CN103966112B - Multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in gentiopicrin biosynthesizing thereof - Google Patents
Multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in gentiopicrin biosynthesizing thereof Download PDFInfo
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- DUAGQYUORDTXOR-WULZUDSJSA-N Gentiopicrin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1[C@@H](C=C)C=2C(C(=O)OCC=2)=CO1 DUAGQYUORDTXOR-WULZUDSJSA-N 0.000 title claims abstract description 58
- DUAGQYUORDTXOR-GPQRQXLASA-N Gentiopicrin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](C=C)C2=CCOC(=O)C2=CO1 DUAGQYUORDTXOR-GPQRQXLASA-N 0.000 title claims abstract description 58
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 56
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 38
- 230000003570 biosynthesizing effect Effects 0.000 title claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims description 36
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 6
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Abstract
The invention provides a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in gentiopicrin biosynthesizing thereof; the method of low energy ion beam implantation mediation bark of ash genome DNA transformed yeast bacterium is mainly used to obtain the recombination microzyme that gentiopicrin is produced in a strain; does is it multiple-shaped nuohan inferior yeast (Hansenula? polymorpha) CGMCC.No.8998; present invention achieves the technological breakthrough utilizing recombination microzyme to carry out biological fermentation production gentiopicrin, providing new way for solving the shortage of gentiopicrin source, protection China's gentianaceae plant resource and Sustainable development thereof.
Description
Technical field
The invention belongs to and obtain the transgenic yeast recombinant bacterium field of producing gentiopicrin by genetic transformation, be specifically related to utilize the ion implantation mediation bark of ash STb gene multiple-shaped nuohan inferior yeast recombinant bacterial strain that genetic transformation obtains in yeast and the application in gentiopicrin biosynthesizing thereof.
Background technology
Gentiopicrin (Gentiopicroside), another name gentiopicroside, molecular formula is C
16h
20o
9, molecular weight is 356.11, and be driffractive ring cyclenes terpene glycosides compound, belonging to sesquiterpenoids material, is the primary medicinal component of medicinal plant bark of ash (Gentianamacrophylla).Research in recent years shows, gentiopicrin has analgesia, anti-inflammatory, antibacterial, antiviral, anti-oxidant, the effect such as hepatic cholagogic, stomach invigorating antiulcer agent, as as first class national new drug injection Qinlongkusu, for the freeze-dried powder of Radix Gentianae Macrophyllae extract gentiopicrin, treat for icteric viral hepatitie.
Gentiopicrin multi-source is in Gentianaceae per nnial herb bark of ash.Bark of ash is Gentianaceae, Gentiana per nnial herb, is one of important Chinese medicinal materials of China.Bark of ash growth is in high altitude localities, and growth cycle is long, is subject to seasonal variation impact.In recent years because bark of ash has very high pharmaceutical use, transition is excavated and is made Wild Gentiana macrophylla resource occur serious scarcity, adds the problem that bark of ash artificial culture survival rate is low, causes the supply of gentiopicrin to be difficult to meet clinical demand.Therefore, in the urgent need to seeking and expanding gentiopicrin source new drugs by way of to meet growing to it clinically demand.
At present, Tiwari etc. use Agrobacterium rhizogenes to induce bark of ash hairly root, but it can not produce gentiopicrin.In addition, Qi Xiangjun etc. use medicine source plant resource bark of ash cells produce gentiopicrin, and its gentiopicrin output is only 0.242mg/g, and its fermentation period is long.Above-mentioned research all fails to obtain desirable result at present.In bark of ash development of resources research, abroad not yet report.Therefore, need badly and seek new Research Thinking and solve gentiopicrin source shortage problem.
In recent years, separating natural product biosynthesis gene, and by yeast cell factory in a large number and economically biosynthesizing phytochemicals production become one of effective way.But plant terpene compound, as the senior terpenes such as monoterpene, sesquiterpene and diterpene not only have special route of synthesis, and has unique enzyme reaction mechanism.Meanwhile, many known pathways metabolisms can not as a reference, and Secondary Metabolism of Plant network is many and complicated, adds that Partial key metabolic enzyme gene is in low expression level, is difficult to obtain expression from protein molecular level.
In recent years, before phytochemicals production biosynthesis gene information and biosynthetic pathway are not yet illustrated, L ü etc. inject mediation Ephedra genome DNA transformation yeast by Ar+ and N+, and 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.Simultaneously, because yeast self exists mevalonic acid (Mevalonate, i.e. MVA) approach, oneself can synthesize terpene substances precursor Isoprenoid, this provides a good basic platform for utilizing Microbe synthesis terpenoid gentiopicrin.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.Specifically in genetic modification yeast bio synthesis gentiopicrin, 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 gentiopicrin biosynthesizing 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.8998.
The screening method of described recombinant bacterial strain, comprises the following steps:
1) low energy ion beam implantation mediation bark of ash genomic dna transforming Hansenula starting strain is used;
2) recombinant bacterial strain primary dcreening operation: picking cultivate in YPD solid medium through step 1) bacterium colony of starting strain after process carries out YPD liquid fermenting, centrifuging and taking fermented liquid, adopt Molish reaction and Fehling test to analyze the fermented liquid of recombinant bacterial strain, initial characterization analyzes in fermented liquid whether there is gentiopicrin;
3) recombinant bacterial strain obtained through qualitative method primary dcreening operation is carried out YPD liquid fermentation and culture further, utilize thin-layer chromatography and efficient liquid phase chromatographic analysis fermentation gained fermented liquid, thus to the further Screening and Identification of recombinant bacterial strain.
Described starting strain is multiple-shaped nuohan inferior yeast H.polymorphaDL-1 (source is ATCCNo.26012).
The condition of described low energy ion beam implantation is: injection ion is N+, and implantation dosage is 1.5 × 10
16~ 2.5 × 10
16ions/cm
2, 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 300r/min condition under cultivate 60 ~ 96h.
The substratum that described fermentation adopts is YPD liquid nutrient medium, and in fermenting process, stream adds the D/W of 0.5%, and flow velocity is 12.5mL/h.
The application of above-mentioned multiple-shaped nuohan inferior yeast recombinant bacterial strain (CGMCCNo.8998) in gentiopicrin biosynthesizing.
Beneficial effect of the present invention is embodied in:
The present invention is transformed in yeast starting strain at random by low energy ion beam implantation technology mediation bark of ash genomic dna, obtain a strain multiple-shaped nuohan inferior yeast recombinant bacterial strain, this recombinant bacterial strain can biosynthesizing gentiopicrin effectively, have and be easy to the advantage that height density fermentation, product are easy to separation and Extraction, for the suitability for industrialized production of gentiopicrin provides new way, provide New methods in working for solving gentiopicrin medicine source shortage problem.
Accompanying drawing explanation
Fig. 1 is that tunning TLC analyzes collection of illustrative plates, wherein, 1: gentiopicrin standard substance, 2: recombinant bacterial strain fermentation broth sample, 3: negative control yeast strain (with TE damping fluid incubation, the wash-out not containing bark of ash genomic dna after low energy ion beam implantation) fermentation broth sample.
Fig. 2 is fermentation production HPLC collection of illustrative plates, wherein, and A: gentiopicrin standard substance, B: recombinant bacterial strain fermentation broth sample, C: negative control yeast strain ferments liquid 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), low energy ion irradiation is utilized to inject the random transformed yeast of mediation bark of ash genomic dna, obtain the restructuring yeast strains producing gentiopicrin by qualitative, quantitative screening, and genetic stability and corresponding fermentation test are carried out to it.
(1) substratum
Consisting of of YPD liquid nutrient medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, pH are 6.5;
Consisting of of YPD solid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, agar 20g/L, pH are 6.5.
(2) method of institute's foundation is screened
(1) iridoid glycosides material detects
Molish reacts: get 800mL fermented liquid, at 100 DEG C after concentrated broth to 5mL (obtaining concentrated broth), add 10mL methanol extraction, extracted by filtration liquid.Get filtrate 3mL, add two Molish reagent (10% naphthyl alcohol ethanolic soln), carefully add the about 1mL vitriol oil (18.4mol/L), be sure not to shake up, observe the colour-change of two-layer liquid level intersection, occur that purple ring is for positive.
Fehling tests: get above-mentioned concentrated broth 3mL and join in 3mLFehling reagent (be made up of first and the second grade volume, first is the aqueous sodium hydroxide solution of 0.1g/mL, and second is the copper sulfate solution of 0.05g/mL).After shaking up, put in boiling water bath and boil 5min, take out cooling, observe precipitation and colour-change, what occur brick-red precipitation is positive.
(2) thin-layer chromatography (TLC) qualitative detection: the point sample on GF254 silica gel thin-layer plate by the above-mentioned concentrated broth of 5 μ L and gentiopicrin standard substance respectively, at developping agent chloroform-methanol (V/V, launch 20:80), observe under ultraviolet lamp after end, record spot, and calculate its Rf value.
(3) HPLC detection by quantitative: get 800mL fermented liquid, be concentrated into 5mL (obtaining concentrated broth) at 100 DEG C after, add 10mL methanol extraction, extracted by filtration liquid, filtrate is to measure the content of gentiopicrin for HPLC method after 0.45 μm of filtering with microporous membrane; HPLC chromatographic condition is: chromatographic column: chemical bond mould assembly octadecyl post (SciC18 chromatographic column); Pump:K-1001; Detecter:K-1501; Moving phase: methanol-water (V/V, 30:70); Column temperature: 25 DEG C.
(3) restructuring and screening process
1) bark of ash genomic dna is extracted: get the fresh blade 5g of road, appropriate Shaanxi real estate bark of ash, 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, pH=8.0) is added in described centrifuge tube; Put into 65 DEG C of water-baths afterwards, after insulation 60min (period jog 5 times), add 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, shaking up; Be placed on 4 DEG C, the centrifugal 10min of 11000rpm, abandon supernatant, precipitate with 70% ethanol rinse 2 times, be positioned in Bechtop and dry up to obtain bark of ash genomic dna.With 1mLTris-EDTA (pH8.0) buffer solution bark of ash genomic dna, and survey its concentration with ultraviolet spectrophotometer, finally put into 4 DEG C of Refrigerator stores.
2) the ion implantation mycoderm of low energy N+: by the starting strain (H.polymorphaDL-1) of this Laboratories Accession in 37 DEG C, on YPD solid medium after line activation 24h, picking fresh colony is inoculated in YPD liquid nutrient medium, in 37 DEG C, cultivate 16h under 110r/min speed conditions and obtain bacterium liquid A, getting appropriate bacterium liquid A sterilized water, to be diluted to concentration be 1.0 × 10
7cFU/mL obtains mycelium dilution liquid, 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, and the vacuum target chamber being then placed in ion beam implanter carries out low energy N+ implantation, Implantation Energy is 20Kev, and implantation dosage is 2.0 × 10
16ions/cm
2, the burst length is 5s, and interval time is 10s, and vacuum tightness is 1.5 × 10
-3pa.
3) solid culture: through step 2) after, the mycoderm after low energy N+ is ion implantation is soaked with the Tris-EDTA damping fluid (pH8.0) that 2mL contains bark of ash genomic dna, after 37 DEG C of incubation 2h, wash-out is carried out with pipettor, bacterium is all eluted to obtain bacterium liquid B, get respectively after 0.1mL bacterium liquid B is spread evenly across YPD solid medium and cultivate 72h in 37 DEG C.
4) recombinant bacterial strain primary dcreening operation: through step 3) after, the bacterium colony of YPD cultured on solid medium is transferred in test tube and carries out liquid culture (37 DEG C, 110rpm/min cultivate 96h), collect fermented liquid in the centrifugal 10min of 10000r/min to concentrate, and then carry out detection analysis.
Gentiopicrin belongs to iridoid glycosides, and the detection of such material mainly adopts Molish to react and Fehling test.First utilizing Molish to react to analyze restructuring bacterial strain fermentation liquor, observing in the reaction of sample liquid whether occur purple ring (positive findings is for occurring purple ring).Whether then, to above-mentioned, recycling Fehling test occurs that the fermented liquid of the recombinant bacterium of purple ring is analyzed further, observe in sample liquid and have brick-red precipitation to generate (positive findings is for occurring brick-red precipitation).Above-mentioned two kinds of positive reaction results show to there is glucoside compound 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 10mLYPD liquid nutrient medium, 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 250mL triangular flask containing 100mLYPD liquid nutrient medium, then in 110r/min, 90h is cultivated at 37 DEG C, then in the centrifugal 10min of 10000r/min, carry out in 85 DEG C of oven dry to constant weights analysis of weighing after the centrifugal thalline sterile distilled water obtained rinses twice, the centrifugal supernatant liquor obtained is qualitative for product, detection by quantitative, main employing thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).
TLC 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 gentiopicrin standard substance point on thin layer plate, then thin layer plate is placed in developping agent chloroform-methanol (V/V, 20:80) to launch.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 gentiopicrin standard substance, its Rf value is 0.3, with gentiopicrin standard substance Rf value (0.29) 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 gentiopicrin standard substance are carried out HPLC analysis, result as shown in Figure 2, retention time is go out peak (Fig. 2 B) in 12.377min place primary dcreening operation recombinant bacterial strain concentrated broth, the retention time 12.370 going out peak (Fig. 2 A) with gentiopicrin 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 biosynthesizing gentiopicrin.Pass through above-mentioned steps, final the present invention's screening obtains a strain recombinant bacterial strain DL-49, 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.8998.
(4) fermentation of recombinant bacterial strain and gentiopicrin separation and Extraction
The zymotechnique of recombinant bacterial strain (CGMCCNo.8998): adopt 15L automatic fermenter to carry out high density fermentation to the recombinant bacterial strain screened (CGMCCNo.8998) and produce gentiopicrin, by recombinant bacterial strain, after seed culture, (acquisition with reference to seed culture fluid above) is inoculated in YPD liquid nutrient medium with the inoculum size of 5% (volume ratio), in 37 DEG C, rotating speed is carry out continuously fermenting 96h under 300r/min, pH is 6.5, in fermentation, stream adds 0.5% (w/v simultaneously, containing 0.5g glucose in every 100mL water) D/W, flow velocity is 12.5mL/h, after fermentation 48h, fermented liquid 500mL is got every 12h, measure yeast cell biomass and gentiopicrin.Result as shown in Figure 3.Known, cell concentration constantly raises along with the increase of incubation time, and after cultivating 72h, cellular biomass is increased to 42.35g/L; Gentiopicrin is a large amount of appearance from 60h, and along with time lengthening, output slightly increases, and during 72h, output reaches the highest, reaches 7.53mg/L.
Gentiopicrin separation and purification: according to above-mentioned zymotechnique, carries out high density fermentation 96h by recombinant bacterial strain (CGMCCNo.8998), gets 500mL fermented liquid respectively carry out separation and purification detection analysis in 48h, 60h, 72h, 84h and 96h.Be specially fermented liquid 500mL after the centrifugal 10min of 12000r/min, supernatant liquor is concentrated into 10mL in 80 DEG C, D301 type macroporous resin is adopted to carry out Static Adsorption enrichment 8h to gentiopicrin, with the aqueous ethanolic solution of 20mL volume fraction 8%, wash-out is carried out to gentiopicrin afterwards, collect after elutriant is concentrated into 1mL in 60 DEG C and carry out HPLC detection analysis, can obtain the product that purity is 84.35%, the rate of recovery of gentiopicrin is 73.15%.
(5) genetic stability of recombinant bacterial strain
Carry out cultivating (37 DEG C, 110r/min) in the triangular flask being inoculated in containing 200mLYPD liquid nutrient medium after being activated by recombinant bacterial strain (CGMCCNo.8998), in Secondary Culture 8 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, gentiopicroside in different morphological is substantially constant, after Secondary Culture 8 generation, gentiopicroside in different morphological is 2.106mg/L, reduce 6.1% compared to the gentiopicroside in different morphological (2.235mg/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 and produces gentiopicrin recombinant bacterial strain, and screening obtains the recombinant bacterial strain (CGMCCNo.8998) that a strain can be used for fermentative production gentiopicrin.In addition; compared with plant extraction method; fermentable biosynthesizing gentiopicrin 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 gentiopicrin source shortage problem, protection gentianaceae plant resource realize its Sustainable development and provide new way.The present invention adopts low energy ion beam implantation technology to mediate bark of ash STb gene random conversion in yeast first and produces gentiopicrin recombinant bacterial strain to screen, and provides practical basis for utilizing microorganisms producing gentiopicrin.
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 (Hansenulapolymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCCNo.8998.
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 60 ~ 96h under being 300r/min condition.
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 YPD liquid nutrient medium, and in fermenting process, stream adds the D/W of 0.5%, and flow velocity is 12.5mL/h.
4. the application of multiple-shaped nuohan inferior yeast recombinant bacterial strain in gentiopicrin biosynthesizing as claimed in claim 1.
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