CN106188093A - A kind of rapamycin structure analog and preparation method thereof - Google Patents

Abstract

The invention provides a kind of rapamycin structure analog and preparation method thereof.Described rapamycin structure analog is as shown in Equation 1.Described preparation method includes: fermentation culture contains the actinoplanes of polyketide synthase rapA mutant gene, obtains from fermentation liquid；Described polyketide synthase rapA mutant gene is as shown in sequence table SEQ ID No.1.The rapamycin structure method for preparing analogue that the present invention provides operates simpler compared with existing chemical synthesis, and cost is lower, and environmental pollution is less, and has good anti-mycotic efficiency by the rapamycin structure analog obtained by this preparation method.

Description

A kind of rapamycin structure analog and preparation method thereof

Technical field

The present invention relates to Combinatorial biosynthesis field, be specifically related to a kind of rapamycin structure analog and Preparation method.

Background technology

Rapamycin is the nitrogenous triene macrolide compounds of a kind of 31 rings, is to apply clinically Important immunosuppressant and antitumor drug research and development intermediate.Temsirolimus, everolimus and Zotarolimus is semisynthetic rapamycin structure analog, respectively at 2004,2005,2007 Year is ratified as antitumor drug and angiocarpy bracket coating medicine list marketing by FDA.

In natural product, except a part of compound self can be with patent medicine, major part is as guide's chemical combination Thing passes through base group modification, transforms its patent medicine character, becomes semisynthetic drug and be applied to clinic.For big portion The native compound of separation structure complexity carries out chemosynthesis or unit structure is modified, and is faced with stereoisomerism With many technological difficulties such as selectivitys.And transform the microbial hosts genome of synthesis compound, utilize it The ability of own biological synthesis produces the analog of structural modification, breaches chemosynthesis to a certain extent Technical bottleneck.Being allowed to low cost, what environmental friendliness mode realized that chemical synthesising technology cannot realize answers The biosynthesis of miscellaneous structural compounds is possibly realized.

The analog study on the synthesis mode of rapamycin is main also with the synthesis that suddenlys change, the life that precursor instructs Thing synthesis and chemistry become main.

Sudden change synthesis (mutasynthesis): L-proline and analog can replace rapamycin to close Natural precursor L-pipecolate needed for one-tenth mixes polyketone chain, produces the new constructions such as prolylrapamycin Compound.The analog of the L-pipecolate of sulfur-bearing can also be as precursor by NRPS114 RapP utilizes thus produces rapamycin derivative

The biosynthesis (Precursor directed biosynthesis) that precursor instructs: the polyketone of rapamycin The load-on module of synthase A has the wide in range property of higher substrate, can be by the analog of different DHCHC Join the analog producing rapamycin in molecule.The 42 of rapamycin loading molecular Position carbon atom is a valuable decorating site, the rapamycin of the listing of FDA approval up to now Analog is the most all the different modifying product in this site.

Chemosynthesis: rapamycin is also by the target spot modified with mTOR binding site.By chemistry Compound WYE-592 and ILS-920 that the synthetic method modification in rapamycin triolefin position obtains, Owing to destroying the integrated structure with mTOR, therefore the immune suppression function of both compounds substantially drops Low, and strengthen with the binding ability of FKBP52, show significant neuroprotective (neuroprotective Activities) activity.

And by knock out streptomyces hygroscopicus rapamycin synthesize rear modification enzyme, have also been obtained a series of not The compound of exhaustive methylation.

At present rapamycin structure analog is carried out chemosynthesis or unit structure is modified and all suffered from The problem of many technological difficulties such as stereoisomerism and selectivity, therefore needs a kind of technique badly simple, low cost, Eco-friendly synthetic method synthesizes the analog of rapamycin.

Summary of the invention

The technical problem to be solved is, in order to overcome, forms of rapamycin analogs carries out chemistry conjunction Become or unit structure modify the problem being faced with many technological difficulties such as stereoisomerism and selectivity, it is provided that A kind of utilization can produce microorganism actinoplanes (the Actinoplanes sp. of rapamycin N902-109) method that own biological synthesis capability produces rapamycin structure analog, and by being somebody's turn to do Method has prepared a kind of rapamycin structure analog.The preparation method that the present invention provides is with existing It is simpler that chemical synthesis compares operation, and cost is lower, and environmental pollution is less, and by this preparation side Rapamycin structure analog obtained by method has good anti-mycotic efficiency.

One of technical solution of the present invention: structure 35,36-bis-dehydrogenation-27-O-demethyl thunder handkerchief as shown in Equation 1 Mycin:

The two of technical solution of the present invention: a kind of aforementioned 35,36-bis-dehydrogenation-27-O-demethyl rapamycin Preparation method, it comprises the steps: that fermentation culture contains the travelling of polyketide synthase rapA mutant gene Actinomycetes N902-109, obtains from fermentation liquid；Described polyketide synthase rapA mutant gene such as sequence table Shown in SEQ ID No.1.

In the present invention, described polyketide synthase rapA mutant gene can come from any source, including from body Interior separation obtains and synthesis obtains.The preparation method of described polyketide synthase rapA mutant gene is this area Conventional method, preferably PCR expand the upstream and downstream in the mutational site of described mutant gene respectively Fragment after with nuclease cutting reconnect and get final product.Described nuclease is this area conventional nucleic acid enzyme, preferably Ground is restriction endonuclease, such as Nhe I.Described ligase is this area routine ligase, more preferably Ground is T4 DNA ligase.The system of the described actinoplanes containing polyketide synthase rapA mutant gene Preparation Method can be the operation that this area is conventional, preferably can comprise the steps: that (1) will contain The genetic fragment stating polyketide synthase rapA mutant gene mutational site is connected on carrier, prepares containing State the recombinant vector of polyketide synthase rapA mutated gene segment；Described containing polyketide synthase rapA sudden change base Because the sequence of the genetic fragment in mutational site is the 5736th～the 10693rd in SEQ ID No.1 sequence Position；(2) step (1) described recombinant vector is transformed into donor bacterium, donor bacterium must be converted；(3) will Step (2) described conversion donor bacterium engages with the mycelia of described actinoplanes N902-109, selects and connects Zygote；(4) by the donor bacterium containing Homing endonucleases expression vector and step (3) described joint Son mixing, selects the double crossing over conjugon with step (1) described mutational site.

Step (1) described carrier can be this area routine carrier, including all kinds of prokaryotic vectors and eucaryon Carrier, preferably prokaryotic vector, be more preferably pLYZL102 carrier.Described recombinant vector can lead to Cross this area conventional method to prepare, preferably can will dash forward containing described polyketide synthase rapA mutant gene The genetic fragment of displacement point is connected on all kinds of conventional carrier in this area obtain.

In step (2), described donor bacterium can be this area routine donor bacterium, preferably large intestine bar Bacterium, is more preferably escherichia coli ET12567 (pUZ8002) bacterial strain.Described conversion can be this area Conventional transformation methods, preferably electrotransformation.

In step (3), preferably can also cultivate with engaging transport medium after described joint, described Engage the culture medium that transport medium can be this area routine, be preferably comprised following component: 0.5% bean Cake powder, 0.5% mannitol, 0.5% 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 mend to 100% water, Described percentage ratio is the quality percent by volume that each constituent mass accounts for described joint transport medium volume.

In step (4), described Homing endonucleases can be the enzyme that this area is conventional, preferably I-SceI. Described donor bacterium can be this area routine strain, preferably escherichia coli, is more preferably escherichia coli ET12567 (pUZ8002) bacterial strain.Described select the routine side that double crossing over conjugon can be this area Method, preferably utilizes antibiotic pressure to select.More preferably for utilizing apramycin pressure to select, As by the escherichia coli of the expression vector of the I-SceI gene containing apramycin promoters driven and step (3) Described conjugon mixes, and cultivates and selects the double crossing over conjugon that apramycin resistance is lost after passing for 2 generations.

In the present invention, described fermentation comprises the steps: to dash forward containing polyketide synthase rapA containing aforementioned The seed culture medium of the actinoplanes N902-109 becoming gene is received in fermentation medium, 28 DEG C 220 Rev/min constant temperature culture 9 days.The described actinoplanes containing polyketide synthase rapA mutant gene The inoculum concentration of the seed culture medium of N902-109 can be the inoculum concentration that this area is conventional, preferably 5-15%, described percentage ratio is the percent by volume accounting for described fermentation medium volume.Described containing polyketone The seed culture medium of the actinoplanes N902-109 of synthase rapA mutant gene can be normal by this area Rule method prepares, and preferably can be prepared by following step: (1) inoculation is described containing polyketide synthase rapA The actinoplanes of mutant gene to actinoplanes ferments slant medium, coating uniformly, 28 DEG C of constant temperature Cultivate 7-9 days；(2) scraping inclined-plane mycelium accesses in primary-seed medium, 28 DEG C, 220 turns of perseverances Temperature is cultivated 4-5 days；(3) take step (2) described primary-seed medium and be inoculated into secondary seed cultivation In base, 28 DEG C of 220 revs/min of constant temperature culture 3-4 days and get final product.Described actinoplanes fermentation slant culture Base is that this area is conventional, is preferably comprised following component: glucose 2%, soluble starch 4%, yeast Extract 0.1-0.2%, enzyme hydrolysis casein 0.5%, CaCO₃0.1% and agar 1.6% and mending to 100% Water, described percentage ratio be each constituent mass account for described actinoplanes fermentation slant medium cumulative volume Quality percent by volume.The preparation method of described actinoplanes fermentation slant medium can be this area Conventional method, is preferably comprised following step: mixed by described raw material components, adjust pH to 7.0～7.2, Sterilizing.Described sterilizing can be this area conventional sterilization procedures, preferably 115 DEG C～121 DEG C of sterilizings 15min～20min, is more preferably 115 DEG C of sterilizing 15min.Described primary-seed medium can be ability Territory conventional medium, is preferably comprised following component: soluble starch 3%, glucose 2%, dry yeast 0.5%, Dried Corn Steep Liquor Powder 0.5%, Semen arachidis hypogaeae dregs 0.5%, CaCO₃0.2% and mend to 100% water, institute State the quality that percentage ratio is described each component and account for the quality volume hundred of described primary-seed medium cumulative volume Proportion by subtraction.The preparation method of described primary-seed medium can be this area conventional method, is preferably comprised Following step: described each component is mixed, adjusts pH to 7.0, sterilizing.Described sterilizing can be this area Conventional sterilization procedures, is more preferably 115 DEG C by preferably 115 DEG C～121 DEG C of sterilizing 15min～20min Sterilizing 15min.Described secondary seed medium can be this area conventional medium, is preferably comprised down State component: soluble starch 3%, glucose 4%, Dried Corn Steep Liquor Powder 0.8%, cottonseed protein powder 0.8%, CaCO₃0.3%, bubble enemy 0.2%, MgSO₄.7H₂O 0.00025%, CoCl₂.6H₂O 0.0001%, VB₁0.02%, VB₁₂0.00002%, folic acid 0.0025% and the water of benefit to 100%, described percentage ratio Quality for described each component accounts for the quality percent by volume of described secondary seed medium cumulative volume.Described The preparation method of secondary seed medium can be this area conventional method, is preferably comprised following step: Described each component is mixed, adjusts pH to 6.9, sterilizing.Described sterilizing is this area conventional sterilization procedures, Preferably 115 DEG C～121 DEG C of sterilizing 15min～20min, be more preferably 115 DEG C of sterilizing 15min.Step (3), in, the inoculum concentration of described primary-seed medium can be conventional inoculum concentration, preferably 5-10%, Described percentage ratio is the percent by volume accounting for described secondary seed medium volume.Described fermentation medium can Think the culture medium that this area is conventional, preferably step (3) described secondary seed medium.

In the present invention, described obtaining described 35 from fermentation liquid, 36-bis-dehydrogenation-27-O-demethyl thunder handkerchief is mould Element comprises the steps: to extract from described fermentation liquid with acetone to obtain described 35,36-bis-dehydrogenation-27-O- Demethyl rapamycin, must concentrate eluted product, extract after eluting, concentrating under reduced pressure after upper macroporous resin Described 35,36-bis-dehydrogenation-27-O-demethyl rapamycin crude extract.Described concentrating under reduced pressure can be ability The operation that territory is conventional, the temperature of described concentrating under reduced pressure is preferably 35 DEG C.Described extraction can be this area Conventional operation, the extractant that described extraction uses can be this area conventional extraction agent, preferably second Acetoacetic ester.The number of times of described extraction can be conventional number of times, preferably 2 times.Described extractant with The volume ratio of described concentration eluted product can be conventional ratio, preferably 1: 1.

The present invention adds the anhydrous sodium sulfate of 5% after preferably can also comprising the steps: described extraction Dehydration, 35 DEG C of concentrating under reduced pressure.35,36-bis-dehydrogenation-27-O-demethyl rapamycin crude extract.

The present invention preferably can also include with the isolated and purified described 35,36-bis-dehydrogenation-27-O-of silica gel nor- Base rapamycin crude extract, it comprises the steps: to dissolve described 35,36-bis-dehydrogenation-27-O-demethyl Rapamycin crude extract, is splined on silica gel, and eluting obtains 35 after purification, 36-bis-dehydrogenation-27-O-demethyl Rapamycin.Described silica gel can be this area routine silica gel, preferably 300-400 mesh silica gel.Institute State that to dissolve the solvent of described 35,36-bis-dehydrogenation-27-O-demethyl rapamycin crude extract can be this area Conventional solvent, preferably volume ratio is dichloromethane and the hexamethylene of 1: 1.Described eluting uses Solvent can be the conventional solvent in this area, preferably volume ratio is the hexamethylene of 10: 1～20: 1 And acetone.

The three of technical solution of the present invention: 35,36-bis-dehydrogenation-27-O-demethyl rapamycin exists as previously mentioned Prepare the application in antifungal composition.

In the present invention, described antifungal medicine composition can be the reagent that this area is conventionally referred, preferably Ground is for suppressing the compositions of the black mould caused by aspergillus niger maybe can treat caused by Candida albicans The compositions of disease, the compositions of the described disease can treated caused by Candida albicans is preferably Can treat by the microbial dermatocandidiasis of Candida albicans, candidiasis of the mucous membranes, internal organs candidiasis, Candidid and the compositions of immune deficiency disorder.

On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, i.e. get Ben Fa Bright each preferred embodiments.

Agents useful for same of the present invention and raw material are the most commercially.

The most progressive effect of the present invention is: the invention provides a kind of Combinatorial biosynthesis method synthesis The approach of rapamycin structure analog, the method technique is simple, with low cost, eco-friendly mode Achieve the biosynthesis of the labyrinth compound that chemical synthesising technology cannot realize, and synthesized Rapamycin structure analog has good anti-mycotic efficiency.

Accompanying drawing explanation

The arrangement of Fig. 1: actinoplanes rapA gene function territory and mutational site design diagram.

Fig. 2: actinoplanes N902-109 single-swap recombinant clone PCR schematic diagram.SC1 and SC2 Represent two single-swap clones respectively；AC: actinoplanes N902-109.

Fig. 3 is: actinoplanes N902-109 double crossing over recombinant clone PCR and PCR primer enzyme action Figure.A1-A7: double crossing over bacterial strain；AC: actinoplanes N902-109.

Fig. 4: rapamycin and analog fermentation liquid HPLC separating spectrum thereof.

Fig. 5: rapamycin and and the ultraviolet absorption peak figure of analog.

Fig. 6: actinoplanes rapA gene ER functional domain point mutation plasmid pLYERIA schematic diagram.

The structure schematic flow sheet of Fig. 7: plasmid pLYERIA.

The structure schematic flow sheet of Fig. 8: plasmid pSETSI.

Detailed description of the invention

Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to Among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to often Rule method and condition, or select according to catalogue.

Reagent used in the present invention and key instrument:

Restricted enzyme is purchased from Thermo Scientific. ligase ligation solution I, T4 DNA Polymerase, restricted enzyme pshAI, 10mM dNTP are purchased from TaKaRa company.pACK4a PCR fragment cloning vehicle is purchased from Suzhou Pan Gu's gene company limited.High-fidelity DNA polymerase Neo KOD-plus is purchased from Toyobo company.Apramycin is purchased from DUCHEFA company.Nalidixic acid, strong Miromycin is purchased from Sheng Gong bio-engineering corporation.Plasmid extraction and agarose gel reclaim test kit and are purchased from Axygen company.Easytaq is purchased from Quan Shijin biotech firm.PCR instrument is Biometra Tprofessional TRIO, centrifuge is Eppendorf 5424 and 5415R, and HPLC chromatogram instrument is Agilent Technologies 1200series type high performance liquid chromatograph, mass spectrograph is Waters company Waters Q-TOF Micromass mass spectrograph, nuclear magnetic resonance analyser is Inova-400 nuclear magnetic resonance analyser, chromatographic column: Waters NaVa-PaK C8 post, specification: (3.9mm × 150mm, 5 μm)；The efficient silicon of HF254 Offset plate is purchased from Yantai Jiang You silica gel development corporation, Ltd..Other reagent are domestic analytical pure, purchased from traditional Chinese medicines Group.Nanjing Jin Sirui Bioisystech Co., Ltd is entrusted in primer synthesis, and gene sequencing service is outstanding by Shanghai Lee biotechnology company provides.

Actinoplanes N902-109 used in the present invention sees below patent documentation: United States Patent (USP) (US5674732), used Candida albicans (Candida albicans), saccharomyces cerevisiae (Saccharomyces cerevisiae) and penicillium (Penicillium Sp.) are purchased from U.S. ATCC, Aspergillus niger (Aspergillus niger) is purchased from CGMCC, and the preserving number of Candida albicans is ATCC11651, The preserving number of saccharomyces cerevisiae is ATCC204508, and the preserving number of penicillium is ATCC32029, aspergillus niger Preserving number be CGMCC3.3928.Used escherichia coli ET12567 (pUZ8002) bacterial strain See 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 To plasmid origin as follows: pSET152 sees below list of references: 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 obtained by pSET152 transformation, sees Fig. 8；PSETSI is by pSET152 Transformation obtains, and sees Fig. 8；PLYZL102 sees below list of references: 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 transformed by pLYZL102 Obtain, see Fig. 7；PLU101 sees below list of references: 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 sees below list of references: 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 America 2003,100(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.

Http:// nrps.igs.umaryland.edu/nrps/_ is utilized to carry out the pre-of polyketide synthase rapA functional domain Surveying, result is as shown in table 1:

Table 1

RapA gene contains a load-on module and four chain extension modules, and the arrangement of each module is shown It is intended to as shown in Figure 1.Act_rapA refers to actinoplanes rapA gene, and CL is carboxylic acid ligase, AT is acyltransferase functional domain, and KS is ketosynthase functional domain, and DH is dehydrogenase function territory, ER is enoyl reductase functional domain, and KR is keto reductase functional domain, and ACP is acyl carrier protein Functional domain.

Embodiment 2

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

Fig. 1 is shown in by the schematic diagram of actinoplanes rapA ER functional domain point mutation.By two pairs of primers ERIAUs-ERIAUas Yu ERIADAs-ERIADAas expands actinoplanes rapA gene respectively Two fragments of ER functional domain mutational site upstream and downstream, then the mutational site by introducing comprise Restricted enzyme NheI two fragments are coupled together.

A) extraction of actinoplanes N902-109 genome:

Take a small amount of actinoplanes N902-109 mycelium and be inoculated in the test tube containing 5mL YEME culture medium In, in 30 DEG C of shaken cultivation about 72 hours.3500rpm is centrifugal collects mycelium, with TES (10mM Tris-HCl pH7.5, EDTA 1mM, NaCl 50mM) wash twice.Add in the mycelium collected 1mL TES (lysozyme final concentration 5mg/mL), vortex to the most homogeneous, 37 DEG C of water-baths 60 minutes, add E.C. 3.4.21.64 (final concentration reaches 0.1mg/mL), 0.1mL 10%SDS, puts into rapidly 55 DEG C of water after mixing Bathe 60 minutes, treat that solution is clarified.It is placed in cooled on ice, adds 0.25mL 5M KAc, cooled on ice 15 minutes.Addition 0.5mL phenol: chloroform (Tris saturated phenol pH7.5: chloroform: isoamyl alcohol is volume ratio 25:24:1), gentle inversion mixes, and is centrifuged 10 minutes in 4 DEG C of 12000rpm.With the 1mL of clip Aqueous phase sucking-off is transferred to new centrifuge tube by rifle head, adds equal amounts of chloroform extracting, 12000rpm, and 4 DEG C are centrifuged 10 Minute, with the 1mL rifle head of clip, aqueous phase sucking-off is transferred to new centrifuge tube, adds 0.1 volume 3M NaAc, the dehydrated alcohol of two volumes, mixing, cotton-shaped DNA occurs.Ticked as newly from In heart pipe, add the washing with alcohol of 0.5mL 70%, exhaust liquid with rifle, the most dry after add 300 μ l TE Dissolve, add RNase (final concentration of 50 μ g/mL), 37 DEG C of water-baths 30 minutes.

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

With actinoplanes N902-109 genome as template, with primer ERIADAas (HindIII) and ERIADAs (NheI), ERIAUAs (XbaI) and ERIAUAas (NheI), carrying out height respectively can property degree Amplification obtains a length of 2846bp, homology arm and upper homology arm fragment under the ER functional domain of 2142bp.Instead System (50 μ l) is answered to include: 5 μ l 10 × KOD NEO plus buffer, 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 polymerase, 50ng genomic DNA, add distilled water to 50 μ l.PCR reaction interval Sequence is: 95 DEG C of 5min, 30 circulations (94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 120s), 72 DEG C of 10min. PCR primer after purification, takes 2 μ l and the mixing of 0.5 μ l pACK4aBS carrier respectively, places 15min for 37 DEG C, Convert escherichia coli DH5a and obtain pACK4a-ERIADA, pACK4a-ERIAUA plasmid.With NheI, HindIII enzyme action pACK4a-ERIADA obtains ERIADA fragment, with NheI, XbaI enzyme cutting PACK4a-ERIAUA obtains ERIAUA fragment, obtains with HindIII, XbaI enzyme cutting pLYZL102 To carrier segments, the connection of three's fragment obtains pLYERIA plasmid, and flow chart is shown in Fig. 7, pLYERIA Plasmid map is shown in Fig. 6.

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

I) acquisition of actinoplanes rapA gene ER functional domain homologous single-crossover bacterial strain

Prepare actinoplanes mycelium suspension: take 3 μ l actinoplanes glycerol stocks liquid coating I-Isp2 Solid medium.Cultivate about 120 hours for 30 DEG C.With aseptic scraper scraping surface thalline, use 15-20mL Sterilized water is washed down at twice and is shaken 6-8 time on vortex agitator by bead, each 10 seconds, By suspension by being equipped with the asepsis injector syringe of cotton plug, filter liquor through 7000 revs/min of centrifugal 10min, Supernatant discarded, obtains mycelium suspension with appropriate 0.4-1.0mL sterilized water is resuspended.

From LB flat board (50 μ g/mL kanamycin, 50 μ g/mL apramycins, 25 μ g/mL chloromycetin) The single bacterium colony of upper picking ET12567 (pUZ8002), the inoculation 3mL LB liquid containing ibid antibiotic Culture medium, overnight incubation.Access three kinds containing ibid concentration with 0.5% (v/v) inoculum concentration next day to resist In the 10mL LB triangular flask of raw element, cultivate to OD 0.4-0.6,4500rpm 5min for 37 DEG C, 4 DEG C from The heart collects thalline, abandons supernatant, washes twice with aseptic 10% glycerol of same volume, be resuspended in about 500 μ L 10% In glycerol and be distributed into 50ul often pipe to be ET12567/pUZ8002 electricity transformed competence colibacillus stand-by.Will 100ng pLYERIA plasmid joins in ET12567/pUZ8002 cell, voltage 2.5kv, resistance 200 Ohm, electroporated, add the aseptic cold LB of 1.0ml resuspended and 37 DEG C cultivate 1hr, be coated with LB flat board (50 μ g/mL kanamycin, 50 μ g/mL apramycins, 25 μ g/mL chloromycetin), screen ET12567 (pUZ8002) pLYERIA transformant.By the escherichia coli ET12567 containing pLYERIA plasmid (pUZ8002), in the joint transfer single bacterium colony of inoculation the previous day to 3mL LB test tube, add 50 μ g/mL kanamycin, 50 μ g/mL apramycins, 25 μ g/mL chloromycetin overnight incubation, next day with 1% (v/v) inoculum concentration accesses containing with in the 10mL LB triangular flask of three kinds of antibiotic of concentration, 37 DEG C Cultivate to OD 0.4-0.6,4500rpm, 5min, 4 DEG C of centrifugal collection thalline, abandon supernatant, use same volume Aseptic LB washes twice, and is resuspended in about 200 μ L LB stand-by.

By abundant with the actinoplanes N902-109 mycelia suspension 100 μ l prepared for Escherichia coli bacteria liquid Mixing, is coated with 2 pieces containing final concentration 20mM MgCl₂M-Isp4 flat board on, cultivate 24 little for 30 DEG C Shi Hou, the every plate evenly laid out 0.7ml of being mixed with sterilized water, 15 μ l 50mg/ml are dissolved in the NaOH of 0.1N In nalidixic acid and the solution of 15 μ l 50mg/ml apramycins.30 DEG C are continued to cultivate 6 days, to Dan Ke Grand grow.Select monoclonal primer apr-V-R and ERIAVAS and carry out PCR inspection, must move about and put Line bacterium rapA gene ER functional domain homologous single-crossover bacterial strain N902ERSC.

Ii) acquisition of actinoplanes rapA gene ER functional domain homologous double-crossover bacterial strain

A) point mutation based on restricted enzyme I-SceI

The structure flow process of I-SceI expression plasmid pSETSI is shown in Fig. 8, and idiographic flow is as follows: pSET152 Use pshAI enzyme action, remove phosphate group with Fast AP enzyme.It is grand mould that pIJ778 XbaI cuts out 1.3kb Element resistance gene fragment, with T4 archaeal dna polymerase filling-in end.It is connected with pSET152pshAI carrier Obtain pSETspc plasmid.Paacs, paacas primer, with pIJ773 as template, uses KOD NEO plus Carry out the amplification of high credibility PCR, it is thus achieved that fragment be apramycin with BamHI, NdeI enzyme action and open Mover Insert Fragment, pLU101 NdeI, EcoRI cut I-SceI Insert Fragment.Apramycin opens The pSETspc carrier of promoter fragment and I-SceI fragment and BamHI, EcoRI enzyme action is connected, it is thus achieved that pSETSI。

B) prepare the competent cell of escherichia coli ET12567 (pUZ8002), convert pSETSI matter Grain, the preparation of competent cell and the method for Plastid transformation are with aforementioned.

C) double crossing over bacterial strain is screened at single-swap bacterial strain N902ERSC intracellular expression I-SceI enzyme

By the escherichia coli ET12567 (pUZ8002) containing pSETSI plasmid, in engaging, transfer is previous It single bacterium colony of inoculation, in 3mL LB test tube, adds 50 μ g/mL kanamycin, and 50 μ g/mL peaces are general mould Element, 25 37 DEG C of overnight incubation of μ g/mL chloromycetin, access containing with dense with 1% (v/v) inoculum concentration next day In the 10mL LB triangular flask of three kinds of antibiotic of degree, cultivate to OD 0.4-0.6,4500rpm for 37 DEG C, 5min, 4 DEG C of centrifugal collection thalline, abandon supernatant, wash twice with the aseptic LB of same volume, be resuspended in about In 200 μ L LB stand-by.

By abundant with the actinoplanes N902-109 mycelia suspension 100ul prepared for Escherichia coli bacteria liquid Mixing, is coated with 2 pieces containing final concentration 20mM MgCl₂M-Isp4 flat board on, cultivate 24 little for 30 DEG C Shi Hou, the every plate evenly laid out 0.7ml of being mixed with sterilized water, 15 μ l 50mg/ml are dissolved in the NaOH of 0.1N In nalidixic acid and the solution of 15 μ l 50mg/ml spectinomycins.30 DEG C are continued to cultivate 6 days, to Dan Ke Grand grow, the homologous double-crossover bacterial strain N902ERSCI clone correctly integrated.Selected clone is at liquid Body culture medium M-Isp2 is cultivated N902ERSCI continuous passage 2 times, dilutes culture, coating M-Isp2 flat board is to growing monoclonal.Monoclonal is rule respectively without apramycin M-Isp2 and contains 30 μ g/ml apramycin M-Isp2 flat boards, choose the clone sensitive to apramycin, with ERIAvs, ERIAvas PCR expands apramycin sensitive clones, reclaims PCR primer, and it is anti-to carry out NheI enzyme action Should, pick out actinoplanes rapA gene ER functional domain mutant strain N902ERIA.

PLYERIA has obtained two monoclonals by engaging transfer conversion actinoplanes N902-109. Extracting genome, carries out PCR checking with primer apr-V-R and ERIAVas, sees Fig. 2.PCR result Illustrating, pLYERIA plasmid inserts in the correct ER functional domain position of actinoplanes rapA gene Genome, single-swap bacterial strain N902ERSC genotype is correct.

PSETSI has I-SceI base by engaging transfer conversion single-swap bacterial strain N902ERSC acquisition integration The bacterial strain N902ERSCI of cause.By being not added with twice subculture of liquid M-Isp 2 of agar, induction is double Commutative ring goes out, and selects double crossing over mutant.The double crossing over bacterium that the seven strain apramycin resistances chosen are lost Strain, with genome as template, carries out PCR amplification checking using ERIAVs and ERIAVas as primer. PCR fragment is with NheI enzyme action, and result is as shown in Figure 3.The ER functional domain of wild type actinoplanes There is not NheI site in site, and the mutational site designed introduces NheI site.Sudden change ER function The NheI feature restriction enzyme mapping in territory is 2.9kb and 1.9kb.According to Fig. 3, it may be said that bright: A3 is double cross Changing bacterial strain and recover wild type, remaining six strain is the double crossing over bacterial strain of ER functional domain sudden change.Sequencing result It is explicitly shown the active site amino A-G-G sudden change of i.e. actinoplanes rapA gene ER functional domain Become aminoacid A-S-P.

Plasmid that is used in table 2 and that build

Primer used in table 3 and sequence

Table 4 uses bacterial strain that is that arrive and that build

Used medium component is as follows:

LB: tryptone 10g, yeast extract 5g, NaCl 10g, add water to 1L, sterilizing.

YEME: sucrose 170g, yeast extract 3g, bacto peptone (Bactopeptone) 5g, Maltose extract 3g, glucose 10g, adjust pH to 7.0, add water to 1L, adds MgCl after sterilizing₂ To final concentration 5mM.

YM culture medium: 0.3% yeast extract (w/v), 0.5% tryptone (w/v), 0.5% maltose (w/v), Moisturizing, to 100%, adjusts pH to 9.0.

M-Isp2: maltose extract 10g, glucose 4g, yeast powder 4g, agar 20g, add water To 1L, pH7.0, sterilizing.

M-Isp4: soybean cake powder 5g, mannitol 5g, soluble starch 5g, tryptone 2g, yeast Extract 1g, NaCl 1g, (NH₄)₂SO₄2g,K₂HPO₃1g,CaCO₃2g, agar powder 20g, nothing Machine salt trace element solution 1ml, adds 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 Thing 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 yeast 5g, Semen Maydis pulp are dry Powder 5g, Semen arachidis hypogaeae dregs 5g and CaCO₃2g, adds water to 1L, pH7.0.

Secondary seed medium: soluble starch 30g, glucose 40g, Dried Corn Steep Liquor Powder 8g, cotton seed Egg albumen 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

Fermentation preparation and the qualification of rapamycin structure analog

A) fermentation of actinoplanes N902-109ERIA

The actinoplanes N902-109ERIA bacterium solution of glycerol pipe preservation is dripped access actinoplanes fermentation The inclined-plane of slant medium, with loop carrier coating uniformly, 28 DEG C of constant temperature culture 7-9 days.Scrape with inoculation shovel Take inclined-plane mycelium and access in 30ml primary-seed medium, 28 DEG C, after 220 turns of constant temperature culture 4-5 days Receiving secondary seed medium by inoculum concentration 10%, described percentage ratio is for accounting for described secondary seed medium body Long-pending percent by volume, 28 DEG C of 220 revs/min of constant temperature culture 3-4 days, then receive by inoculum concentration 7.5%-10% Equipped with in the fermentation flask of secondary seed medium, described percentage ratio is for accounting for secondary seed training in described fermentation flask Support the percent by volume that matrix is long-pending, 220 revs/min of 28 DEG C of constant temperature culture of shaking speed 9 days.

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

Proportionally adding 2.5% super-cell in fermentation liquid to stir 5 minutes, described percentage ratio is Described kieselguhr quality accounts for the quality percent by volume of described fermentating liquid volume, vacuum filtration；Collect filter cake, In filter cake, add the stirring of isopyknic 60% (v/v) acetone, soak 2h；Sucking filtration, collects filtrate. Re-use 60% (v/v) acetone to extract equally once, merge twice filtrate.

Acetone extract adds the water dilution acetone concentration of two volumes, upper X-5 after mix homogeneously (4cm × 40cm) macroporous resin column；After loading, successively with water, 30% (v/v) acetone, 40% (v/v) acetone, 50% (v/v) acetone, 60% (v/v) acetone each 2L gradient elution, TLC analyzes, Merge the eluent containing target product, 35 DEG C of concentrating under reduced pressure, it is thus achieved that remove the emulsion of acetone, described Percentage ratio is the percent by volume that acetone volume accounts for aqueous acetone solution cumulative volume.

In emulsion, add isopyknic ethyl acetate stirring extraction 2 times, merge twice ethyl acetate Extract, adds the anhydrous sodium sulfate dehydration of 5%, and described percentage ratio is that described anhydrous sodium sulfate quality accounts for The percent by volume of water volume, 35 DEG C are evaporated to slurry, it is thus achieved that containing the crude extract of target product.

Isolated and purified: to weigh 300-400 mesh silica gel appropriate, add hexamethylene wet method dress post (3cm × 32cm). After adding 1.5mL dichloromethane and 1.5mL hexamethylene dissolving crude extract in sample, it is splined on silicagel column. Eluting solvent is hexamethylene and acetone, and ratio is respectively 20:1,18:1,15:1,12:1,10:1, each The each 250ml of ratio, to isocratic elution during gradient 10:1, collects eluent, by thin layer chromatography TLC method Liquid is collected in detection, is merged by the purer collection liquid containing target product, and 35 DEG C are evaporated to slurry. Concentrate dichloromethane is dissolved, with HPLC half preparation, collects highly purified target product, concentrate, Vacuum drying.Take sample segment dichloromethane to dissolve, carry out normal-phase HPLC analysis.

Analysis method

TLC analyzes:

Draw testing sample with capillary tube, point sample on HF254 high-efficient silica gel plate, after in developing solvent open up Opening, when solvent front runs to distance silica gel plate upper end 0.5cm, take out silica gel plate, cold wind dries up. A moment in the hermetic container containing pure iodine put into by silica gel plate after air-drying, until spot development.Developing solvent System: hexamethylene: acetone=1:1.

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

The positive facies analysis of HPLC:

Instrument: Waters 515 type high performance liquid chromatograph, including the s996 type UV-detector of band PDA； Chromatographic column: Welch Ultimate XB-CN post (4.6mm × 250mm, 5 μm)；Sample size: 20 μ l； Detection detection wavelength: 278nm；Flowing phase: hexamethylene: isopropanol=95:5；Flow velocity: 1ml/min； Column temperature: room temperature.

The fermentation liquid of actinoplanes N902-109 detects rapamycin and demethyl rapamycin, Actinoplanes N902-109ERIA bacterial strain then synthesizes a small amount of rapamycin, and occurs in that one the most not Knowing the peak of product, result is as shown in Figure 4.This compound is named as SIPI-Rapxin.This noval chemical compound Ultra-violet absorption spectrum be sufficiently close to rapamycin, as shown in Figure 5, thus it is speculated that it should be rapamycin Analog.

Mass spectrum measures with NMR data:

The sample determination MS of the rapamycin structure analog SIPI-Rapxin that isolated and purified acquisition is new and NMR spectra data.

Result shows, detects that SIPI-Rapxin (compound 3) (changes with 27-demethyl rapamycin Compound 1) ultraviolet spectra basically identical, show two compound structures be similar to.According to compound 3 , there are 920 (M+Na in ESI-MS collection of illustrative plates in MS collection of illustrative plates⁺) peak, therefore conclude that its molecular weight is 897, Fewer than rapamycin molecule amount 913 16, thus it is speculated that-a CH may be lacked₂-methylene group and 2 hydrogen Proton, the possible molecular formula of compound 3 is C₄₉H₇₃NO₁₃。

Analysis of compounds 3¹H NMR and¹³C NMR spectra, it only has two to the results are shown in Table 6 discoveries Individual methoxyl group 3H peak (3.13 and 3.17ppm) and the carbon atom peak (55.8 and 56.4 of two methoxyl groups Ppm), and in 27-O-demethyl rapamycin molecular structure containing two methoxyl groups (be connected to 16, On 39 carbon atoms, chemical shift is respectively as follows: 55.9,56.8ppm).Compound 3 is nor-with 27-O- The methoxyl group of base rapamycin is completely the same.Thus it is speculated that compound 2 may be 27-O-demethyl thunder Handkerchief mycin analog.Analyze further and find compound 3 ratio more than 1 double bonds of compound, be present in 35 With 36 carbon atoms, build consistent with engineering bacteria.Therefore, 3 compound structures are 35,36-bis-dehydrogenation -27-O-demethyl rapamycin.

Table 6 27-O-demethyl rapamycin (compound 1) and SIPI-Rapxin's (compound 3)¹³C-NMR Data Comparison

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

Effect example 1

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

(1) the YM liquid yeast culture (cultivating 20h for 30 DEG C) of 200ul adds 100ml warm YM culture medium, 0.8% agar, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, every plate about 15-20ml.

(2) mycete YM liquid culture (cultivating 36hr for 30 DEG C) 630ul adds 100ml warm YM culture medium, 0.8% agar, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, every plate about 15-20ml.

(3) rapamycin is dissolved in methanol, gradient dilution to 20,10,5,2.5 and 1.25 μ g/mL, By 35,36-bis-dehydrogenation-27-O-demethyl rapamycin is dissolved in methanol, is diluted to 20 and 10 μ g/mL.Will The sample of each concentration respectively takes 100ul and is added on the filter paper dick of diameter 1cm above, and room temperature places 10 Minute by methanol volatilize, filter paper froth is fitted in four quadrants of flat board, room temperature place 30 points Clock makes medicine spread in the medium.Then put 30 DEG C to cultivate 2-3 days until inhibition zone produces.

(4) diameter of inhibition zone is measured: with logarithm and the antibacterial circle diameter mapping of drug concentration, by one The antibacterial circle diameter of the 35,36-bis-dehydrogenation-27-O-demethyl rapamycin generation determining concentration is bent according to standard Line computation obtains the concentration of the rapamycin with diameter inhibition zone, by 35,36-bis-dehydrogenation-27-O-demethyl The ratio of rapamycin concentrations/rapamycin concentrations, as the sign of relative bacteriostatic activity, the results are shown in Table 7.

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

According to the result of table 7,35,36-bis-dehydrogenation-27-O-demethyl rapamycins are to Candida albicans, wine The inhibition of brewer yeast, aspergillus niger and penicillium is significantly stronger than rapamycin.

Should be understood that, after the foregoing having read the present invention, those skilled in the art can be to this Bright correlated condition makes various changes or modifications, and these equivalent form of values fall within right appended by the application equally and want Seek book limited range.

Claims (10)

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

2. the preparation side of a 35,36-bis-dehydrogenation-27-O-demethyl rapamycin as claimed in claim 1 Method, it is characterised in that it comprises the steps: that fermentation culture contains polyketide synthase rapA mutant gene Actinoplanes N902-109, obtain from fermentation liquid；Described polyketide synthase rapA mutant gene is such as Shown in sequence table SEQ ID No.1.

3. preparation method as claimed in claim 2, it is characterised in that described containing polyketide synthase rapA The preparation method of the actinoplanes N902-109 of mutant gene comprises the steps:

(1) it is connected to the genetic fragment containing described polyketide synthase rapA mutant gene mutational site carry On body, prepare the recombinant vector containing described polyketide synthase rapA mutated gene segment；Described containing poly- The sequence of the genetic fragment in ketone synthase rapA mutant gene mutational site is sequence shown in SEQ ID No.1 In the 5736th～the 10693rd；

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

(3) by the mycelia of step (2) described conversion donor bacterium Yu described actinoplanes N902-109 Engage, select conjugon；

(4) by the donor bacterium containing Homing endonucleases expression vector and step (3) described conjugon Mixing, selects the double crossing over conjugon with step (1) described mutational site.

4. preparation method as claimed in claim 3, it is characterised in that step (2) described donor bacterium is Escherichia coli；And/or, step (4) described Homing endonucleases is I-SceI；And/or, step (4) institute Stating donor bacterium is escherichia coli.

5. preparation method as claimed in claim 2, it is characterised in that described fermentation comprises the steps: By containing the actinoplanes containing polyketide synthase rapA mutant gene as claimed in claim 2 The seed liquor of N902-109 is received in fermentation medium, 28 DEG C of 220 revs/min of constant temperature culture 9 days.

6. as claimed in claim 2 preparation method, it is characterised in that described obtain institute from fermentation liquid State 35,36-bis-dehydrogenation-27-O-demethyl rapamycin to comprise the steps: with acetone from described fermentation liquid Middle extraction obtains described 35,36-bis-dehydrogenation-27-O-demethyl rapamycin, eluting after upper macroporous resin, Eluted product must be concentrated after concentration, ethyl acetate extract described 35,36-bis-dehydrogenation-27-O-demethyl thunder Handkerchief mycin crude extract.

7. preparation method as claimed in claim 6, it is characterised in that the number of times of described extraction is 2 times； And/or, described extractant is 1: 1 with the volume ratio of described concentration eluted product；And/or, described extraction After be additionally added 5% anhydrous sodium sulfate dehydration, 35 DEG C of concentrating under reduced pressure.

8. preparation method as claimed in claim 6, it is characterised in that it also comprises the steps: molten Solve described 35,36-bis-dehydrogenation-27-O-demethyl rapamycin crude extract, it is splined on silica gel, eluting obtains pure 35,36-bis-dehydrogenation-27-O-demethyl rapamycin after change.

9. preparation method as claimed in claim 8, it is characterised in that described silica gel is 300-400 mesh Silica gel；And/or, dissolving described 35, the solvent of 36-bis-dehydrogenation-27-O-demethyl rapamycin crude extract is Volume ratio is dichloromethane and the hexamethylene of 1: 1；And/or, eluting solvent be volume ratio be 10: 1～20: The hexamethylene of 1 and acetone.

10. 35,36-bis-dehydrogenation-27-O-demethyl rapamycin is anti-true in preparation as claimed in claim 1 Application in bacteria composition.