R, 6R, 8S, 12R, 13S, 16R,
23S.
Parker spy's acid amides E (pactamide E) structure elucidation (table 6-7, Figure 15,38-44)：
According to HRESIMS (m/z 499.2581 [M+Na]+, calcd 499.2657) and by the molecular formula of pactamide E
It is defined as C29H36N2O4.The H NMR spectroscopy of pactamide E is similar to pactamide C's, and unlike pactamide C,
Pactamide E has 12 olefinic carbons, and this explanation pactamide E only contains 5 yuan of rings.By belonging to pactamide E
2D H NMR spectroscopy confirm this supposition.The relative configuration of pactamide E is by coupling constant (E Δ2,3J2,315.0Hz；E
Δ4,5J4,514.5Hz；EΔ6,7J6,715.0Hz；EΔ13,14J13,1415Hz；EΔ15,16J15,1615.0Hz；EΔ17,18J17,18
14.5Hz) related with NOESY belonging to.Related according to NOESY, H-8/H-10/H-12/H3-31 is attributed to homonymy.Because
Pactamide E is the biosynthesis precursor of pactamied C, is pushed away the absolute configuration of pactamide E according to biosynthesis
Survey as 8S, 10S, 11R, 12S.
Parker spy's acid amides F (pactamide F) structure elucidation (table 6-7, Figure 45-51)：
The molecular formula of pactamide F is defined as by C according to HRESIMS29H39ClN2O5(m/z 531.2606[M+H]+,
calcd 531.2626).The mass spectrographic isotopic peak of pactamide F is consistent containing 1 chlorine atom with molecular formula.
The nuclear magnetic data of pactamide F is much like with the nuclear magnetic data of pactamide A, and different places is that pactamide F lacks
Few bimodal methyl, how a methylene is thus it is speculated that H in pactamide F3In -31,1 H is replaced by OH or Cl atom.OH-11
To C-10, the HMBC of C-12 is related to be positioned OH for C-11, and this illustrates H3H in -31 is replaced by Cl atom.pactamide F
Relative configuration by coupling constant (Z Δ2,3J2,310.6Hz；EΔ17,18J17, 18 15.2Hz) related with NOESY belonging to.Root
Related according to NOESY, H-5/H-13 is attributed to the same side, H-6/H-8/H-10/H-12/H-16/H3- 31 are attributed to opposite side.Logical
Cross the CD spectrum comparing pactamide F and lysobacteramide B, the absolute configuration of pactamide F be defined as 5R,
6S, 8R, 10S, 11S, 12S, 13S, 16R, 23S.
No.11, No.12, No.13 compound in table 4 and table 5 represents pactamide A, pactamide successively respectively
B、pactamide C.No.14, No.15, No.16 compound in table 6 and table 7 successively respectively represent pactamide D,
pactamide E、pactamide F.
Table 4pactamide A-C's1H (500MHz) NMR data
aMeasured in 90%CDCl3/methanol-d4；bmeasured in DMSO-d6.
Table 5pactamide A-C's13C (125MHz) NMR data
aMeasured in 90%CDCl3/methanol-d4；bmeasured in DMSO-d6.
Table 6pactamide D-F's1H NMR data
ameasured at 500MHz in DMSO-d6；bmeasured at 600MHz in DMSO-d6.
Table 7pactamide D-F's13C NMR data
ameasured at 500MHz in DMSO-d6；bmeasured at 600MHz in DMSO-d6.
Thereby determine that the structure of compound pactamide A-F is shown below：
9. the structure of cyclase PtmC vitro enzyme catalyst system and catalyzing
(the primer is to connect equimolecular biological method using round pcr, digestion：EptmCF/EptmCR, restricted
Restriction endonuclease is NdeI and BamHI；It is shown in Table 2), by ptmC gene cloning to coli expression carrier pET-28a, obtain cyclase
The expression vector pET-28a (pCSG3641) of PtmC.By the expression vector pET-28a (pCSG3641) of cyclase PtmC convert to
After abduction delivering in E.coli BL21 (DE3), collect cell, ultrasonication obtains crude enzyme liquid；Affinity chromatography purifying,
SDS-PAGE and Bradford method determination of protein concentration etc., final acquisition high-purity PtmC recombinase (Fig. 5).Every 100 μ l PtmC
The compound pactamide C (compound 13) that enzymatic system reaction system comprises 200 μm, 2mM NADPH, 10 μ g purify
PtmC recombinase.This reacts on 28 DEG C of incubations in the phosphate buffer of pH8.0 and completes for 10 hours.Successfully will be front using this system
Body compound 13 (pactamide C) is catalyzed into compound 11 (pactamide A) (Fig. 6).
The cytotoxic activity to tumour cell for the 10.pactamide A-F
The present inventor is found through experiments, and compound pactamide A F includes breast cancer MCF-7, people to human cancer cell
Liver cancer HepG2, human lung cancer H460 and human glioma cell SF-268 have cytotoxic activity (table 8), wherein pactamide A
Human cancer cell is included with breast cancer MCF-7, human lung cancer H460 cell, human liver cancer HepG2 and human glioma cell SF-268
IC50It is 0.26 μM, 0.24 μM, 0.37 μM and 0.51 μM respectively.
There is provided embodiment further below, these embodiments contribute to understanding the present invention, be used only as illustrating and do not limit
The range of application of the present invention processed.
Embodiment 1
The extraction of streptomycete (Streptomyces pactum) SCSIO 02999 STb gene：
The mycelium of fresh streptomycete (Streptomyces pactum) SCSIO 02999 is connect according to 5% inoculum concentration
Plant in TSB culture medium (tryptone 17g, phytone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, the glucose of 50mL
2.5g, adds water to 1L, pH 7.2-7.4) in, 28-30 DEG C, shaken cultivation about 3-4 days, 4000rpm is centrifuged 10 minutes and collects mycelia
Body.Mycelium is washed twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), the mycelium to after washing
Middle addition 30mL STE solution and the lysozyme of final concentration 3mg/mL, are vortexed uniformly, and 37 DEG C of temperature are bathed 3 hours, add to final concentration
The Proteinase K of 0.1-0.2mg/mL, mixes, and 37 DEG C of temperature are bathed 10 minutes, adds to the SDS of final concentration 1-2%, mixes, put into 55
DEG C water-bath about 1 hour, period overturns for several times.Add isopyknic phenol-chloroform-isoamyl alcohol (V/V/V=25:24:1), mix all
Even, it is placed in cooled on ice 30 minutes.12000rpm, 4 DEG C are centrifuged 10 minutes, and then the heavy caliber pipette tips with cutting are careful draws
Clear repeatedly process 3 times with same method in new centrifuge tube, then with isopyknic chloroform twice, 12000rpm,
4 DEG C are centrifuged 10 minutes.With the heavy caliber pipette tips cut, aqueous phase is suctioned out and be transferred to new centrifuge tube, add 1/10 volume 3mol/L
NaAc (pH5.2), adds isopyknic isopropanol, places on ice after mixing after mixing, precipitate DNA.Carefully use glass bar
DNA fiber group is transferred in new centrifuge tube, is washed twice with 70% ethanol, liquid is poured out, slightly dry at 37 DEG C,
Plus 5mL TE dissolving, and add the RNase of 3-5U.Thus obtain streptomycete (Streptomyces pactum) SCSIO 02999
STb gene.
Embodiment 2
The foundation in streptomycete (Streptomyces pactum) SCSIO 02999 full-length genome library：
First pass through a series of dilution experiment to determine the consumption of restriction endonuclease Sau3A I, in 20 μ L bodies
In system, the streptomycete containing 17 μ L (Streptomyces pactum) SCSIO 02999 STb gene, 10 × reaction buffering of 2 μ L
Liquid and the different dilution Sau3A I of 1 μ L, its terminating reaction is 4 μ L 0.5mol/L EDTA and suitable sample-loading buffer.Logical
Cross grope to determine 0.025-0.05U enzyme-activity unit proper.On this basis pass through in a large number partially digested obtain bigger
In the genomic DNA fragment of 40kb, spend phosphorylase and carry out dephosphorylation process.
Carrier S uperCos l plasmid for building library first uses restriction endonuclease Xba I from two cos sequences
Cut in the middle of row, then carry out dephosphorylation process, then cut with restriction endonuclease Bam HI at MCS,
Obtain two arms.Carrier after process is connected overnight with the genomic DNA fragment of the partially digested about 40kb preparing before, even
Junctor system be 10 μ L, containing 1.25 μ g preparation genomic DNA and 0.5 μ g process after SuperCos 1 plasmid, 1 μ L 10 ×
The ligase of Buffer, 0.3U.Connection product and 65 DEG C are processed 15 minutes, so that ligase is inactivated.One is taken out from -80 DEG C of refrigerators
Tube packaging mixture (50 μ L) is placed on ice, and package combination is melted rapidly between referring to, careful absorption half package combination
(25 μ L), to a new centrifuge tube, adds the connection product after 10 μ L heat treatments, and remaining package combination is protected in -80 DEG C
Deposit.Careful mixing, 30 DEG C of temperature are bathed 90 minutes, add other half package combination (25 μ L), and 30 DEG C of temperature are bathed and continued 90 minutes.Plus
Enter 500 μ L bacteriophage dilution buffer (100mmol/L NaCl, 10mmol/L MgCl2,10mmol/L pH 8.3Tris-
HCl), add 25 μ L chloroforms, gently mix, preserve in 4 DEG C.
The frozen bacterial strain E.coli LM392MP in -80 DEG C is coated on recovery on LB culture medium.Packaging reaction the previous day,
Picking monoclonal is inoculated in LB culture medium and (adds 0.2% maltose and 10mM MgSO4), 37 DEG C of shaken cultivation overnight, are packed
On the reaction same day, take the bacterium solution of 5mL incubated overnight to be added in the fresh LB culture medium of 50mL and (add 0.2% maltose and 10mM
MgSO4), 37 DEG C, 200rpm vibrates to culture OD600When reaching 0.8-1,4 DEG C save backup.Take the place that 100 μ L are processed as above
The packaging liquid of main bacterium solution and 100 μ L appropriateness dilution gently mixes, and in 37 DEG C of temperature baths 15 minutes, then coats containing 100 μ g/mL
On the LB flat board of ampicillin and 50 μ g/mL kanamycins, 37 DEG C of overnight incubation.By longer single clone, with no
Bacterium toothpick dibbling in 25 pieces contain suitable antibiotic LB 96 orifice plates on, 37 DEG C of overnight incubation, add final concentration of 20% sweet
Oil, mixes, and is placed in -80 DEG C of preservations.Thus obtain streptomycete (Streptomyces pactum) the full base of SCSIO 02999
Because organizing library.
Embodiment 3：The determination of cytotoxic activity of pactamide A-F
Pactamide A-F is directed to four kinds of tumor cell lines：MCF-7 Human Breast Cancer Cells, human liver cancer cell HepG2, people
Lung cancer H460 cell and human glioma cell SF-268 have carried out determination of activity, experimental technique bibliography [Wu, Z.C.；
Li,D.L.；Chen,Y.C.；Zhang,W.M.,A new isofuranonaphthalenone and benzopyrans
from the endophytic fungus Nodulisporium sp.A4from Aquilaria
Sinensis.Helv.Chim.Acta 2010,93, (5), 920-924.], using cis-platinum (Cisplatin) as comparison, it is right
Human cancer cell includes breast cancer MCF-7, human lung cancer H460 cell, human liver cancer HepG2 and human glioma cell SF-268's
IC50It is 9.2 μM, 1.5 μM, 1.4 μM and 3.9 μM respectively.Result shows that pactamide A includes breast cancer MCF- to human cancer cell
7th, the IC of human lung cancer H460 cell, human liver cancer HepG2 and human glioma cell SF-268500.26 μM respectively, 0.24 μM,
0.37 μM and 0.51 μM；Pactamide B includes breast cancer MCF-7, human lung cancer H460 cell, human liver cancer to human cancer cell
The HepG2 and IC of human glioma cell SF-26850It is 24.4 μM, 21.9 μM, 26.1 μM and 25.5 μM respectively；
Pactamide C includes breast cancer MCF-7, human lung cancer H460 cell, human liver cancer HepG2 and human glioma to human cancer cell
The IC of cell SF-26850It is 0.71 μM, 0.74 μM, 1.71 μM and 2.42 μM respectively；Pactamide D includes breast to human cancer cell
Gland cancer MCF-7, the IC of human lung cancer H460 cell, human liver cancer HepG2 and human glioma cell SF-26850It is 14.56 μ respectively
M, 17.4 μM, 17.2 μM and 19.2 μM；Pactamide E human cancer cell is included breast cancer MCF-7, human lung cancer H460 cell,
Human liver cancer HepG2 and the IC of human glioma cell SF-26850It is 5.1 μM, 5.2 μM, 6.8 μM and 8.7 μM respectively；
Pactamide F includes breast cancer MCF-7, human lung cancer H460 cell, human liver cancer HepG2 and human glioma to human cancer cell
The IC of cell SF-26850It is 2.66 μM, 2.85 μM, 2.66 μM and 2.65 μM respectively；Pactamide A-F is thin to four kinds being surveyed
Born of the same parents are presented with cytotoxic activity, are expected to be developed into anti-cancer agent by bioengineering or chemical modification.
The cytotoxic activity of table 8pactamide A-F
Claims (14)
1. a kind of Parker spy acid amides biological synthesis gene cluster it is characterised in that its nucleotide sequence such as SEQ ID NO.1 the
Shown in the base sequence of 14290-29055 position.
2. the biological synthesis gene cluster of the Parker's spy's acid amides described in claim 1 is in preparation Parker's spy's acid amides or its analog
Application.
3. application according to claim 2 is it is characterised in that be in preparing the arbitrary compound of pactamide A-F
Application,
4. a kind of cyclase gene ptmC is it is characterised in that the 28000-29055 of its nucleotide sequence such as SEQ ID NO.1
Shown in the base sequence of position.
5. the cyclase PtmC of the cyclase gene ptmC coding described in a kind of claim 4.
6. the cyclase PtmC described in claim 5 becomes in compound pactamide A in catalytic cpd pactamide C-shaped
Application,
7. the promoter engineered mutant of biological synthesis gene cluster of Parker's spy's acid amides described in a kind of claim 1 and its and right
The cyclase gene ptmC deletion mutation described in 4, oxide-reductase gene ptmB1 deletion mutation or phytoene is required to take off
The genetic engineering bacterium of hydrogenase gene ptmB2 deletion mutation；Described oxide-reductase gene ptmB1, its nucleotide sequence such as SEQ
Shown in the base sequence of 24663-26348 position of ID NO.1；Described Phytoene dehydrogenase gene ptmB2, its core
Shown in the base sequence of the 26360-28003 position as SEQ ID NO.1 for the nucleotide sequence.
8. genetic engineering bacterium according to claim 7 is it is characterised in that the biosynthesis gene of described Parker spy's acid amides
The genetic engineering bacterium of the promoter engineered mutant of cluster is Streptomyces pactum SCSIO 02999PTMp1.
9. application in preparation Parker's spy's acid amides or its analog for the genetic engineering bacterium described in claim 7.
10. compound pactamide A, B, C, D, E or F,
Application in preparing antineoplastic for compound pactamide A, B, C, D, E or the F described in 11. claims 10.
12. applications according to claim 11 it is characterised in that described antineoplastic be anti-breast cancer, human lung cancer,
Human liver cancer or the medicine of human glioma.
A kind of 13. antineoplastics are it is characterised in that comprise the compound pactamide described in claim 10 of effective dose
A, B, C, D, E or F are as active ingredient.
14. antineoplastics according to claim 13 it is characterised in that described antineoplastic be anti-breast cancer,
The medicine of human lung cancer, human liver cancer or human glioma.
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| CN107794286A (en) * | 2017-10-09 | 2018-03-13 | 中国科学院南海海洋研究所 | A kind of cyclic lipopeptide compound biological synthesis gene cluster and its Activiation method and application |
| CN108623607A (en) * | 2017-03-24 | 2018-10-09 | 中国海洋大学 | Polycyclic compounds of macrolactams containing tetramic acid of 5,5,6- and its preparation method and application |
| CN109943493A (en) * | 2019-04-17 | 2019-06-28 | 天津大学 | Realize the mutant strain and its construction method of general enzymatic functional diversity |
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| CN108623607A (en) * | 2017-03-24 | 2018-10-09 | 中国海洋大学 | Polycyclic compounds of macrolactams containing tetramic acid of 5,5,6- and its preparation method and application |
| CN108623607B (en) * | 2017-03-24 | 2021-04-27 | 中国海洋大学 | 5,5, 6-polycyclic tetramic acid-containing macrocyclic lactam compound and preparation method and application thereof |
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| CN110305881B (en) * | 2019-04-16 | 2021-04-02 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of polyketide neoenterocins and application thereof |
| CN109943493A (en) * | 2019-04-17 | 2019-06-28 | 天津大学 | Realize the mutant strain and its construction method of general enzymatic functional diversity |
| CN110642863A (en) * | 2019-09-23 | 2020-01-03 | 中国科学院南海海洋研究所 | 5,5,6 type PTM compound and preparation method and application thereof |
| CN111253408A (en) * | 2020-02-11 | 2020-06-09 | 中国科学院南海海洋研究所 | Antibiotic pactamide G, preparation method thereof and application thereof in preparation of antibacterial drugs |
| CN114540398A (en) * | 2022-03-17 | 2022-05-27 | 浙江大学 | Method for screening streptomycete antibiotic related regulatory gene |
| CN114540398B (en) * | 2022-03-17 | 2023-10-24 | 浙江大学 | Streptomyces antibiotic related regulation gene screening method |
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