CN103146594B

CN103146594B - Sorangiumcellulosum strain and application thereof to synthesis of epothilone

Info

Publication number: CN103146594B
Application number: CN201210441677.XA
Authority: CN
Inventors: 赵林; 刘新利; 李越中; 李鹏飞; 孙欣
Original assignee: Shandong Institute of Light Industry
Current assignee: Shandong Institute of Light Industry
Priority date: 2012-11-08
Filing date: 2012-11-08
Publication date: 2015-04-01
Anticipated expiration: 2032-11-08
Also published as: CN103146594A

Abstract

The invention belongs to the fields of bioengineering and biological pharmacy, and in particular to a Sorangiumcellulosum So2163 strain, and application of the strain to synthesis of epothilone. The Sorangiumcellulosum So2163 strain was preserved on June 10, 2012 in China Center for Type Culture Collection, and has a collection number CCTCC M 2012211. The Sorangiumcellulosum So2163 strain involved in the invention provides new strain resource for industrialization production of epothilone.

Description

Sorangium cellulosum strain and the application in ebormycine synthesis thereof

Technical field

The invention belongs to biotechnology and field of biological pharmacy, be specifically related to sorangium cellulosum strain sorangium cellulosumso2163, also relates to the application of this bacterial strain in ebormycine synthesis.

Background technology

Cancer is common disease and the frequently-occurring disease of a kind of serious threat human health and life, is the important scientific research task in the field of medical science of countries in the world, there is no satisfied remedy measures at present.Taxol is as the antitumor drug be most widely used at present, and within its year, sales volume reaches 2,000,000,000 dollars, accounts for 10% of the antitumor drug market share.Due to the good treatment prospect of its anticancer general expansion and non-neoplastic disease aspect, estimate that world demand amount will reach annual 2000 kilograms from now on, and the price of BMS company of U.S. formulation for paclitaxel is per kilogram 500-1000 ten thousand dollars.

Ebormycine has a class macrolides compound of identical antitumor mechanism as with taxol, can suppress the formation of cancer of late stage spindle body and spindle fibre, thus suppresses mitotic division, and tissue cancer cell rises in value, and also has the anticancer therapeutic of spectrum.Ebormycine also possesses following advantage compared with taxol simultaneously: good is water-soluble; Wider anticancer spectrum; The stronger rejection ability to resistant tumors; And the most important---it is the uniquely a kind of short microtubule polymerization active compound produced by fermentable found at present, possesses potentiality prepared by large scale fermentation.Since 1996 are found, become the study hotspot of antitumor drug gradually, a lot of epothilones has been had to enter the clinical assessment stage of different levels at present, and one of them---ixabepilong, the great trouble lactam structure analogue of ebormycine, go on the market through U.S. food Drug Administration (FDA) approval in October, 2007, illustrate good therapeutic action.Ebormycine class medicine is also considered to the renewal product of taxol, and its market capacity is not less than 1,000,000,000 dollars/year, and the annual sales amount of China also will more than 100,000,000 dollars.

The preparation method of Epothilone mainly contains three kinds at present, is chemical complete synthesizing process, biological synthesis process and chemical modification method respectively.

In view of its simple chemical structure and good antitumor curative effect, epothilone is once finding just to become the focus that bio-organic scholars pay close attention to, tens of complete synthesis routes have been had in succession to report at present, and comparatively detailed Structure-activity relationship is also derived, the complete synthesis work of epothilone is also carried out in last century Mo in China, but due to the structure of ebormycine relatively still comparatively complicated, chemical complete synthesis step is tediously long, productive rate is very low, does not possess the prospect of suitability for industrialized production.

People by the structure activity study of epothilone is found acyl moiety in its 16 membered macrolide structure and its biological activity closely related; and the modification of O-moieties is little to its activity influence, therefore develops new analog have a good application prospect by carrying out chemically modified to natural epothilone.Ixabepilone and the BMS-310705 being in clinical assessment of current approval listing are all analogs of chemically modified.But the supply that chemical modification method also must depend on large content of starting materials can be implemented.Therefore biosynthesizing remains unique effective way that epothilone produces.

What Epothilone producing strains was reported the earliest is come from the SMP44 bacterial strain of Emporia state university of the U.S. and come from the So ce90 bacterial strain of German National biotech research center GBF, and the output of its epothilone A is about 20mg/L.Domestic only have a few unit at present in the fermentative production of research ebormycine, the bacterial strain rare numbers that can apply and mostly be wild strain, therefore exploitation is found the new microorganism strains that can synthesize ebormycine and is had important theory significance and using value.

Summary of the invention

In order to solve above-mentioned technical problem, the invention provides can synthesize ebormycine sorangium cellulosum ( sorangium cellulosum) So2163, additionally provide the method utilizing this strain fermentation to produce ebormycine.

Sorangium cellulosum strain of the present invention ( sorangium cellulosum) So2163, this bacterial strain is preserved in China typical culture collection center, preservation address on June 10th, 2012: Wuhan University of Wuhan, China city, deposit number: CCTCC NO. M 2012211.

The 16S rDNA sequence fragment length of above-mentioned bacterial strains is 1466bp.

Its cultural method of sorangium cellulosum strain, comprises following step: adopt CNST to cultivate and cultivate 5 days based on 30 DEG C, and expand fully until bacterium colony and after forming sporophore structure, proceed to EMP fermention medium, culture temperature is 30 DEG C, rotating speed 200rpm, incubation time 7-8 days.

In the cultural method of sorangium cellulosum strain, in the EPM substratum that this bacterial strain of fermentation is used, with the addition of macropore class polymeric adsorbent, be preferably the XAD-16 macroporous adsorbent resin of 1%.

With Chromatographic Pure Methanol lixiviate resin absorption product after cultivation completes.

The present invention also will protect, sorangium cellulosum strain is producing antineoplastic medicament epothilones, at epothilones and take ebormycine as the structural derivative of parent nucleus, ebomycin A, epothilone B produce in application.

Beneficial effect of the present invention is, sorangium cellulosum strain So2163 of the present invention, it is the natural ebormycine producing strains that a strain is separated from pedotheque, ebormycine can be synthesized under liquid fermentation condition, therefore possess and be developed to the potentiality that medicinal raw material produces bacterial classification, huge economic benefit and marketable value can be brought.

Accompanying drawing explanation

The cell picture of bacterial strain in Fig. 1 embodiment 2;

Bacterial strain colonial morphology photo in Fig. 2 embodiment 2;

In Fig. 3 embodiment 4, the HPLC of strain fermentation crude extract detects spectrogram;

In Fig. 4 embodiment 4, the LC-MS of the ebomycin A of bacterial strain synthesis detects spectrogram.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art more understands the present invention, but does not limit the present invention with this.

Bacterial strain sorangium cellulosum ( sorangium cellulosum) So2163, this bacterial strain is preserved in China typical culture collection center, deposit number on June 10th, 2012: CCTCC M 2012211.

Raw materials usedly in embodiment all to buy from market, the experimental technique of unreceipted actual conditions in embodiment, all with reference to normal condition as the people such as Sambrook " Molecular Cloning: A Laboratory room handbook " (New York:Cold Spring Harbor Laboratory Press of showing, 1989), in embodiment, nuclear magnetic resonance analyser used is Bruker Avance600 type nuclear magnetic resonance spectrometer.

Embodiment 1

The separation method of bacterial strain So2163

Ground by pedotheque and be evenly sprinkled upon on CNST substratum afterwards, cultivate after 7-10 days for 30 DEG C and observe, after tentatively growing bacterium colony, picking is inoculated in new CNST substratum and carries out purification storage.

CNST culture medium prescription (/L): KNO ₃0.5g/L; Na ₂hPO ₄0.25g/L; MgSO ₄1g/L; PH 7.5; Solid medium adds the agar powder of 1.2%, and the filter paper of lay sterilising treatment does carbon source; For subsequent use after 102.34 kPa 30min moist heat sterilizations.

Embodiment 2

The Species estimation of bacterial strain So2163

(1) morphological method qualification

Adopt CNST to cultivate bacterial strain So2163 to cultivate 5 days based on 30 DEG C, expand fully until bacterium colony and carry out microscopy after forming sporophore structure, observe its somatic cells, sporophore shape and expansion mycoderm form (experimental result is shown in accompanying drawing 1) respectively, result shows, this bacterial strain possess typical sorangium cellulosum ( sorangium cellulosum) morphological feature.

(2) molecular biology method qualification

By design 16S rDNA bacterium universal amplification primer 2 7F(5 '-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R(5 '-TAC CTT GTT ACG ACT T-3 '), with bacterial strain So2163 genome for template carries out pcr amplification, obtain the 16S rDNA fragment that length is 1466bp, compare with sequence in GeneBank after checking order, find with sorangium cellulosum16S rDNA sequence homology reaches more than 99% (sequence results is shown in sequence table 1).

According to the result of embodiment 2, by this microorganism identification be sorangiumbelong to, called after So2163.

Embodiment 3

The cultural method of bacterial strain So2163 and tunning pretreatment process

Synthesis ebormycine adopts EPM substratum, culture temperature is 30 DEG C, rotating speed 200rpm, incubation time 7-8 days, XAD-16 macroporous adsorbent resin is collected after fermentation ends, 2 times of pure methyl alcohol lixiviates of volumetric chromatography 2 hours are adopted, after 10000rpm centrifugal treating 10min and obtain the crude extract that ferments after distilled water wash 2-3 time.

EPM culture medium prescription (/L): yam starch 2.0 g, glucose 2.0 g, soybean cake powder 2.0 g; Skimmed milk 1.0 g, MgSO ₄1.0 g, CaCl ₂1.0 g, VB ₁₂0.5 mg, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v), regulates pH 7.2,121 DEG C of sterilising treatment 20min.

Embodiment 4

The analysis and identification method of contained ebormycine in bacterial strain So2163 tunning

(1) thin-layer chromatography (TLC)

Use sherwood oil: acetone=4:1 developping agent system, So2163 fermentation crude extract is launched, GF254 silica-gel plate observes obvious dim spot by UV-irradiation, and compares with ebormycine standard substance, both discoveries have identical Rf value, and both preliminary proofs are same substance.

(2) high performance liquid chromatography (HPLC)

Selection analysis C-18 ODS chromatographic column (4.6 × 250 mm, Kromasil), chromatographic condition: determined wavelength 249nm, flow velocity 0.8 mL/min, moving phase is methyl alcohol: water=7:3, experiment shows that (retention time of ebomycin A is 7.5 min, and the retention time of epothilone B is 8.7 min) can calculate the content of ebormycine in tunning according to peak area containing the material (see accompanying drawing 3) identical with ebormycine standard substance retention time in tunning.

(3) LC-MS mass spectrum (LC-MS)

A small amount of fermented sample after preparative liquid chromatography purifying is used to carry out LC-MS detection in embodiment.Result shows that the main component molecular weight in sample is 493.4(+1 H ⁺), identical with ebomycin A standard substance molecular weight (as accompanying drawing 4), both explanations are same substance.

(4) nucleus magnetic resonance (NMR)

Use a certain amount of sample after HPLC purifying to carry out magnetic resonance detection in embodiment, show by experiment, fermented sample has identical response signal with epothilone B standard substance, is same substance both molecular structure proves.

Claims

1. sorangium cellulosum strain ( sorangium cellulosum) So2163, this bacterial strain is preserved in China typical culture collection center, deposit number on June 10th, 2012: CCTCC M 2012211; The 16S rDNA sequence fragment length of this bacterial strain is 1466bp.

Sorangium cellulosum strain 2. as described in claim 1 ( sorangium cellulosum) cultural method of So2163, comprise following step: adopt CNST to cultivate and cultivate 5 days based on 30 DEG C, until bacterium colony expansion fully and after forming sporophore structure, proceed to EMP fermention medium, culture temperature is 30 DEG C, rotating speed 200rpm, incubation time 7-8 days;

CNST culture medium prescription (/L): KNO ₃0.5g/L; Na ₂hPO ₄0.25g/L; MgSO ₄1g/L; PH 7.5; Solid medium adds the agar powder of 1.2%, and the filter paper of lay sterilising treatment does carbon source; For subsequent use after 102.34 kPa 30min moist heat sterilizations;

EPM culture medium prescription (/L): yam starch 2.0 g, glucose 2.0 g, soybean cake powder 2.0 g; Skimmed milk 1.0 g, MgSO ₄1.0 g, CaCl ₂1.0 g, VB12 0.5 mg, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v), regulate pH 7.2,121 DEG C of sterilising treatment 20min;

Ferment in this bacterial strain EPM substratum used and with the addition of macropore class polymeric adsorbent;

Described macropore class polymeric adsorbent is the XAD-16 macroporous adsorbent resin of 1%;

3. sorangium cellulosum strain as claimed in claim 1 ( sorangium cellulosum) application of So2163 in production antineoplastic medicament epothilones.

Sorangium cellulosum strain 4. as described in claim 1 ( sorangium cellulosum) So2163 is at epothilones and take ebormycine as application during the structural derivative of parent nucleus is produced.

Sorangium cellulosum strain 5. as described in claim 1 ( sorangium cellulosum) application of So2163 in ebomycin A, B produce.