CN101402929B

CN101402929B - A alkali-fast sorangium cellulosum and uses of the same in producing epothilone

Info

Publication number: CN101402929B
Application number: CN2008101575157A
Authority: CN
Inventors: 李越中; 胡玮; 刘红
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2008-10-06
Filing date: 2008-10-06
Publication date: 2010-10-13
Anticipated expiration: 2028-10-06
Also published as: CN101402929A

Abstract

The invention discloses an alkali proof Sorangium cellulosum strain named Sorangium cellulosum So0157-2, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC No. M208078 on May 27, 2008. The invention also discloses the application of the alkali proof Sorangium cellulosum to the preparation of Epothilones. The Epothilones generated by the fermentation of the alkali proof Sorangium cellulosum has high yield stable fermenting property of strains and short fermentation period, is not easy to cause bacteria contaminants and does not generate Epothilones C, D, E, F and other homologues, thus greatly reducing the difficulty in subsequent separation and purification and being an Epothilones producing strain with high value in research and development.

Description

One strain alkali proof fiber heap capsule bacterium and the application in the preparation ebormycine thereof

Technical field

The present invention relates to a strain sorangium cellulosum and an application thereof, but relate in particular to the alkali proof fiber heap capsule bacteria strain and the application in the preparation ebormycine thereof of a strain high yield ebormycine, belong to biological technical field.

Background technology

Short microtubule polymerization compounds taxol (Paclitaxel, ) and analogue Docetaxel

It is present the most successful clinically antineoplastic chemotherapy medicine, be used to the treatment of solid carcinomas such as ovarian cancer, thymic carcinoma, colorectal carcinoma, lung cancer and liver cancer, be U.S. food and medicine surveillance authority (U.S.Food and DrugAdministration, FDA) choice drug of the solid tumor chemotherapy of Ren Dinging.Having in the multiple natural compounds of same or similar biological function, has only ebormycine (Epothilones), [1S-[1R with taxol ^*, 3R ^*(E), 7R ^*, 10S ^*, 11R ^*, 12R ^*]]-7,11-dihydroxyl-8,8,10,16-tetramethyl--3-[1-methyl-2-(2-methyl-4-thiazolyl) vinyl]-4-azepine-17-oxabicyclo [14.1.0] heptadecane-5, the 9-diketone), derive from the tunning of microorganism, and be considered to the renewal product of taxol, be better cancer therapy drug.So far, existing five kinds of ebormycine compounds have entered the clinical trial of different steps, comprise two the semi-synthetic analogue BMS-247550 and the BMS-310705 of the epothilone B of Bristol-Myers Squibb company (BMS); The epothilone B of Novartis company (patupilone); The ebormycine D (KOS-862) of Roche company; And the ZK-EPO that has just entered the Berlex company of clinical trial recently.They are the tunning of sorangium cellulosum (Sorangiumcellulosum) in the slime bacteria (myxobacteia) or the simple chemical derivative after the fermentation.U.S. food and medicine surveillance authority have ratified Bristol-Myers Squibb company in October, 2007

(ixabepilone, BMS-247550) medicine as treatment mammary cancer goes on the market.

At present, the preparation method of ebormycine mainly contains chemical synthesis, biological synthesis process and chemical modification method three classes.The chemical complete synthesis route of ebormycine has tens kinds, has 6 kinds as the synthetic route of ebomycin A, and epothilone B has 11 kinds, and the complete synthesis route of ebormycine C and D has 2 kinds.But because the complete synthesis step of ebormycine chemistry is various, yield is lower, compares with other method still not possess advantage, does not have industrial production foreground.Carry out chemically modified by natural ebormycine and form multiple Aibomycin analogue, shown good application prospects fermentative production.But this method also must depend on biological synthesis process provides a large amount of precursor raw materials to implement.Therefore, biological synthesis process prepares the main flow direction that ebormycine becomes the research of ebormycine technology for producing.

Slime bacteria is the slidably unicellular gram negative bacterium of a class, has the special sociology behavioural characteristic and the complicated growth life history.By sliding motion, it can particularly grow the thin membranaceous expansion bacterium colony of formation on the barren substratum on solid medium.Under certain condition, the simple or complicated hypopus of sporophore structure-be made up of the cell and the myxospore of the different differentiation of flintiness can be assembled and form to the vegetative cell of slime bacteria.The generation bacterium of ebormycine is the sorangium cellulosum (Sorangium cellulosum) that belongs to slime bacteria, it is the unique monoid that can degraded cellulose class substrate in the slime bacteria, but the novel active compound that is separated to from slime bacteria so far derives from this monoid near half, and the secondary metabolite that sorangium cellulosum produces has stronger bacterial strain specificity, and is not only species specificity.Pile the capsule Pseudomonas in the chmosynthetic heterotrophs aerobic bacteria, some substratum commonly used, as Semen Maydis powder, soyflour, yeast cell, Zulkovsky starch, skim-milk, various peptones all are reasonable nutrient raw material.Cell concn can reach 10 for every milliliter during fermentation ⁹To 10 ¹⁰Individual, every liter of about 1-3 gram of dry cell weight, the temperature range of fermentation is 28-30 ℃, pH is 6.8-7.8.But,, utilize sorangium cellulosum to carry out the fermentative preparation ebormycine and still be faced with many problem demanding prompt solutions as the special case in the bacterium.

1. the sorangium cellulosum speed of growth is very slow, Dai Shida is more than 16 hours (be at present in all slime bacterias for the time the longest monoid), therefore fermentation period is longer, causes microbiological contamination when liquid state is cultivated easily, and for example the cycle of ebormycine fermentation is usually about 14 days;

2. the rate ratio of wild sorangium cellulosum secondary metabolite is lower, is generally 0.5-20mg/L, for example, utilizes the output of sorangium cellulosum So ce90 strain fermentation ebomycin A to be about 23mg/L;

3. by clone's ebormycine synthase gene bunch and thereby it is expressed the fermentative production ebormycine in other host also be the important directions of a research.But, with the test of streptomyces coelicolor (Streptomyces coelicolor), because ebormycine is for host's cytotoxicity and the result is undesirable as the host.2002, Julien bunch transfers to the synthase gene of ebormycine to express in the yellow myxococcus (Myxococcus xanthus) again and produces ebormycine D, the output of the synthetic ebormycine of this genetic engineering bacterium M.xanthus K111-40-1 fermentation is: batch fermentation was up to 30mg/L in 14 days, the 30 days production peaks of continuously fermenting reach 80mg/L, and 22 days production peaks of semicontinuous fermentation are 85mg/L.Report such as Boddy in 2004 produces ebormycine C with back four modules successful expression in intestinal bacteria of ebormycine synthetic gene; but necessary complex substrate to the artificial chemosynthesis of the outer confession of host bacterium (the pulsating N-acetyl-cysteine of C9-C21 thioesters, SNAC).2006, Mutka SC etc. reported whole ebormycine NRPS/PKS gene cluster produced ebormycine C and D at expression in escherichia coli, but output be LC-MS just can detected level.The huge difficult problem that these work faced is: ebormycine has the intensive cytotoxic activity for non-sorangium cellulosum host, and the resistance mechanism of ebormycine is indeterminate, and genes involved does not find, and makes that heterogenous expression output is lower;

4. in the separation and purification process of ebormycine, another problem that runs into is that homologue is too much, for example So ce90 bacterial strain can produce the ebormycine near 50 kinds of similar during the fermentation, and wherein, having only minority is to possess better activity can be developed to and be medicine.

So for the biosynthetic various trials of ebormycine, the key problem that solve all is how to improve output, reduce cost at present, realize industrialized extensive biological preparation.

It is generally acknowledged that slime bacteria is the bacterium of a class neutrophilia pH, its growth pH scope is generally at 6.5-8.0.In the outstanding Bacteria Identification handbook of uncle (the 8th edition), the suitableeest growth pH of sorangium cellulosum is decided to be 6.8-7.2 (this moment, this bacterium was named as many capsules of fiber bacterium, Polyangium cellulosum); In prokaryotic organism (III) book, the culture condition of the sorangium cellulosum of recommendation (as the VY/2 substratum) is neutral pH (being generally pH7.2); Learn to do in the volume (second edition) at the outstanding bacterial system of uncle, think that sorangium cellulosum can stand lower pH culture condition (5.8-6.2).From the microbial ecological angle, people separate the slime bacteria that has obtained the part kind from minority slant acidity environment (pH3.7,5.0 even 2.5), as myxococcus (Myxococcus), coral coccus (Corallococcus) and many capsules bacterium (Polyangium) etc.Under alkaline environment, just in the environmental sample of pH8.1-8.2, separate having obtained myxococcus (Myxococcusfulvus and Myxococcus stipitatus), do not identify whether they can grow under alkaline environment.Simultaneously, (pH7.9-8.1) also can detect the existence of capsule coccus (Angiococcus disclformis) in weakly alkaline environment.But, do not see any report about alkali proof fiber heap capsule bacterium.

Utilize alkali proof fiber heap capsule bacterium as fermentation strain, can overcome some drawbacks of art methods, realize the high yield of ebormycine.Yet the retrieval through document and patent utilizes the correlation technique of this thinking and method not to see document or patent report so far both at home and abroad.

Reference:

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Summary of the invention

At the deficiencies in the prior art, but the problem to be solved in the present invention provides the alkali proof fiber heap capsule bacteria strain and the application in the preparation ebormycine thereof of a strain high yield ebormycine.Utilize bacterial strain of the present invention to ferment effectively and synthesize the ebormycine compounds, shorten fermentation period, reduce living contaminants, improve the generation of fermentation yield and minimizing homologue, realization obtains the hope of efficient production ebormycine by microbe fermentation method.

The alkali proof fiber of high yield ebormycine of the present invention heap capsule bacterium (Sorangium cellulosum) is separation and purification from Yunnan Cheng Hai loke shore limit pedotheque and the sorangium cellulosum bacterial strain So0157-2 that obtains through subsequently seed selection, this bacterial strain is preserved in " Chinese typical culture collection center (CCTCC) " on May 27th, 2008, and its deposit number is: CCTCCNO:M208078.

The biological property of above-mentioned alkali proof fiber heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M208078 is: the Gram-negative aerobic bacteria, vegetative cell is harder rod-short 6-7 * 1-1.2 μ m, the blunt circle in two ends (Fig. 1-A), can carry out typical sliding motion (A and S motion) (Fig. 1-B) at solid surface, and can utilize the cellulose family substrate as the sole carbon source (Fig. 1-C), be most that growth temperature is 28-32 ℃ that grows.This bacterial strain can be grown (Fig. 2) under the culture condition of pH5-14, is a strain alkaline-resisting (facultative have a liking for alkali) bacterium.Along with the difference of culture condition (mainly being pH), its cell behavior and morphological specificity generation considerable change.When the pH value is 7.0-8.0, on the minimal medium that only contains filter paper (CNST substratum), vegetative cell shows intensive extended capability (Fig. 1-D) at early growth period, it is faint yellow that colony edge is, the filter paper that the cell expansion touches is almost completely degraded, and the filter paper fibre that neighbour cell does not touch is kept perfectly.To late stage of culture, vegetative cell is piled up the cell heap that forms similar sporophore (Fig. 1-E), present pale pink, but do not have sophisticated sporophore to occur in bacterium colony central authorities.When the pH value is 9.0-12.0, on the minimal medium that only contains filter paper (CNST substratum), vegetative cell shows very strong extended capability equally, and in late stage of culture, when cells contacting to filter paper when being utilized totally, the growth of cell has also just stopped, and enters the follow-up differentiation and development stage, can form typical orange sporophore structure (Fig. 1-F).Its sporophore is the accumulation product of many microsporangiums, sporangiocyst is spherical (Fig. 1-G), diameter 20-30 μ m, sporangiosorus does not have mucus bag quilt outward, and a large amount of myxospores (Fig. 1-H) exist is arranged in the sporangiocyst, shape and vegetative cell are similar, size is 2-3 * 1 μ m, is placed on 55 ℃ of heating in water bath processing 10min in the sterile distilled water, inoculation culture flat board (CNST substratum), can observe the sprouting of spore and form cell heap (Fig. 1-I), prove ripe myxospore of group with degeneration-resistant proterties.

The G+C content of bacterial strain alkali proof fiber heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M208078 of the present invention is 69.31mol%, the result who measures the gene order of 16S rRNA shows, its gene order length is 1555bp, and nucleotide sequence is shown in SEQ ID NO.1.

By using (the National Center for Biotechnology Information of U.S. biotechnology information center, NCBI) BLASTN program comparison, the gene order of many strains sorangium cellulosum (Sorangium cellulosum) 16S rRNA of the gene order of CCTCC NO:M208078 bacterial strain 16S rRNA of the present invention and NCBI registration has higher homology (98-99%).Further carry out system's generation analysis discovery by drawing evolutionary tree (Fig. 3), though CCTCCNO:M208078 bacterial strain of the present invention should classify as sorangium cellulosum (Sorangium cellulosum), but the evolutionary distance of itself and other sorangium cellulosum bacterial strain is all greater than 2.0%, for example the evolutionary distance with Sorangiumcellulosum Soce26 is 2.8%, with the evolutionary distance of Sorangium cellulosum So9741 be 4.3%, consider the alkaline-resisting growth characteristics of this bacterial strain simultaneously, judge that alkali proof fiber heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M 208078 bacterial strains of the present invention may be new classifications of sorangium cellulosum (Sorangium cellulosum).

The above-mentioned substratum that is used for thalli morphology observation and growth analysis is formed: KNO ₃0.5g/L, Na ₂HPO ₄0.25g/L, MgSO ₄7H ₂O1.0g/L, FeCl ₃0.01g/L, agar 1.6%, one of 6 centimetres of circular filter paper of diameter, TE solution 1.0ml/L looks requiring to transfer to different pH; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L.

The experimental technique of above-mentioned observation of morphological is with reference to Garrity GM, et al.Bergey ' s Manual ofSystematic Bacteriology (2nd edition), 2001,2,1132-1136.

Above-mentioned 16s rRNA genic system generation analytical procedure is with reference to C.Spr

Er, et al.The correlationbetween morphological and phylogenetic classification of myxobacterial, Int.J.Syst.Bacteriol.1999,49,1255-1262.

Alkali proof fiber heap capsule bacterium (Sorangium cellulosum) the So0157-2CCTCC NO:M208078 strain separating purifying of high yield ebormycine of the present invention and the basic skills of inducing and acclimating are:

Isolating pedotheque comes from loke shore limit, the journey sea soil at Yongsheng County, Lijiang City, Yunnan Province middle part, 1503 meters of sampling site height above sea level, and 18.7 ℃ of temperature on average, annual frostless, water quality alkalescence (pH8.6), the illumination abundance, pedotheque pH is 9.2 air-dry standby.Aseptic filter paper is layered on the CNST flat board, medium pH is 7.2, and to have added concentration be the filtration sterilization cycloheximide of 25 μ g/ml, just putting cultivation for 30 ℃, observe the spread scenarios of slime bacteria every day after 2 days under anatomical lens, and choose and pick out the expansion of existing slime bacteria and touch culture purified to the fresh CNST substratum, transfer to 10.0 with medium pH this moment.Just putting cultivation for 30 ℃, the sporophore structure appearred after 5 days, again longer sporophore is inoculated in the WCX flat board that is added with the 250ug/ml sulphuric acid kanamycin on the sterilized colibacillary bacterium line, to remove most of bacteriums, Mierocrystalline cellulose heap capsule bacterium with the group edge is transferred on the dull and stereotyped filter paper of CNST (pH10.0) at last, finishes purifying.Mature purifying slime bacteria sporophore on the flat board is scraped, forward on the aseptic filter paper of 1.5 * 3cm size, again filter paper is put in the aseptic culture presevation pipe kept dry.According to this flow process, obtained to produce the starting strain of the alkali proof fiber heap capsule bacterium 0157 of ebormycine as follow-up strain improvement.

The alkali proof fiber heap capsule bacterium 0157 that produces ebormycine with a strain is a starting strain, cultivates the inducing and acclimating that carries out bacterial strain by solid-liquid consecutive intervals repeatedly.Specific practice is: bacterial strain is carried out 30 ℃ earlier on the solid CNST of pH9.0 substratum and be inverted cultivation, can be observed sporophore after 7 days forms, the comparatively fresh cell inoculation in picking colony edge is in the liquid VY/2 of 100ml volume substratum, 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum that the 10ml fermented liquid is inoculated in the 90ml volume then, 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum that the 10ml fermented liquid is inoculated in the 90ml volume again, 30 ℃, the 200rpm concussion was cultivated 5 days, finished the cultivation flow process of an inducing and acclimating.The fermented liquid 1ml that cultivates for the last time is inoculated in carries out 30 ℃ on the solid CNST substratum of pH9.0 and be inverted and cultivate, begin second cultivation flow process.Repeat a plurality of flow processs in this way, the bacterial strain that obtains is carried out shake flask fermentation, by assessing the yields screening purpose bacterial strain of its growth conditions and mensuration ebormycine, final screening obtains the sorangium cellulosum bacterial strain of high yield ebormycine.

The above-mentioned CNST substratum that is used for strains separation and inducing and acclimating consists of: KNO ₃0.5g/L, Na ₂HPO ₄0.25g/L, MgSO ₄7H ₂O1.0g/L, FeCl ₃0.01g/L, TE solution 1.0ml/L, agar 16g/L looks requiring to transfer to different pH; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L.

The above-mentioned WCX substratum that is used for the bacterial strain purifying consists of: CaCl ₂2H ₂O1.5g/L; Agar 16g/L; KOH transfers to pH7.0.After the sterilization, add the cycloheximide of filtration sterilization by 25 μ g/ml amount.After making flat board, at the bait of the intensive line of media surface intestinal bacteria viable bacteria body as the formation of slime bacteria sporophore.Intestinal bacteria are by the LB medium preparation.

The above-mentioned VY/2 substratum that is used for the bacterial strain inducing domestication consists of: live yeast 5g/L; CaCl ₂0.8g/L; VB ₁₂0.5 μ g/ml; PH9.0.

The application of alkali proof fiber heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M208078 of the present invention in the preparation ebormycine.

Wherein, the sequence of steps that relates to of described application method is as follows:

(1) bacterial classification is selected: select sorangium cellulosum of the present invention (Sorangium cellulosum) So0157-2CCTCC NO:M208078 for use;

(2) slant culture activation: with bacterial classification inoculation in slant medium, under the 28-32 ℃ of condition, static cultivation 96-168 hour, standby;

(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, scrape with whole thalline of transfering loop with an inclined-plane, be inoculated in the 250mL triangular flask that the 30-50mL liquid seed culture medium is housed, putting the rotation rotating speed is that 180～220 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 72-120 hour for 28-32 ℃, get seed liquor;

(4) fermentation culture:

Shake flask fermentation: the inoculum size with the volume ratio of 10-20% is inoculated in the 500mL that the 80-120mL fermention medium is housed with seed liquor and shakes in the bottle, 28-32 ℃, rotating speed is 160-200 rev/min, shake flask fermentation 168-288 hours when thalline color in the fermented liquid from depth to shallow the time, stops fermentation;

The 5L ferment tank: the inoculum size with the volume ratio of 15-25% is inoculated in seed liquor in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, automatically monitor fermented liquid dissolved oxygen, temperature, pH value, mixing speed, liquid amount 3L, ventilation 1.0-2.0L/min, mixing speed 180-220rpm in 48 hours, mixing speed is controlled at 90-120rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 8.0-10.0; In order to shorten the vegetative period of thalline, as early as possible enter the secondary metabolism stage, can proceed to beginning in 25-35 hour in fermentation and in substratum, add glucose with the flow velocity of 1.8-2.2g/L and quicken thalli growth, proceed to 65-75 in fermentation and as a child stopped stream and add glucose; Total fermentation time is 168-220 hour, and the interval timing sampling is measured the ebormycine concentration in the fermented liquid in the fermenting process, when ebormycine concentration no longer rises in the fermented liquid, finishes fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 2～4 times of volume distilled water washs three times, seasoning; With dried fermentation resin with 8～10 times of volumes methanol vibration lixiviates; Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing; Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, measure the kind (Fig. 4) and the content of various ebormycine compounds in the fermented liquid with high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling.

Above-mentioned slant medium consists of: potato starch 8g/L, soy peptone 2g/L, glucose 2g/L, yeast extract paste 2g/L, MgSO ₄1g/L, CaCL ₂1g/L, TE solution 1.0ml/L, agar 1.5%, pH8.0; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L;

The aforesaid liquid seed culture medium is formed: yam starch 2g/L, glucose 2g/L, yeast extract paste 1g/L, peptone 1g/L, corn steep liquor 1g/L, sal epsom 0.5g/L, calcium chloride 0.15g/L, FeCl ₃-EDTA0.02g/L, TE solution 1ml/L, HEPES1%, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v) is transferred pH to 8.0,121 ℃ of sterilizations with potassium hydroxide solution; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L;

Consisting of of above-mentioned fermention medium: yam starch 4g/L, glucose 2g/L, peptone 4g/L, soybean cake powder 2g/L, skim-milk 1g/L, Na ₂HPO ₄0.15g/L, K ₂HPO ₄0.15g/L, MgSO ₄1.5g/L, CaCl ₂1.5g/L, FeCl ₃-EDTA0.01g/L, Thr5mg/L, VB ₁₂1mg/L, initial pH adjusts to 9.0.

In the above-mentioned application, preferred 30 ℃ ± 0.2 ℃ of step (2), (3), (4) described culture temperature.

In the above-mentioned application, preferred 120 hours of the described incubation time of step (2).

In the above-mentioned application, the preferred 40mL of the described substratum consumption of step (3) (250mL triangular flask)

In the above-mentioned application, preferred 200 rev/mins of the rotation rotating speed of the described shaking table of step (3).

In the above-mentioned application, preferred 96 hours of the described incubation time of step (3).

In the above-mentioned application, preferred 15% volume ratio of inoculum size of seed liquor during the described shake flask fermentation of step (4).

In the above-mentioned application, the preferred 100mL of fermention medium consumption (500mL triangular flask) during the described shake flask fermentation of step (4).

In the above-mentioned application, preferred 180 rev/mins of the rotation rotating speed of shaking table during the described shake flask fermentation of step (4).

In the above-mentioned application, incubation time is preferred 216 hours during the described shake flask fermentation of step (4).

In the above-mentioned application, preferred 20% volume ratio of inoculum size of seed liquor during the described 5L ferment tank of step (4).

In the above-mentioned application, the preferred 1.5L/min of ventilation during the described 5L ferment tank of step (4).

In the above-mentioned application, preferred 200 rev/mins of the mixing speed during the described 5L ferment tank of step (4) in 48 hours.

In the above-mentioned application, preferred 100 rev/mins of the mixing speed during the described 5L ferment tank of step (4) after 48 hours.

In the above-mentioned application, the fermented liquid pH preferred 9.0 ± 0.3 during the described 5L ferment tank of step (4) in the fermenting process.

In the above-mentioned application, the time opening that stream adds glucose in the fermenting process during the described 5L ferment tank of step (4) preferably ferments and proceeds to 30 hours.

In the above-mentioned application, the concluding time that stream adds glucose in the fermenting process during the described 5L ferment tank of step (4) preferably ferments and proceeds to 70 hours.

In the above-mentioned application, during the described 5L ferment tank of step (4) in the fermenting process stream add the preferred 2.0g/L of flow velocity of glucose.

In the above-mentioned application, total fermentation time is preferred 192 hours during the described 5L ferment tank of step (4).

The kind of ebormycine and concentration are measured as follows in the above-mentioned fermented liquid:

Utilize high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to carry out the qualitative and quantitative analysis for the ebormycine compounds.

The HPLC analysis condition: chromatographic column is Shim-pack VP-ODS, 4.6 * 250mm, 4.60 ± 0.3 μ m; Applied sample amount is 10 μ l; Moving phase be methyl alcohol and 0.2% acetic acid aqueous solution (65:35, v:v); Flow velocity is 1.0ml/min; Column temperature is 28 ℃.

The DAD testing conditions: will detect wavelength is 249nm, can measure the residence time (Rt) of ebormycine compounds, and compare in order to tentatively qualitative with standard substance: the residence time of ebomycin A is about 12.6 minutes, and the elution time of epothilone B is (Fig. 5 A) about 15.2 minutes.Utilize external standard method, the use standard is joined the drawing standard curve, can utilize the area at fermented liquid related compound peak in the color atlas under the 249nm that the ebormycine compounds is carried out quantitative analysis.Simultaneously, can utilize the full wavelength scanner function of DAD detector, the uv atlas (UV Spectra) that obtains compound is used for further qualitative: the ebormycine compounds all has two absorption peaks in the 200-400nm scope, the λ max at peak 1 is 210nm, log ε=4.17; The λ max at peak 2 is 249nm, and log ε=3.97 (Fig. 5 B, C).

The MS testing conditions: adopt one pole quadrupole mass spectrum, utilize electrospray ionization source (ESI) to be connected with LC, the mass spectrogram of mensuration ebormycine compounds is used for carrying out qualitative fully, and analysis condition is: 500 ℃ of ESI probe temperature; Vertebral foramen voltage is 30V; Nitrogen flow rate is 1ml/min; Mass scanning scope m/z is 100-1000, positive ion mode.The spectrogram result who obtains should be: the molecular ion peak of ebomycin A is [M+Na] ⁺516m/Z (Fig. 5 D); The molecular ion peak of epothilone B is [M+Na] ⁺530m/Z (Fig. 5 E).

Alkali proof fiber of the present invention heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M208078 can ferment, and to generate the purpose ebormycine (mainly be ebomycin A and B, Fig. 4 A-B), ebormycine productive rate height in the unit volume fermented liquid, the strain fermentation stable performance, be difficult for taking place living contaminants, fermentation period is short, and do not produce ebormycine C, D, homologue such as E and F (Fig. 4 C-D), greatly having reduced follow-up separation purification difficulty, is that a strain has the ebormycine production bacterial strain that research and development are worth.

Through experiment confirm: utilize alkali proof fiber heap capsule bacterium (Sorangium cellulosum) So0157-2CCTCC NO:M208078 of the present invention fermentation, the 500ml triangular flask carries out shake flask fermentation and reaches as high as more than the 98mg/L through 216 hours ebomycin A yields, and epothilone B output reaches as high as more than the 52mg/L; The 5L ferment tank reaches as high as more than the 117mg/L through 196 hours ebomycin A yields, and epothilone B output reaches as high as more than the 61mg/L.

The inventor does not retrieve and utilizes alkali proof fiber heap capsule bacterium is the bibliographical information of purpose product to produce ebormycine, does not also retrieve the fibrous capsule bacterium and produces ebormycine and the identical high bibliographical information of high yield, short period and purity of alkali proof fiber heap capsule bacterium (Sorangiumcellulosum) So0157-2CCTCC NO:M208078 bacterial strain of the present invention.

Description of drawings

Bacterial strain sorangium cellulosum provided by the invention (Sorangium cellulosum) So0157-2 has been preserved in " Chinese typical culture collection center (CCTCC) " on May 27th, 2008, preservation address: Wuhan City Wuhan University China typical culture collection center, postcode: 430072, its deposit number is: CCTCC NO:M208078.

Fig. 1 shows the grown form feature of sorangium cellulosum CCTCC NO:M208078.Wherein A illustrates the vegetative cell form (* 5000) of CCTCC NO:M208078 bacterial strain; The cell movement process (* 320) of B diagram CCTCC NO:M208078 bacterial strain; C diagram CCTCC NO:M208078 strain cell is for the degradation capability (* 5000) of filter paper medium; The spread scenarios (* 6) of D diagram CCTCC NO:M208078 bacterial strain on the CNST substratum; Cell heap group and bacterium colony (* 4) that E diagram CCTCC NO:M208078 bacterial strain forms on the CNST of pH7 substratum; The sporophore structure (* 20) that F diagram CCTCC NO:M208078 bacterial strain forms on the CNST of pH9 substratum; Microsporangium (* 800) in the sporophore structure that G diagram CCTCC NO:M208078 bacterial strain forms on the CNST of pH9 substratum; Myxospore (* 5000) in the sporophore structure that H diagram CCTCCNO:M208078 bacterial strain forms on the CNST of pH9 substratum; The cell heap group (* 40) that the myxospore of I diagram CCTCC NO:M208078 bacterial strain forms when sprouting again.

Fig. 2 shows that sorangium cellulosum CCTCC NO:M208078 is at the growing state of 30 ℃ of cultivations after 10 days on the CNST of the pH4-14 substratum.

Fig. 3 shows the evolutionary tree of drawing based on 16s rRNA gene ordered series of numbers, has shown the relation of sorangium cellulosum CCTCC NO:M208078 and other slime bacteria.Scale is an evolutionary distance unit, the numeral in the branch the nested number of support from 2000 samples.

Fig. 4 shows the chemical structure of several main ebormycine compounds.Wherein A illustrates the chemical structure of ebomycin A; Wherein B illustrates the chemical structure of epothilone B; Wherein C illustrates the chemical structure of ebormycine C; Wherein D illustrates the chemical structure of ebormycine D; Wherein E illustrates the chemical structure of ebormycine E; Wherein F illustrates the chemical structure of ebormycine F.

Fig. 5 shows the HPLC-DAD-MS analytical results of sorangium cellulosum CCTCC NO:M208078 tunning.Wherein scheme A and show the HPLC analytical results, the detection wavelength is 249nm, and the residence time of ebomycin A is 12.6 minutes, and the residence time of epothilone B is 15.2; Figure B shows the uv atlas of the ebomycin A that the full wavelength scanner function of utilizing the DAD detector is obtained, and two absorption peaks (210nm and 249nm) are all arranged in the 200-400nm scope; Figure C shows the uv atlas of the epothilone B that the full wavelength scanner function of utilizing the DAD detector is obtained, and two absorption peaks (210nm and 249nm) are all arranged in the 200-400nm scope; Figure D shows the mass spectrum that utilizes the MS detector to measure ebomycin A, and molecular ion peak is [M+Na] ⁺516.3m/Z; Figure D shows the mass spectrum that utilizes the MS detector to measure epothilone B, and molecular ion peak is [M+Na] ⁺530.3m/Z.

Embodiment

Embodiment 1 utilizes inducing and acclimating to obtain the alkali proof fiber heap capsule bacteria strain of high yield ebormycine

The separation of starting strain and purifying: isolating pedotheque comes from loke shore limit, the journey sea soil at middle part, Yongsheng County, Lijiang City, Yunnan Province, 1503 meters of sampling site height above sea level, 18.7 ℃ of temperature on average, annual frostless, water quality alkalescence (pH8.6), the illumination abundance, pedotheque pH is 9.2 air-dry standby.Aseptic filter paper is layered on the CNST flat board, medium pH is 7.2, and to have added concentration be the filtration sterilization cycloheximide of 25 μ g/ml, just putting cultivation for 30 ℃, observe the spread scenarios of slime bacteria every day after 2 days under anatomical lens, and choose and pick out the expansion of existing slime bacteria and touch culture purified to the fresh CNST substratum, transfer to 10.0 with medium pH this moment.Just putting cultivation for 30 ℃, the sporophore structure appearred after 5 days, again longer sporophore is inoculated in the WCX flat board that is added with the 250ug/ml sulphuric acid kanamycin on the sterilized colibacillary bacterium line, to remove most of bacteriums, Mierocrystalline cellulose heap capsule bacterium with the group edge is transferred on the dull and stereotyped filter paper of CNST (pH10.0) at last, finishes purifying.Mature purifying slime bacteria sporophore on the flat board is scraped, forward on the aseptic filter paper of 1.5 * 3cm size, again filter paper is put in the aseptic culture presevation pipe kept dry.According to this flow process, obtained to produce the starting strain of the alkali proof fiber heap capsule bacterium 0157 of ebormycine as follow-up strain improvement.

The alkali proof fiber heap capsule bacterium 0157 that produces ebormycine with a strain is a starting strain, cultivates the inducing and acclimating that carries out bacterial strain by solid-liquid consecutive intervals repeatedly.

Specific practice is: bacterial strain is carried out 30 ℃ earlier on the solid CNST of pH9.0 medium slant and be inverted cultivation, can be observed sporophore after 7 days forms, the comparatively fresh cell inoculation in picking colony edge is in the liquid VY/2 of 100ml volume substratum (500ml triangular flask), 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum (500ml triangular flask) that the 10ml fermented liquid is inoculated in the 90ml volume then, 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum (500ml triangular flask) that the 10ml fermented liquid is inoculated in the 90ml volume again, 30 ℃, the 200rpm concussion was cultivated 5 days, finished the cultivation flow process of an inducing and acclimating.The fermented liquid 1ml that cultivates for the last time is inoculated in carries out 30 ℃ on the solid CNST substratum (9cm flat board) of pH9.0 and be inverted and cultivate, can be observed sporophore after 7 days forms, the comparatively fresh cell inoculation in picking colony edge is in the liquid VY/2 of 100ml volume substratum (500ml triangular flask), 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum (500ml triangular flask) that the 10ml fermented liquid is inoculated in the 90ml volume then, 30 ℃, the 200rpm concussion was cultivated 5 days, get the liquid VY/2 substratum (500ml triangular flask) that the 10ml fermented liquid is inoculated in the 90ml volume again, 30 ℃, the 200rpm concussion was cultivated 5 days, finished the cultivation flow process of an inducing and acclimating.The fermented liquid 1ml that cultivates for the last time is inoculated in carries out 30 ℃ on the solid CNST substratum (9cm flat board) of pH9.0 and be inverted and cultivate, begin the 3rd and cultivate flow process.Repeat a plurality of flow processs in this way, the bacterial strain that obtains is carried out shake flask fermentation, by assessing the yields screening purpose bacterial strain of its growth conditions and mensuration ebormycine.

The output of initial 0157 bacterial strain ebomycin A is 9.2mg/L, and the output of epothilone B is 2.7mg/L; After having carried out the 10th domestication flow process, the output that obtains bacterial strain 0157-1-10 ebomycin A is 15.9mg/L, and the output of epothilone B is 8.3mg/L; After having carried out 21 inducing and acclimating flow processs, measuring the fermentation rate of finding bacterial strain accelerates greatly, ebormycine output obviously increases, final contriver therefrom screens the sorangium cellulosum bacterial strain So0157-2 that has obtained plant height product ebormycine, the output of its ebomycin A is more than the 30mg/L, the output of epothilone B is more than the 12mg/L, carries out follow-up inducing and acclimating flow process the ebormycine generation ability of bacterial strain is improved once more; Described bacterial strain So0157-2 is preserved in Chinese typical culture collection center (Wuhan University, Chinese Wuhan) on May 27th, 2008, and the preservation center is numbered: sorangium cellulosum (Sorangium cellulosum) So0157-2CCTCC NO:M208078.

The above-mentioned CNST substratum that is used for strains separation and inducing and acclimating consists of: KNO ₃0.5g/L, Na ₂HPO ₄0.25g/L, MgSO ₄7H ₂O1.0g/L, FeCl ₃0.01g/L, TE solution 1.0ml/L, agar 1.6% looks requiring to transfer to different pH; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L.

The above-mentioned WCX substratum that is used for the bacterial strain purifying consists of: CaCl ₂2H ₂O0.15%; Agar 1.6%; KOH transfers to pH7.0.After the sterilization, add the cycloheximide of filtration sterilization by 25 μ g/ml amount.After making flat board, at the bait of the intensive line of media surface intestinal bacteria viable bacteria body as the formation of slime bacteria sporophore.Intestinal bacteria are by the LB medium preparation.

The above-mentioned VY/2 substratum that is used for the bacterial strain inducing domestication consists of: live yeast 0.5%; CaCl ₂0.08%; VB ₁₂0.5 μ g/ml; PH9.0.

The order-checking of embodiment 2 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 bacterial strain 16S rRNA gene

The alkali proof fiber heap capsule bacterium So0157-2 of the high yield ebormycine that embodiment 1 inducing and acclimating is obtained, be that (Shanghai Sangon BiologicalEngineering Technology and Service Co. Ltd.) carries out 16S rRNA gene sequencing in CCTCCNO:M208078 bacterial strain trust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

Experimental technique is: the thalline of CCTCC NO:M208078 bacterial strain is beaten with rolling bottle even, centrifugal, and with STE solution suspend (about every 0.1g thalline suspends with 0.5ml STE solution); The 0.5ml thalline is added in the centrifuge tube of 5ml, use the vibrator mixing, add 400 μ l10%SDS, the Proteinase K of 30 μ l20mg/ml, abundant mixing, 37 ℃ are incubated 1 hour; Add 0.4ml5M NaCl, left standstill behind the mixing 10 minutes, add 0.4ml CTAB/NaCl then, mixing, 65 ℃ are incubated 20 minutes; Phenol-chloroform/primary isoamyl alcohol (25:24:1) extracting of taking-up centrifuge tube and usefulness equal-volume (2.4ml) 0.5-1 hour, the centrifugal 10min of 7000rpm, supernatant goes to clean centrifuge tube; Repeat above-mentioned chloroform/primary isoamyl alcohol extracting, the centrifugal 10min of 7000rpm.Supernatant is gone to clean centrifuge tube; Add the dehydrated alcohol (or Virahol of 1 times of volume) of 2 times of volumes, put upside down mixing, room temperature left standstill 10 minutes, and the centrifugal 20min of 5000rpm abandons supernatant; After empty slightly the doing, DNA is dissolved in 500 μ lSSC liquid (in the Eppendorf pipe of the preparation of SSC liquid: 1.5ml, adds 20 * SSC liquid of 50 μ l, the ddH of 950 μ l ₂O, mixing) in, adds 2% volume RNA enzyme (1mg/ml, final concentration to 50 μ g/ml-10 μ g/ml), 37 ℃ of water-bath effects 1-2 hour; Add isopyknic chloroform/primary isoamyl alcohol extracting, 30min.The centrifugal 10min of 7000rpm.Supernatant is gone to clean Eppendorf pipe; Add 1/10 volume 3M NaAc, the dehydrated alcohol of 2 times of volumes (or Virahol of 1 times of volume), the centrifugal 20min of 5000rpm; Remove supernatant, use 70% washing with alcohol, the centrifugal 10min of 5000rpm, drying at room temperature to ethanol volatilizees fully, and chromosomal DNA is dissolved in 80-100 μ lddH ₂Among the O ,-20 ℃ of preservations.

The genomic dna that extracts carries out electrophoresis detection and concentration purity check: get 3 μ l DNA samples and carry out the agarose electrophoresis detection, use 0.8% sepharose, every centimetre of voltage is no more than 2 volts.

Utilize PCR method amplification bacterial strain 16S rRNA gene order, and gene is carried out sequencing: require chromosomal DNA template length greater than 20kb, 25 μ l system template amounts are about 10-100ng; The primer is bacterial 16 S rRNA gene universal amplification primer GAGTTTGATCCTGGCTCAG (F primer) and AGAAAGGAGGTGATCC AGCC (R primer)

Reaction system is:

Reaction process:

96℃，3min

72℃，10min

4℃，hold

Get PCR reaction product 5 μ l and carry out electrophoresis detection, 0.8% sepharose, 100V electrophoresis.Gained PCR product is cut glue with Omega Gel extraction kit and is reclaimed order-checking, sequencing primer F and R.

The result who measures the gene order of 16S rRNA shows that its gene order length is 1555bp, and nucleotide sequence is shown in SEQ ID NO.1.

By using (the National Center for Biotechnology Information of U.S. biotechnology information center, NCBI) BLASTN program comparison, the gene order of many strains sorangium cellulosum (Sorangium cellulosum) 16S rRNA of the gene order of CCTCC NO:M 208078 bacterial strain 16S rRNA of the present invention and NCBI registration has higher homology (98-99%).Further carry out system's generation analysis discovery by drawing evolutionary tree (Fig. 3), though CCTCCNO:M 208078 bacterial strains of the present invention should classify as sorangium cellulosum (Sorangium cellulosum), but the evolutionary distance of itself and other sorangium cellulosum bacterial strain is all greater than 2.0%, for example the evolutionary distance with Sorangium cellulosum Soce26 is 2.8%, with the evolutionary distance of Sorangium cellulosum So9741 be 4.3%, consider the alkaline-resisting growth characteristics of this bacterial strain simultaneously, judge that CCTCC NO:M 208078 bacterial strains of the present invention may be new classifications of sorangium cellulosum (Sorangiumcellulosum).

Above-mentioned 16s rRNA genic system generation analytical procedure is with reference to C.

, et al.The correlationbetween morphological and phylogenetic classification of myxobacterial, Int.J.Syst.Bacteriol.1999,49,1255-1262.

The application of embodiment 3 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(1) bacterial classification is selected: select sorangium cellulosum of the present invention (Sorangium cellulosum) So0157-2CCTCCNO:M208078 for use;

(2) slant culture activation: with bacterial classification inoculation in slant medium, under 32 ℃ of conditions, static cultivation 96 hours, standby;

(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, scrape with whole thalline of transfering loop with an inclined-plane, be inoculated in 50mL (250mL triangular flask) liquid seed culture medium, putting the rotation rotating speed is that 180 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 72 hours for 32 ℃, get seed liquor;

(4) fermentation culture:

Shake flask fermentation: the inoculum size of volume ratio with 10%, with seed liquor be inoculated in 120mL (500mL triangular flask) fermention medium is housed shake in the bottle 32 ℃, rotating speed is 160 rev/mins, shake flask fermentation 168 hours when thalline color in the fermented liquid from depth to shallow the time, stops fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content that utilizes high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to measure ebomycin A in the fermented liquid is 62mg/L, and the content of epothilone B is 27mg/L.

Above-mentioned slant medium consists of: potato starch 8g/L, soy peptone 2g/L, glucose 2g/L, yeast extract paste 2g/L, MgSO ₄1g/L, CaCL ₂1g/L, TE solution 1.0ml/L, agar 15g/L, pH8.0; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L;

The application of embodiment 4 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(2) slant culture activation: with bacterial classification inoculation in slant medium, under 28 ℃ of conditions, static cultivation 168 hours, standby;

(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, scrape with whole thalline of transfering loop with an inclined-plane, be inoculated in 30mL (250mL triangular flask) liquid seed culture medium, putting the rotation rotating speed is that 220 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 100 hours for 28 ℃, get seed liquor;

(4) fermentation culture:

Shake flask fermentation: the inoculum size of volume ratio with 20%, with seed liquor be inoculated in 100mL (500mL triangular flask) fermention medium is housed shake in the bottle 28 ℃, rotating speed is 200 rev/mins, shake flask fermentation 288 hours when thalline color in the fermented liquid from depth to shallow the time, stops fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content that utilizes high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to measure ebomycin A in the fermented liquid is 79.5mg/L, and the content of epothilone B is 46mg/L.

The aforesaid liquid seed culture medium is formed: yam starch 2g/L, glucose 2g/L, yeast extract paste 1g/L, peptone 1g/L, corn steep liquor 1g/L, sal epsom 0.5g/L, calcium chloride 0.15g/L, FeCl ₃-EDTA0.02g/L, TE solution 1ml/L, HEPES1%, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v) is transferred pH to 8.0,12l ℃ of sterilization with potassium hydroxide solution; Wherein TE solution consists of MnCl ₂4H ₂O100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl5mg/L, SnCl ₂2H ₂O5mg/L, H ₃BO ₃10mg/L, KBr20mg/L, KI20mg/L;

The application of embodiment 5 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(2) slant culture activation: with bacterial classification inoculation in slant medium, under 30 ℃ of conditions, static cultivation 120 hours, standby;

(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, scrape with whole thalline of transfering loop with an inclined-plane, be inoculated in 40mL (250mL triangular flask) liquid seed culture medium, putting the rotation rotating speed is that 200 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 96 hours for 30 ℃, get seed liquor;

(4) fermentation culture:

Shake flask fermentation: the inoculum size of volume ratio with 15%, with seed liquor be inoculated in 100mL (500mL triangular flask) fermention medium is housed shake in the bottle 30 ℃, rotating speed is 180 rev/mins, shake flask fermentation 216 hours when thalline color in the fermented liquid from depth to shallow the time, stops fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content that utilizes high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to measure ebomycin A in the fermented liquid is 98mg/L, and the content of epothilone B is 52mg/L.

The application of embodiment 6 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(4) fermentation culture:

5L ferment tank: with 15% inoculum size seed culture fluid is inoculated in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, monitors fermented liquid dissolved oxygen, temperature, pH value, mixing speed automatically.Liquid amount 3L, ventilation 1.0L/min, mixing speed 180rpm in 48 hours, mixing speed is controlled at 90rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 8.0.Total fermentation time is 168 hours, and the interval timing sampling is measured the ebormycine concentration in the fermented liquid in the fermenting process, when ebormycine concentration no longer rises in the fermented liquid, finishes fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content that utilizes high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to measure ebomycin A in the fermented liquid is 57mg/L, and the content of epothilone B is 26mg/L.

The application of embodiment 7 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(3) seed culture: with the bacterial strain of step (2) cultivation, under aseptic condition, scrape with whole thalline of transfering loop with an inclined-plane, be inoculated in 30mL (250mL triangular flask) liquid seed culture medium, putting the rotation rotating speed is that 220 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 120 hours for 28 ℃, get seed liquor;

(4) fermentation culture:

5L ferment tank: with 25% inoculum size seed culture fluid is inoculated in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, monitors fermented liquid dissolved oxygen, temperature, pH value, mixing speed automatically.Liquid amount 3L, ventilation 2.0L/min, mixing speed 220rpm in 48 hours, mixing speed is controlled at 120rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 10.0.In order to shorten the vegetative period of thalline, as early as possible enter the secondary metabolism stage, can proceed to beginning in 25 hours in fermentation and in substratum, add glucose with the flow velocity of 2.2g/L and quicken thalli growth, proceed to 75 in fermentation and as a child stopped stream and add glucose.Total fermentation time is 220 hours, and the interval timing sampling is measured the ebormycine concentration in the fermented liquid in the fermenting process, when ebormycine concentration no longer rises in the fermented liquid, finishes fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content that utilizes high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling to measure ebomycin A in the fermented liquid is 91mg/L, and the content of epothilone B is 53mg/L.

The application of embodiment 8 sorangium cellulosums (Sorangium cellulosum) So0157-2CCTCC NO:M208078 in the preparation ebormycine

(4) fermentation culture:

5L ferment tank: with 20% inoculum size seed culture fluid is inoculated in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, monitors fermented liquid dissolved oxygen, temperature, pH value, mixing speed automatically.Liquid amount 3L, ventilation 1.5L/min, mixing speed 200rpm in 48 hours, mixing speed is controlled at 100rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 9.0.In order to shorten the vegetative period of thalline, as early as possible enter the secondary metabolism stage, can proceed to beginning in 30 hours in fermentation and in substratum, add glucose with the flow velocity of 2.0g/L and quicken thalli growth, proceed to 70 in fermentation and as a child stopped stream and add glucose.Total fermentation time is 192 hours, and the interval timing sampling is measured the ebormycine concentration in the fermented liquid in the fermenting process, when ebormycine concentration no longer rises in the fermented liquid, finishes fermentation;

(5) product detects: get above-mentioned fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin (Rohm and Haas company), resin is with 3 times of volume distilled water washs three times, seasoning.With dried fermentation resin with 10 times of volumes methanol vibration lixiviates.Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing.Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, the content of measuring ebomycin A in the fermented liquid with high performance liquid chromatography (HPLC)-diode-array detector (DAD)-mass detector (MS) coupling is 117mg/L, and the content of epothilone B is 61mg/L.

Above-mentioned slant medium consists of: potato starch 0.8%, soy peptone 0.2%, glucose 0.2%, yeast extract paste 0.2%, MgSO ₄0.1%, CaCL ₂0.1%, TE solution 1.0ml/L, agar 1.5%, pH8.0;

The aforesaid liquid seed culture medium is formed: yam starch 0.2%, glucose 0.2%, yeast extract paste 0.1%, peptone 0.1%, corn steep liquor 0.1%, sal epsom 0.05%, calcium chloride 0.15%, FeCl ₃-EDTA0.002%, TE solution 1ml/L, HEPES1%, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v) is transferred pH to 8.0,121 ℃ of sterilizations with potassium hydroxide solution;

Consisting of of above-mentioned fermention medium: yam starch 0.4%, glucose 0.2%, peptone 0.4%, soybean cake powder 0.2%, skim-milk 0.1%, Na ₂HPO ₄0.015%, K ₂HPO ₄0.015%, MgSO ₄0.15%, CaCl ₂0.15%, FeCl ₃-EDTA0.001%, Thr5mg/L, VB ₁₂1mg/L, initial pH adjusts to 9.0.

The kind and the concentration of the described ebormycine of the foregoing description are measured as follows:

The DAD testing conditions: will detect wavelength is 249nm, can measure the residence time (Rt) of ebormycine compounds, and compare in order to tentatively qualitative with standard substance: the residence time of ebomycin A is about 12.6 minutes, and the elution time of epothilone B is (Fig. 5 A) about 15.2 minutes.Utilize external standard method, the use standard is joined the drawing standard curve, and equation of linear regression is:

The concentration of ebomycin A=1.31471 * 10 ^-7The peak area R of * ebomycin A ²=0.998528

The concentration of epothilone B=1.23976 * 10 ^-7The peak area R of * epothilone B ²=0.994916

Thereby can utilize the area at fermented liquid related compound peak in the color atlas under the 249nm that the ebormycine compounds is carried out quantitative analysis.

Simultaneously, can utilize the full wavelength scanner function of DAD detector, the uv atlas (UV Spectra) that obtains compound is used for further qualitative: the ebormycine compounds all has two absorption peaks in the 200-400nm scope, the λ max at peak 1 is 210nm, log ε=4.17; The λ max at peak 2 is 249nm, and log ε=3.97 (Fig. 5 B, C).

The MS testing conditions: adopt one pole quadrupole mass spectrum, utilize electrospray ionization source (ESI) to be connected with LC, the mass spectrogram of mensuration ebormycine compounds is used for carrying out qualitative fully, and analysis condition is: 500 ℃ of ESI probe temperature; Vertebral foramen voltage is 30V; Nitrogen flow rate is 1ml/min; Mass scanning scope m/z is 100-1000, positive ion mode.

The spectrogram result who obtains should be: the molecular ion peak of ebomycin A is [M+Na] ⁺(516.3m/Z Fig. 5 D); The molecular ion peak of epothilone B is [M+Na] ⁺(530.3m/Z Fig. 5 E).

Sequence table

＜110〉Shandong University

＜120〉strain alkali proof fiber heap capsule bacterium and the application in the preparation ebormycine thereof

<141>2008-9-21

<160>1

<210>1

<211>1555

<212>DNA

＜213〉sorangium cellulosum (Sorangium cellulosum)

＜221〉sorangium cellulosum (Sorangium cellulosum) So0157-2CCTCCNO:M20807816S rRNA gene

<222>(1)…(1555)

<400>1

Claims

1. a strain alkali proof fiber is piled the capsule bacterium, it is characterized in that: this bacterial strain is called sorangium cellulosum (Sorangium cellulosum) So0157-2, this bacterial strain is preserved in " Chinese typical culture collection center (CCTCC) " on May 27th, 2008, and its deposit number is: CCTCC NO:M 208078.

2. the application of the described alkali proof fiber heap of claim 1 capsule bacterium in the preparation ebormycine, it is characterized in that: the sequence of steps of described application is as follows:

(1) bacterial classification is selected: select sorangium cellulosum (Sorangium cellulosum) So0157-2CCTCC NO:M208078 for use;

(4) fermentation culture:

Shake flask fermentation: the inoculum size with the volume ratio of 10-20% is inoculated in the 500mL that the 80-120mL fermention medium is housed with seed liquor and shakes in the bottle, 28-32 ℃, rotating speed is 160-200 rev/min, shake flask fermentation 168-288 hour,, stop fermentation when thalline color in the fermented liquid from depth to shallow the time;

The 5L ferment tank: the inoculum size with the volume ratio of 15-25% is inoculated in seed liquor in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, automatically monitor fermented liquid dissolved oxygen, temperature, pH value, mixing speed, liquid amount 3L, ventilation 1.0-2.0L/min, mixing speed 180-220rpm in 48 hours, mixing speed is controlled at 90-120rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 8.0-10.0; Proceed to beginning in 25-35 hour in fermentation and in substratum, add glucose with the flow velocity of 1.8-2.2g/L and quicken thalli growth, after fermentation proceeds to 65-75 hour, stop stream and add glucose; Total fermentation time is 168-220 hour, and the interval timing sampling is measured the ebormycine concentration in the fermented liquid in the fermenting process, when ebormycine concentration no longer rises in the fermented liquid, finishes fermentation;

(5) product detects: get step (4) fermented liquid and filter with 100 mesh sieve sand core funnels and collect Amberlite XAD-16 macroporous adsorbent resin, resin is with 2～4 times of volume distilled water washs three times, seasoning; With dried fermentation resin with 8～10 times of volumes methanol vibration lixiviates; Lixiviate is abandoned resin, methyl alcohol vat liquor constant volume after finishing; Get the above-mentioned methyl alcohol vat liquor of 200 μ l with the millipore filtration membrane filtration of 0.22 μ m after as the sample of efficient liquid phase chromatographic analysis, measure the kind and the content of various ebormycine compounds in the fermented liquid with high performance liquid chromatography-diode-array detector-mass detector coupling;

Above-mentioned slant medium consists of: potato starch 8g/L, soy peptone 2g/L, glucose 2g/L, yeast extract paste 2g/L, MgSO ₄1g/L, CaCL ₂1g/L, TE solution 1.0ml/L, agar 15g/L, pH 8.0;

The aforesaid liquid seed culture medium is formed: yam starch 2g/L, glucose 2g/L, yeast extract paste 1g/L, peptone 1g/L, corn steep liquor 1g/L, sal epsom 0.5g/L, calcium chloride 1.5g/L, FeCl ₃-EDTA 0.02g/L, TE solution 1ml/L, HEPES 10g/L, Amberlite XAD-16 macroporous adsorbent resin 2% (v/v), pH 8.0;

Consisting of of above-mentioned TE solution: MnCl ₂4H ₂O 100mg/L, CoCl ₂20mg/L, CuSO ₄10mg/L, Na ₂MoO ₄2H ₂O10mg/L, ZnCl ₂20mg/L, LiCl 5mg/L, SnCl ₂2H ₂O 5mg/L, H ₃BO ₃10mg/L, KBr 20mg/L, KI 20mg/L;

Consisting of of above-mentioned fermention medium: yam starch 4g/L, glucose 2g/L, peptone 4g/L, soybean cake powder 2g/L, skim-milk 1g/L, Na ₂HPO ₄0.15g/L, K ₂HPO ₄0.15g/L, MgSO ₄1.5g/L, CaCl ₂1.5g/L, FeCl ₃-EDTA 0.01g/L, Thr 5mg/L, VB ₁₂1mg/L, initial pH adjusts to 9.0.

3. application as claimed in claim 2 is characterized in that, the described slant culture activation temperature of step (2) is 30 ± 0.2 ℃, and static incubation time is 120 hours.

4. application as claimed in claim 2, it is characterized in that, the described seed culture of step (3) is that inoculation that step (2) is cultivated is in the 250mL triangular flask that the 40mL liquid seed culture medium is housed, putting the rotation rotating speed is that 200 rev/mins, rotation radius are on the shaking table of 35mm, cultivated 96 hours for 30 ± 0.2 ℃, get seed liquor.

5. application as claimed in claim 2 is characterized in that, the described shake flask fermentation of step (4) is the inoculum size with 15% volume ratio, seed liquor is inoculated in the 500mL that the 100mL fermention medium is housed shakes in the bottle, 30 ± 0.2 ℃, rotating speed is 180 rev/mins, shake flask fermentation 216 hours.

6. application as claimed in claim 2, it is characterized in that, the described 5L ferment tank of step (4) is that the inoculum size with 20% volume ratio is inoculated in seed liquor in 5 liters of glass fermentor tanks of full-automatic magnetic agitation, liquid amount 3L, ventilation 1.5L/min, mixing speed 200rpm in 48 hours, mixing speed is controlled at 100rpm after the 48h, and KOH or HCl by auto-feeding 5mol/L make the pH value constant in 9.0 ± 0.3; Proceed to beginning in 30 hours in fermentation and in substratum, add glucose with the flow velocity of 2.0g/L and quicken thalli growth, after fermentation proceeds to 70 hours, stop stream and add glucose; Total fermentation time is 192 hours.