CN104611303A - Fusion protein capable of improving dammarenediol conversion efficiency, construction method and application - Google Patents

Abstract

The invention discloses fusion protein capable of improving dammarenediol conversion efficiency, a construction method and application. The construction steps comprise: excising first 138 bases at 5' end of arabidopsis thaliana cytochrome-NADPH-reductase 1 gene AtCPR1, so as to obtain a sequence shown as SEQ ID NO. 2; (2) removing a termination codon TAA at 3' end of ginseng protopanaxadiol synthase gene PPDS with a sequence shown as SEQ ID NO. 3, and connecting with the base sequence which at 5' end of the sequence shown as SEQ ID NO. 2 and used for coding polypeptide GSTSSGSG, so as to construct a gene member of fusion protein; and (3) connecting the gene member of the fusion protein with saccharomyces cerevisiae cell endogenous promoter and terminator, so as to construct a fusion protein gene expression cassette, and transforming into saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method is capable of improving the conversion rate of dammarenediol to protopanaxadiol.

Description

A kind of can improve dammarenediol transformation efficiency fused protein and construction process and application

Technical field

The present invention relates to biological technical field, particularly relate to the fused protein and construction process and application that a kind ofly can improve dammarenediol transformation efficiency.

Background technology

Ginsenoside is the main active ingredient of Chinese medicine ginseng, has protection cardiovascular, antifatigue, anti-ageing and anticancer pharmacological action.The plant tissue in traditional culture of ginseng, tissue culture and downstream extracts and has been difficult to meet the demand of market to ginsenoside.

As a kind of triterpene compound, the synthesis that yeast entogenous pathways metabolism can be ginsenoside provides precursor 2,3-oxidosqualene.Epoxidation subsequently, oxidation and glycosyl interpolation are completed by dammarenediol synthetic enzyme, protopanoxadiol synthetic enzyme and glycosyltransferase respectively.Dammarenediol synthase gene was found in 2006.Tansakul etc. report that PNA (DDBJ database, sequence number AB265170) is the gene of catalysis dammarenediol synthesis.Han etc. report another one DDS (DDBJ/EMBL/GenBanK database, sequence number AB122080).2011, Jung-Yeon Han etc. carried out cDNA by the ginseng adventitious root of inducing methyl jasmonate and builds storehouse, to after transcript profile order-checking through a series of screening, determine that PPDS gene (cyp716a47) is encoded protopanoxadiol synthetic enzyme.2014, the screening of GT also obtained certain progress, and the information of the ginseng transcript profile that Zhou Zhihua etc. have comprehensively delivered, filters out the glycosyltransferase that protopanoxadiol can be converted into ginsenoside compound K (CK).

Based on the excavation of genes involved in above ginsenoside route of synthesis, Zhang Xueli carries out through engineering approaches to yeast saccharomyces cerevisiae, introduce the AtCPR1 in DS, PPDS and Arabidopis thaliana, and process LAN is carried out to yeast mevalonate pathway speed limit process, construct the artificial yeast's cell producing protopanoxadiol.Zhou Zhihua, by the GT gene insertion yeast saccharomyces cerevisiae that screens, obtains the yeast cell that can produce CK.

PPDS is a kind of cytochrome P 450 monooxygenases.The research in early stage finds, in artificial constructed protopanoxadiol yeast route of synthesis, PPDS is the step of a speed limit, and simple increase PPDS and the copy of reductase enzyme thereof can not be dealt with problems, and cause the transformation efficiency of dammarenediol relatively low.

Summary of the invention

The object of the invention is to overcome deficiency of the prior art, a kind of fused protein that can improve dammarenediol transformation efficiency is provided.

Second object of the present invention is to provide a kind of construction process that can improve the fused protein of dammarenediol transformation efficiency.

3rd object of the present invention is to provide a kind of application that can improve the fused protein of dammarenediol transformation efficiency.

Technical scheme of the present invention is summarized as follows:

A construction process for the fused protein of dammarenediol transformation efficiency can be improved, comprise the steps:

1. front 138 base excision held 5 ' of cytopigment-NADPH-reductase enzyme 1 Gene A tCPR1 in Arabidopis thaliana, obtain sequence shown in SEQ ID NO.2, and in described Arabidopis thaliana, AtCPR1 gene is with shown in SEQ ID NO.1;

2. remove with 3 ' of protopanoxadiol synthase gene PPDS in the ginseng shown in SEQ ID NO.3 end terminator codon TAA, hold with 5 ' of sequence shown in SEQ ID NO.2 and be connected with the base sequence of coded polypeptide GSTSSGSG, construct the Genetic elements of PPDS-linker1-AtCPR1 fused protein;

3. the Genetic elements of PPDS-linker1-AtCPR1 fused protein is connected with brewing yeast cell endogenesis promoter and terminator, builds PPDS-linker1-AtCPR1 fused protein expression casette, and conversion enters brewing yeast cell expression.

Concrete building process is shown in embodiment 3.

The fused protein PPDS-linker1-AtCPR1 that can improve dammarenediol transformation efficiency that aforesaid method builds.

Above-mentioned fused protein synthesizes the application in ginsenoside in yeast saccharomyces cerevisiae.

The base sequence of coded polypeptide GSTSSGSG as 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ', but is not limited only to this sequence.

Yeast saccharomyces cerevisiae endogenesis promoter as PGK1p (phosphoglyceric kinase 1 promotor), sequence SEQ ID NO.7.

Yeast saccharomyces cerevisiae endogenous terminator is as ADH3t (ethanol dehydrogenase 3) sequence SEQ ID NO.12.

AtCPR1 gene order shown in SEQ ID NO.1 is the majorizing sequence of gene for the codon of yeast saccharomyces cerevisiae of Arabidopis thaliana NADPH-cytochrome P450 reductase 1.

Gene order shown in SEQ ID NO.2 is obtain after 138bp before sequence SEQ ID NO.1 removes.

PPDS gene order shown in SEQ ID NO.3 is the majorizing sequence of gene for the codon of yeast saccharomyces cerevisiae of ginseng Central Plains panoxadiol synthetic enzyme.

Advantage of the present invention:

Experiment proves, PPDS and the AtCPR1 catalytic efficiency of the PPDS-linker1-AtCPR1 fused protein that can improve dammarenediol transformation efficiency than Individual existence in yeast saccharomyces cerevisiae that the present invention builds is high.The fusion rotein that this construction process obtains improves the transformation efficiency of dammarenediol, is conducive to the efficient synthesis of ginsenoside in microorganism.

Accompanying drawing explanation

Fig. 1 .PGK1p-PPDS-linker1-AtCPR1-ADH3t Fusion Module electrophorogram.

Fig. 2 .PPDS and the schematic diagram of AtCPR1 protein in yeast cell.Fig. 2 A is the schematic diagram of two kinds of protein in yeast cell under natural condition; The PPDS-linker1-AtCPR1 fused protein schematic diagram yeast cell in of Fig. 2 B constructed by the present invention.

Fig. 3. dammarenediol and protopanoxadiol LC-MS analyze.

Fig. 4. PPDS and AtCPR1 and PPDS-linker1-AtCPR1 fusion rotein catalytic capability contrast under natural condition.

Fig. 5. the yield comparison of each bacterial strain dammarenediol and protopanoxadiol.

Embodiment

Below by specific embodiment, the present invention is further illustrated.

The experimental technique used in embodiment below if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Embodiment 1

The structure of synthesis dammarenediol brewing yeast cell

One, module construction

According to the aminoacid sequence of dammarenediol synthase in ginseng, that carries out for yeast saccharomyces cerevisiae is codon optimized, is then SEQ ID NO.4 by the method (synthesis of Jin Wei intelligence bio tech ltd) of chemosynthesis obtain the encoding gene DS of dammarenediol synthetic enzyme; The tHMG1 (SEQ ID NO.5) that yeast saccharomyces cerevisiae is endogenous, erg1 (SEQ ID NO.6), and promotor 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) are all from yeast saccharomyces cerevisiae w303-1a genome; Riddled basins leu2 is from plasmid prs405 (U.S. ATCC), and his3 is from plasmid pxp320 (purchased from Addgene, Inc.WWW.addgene.org).

With the yeast saccharomyces cerevisiae W303-1a (U.S., ATCC) genome is template, with PGK1p-DS-F (SEQ ID NO.13), (with PGK1p-DS-R (SEQ ID NO.14) and DS-CYC1t-F (SEQ ID NO.17) and DS-CYC1t-R (SEQ ID NO.18) for primer, increase PGK1p promotor and CYC1t terminator respectively.

With dammarenediol synthase gene DS (SEQ ID NO.4) for template, with DS-F (SEQ ID NO.15) and DS-R (SEQ ID NO.16) for primer amplification DS gene.The method of PGK1p promotor, DS gene and CYC1t terminator fusion DNA vaccine is fused into the expression module PGK1p-DS-CYC1t of DS; With yeast saccharomyces cerevisiae W303-1a genome for template, with TEF1p-erg1-F (SEQ ID NO.19) and TEF1p-erg1-R (SEQ ID NO.20) and erg1-ADH1t-F (SEQ IDNO.23) and erg1-ADH1t-R (SEQ ID NO.24) for primer, increase TEF1p promotor and ADH1t terminator respectively.With W303-1a genome for template, with erg1-F (SEQ ID NO.21) and erg1-R (SEQ ID NO.22) for primer amplification erg1 gene fragment.The method of TEF1p promotor, erg1 gene fragment and ADH1t terminator fusion DNA vaccine is fused into the expression module TEF1p-erg1-ADH1t of erg1, sequence used is shown in sequence table.

With yeast saccharomyces cerevisiae W303-1a genome for 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) for primer, increase TDH3p promotor and ADH3t terminator respectively.

With W303-1a genome for template, with tHMG1-F (SEQ ID NO.27) and tHMG1-R (SEQ ID NO.28) for primer amplification tHMG1 gene fragment.The method of TDH3p promotor, tHMG1 gene fragment and ADH3t terminator fusion DNA vaccine is fused into the expression module TDH3p-tHMG1-ADH3t of tHMG1, sequence used is shown in sequence table.

In addition, take prs405 as template, with leu-F (SEQ ID NO.33) and leu-R (SEQ ID NO.34) for primer amplification leu2 marker gene, with yeast saccharomyces cerevisiae W303-1a genome for template, with δ 1-F (SEQ ID NO.39) and δ 1-R (SEQ ID NO.40) for primer, amplification δ Post section fragment, and adopt the method for fusion DNA vaccine to build leu2-δ 1 conversion elements; Take pxp320 as template, with his-F (SEQ ID NO.31) and his-R (SEQ ID NO.32) for primer amplification his3 marker gene, with yeast saccharomyces cerevisiae W303-1a genome for template, with rDNA1-F (SEQ ID NO.35) and rDNA1-R (SEQ ID NO.36) for primer amplification rDNA Partial Fragment, and the method for fusion DNA vaccine is adopted to build his3-rDNA1 conversion elements.The self-assembly of module transforms also to be needed with genes of brewing yeast group for 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) for primer amplification rDNA2 and δ 2 fragment.The each module of pcr amplification after module construction completes, each conversion module of method purifying that blend compounds reclaims.

The present invention's PCR enzyme used is the pfu polysaccharase of Beijing Quanshijin Biotechnology Co., Ltd, and the PCR amplification system of 50 μ L is as follows: DNA profiling, 1 μ L; Before draw (10 μMs) and after draw (10 μMs) each 1 μ L; DNTP (2.5mM), 5 μ L; 10 × Buffer, 10 μ L; Pfu polysaccharase, 1 μ L; Finally use distilled water polishing 50 μ L.PCR instrument arranges amplification program.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation); 98 DEG C of sex change 10 seconds, annealing 10 seconds, 72 DEG C extend 1 minute (32 circulations); 72 DEG C extend 8 minutes (1 circulation).

The present invention's fusion DNA vaccine used system is as follows: DNA fragmentation total amount 800ng, mol ratio 1:1; DNTP (2.5mM), 5 μ L; 10 × Buffer, 10 μ L; Pfu polysaccharase, 1 μ L; Finally use distilled water polishing 50 μ L.PCR instrument arranges amplification program.Amplification condition is 95 DEG C of denaturations 2 minutes (1 circulation); 95 DEG C of sex change 10 seconds, annealing 55 DEG C 30 seconds, 72 DEG C extend 1 minute (11 circulations), 72 DEG C of extensions 5 minutes (1 circulation).

Two, yeast conversion

The conversion of above-mentioned module divides two groups to carry out.The yeast saccharomyces cerevisiae W303-1a that sets out cultivates and gets 200 μ L after 12 hours and add in the fresh YPD substratum of 2mL in YPD substratum, cultivates 5 hours.Under 3000 turns of normal temperature, centrifugal 5min collects thalline, with the ddH of sterilizing after supernatant discarded ₂o rinses thalline, and under 3000 turns of normal temperature, centrifugal 5min collects thalline, supernatant discarded.Then add in thalline by the Lithium Acetate of 1mL100mM, after ambient temperatare puts 5min, under 3000 turns of normal temperature, centrifugal 5min collects thalline, prepares competent yeast cells.Transform mixed system and comprise 240 μ L PEG (50%W/V), 36 μ L 1.0M Lithium Acetates, 10 μ L ss-DNA, transform each 200ng of fragment PGK1p-DS-CYC1t, TEF1p-erg1-ADH1t, his3-rDNA1 and rDNA2 fragment, finally use ddH ₂o polishing to 360 μ L.Add in the Saccharomyces cerevisiae competent cell of just preparation by said sequence successively by every, centrifugal vortex 1min, at 42 DEG C of water-bath 30min, centrifugal 2min under 4000 turns of normal temperature, adds 1mLYPD substratum after removing supernatant, and 30 DEG C of 150rpm cultivate 2h.Centrifugal 5min under 4000 turns of normal temperature, supernatant discarded, sterilized water washs 2 times, and with 100 μ L sterilized water re-suspended cells, is coated with the flat board lacking Histidine and screens.Screening and culturing condition is 30 DEG C, cultivates more than 48h.Obtain the S. cervisiae W1 transforming DS and process LAN erg1.

Take W1 as starting strain, process LAN DS and tHMG1.Take PGK1p-DS-CYC1t as template, with leu-PGKp-F (SEQID NO.72) and DS-CYC1t-R (SEQ ID NO.18) for primer, amplification PGK1p-DS-CYC1t expression cassette.The preparation method of Saccharomyces cerevisiae competent cell is same as above, transforms fragment PGK1p-DS-CYC1t, TDH3p-tHMG1-ADH3t, leu2-δ 1, and each 200ng of δ 2, method for transformation and screening method described above.Then, after carrying out bacterium colony PCR checking, the Wine brewing yeast strain W2 of process LAN DS and tHMG1 is obtained.

The structure of embodiment 2, synthesis protopanoxadiol brewing yeast cell W3 and W3plus

According to the aminoacid sequence of cytopigment-NADPH-reductase enzyme 1 in protopanoxadiol synthase in ginseng and Arabidopis thaliana, that carries out for yeast saccharomyces cerevisiae is codon optimized, is then that in sequence table, in SEQ ID NO.3 and Arabidopis thaliana, Gene A tCPR1 is the gene order SEQ ID NO.1 in sequence table by the method (synthesis of Jin Wei intelligence bio tech ltd) of chemosynthesis obtain the encoding gene PPDS of ginseng Central Plains panoxadiol synthetic enzyme.TDH3p, PGK1p and terminator CYC1t, ADH3t are all from yeast saccharomyces cerevisiae w303-1a genome; Riddled basins ura3 is from plasmid pxp218 (Addgene, Inc.WWW.addgene.org).

With yeast saccharomyces cerevisiae W303-1a genome for 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) for primer, increase TDH3p promotor and ADH3t terminator respectively.With AtCPR1 (SEQ ID NO.1) for template, with AtCPR1-F (SEQ ID NO.45) and AtCPR1-R (SEQ ID NO.46) for primer amplification AtCPR1 gene fragment.The method of TDH3p promotor, AtCPR1 gene fragment and ADH3t terminator fusion DNA vaccine is fused into the expression module TDH3p-AtCPR1-ADH3t of AtCPR1, the method for PCR and fusion DNA vaccine is with embodiment 1.

With yeast saccharomyces cerevisiae W303-1a genome for 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) for primer, increase PGK1p promotor and CYC1t terminator respectively.With PPDS (SEQ ID NO.3) for template, with PPDS-F (SEQ ID NO.51) and PPDS-R (SEQ ID NO.52) for primer amplification PPDS gene fragment.The method of PGK1p promotor, PPDS gene fragment and CYC1t terminator fusion DNA vaccine is fused into the expression module PGK1p-PPDS-CYC1t of PPDS, the method for PCR and fusion DNA vaccine is with embodiment 1.

In addition, take pxp218 as template, with ura-F (SEQ ID NO.55) and ura-R (SEQ ID NO.56) for primer amplification ura3 marker gene, with yeast saccharomyces cerevisiae W303-1a genome for template, with δ 1-F2 (SEQ ID NO.57) and δ 1-R2 (SEQ ID NO.58) for primer amplification δ Post section fragment, and the method for fusion DNA vaccine is adopted to build ura3-δ 1 conversion elements; With W303 genome for template, with δ 2-F (SEQ ID NO.59) and δ 2-R (SEQ ID NO.60) for primer, amplification conversion elements δ 2.Adopt the method for transformation of embodiment 1, by PGK1p-PPDS-CYC1t, TDH3p-AtCPR1-ADH3t, ura3-δ 1 and δ 2 transforms fragment and to be incorporated in genes of brewing yeast group on δ site in the mode of yeast self-assembly, carry out bacterium colony PCR checking after obtaining transformant, obtain the Wine brewing yeast strain W3 synthesizing protopanoxadiol.

On the basis of W3, increase the copy that PPDS and AtCPR1 expresses module, main method is as follows.With yeast saccharomyces cerevisiae BY4742 (U.S. ATCC) genome for template, with Ade2-F (SEQ ID NO.63) and Ade2-R (SEQ ID NO.64) for primer amplification ade2 marker gene Expression element, with prs405 plasmid for template, with leu1-F2 (SEQ ID NO.65) and leu1-R2 (SEQ ID NO.66) for primer amplification leu2 Post section fragment, and the method for fusion DNA vaccine is adopted to build ade2-leu1 conversion elements; With prs405 plasmid for template, with leu2-F (SEQ ID NO.67) and leu2-R (SEQ ID NO.68) for primer, amplification conversion elements leu2.Meanwhile, take PGK1p-PPDS-CYC1t as template, with PGK1P-PDDS-F2 (SEQ IDNO.61) and PDDS-CYC1t-R (SEQ ID NO.54) for primer amplification PGK1p-PPDS-CYC1t module; With TDH3p-AtCPR1-F (SEQ ID NO.43) and TDH3p-AtCPR1-R2 (SEQ ID NO.62) for primer amplification TEF1p-AtCPR1-ADH3t module.Adopt the method for transformation of embodiment 1, by PGK1p-PPDS-CYC1t, TDH3p-AtCPR1-ADH3t, ade2-leu1 and leu2 transforms each 200ng of fragment and is incorporated into Yeast genome leu2 site in the mode of yeast self-assembly, after bacterium colony PCR verifies, obtain the bacterial strain W3plus increasing PPDS and AtCPR1 module copy.

Embodiment 3, a kind of structure that can improve the fused protein of dammarenediol transformation efficiency

According to the gene order of cytopigment-NADPH-reductase enzyme 1 Gene A tCPR1 in protopanoxadiol synthase gene PPDS in ginseng and Arabidopis thaliana, that carries out for yeast saccharomyces cerevisiae is codon optimized, then obtains gene fragment by the method (synthesis of Jin Wei intelligence bio tech ltd) of chemosynthesis.In ginseng, PPDS gene is with shown in SEQ ID NO.3, and in Arabidopis thaliana, AtCPR1 gene is with shown in SEQ ID NO.1; By front 138 base excision of AtCPR1 gene 5 ' end in Arabidopis thaliana, obtain sequence shown in SEQ ID NO.2.Endogenesis promoter PGK1p (SEQ ID NO.7) and endogenous terminator ADH3t (SEQ ID NO.12) is all from yeast saccharomyces cerevisiae w303-1a genome; Riddled basins ura3 is from plasmid pxp218.

Two protein connect with the base sequence of coded polypeptide GSTSSGSG, the base sequence of linker1 of encoding in the present embodiment is 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ' (SEQ ID NO.71), this base sequence to be designed in primer and to adopt the method for fusion DNA vaccine to realize the connection of two gene fragments, specific implementation process is: with protopanoxadiol synthase gene PPDS in ginseng (SEQ ID NO.3) for template, with PPDS-F in sequence table (SEQ ID NO.51) and PPDS-linker1-R (SEQ ID NO.69) for primer, amplification PPDS-linker1 fragment, this fragment eliminates 3 ' the end terminator codon TAA of protopanoxadiol synthase gene PPDS in ginseng, base sequence 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ' (SEQ ID NO.71) simultaneously also containing coded polypeptide GSTSSGSG, with front 138 base excision sequence (SEQ ID NO.2) of cytopigment-NADPH-reductase enzyme 1 Gene A tCPR1 gene 5 ' end in Arabidopis thaliana for template, with linker1-AtCPR1-F (SEQ ID NO.70) and AtCPR1-R (SEQ ID NO.46) for primer, amplification linker1-AtCPR1 fragment, this fragment comprises the base sequence 5 '-GGTTCTACTTCTTCAGGTTCAGGT-3 ' (SEQID NO.71) of coded polypeptide GSTSSGSG and removes the AtCPR1 gene order (SEQ ID NO.2) of 5 ' end 138bp.The method of PPDS-linker1 and linker1-AtCPR1 gene fragment fusion DNA vaccine is connected into the Genetic elements of PPDS-linker1-AtCPR1, this element is the Genetic elements of PPDS-linker1-AtCPR1 fused protein.The method of PCR and fusion DNA vaccine is with embodiment 1.

The Genetic elements of PPDS-linker1-AtCPR1 fused protein is connected with brewing yeast cell endogenesis promoter and terminator, build PPDS-linker1-AtCPR1 fused protein expression casette, concrete grammar is: with PGK1p-PPDS-F (SEQID NO.49) and PGK1p-PPDS-R (SEQ ID NO.50) for primer, with W303-1a genome for template, amplification PGK1p promotor; With AtCPR1-ADH3t-F (SEQ ID NO.47) and AtCPR1-ADH3t-R (SEQ ID NO.48) for primer, with W303-1a genome for template, amplifying ADH 3t terminator.Genetic elements PPDS-linker1-AtCPR1 PPDS-linker1-AtCPR1 being merged enzyme is connected with brewing yeast cell promotor PGK1p and terminator ADH3t fusion DNA vaccine, builds PPDS-linker1-AtCPR1 and merges enzyme gene expression box PGK1p-PPDS-linker1-AtCPR1-ADH3t (Fig. 1).

Expression cassette PGK1p-PPDS-linker1-AtCPR1-ADH3t is transformed and enters brewing yeast cell expression, concrete grammar is: take pxp218 as template, with ura-F (SEQ ID NO.55) and ura-R (SEQ ID NO.56) for primer amplification ura3 marker gene, with yeast saccharomyces cerevisiae W303-1a genome for template, with δ 1-F2 (SEQ ID NO.57) and δ 1-R2 (SEQ ID NO.58) for primer amplification δ Post section fragment, and the method for fusion DNA vaccine is adopted to build ura3-δ 1 conversion elements; With W303-1a genome for template, with δ 2-F (SEQ ID NO.59) and δ 2-R (SEQ ID NO.60) for primer, amplification conversion elements δ 2.Adopt the method for transformation of embodiment 1, by PGK1p-PPDS-linker1-AtCPR1-ADH3t expression cassette, ura3-δ 1 and δ 2 transforms each 200ng of fragment and transforms in the mode of yeast self-assembly and enter on the genome δ site of the brewing yeast cell W2 that embodiment 2 obtains, after carrying out bacterium colony PCR checking after obtaining transformant, obtain the yeast strain W3a synthesizing protopanoxadiol.

The catalytic capability of embodiment 4, fusion PPPDS enzyme measures

One, the MC preparation of yeast saccharomyces cerevisiae

Embodiment 1,2, the brewing yeast cell W2 obtained in 3, W3 and W3a carry out cultivating (30 DEG C, 220rmp) respectively in YPD substratum.Centrifugal (3000rmp, 5min) collecting cell after cultivation 24h.Brewing yeast cell is resuspended with TEK damping fluid (100mM KCl, 50mM Tris-HCl, 1mM EDTA), the centrifugal 3min of 6100g.Remove supernatant, add the granulated glass sphere with biomass equivalent, and add Extraction buffer (20mM beta-mercaptoethanol, 1%BSA, 0.6M sorbyl alcohol, 50mM Tris-HCl, 1mM EDTA).After concuss 20min, the centrifugal 15min of 6100g, is transferred in clean centrifuge tube after supernatant being crossed film, adds MgCl ₂(50mM) 1h depositing particles body is placed after on ice.Then the centrifugal 20min of 12500g.After supernatant is removed, the microsome bottom centrifuge tube is transferred to (30% glycerine, 50mM Tris-HCl, 1mMEDTA) in TEG damping fluid, and grinds fast carry out homogenization process with grinding pestle.

Two, CO differential method measures P450 enzyme

Get 4mL yeast cell Microsome preparation liquid, add 80 μ L 0.5mmol/L V-Brite B mixings, in ice bath, place 2min.Above-mentioned for 4mL liquid of preparing is divided and proceeds in two cuvettes, a copy of it CO gas sparging 1min, another part not bubbling, measures the ultra-violet absorption spectrum of two parts of suspensions respectively.Instrument is TU-1810 type ultraviolet-visible pectrophotometer (cuvette light path 1cm), scanning wavelength scope 400-500nm.With the suspension of logical CO bubbling for sample liquid, the suspension of illogical CO is reference liquid, draws differential uv absorption spectra.The absorption value at record 45Onm and 490nm wavelength place, (molar extinction coefficient ε is 91cm to calculate P450 content as follows ^-1mM ^-1): P450 (mM)=(A _450nm-A _490nm)/91.

Three, the mensuration of the enzyme activity of P450 enzyme

10mg microsome is added at 500 μ L 100mM potassium phosphate buffers (pH 7.4).Dammarenediol concentration 500 μMs, incubation in 30 DEG C of water-baths, add NADPH (1mM) and start reaction, a certain amount of mixed solution is got every 5min, add isopyknic n-hexane extraction, cross 0.22 organic membrane after the centrifugal 2min of 12000rmp for subsequent use, finally measure the content of protopanoxadiol with HPLC.Liquid phase chromatogram condition: sample size 20 μ L, Agilent ZORBAX SB-Aq; Moving phase is methyl alcohol: acetonitrile=4:6, flow velocity: 1mL/min, UV-detector wavelength 203nm.

Result: the content W3 of PPDS enzyme, and in w3a microsome, the content of P450 enzyme deducts the content gained of P450 enzyme in W2.As calculated, in W3, the content of PPDS is 9.82 ± 0.83 μMs/g microsome, and in W3a, the content of PPDS is 10.04 ± 0.79 μMs/g microsome.It can thus be appreciated that the PPDS concentration difference in yeast saccharomyces cerevisiae microsome is less, and their enzyme activity determination result as shown in Figure 4.In Fig. 4, the catalytic efficiency of the PPDS-linker1-AtCPR1 fused protein constructed by this experiment is apparently higher than the catalytic efficiency of PPDS and AtCPR1 under natural condition.

The fermentation of embodiment 5, engineering strain and the LC-MS of tunning detect.

By embodiment 2, the synthetic brewing yeast cell W3 obtained in 3, W3plus and W3a carry out cultivating (30 DEG C, 220rmp) respectively in YPD substratum.Centrifugal collecting cell after 6 days, adds acetone, in ice-water bath, carry out ultrasonic disruption 10min, then centrifugal 2min under 12000 turns/min normal temperature condition, get supernatant cross 0.22mm organic membrane after for subsequent use.

The qualitative LC-MS of dammarenediol and protopanoxadiol carries out.Liquid phase chromatogram condition: sample size 5 μ L, Agilent ZORBAX SB-Aq; Moving phase is 90% acetonitrile, flow velocity: 0.2mL/min.Mass Spectrometry Conditions: atomization gas and dry gas are all N ₂; Collision voltage :-70V; Spray voltage: 3.8kV; Ion source: APCI; Ion source temperature: 120 DEG C; Precipitation temperature: 300 DEG C; After post, effluent imports ion source speed: 5 μ L/min; Scanning of the mass spectrum total mass number scope: 200-1000Da.

The quantitative HPLC of dammarenediol and protopanoxadiol carries out, liquid phase chromatogram condition: sample size 20 μ L, Agilent ZORBAX SB-Aq; Moving phase is methyl alcohol: acetonitrile=4:6, flow velocity: 1mL/min.Dammarenediol is purchased from Yunnan Province Xili Bioisystech Co., Ltd (WWW.biobiopha.com).Protopanoxadiol is purchased from Beijing Suo Laibao Science and Technology Ltd. (http://www.solarbio.cn/).

Result is as shown in Figure 5: the output of W3 dammarenediol and protopanoxadiol is respectively 96.8mg/L and 155.6mg/L; W3plus adds the copy number that PPDS and AtCPR1 expresses module, and the output of dammarenediol and protopanoxadiol is respectively 102.5mg/L and 150.6mg/L; The output of W3a dammarenediol and protopanoxadiol is respectively 13.2mg/L and 265.6mg/L.

Experiment proves, in yeast saccharomyces cerevisiae W303-1a host, the copy number of simple increase PPDS and AtCPR1 expression module can not improve the transformation efficiency of dammarenediol.Constructed PPDS-linker1-AtCPR1 fusion rotein mass-energy effectively improves the transformation efficiency of dammarenediol, thus improves the output of protopanoxadiol.

Claims (3)

1. can improve a construction process for the fused protein of dammarenediol transformation efficiency, it is characterized in that comprising the steps:

1. front 138 base excision held 5 ' of cytopigment-NADPH-reductase enzyme 1 Gene A tCPR1 in Arabidopis thaliana, obtain sequence shown in SEQ ID NO.2, and in described Arabidopis thaliana, AtCPR1 gene is with shown in SEQ ID NO.1;

2. remove with 3 ' of protopanoxadiol synthase gene PPDS in the ginseng shown in SEQ ID NO.3 end terminator codon TAA, hold with 5 ' of sequence shown in SEQ ID NO.2 and be connected with the base sequence of coded polypeptide GSTSSGSG, construct the Genetic elements of PPDS-linker1-AtCPR1 fused protein;

3. the Genetic elements of PPDS-linker1-AtCPR1 fused protein is connected with brewing yeast cell endogenesis promoter and terminator, builds PPDS-linker1-AtCPR1 fused protein expression casette, and conversion enters brewing yeast cell expression.

2. the fused protein that can improve dammarenediol transformation efficiency of the method structure of claim 1.

3. the fused protein of claim 2 synthesizes the application in ginsenoside in yeast saccharomyces cerevisiae.