CN103923848B - Multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in Taxol biosynthesis thereof - Google Patents
Multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in Taxol biosynthesis thereof Download PDFInfo
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- CN103923848B CN103923848B CN201410175122.4A CN201410175122A CN103923848B CN 103923848 B CN103923848 B CN 103923848B CN 201410175122 A CN201410175122 A CN 201410175122A CN 103923848 B CN103923848 B CN 103923848B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention provides a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in Taxol biosynthesis thereof; low energy ion beam implantation mediation Ramulus et folium taxi cuspidatae genome DNA transformed yeast is used to obtain the paclitaxel produced multiple-shaped nuohan inferior yeast (Hansenula of a strain? polymorpha) recombinant bacterial strain CGMCC.No.8999; present invention achieves the technological breakthrough utilizing recombination microzyme to carry out biological fermentation production taxol, providing new way for solving the shortage of taxol source, protection China's Chinese yew resource and Sustainable development thereof.
Description
Technical field
The invention belongs to and obtain paclitaxel produced transgenic yeast recombinant bacterium field by genetic transformation, be specifically related to utilize low energy ion beam implantation to mediate the Ramulus et folium taxi cuspidatae genome DNA multiple-shaped nuohan inferior yeast recombinant bacterial strain that genetic transformation obtains in yeast and the application in Taxol biosynthesis thereof.
Background technology
Taxol (Paclitaxel, trade(brand)name Taxo1) be the world today generally acknowledge wide spectrum, determined curative effect natural anti-cancer drugs, its molecular structure is by baccatin iii (baccatin III) and is connected to the tricyclic diterpene Alkaloid that the phenylalanine derivative on 13 carbon forms.Taxol is that uniquely a kind of that current people understand can promote microtubule polymerization and the stable medicine being polymerized microtubule, at the microtubule that thin intracellular accumulation is a large amount of after cells contacting taxol, and the accumulation of these microtubules disturbs the various functions of cell, particularly make cell fission stop at m period, thus block the proper splitting of cell.1992, FDA (Food and Drug Adminstration) (FDA) official approval taxol went on the market, and is used for the treatment of ovarian cancer and mammary cancer.After this, in succession find that taxol is to colon and rectum carcinoma, bladder cancer, metastatic breast cancer clinically, and all show certain curative effect to lung cancer, chromoma and sarcoma etc., taxol has become one of international antineoplastic mainstream medicine.
At present, taxol is mainly obtained by the method extracting taxol or its intermediate from Ramulus et folium taxi cuspidatae raw material.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 about 0.015%) in addition, producing 1kg taxol probably needs 7000kg, is equivalent to 2000-2500 adult Ramulus et folium taxi cuspidatae.Owing to day by day increasing the demand of taxol clinically, cause excessively developing Ramulus et folium taxi cuspidatae, natural Ramulus et folium taxi cuspidatae is caused to face deficient and exhausted, in addition introduction and acclimatization difficulty, artificial culture cycle length, active ingredient yield poorly, and make traditional mode of extracting directly 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 Ramulus et folium taxi cuspidatae is classified as one-level Precious, Rare, Endangered by China, 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 stereochemical characteristics in molecule.Although just obtain successfully as far back as the full chemosynthesis of taxol in 1994, because its reactions steps is many, need use chiral reagent in a large number, reaction conditions is extremely difficult to be controlled, and preparation cost is very expensive, is difficult to applicable large-scale commercial production.
Modern microbiological fermentation technique has unique advantage because of it seeking taxol novel medicine source side mask, becomes one of study hotspot of current international crude drug educational circles gradually.But current Taxol biosynthesis approach and genes involved thereof are not yet illustrated clear.
In recent years, before phytochemicals production 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 beam implantation Mediated Glycyrrhiza uralensis genomic dna transformed yeast, obtain 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 utilizing low energy ion beam implantation to mediate the recombinant bacterium of medicinal plant genomic dna transformed yeast technique construction product effective medicinal components abroad, in genetic modification yeast bio taxol biosynthesis, all there is not been reported 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 biosynthesis thereof.
For achieving the above object, present invention employs 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 (Hansenulapolymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCCNo.8999.
The screening method of described recombinant bacterial strain comprises the following steps:
1) low energy ion beam implantation mediation Ramulus et folium taxi cuspidatae genomic dna transforming Hansenula starting strain is used;
2) recombinant bacterial strain primary dcreening operation: by through step 1) starting strain after process coats YPD solid medium and cultivates, and after picking fresh colony carries out liquid culture fermentation, adopts Vanillin reaction and FeCl
3test is analyzed the fermented liquid of recombinant bacterial strain, whether there is taxol in qualification fermented liquid;
3) the further fermentation culture of recombinant bacterial strain will obtained through qualitative method primary dcreening operation, utilizes 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.polymorphaDL-1 (source is ATCCNo.26012).
Described low energy ion beam implantation adopts low energy N+ as injection 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 interval time is 5 ~ 10s, and vacuum tightness is 1.5 ~ 2.0 × 10
-3pa.
The fermentation process of described recombinant bacterial strain comprises the following steps: in 37 DEG C, rotating speed be 200r/min under cultivate 72 ~ 120h.
The substratum that described fermentation adopts is liquid inorganic substratum, and in fermentation, stream adds the aqueous glycerin solution of 0.5% simultaneously, and flow velocity is 12.5mL/h.
The application of above-mentioned multiple-shaped nuohan inferior yeast recombinant bacterial strain (CGMCCNo.8999) in Taxol biosynthesis.
Beneficial effect of the present invention is embodied in:
The present invention is transformed in starting strain at random by low energy ion beam implantation technology mediation Ramulus et folium taxi cuspidatae genomic dna, obtain a strain multiple-shaped nuohan inferior yeast recombinant bacterial strain, this recombinant bacterial strain can Taxol Biosynthesis effectively, have be easy to high density fermentation, advantage that product is 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.
Accompanying drawing explanation
Fig. 1 is that tunning TLC analyzes collection of illustrative plates, wherein, 1: negative control yeast strain (with TE damping fluid incubation, the wash-out not containing Ramulus et folium taxi cuspidatae genomic dna after low energy ion beam implantation) fermentation broth sample, 2: recombinant bacterial strain fermentation broth sample, 3: Taxol Standard.
Fig. 2 is fermentation production HPLC collection of illustrative plates, wherein, and A: Taxol Standard, B: negative control yeast strain ferments liquid 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 take H.polymorphaDL-1 as starting strain (source is ATCCNo.26012), utilize low energy ion irradiation to inject mediation Ramulus et folium taxi cuspidatae genomic dna to transform 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 substratum: 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, is settled to 1L with water, and pH is 6.5;
Wherein each solution batching is as follows:
Iron salt solutions: FeCl
36H
2o30g, is settled to 1L by water dissolution;
Thiamine salt acid salt solution: thiamine salt hydrochlorate 4g, is settled to 1L by water dissolution;
PTM trace element solution: KI0.1g, Na
2moO
42H
2o0.2g, MnSO
4h
2o0.303g, CuSO
45H
2o0.04g, ZnSO
47H
2o0.4g, is settled to 1L by water dissolution.
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 are 6.5.
(2) screening method
1) terpene substances detects
Vanillin reacts: get 800mL fermented liquid, at 100 DEG C after concentrated broth to 5mL (obtaining concentrated broth), add methyl alcohol 10mL and extract, extracted by filtration liquid.Get filtrate 1mL, add 5% (massfraction) Vanillin concentrated sulfuric acid solution 0.5mL, heating 2min, observe solution colour change, present lilac for positive.
FeCl
3test: put multiple sampling point with sample introduction needle pipette samples on filter paper, spray with 3% (massfraction) FeCl
3the aqueous solution, observes sampling point colour-change, and what occur punctation is the positive.
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 respectively, at developping agent chloroform-methanol (V/V, launch 90:10), observe under ultraviolet lamp after end, record spot, and calculate its Rf value.
3) HPLC detection by quantitative: get fermented liquid 800mL, at 100 DEG C after concentrated broth to 5mL (obtaining concentrated broth), add methyl alcohol 10mL to extract, extracted by filtration liquid, filtrate is to measure the content of taxol for HPLC method after 0.45 μm of 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) Ramulus et folium taxi cuspidatae genomic dna is extracted: get Shaanxi as the fresh blade 50g of real estate Ramulus et folium taxi cuspidatae, after being ground to powder rapidly with liquid nitrogen, loaded in the centrifuge tube of 50mL; Then the rapid CTAB extracting solution by 3mL preheating (2%CTAB, 1.4MNaCl, 0.02MEDTA, 0.1MTris-HCl, 0.2% mercaptoethanol, pH8.0) joins and is equipped with in the 50mL centrifuge tube of powder; Put into 65 DEG C of water-baths afterwards, after insulation 30 ~ 60min (period jog 5 times), add 3 ~ 4mL chloroform-isoamyl alcohol (24:1), jog mixing, 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, being placed in 4 DEG C, the centrifugal 10min of 11000rpm, abandoning supernatant, precipitating with 70% ethanol rinse 2 times, be positioned in Bechtop and dry up to obtain Ramulus et folium taxi cuspidatae genomic dna.Finally add 1mLTris-EDTA (pH8.0) buffer solution genomic dna, and survey its concentration with ultraviolet spectrophotometer, put into 4 DEG C of Refrigerator stores.
2) low energy ion beam implantation mycoderm: by the starting strain (H.polymorphaDL-1) of this Laboratories Accession in 37 DEG C, on YPD organic solid substratum, activate 12h after, picking colony is inoculated in liquid inorganic substratum, and in 37 DEG C, cultivate 16h under 110r/min speed conditions 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, and get the sterilized petri dishes central authorities that 0.1mL mycelium dilution liquid is spread evenly across diameter 90mm, 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 of Ramulus et folium taxi cuspidatae genomic dna is contained with 2mL, pH8.0) soak ion implantation after mycoderm, then after 37 DEG C of incubation 2h, wash-out is carried out with pipettor, collect elutriant and with aseptic spatula, bacterium all scraped to obtain bacterium liquid B, getting respectively after 0.1mL bacterium liquid B is spread evenly across YPD organic solid substratum and cultivate 72h in 37 DEG C.
4) primary dcreening operation: through step 3) after, transfer to carry out liquid culture (37 DEG C, 110rpm/min cultivate 120h) in test tube by cultivating the bacterium colony obtained, then collect fermented liquid in the centrifugal 10min of 10000r/min and carry out detection analysis.The detection of terpene substances mainly adopts Vanillin to react and FeCl
3test.First utilize Vanillin to react to analyze the fermented liquid of recombinant bacterial strain, observe the colour-change (positive findings is lavender) of sample liquid.Then, FeCl is recycled
3test is analyzed further to the fermented liquid of the lilac recombinant bacterial strain of above-mentioned appearance, observes whether occur punctation (positive findings is for occurring punctation).Above-mentioned two kinds of positive reaction results show to there is 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 substratum, and in 37 DEG C, cultivate 12h under 110r/min and obtain seed culture fluid, with volume be 5% inoculum size seed culture fluid is inoculated in the 500mL triangular flask containing 100mL liquid inorganic substratum, then in 110r/min, 90h is cultivated at 37 DEG C, then in the centrifugal 10min of 7000r/min, dry to constant weights in 85 DEG C after the centrifugal thalline distilled water flushing obtained twice and carry out analysis of weighing, the centrifugal supernatant liquor obtained is qualitative for product, detection by quantitative, main employing thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).
Thin layer chromatography Qualitative Identification: respectively with kapillary by the primary dcreening operation recombinant bacterial strain concentrated broth of 5 μ L, the concentrated broth of negative control yeast strain and Taxol Standard point on thin layer plate, then thin layer plate is launched in developping agent chloroform-methanol (V/V, 90:10).Result as shown in Figure 1, same spot is there is in primary dcreening operation recombinant bacterial strain concentrated broth on the position corresponding with Taxol Standard, its Rf value is 0.435, with Taxol Standard Rf value (0.438) closely, 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 to synthesize.
HPLC standard measure is identified: respectively the concentrated broth of primary dcreening operation recombinant bacterial strain concentrated broth, negative control yeast strain and Taxol Standard are carried out HPLC analysis, result as shown in Figure 2, retention time is go out peak (Fig. 2 C) in 20.441min place primary dcreening operation recombinant bacterial strain concentrated broth, the retention time 20.550min going out peak (Fig. 2 A) with Taxol Standard 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 obtains 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 (Hansenulapolymorpha), 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 CGMCCNo.8999.
(4) fermentation of recombinant bacterial strain and taxol separation and Extraction
The zymotechnique of recombinant bacterial strain (CGMCCNo.8999): adopt 5L automatic fermenter to carry out high density fermentation to the recombinant bacterial strain screened (CGMCCNo.8999) and produce taxol, recombinant bacterial strain is inoculated in liquid inorganic substratum with the inoculum size of 5% (volume ratio) after seed culture (culture condition with reference to above-mentioned seed culture fluid), in 37 DEG C, rotating speed is carry out continuously fermenting 120h under 200r/min, in fermentation, stream adds 0.5% (w/v simultaneously, containing 0.5g glycerine in 100mL water) aqueous glycerin solution, flow velocity is 12.5mL/h, after fermentation 60h, fermented liquid 500mL is got every 12h, measure yeast cell biomass and taxol.As shown in Figure 3, cell concentration constantly raises along with the increase of incubation time result, and after cultivating 96h, cellular biomass reaches and is up to 39.92g/L, then enters the growth stage of stable development; Taxol occurs from 72h, and along with time lengthening, output increases thereupon, and during 108h, output reaches the highest, reaches 1.106mg/L.
Purification of Taxol: according to above-mentioned zymotechnique, carries out high density fermentation 120h by recombinant bacterial strain (CGMCCNo.8999), and in 60h, 72h, 84h, 96h and 120h gets 500mL fermented liquid respectively 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 be separated taxol.It is that the solution of the methanol-water (volume ratio of methyl alcohol and water is 50:50) of 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 to show, in sample, the taxol rate of recovery reaches 85%.
(5) genetic stability of recombinant bacterial strain
Carry out shake-flask culture (37 DEG C, 160r/min) in the 2000mL triangular flask being inoculated in containing 500mL liquid inorganic substratum after being activated by recombinant bacterial strain (CGMCCNo.8999), in Secondary Culture 10 generation, measures the genetic stability of recombinant bacterial strain.Result as shown in Figure 4, show that in Secondary Culture process, in recombinant bacterial strain fermented liquid, content of taxol is substantially constant, after Secondary Culture 10 generation, content of taxol is 1.081mg/L, reduce 1.8% compared to the content of taxol (1.106mg/L) of first-generation recombinant bacterial strain, this recombinant bacterial strain inheritance stability is described.
In a word; the present invention utilizes low energy ion beam implantation technical transform yeast to build paclitaxel produced recombination microzyme; fermentable Taxol Biosynthesis have production cost low, with short production cycle, do not affect by natural climate, the advantage such as Economization on land, extracellular products separation and Extraction are simple, this is for solving taxol source shortage problem, protection taxaceae plant resources realize its Sustainable development and provide new way.
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 be multiple-shaped nuohan inferior yeast (
hansenulapolymorpha), described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCCNo.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: in 37 DEG C, rotating speed cultivates 72 ~ 120h under being 200r/min.
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 substratum, and in fermentation, stream adds the aqueous glycerin solution of 0.5% simultaneously, and flow velocity is 12.5mL/h.
4. the application of multiple-shaped nuohan inferior yeast recombinant bacterial strain in Taxol biosynthesis as claimed in claim 1.
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