CN103923848A - hansenula polymorpha recombinant strain and application thereof in biosynthesis of paclitaxel - Google Patents
hansenula polymorpha recombinant strain and application thereof in biosynthesis of paclitaxel Download PDFInfo
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- CN103923848A CN103923848A CN201410175122.4A CN201410175122A CN103923848A CN 103923848 A CN103923848 A CN 103923848A CN 201410175122 A CN201410175122 A CN 201410175122A CN 103923848 A CN103923848 A CN 103923848A
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- taxol
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- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 57
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 57
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- 150000002500 ions Chemical class 0.000 abstract description 13
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention provides a hansenula polymorpha recombinant strain and an application thereof in biosynthesis of paclitaxel. A total DNA transformation yeast of mediated taxus chinensis genome is injected to obtain a hansenula polymorpha recombinant strain CGMCC No.8999 for producing paclitaxel by using low-energy ions. The technical breakthrough for biological fermentation to produce paclitaxel by using the recombined yeast is achieved, and a new way is provided for solving paclitaxel source shortage, protecting taxus chinensis plant resources in China and sustainable development thereof.
Description
Technical field
The invention belongs to by genetic transformation and obtain paclitaxel produced transgenic yeast recombinant bacterium field, be specifically related to utilize low energy ion to inject multiple-shaped nuohan inferior yeast recombinant bacterial strain that mediation Ramulus et folium taxi cuspidatae genome DNA obtains at yeast genetic transformation and the application in taxol biosynthesizing thereof.
Background technology
Taxol (Paclitaxel, trade(brand)name Taxo1) be the natural anti-cancer drugs of the generally acknowledged wide spectrum in the world today, determined curative effect, its molecular structure is by baccatin iii (baccatin III) and is connected to the tricyclic diterpene Alkaloid that 13 phenylalanine derivatives on carbon form.Taxol is that unique a kind of that current people understand can promote microtubule polymerization and the stable medicine of polymerization microtubule, after cells contacting taxol at the thin a large amount of microtubule of intracellular accumulation, and the various functions of cell have been disturbed in the accumulation of these microtubules, particularly make cell fission stop at m period, thereby blocked the proper splitting of cell.1992, FDA (Food and Drug Adminstration) (FDA) official approval taxol listing, was used for the treatment of ovarian cancer and mammary cancer.After this, in succession find that clinically taxol is to colon and rectum carcinoma, bladder cancer, metastatic breast cancer, and lung cancer, chromoma and sarcoma etc. are all shown to certain curative effect, taxol has become one of international antineoplastic main flow medicine.
At present, taxol is mainly that method by extract taxol or its intermediate from Ramulus et folium taxi cuspidatae raw material obtains.It should be noted that Ramulus et folium taxi cuspidatae becomes tree generally to need 100-250, the content of taxol in bark lower (average content approximately 0.015%) in addition, producing 1kg taxol probably needs 7000kg, is equivalent to 2000-2500 adult Ramulus et folium taxi cuspidatae.Because the demand to taxol clinically increases day by day, cause Ramulus et folium taxi cuspidatae excessively to be developed, cause natural Ramulus et folium taxi cuspidatae to face deficient and exhausted, in addition introduction and acclimatization difficulty, artificial culture cycle length, active ingredient yield poorly, and make traditional mode of directly extracting taxol from Ramulus et folium taxi cuspidatae plant be subject to restriction greatly and challenge.
Therefore, the resource problem of Chinese yew genus plants has caused the great attention of various countries environment specialist and government, and China has classified Ramulus et folium taxi cuspidatae as one-level Precious, Rare, Endangered, is equivalent to " giant panda in plant ", forbids to cut down.In addition, the molecular structure of taxol is very complicated, has numerous functional groups and stereochemistry feature in molecule.Although the full chemosynthesis as far back as taxol in 1994 has just obtained success, because its reactions steps is many, need a large amount of chiral reagents that use, reaction conditions is difficult control extremely, and preparation cost is very expensive, is difficult to be applicable to large-scale commercial production.
Modern microbial fermentation technology is because it has unique advantage seeking taxol novel medicine source side mask, becomes gradually one of study hotspot of current international crude drug educational circles.But taxol biosynthetic pathway and genes involved thereof are not yet illustrated clear at present.
In recent years, before natural plant product biosynthesis gene information and biosynthetic pathway are not yet illustrated, L ü etc. passes through Ar
+and N
+inject mediation Ephedra genome DNA transformation yeast, obtain inheritance stability Yeast engineering bacteria, ephedrine and pseudoephedrine output are respectively 18.85mg/L and 4.11mg/L.Jin etc. utilize low energy ion to inject Mediated Glycyrrhiza uralensis genomic dna transformed yeast, have obtained the Yeast engineering bacteria of inheritance stability, and the production peak of its pentacyclic triterpene glycoside material Potenlini reaches 114.49mg/L.
At present, there is not yet the research report that utilizes low energy ion to inject the recombinant bacterium of mediation medicinal plant genomic dna transformed yeast technique construction product effective medicinal components abroad, aspect genetic modification yeast bio taxol biosynthesis, all there is not yet report both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in taxol biosynthesizing thereof.
For achieving the above object, the present invention has adopted following technical scheme.
A kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain, the Classification And Nomenclature of this recombinant bacterial strain is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCC No.8999.
The screening method of described recombinant bacterial strain comprises the following steps:
1) use low energy ion to inject mediation Ramulus et folium taxi cuspidatae genomic dna and transform multiple-shaped nuohan inferior yeast starting strain;
2) recombinant bacterial strain primary dcreening operation: will be through step 1) starting strain after treatment coats YPD solid medium and cultivates, and the fresh bacterium colony of picking carries out after liquid culture fermentation, adopts Vanillin reaction and FeCl
3test is analyzed the fermented liquid of recombinant bacterial strain, in qualification fermented liquid, whether has taxol;
3) by the further fermentation culture of recombinant bacterial strain obtaining through qualitative method primary dcreening operation, utilize thin-layer chromatography and high performance liquid chromatography to analyze its fermented liquid, further Screening and Identification recombinant bacterial strain.
Described starting strain is multiple-shaped nuohan inferior yeast H.polymorpha DL-1 (source is ATCCNo.26012).
Described low energy ion injects and adopts low energy N+ as injecting ion, and best implantation dosage is 1.5 × 10
16～2.5 × 10
16ions/cm
2, best Implantation Energy is 15～25KeV, and the burst length is 5～10s, and be 5～10s interval time, and vacuum tightness is 1.5～2.0 × 10
-3pa.
The fermentation process of described recombinant bacterial strain comprises the following steps: be to cultivate 72～120h under 200r/min in 37 DEG C, rotating speed.
The substratum that described fermentation adopts is liquid inorganic medium, and in fermentation, stream adds 0.5% aqueous glycerin solution simultaneously, and flow velocity is 12.5mL/h.
The application of above-mentioned multiple-shaped nuohan inferior yeast recombinant bacterial strain (CGMCC No.8999) in taxol biosynthesizing.
Beneficial effect of the present invention is embodied in:
The present invention transforms in starting strain at random by low energy ion implantttion technique mediation Ramulus et folium taxi cuspidatae genomic dna, obtain a strain multiple-shaped nuohan inferior yeast recombinant bacterial strain, this recombinant bacterial strain Taxol Biosynthesis effectively, have advantages of that the high density fermentation of being easy to, product are easy to separation and Extraction, for the suitability for industrialized production of taxol provides new way, provide New methods in working for solving medicine source of Taxol shortage problem.
Brief description of the drawings
Fig. 1 is that tunning TLC analyzes collection of illustrative plates, wherein, 1: negative control yeast strain (low energy ion is used not TE damping fluid incubation, the wash-out containing Ramulus et folium taxi cuspidatae genomic dna after injecting) fermentation broth sample, 2: recombinant bacterial strain fermentation broth sample, 3: taxol standard substance.
Fig. 2 is fermentation production HPLC collection of illustrative plates, wherein, and A: taxol standard substance, B: negative control yeast strain fermentation broth sample, C: recombinant bacterial strain fermentation broth sample.
Fig. 3 is the fermentograph of recombinant bacterial strain.
Fig. 4 is the genetic stability collection of illustrative plates of recombinant bacterial strain.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
The present invention is (source is ATCC No.26012) taking H.polymorpha DL-1 as starting strain, utilizing low energy ion irradiation to inject mediation Ramulus et folium taxi cuspidatae genomic dna transforms starting strain, obtain paclitaxel produced recombinant bacterial strain by screening, and carry out genetic stability and corresponding fermentation test.
(1) substratum
The composition of liquid inorganic medium: glycerol 19g, (NH
4)
2sO
46g, K
2hPO
40.24g, MgSO
47H
2o1.4g, CaCl
20.38g, NaCl0.6g, NaNO
30.58g, iron salt solutions 0.2mL, thiamine salt acid salt solution 1mL, PTM trace element solution 1mL, water is settled to 1L, and pH is 6.5;
Wherein each solution batching is as follows:
Iron salt solutions: FeCl
36H
2o30g, by water dissolution and be settled to 1L;
Thiamine salt acid salt solution: thiamine salt hydrochlorate 4g, by water dissolution and be settled to 1L;
PTM trace element solution: KI0.1g, Na
2moO
42H
2o0.2g, MnSO
4h
2o0.303g, CuSO
45H
2o0.04g, ZnSO
47H
2o0.4g, by water dissolution and be settled to 1L.
Consisting of of YPD organic solid substratum: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, agar 20g/L, pH is 6.5.
(2) screening method
1) terpene substances detects
Vanillin reaction: get 800mL fermented liquid, concentrated broth, to 5mL (obtaining concentrated broth), adds methyl alcohol 10mL extraction at 100 DEG C, filters extraction liquid.Get filtrate 1mL, add 5% (massfraction) Vanillin concentrated sulfuric acid solution 0.5mL, heating 2min, observes solution colour and changes, and presents lilac positive.
FeCl
3test: draw sample by sample introduction needle and put multiple sampling points on filter paper, spray is with 3% (massfraction) FeCl
3the aqueous solution, observes sampling point colour-change, occurs the positive of punctation.
2) thin-layer chromatography (TLC) qualitative detection: the point sample on GF254 silica gel thin-layer plate by 5 μ L concentrated broths and taxol standard substance respectively, at developping agent chloroform-methanol (V/V, 90:10), launch, after end, under ultraviolet lamp, observe, record spot, and calculate its Rf value.
3) HPLC detection by quantitative: get fermented liquid 800mL, at 100 DEG C, concentrated broth is to 5mL (obtaining concentrated broth), add methyl alcohol 10mL extraction, filter extraction liquid, filtrate is measured the content of taxol for HPLC method after with 0.45 μ m filtering with microporous membrane; HPLC chromatographic condition is: chromatographic column is chemical bond mould assembly octadecyl post (SciC
18chromatographic column); Pump:K-1001; Detecter:K-1501; Moving phase: methanol-water (V/V, 68:32); Column temperature: 25 DEG C.
(3) restructuring and screening process
1) extract Ramulus et folium taxi cuspidatae genomic dna: get Shaanxi as the fresh blade 50g of real estate Ramulus et folium taxi cuspidatae, be ground to rapidly after powder with liquid nitrogen, packed in the centrifuge tube of 50mL; Then rapidly the CTAB extracting solution of 3mL preheating (2%CTAB, 1.4MNaCl, 0.02MEDTA, 0.1MTris-HCl, 0.2% mercaptoethanol, pH8.0) is joined in the 50mL centrifuge tube that powder is housed; Put into afterwards 65 DEG C of water-baths, after insulation 30～60min (jog 5 times during this time), add 3～4mL chloroform-primary isoamyl alcohol (24:1), jog mixes to oyster white, is placed in 4 DEG C, the centrifugal 15min of 11000rpm; Get supernatant liquor and add the Virahol of 0.6 times of volume, after shaking up, be placed in 4 DEG C, the centrifugal 10min of 11000rpm, abandon supernatant, 70% ethanol rinsing 2 times for precipitation, is positioned over and in Bechtop, dries up to obtain Ramulus et folium taxi cuspidatae genomic dna.Finally add 1mLTris-EDTA (pH8.0) damping fluid to dissolve genomic dna, and survey its concentration with ultraviolet spectrophotometer, put into 4 DEG C of Refrigerator stores.
2) low energy ion inject mycoderm: by the starting strain of this laboratory preservation (H.polymorpha DL-1) in 37 DEG C, on YPD organic solid substratum, activate after 12h, picking colony is inoculated in liquid inorganic medium, and under 37 DEG C, 110r/min speed conditions, cultivate 16h and obtain bacterium liquid A, getting appropriate bacterium liquid A, to be diluted to concentration be 1.0 × 10
7cFU/mL obtains mycelium dilution liquid, gets 0.1mL mycelium dilution liquid and evenly coat the aseptic plate central authorities of diameter 90mm, and sterile wind dries up makes mycoderm, is then placed in ion implanter vacuum target chamber and carries out N
+inject, Implantation Energy is 20KeV, and dosage is 2.0 × 10
16ions/cm
2, burst length 6s, interval time 10s, vacuum tightness is 1.5 × 10
-3pa.
3) solid culture: through step 2) after, Tris-EDTA damping fluid (the TE that contains Ramulus et folium taxi cuspidatae genomic dna with 2mL, pH8.0) soak the mycoderm after ion implantation, then after 37 DEG C of incubation 2h, carry out wash-out with pipettor, collect elutriant and also with aseptic spatula, bacterium is all scraped to obtain to bacterium liquid B, get respectively after 0.1mL bacterium liquid B evenly coats YPD organic solid substratum and cultivate 72h in 37 DEG C.
4) primary dcreening operation: through step 3) after, transfer and carry out liquid culture (37 DEG C, 110rpm/min cultivate 120h) in test tube cultivating the bacterium colony obtaining, then detect analysis in the centrifugal 10min collection of 10000r/min fermented liquid.The detection of terpene substances mainly adopts Vanillin reaction and FeCl
3test.First utilize Vanillin reaction to analyze the fermented liquid of recombinant bacterial strain, observe the colour-change (positive findings is lavender) of sample liquid.Then, recycling FeCl
3test is further analyzed the fermented liquid of the lilac recombinant bacterial strain of above-mentioned appearance, observes and whether occurs punctation (positive findings is for occurring punctation).Above-mentioned two kinds of positive reaction results show to have terpenoid in the fermented liquid of recombinant bacterial strain, can be used as the primary dcreening operation recombinant bacterial strain of subsequent analysis.
5) qualitative, detection by quantitative: primary dcreening operation recombinant bacterial strain is inoculated in the test tube containing 10mL liquid inorganic medium, and in 37 DEG C, under 110r/min, cultivate 12h and obtain seed culture fluid, taking volume as 5% inoculum size, seed culture fluid is inoculated in the 500mL triangular flask containing 100mL liquid inorganic medium, then in 110r/min, at 37 DEG C, cultivate 90h, then in the centrifugal 10min of 7000r/min, the centrifugal thalline obtaining is with drying to the constant weight analysis of weighing in 85 DEG C after twice of distilled water flushing, the centrifugal supernatant liquor obtaining is qualitative for product, detection by quantitative, main thin-layer chromatography (TLC) and the high performance liquid chromatography (HPLC) of adopting.
Thin layer chromatography Qualitative Identification: with kapillary, concentrated broth and the taxol standard substance of the primary dcreening operation recombinant bacterial strain concentrated broth of 5 μ L, negative control yeast strain are put on thin layer plate respectively, then thin layer plate is launched in developping agent chloroform-methanol (V/V, 90:10).Result as shown in Figure 1, there is same spot with on the corresponding position of taxol standard substance in primary dcreening operation recombinant bacterial strain concentrated broth, its Rf value is 0.435, very approaching with taxol standard substance Rf value (0.438), and spot does not appear in the concentrated broth of negative control yeast strain at this place, show that primary dcreening operation recombinant bacterial strain fermenting process has novel substance synthetic.
HPLC standard measure qualification: respectively the concentrated broth of primary dcreening operation recombinant bacterial strain concentrated broth, negative control yeast strain and taxol standard substance are carried out to HPLC analysis, result as shown in Figure 2, retention time is in 20.441min place primary dcreening operation recombinant bacterial strain concentrated broth, to go out peak (Fig. 2 C), the retention time 20.550min that goes out peak (Fig. 2 A) with taxol standard substance is basically identical, and the concentrated broth of negative control yeast strain does not go out peak at this place, show that primary dcreening operation recombinant bacterial strain has the ability of Taxol Biosynthesis.Pass through above-mentioned steps, final the present invention's screening has obtained a strain recombinant bacterial strain DL-781, the Classification And Nomenclature that the present invention screens the recombinant bacterial strain of acquisition is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), this recombinant bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), and preservation date is on April 3rd, 2014; Deposit number is CGMCC No.8999.
(4) fermentation of recombinant bacterial strain and taxol separation and Extraction
The zymotechnique of recombinant bacterial strain (CGMCC No.8999): adopt 5L automatic fermenter to carry out high density fermentation to the recombinant bacterial strain screening (CGMCC No.8999) and produce taxol, recombinant bacterial strain is inoculated in liquid inorganic medium with the inoculum size of 5% (volume ratio) after seed culture (with reference to the culture condition of above-mentioned seed culture fluid), in 37 DEG C, rotating speed is the 120h that continuously ferments under 200r/min, in fermentation, stream adds 0.5% (w/v simultaneously, in 100mL water containing 0.5g glycerine) aqueous glycerin solution, flow velocity is 12.5mL/h, after fermentation 60h, get fermented liquid 500mL every 12h, measure yeast cell biomass and taxol.As shown in Figure 3, cell concentration is along with the increase of incubation time constantly raises for result, and after cultivation 96h, cellular biomass reaches and is up to 39.92g/L, then enters the growth stage of stable development; Taxol starts to occur from 72h, and along with time lengthening, output increases thereupon, and when 108h, output reaches the highest, reaches 1.106mg/L.
Purification of Taxol: according to above-mentioned zymotechnique, recombinant bacterial strain (CGMCC No.8999) is carried out to high density fermentation 120h, in 60h, 72h, 84h, 96h and 120h get respectively 500mL fermented liquid and carry out separation and purification detection analysis.Be specially fermented liquid 500mL after the centrifugal 10min of 12000r/min, supernatant liquor is concentrated into 10mL in 80 DEG C, adopt H60 macroporous resin to separate taxol.It is that the solution of 50% methanol-water (volume ratio of methyl alcohol and water is 50:50) is as sample solution that supernatant liquor after concentrated is made into volume fraction, flow velocity is 1.5mL/min loading, make elutriant with the aqueous ethanolic solution that volume fraction is 80% again, flow velocity is 0.5mL/min wash-out.Utilize HPLC to carry out quantitative analysis and show, in sample, the taxol rate of recovery reaches 85%.
(5) genetic stability of recombinant bacterial strain
To after recombinant bacterial strain (CGMCC No.8999) activation, be inoculated in the 2000mL triangular flask that contains 500mL liquid inorganic medium and carry out shake-flask culture (37 DEG C, 160r/min), in 10 generations of cultivation of going down to posterity, be measured the genetic stability of recombinant bacterial strain.Result as shown in Figure 4, the content of taxol that shows to go down to posterity in culturing process in recombinant bacterial strain fermented liquid is substantially constant, going down to posterity, to cultivate content of taxol after 10 generations be 1.081mg/L, content of taxol (1.106mg/L) than first-generation recombinant bacterial strain has reduced by 1.8%, and this recombinant bacterial strain inheritance stability is described.
In a word; the present invention utilizes low energy ion implantttion technique transformed yeast to build paclitaxel produced recombination microzyme; microorganism fermenting organism taxol biosynthesis have production cost low, with short production cycle, be not subject to natural climate to affect, save the advantages such as soil, extracellular products separation and Extraction are simple, this provides new way for solving taxol source shortage problem, protection taxaceae plant resources and realizing its Sustainable development.
Claims (4)
1. a multiple-shaped nuohan inferior yeast recombinant bacterial strain, is characterized in that: the Classification And Nomenclature of this recombinant bacterial strain is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCC No.8999.
2. a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain according to claim 1, is characterized in that: the fermentation process of described recombinant bacterial strain comprises the following steps: be to cultivate 72～120h under 200r/min in 37 DEG C, rotating speed.
3. a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain according to claim 2, is characterized in that: the substratum that described fermentation adopts is liquid inorganic medium, and in fermentation, stream adds 0.5% aqueous glycerin solution simultaneously, and flow velocity is 12.5mL/h.
4. the application of multiple-shaped nuohan inferior yeast recombinant bacterial strain in taxol biosynthesizing as claimed in claim 1.
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| CN107189953A (en) * | 2017-05-19 | 2017-09-22 | 宁波市疾病预防控制中心 | Multiple-shaped nuohan inferior yeast recombinant bacterial strain and its application in glycerine biosynthesis |
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