CN104450769B - The fused protein and construction method of dammarendiol transformation efficiency can be improved - Google Patents
The fused protein and construction method of dammarendiol transformation efficiency can be improved Download PDFInfo
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Abstract
The invention discloses the fused protein and construction method that can improve dammarendiol transformation efficiency, construction method step is：1. by preceding 138 base excisions at the Gene A tCPR1 of cytochromes NADPH reductases 15 ' ends in arabidopsis, sequence shown in SEQ ID NO.2 is obtained；2. it will be removed with 3 ' the end terminator codon TAA of protopanoxadiol synthase gene PPDS in the ginseng shown in SEQ ID NO.3, be connected with 5 ' terminal sequences of sequence shown in SEQ ID NO.2, construct the Genetic elements of fused protein；3. the Genetic elements of fused protein are connected with brewing yeast cell endogenesis promoter and terminator, construction of fusion protein matter expression casette, and converted into brewing yeast cell expression.The fused protein that the method for the present invention is built, can improve the transformation efficiency of dammarendiol to protopanoxadiol.
Description
Technical field
The present invention relates to biological technical field, more particularly to can improve dammarendiol transformation efficiency fused protein and
Construction method.
Background technology
Ginsenoside is the main active of Chinese medicine ginseng, cardiovascular with protection, antifatigue, anti-aging and anticancer
Pharmacological action.It is difficult to meet market to ginsenoside that traditional culture of ginseng, tissue cultures and plant tissue in downstream, which is extracted,
Demand.
As a kind of triterpene compound, yeast entogenous metabolic pathway can provide precursor 2,3- oxygen for the synthesis of ginsenoside
Change MF59.Subsequent epoxidation, oxidation and glycosyl addition respectively by dammarendiol synzyme, protopanoxadiol synzyme and
Glycosyl transferase is completed.Dammarendiol synthase gene was found in 2006.Tansakul etc. reports PNA (DDBJ data
Storehouse, sequence number AB265170) it is the gene for being catalyzed dammarendiol synthesis.Han etc. reports another DDS (DDBJ/EMBL/
GenBanK databases, sequence number AB122080).2011, Jung-Yeon Han etc. passed through the ginseng that induces methyl jasmonate
Adventitious root carries out cDNA and builds storehouse, by a series of screenings after transcript profile is sequenced, and determines that PPDS genes (cyp716a47) coding is former
Panoxadiol synzyme.2014, GT screening also obtained certain progress, and Zhou Zhihua etc. integrates the ginseng transcript profile delivered
Information, protopanoxadiol can be converted into ginsenoside compound K (CK) glycosyl transferase by filtering out.
Based on the excavation of related gene in above ginsenoside route of synthesis, Zhang Xueli is engineered to saccharomyces cerevisiae,
The AtCPR1 being introduced into DS, PPDS and arabidopsis, and yeast mevalonate pathway speed limit process is overexpressed, construct production
Artificial yeast's cell of protopanoxadiol.Zhou Zhihua has obtained to produce CK's by the GT gene insertion saccharomyces cerevisiaes screened
Yeast cells.
PPDS is a kind of cytochrome P 450 monooxygenases.The research of early stage is found, in artificial constructed protopanoxadiol
In yeast route of synthesis, the step of PPDS is a speed limit, simple increase PPDS and its copy of reductase can not be solved
Problem, causes the transformation efficiency of dammarendiol relatively low.
The content of the invention
The purpose of the present invention overcomes deficiency of the prior art to improve dammarendiol transformation efficiency there is provided a kind of
Fused protein.
Second object of the present invention is to provide a kind of structure for the fused protein that can improve dammarendiol transformation efficiency
Construction method.
Third object of the present invention is to provide the application for the fused protein that can improve dammarendiol transformation efficiency.
Technical scheme is summarized as follows：
The construction method of the fused protein of dammarendiol transformation efficiency can be improved, is comprised the following steps：
1. by preceding 138 base excisions at the Gene A tCPR1 of cytochromes-NADPH- reductases 15 ' ends in arabidopsis,
AtCPR1 genes are obtained in sequence shown in SEQ ID NO.2, the arabidopsis with shown in SEQ ID NO.1；
2. by with 3 ' the end terminator codons of protopanoxadiol synthase gene PPDS in the ginseng shown in SEQ ID NO.3
TAA is removed, and is connected with 5 ' terminal sequences of sequence shown in SEQ ID NO.2, is constructed the gene of PPDS-AtCPR1 fused proteins
Element；
3. the Genetic elements of PPDS-AtCPR1 fused proteins and brewing yeast cell endogenesis promoter and terminator are connected
Connect, build PPDS-AtCPR1 fused protein expression casettes, and convert into brewing yeast cell expression.
Specific building process is shown in embodiment 3.
What the above method was built can improve the fused protein of dammarendiol transformation efficiency.
Above-mentioned fused protein synthesizes the application in ginsenoside in saccharomyces cerevisiae.
The saccharomyces cerevisiae endogenesis promoter such as PGK1p (promoter of phosphoglyceric kinase 1), sequence SEQ ID NO.7.
Saccharomyces cerevisiae endogenous terminator such as ADH3t (alcohol dehydrogenase 3) the sequence SEQ ID NO.12.
AtCPR1 gene orders shown in SEQ ID NO.1 are the base of arabidopsis NADPH- cytochrome P450 reductases 1
Because of the optimization of the codon for saccharomyces cerevisiae.
Obtained before gene order shown in SEQ ID NO.2 removes for sequence SEQ ID NO.1 after 138bp.
PPDS gene orders shown in SEQ ID NO.3 are directed to wine brewing ferment for the gene of ginseng Central Plains panoxadiol synzyme
The optimization of female codon.
Advantages of the present invention：
It is demonstrated experimentally that the present invention build can improve the PPDS-AtCPR1 fused proteins of dammarendiol transformation efficiency
Than PPDS the and AtCPR1 high catalytic efficiencies of individualism in saccharomyces cerevisiae.The fusion protein that this construction method is obtained is improved
The transformation efficiency of dammarendiol, is conducive to ginsenoside efficiently synthesizing in microorganism.
Brief description of the drawings
Fig. 1 .PGK1p-PPDS-AtCPR1-ADH3t Fusion Module electrophoretograms.
Schematic diagram of Fig. 2 .PPDS and AtCPR1 protein in yeast cells.Fig. 2A is two kinds of protein under natural conditions
Schematic diagram in yeast cells；Fig. 2 B are the PPDS-AtCPR1 fused proteins constructed by the present invention in yeast cells
Schematic diagram.
Fig. 3 dammarendiols and protopanoxadiol LC-MS analyses.
PPDS and AtCPR is contrasted with PPDS-AtCPR1 fusion proteins catalytic capability under Fig. 4 natural conditions.
The yield comparison of each bacterial strain dammarendiols of Fig. 5 and protopanoxadiol.
Embodiment
Below by specific embodiment, the present invention is further illustrated.
Experimental method used in example below is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1
Synthesize the structure of dammarendiol brewing yeast cell
First, module construction
According to the amino acid sequence of dammarendiol synthase in ginseng, the codon optimization for saccharomyces cerevisiae is carried out, so
Obtain encoding the base of dammarendiol synzyme by the method (synthesis of Jin Wei intelligence bio tech ltd) of chemical synthesis afterwards
Because DS is SEQ ID NO.4；The endogenous tHMG1 of saccharomyces cerevisiae (SEQ ID NO.5), erg1 (SEQ ID NO.6), and start
Sub- PGK1p (SEQ ID NO.7), TEF1p (SEQ ID NO.8), TDH3p (SEQ ID NO.9) and terminator CYC1t (SEQ
ID NO.10), ADH1t (SEQ ID NO.11), ADH3t (SEQ ID NO.12) is all from saccharomyces cerevisiae w303-1a genomes；
Riddled basins leu2 come from plasmid prs405 (U.S. ATCC), his3 from plasmid pxp320 (be purchased from Addgene,
Inc.WWW.addgene.org)。
With saccharomyces cerevisiae W303-1a (U.S., ATCC) genome for template, with PGK1p-DS-F (SEQ ID NO.13)
(and PGK1p-DS-R (SEQ ID NO.14) and DS-CYC1t-F (SEQ ID NO.17) and DS-CYC1t-R (SEQ ID
NO.18) it is primer, PGK1p promoters and CYC1t terminators is expanded respectively.
With dammarendiol synthase gene DS (SEQ ID NO.4) for template, with DS-F (SEQ ID NO.15) and DS-
R (SEQ ID NO.16) is that primer expands DS genes.By PGK1p promoters, DS genes and CYC1t terminator fusion DNA vaccines
Method is fused into DS expression module PGK1p-DS-CYC1t；Using saccharomyces cerevisiae W303-1a genomes as template, with TEF1p-
Erg1-F (SEQ ID NO.19) and TEF1p-erg1-R (SEQ ID NO.20) and erg1-ADH1t-F (SEQ IDNO.23)
It is primer with erg1-ADH1t-R (SEQ ID NO.24), TEF1p promoters and ADH1t terminators is expanded respectively.
Using W303-1a genomes as template, it is with erg1-F (SEQ ID NO.21) and erg1-R (SEQ ID NO.22)
Primer expands erg1 genetic fragments.The method of TEF1p promoters, erg1 genetic fragments and ADH1t terminator fusion DNA vaccines is melted
Erg1 expression module TEF1p-erg1-ADH1t is synthesized, sequence used is shown in sequence table.
Using saccharomyces cerevisiae W303-1a genomes as template, with TDH3p-tHMG1-F (SEQ ID NO.25) and TDH3-
THMG1-R (SEQ ID NO.26) and tHMG1-ADH3t-F (SEQ ID NO.29) and tHMG1-ADH3t-R (SEQ ID
NO.30) it is primer, TDH3p promoters and ADH3t terminators is expanded respectively.
Using W303-1a genomes as template, with tHMG1-F (SEQ ID NO.27) and tHMG1-R (SEQ ID NO.28)
THMG1 genetic fragments are expanded for primer.By the side of TDH3p promoters, tHMG1 genetic fragments and ADH3t terminator fusion DNA vaccines
Method is fused into tHMG1 expression module TDH3p-tHMG1-ADH3t, and sequence used is shown in sequence table.
In addition, using prs405 as template, with leu-F (SEQ ID NO.33) and leu-R (SEQ ID NO.34) for primer
Leu2 marker gene is expanded, using saccharomyces cerevisiae W303-1a genomes as template, with δ 1-F (SEQ ID NO.39) and δ 1-R (SEQ
ID NO.40) it is primer, δ Post section fragments are expanded, and the conversion elements of leu2- δ 1 are built using the method for fusion DNA vaccine；With
Pxp320 is template, is primer amplification his3 mark bases with his-F (SEQ ID NO.31) and his-R (SEQ ID NO.32)
Cause, using saccharomyces cerevisiae W303-1a genomes as template, with rDNA1-F (SEQ ID NO.35) and rDNA1-R (SEQ ID
NO.36 rDNA Partial Fragments) are expanded for primer, and his3-rDNA1 conversion elements are built using the method for fusion DNA vaccine.Module
Self assembly conversion is also needed to using saccharomyces cerevisiae genome as template, respectively with rDNA2-F (SEQ ID NO.37), rDNA2-R
(SEQ ID NO.38) and δ 2-F (SEQ ID NO.41), δ 2-R (SEQ ID NO.42) are that primer expands rDNA2 and the fragments of δ 2.
PCR expands each module after the completion of module construction, and the method that blend compounds are reclaimed purifies each conversion module.
PCR enzymes used of the invention are the pfu polymerases of Beijing Quanshijin Biotechnology Co., Ltd, 50 μ L PCR amplifications
System is as follows：DNA profiling, 1 μ L；Leading (10 μM) and after draw (10 μM) each 1 μ L；DNTP (2.5mM), 5 μ L；10 × Buffer,
10μL；Pfu polymerase, 1 μ L；Finally use the μ L of distilled water polishing 50.Amplification program is set in PCR instrument.Amplification condition is 98 DEG C
Pre-degeneration 2 minutes (1 circulation)；98 DEG C be denatured 10 seconds, annealing extends 1 minute (32 circulation) for 10 seconds, 72 DEG C；72 DEG C of extensions 8
Minute (1 circulation).
Fusion DNA vaccine system used of the invention is as follows：DNA fragmentation total amount 800ng, mol ratio 1:1；DNTP (2.5mM), 5 μ L；
10 × Buffer, 10 μ L；Pfu polymerase, 1 μ L；Finally use the μ L of distilled water polishing 50.Amplification program is set in PCR instrument.Amplification
Condition is 95 DEG C of pre-degenerations 2 minutes (1 circulation)；95 DEG C be denatured 10 seconds, annealing 55 DEG C 30 seconds, 72 DEG C extension (11 are followed within 1 minute
Ring), 72 DEG C extend 5 minutes (1 circulation).
2nd, yeast conversion
The conversion of above-mentioned module is divided to two groups of progress.After the saccharomyces cerevisiae W303-1a that sets out is cultivated 12 hours in YPD culture mediums
Take 200 μ L to add in the fresh YPD culture mediums of 2mL, cultivate 4-5 hours.5min is centrifuged under 3000 turns of normal temperature and collects thalline, is discarded
With the ddH of sterilizing after supernatant2O rinses thalline, and 5min is centrifuged under 3000 turns of normal temperature and collects thalline, supernatant discarding.Then by 1mL
100mM lithium acetate is added in thalline, places centrifuge 5min collection thalline after 5min under 3000 turns of normal temperature at room temperature, prepare ferment
Female competent cell.Convert mixed system include 240 μ L PEG (50%W/V), 36 μ L 1.0M lithium acetates, 10 μ L ss-DNA,
Fragment PGK1p-DS-CYC1t, TEF1p-erg1-ADH1t, his3-rDNA1 and rDNA2 each 200ng of fragment is converted, is finally used
ddH2O polishings are to 360 μ L.Items are sequentially added in the Saccharomyces cerevisiae competent cell just prepared by said sequence, whirlpool is centrifuged
Whirlpool 1min, 2min is centrifuged under 42 DEG C of water-bath 30min, 4000 turns of normal temperature, removes addition 1mLYPD culture mediums, 30 DEG C after supernatant
150rpm cultivates 2h.Centrifuge 5min under 4000 turns of normal temperature, supernatant discarding, sterile water washing 2 times, and be resuspended with 100 μ L sterilized waters
Cell, the flat board for applying missing histidine is screened.Screening and culturing condition is 30 DEG C, cultivates more than 48h.Obtain conversion DS and mistake
Express erg1 S. cervisiae W1.
Using W1 as starting strain, DS and tHMG1 is overexpressed.Using PGK1p-DS-CYC1t as template, with leu-PGKp-F
(SEQID NO.72) and DS-CYC1t-R (SEQ ID NO.18) are primer, expand PGK1p-DS-CYC1t expression cassettes.Yeast sense
Preparation method by state cell is same as above, conversion fragment PGK1p-DS-CYC1t, TDH3p-tHMG1-ADH3t, leu2- δ 1,
With each 200ng of δ 2, method for transformation and screening technique are as described above.Then carry out bacterium colony PCR checkings after, obtain be overexpressed DS and
THMG1 Wine brewing yeast strain W2.
The structure of embodiment 2, synthesis protopanoxadiol brewing yeast cell W3 and W3plus
According to the amino acid sequence of cytochromes-NADPH- reductases 1 in protopanoxadiol synthase and arabidopsis, enter the hand-manipulating of needle
Codon optimization to saccharomyces cerevisiae, is then obtained by the method (synthesis of Jin Wei intelligence bio tech ltd) of chemical synthesis
It is AtCPR1 in SEQ ID NO.3 in sequence table and arabidopsis to encode the gene PPDS genes of ginseng Central Plains panoxadiol synzyme
Gene is the gene order SEQ ID NO.1 in sequence table.TDH3p, PGK1p and terminator CYC1t, ADH3t are all from wine brewing
Yeast w303-1a genomes；Riddled basins ura3 from plasmid pxp218 (Addgene,
Inc.WWW.addgene.org)。
Using saccharomyces cerevisiae W303-1a genomes as template, with TDH3p-AtCPR1-F (SEQ ID NO.43) and TDH3p-
AtCPR1-R (SEQ ID NO.44) and AtCPR1-ADH3T-F (SEQ ID NO.47) and AtCPR1-ADH3T-R (SEQ ID
NO.48) it is primer, TDH3p promoters and ADH3t terminators is expanded respectively.With AtCPR1 (SEQ ID NO.1) for template, with
AtCPR1-F (SEQ ID NO.45) and AtCPR1-R (SEQ ID NO.46) is that primer expands AtCPR1 genetic fragments.Will
TDH3p promoters, AtCPR1 genetic fragments and ADH3T terminators are fused into AtCPR1 expression module with the method for fusion DNA vaccine
TDH3p-AtCPR1-ADH3t, PCR and fusion DNA vaccine method be the same as Example 1.
Using saccharomyces cerevisiae W303-1a genomes as template, with PGK1p-PPDS-F (SEQ ID NO.49) and PGK1p-
PPDS-R (SEQ ID NO.50) and PPDS-CYC1t-F (SEQ ID NO.53) and PPDS-CYC1t-R (SEQ ID
NO.54) it is primer, PGK1p promoters and PPDS terminators is expanded respectively.With PPDS (SEQ ID NO.3) for template, with
PPDS-F (SEQ ID NO.51) and PPDS-R (SEQ ID NO.52) is that primer expands PPDS genetic fragments.PGK1p is started
Son, PPDS genetic fragments and CYC1t terminators are fused into PPDS expression module PGK1p-PPDS- with the method for fusion DNA vaccine
CYC1t, PCR and fusion DNA vaccine method be the same as Example 1.
In addition, using pxp218 as template, with ura-F (SEQ ID NO.55) and ura-R (SEQ ID NO.56) for primer
Ura3 marker gene is expanded, using saccharomyces cerevisiae W303-1a genomes as template, with δ 1-F2 (SEQ ID NO.57) and δ 1-R2
(SEQ ID NO.58) is that primer expands δ Post section fragments, and builds the conversion elements of ura3- δ 1 using the method for fusion DNA vaccine；
Using W303 genomes as template, with δ 2-F (SEQ ID NO.59) and δ 2-R (SEQ ID NO.60) for primer, amplification conversion member
Part δ 2.Using the method for transformation of embodiment 1, by PGK1p-PPDS-CYC1t, TDH3p-AtCPR1-ADH3t, ura3- δ 1 and δ 2
Conversion fragment is incorporated on Yeast genome in the way of yeast self assembly on δ sites, is obtained progress bacterium colony PCR after transformant and is tested
Card, obtains synthesizing the Wine brewing yeast strain W3 of protopanoxadiol.
On the basis of W3, the copy of increase PPDS and AtCPR1 expression modules, main method is as follows.With saccharomyces cerevisiae
BY4742 (U.S. ATCC) genome is template, is to draw with Ade2-F (SEQ ID NO.63) and Ade2-R (SEQ ID NO.64)
Thing expands ade2 marker gene Expression elements, using prs405 plasmids as template, with leu1-F2 (SEQ ID NO.65) and leu1-
R2 (SEQ ID NO.66) is that primer expands leu2 Post section fragments, and builds ade2-leu1 turns using the method for fusion DNA vaccine
Change element；Using prs405 plasmids as template, with leu2-F (SEQ ID NO.67) and leu2-R (SEQ ID NO.68) for primer,
Expand conversion elements leu2.Meanwhile, using PGK1p-PPDS-CYC1t as template, with PGK1P-PDDS-F2 (SEQ IDNO.61) and
PDDS-CYC1t-R (SEQ ID NO.54) is that primer expands PGK1p-PPDS-CYC1t modules；With TDH3p-AtCPR1-F (SEQ
ID NO.43) and TDH3p-AtCPR1-R2 (SEQ ID NO.62) be primer expand TEF1p-AtCPR1-ADH3t modules.Using
The method for transformation of embodiment 1, by PGK1p-PPDS-CYC1t, TDH3p-AtCPR1-ADH3t, ade2-leu1 and leu2 conversion plates
Each 200ng of section is incorporated into Yeast genome leu2 sites in the way of yeast self assembly, after bacterium colony PCR checkings, is increased
The bacterial strain W3plus of PPDS and AtCPR1 modules copy.
Embodiment 3, the fused protein structure that dammarendiol transformation efficiency can be improved
According to the gene of cytochromes-NADPH- reductases 1 in protopanoxadiol synthase gene PPDS in ginseng and arabidopsis
AtCPR1 amino acid sequence, carries out the codon optimization for saccharomyces cerevisiae, then passes through the method (Jin Weizhi of chemical synthesis
Bio tech ltd is synthesized) obtain genetic fragment.PPDS genes are with shown in SEQ ID NO.3 in ginseng, in arabidopsis
AtCPR1 genes are with shown in SEQ ID NO.1；By preceding 138 base excisions at AtCPR1 gene 5 's end in arabidopsis, SEQ is obtained
Sequence shown in ID NO.2.Endogenesis promoter PGK1p (SEQ ID NO.7) and endogenous terminator ADH3t (SEQ ID NO.12) is
From saccharomyces cerevisiae w303-1a genomes；Riddled basins ura3 comes from plasmid pxp218.
The connection of two genetic fragments is realized using the method for fusion DNA vaccine, specific implementation process is：With PPDS genes (SEQ
IDNO.3 it is) template, is with PPDS-F in sequence table (SEQ ID NO.51) and PPDS-nolinker-R (SEQ ID NO.69)
Primer, expands PPDS-nolinker fragments, and the 3 ' ends that the fragment eliminates protopanoxadiol synthase gene PPDS in ginseng are terminated
Codon TAA；Preceding 138 base excision sequences held with the Gene A tCPR15 ' of cytochromes-NADPH- reductases 1 in arabidopsis
(SEQ ID NO.2) is template, with nolinker-AtCPR1-F (SEQ ID NO.70) and AtCPR1-R (SEQ IDNO.46)
For primer, nolinker-AtCPR1 fragments are expanded, the fragment holds 138bp AtCPR1 gene orders (SEQ ID for removal 5 '
NO.2).PPDS-nolinker and nolinker-AtCPR1 genetic fragments are connected into PPDS- with the method for fusion DNA vaccine
AtCPR1 Genetic elements, the element is the Genetic elements of PPDS-AtCPR1 fused proteins.The method of PCR and fusion DNA vaccine is same
Embodiment 1.
The Genetic elements of PPDS-AtCPR1 fused proteins and brewing yeast cell endogenesis promoter and terminator are connected
Connect, build PPDS-AtCPR1 fused protein expression casettes, embodiment is：With PGK1p-PPDS-F (SEQ ID
NO.49) and PGK1p-PPDS-R (SEQ ID NO.50) be primer, using W303-1a genomes as template, amplification PGK1p start
Son；With AtCPR1-ADH3t-F (SEQ ID NO.47) and AtCPR1-ADH3t-R (SEQ ID NO.48) for primer, with W303-
1a genomes are template, amplifying ADH 3t terminators.PPDS-AtCPR1 is merged to Genetic elements PPDS-AtCPR1 and the wine brewing of enzyme
Yeast cells promoter PGK1p and terminator ADH3t are connected with fusion DNA vaccine, build PPDS-AtCPR1 fusion enzyme gene expression boxes
PGK1p-PPDS-AtCPR1-ADH3t (Fig. 1).
Using pxp218 as template, expanded with ura-F (SEQ ID NO.55) and ura-R (SEQ ID NO.56) for primer
Ura3 marker gene, using saccharomyces cerevisiae W303-1a genomes as template, with δ 1-F2 (SEQ ID NO.57) and δ 1-R2 (SEQ
ID NO.58) δ Post section fragments are expanded for primer, and the conversion elements of ura3- δ 1 are built using the method for fusion DNA vaccine；With
W303-1a genomes are template, with δ 2-F (SEQ ID NO.59) and δ 2-R (SEQ ID NO.60) for primer, amplification conversion member
Part δ 2.Using the method for transformation of embodiment 1, by PGK1p-PPDS-AtCPR1-ADH3t expression cassettes, ura3- δ 1 and the conversion plates of δ 2
Each 200ng of section converts the brewing yeast cell W2 obtained into embodiment 2 genome δ sites in the way of yeast self assembly
On, obtain carrying out after bacterium colony PCR checkings after transformant, obtain synthesizing the yeast strain W3d of protopanoxadiol.
Embodiment 4, the catalytic capability measure for merging P450 enzymes
First, the preparation of saccharomyces cerevisiae microsome
The brewing yeast cell W2, W3 and W3d obtained in embodiment 1,2,3 is cultivated (30 in YPD culture mediums respectively
℃,220rmp).Cultivate centrifugation (3000rmp, 5min) after 24h and collect cell.Brewing yeast cell TEK buffer solutions (100mM
KCl, 50mM Tris-HCl, 1mM EDTA) it is resuspended, 6100g centrifugations 3min.Supernatant is removed, the glass with biomass equivalent is added
Pearl, and add Extraction buffer (20mM beta -mercaptoethanols, 1%BSA, 0.6M sorbierites, 50mM Tris-HCl, 1mM EDTA).
Acutely after concussion 20min, supernatant is crossed after film and is transferred in clean centrifuge tube, adds MgCl by 6100g centrifugation 15min2
1h depositing particles bodies are being placed on ice after (50mM).Then 12500g centrifuges 20min.After supernatant is removed, the micro- of bottom of the tube is centrifuged
Plastochondria is transferred in TEG buffer solutions (30% glycerine, 50mM Tris-HCl, 1mMEDTA), and is quickly ground and carried out with grinding pestle
Homogenization is handled.
2nd, CO shows that poor method determines P450 enzymes
4mL yeast cells microsome preparation solutions are taken, 80 μ L 0.5mmol/L sodium dithionites is added and mixes, in ice bath
Place 2min.By 4mL it is above-mentioned prepare liquid and be divided to be transferred in two cuvettes, a copy of it CO gas sparging 1min, another
Bubbling, does not determine the ultra-violet absorption spectrum of two parts of suspensions respectively.Instrument is TU-1810 type ultraviolet-uisible spectrophotometers
(cuvette light path 1cm), scanning wavelength scope 400-500nm.Suspension using logical CO bubblings is sample liquid, and the suspension that CO is not led to is
Poor uv absorption spectra is shown in reference liquid, drafting.The absorption value at 45Onm and 490nm wavelength is recorded, is calculated as follows
(molar extinction coefficient ε is 91cm to P450 contents-1mM-1):P450 (mM)=(A450nm-A490nm)/91。
3rd, the measure of the enzyme activity of P450 enzymes
10mg microsomes are added in 500 μ L 100mM potassium phosphate buffers (pH 7.4).Dammarendiol concentration 500
μM, incubated in 30 DEG C of water-baths, add NADPH (1mM) and start reaction, a certain amount of mixed liquor is taken every 5min, the body such as add
0.22 organic film excessively is standby after long-pending n-hexane extraction, 12000rmp centrifugations 2min, finally determines protopanoxadiol with HPLC
Content.Liquid phase chromatogram condition：Sample size 20 μ L, Agilent ZORBAX SB-Aq；Mobile phase is methanol：Acetonitrile=4:6, flow velocity：
1mL/min, UV-detector wavelength 203nm.
As a result：The content W3 of PPDS enzymes, and the content of P450 enzymes subtracts the content institute of P450 enzymes in W2 in W3d microsomes
.Be computed, PPDS content is 9.82 ± 0.83 μM/g microsomes in W3, in W3d PPDS content for 8.81 ± 0.71 μM/
G microsomes.It follows that the PPDS concentration difference in saccharomyces cerevisiae microsome is smaller, their enzyme activity determination result such as Fig. 4 institutes
Show.In Fig. 4, the catalytic efficiencies of the constructed PPDS-AtCPR1 fused proteins of this experiment is apparently higher than PPDS under natural conditions
With AtCPR1 catalytic efficiency.
The LC-MS detections of embodiment 5, the fermentation of engineered strain and tunning.
By artificial synthesized the brewing yeast cell W3, W3plus and W3d that are obtained in embodiment 2,3 respectively in YPD culture mediums
It is middle to be cultivated (30 DEG C, 220rmp).Cell is collected by centrifugation after 6 days, adds acetone, ultrasonic disruption is carried out in ice-water bath
10min, then centrifuges 2min under 12000 turns/min normal temperature conditions, takes supernatant to cross standby after 0.22mm organic films.
Dammarendiol and the qualitative of protopanoxadiol are carried out with LC-MS.Liquid phase chromatogram condition：The μ L of sample size 5, Agilent
ZORBAX SB-Aq；Mobile phase is 90% acetonitrile, flow velocity：0.2mL/min.Mass Spectrometry Conditions：Atomization gas and dry gas are all N2；Touch
Hit voltage：-70V；Spray voltage：3.8kV；Ion gun：APCI；Ion source temperature：120℃；Precipitation temperature：300℃；Flowed after post
Go out thing and import ion gun speed：5μL/min；Scanning of the mass spectrum mass number scope：200-1000Da.
Dammarendiol and the quantitative of protopanoxadiol are carried out with HPLC, liquid phase chromatogram condition：The μ L of sample size 20, Agilent
ZORBAX SB-Aq；Mobile phase is methanol：Acetonitrile=4:6, flow velocity：1mL/min.Dammarendiol is purchased from Yunnan ethylmercuric cloride thing skill
Art Co., Ltd (WWW.biobiopha.com).Protopanoxadiol is purchased from Beijing Suo Laibao Science and Technology Ltd (http://
www.solarbio.cn/)。
As a result as shown in Figure 5：The yield of W3 dammarendiols and protopanoxadiol be respectively 96.8mg/L and
155.6mg/L；W3plus adds the copy number that PPDS and AtCPR1 expresses module, the production of dammarendiol and protopanoxadiol
Amount is respectively 102.5mg/L and 150.6mg/L；The yield of W3d dammarendiols and protopanoxadiol be respectively 10.1mg/L and
269.4mg/L。
It is demonstrated experimentally that in saccharomyces cerevisiae W303-1a host, the copy of simple increase PPDS and AtCPR1 expression modules
Number can not improve the conversion ratio of dammarendiol.Constructed PPDS-AtCPR1 fusion protein mass-energy is effectively improved up to agate alkene
The transformation efficiency of glycol, so as to improve the yield of protopanoxadiol.
Claims (3)
1. the construction method of the fused protein of dammarendiol transformation efficiency can be improved, it is characterized in that comprising the following steps：
1. by preceding 138 base excisions at the Gene A tCPR1 of cytochromes-NADPH- reductases 15 ' ends in arabidopsis, obtain
AtCPR1 genes are with shown in SEQ ID NO.1 in sequence shown in SEQ ID NO.2, the arabidopsis；
2. it will be gone with 3 ' the end terminator codon TAA of protopanoxadiol synthase gene PPDS in the ginseng shown in SEQ ID NO.3
Remove, be connected with 5 ' terminal sequences of sequence shown in SEQ ID NO.2, construct the Genetic elements of PPDS-AtCPR1 fused proteins；
3. the Genetic elements of PPDS-AtCPR1 fused proteins are connected with brewing yeast cell endogenesis promoter and terminator,
PPDS-AtCPR1 fused protein expression casettes are built, and are converted into brewing yeast cell expression.
What 2. the method for claim 1 was built can improve the fused protein of dammarendiol transformation efficiency.
3. the fused protein of claim 2 synthesizes the application in ginsenoside in saccharomyces cerevisiae.
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Non-Patent Citations (2)
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| Ginsenosides chemistry,biosynthesis,analysis,and potential health effects;Christensen LP;《Adv Food Nutr Res》;20091231(第55期);第1-99页 * |
| 登录号：JX268037.1;Koopman,F.等;《GENBANK》;20121220;第1页 * |
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