CN104611303B - A kind of fused protein that can improve dammarendiol transformation efficiency and construction method and application - Google Patents

A kind of fused protein that can improve dammarendiol transformation efficiency and construction method and application Download PDF

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CN104611303B
CN104611303B CN201410735927.XA CN201410735927A CN104611303B CN 104611303 B CN104611303 B CN 104611303B CN 201410735927 A CN201410735927 A CN 201410735927A CN 104611303 B CN104611303 B CN 104611303B
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atcpr1
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卢文玉
赵方龙
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Tianjin University
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Abstract

The invention discloses a kind of fused protein that can improve dammarendiol transformation efficiency and construction method and application, construction step to be：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, it is connected with 5 ' ends of sequence shown in SEQ ID NO.2 with coded polypeptide GSTSSGSG base sequence, constructs 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 convert and expressed into brewing yeast cell.The fused protein of the method structure of the present invention, can improve the transformation efficiency of dammarendiol to protopanoxadiol.

Description

A kind of fused protein that can improve dammarendiol transformation efficiency and construction method and Using
Technical field
The present invention relates to biological technical field, more particularly to can a kind of fusion egg that can improve dammarendiol transformation efficiency White matter and construction method and application.
Background technology
Ginsenoside is the main active of Chinese medicine ginseng, has that protection is cardiovascular, antifatigue, anti-aging and anticancer Pharmacological action.Traditional culture of ginseng, tissue cultures and extraction of the plant tissue in downstream has been difficult to meet market to ginsenoside 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, determines that PPDS genes (cyp716a47) coding is former Panoxadiol synzyme.2014, GT screening also obtained the comprehensive ginseng transcript profile delivered such as certain progress, Zhou Zhihua Information, filter out the glycosyl transferase that protopanoxadiol can be converted into ginsenoside compound K (CK).
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, the copy of simple increase PPDS and its reductase can not solve Problem, cause the transformation efficiency of dammarendiol relatively low.
The content of the invention
The purpose of the present invention is to overcome deficiency of the prior art, there is provided a kind of dammarendiol transformation efficiency that can improve 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 a kind of answering for fused protein that can improve dammarendiol transformation efficiency With.
Technical scheme is summarized as follows：
A kind of construction method for the fused protein that can improve dammarendiol transformation efficiency, comprises the following steps：
1. preceding 138 base excisions that 5 ' of the Gene A tCPR1 of cytochromes-NADPH- reductases 1 in arabidopsis are held, Sequence shown in SEQ ID NO.2 is obtained, AtCPR1 genes are with shown in SEQ ID NO.1 in the arabidopsis；
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, constructed with coded polypeptide GSTSSGSG base sequence with 5 ' ends of sequence shown in SEQ ID NO.2 The Genetic elements of PPDS-linker1-AtCPR1 fused proteins；
3. by the Genetic elements of PPDS-linker1-AtCPR1 fused proteins and brewing yeast cell endogenesis promoter and Terminator connects, and builds PPDS-linker1-AtCPR1 fused protein expression casettes, and convert into brewing yeast cell Expression.
Specific building process is shown in embodiment 3.
The fused protein PPDS-linker1- that can improve dammarendiol transformation efficiency of above method structure AtCPR1。
Above-mentioned fused protein synthesizes the application in ginsenoside in saccharomyces cerevisiae.
Coded polypeptide GSTSSGSG base sequence such as 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ', but be not limited only to This sequence.
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) 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 PPDS-linker1-AtCPR1 that can improve dammarendiol transformation efficiency that the present invention is built melts Hop protein matter is in saccharomyces cerevisiae than PPDS the and AtCPR1 high catalytic efficiencies of individualism.The fusion that this construction method obtains Albumen improves the transformation efficiency of dammarendiol, is advantageous to ginsenoside efficiently synthesizing in microorganism.
Brief description of the drawings
Fig. 1 .PGK1p-PPDS-linker1-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-linker1-AtCPR1 fused proteins constructed by the present invention in yeast Intracellular schematic diagram.
Fig. 3 dammarendiols and protopanoxadiol LC-MS analyses.
PPDS and AtCPR1 contrasts with PPDS-linker1-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 used, reagent 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 comes 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, expands PGK1p promoters and CYC1t terminators 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 ID NO.23) and erg1-ADH1t-R (SEQ ID NO.24) is primer, expands TEF1p promoters and ADH1t terminators respectively.With W303-1a genomes are template, are expanded with erg1-F (SEQ ID NO.21) and erg1-R (SEQ ID NO.22) for primer Erg1 genetic fragments.TEF1p promoters, erg1 genetic fragments and ADH1t terminators are fused into erg1 with the method for fusion DNA vaccine Expression module TEF1p-erg1-ADH1t, 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, expands TDH3p promoters and ADH3t terminators 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 using the conversion elements of method structure leu2- δ 1 of fusion DNA vaccine；With Pxp320 is template, is that primer expands 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 using the method structure his3-rDNA1 conversion elements of 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 for blend compounds recovery purifies each conversion module.
PCR enzymes used in the present invention are the pfu polymerases of Beijing Quanshijin Biotechnology Co., Ltd, and 50 μ L PCR is expanded 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 is as follows used in the present invention：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, 55 DEG C of annealing 30 seconds, (11 are followed within 1 minute for 72 DEG C of extensions 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 5 hours.5min is centrifuged under 3000 turns of normal temperature and collects thalline, is discarded With the ddH of sterilizing after clear2O rinses thalline, and 5min is centrifuged under 3000 turns of normal temperature and collects thalline, supernatant discarding.Then will 1mL100mM lithium acetate is added in thalline, and 5min collection thalline, system are centrifuged under 3000 turns of normal temperature after placing 5min at room temperature Standby competent yeast cells.Conversion mixed system includes 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, finally Use ddH2O polishings are to 360 μ L.Items are sequentially added in the Saccharomyces cerevisiae competent cell just prepared by said sequence, centrifuged Whirlpool 1min, 2min is centrifuged under 42 DEG C of water-bath 30min, 4000 turns of normal temperature, remove and 1mLYPD culture mediums are added after supernatant, 30 DEG C 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 are screened.Screening and culturing condition is 30 DEG C, cultivates more than 48h.Obtain converting 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.Wine brewing ferment The preparation method of female competent cell is same as above, conversion fragment PGK1p-DS-CYC1t, TDH3p-tHMG1-ADH3t, leu2- δ 1, and 2 each 200ng of δ, method for transformation and screening technique are as described above.Then after carrying out bacterium colony PCR checkings, obtain being overexpressed DS With 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 in ginseng and arabidopsis, The codon optimization for saccharomyces cerevisiae is carried out, then (is closed Jin Wei intelligence bio tech ltd by the method for chemical synthesis Into) the gene PPDS that obtains encoding ginseng Central Plains panoxadiol synzyme is base in SEQ ID NO.3 and arabidopsis in sequence table Because AtCPR1 is the gene order SEQ ID NO.1 in sequence table.TDH3p, PGK1p and terminator CYC1t, ADH3t are all from Saccharomyces cerevisiae 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, expands TDH3p promoters and ADH3t terminators 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 The method of TDH3p-AtCPR1-ADH3t, PCR and fusion DNA vaccine is the same as embodiment 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, expands PGK1p promoters and CYC1t terminators 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 The method of CYC1t, PCR and fusion DNA vaccine is the same as embodiment 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 using the conversion elements of method structure ura3- δ 1 of 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 into saccharomyces cerevisiae genome in a manner of yeast self assembly on δ sites, and bacterium colony is carried out after obtaining transformant PCR is verified, obtains synthesizing the Wine brewing yeast strain W3 of protopanoxadiol.
On the basis of W3, increase the copy of 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 is turned using the method structure ade2-leu1 of 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 ID NO.61) It is that primer expands PGK1p-PPDS-CYC1t modules with PDDS-CYC1t-R (SEQ ID NO.54)；With TDH3p-AtCPR1-F (SEQ ID NO.43) and TDH3p-AtCPR1-R2 (SEQ ID NO.62) are that primer expands TEF1p-AtCPR1-ADH3t moulds Block.Using the method for transformation of embodiment 1, by PGK1p-PPDS-CYC1t, TDH3p-AtCPR1-ADH3t, ade2-leu1 and Leu2 conversions each 200ng of fragment is incorporated into Yeast genome leu2 sites in a manner of yeast self assembly, after bacterium colony PCR checkings, Obtain increasing the bacterial strain W3plus of PPDS and AtCPR1 modules copy.
Embodiment 3, a kind of structure for the fused protein that can improve dammarendiol transformation efficiency
According to the gene of cytochromes-NADPH- reductases 1 in protopanoxadiol synthase gene PPDS in ginseng and arabidopsis AtCPR1 gene order, the codon optimization for saccharomyces cerevisiae is carried out, then pass through method (the gold only intelligence life of chemical synthesis Thing Science and Technology Ltd. synthesizes) 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 equal From saccharomyces cerevisiae w303-1a genomes；Riddled basins ura3 comes from plasmid pxp218.
Two protein are used to be connected with coded polypeptide GSTSSGSG base sequence, encodes linker1's in the present embodiment Base sequence is 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ' (SEQ ID NO.71), and the base sequence is designed into primer In and the connections of two genetic fragments realized using the method for fusion DNA vaccine, specific implementation process is：With protopanoxadiol in ginseng Synthase gene PPDS (SEQ ID NO.3) is template, with PPDS-F in sequence table (SEQ ID NO.51) and PPDS-linker1- R (SEQ ID NO.69) is primer, expands PPDS-linker1 fragments, and the fragment eliminates protopanoxadiol synthase base in ginseng Terminator codon TAA are held because of the 3 ' of PPDS, while the base sequence 5 ' also containing coded polypeptide GSTSSGSG- GGTTCTACTTCTTCAGGTTCAGGT-3’(SEQ ID NO.71)；With the base of cytochromes-NADPH- reductases 1 in arabidopsis Because preceding 138 base excision sequences (SEQ ID NO.2) at AtCPR1 gene 5 's end are template, with linker1-AtCPR1-F (SEQ ID NO.70) and AtCPR1-R (SEQ ID NO.46) are primer, expand linker1-AtCPR1 fragments, the fragment bag - the GGTTCTACTTCTTCAGGTTCAGGT-3 ' of base sequence 5 ' (SEQ ID NO.71) of the GSTSSGSG containing coded polypeptide and go Except 5 ' end 138bp AtCPR1 gene orders (SEQ ID NO.2).By PPDS-linker1 and linker1-AtCPR1 gene pieces Section connects into PPDS-linker1-AtCPR1 Genetic elements with the method for fusion DNA vaccine, and the element is PPDS-linker1- The Genetic elements of AtCPR1 fused proteins.The method of PCR and fusion DNA vaccine is the same as embodiment 1.
By the Genetic elements of PPDS-linker1-AtCPR1 fused proteins and brewing yeast cell endogenesis promoter and end Only son connection, builds PPDS-linker1-AtCPR1 fused protein expression casettes, and specific method is：With PGK1p-PPDS- F (SEQ ID NO.49) and PGK1p-PPDS-R (SEQ ID NO.50) is primer, using W303-1a genomes as template, amplification PGK1p promoters；It is to draw with AtCPR1-ADH3t-F (SEQ ID NO.47) and AtCPR1-ADH3t-R (SEQ ID NO.48) Thing, using W303-1a genomes as template, amplifying ADH 3t terminators.By the gene member of PPDS-linker1-AtCPR1 fusion enzymes Part PPDS-linker1-AtCPR1 is connected with brewing yeast cell promoter PGK1p and terminator ADH3t with fusion DNA vaccine, structure PPDS-linker1-AtCPR1 fusion enzyme gene expression box PGK1p-PPDS-linker1-AtCPR1-ADH3t (Fig. 1).
Expression cassette PGK1p-PPDS-linker1-AtCPR1-ADH3t conversions are entered into brewing yeast cell to express, specifically Method is：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) it is that primer expands δ Post section fragments, and using the conversion elements of method structure ura3- δ 1 of 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-linker1-AtCPR1-ADH3t expression cassettes, ura3- δ 1 and δ 2 conversion each 200ng of fragment convert the brewing yeast cell W2 obtained into embodiment 2 genome in a manner of yeast self assembly On δ sites, obtain after carrying out bacterium colony PCR checkings after transformant, obtain synthesizing the yeast strain W3a of protopanoxadiol.
Embodiment 4, the catalytic capability measure for merging PPPDS enzymes
First, the preparation of saccharomyces cerevisiae microsome
The brewing yeast cell W2, W3 and W3a obtained in embodiment 1,2,3 is cultivated (30 in YPD culture mediums respectively ℃,220rmp).(3000rmp, 5min) collection cell is centrifuged after cultivating 24h.Brewing yeast cell TEK buffer solutions (100mM KCl, 50mM Tris-HCl, 1mM EDTA) it is resuspended, 6100g centrifugations 3min.Supernatant is removed, adds the glass with biomass equivalent Pearl, and add Extraction buffer (20mM beta -mercaptoethanols, 1%BSA, 0.6M sorbierites, 50mM Tris-HCl, 1mM EDTA). Acutely after concussion 20min, 6100g centrifugation 15min, supernatant is crossed after film and is transferred in clean centrifuge tube, adds MgCl2 1h depositing particles bodies are being placed on ice after (50mM).Then 12500g centrifuges 20min.After supernatant removes, 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 poor method measure 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 Not bubbling, the ultra-violet absorption spectrum of two parts of suspensions is determined respectively.Instrument is TU-1810 type ultraviolet-uisible spectrophotometers (cuvette light path 1cm), scanning wavelength scope 400-500nm.Using the suspension of logical CO bubblings as sample liquid, illogical CO suspension 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 to react, the body such as a certain amount of mixed liquor, addition is taken every 5min Long-pending n-hexane extraction, 0.22 organic film excessively is standby after 12000rmp centrifuges 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 w3a microsomes .It is computed, PPDS content is 9.82 ± 0.83 μM/g microsomes in W3, and PPDS content is 10.04 ± 0.79 μ in W3a M/g microsomes.It follows that the PPDS concentration difference in saccharomyces cerevisiae microsome is smaller, their enzyme activity determination result such as Fig. 4 It is shown.In Fig. 4, the catalytic efficiencies of the constructed PPDS-linker1-AtCPR1 fused proteins of this experiment is apparently higher than nature Under the conditions of PPDS and 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 W3a that are obtained in embodiment 2,3 respectively in YPD culture mediums In 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, 2min then is centrifuged under 12000 turns/min normal temperature conditions, take 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 of PPDS and AtCPR1 expression modules, the production of dammarendiol and protopanoxadiol Amount is respectively 102.5mg/L and 150.6mg/L；The yield of W3a dammarendiols and protopanoxadiol be respectively 13.2mg/L and 265.6mg/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-linker1-AtCPR1 fusion protein mass-energy effectively carries The transformation efficiency of high dammarendiol, so as to improve the yield of protopanoxadiol.

Claims (3)

  1. A kind of 1. construction method for the fused protein that can improve dammarendiol transformation efficiency, it is characterized in that including following step Suddenly：
    1. by preceding 138 base excisions at the Gene A tCPR1 of cytochromes-NADPH- reductases 15 ' ends in arabidopsis, obtain Sequence shown in SEQ ID NO.2, AtCPR1 genes are with shown in SEQ ID NO.1 in 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 ' ends of sequence shown in SEQ ID NO.2 with coded polypeptide GSTSSGSG base sequence, construct PPDS- The Genetic elements of linker1-AtCPR1 fused proteins；
    3. by the Genetic elements of PPDS-linker1-AtCPR1 fused proteins and brewing yeast cell endogenesis promoter and termination Son connection, builds PPDS-linker1-AtCPR1 fused protein expression casettes, and convert into brewing yeast cell table Reach.
  2. 2. the fused protein that can improve dammarendiol transformation efficiency of the method structure of claim 1.
  3. 3. the fused protein of claim 2 synthesizes the application in ginsenoside in saccharomyces cerevisiae.
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