CN106188093B - A kind of rapamycin structure analog and preparation method thereof - Google Patents

Abstract

The present invention provides a kind of rapamycin structure analogs and preparation method thereof.The rapamycin structure analog is as shown in Equation 1.The preparation method includes：Fermented and cultured contains the actinoplanes of polyketide synthase rapA mutators, is obtained from zymotic fluid；The polyketide synthase rapA mutators are as shown in sequence table SEQ ID No.1.Rapamycin structure method for preparing analogue provided by the invention operates simpler compared with existing chemical synthesis, and cost is lower, and environmental pollution is smaller, and has good anti-mycotic efficiency by the rapamycin structure analog obtained by the preparation method.

Description

A kind of rapamycin structure analog and preparation method thereof

Technical field

The present invention relates to Combinatorial biosynthesis fields, and in particular to a kind of rapamycin structure analog and its preparation side Method.

Background technology

Rapamycin is a kind of nitrogenous triene macrolide class compound of 31 round rings, be clinically apply it is important immune Inhibitor and the intermediate of antitumor drug research and development.Temsirolimus, everolimus and zotarolimus are semi-synthetic Rapamycin structure analog, respectively at 2004,2005,2007 by FDA approvals as antitumor drug and angiocarpy Bracket coating drug list marketing.

In natural products, except a part of combound itself can be largely to pass through base as lead compound with patent medicine Group's modification, is transformed its patent medicine property, becomes semisynthetic drug and is applied to clinic.For most of complicated native compound Chemical synthesis or unit structure modification are carried out, is faced with many technological difficulties such as alloisomerism and selectivity.And synthesis is transformed The microbial hosts genome of compound, using the analog of the ability production structural modification of its own biosynthesis, from certain The technical bottleneck of chemical synthesis is breached in degree.It is allowed to low cost, environmental-friendly mode realizes that chemical synthesising technology can not The biosynthesis of the labyrinth compound of realization is possibly realized.

The analogue study on the synthesis mode of rapamycin mainly also be mutated synthesis, precursor guidance biosynthesis and Chemistry becomes master.

Mutation synthesis (mutasynthesis)：L-proline and analogue can be replaced needed for rapamycin synthesis Natural precursor L-pipecolate incorporation polyketone chains, generate the novel compounds such as prolylrapamycin.The L- of sulfur-bearing The analog of pipecolate can also be used as precursor and be utilized by non-ribosomal peptide synthetase rapP to spread out so as to generate rapamycin Biology

The biosynthesis (Precursor directed biosynthesis) of precursor guidance：The polyketone of rapamycin closes The load-on module of enzyme A has the higher wide in range property of substrate, and the analog of different DHCHC can be added in molecule and generates thunder The analogue of pa mycin.42 carbon atoms of rapamycin loading molecular are a valuable modification positions The analogue of the rapamycin of point, so far FDA approval listing is the different modifying product in the site without exception.

Chemical synthesis：Rapamycin and mTOR binding sites are also the target spot modified.Existed by chemical synthesis process The compound WYE-592 and ILS-920 that the modification of rapamycin triolefin position obtains, due to destroying the combination knot with mTOR Structure, therefore the immune suppression function of both compounds is substantially reduced, and enhance with the binding ability of FKBP52, it shows significantly Neuroprotection (neuroprotective activities) activity.

And the rear modification enzyme synthesized by knocking out the rapamycin of streptomyces hygroscopicus, also obtain a series of incomplete methyl The compound of change.

Chemical synthesis is carried out to rapamycin structure analog at present or unit structure modification all suffers from alloisomerism The problem of with many technological difficulties such as selectivity, therefore there is an urgent need for a kind of simple for process, low cost, environmental-friendly synthetic method is come Synthesize the analogue of rapamycin.

Invention content

The technical problems to be solved by the invention are to carry out chemical synthesis or base to forms of rapamycin analogs to overcome Group's structural modification is faced with the problem of many technological difficulties such as alloisomerism and selectivity, and thunder can be produced by providing a kind of utilization Microorganism actinoplanes (Actinoplanes sp.N902-109) the own biological synthesis capability of pa mycin produces thunder pa The method of mycin analogue, and pass through this method and a kind of rapamycin structure analog has been prepared.The present invention provides Preparation method operated compared with existing chemical synthesis simpler, cost is lower, and environmental pollution is smaller, and passes through the system Rapamycin structure analog obtained by Preparation Method has good anti-mycotic efficiency.

One of technical solution of the present invention：Structure bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- as shown in Equation 1：

The two of technical solution of the present invention：A kind of preparation side of bis- dehydrogenation -27-O- demethyl rapamycins of aforementioned 35,36- Method includes the following steps：Fermented and cultured contains the actinoplanes N902-109 of polyketide synthase rapA mutators, from fermentation It is obtained in liquid；The polyketide synthase rapA mutators are as shown in sequence table SEQ ID No.1.

In the present invention, the polyketide synthase rapA mutators can come from any source, including being obtained from internal separation It is obtained with synthesis.The preparation method of the polyketide synthase rapA mutators is this field conventional method, and preferably PCR distinguishes It is reconnected with nucleic acid cleavage after expanding the segment of the upstream and downstream in the mutational site of the mutator to obtain the final product.The nucleic acid Enzyme is this field conventional nucleic acid enzyme, preferably restriction endonuclease, such as Nhe I.The ligase is conventional for this field Ligase is more preferably T4 DNA ligases.The preparation side of the actinoplanes containing polyketide synthase rapA mutators Method can be the operation of this field routine, can preferably include the following steps：(1) the polyketide synthase rapA will be contained to be mutated The genetic fragment of gene mutation site is connected on carrier, and the recombination containing the polyketide synthase rapA mutated gene segments is made Carrier；The sequence of the genetic fragment containing polyketide synthase rapA mutators mutational site is in SEQ ID No.1 sequences The 5736th~the 10693rd；(2) step (1) described recombinant vector is transformed into donor bacterium, donor bacterium must be converted；(3) will Step (2) the conversion donor bacterium engages with the mycelia of the actinoplanes N902-109, selects joint element；(4) will contain The donor bacterium of Homing endonucleases expression vector mixes with step (3) described joint element, selects with step (1) mutation The double crossing over joint element in site.

Step (1) described carrier can be this field routine carrier, including all kinds of prokaryotic vectors and eukaryotic vector, preferably It is more preferably pLYZL102 carriers for prokaryotic vector.The recombinant vector can be made by this field conventional method, preferably Genetic fragment containing the polyketide synthase rapA mutators mutational site can be connected to all kinds of conventional carriers in this field On obtain.

In step (2), the donor bacterium can be this field routine donor bacterium, preferably Escherichia coli, more preferably for Escherichia coli ET12567 (pUZ8002) bacterial strain.The conversion can be this field conventional transformation methods, and preferably electricity converts Method.

In step (3), engagement transport medium culture, the engagement transfer culture can also be preferably used after the engagement Base can be the culture medium of this field routine, be preferably comprised following components：0.5% beancake powder, 0.5% mannitol, 0.5% can Soluble starch, 0.2% tryptone, 0.1% yeast extract, 0.1%NaCl, 0.2% (NH4)₂SO₄, 0.1%K₂HPO₃, 0.2%CaCO₃, 0.2%MgCl₂, 2% agar powder, 0.0001%ZnSO₄, 0.0001%FeSO₄, 0.0001%MnSO₄With benefit extremely 100% water, the percentage account for the quality percent by volume of the engagement transport medium volume for each component quality.

In step (4), the Homing endonucleases can be the enzyme of this field routine, preferably I-SceI.The donor Bacterium can be this field conventional bacterial classification, preferably Escherichia coli, be more preferably Escherichia coli ET12567 (pUZ8002) bacterial strain. It is described to select the conventional method that double crossing over joint element be this field, preferably selected using antibiotic pressure.More Goodly to be selected using apramycin pressure, such as by the expression vector of I-SceI genes driven containing apramycin promoter Escherichia coli mixed with step (3) described joint element, culture pass 2 generations after select apramycin resistance loss double crossing over engagement Son.

In the present invention, the fermentation includes the following steps：The aforementioned trip containing polyketide synthase rapA mutators will be contained The seed culture medium of dynamic actinomyces N902-109 is connected in fermentation medium, 28 DEG C of 220 revs/min of constant temperature incubations 9 days.It is described containing The inoculum concentration of the seed culture medium of the actinoplanes N902-109 of polyketide synthase rapA mutators can be that this field is conventional Inoculum concentration, preferably 5-15%, the percentage is the percent by volume for accounting for the fermentation medium volume.It is described containing The seed culture medium of the actinoplanes N902-109 of polyketide synthase rapA mutators can pass through this field conventional method system , it can be preferably made by following step：(1) actinoplanes containing polyketide synthase rapA mutators are inoculated with It ferments slant medium to actinoplanes, coating is uniform, 28 DEG C of constant temperature incubations 7-9 days；(2) scraping inclined-plane mycelium access one In grade seed culture medium, 28 DEG C, 220 turns of constant temperature incubations 4-5 days；(3) step (2) described primary-seed medium is taken to be inoculated into two In grade seed culture medium, 28 DEG C of 220 revs/min of constant temperature incubations 3-4 days to obtain the final product.The actinoplanes fermentation slant medium is this Field is conventional, is preferably comprised following components：Glucose 2%, soluble starch 4%, yeast extract 0.1-0.2%, enzyme water Solve casein 0.5%, CaCO₃It 0.1% and agar 1.6% and mends to 100% water, the percentage accounts for institute for each component quality State the quality percent by volume of actinoplanes fermentation slant medium total volume.The actinoplanes fermentation slant medium Preparation method can be this field conventional method, be preferably comprised following step：The raw material components are mixed, adjust pH extremely 7.0~7.2, sterilizing.The sterilizing can be this field conventional sterilization procedures, preferably 115 DEG C~121 DEG C sterilizing 15min ~20min is more preferably 115 DEG C of sterilizing 15min.The primary-seed medium can be this field conventional medium, preferably Ground includes following components：Soluble starch 3%, glucose 2%, dry ferment 0.5%, Dried Corn Steep Liquor Powder 0.5%, peanut meal 0.5%th, CaCO₃It 0.2% and mends to 100% water, the percentage accounts for first order seed training for the quality of each component Support the quality percent by volume of base total volume.The preparation method of the primary-seed medium can be this field conventional method, It is preferably comprised following step：The each component is mixed, adjusts pH to 7.0, sterilizing.The sterilizing can be that this field is routinely gone out Bacterium method, preferably 115 DEG C~121 DEG C sterilizing 15min~20min are more preferably 115 DEG C of sterilizing 15min.The two level kind Sub- culture medium can be this field conventional medium, be preferably comprised following components：Soluble starch 3%, glucose 4%, jade Rice ＆ peanut milk dry powder 0.8%, cottonseed protein powder 0.8%, CaCO₃0.3%th, bubble enemy 0.2%, MgSO₄.7H₂O 0.00025%, CoCl₂.6H₂O 0.0001%, VB₁0.02%th, VB₁₂0.00002%th, folic acid 0.0025% and the water of benefit to 100%, described hundred Divide the quality percent by volume than accounting for the secondary seed medium total volume for the quality of each component.The secondary seed The preparation method of culture medium can be this field conventional method, be preferably comprised following step：The each component is mixed, adjusts pH To 6.9, sterilize.The sterilizing is this field conventional sterilization procedures, preferably 115 DEG C~121 DEG C sterilizing 15min~20min, It is more preferably 115 DEG C of sterilizing 15min.In step (3), the inoculum concentration of the primary-seed medium can be conventional inoculation Amount, preferably 5-10%, the percentage is the percent by volume for accounting for the secondary seed medium volume.The fermentation training Foster base can be the culture medium of this field routine, preferably step (3) described secondary seed medium.

It is described to be obtained from zymotic fluid under 35,36-, the bis- dehydrogenation -27-O- demethyl rapamycins include in the present invention State step：It is extracted from the zymotic fluid with acetone and obtains 35,36-, the bis- dehydrogenation -27-O- demethyl rapamycins, it is upper big It is eluted after the resin of hole, must concentrate eluted product after reduced pressure, extract to obtain 35,36-, the bis- dehydrogenation -27-O- demethyl thunder pas Mycin crude extract.The reduced pressure can be the operation of this field routine, and the temperature of the reduced pressure is preferably 35 DEG C. The extraction can be the operation of this field routine, and the extractant used that extracts can be this field conventional extraction agent, compared with It is ethyl acetate goodly.The number of the extraction can be conventional number, preferably 2 times.The extractant with it is described dense The volume ratio of contracting eluted product can be conventional ratio, preferably 1: 1.

The present invention can also preferably include the following steps：The anhydrous sodium sulfate dehydration of addition 5% after the extraction, 35 DEG C It is concentrated under reduced pressure.Bis- dehydrogenation -27-O- demethyl rapamycin crude extracts of 35,36-.

The present invention can also preferably include isolating and purifying the bis- dehydrogenation -27-O- demethyl thunder pas of 35,36- with silica gel Mycin crude extract, includes the following steps：Dissolve 35,36-, the bis- dehydrogenation -27-O- demethyl rapamycin crude extracts, loading In silica gel, 35,36-, the bis- dehydrogenation -27-O- demethyl rapamycins for eluting after purification.The silica gel can be that this field is normal Advise silica gel, preferably 300-400 mesh silica gel.The dissolving bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- slightly carry The solvent of object can be the solvent of this field routine, and preferably volume ratio is 1: 1 dichloromethane and hexamethylene.The elution The solvent used can be the solvent of this field routine, and preferably volume ratio is 10: 1~20: 1 hexamethylene and acetone.

The three of technical solution of the present invention：Bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- are anti-true in preparation as previously described Application in bacteria composition.

In the present invention, the antifungal medicine composition can be the conventionally referred reagent in this field, preferably can be with Inhibit the composition of the black mould caused by aspergillus niger or the composition of the disease caused by Candida albicans can be treated, it is described can It can preferably be treated by the microbial skin thought of Candida albicans with treating the composition of the disease caused by Candida albicans Pearl bacterium disease, candidiasis of the mucous membranes, internal organ candidiasis, candidid and immune deficiency disorder composition.

On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is：The present invention provides a kind of Combinatorial biosynthesis methods to synthesize rapamycin The approach of analogue, this method is simple for process, and with low cost, environmental-friendly mode realizes chemical synthesising technology can not The biosynthesis of the labyrinth compound of realization, and synthesized rapamycin structure analog is with antimycotic well Effect.

Description of the drawings

Fig. 1：Actinoplanes rapA gene functions domain arranges and mutational site design diagram.

Fig. 2：Actinoplanes N902-109 single-swap recombinant clone PCR schematic diagrames.SC1 and SC2 represents two lists respectively Exchange clone；AC：Actinoplanes N902-109.

Fig. 3 is：Actinoplanes N902-109 double exchange reorganizations clone PCR and PCR product cleavage map.A1-A7：Double crossing over Bacterial strain；AC：Actinoplanes N902-109.

Fig. 4：Rapamycin and its analogue zymotic fluid HPLC separating spectrums.

Fig. 5：Rapamycin and and its analogue UV absorption peak figure.

Fig. 6：Actinoplanes rapA gene ER functional domain point mutation plasmid pLYERIA schematic diagrames.

Fig. 7：The structure flow diagram of plasmid pLYERIA.

Fig. 8：The structure flow diagram of plasmid pSETSI.

Specific embodiment

It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.Test method without specific conditions in the following example, according to conventional methods and conditions or according to quotient Product specification selects.

Reagent and key instrument used in the present invention：

Restriction enzyme is purchased from Thermo Scientific. ligase ligation solution I, T4 DNApolymerase, restriction enzyme pshAI, 10mM dNTP are purchased from TaKaRa companies.PACK4aPCR segment cloning vectors Purchased from Suzhou Pan Gu's gene Co., Ltd.High-fidelity DNA polymerase Neo KOD-plus are purchased from Toyobo companies.Apramycin is purchased From DUCHEFA companies.Nalidixic acid, spectinomycin are purchased from Sheng Gong bio-engineering corporations.Plasmid extraction and Ago-Gel return It receives kit and is purchased from Axygen companies.Easytaq is purchased from Quan Shijin biotech firms.PCR instrument is Biometra Tprofessional TRIO, centrifuge are Eppendorf 5424 and 5415R, and HPLC chromatogram instrument is Agilent Technologies 1200series type high performance liquid chromatographs, mass spectrograph are Waters companies Waters Q-TOF Micromass mass spectrographs, Nuclear Magnetic Resonance be Inova-400 Nuclear Magnetic Resonance, chromatographic column：Waters NaVa-PaK C8 columns, Specification：(3.9mm × 150mm, 5 μm)；HF254 high-efficient silica gels plate is purchased from Yantai Jiang You silica gel development corporation, Ltd..Other reagents It is that domestic analysis is pure, purchased from Chinese medicines group.Primer synthesis commission Nanjing Jin Sirui Bioisystech Co., Ltd, gene sequencing clothes Business is provided by Shanghai Jie Li biotechnologies company.

Actinoplanes N902-109 used in the present invention sees below patent document：United States Patent (USP) (US5674732), institute Candida albicans (Candida albicans), saccharomyces cerevisiae (Saccharomyces cerevisiae) and the Penicillium notatum used (Penicillium Sp.) is purchased from U.S. ATCC, and aspergillus niger (Aspergillus niger) is purchased from CGMCC, Candida albicans Preserving number for ATCC11651, the preserving number of saccharomyces cerevisiae is ATCC204508, and the preserving number of Penicillium notatum is ATCC32029, black The preserving number of aspergillus is CGMCC3.3928.Used Escherichia coli ET12567 (pUZ8002) bacterial strain sees below document： Macneil DJ,Gewain KM,Ruby CL,Dezeny G,Gibbons PH,Macneil T.Analysis of Streptomyces avermitilis genes required for Avermectin biosynthesis utilizing a novel integration vector.Gene.1992,111(1):61-68；Used plasmid origin is as follows： PSET152 sees below bibliography：Bierman M,Logan R,Obrien K,Seno ET,Rao RN,Schoner BE: Plasmid Cloning Vectors for the Conjugal Transfer of DNA from Escherichia.coli to Streptomyces Spp.Gene 1992,116(1):43-49；PSETspc is by pSET152 Transformation obtains, and sees Fig. 8；PSETSI is transformed to obtain by pSET152, sees Fig. 8；PLYZL102 sees below bibliography：Shen Y, Huang H,Zhu L,Luo M,Chen D.Type II thioesterase gene(ECO-orf27)from Amycolatopsis orientalis influences production of the polyketide antibiotic, ECO-0501(LW01).Biotechnology Letters.2012,34(11):2087-2091.；PLYERIA is by pLYZL102 Transformation obtains, and sees Fig. 7；PLU101 sees below bibliography：Lu Z,Xie P,Qin Z:Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor(dagger).Acta Biochimica Et Biophysica Sinica 2010,42(10):717-721； PIJ773 and pIJ778 see below bibliography：Gust B,Challis GL,Fowler K,Kieser T,Chater KF: PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.Proceedings of the National Academy of Sciences of the United States of America2003,100(4):1541- 1546；PACK4aBS is purchased from Pan Gu's gene (Suzhou) Science and Technology Ltd..

Embodiment 1

The functional domain structural analysis of actinoplanes polyketide synthase rapA.

Utilize https://nrps.igs.umaryland.edu/nrps/_ carries out the prediction of polyketide synthase rapA functional domains, The results are shown in Table 1：

Table 1

RapA genes contain a load-on module and four chain extension modules, arrangement schematic diagram such as Fig. 1 of each module It is shown.Act_rapA refers to actinoplanes rapA genes, and CL is carboxylic acid ligase, and AT is acyltransferase functional domain, and KS is ketone Base synthetase functional domain, DH be dehydrogenase function domain, ER be enoyl reductase functional domain, KR be keto reductase functional domain, ACP For acyl carrier protein functional domain.

Embodiment 2

The acquisition of actinoplanes rapA ER functional domain point mutation homologous double-crossover bacterial strains.

The schematic diagram of actinoplanes rapA ER functional domain point mutation is shown in Fig. 1.Pass through two couples of primer ERIAUs-ERIAUas Expand two of actinoplanes rapA gene ER functional domains mutational site upstream and downstream respectively with ERIADAs-ERIADAas The restriction enzyme NheI included in segment, then the mutational site by being introduced into connects two segments.

A) the extraction of actinoplanes N902-109 genomes：

A small amount of actinoplanes N902-109 mycelium is taken to be inoculated in the test tube of the culture mediums of YEME containing 5mL, are shaken in 30 DEG C Swing culture about 72 hours.Mycelium is collected by centrifugation in 3500rpm, with TES (10mM Tris-HCl pH7.5, EDTA 1mM, NaCl It 50mM) washes twice.1mL TES (lysozyme final concentration 5mg/mL) are added in into the mycelium of collection, are vortexed to uniform, 37 DEG C Water-bath 60 minutes, adds in Proteinase K (final concentration reaches 0.1mg/mL), and 0.1mL 10%SDS are put into rapidly 55 DEG C of water after mixing Bath 60 minutes treats that solution is clarified.Cooled on ice is placed in, adds in 0.25mL 5M KAc, cooled on ice 15 minutes.Add in 0.5mL Phenol：Chloroform (Tris saturated phenols pH7.5：Chloroform：Isoamyl alcohol is volume ratio 25：24：1), gentle inversion mixing, in 4 DEG C 12000rpm is centrifuged 10 minutes.Water phase suction is transferred to new centrifuge tube with the 1mL pipette tips of clip, equal amounts of chloroform is added to extract, 12000rpm, 4 DEG C centrifuge 10 minutes, and water phase suction is transferred to new centrifuge tube with the 1mL pipette tips of clip, adds in 0.1 volume 3M NaAc, the absolute ethyl alcohol of two volumes, there is cotton-shaped DNA in mixing.It is ticked as in new centrifuge tube, is added in The ethyl alcohol washing of 0.5mL 70%, with rifle exhaustion liquid, the slightly dry rear 300 μ l TE that add in dissolve, and add in RNA enzyme (final concentration of 50 μ g/mL), 37 DEG C of water-baths 30 minutes.

B) the structure of enoyl reductase functional domain rite-directed mutagenesis plasmid pLYERIA：

Using actinoplanes N902-109 genomes as template, with primer ERIADAas (HindIII) and ERIADAs (NheI), ERIAUAs (XbaI) and ERIAUAas (NheI), carry out respectively height can property degree expand to obtain length as 2846bp, Homology arm and upper homology arm segment under the ER functional domains of 2142bp.Reaction system (50 μ l) includes：5μl 10×KOD NEO Plus buffer solutions, 3 μ l 25mM MgCl₂, 1.5 μ l 2.5mmol/L dNTP solution, 2.5 μ l DMSO, 30pmol P1, 30pmol P2,1U KOD NEO plus archaeal dna polymerases, 50ng genomic DNAs add distilled water to 50 μ l.PCR response procedures For：95 DEG C of 5min, 30 cycles (94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 120s), 72 DEG C of 10min.PCR product after purification, takes respectively 2 μ l are mixed with 0.5 μ l pACK4aBS carriers, and 37 DEG C of placement 15min, conversion escherichia coli DH5a obtains pACK4a-ERIADA, PACK4a-ERIAUA plasmids.With NheI, HindIII digestions pACK4a-ERIADA obtains ERIADA segments, with NheI, XbaI enzymes It cuts pACK4a-ERIAUA and obtains ERIAUA segments, with HindIII, XbaI enzyme cutting pLYZL102 obtains carrier segments, three's segment Connection obtains pLYERIA plasmids, and flow chart is shown in Fig. 7, and pLYERIA plasmid maps are shown in Fig. 6.

C) actinoplanes N902-109 engages transfer with Escherichia coli:

I) acquisition of actinoplanes rapA genes ER functional domains homologous single-crossover bacterial strain

Prepare actinoplanes mycelium suspension：3 μ l actinoplanes glycerol stocks liquid is taken to be coated with I-Isp2 solid cultures Base.30 DEG C are cultivated 120 hours or so.Surface thalline is scraped with sterile scraper, is washed down and passed through in two times with 15-20mL sterile waters Bead shakes 6-8 times on vortex oscillator, 10 seconds every time, by suspension by being equipped with the asepsis injector needle of cotton plug Cylinder, filter liquor discard supernatant by 7000 revs/min of centrifugation 10min, are resuspended to obtain mycelium with appropriate 0.4-1.0mL sterile waters Suspension.

From picking ET12567 on LB tablets (50 μ g/mL kanamycins, 50 μ g/mL apramycins, 25 μ g/mL chloramphenicol) (pUZ8002) single bacterium colony, 3mL LB fluid nutrient medium of the inoculation containing ibid antibiotic, overnight incubation.Next day is with 0.5% (v/ V) containing in the ibid 10mL LB triangular flasks of three kinds of antibiotic of concentration, 37 DEG C are cultivated to OD 0.4-0.6 for inoculum concentration access, 4500rpm 5min, 4 DEG C thalline were collected by centrifugation, abandons supernatant, is washed twice with sterile 10% glycerine of same volume, is resuspended in about 500 μ In 10% glycerine of L and be distributed into 50ul often manage it is for use as ET12567/pUZ8002 electricity transformed competence colibacillus.By 100ng PLYERIA plasmids are added in ET12567/pUZ8002 cells, voltage 2.5kv, 200 ohm of resistance, electroporated, are added in The sterile cold LB of 1.0ml are resuspended and 37 DEG C of culture 1hr, apply LB tablets (50 μ g/mL kanamycins, 50 μ g/mL apramycins, 25 μ g/ ML chloramphenicol), the pLYERIA transformants of screening ET12567 (pUZ8002).By the Escherichia coli containing pLYERIA plasmids ET12567 (pUZ8002) is fallen in engagement transfer the previous day inoculation single bacterium in 3mL LB test tubes, and adding in 50 μ g/mL cards, that is mould Element, 50 μ g/mL apramycins, 25 μ g/mL chloramphenicol overnight incubations, next day are contained with the access of 1% (v/v) inoculum concentration with concentration In the 10mL LB triangular flasks of three kinds of antibiotic, to OD bacterium is collected by centrifugation for 0.4-0.6,4500rpm, 5min, 4 DEG C in 37 DEG C of cultures Body abandons supernatant, is washed twice with the sterile LB of same volume, is resuspended in about 200 μ L LB for use.

Escherichia coli bacteria liquid is sufficiently mixed with the 100 μ l of actinoplanes N902-109 mycelia suspension prepared, coating 2 Block contains final concentration 20mM MgCl₂M-Isp4 tablets on, after 30 DEG C of cultures 24 hours, is mixed with 0.7ml per plate is evenly laid out Sterile water, 15 μ l 50mg/ml are dissolved in the solution of the nalidixic acid and 15 μ l 50mg/ml apramycins in the NaOH of 0.1N. 30 DEG C are continued culture 6 days, until monoclonal is grown.It selects monoclonal and carries out PCR inspections with primer apr-V-R and ERIAVAS, must swim Dynamic actinomyces rapA gene ER functional domain homologous single-crossover bacterial strains N902ERSC.

Ii) the acquisition of actinoplanes rapA genes ER functional domains homologous double-crossover bacterial strain

A) point mutation based on restriction enzyme I-SceI

The structure flow of I-SceI expression plasmids pSETSI is shown in Fig. 8, and idiographic flow is as follows：PSET152 pshAI digestions, Phosphate group is removed with Fast AP enzymes.PIJ778 cuts out 1.3kb spectinomycin resistance gene segments with XbaI, is polymerize with T4 DNA Enzyme filling-in end.It is connect with pSET152pshAI carriers and obtains pSETspc plasmids.Paacs, paacas primer are using pIJ773 as mould Plate carries out high confidence level PCR amplification using KOD NEO plus, and for the segment of acquisition with BamHI, NdeI digestions are apramycin Promoter Insert Fragment, pLU101 NdeI, EcoRI cut I-SceI Insert Fragments.Apramycin promoter fragment and I- SceI segments and the pSETspc carriers of BamHI, EcoRI digestion are connected, and obtain pSETSI.

B) competent cell of Escherichia coli ET12567 (pUZ8002) is prepared, converts pSETSI plasmids, competent cell The method that is converted with plasmid of preparation with aforementioned.

C) double crossing over bacterial strain is screened in single exchange strains N902ERSC intracellular expression I-SceI enzymes

By the Escherichia coli ET12567 (pUZ8002) containing pSETSI plasmids, single bacterium colony is inoculated in engagement transfer the previous day Into 3mL LB test tubes, 50 μ g/mL kanamycins of addition, 50 μ g/mL apramycins, 25 37 DEG C of overnight incubations of μ g/mL chloramphenicol, Next day, 37 DEG C were cultivated to OD with the access of 1% (v/v) inoculum concentration containing in the 10mL LB triangular flasks with three kinds of antibiotic of concentration 0.4-0.6,4500rpm, 5min, 4 DEG C thalline were collected by centrifugation, abandons supernatant, is washed twice, is resuspended in about with the sterile LB of same volume It is for use in 200 μ L LB.

Escherichia coli bacteria liquid is sufficiently mixed with the actinoplanes N902-109 mycelia suspensions 100ul prepared, coating 2 Block contains final concentration 20mM MgCl₂M-Isp4 tablets on, after 30 DEG C of cultures 24 hours, is mixed with 0.7ml per plate is evenly laid out Sterile water, 15 μ l 50mg/ml are dissolved in the solution of the nalidixic acid and 15 μ l 50mg/ml spectinomycins in the NaOH of 0.1N. 30 DEG C are continued culture 6 days, until monoclonal is grown, the homologous double-crossover bacterial strain N902ERSCI clones correctly integrated.It selects gram N902ERSCI simultaneously continuous passage 2 times are cultivated in grand liquid medium within M-Isp2, dilute culture, coating M-Isp2 tablets are extremely Grow monoclonal.Monoclonal is crossed respectively no apramycin M-Isp2 and containing 30 μ g/ml apramycin M-Isp2 tablets, is chosen Go out the clone to apramycin sensitivity, with ERIAvs, ERIAvas PCR amplification apramycin sensitive clones, recycle PCR product, And NheI endonuclease reactions are carried out, pick out actinoplanes rapA gene ER functional domain mutant strains N902ERIA.

PLYERIA has obtained two monoclonals by engaging transfer conversion actinoplanes N902-109.Extract genome, PCR verifications are carried out with primer apr-V-R and ERIAVas, see Fig. 2.PCR results illustrate that pLYERIA plasmids are in actinoplanes Genome is inserted into the correct ER functional domains position of rapA genes, and single exchange strains N902ERSC genotype is correct.

PSETSI is by engaging the bacterial strain for shifting the N902ERSC acquisitions of conversion single exchange strains and being integrated with I-SceI genes N902ERSCI.By being not added with the liquid M-Isp 2 of agar, subculture, induction double cross ring change go out twice, select double crossing over mutation Strain.To the double crossing over bacterial strain that the seven plants of apramycin resistances chosen are lost, using genome as template, with ERIAVs and ERIAVas PCR amplification verification is carried out as primer.PCR fragment is with NheI digestions, and the results are shown in Figure 3.The ER of wild type actinoplanes NheI sites are not present in functional domain site, and the mutational site designed introduces NheI sites.The NheI for being mutated ER functional domains is special Sign restriction enzyme mapping is 2.9kb and 1.9kb.According to fig. 3, it may be said that bright：A3 restores wild type for double crossing over bacterial strain, remaining six plants Double crossing over bacterial strain for the mutation of ER functional domains.Sequencing result is explicitly shown the activity of i.e. actinoplanes rapA genes ER functional domains Site amino acids A-G-G mutation become amino acid A-S-P.

Used in table 2 and structure plasmid

Primer and sequence used in table 3

Bacterial strain that table 4 uses and structure

Used medium component is as follows：

LB：Tryptone 10g, yeast extract 5g, NaCl 10g, adds water to 1L, sterilizing.

YEME：Sucrose 170g, yeast extract 3g, bacto peptone (Bactopeptone) 5g, malt sugar extract 3g, Glucose 10g adjusts pH to 7.0, adds water to 1L, MgCl is added in after sterilizing₂To final concentration 5mM.

YM culture mediums：0.3% yeast extract (w/v), 0.5% tryptone (w/v), 0.5% maltose (w/v), moisturizing is extremely 100%, adjust pH to 9.0.

M-Isp2：Malt sugar extract 10g, glucose 4g, dusty yeast 4g, agar 20g add water to 1L, pH7.0, sterilizing.

M-Isp4：Beancake powder 5g, mannitol 5g, soluble starch 5g, tryptone 2g, yeast extract 1g, NaCl 1g, (NH₄)₂SO₄2g,K₂HPO₃1g,CaCO₃2g, agar powder 20g, inorganic salt microelement solution 1ml, add water to 1L, pH7.0, Sterilizing.Inorganic salt microelement solution：ZnSO₄1g, FeSO₄1g, MnSO₄1g adds water to 1L.

Actinoplanes fermentation slant medium：Glucose 20g, soluble starch 40g, yeast extract 5g, enzyme hydrolysis Casein 5g, CaCO₃1g and agar 16g, adds water to 1L, pH7.0-7.2.

Primary-seed medium：Soluble starch 30g, glucose 20g, dry ferment 5g, Dried Corn Steep Liquor Powder 5g, peanut meal 5g And CaCO₃2g adds water to 1L, pH7.0.

Secondary seed medium：Soluble starch 30g, glucose 40g, Dried Corn Steep Liquor Powder 8g, cottonseed protein powder 8g, CaCO₃3g, bubble enemy 2g, MgSO₄.7H₂O 0.0025g、CoCl₂.6H₂O 0.001g、VB₁0.2g、VB₁₂0.0002g and folic acid 0.025g adds water to 1L, pH6.9.

Embodiment 3

The fermentation of rapamycin structure analog prepares and identification

A) the fermentation of actinoplanes N902-109ERIA

By the actinoplanes N902-109ERIA bacterium solutions drop access actinoplanes fermentation inclined-plane culture of glycerol tube preservation The inclined-plane of base, 28 DEG C constant temperature incubation 7-9 days uniform with inoculation rod coating.Scraping inclined-plane mycelium, which is shoveled, with inoculation accesses 30ml mono- In grade seed culture medium, 28 DEG C, 220 turns of constant temperature incubations are connected to secondary seed medium after 4-5 days by inoculum concentration 10%, and described hundred Point compare the percent by volume for accounting for the secondary seed medium volume, 28 DEG C of 220 revs/min of constant temperature incubations 3-4 days, then press and be inoculated with Amount 7.5%-10% is connected in the fermentation flask equipped with secondary seed medium, and the percentage is accounts for two level kind in the fermentation flask The percent by volume of sub- culture volume, 220 revs/min of shaking speed, 28 DEG C of constant temperature incubations 9 days.

B) the extraction of bis- dehydrogenation -27-O- demethyl rapamycins of 35,36-

It proportionally adds in 2.5% super-cell in zymotic fluid to stir 5 minutes, the percentage is the diatomite Quality accounts for the quality percent by volume of the fermentating liquid volume, vacuum filtration；Filter cake is collected, is added in equal volume in filter cake 60% (v/v) acetone stirs, and impregnates 2h；It filters, collects filtrate.It is primary to reuse the equally extraction of 60% (v/v) acetone, merges two Secondary filtrate.

The water dilution acetone concentration of two volumes is added in acetone extract, upper X-5 (4cm × 40cm) is big after mixing Hole resin column；After loading, successively with water, 30% (v/v) acetone, 40% (v/v) acetone, 50% (v/v) acetone, 60% (v/v) each 2L gradient elutions of acetone, TLC analyses merge the eluent containing target product, and 35 DEG C of reduced pressures are removed The emulsion of acetone, the percentage account for the percent by volume of aqueous acetone solution total volume for acetone volume.

Isometric ethyl acetate stirring extraction 2 times is added in into emulsion, merges the extract liquor of ethyl acetate twice, adds Enter 5% anhydrous sodium sulfate dehydration, the percentage accounts for the percent by volume of water volume for the anhydrous sodium sulfate quality, 35 DEG C Slurry is concentrated under reduced pressure into, obtains the crude extract containing target product.

It isolates and purifies：It is appropriate to weigh 300-400 mesh silica gel, adds in hexamethylene wet method dress post (3cm × 32cm).Into sample After adding in 1.5mL dichloromethane and 1.5mL hexamethylenes dissolving crude extract, it is splined on silicagel column.Eluting solvent is hexamethylene and third Ketone, ratio are respectively 20:1、18:1、15:1、12:1、10:1, each each 250ml of ratio, until gradient 10：Isocratic elution when 1 is received Collect eluent, detect collection liquid with thin-layer chromatography TLC methods, the purer collection liquid containing target product is merged, 35 DEG C of decompressions are dense It is reduced to slurry.Concentrate with dichloromethane is dissolved, is prepared with HPLC half, collects the target product of high-purity, concentration, vacuum It is dry.Sample segment is taken to be dissolved with dichloromethane, carries out normal-phase HPLC analysis.

Analysis method

TLC is analyzed：

Sample to be tested is drawn with capillary, in point sample on HF254 high-efficient silica gel plates, after being unfolded in solvent, works as expansion When agent forward position is run at the 0.5cm of silica gel plate upper end, silica gel plate, cold wind drying are taken out.Silica gel plate after air-drying is put into and is contained There is a moment in the closed container of pure iodine, until spot development.Solvent system：Hexamethylene：Acetone=1:1.

The physico-chemical property and thin-layer chromatography mobility of 5 35,36- of table, bis- dehydrogenation -27-O- demethyl rapamycins

The positive facies analyses of HPLC：

Instrument:515 type high performance liquid chromatographs of Waters, including the s996 type UV detector with PDA；Chromatographic column： Welch Ultimate XB-CN columns (4.6mm × 250mm, 5 μm)；Sample size：20μl；Detect Detection wavelength：278nm；Flowing Phase：Hexamethylene：Isopropanol=95:5；Flow velocity：1ml/min；Column temperature：Room temperature.

Rapamycin and demethyl rapamycin, actinoplanes are detected in the zymotic fluid of actinoplanes N902-109 N902-109ERIA bacterial strains then synthesize a small amount of rapamycin, and the peak of a new unknown product occur, as a result such as Fig. 4 institutes Show.The compound is named as SIPI-Rapxin.The ultra-violet absorption spectrum of the noval chemical compound is sufficiently close to rapamycin, such as Shown in Fig. 5, thus it is speculated that it should be the analogue of rapamycin.

Mass spectrum is measured with NMR data：

It isolates and purifies and obtains the sample measure MS of new rapamycin structure analog SIPI-Rapxin and NMR spectra number According to.

The results show that detect the ultraviolet of SIPI-Rapxin (compound 3) and 27- demethyl rapamycins (compound 1) Spectrum is basically identical, shows that two compound structures are similar.According to the ESI-MS collection of illustrative plates of compound 3, there are 920 (M in MS collection of illustrative plates +Na⁺) peak, it is inferred that its molecular weight is 897, than rapamycin molecule amount 913 few 16, thus it is speculated that may lack-a CH₂It is sub- Methyl group and 2 Hydrogen Protons, 3 possible molecular formula of compound are C₄₉H₇₃NO₁₃。

Analysis of compounds 3¹H NMR and¹³C NMR spectras, the results are shown in Table 6 discoveries its only there are two methoxyl group 3H peaks The carbon atom peak of (3.13 and 3.17ppm) and two methoxyl groups (55.8 and 56.4ppm), and 27-O- demethyl rapamycins point It (is connected on 16,39 carbon atoms, chemical shift is respectively containing two methoxyl groups in minor structure：55.9、 56.8ppm).The methoxyl group of compound 3 and 27-O- demethyl rapamycins is completely the same.Thus it is speculated that compound 2 may be 27-O- demethyl rapamycin analogs.Further analysis finds that compound 3 than more than 1 double bonds of compound, is present in 35 Hes 36 carbon atoms, it is consistent with engineering bacteria structure.Therefore, 3 compound structures are mould for 35,36-, bis- dehydrogenation -27-O- demethyl thunder pas Element.

6 27-O- demethyl rapamycins (compound 1) of table and SIPI-Rapxin's (compound 3)¹³C-NMR data pair Than

Note：1 is the 27- demethyl rapamycin NMR datas of reported in literature；3 be the NMR data data of SIPI-Rapxin.

Effect example 1

The antifungal activity of bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- measures

(1) the YM culture mediums of YM liquid yeast cultures (30 DEG C of culture 20h) addition 100ml warms of 200ul, 0.8% Agar, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, per plate about 15-20ml.

(2) mould YM liquid cultures (30 DEG C of culture 36hr) 630ul adds in the YM culture mediums of 100ml warms, 0.8% fine jade Fat, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, per plate about 15-20ml.

(3) rapamycin is dissolved in methanol, the μ g/mL of gradient dilution to 20,10,5,2.5 and 1.25 are de- by 35,36- bis- Hydrogen -27-O- demethyl rapamycins are dissolved in methanol, are diluted to 20 and 10 μ g/mL.The sample of above each concentration is respectively taken 100ul is added on the filter paper dick of diameter 1cm, is placed at room temperature for 10 minutes and methanol volatilizees, and filter paper froth is fitted in tablet Four quadrants in, be placed at room temperature for 30 minutes and make drug diffusion in the medium.Then 30 DEG C are put to cultivate 2-3 days until inhibition zone It generates.

(4) diameter of inhibition zone is measured：It is mapped with the logarithm of drug concentration and antibacterial circle diameter, by certain density 35, The antibacterial circle diameter that bis- dehydrogenation -27-O- demethyl rapamycins of 36- generate is calculated according to standard curve and is obtained with diameter inhibition zone Rapamycin concentration, the ratio of 35,36-, bis- dehydrogenation -27-O- demethyl rapamycins concentration/rapamycin concentrations is made For the characterization of opposite bacteriostatic activity, 7 are the results are shown in Table.

The opposite antifungal activity of bis- dehydrogenation -27-O- demethyl rapamycins of 7 rapamycin of table and 35,36-

According to table 7 as a result, 35,36- bis- dehydrogenation -27-O- demethyl rapamycins to Candida albicans, saccharomyces cerevisiae, The inhibition of aspergillus niger and Penicillium notatum is significantly stronger than rapamycin.

It should be understood that after the above for having read the present invention, those skilled in the art can be to the correlation of the present invention Condition makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims (10)

1. structure bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- as shown in Equation 1：

2. a kind of preparation method of 35,36-, bis- dehydrogenation -27-O- demethyl rapamycins as described in claim 1, feature exist In including the following steps：Fermented and cultured contains the actinoplanes N902-109 of polyketide synthase rapA mutators, from fermentation It is obtained in liquid；The polyketide synthase rapA mutators are as shown in sequence table SEQ ID No.1.

3. preparation method as claimed in claim 2, which is characterized in that the travelling containing polyketide synthase rapA mutators is put The preparation method of line bacterium N902-109 includes the following steps：

(1) genetic fragment containing the polyketide synthase rapA mutators mutational site is connected on carrier, is made and contains The recombinant vector of the polyketide synthase rapA mutated gene segments；It is described containing polyketide synthase rapA mutators mutational site The sequence of genetic fragment is the 5736th~the 10693rd in sequence shown in SEQ ID No.1；

(2) step (1) described recombinant vector is transformed into donor bacterium, donor bacterium must be converted；

(3) step (2) the conversion donor bacterium with the mycelia of the actinoplanes N902-109 is engaged, selects joint element；

(4) the donor bacterium containing Homing endonucleases expression vector with step (3) described joint element is mixed, selected with step Suddenly the double crossing over joint element in (1) described mutational site.

4. preparation method as claimed in claim 3, which is characterized in that step (2) the donor bacterium is Escherichia coli；And/or step Suddenly (4) described Homing endonucleases are I-SceI；And/or step (4) the donor bacterium is Escherichia coli.

5. preparation method as claimed in claim 2, which is characterized in that the fermentation includes the following steps：Right such as will be contained will The seed liquor of the 2 actinoplanes N902-109 containing polyketide synthase rapA mutators is asked to be connected in fermentation medium, 28 DEG C of 220 revs/min of constant temperature incubations 9 days.

6. preparation method as claimed in claim 2, which is characterized in that it is described obtained from zymotic fluid 35,36-, bis- dehydrogenations- 27-O- demethyl rapamycins include the following steps：Extracted from the zymotic fluid with acetone obtain bis- dehydrogenations of 35,36-- 27-O- demethyl rapamycins must concentrate eluted product after upper macroreticular resin after elution, concentration, and ethyl acetate extracts described Bis- dehydrogenation -27-O- demethyl rapamycin crude extracts of 35,36-.

7. preparation method as claimed in claim 6, which is characterized in that the number of the extraction is 2 times；And/or the extractant Volume ratio with the concentration eluted product is 1: 1；And/or the concentration is 35 DEG C of reduced pressures.

8. preparation method as claimed in claim 6, which is characterized in that it further includes following step：It is de- to dissolve the 35,36- bis- Hydrogen -27-O- demethyl rapamycin crude extracts, are splined on silica gel, and 35,36-, the bis- dehydrogenation -27-O- for eluting after purification go first Base rapamycin.

9. preparation method as claimed in claim 8, which is characterized in that the silica gel is 300-400 mesh silica gel；And/or dissolving institute The solvent for stating bis- dehydrogenation -27-O- demethyl rapamycin crude extracts of 35,36- is the dichloromethane and hexamethylene that volume ratio is 1: 1 Alkane；And/or eluting solvent is the hexamethylene and acetone that volume ratio is 10: 1~20: 1.

10. bis- dehydrogenation -27-O- demethyl rapamycins of 35,36- as described in claim 1 are in antifungal composition is prepared Using.