CN104830893A - Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast - Google Patents
Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast Download PDFInfo
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Abstract
The invention relates to a vector used for multigene stacking cis-form expression. The vector used for the multigene stacking cis-form expression is characterized by containing restriction enzyme cutting site sequences of two pairs of isocaudarners, and multiple genes can be stacked in one vector by virtue of the two pairs of isocaudarners. The construction method of the vector used for multigene stack cis-form expression comprises the step of respectively introducing corresponding restriction enzyme identification sequences at upstream of a promoter and downstream of a terminator through PCR site-specific mutagenesis. In the expression carrier, each gene carries an independent promoter and an independent terminator, an independent expression element is formed, then the isocaudarners are utilized for realizing stacking assembling of the independent element, and expression of each gene is relatively independent. The vector can conveniently and rapidly realize multigene stacking, namely only a target gene is respectively cloned to a polyclonal site of the expression carrier, and then the two pairs of isocaudarners are utilized for completing subsequent assembling.
Description
Technical field
The invention belongs to technological field of biochemistry, relate to the yeast vector that a kind of multiple gene stacking cis is expressed, the invention still further relates to the construction process of this carrier, and the application of this carrier in synthetic biology in the expression of multiple gene stacking cis.
Background technology
Yeast is the most frequently used genetic engineering Host Strains in a kind of laboratory, can be used for expressing eukaryote foreign gene.Conventional Yeast expression carrier pRS425, genotype is ori (f1)-lacZ-T7promoter-MCS (KpnI-SacI)-T3promoter-lacI-ori (pMB1)-ampR-ori (2micron)-LEU2, it is E. coli-Yeast bacterium shuttle plasmid, pMB1 replication origin wherein and 2micron replication origin control carrier respectively and copy in E.coli and yeast, carrier, with ampicillin resistance gene and leucine nutritional marker thing, can be used for screening.
Yeast expression vector common at present, comprise above-mentioned pRS425, substantially can meet the routine of single foreign gene in yeast to express, but, a lot of biological function is not controlled by term single gene and complete, need multiple genes involved acting in conjunction, such as in synthetic biology field, cis is often needed to express multiple genes in a whole set of secondary metabolite route of synthesis, its biological function could be realized, therefore, the yeast vector multiple gene stacking cis can expressed is needed in practice.
At present, there is not yet the report building multiple gene stacking cis expression yeast vector.
In addition, the multiple gene stackings realized by two pairs of isocaudarners are on same carrier, and also there is not been reported.
Summary of the invention
The object of the invention is the yeast vector building a kind of multiple gene stacking cis expression, be applied to multiple gene stacking cis and express.
The present invention constructs a kind of novel multiple gene stacking cis and expresses yeast vector, reaches the object of multiple gene stacking on same Yeast expression carrier.
At present, generally realize multiple gene stacking cis expression strategy by multiple plasmid cotransformation system, normally multiple foreign gene is cloned into respectively in the carrier of different screening resistance or nutritional marker thing, add Multiple Classes of Antibiotics after the multiple plasmid of cotransformation simultaneously, or remove multiple nutrients mark, the cis realizing multiple gene is expressed.Owing to being subject to the restriction of resistance and nutrition mark kind and array mode, the cis that this method only can realize a few gene is expressed.And, if multiple plasmids of cotransformation use identical dubbing system, namely use identical replication orgin, these plasmids just belong to incompatible plasmids, when their corotation enter same cell, can contend with one other in the process copying and be assigned to subsequently daughter cell, cause plasmid loss.If use not identical dubbing system, duplicate copy number can be caused different, affect subsequent experimental efficiency.
There is many defects in multiple plasmid cotransformation system, so often then multiple gene stacking is carried out single Plastid transformation on a carrier in practice.Multiple gene stacking also can realize by cutting connection at multiple clone site place enzyme on a carrier, but, the restriction enzyme value volume and range of product at multiple clone site place is limited, often increase a gene, next step available restriction enzyme value volume and range of product will reduce, therefore, a few gene stacking can only be realized on carrier.And, when selectional restriction restriction endonuclease kind, also to consider whether gene internal has the sequence of this restriction enzyme, and experimental design bothers very much, the carrier that a kind of multiple gene stacking cis that the present invention relates to are expressed, can very conveniently overcome the above problems efficiently.
The feature of multiple gene stacking cis expression vector of the present invention is:
Can be used for the Yeast expression carrier that multiple gene stacking cis is expressed, it is characterized by: the restriction enzyme digestion sites sequence containing two pairs of isocaudarners, multiple gene stacking can be realized on same carrier by these two pairs of isocaudarners.
Described two pairs of isocaudarners can be selected from two couple of following isocaudarner centering: Xho I/Sal I, BamH I/Bgl II, XbaI/Nhe I, Spe I/Nhe I or Xba I/Spe I etc., also can be implemented in identical carrier the object superposing multiple gene.
Described expression vector is selected from the prokaryotic expression carrier of Yeast expression carrier, pET series, or carrier for expression of eukaryon pEGFP.
Described Yeast expression carrier preferred pRS425, pRS423, pRS426, pRS316 or pRS403 etc.
Select the corresponding promotor of vector gene expression cassette and terminator according to actual needs, in one embodiment of the present of invention, have chosen promotor ADH2p and the terminator ADH2t of ethanol dehydrogenase ADH2, for the expression of fungi poly ketone secondary metabolite.In practice, other corresponding promotor and terminator can also be chosen to complete corresponding function.
In an example of the present invention, by carrying out directional transformation to Yeast expression carrier pRS425, construct the yeast vector that a kind of novel multiple gene stacking cis are expressed, called after pBDL6063.
The following experiment of the present invention confirms the function of described multiple gene stacking cis expression vector pBDL6063:
Have chosen 3 genes in fungi poly ketone compounds lasiodiplodin biosynthetic pathway, be respectively reduced form polyketide synthase LtLasS1, Non polyketide synthase LtLasS2, oxygen methyltransgerase LtOMT, 3 genomes are filled on pBDL6063 carrier, transformed yeast heterogenous expression obtains compound lasiodiplodin (also comprising not containing the lasiodiplodin analogue of methyl group), and the common cis achieving 3 genes is expressed.Oxygen methyltransgerase HsOMT and above-mentioned 3 gene derived from fungi poly ketone compounds Hypothemycin biosynthetic pathway is also assembled in pBDL6063 carrier by the present invention jointly, transformed yeast heterogenous expression obtains 4 kinds of compounds, be respectively the lasiodiplodin of methyl group on 3 atom side chain hydroxyls, not containing the lasiodiplodin analogue of methyl group, the lasiodiplodin analogue of methyl group on 5 atom side chain hydroxyls, 3 and 5 atom side chain hydroxyls are contained the lasiodiplodin analogue of methyl group, the common cis achieving 4 genes is expressed.Result shows, the carrier of structure is practical, and the strategy utilizing two pairs of isocaudarners to superpose multiple gene in identical carrier is feasible.Although the present inventor not yet carries out the superposition cis that quantity is greater than 4 genes express experiment, see theoretically, those skilled in the art can reasonable prediction, and the gene dosage that this carrier can carry and gene order length are on these 4 genes.Because this carrier realizes the superposition of multiple gene by cutting the operations such as connection to the enzyme of two pairs of isocaudarners, limited screening resistance or nutritional marker thing is not related in experimentation, so this carrier can superpose the gene of unlimited amount in theory, how many genes can be carried at most and need to be verified further in actual applications.
The building mode of multiple gene stacking cis expression vector of the present invention, is characterized in that: introduce restriction endonuclease recognition sequence respectively in promotor upstream and terminator downstream.
Concrete grammar is: first on expression vector to be rebuilt, add promotor and terminator, then introduces restriction endonuclease recognition sequence in promotor upstream and terminator downstream respectively by the method for PCR rite-directed mutagenesis.
Be performed such in the present invention's example: on Yeast expression carrier pRS425, first add ethanol dehydrogenase ADH2 promotor ADH2p and terminator ADH2t, Sal I endonuclease recognition sequence is introduced in promotor ADH2p upstream again by the method for PCR rite-directed mutagenesis, introduce BamH I and Bgl II two endonuclease recognition sequence in terminator ADH2t downstream, finally obtain transforming successful Yeast expression carrier pBDL6063.Multiple gene stacking cis expression vector pBDL6063 that the present invention builds can be applicable to synthetic biology field, express the multiple genes in a whole set of secondary metabolite route of synthesis, even integrate the multiple genes in different route of synthesis, also can be applicable to all experiments needing superposition cis to express multiple gene such as the expression and purification of albumen composition.
In described multiple gene stacking cis expression vector, each gene, with independently promotor and terminator, forms independently Expression element, and recycling isocaudarner realizes the superposition assembling of independent Expression element, and the expression of each gene is relatively independent.Utilize the feature of Xho I/Sal I and BamH I/Bgl II isocaudarner each other, the efficient cis realizing multiple genes of single plasmid mediation is expressed.In carrier, the superposition of each Expression element is very convenient, only goal gene conventionally need be cloned into respectively the multiple clone site place of expression vector, recycle two pairs of isocaudarners and complete follow-up assembling.When second gene stacking is assembled on carrier, corresponding two pairs of isocaudarner sites disappear, and meanwhile, introduced again the Xho I on second gene and BamH I two restriction enzyme sites, the gene stacking for next round is assembled.So pBDL6063 carrier can be carried on its unlimited multiple gene accepting in scope in theory.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of pRS425-ADH2p-ADH2t-Sal I carrier qualification, wherein:
1)Marker(8000bp、5000bp、3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp)；
2) Sal I restriction enzyme qualification, obtains the band that size is 8696bp;
3) Pvu II restriction enzyme qualification, obtains three bands that size is 6401bp, 1278bp, 1017bp;
4) pRS425-ADH2p-ADH2t-Sal I plasmid;
Fig. 2 is the electrophorogram of pBDL6063 carrier qualification, wherein:
1) BamH I restriction enzyme qualification, obtains the band that size is 8706bp;
2)Marker(8000bp、5000bp、3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp)；
3) Bgl II restriction enzyme qualification, obtains the band that size is 8706bp;
Fig. 3 is pBDL6063 Vector map (the GeneA gene order containing laboratory clone in early stage);
Fig. 4 is the combined strategy schematic diagram that pBDL6063 carrier superposes 3 genes;
Fig. 5 is the genetic expression checking HPLC collection of illustrative plates of pBDL6063 carrier, a () is the expression checking HPLC collection of illustrative plates of 3 genes of biosynthesizing lasiodiplodin, b () is the expression checking HPLC collection of illustrative plates that 3 genes of biosynthesizing lasiodiplodin add the HsOMT gene of biosynthesizing Hypothemycin, wherein:
1) lasiodiplodin (methyl group is on 3 atom side chain hydroxyls)
2) not containing the lasiodiplodin analogue of methyl group
3) the lasiodiplodin analogue of methyl group on 5 atom side chain hydroxyls
4) on 3 and 5 atom side chain hydroxyls, all contain the lasiodiplodin analogue of methyl group
Embodiment
The structure of embodiment 1pBDL6063 carrier
1. experiment material
Yeast expression carrier pRS425 is that carrier is preserved in laboratory; Restriction enzyme, T4DNA ligase enzyme are purchased from NEB company; Archaeal dna polymerase and DNAmarker are purchased from Beijing Quan Shijin biotech firm; The little extraction reagent kit of plasmid, gel reclaim test kit purchased from Tian Gen company; TOP 10 competence is purchased from Kang Wei ShiJi Co., Ltd; Other reagent is domestic analytical pure product.
2. experimental technique and result
First on commercial yeast expression vector pRS425, add promotor ADH2p and the terminator ADH2t of ethanol dehydrogenase ADH2, two genes are preserved by this laboratory.First the corresponding restriction enzyme of ADH2p gene is carried out double digestion reaction, the same endonuclease digestion of expression vector pRS425, connect also routine transformation and enter E. coli competent TOP10, screening positive clone, obtain correct pRS425-ADH2p carrier.Use the same method again and connect ADH2t gene, obtain correct pRS425-ADH2p-ADH2t carrier.
With reference to pRS425-ADH2p-ADH2t sequence and restriction enzyme site, design primer introduces restriction enzyme site.Before promotor ADH2p, add Sal I restriction enzyme site, after terminator ADH2t, add BamH I and Bgl II restriction enzyme site.Primer sequence is in table 1, and wherein introducing sudden change in 425F1 sequence increases Sal I restriction enzyme site, and introducing sudden change in 425R2 sequence increases BamH I and Bgl II restriction enzyme site.Concrete grammar is as follows:
With pRS425-ADH2p-ADH2t carrier for template, 425F1/425R1 is primer, and pcr amplification goes out to introduce the fragment 1 of Sal I restriction enzyme site; Meanwhile, 425F2/425R2 and 425F3/425R3 is primer, and pcr amplification goes out to introduce fragment 2 and the fragment 3 of BamHI and Bgl II restriction enzyme site, then fragment 2 and fragment 3 is merged by the method for fusion DNA vaccine, called after fragment 23.Above-mentioned fragment is connected to pJET1.2 cloning vector, and routine transformation enters E. coli competent TOP10, screening positive clone, send company to check order, and the plasmid checking order correct is called after pJET-1 and pJET-23 respectively.
Respectively by pJET-1 and pRS425-ADH2p-ADH2t plasmid vector Not I and the process of Nde I double digestion, reclaim fragment and carrier, connect and transform, screening positive clone, enzyme cuts the carrier pRS425-ADH2p-ADH2t-Sal I (Fig. 1) identifying and obtain containing Sal I restriction enzyme site; Again respectively by pJET-23 and pRS425-ADH2p-ADH2t-Sal I plasmid vector Xho I and the process of Dra III double digestion, subsequent experimental procedure is the same, final acquisition verifies correct carrier pRS425-ADH2p-ADH2t-Sal I-BamH I-Bgl II (Fig. 2), and called after pBDL6063 (Fig. 3).
Table 1 pRS425M vector construction primer
In table, underlined sequences represents restriction endonuclease recognition sequence.
Embodiment 2 applies the biosynthesizing that pBDL6063 carrier realizes lasiodiplodin
1. experiment material
The gene of expressing for superposing cis is this laboratory clone and obtains (table 2).All the other reagent are with embodiment 1.
The gene that table 2 is expressed for superposing cis
| Gene Name | Length (bp) | Function | Source |
| LtLasS1 | 7173 | Reduced form polyketide synthase | Lasiodiplodia theobromae |
| LtLasS2 | 6252 | Non polyketide synthase | Lasiodiplodia theobromae |
| LtOMT | 1197 | Oxygen methyltransgerase | Lasiodiplodia theobromae |
| HsOMT | 1202 | Oxygen methyltransgerase | Hypomyces subiculosus |
2. experimental technique and result
Fungi poly ketone compounds Lasiodiplodin can cell death inducing, suppress breast cancer cell growth, have important pharmaceutical use, it is also plant poison, people's coagulation factor VIII inhibitor, mineralcorticoid receptor etc. simultaneously, has multiple important biomolecule active.3 of this programme choice experiment room clone derive from the gene of lasiodiplodin biosynthetic pathway, be cloned on pBDL6063 carrier respectively, utilize the characteristic of Xho I/Sal I and BamH I/Bgl II two pairs of isocaudarners, by the combined strategy shown in Fig. 4, cut by enzyme and be connected the stack combinations realizing 3 genes, obtaining restructuring superposition plasmid pBDL6063-LtLasS1-LtLasS2-LtOMT.Concrete grammar is as follows.First LtLasS1, LtLasS2, LtOMT3 gene is recombinated respectively to plasmid pBDL6063, obtain 3 recombinant plasmid pBDL6063-LtLasS1, pBDL6063-LtLasS2, pBDL6063-LtOMT.By pBDL6063-LtLasS1 plasmid Xho I and BamH I double digestion, reclaim carrier; By pBDL6063-LtLasS2 plasmid Sal I and Bgl II double digestion, reclaim fragment, be connected with fragment by above-mentioned carrier and routine transformation intestinal bacteria, screening positive clone, enzyme is cut qualification and is obtained plasmid pBDL6063-LtLasS1-LtLasS2.Again by pBDL6063-LtLasS1-LtLasS2 plasmid Xho I and BamH I double digestion, reclaim carrier; By pBDL6063-LtOMT plasmid Sal I and Bgl II double digestion, reclaim fragment, carrier is connected with fragment and routine transformation intestinal bacteria, screening positive clone, and extract plasmid, enzyme is cut qualification and obtained plasmid pBDL6063-LtLasS1-LtLasS2-LtOMT.
Plasmid pBDL6063-LtLasS1-LtLasS2-LtOMT expression identification.Lasiodiplodin to be cooperated biosynthesizing core skeleton by reduced form polyketide synthase LtLasS1 and Non polyketide synthase LtLasS, then on the hydroxyl of the 3rd carbon atom, increases a methyl by LtOMT and form Lasiodiplodin.Being proceeded to by pBDL6063-LtLasS1-LtLasS2-LtOMT plasmid to synthesize in leucic yeast, ferment after 2 days, extract tunning by ethyl acetate, can find that allos has been synthesized Lasiodiplodin and do not contained the lasiodiplodin analogue (Fig. 5-a) of methyl group in yeast.Meanwhile, also demonstrate this carrier 3 common cis of gene to be expressed.
Embodiment 3 applies the modification that pBDL6063 carrier realizes lasiodiplodin
Some oxygen Methyl transporters zymolyte is very extensive, less demanding to the specificity of substrate, is well suited for carrying out molecular structure alteration to various active compound, increases the diversity of compound library, for the screening operation of lead drug.
The present invention have chosen the oxygen methyltransgerase HsOMT derived from fungi poly ketone compounds Hypothemycin biosynthetic pathway, first by HsOMT gene recombination to plasmid pBDL6063, obtain recombinant plasmid pBDL6063-HsOMT.By pBDL6063-LtLasS1-LtLasS2-LtOMT plasmid Xho I and BamH I double digestion, reclaim carrier; By pBDL6063-HsOMT plasmid Sal I and Bgl II double digestion, reclaim fragment, above-mentioned carrier is connected with fragment and routine transformation intestinal bacteria, screening positive clone, extract plasmid, enzyme is cut qualification and is obtained plasmid pBDL6063-LtLasS1-LtLasS2-LtOMT-HsOMT.Being proceeded to by pBDL6063-LtLasS1-LtLasS2-LtOMT-HsOMT plasmid to synthesize in leucic yeast, ferment after 2 days, tunning is extracted by ethyl acetate, carry out HPLC analysis, find except the lasiodiplodin of methyl group on 3 atom side chain hydroxyls, not containing except the lasiodiplodin analogue of methyl group, have also appeared 2 peaks.Further structural analysis proves, other 2 compounds are respectively methyl group not to be had the lasiodiplodin analogue of Methyl groups, on 3 and 5 atom side chain hydroxyls, all contain the lasiodiplodin analogue (Fig. 5-b) of methyl group on 5 atom side chain hydroxyls on 3 atom side chain hydroxyls.Prove that the oxygen methyltransgerase HsOMT derived from Hypothemycin biosynthetic pathway also can modify lasiodiplodin, its substrate selective is extensive, not high to the susceptibility of substrate, may be used for the molecular structure alteration to other compounds.Meanwhile, also demonstrate this carrier 4 common cis of gene to be expressed.
Table 3 sequence table for information about
Claims (8)
1. can be used for the carrier that multiple gene stacking cis is expressed, it is characterized in that: the restriction enzyme digestion sites sequence containing two pairs of isocaudarners, and multiple gene stacking can be realized on same expression vector by these two pairs of isocaudarners; Expression vector used comes from Yeast expression carrier, the prokaryotic expression carrier of pET series or carrier for expression of eukaryon pEGFP.
2. carrier according to claim 1, described two pairs of isocaudarners are selected from two couple of following isocaudarner centering: Xho I/Sal I, BamH I/Bgl II, Xba I/Nhe I, Spe I/Nhe I or Xba I/Spe I.
3. carrier according to claim 1, expression vector used is Yeast expression carrier, is selected from pRS425, pRS423, pRS426, pRS316 or pRS403.
4. carrier according to claim 3, the promotor that vector gene expression cassette is used and terminator are promotor ADH2p and the terminator ADH2t of ethanol dehydrogenase ADH2.
5. carrier according to claim 4, expression vector used is Yeast expression carrier pRS425.
6. build a method for multiple gene stacking cis expression vector, it is characterized in that: introduce restriction endonuclease recognition sequence respectively in promotor upstream and terminator downstream.
7. method according to claim 6, first adds promotor and terminator on expression vector to be rebuilt, then introduces restriction endonuclease recognition sequence in promotor upstream and terminator downstream respectively by the method for PCR rite-directed mutagenesis.
8. method according to claim 6, first on Yeast expression carrier pRS425, add promotor ADH2p and the terminator ADH2t of ethanol dehydrogenase ADH2, Sal I endonuclease recognition sequence is introduced in promotor ADH2p upstream again by the method for PCR rite-directed mutagenesis, introduce BamH I and Bgl II two endonuclease recognition sequence in terminator ADH2t downstream, finally obtain the Yeast expression carrier of expressing for multiple gene stacking cis.
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| CN107794273A (en) * | 2017-11-02 | 2018-03-13 | 河北师范大学 | A kind of three gene co-expressing carriers of synthesis DL alanine and application |
| CN111088254A (en) * | 2019-11-05 | 2020-05-01 | 中国农业科学院生物技术研究所 | Exogenous gene fixed-point quantitative timing expression system based on fungal operon |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105647959A (en) * | 2016-03-24 | 2016-06-08 | 华中科技大学 | Method for constructing yeast multi-copy expression vector |
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| CN107475282A (en) * | 2017-09-13 | 2017-12-15 | 河北师范大学 | A kind of three gene co-expressing carriers of tetrahydrobiopterin synthesis pyrimidine and application |
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| CN111088254A (en) * | 2019-11-05 | 2020-05-01 | 中国农业科学院生物技术研究所 | Exogenous gene fixed-point quantitative timing expression system based on fungal operon |
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