CN100400665C - Constitution type expression carrier and its application - Google Patents
Constitution type expression carrier and its application Download PDFInfo
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- CN100400665C CN100400665C CNB2005100829928A CN200510082992A CN100400665C CN 100400665 C CN100400665 C CN 100400665C CN B2005100829928 A CNB2005100829928 A CN B2005100829928A CN 200510082992 A CN200510082992 A CN 200510082992A CN 100400665 C CN100400665 C CN 100400665C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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Abstract
The present invention discloses a constitution type expression carrier and the application thereof. The purpose of the present invention is to provide a constitution type expression carrier for constitutively expressing foreign genes in a Hansen yeast and a method for expressing high temperature resisting xylanase by using the constitution type expression carrier. The constitution type expression carrier comprises a PMA1 promoter sequence, a Hansen polymorphic yeast rDNA sequence and a eukaryotic cell expression carrier used for secreting a signal peptide coding gene sequence. The method for expressing the high temperature resisting xylanase comprises the following steps: 1) high temperature resisting xylanase coding genes are inserted in the constitution type expression carrier in right requirements 1, 2, 3 to obtain a high temperature resisting xylanase constitution type expression carrier; 2) the high temperature resisting xylanase constitution type expression carrier is inducted in the Hansen polymorphic yeast to obtain engineering enzyme used for expressing the high temperature resisting xylanase; 3) the engineering enzyme is cultivated for 72 to 80 hours to obtain the high temperature resisting xylanase.
Description
Technical field
The present invention relates to a kind of constitutive expression carrier and application thereof in the biological technical field.
Background technology
Zytase is mainly produced by saprophytic fungus and bacterium, its xylan composition in can degrading plant cell walls, and be proved in agricultural and industrial everyway and have important use value.Xylan has heterology, thereby complete degradation of xylan needs the acting in conjunction of plurality of enzymes, wherein β-1, the 4-D-zytase is wherein a kind of key enzyme, it can interrupt the β between the D-xylose residues-1,4 glycosidic link on the main chain, thereby the skeleton of polysaccharide is degraded into the wood oligose of short chain.So β-1, the 4-D-zytase has a very wide range of applications in feed and the paper industry at food.Wherein, fire resistant xylanase XynB clone is from a kind of heat-resisting eubacterium-Hai hot born of the same parents bacterium (Thermotoga maritima) MSB8 of dwelling, the optimal reactive temperature of this enzyme is 90 ℃, optimal reaction pH value is 6.2, and it is stable under alkaline condition, therefore industrial, particularly on paper pulp bleaching process, has good using value (Winterhalter C, Liebl W.Two extremely thermostable xylanasesof the hyperthermophilic bacterium Thermotoga maritima MSB8.Applied andEnvironmental Microbiology, 1995,61 (5): 1810-1815).
Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) has many advantages, is one of current ideal heterologous gene expression system of generally acknowledging in the world, and many genes are efficiently expressed in its cell.The multiple-shaped nuohan inferior yeast optimum growth temperature is 37 ℃-43 ℃ (other methanol yeast is 30 ℃), and the speed of growth is fast than other yeast.Because it contains special methanol metabolic pathway, the key enzyme of this approach is methanol oxidase (MOX), formaldehyde dehydrogenase (FMD), dihydro acetone synthetase (DHAS) etc.The synthetic of these key enzymes all is subjected to inducing of methyl alcohol, and when being sole carbon source with methyl alcohol, these enzymes are by great expression.Therefore the promotor of these enzyme genes all is strong inducible promoter, utilizes it as exogenous gene promoter, can make foreign gene great expression in debaryomyces hansenii.Wherein the promotor of MOX, FMD is widely used.
H on the debaryomyces hansenii plasma membrane
+The promotor of-ATP enzyme gene (PMA1) is the efficient constitutive promoter of newfound in recent years debaryomyces hansenii.
Summary of the invention
The purpose of this invention is to provide a kind of can be in debaryomyces hansenii the constitutive expression carrier of constitutive expression foreign gene.
Constitutive expression carrier provided by the present invention is to contain the PMA1 promoter sequence, the eukaryotic expression vector of multiple-shaped nuohan inferior yeast rDNA sequence and secreting signal peptide coding gene sequence.
Described PMA1 promotor is H on the debaryomyces hansenii plasma membrane
+The promotor of-ATP enzyme gene (PMA1).It can have the nucleotide sequence of sequence 1 in the sequence table or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
Described multiple-shaped nuohan inferior yeast rDNA sequence can have the nucleotide sequence of sequence 2 in the sequence table or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
Secreting signal peptide can be the yeast saccharomyces cerevisiae alpha factor (leading peptide of α-factor).
Described secretion signal dna encoding peptide can have the nucleotide sequence of sequence 3 in the sequence table or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Described eukaryotic expression vector is preferably Yeast expression carrier, as Yeast expression carrier pPIC3, and pPIC3K, pHIL-D1, pA0804, pA0815, pPSC3K.
Above-mentioned constitutive expression carrier can make up according to ordinary method.
Constitutive expression carrier of the present invention is particularly suitable for expressing fire resistant xylanase XynB in debaryomyces hansenii.
Fire resistant xylanase XynB encoding gene mxynB
(64)(retrieval at GenBank is numbered: AY340789).
The constitutive expression carrier that contains fire resistant xylanase encoding gene mxynB is pPMA1-xynB
(64)
Contain pPMA1-xynB
(64)Yeast strain also belong to protection scope of the present invention.
Described yeast strain is preferably the multiple-shaped nuohan inferior yeast bacterial strain, especially is preferably multiple-shaped nuohan inferior yeast ATCC34438.
The described pPMA1-xynB that contains
(64)Yeast strain be preferably HpA16-pPMA1-xynB
(64)9#.
It is simple that another object of the present invention provides a kind of culture condition, the method for the expression fire resistant xylanase that fermentation time is short.
The method of expression fire resistant xylanase provided by the present invention may further comprise the steps:
1) the fire resistant xylanase encoding gene is inserted obtains the fire resistant xylanase constitutive expression carrier in the above-mentioned constitutive expression carrier;
2) described fire resistant xylanase constitutive expression carrier is imported in the multiple-shaped nuohan inferior yeast, obtain expressing the engineering bacteria of fire resistant xylanase;
3) cultivated described engineering bacteria 72-80 hour, obtain fire resistant xylanase.
Described fire resistant xylanase constitutive expression carrier can be pPMA1-xynB
(64)Described engineering bacteria can be and contains pPMA1-xynB
(64)Multiple-shaped nuohan inferior yeast ATCC 34438 strains, be preferably HpA16-pPMA1-xynB
(64)9#; The carbon source of cultivating described engineering bacteria can be glycerine.
Constitutive expression carrier of the present invention has been owing to adopted constitutive promoter PMA1 promotor, just can expression alien gene in as the methanol yeast of carbon source with glycerine, need not inducing of methyl alcohol; Multiple-shaped nuohan inferior yeast rDNA is the tumor-necrosis factor glycoproteins of multiple copied, can improve to transform the copy number of plasmid on yeast chromosomal, thereby increase the copy number of goal gene, more helps expression of exogenous gene; (leader peptide sequences of α-factor), bootable foreign protein of expressing in multiple-shaped nuohan inferior yeast is secreted into the extracellular, more helps follow-up purifying and application, simplifies and extracts proteic operation to utilize the yeast saccharomyces cerevisiae alpha factor.
Utilize constitutive expression carrier of the present invention to express foreign protein in debaryomyces hansenii, culture condition is simple, and fermentation time is short, and expression product can be secreted into outside the born of the same parents, is beneficial to the extraction of purpose product, is fit to industrial applications.
The present invention utilizes constitutive expression carrier pPMA1-xynB
(64)Made up the engineering bacteria of expressing fire resistant xylanase.This project bacterium is cultivated shaking on the bottle, and the outer fire resistant xylanase secretory volume of born of the same parents reaches 15mg/L, and the outer fire resistant xylanase vigor of born of the same parents reaches 800U/L.The optimal reactive temperature of this fire resistant xylanase is 90 ℃, handles after 30 minutes in boiling water bath, still can keep 70% enzyme activity, decomposes the xylan activity under 90 ℃, and the pH value is stable in the 6.2-10.0 scope.Constitutive expression carrier of the present invention and engineering bacteria thereof have broad application prospects in the production of fire resistant xylanase.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment is the quality percentage composition.
Embodiment 1, reorganization multiple-shaped nuohan inferior yeast expression vector pPMA1-xynB
(64)Structure
(1) clone of debaryomyces hansenii PMA1 promotor
According to plasmid pATP7 (Helen Cox, David Mead, Peter Sudbery, the sequence of the PMA1 promotor et al.Constitutive expression of recombinant proteins in the methylotrophicyeast Hansenula polymorpha using the PMA1 promoter.2000 Yeast16:1191-1203) and the feature of the restriction enzyme digestion sites on the expression vector pPIC3.5K (available from Inventrogen company), the design synthetic primer:
FW:5 '-
GtttaaacCAATTGGGTCTGGTTTGCCA-3 ' (base of band underscore is a Pme I recognition sequence)
REV:5 '-
GgatccCCAATTGAACTGAAAGAAAAAAAGA-3 ' (base of band underscore is a BamH I recognition sequence)
With plasmid pATP7 is template, amplifies PMA1 promoter sequence (1976bp), and amplification condition is: 94 ℃ of earlier pre-sex change 5 minutes; Carry out 25 circulations again: 56 ℃ of 94 ℃ of sex change 30 seconds, annealing 30 seconds, extend 72 ℃ 2 minutes; Last 72 ℃ were extended 10 minutes.Amplified production (1976bp) reclaims and is connected on the carrier pGEM-T Easy, picks out the carrier pGEM-T-PMA1 that contains the PMA1 promoter sequence
PPositive transformant carry out the sequencing analysis, the result shows pGEM-T-PMA1
PIn the PMA1 promoter sequence correct.PGEM-T-PMA1
PBehind Pme I and BamH I double digestion, the PMA1 promoter sequence is cloned between the Pme I and BamH I recognition sequence of expression vector pPIC3.5k, obtain containing the expression vector pPIC3.5k-PMA1 of PMA1 promoter sequence
P
(2) clone of debaryomyces hansenii rDNA sequence
With restriction enzyme Not I digested plasmid pATP7, obtain debaryomyces hansenii rDNA fragment, this fragment is connected to the pPIC3.5k-PMA1 that cuts with Not I enzyme
PIn the plasmid, obtain containing the expression vector pPIC3.5k-PMA1 of PMA1 promoter sequence and debaryomyces hansenii rDNA sequence
P-rDNA.
(3) clone of high temperature resistant xylanase gene
According to yeast expression vector pPIC9K-xynB
(64)(according to application number is that 200310113562.9 Chinese patent application prepares pPIC9K-xynB
(64)Method obtain) sequence at gene two ends, the synthetic following primer of design:
FW:5 '-CCC
GgatccAAACGATGAGATTT-3 ' (base of band underscore is a BamH I recognition sequence)
REV:5 '-CCC
GgatccCGATAAGCTTGCACAAAC-3 ' (base of band underscore is a BamH I recognition sequence)
The restriction enzyme site of the synthetic BamH I of primer two ends design is with yeast expression vector pPIC9K-xynB
(64)As template, amplify 5 ' end by PCR method and have secretion signal peptide sequence-yeast saccharomyces cerevisiae alpha factor leader peptide sequences, 3 ' end has fire resistant xylanase (XynB) gene of pichia spp alcohol oxidase (AOX) gene termination sequence.Amplification condition is: 94 ℃ of pre-sex change 5 minutes; Carry out 25 circulations again: 94 ℃ of sex change 30 seconds, anneal 55 ℃ 1 minute, extend 72 ℃ 2 minutes; Last 72 ℃, extended 10 minutes.Amplified production (1705bp) reclaims and is connected on the carrier pGEM-T Easy, picks out the carrier pGEM-T-xynB that contains the XynB gene
(64)Positive transformant carry out the sequencing analysis, the result shows pGEM-T-xynB
(64)In the XynB gene order correct.PGEM-T-xynB
(64)After BamH I enzyme is cut, the XynB gene clone is carried pPIC3.5k-PMA1 to expressing
PBetween two BamH I recognition sequences of-rDNA, obtain containing the PMA1 promoter sequence, debaryomyces hansenii rDNA sequence, yeast saccharomyces cerevisiae alpha factor leader peptide sequences, the expressed by Hansenula yeast carrier pPMA1-xynB of pichia spp alcohol oxidase (AOX) gene termination sequence and fire resistant xylanase (XynB) gene
(64)
The expression of embodiment 2, fire resistant xylanase
(1) reorganization expressed by Hansenula yeast carrier pPMA1-xynB
(64)Conversion and the screening of high copy number transformant
Reorganization expressed by Hansenula yeast carrier pPMA1-xynB
(64)After the linearizing of NotI complete degestion, electric shocking method transforms multiple-shaped nuohan inferior yeast ATCC 34438, transforms the back and screen positive transformant on the YPD that contains 0.1mg/ml G418 (2% glucose, pH 5.8 for 1% yeast extract, 2% peptone) flat board.
Being connected on the YPD flat board of the G418 that contains 0.8mg/ml containing the bacterium colony point of growing on the YPD flat board of 0.1mg/ml G418, select totally 30 of transformants, cultivate, wherein bacterial strain HpA16-pPMA1-xynB
(64)9# expression-secretion amount is higher.
(2) expression of fire resistant xylanase and extraction
Bacterial strain HpA16-pPMA1-xynB
(64)9# is inoculated in the 5mlYPD substratum, and 37 ℃ of shaking tables are cultured to OD
600Be about 8.0, be transferred to 5ml BMGY substratum [1% yeast extract with 10% inoculum size again, 2% peptone, 100mmol/l potassium phosphate buffer, 1.34%YNB (Yeast Nitrogen Base, yeast nitrogen), 2% glycerine, pH6.0], cultured continuously 2 days, centrifugal, collect supernatant liquor and somatic cells.Contain in the supernatant liquor and express justacrine, supernatant liquor was handled 10 minutes in 70 ℃ of water-baths, remove foreigh protein removing, obtain the outer crude enzyme liquid of born of the same parents to the outer fire resistant xylanase of born of the same parents.Collect 1ml bacterium liquid cell, with twice of 500 μ l sterilized waters washing, MES damping fluid (pH6.14) suspension cell with 100 μ l 50mM, add 20 μ l 6mg/mL Zymolyase solution, the rearmounted 37 ℃ of reactions of mixing 50-60 minute are put 70 ℃ of water-baths 10 minutes, 12, centrifugal 10 minutes of 000rpm, supernatant liquor are crude enzyme liquid in the cell.Crude enzyme liquid is through SDS-PAGE electrophoresis detection expressing quantity in outer crude enzyme liquid of these born of the same parents and the born of the same parents, electrophoresis result shows outside the born of the same parents and the interior crude enzyme liquid of born of the same parents can both obtain electrophoretically pure fire resistant xylanase XynB after treatment, its relative molecular weight is 42kDa, matches with the molecular weight (42.067KDa) of theoretical calculate.Fire resistant xylanase XynB secretory volume can reach the 15mg/L nutrient solution.
(3) fire resistant xylanase determination of activity
Oat xylan (oat spelts xylan with 0.25% (w/v), Sigma Chemical St.Louis, MO.USA) solution is substrate, adopts P-hydroxybenzoic acid hydrazides method (p-hydroxybenzoic acidhydrazide, PHBAH method) to measure the fire resistant xylanase vigor.Concrete operations are as follows:
1). get storage liquid 0.5mol/L Trisodium Citrate in order respectively, 1mol/L S-WAT, 0.2mol/L calcium chloride, each 10ml of 5mol/L sodium hydroxide adds while stirring, uses the distilled water constant volume to 100ml, add 1.52 gram PHBAH again, the stirring that does not stop, the necessary matching while using of this solution;
2). weighing 1 gram oat xylan is dissolved in the 100ml 50mmol/L MES damping fluid (pH 6.2), after fully stirring, the centrifugal precipitation of removing, supernatant liquor is 0.25% fire resistant xylanase reaction substrate;
3). get the oat xylan substrate step 2 of 190 μ l0.25%) supernatant liquor in 70 ℃ of following insulation 10min, step (2) the fire resistant xylanase solution that adds 10 μ l dilution, in 70 ℃ of following accurate response 10min, add 400 μ l PHBAH solution again, in boiling water bath, keep 6min behind the mixing;
4). after the sample cooling, with spectrophotometric determination absorbance (OD
405).The barren operation replaces the enzyme solution of dilution rationally with above-mentioned the same with 10 μ l water.1 enzyme activity unit (unit) is defined as: under above-mentioned experiment condition, it is 1 enzyme activity unit that per minute produces the required enzyme amount of 1 μ mol wood sugar, and OD
405Be equivalent to 0.067 μ mol wood sugar at=1 o'clock.The result shows that it is the outer crude enzyme liquids of 800U/L born of the same parents that fire resistant xylanase XynB enzyme is lived.
Utilize the standard method of known protein materialization to measure the fundamental characteristics of this fire resistant xylanase, measurement result shows that the optimum temperature of this fire resistant xylanase is 90 ℃; Decompose the xylan activity at 90 ℃, the pH value is that the scope of 6.2-8.0 is interior stable; In boiling water bath, handle after 30 minutes, still can keep 70% enzyme activity.
Sequence table
<160>3
<210>1
<211>1960
<212>DNA
＜213〉multiple-shaped nuohan inferior yeast (Hansenula polymorpha)
<400>1
caattgggtc?tggtttgcca?tggaattagg?aagcaacggt?gagttgtttg?acaagatcga 60
gcctgatctg?ggagttgacg?aggatgtcgc?tcaattttat?ttcaaacagc?tcataaatgc 120
agtttctttt?attcattcca?aaggtgttgc?acaccgggat?atcaagccag?aaaacctagt 180
tatcgataag?agcggaaacc?tgaagctgac?ggactttggc?cttgccagtg?tgtttaaaaa 240
gaaaaacggt?tccaaaagaa?tgtgcaccac?agcatgcggc?tctcctcctt?accttgctcc 300
ggaggtggtc?tctcaaaaat?atgatcccga?gccctcggac?gtgtggtcct?gcggcatagt 360
tttgtttgtg?ctcctcacag?ggcagatagc?gtgggagatg?ccacacgagg?aagacacaga 420
cttccgctac?tacatggaga?agaagggaga?agtgctgatt?tccccatgga?acaagatccc 480
tctaggtgca?ctttccctgc?tgcggaaggt?gctagtcttg?gagccgtttc?gtcgacgcat 540
cgaagagatt?aagcagcatc?cttgggtggc?caagaaaaac?aagtttgcgg?accaaaatga 600
tatgtgcaaa?gacccaatgc?gtctcaccac?gagcctgctg?gtgaaccttt?acatcaacct 660
ttcggacgca?gagttcaaca?agatcagcca?gatgtccacc?cagatcagca?agccggcagc 720
aggaacgcaa?cccccaaaat?acgtgatgga?cgatacgcag?ccggcagaga?tcctgtcgag 780
cgacagcacg?aggagtggac?ggagtaaacc?agcggcgagt?ttgggttata?agttttcggt 840
tataggagga?taaatggaac?tgtatgaata?gtgaggatcg?ttatctgtaa?ttaaaaatgc 900
agaattatta?gctggcctcg?gtgcagtgca?gtcaggagta?gtaatcacca?aatttaccta 960
attagtgcta?tgaaacagga?ccaccctgga?tacacgcaag?gcaagtctcc?gagagggtct 1020
atgtcacatg?gggcatcaaa?aggtacagta?ctgagggcca?tgacaacaag?actgtcgtga 1080
tccggggtat?cttatttggg?taagcaagtt?tcccaatagg?gaacaatgcg?gtctaaaaac 1140
agaacacagg?catttttagg?cggtggatgc?ggaatgaata?aatggctgga?aagatcggct 1200
cggttttggc?aagttgagtg?aaaaaaaacc?tggccgaaaa?aacctcaaaa?gttatgattt 1260
cgcatataac?cggtcttggt?ccgaaaccgg?tgcagaaaat?atgctcttgg?ccgtagttgt 1320
agaagctggg?gatgagttgc?aaagaggcca?tttggcgttg?acgggtgagg?gcggatctcc 1380
aggaacagta?catggacaga?agccagttct?agattcgcat?tttcccgagt?ttgagctgtc 1440
cctgtctggg?tcctgcgact?tttcccctgg?attgctaaaa?aggtaaacaa?tagcgtacag 1500
aaagaaacga?tagcgtagaa?aaggcgggta?agatacactt?aataaaacgt?ctccgcgaaa 1560
gcagcatcca?gataaccggg?tcagatggag?gagctcacgg?ctagattgag?tgcagcggtt 1620
tgctcggcac?ggtcgagcca?gtatcggcaa?tgtcccggtg?tccaattttc?acgcaaatta 1680
cgtcttcccg?gatattccgt?tgctggaaaa?caatagaaat?cggttttgtt?aacgatatcg 1740
accagtgaaa?atacagctaa?ataactcgtt?ggtcggaaac?ttaattgtgg?tttgtgtttc 1800
ttttttaccc?ttttggtacc?gattgttgtt?tactgaattt?ggacaaacaa?agtcgctcgt 1860
tgcaaggtgc?ataatttgca?tccacctctt?aaaaatgcag?tatctttcct?tcgtttttct 1920
ccatccaact?ttatattctt?tttttctttc?agttcaattg 1960
<210>2
<211>2962
<212>DNA
＜213〉multiple-shaped nuohan inferior yeast (Hansenula polymorpha)
<400>2
gcggccgcaa?gcttctttca?aaaattggct?ctgccttaca?aactttaatc?ataaagtttg 60
acctcaaatc?aggtaggatt?acccgctgaa?cttaagcata?tcaataagcg?gaggaaaaga 120
aaccaacagg?gattgcctta?gtagcggcga?gtgaagcggc?aagagctcaa?atttgaaatc 180
tggtaccttc?ggtgcccgag?ttgtaatttg?aagaaagcta?tcttggaggt?ggcctttgtc 240
tatgttcctt?ggaacaggac?gtcatggagg?gtgagaatcc?cgtgtgatga?ggtgtccatt 300
tccgtgtaag?atgctttcga?agagtcgagt?tgtttgggaa?tgcagctcaa?agtgggtggt 360
aaattccatc?taaagctaaa?tattggcgag?agaccgatag?cgaacaagta?ctgtgaagga 420
aagatgaaaa?gaactttgaa?aagagagtga?aaaagtacgt?gaaattgttg?aaagggaagg 480
gtatttgatc?agacttggta?tttagctatc?atcgctcctt?gtgggtggtg?ctctagcttt 540
ttactgggcc?agcatcagtt?ttggtggcaa?gataatgaca?gttgaatgtg?gctcctcgga 600
gtgttatagc?ttctgttgat?gttgcctacc?gagactgagg?tctgcggctt?ttgcctagga 660
tgctggcgta?atgatccaat?accgcccgtc?ttgaaacatg?gaccaaggag?tctaacgtct 720
atgcgagtgt?ttgggtgtaa?aacccgtacg?cgtaatgaaa?gtgaacgtag?gtcagggccc 780
gcaagggtgc?atgatcgacc?gatcctgatg?ttctcggatg?gatttgagta?agagcatagc 840
tgttgggacc?cgaaagatgg?tgaactatgc?ctgaataggg?tgaagccaga?ggaaactctg 900
gtggaggctc?gtagcggttc?tgacgtgcaa?atcgatcgtc?gaatttgggt?ataggggcga 960
aagactaatc?gaaccatcta?gtagctggtt?cctgccgaag?tttccctcag?gatagcagaa 1020
gctcgtatca?gttttatgag?gtaaagcgaa?tgattagagg?tcttggggtc?gaaatgacct 1080
tagcctattc?tcaaacttta?aatatgtaag?aagtccttgt?tgcttaattg?aacgtggaca 1140
tttgaatgaa?gagcttttag?tgggccattt?ttggtaagca?gaactggcga?tgcgggatga 1200
accgaacgtg?aagttaaggt?gccggaatgc?acgctcatca?gacaccacaa?aaggtgttag 1260
ttcatctaga?cagccggacg?gtggccatgg?aagtcggaat?ccgctaagga?gtgtgtaaca 1320
actcaccggc?cgaatgaact?agccctgaaa?atggatggcg?ctcaagcgtg?ttacctatac 1380
ttcaccgtca?tggtttttat?gatgccatga?cgagtaggca?ggcgtggagg?tcagtgacga 1440
agcctttggt?gtgaacctgg?gtagaacggc?ctctagtgca?gatcttggtg?gtagtagcaa 1500
atattcaaat?gagatctttg?aagactgaag?tggggaaagg?ttccacatca?acagcagttg 1560
gatgtgggtt?agtcgatcct?aagagatggg?gaagctccgt?ttcaaagtgc?ttgatttttc 1620
aagccaccat?cgaaagggaa?tccggttaat?attccggaac?ttggatatgg?attcttcacg 1680
gtaacgtaac?tgaatgtgga?gacgtcggca?tgagccctgg?gaggagttat?cttttcttct 1740
taacagctta?tcaccctgga?attggtttat?ccggagatag?ggtcttatgg?ctggaagagc 1800
gtaatatctt?tgttacgtcc?ggtgcgctca?tgacggccct?tgaaaatcca?caggaaggaa 1860
tagttttcat?gccaagtcgt?actcataacc?gcagcaggtc?tccaaggtta?acagcctcta 1920
gttgatagaa?taatgtagat?aagggaagtc?ggcaaaatag?atccgtaact?tcgggataag 1980
gattggctct?aagggtcggg?tgtcttgggc?ctttttcaga?cgccattccg?cattgggatc 2040
tgctttcggg?tggatctctt?tgtggtttgg?ttgacgaatt?tggtaggtct?tcatggccgt 2100
ccggggcaca?attaacgacc?aacttagaac?tggtacggac?aaggggaatc?tgactgtcta 2160
attaaaacat?agcattgcga?tggtcataaa?atgatgttga?cgcaatgtga?tttctgccca 2220
gtgctctgaa?tgtcaaagtg?aagaaattca?accaagcgcg?ggtaaacggc?gggagtaact 2280
atgactctct?taaggtagcc?aaatgcctcg?tcatctaatt?agtgacgcgc?atgaatggat 2340
taacgagatt?cccactgtcc?ctatctacta?tctagcgaaa?ccacagccaa?gggaacgggc 2400
ttggcagaat?cagcggggaa?agaagaccct?gttgagcttg?actctagttt?gacattgtga 2460
aaagacatag?agggtgtaga?ataagtggga?gcttcggcgc?cagtgaaata?ccactacctt 2520
tatcgttttt?ttacttattc?aattaagcgg?agctggactt?caccgtccac?gttctagatt 2580
taaggtctct?tgtaggctga?tccgggttga?agacattgtc?aggtggggag?tttggctggg 2640
gcggcacatc?tgttaaacga?taacgcaggt?gtcctaaggg?ggactcatgg?agaacagaaa 2700
tctccagtag?aacaaaaggg?taaaagtccc?cttgattttg?attttcagtg?tgaatacaaa 2760
ccatgaaagt?gtggcctatc?gatcctttag?tccctcggaa?tttgaggcta?gaggtgccag 2820
aaaagttacc?acagggataa?ctggcttgtg?gcagtcaagc?gttcatagcg?acattgcttt 2880
ttgattcttc?gatgtcggct?cttcctatca?taccgaagca?gaattctaga?gtcgacctgc 2940
aggcatgcaa?gcttgcggcc?gc 2962
<210>3
<211>293
<212>DNA
＜213〉yeast saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400>3
atgagatttc?cttcaatttt?tactgcagtt?ttattcgcag?catcctccgc?attagctgct 60
ccagtcaaca?ctacaacaga?agatgaaacg?gcacaaattc?cggctgaagc?tgtcatcggt 120
tactcagatt?tagaagggga?tttcgatgtt?gctgttttgc?cattttccaa?cagcacaaat 180
aacgggttat?tgtttataaa?tactactatt?gccagcattg?ctgctaaaga?agaaggggta 240
tctctcgaga?aaagagaggc?tgaagcttac?gtagaattcc?ctagggcggc?cgc 293
Claims (5)
1. the constitutive expression carrier of a multiple-shaped nuohan inferior yeast, this carrier contains the PMA1 promoter sequence successively, multiple-shaped nuohan inferior yeast rDNA sequence, yeast saccharomyces cerevisiae alpha factor leader peptide sequences encoding gene, pichia spp alcohol oxidase gene termination sequence and high temperature resistant xylanase gene; The base sequence of described PMA1 promotor is shown in SEQ IDNO:1, and the base sequence of multiple-shaped nuohan inferior yeast rDNA sequence is shown in SEQ ID NO:2, and the base sequence of yeast saccharomyces cerevisiae alpha factor leader peptide sequences encoding gene is shown in SEQ ID NO:3.
2. the multiple-shaped nuohan inferior yeast bacterial strain that contains the described constitutive expression carrier of claim 1.
3. yeast strain according to claim 2 is characterized in that: described multiple-shaped nuohan inferior yeast bacterial strain is multiple-shaped nuohan inferior yeast ATCC 34438.
4. a method of expressing fire resistant xylanase is cultivation claim 2 or 3 described multiple-shaped nuohan inferior yeast bacterial strains 72-80 hour, obtains fire resistant xylanase.
5. method according to claim 4 is characterized in that: the carbon source of cultivating described multiple-shaped nuohan inferior yeast bacterial strain is a glycerine.
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| CN101372694B (en) * | 2007-08-22 | 2013-03-27 | 中国农业大学 | Expression of high temperature resistant xylanase gene in Kluyveromyces lactis |
| CN101560516B (en) * | 2009-01-19 | 2010-12-08 | 广西大学 | Constitutive expression promoter in escherichia coli and applications in escherichia coli |
| CN103194480A (en) * | 2012-01-06 | 2013-07-10 | 中国科学技术大学 | High-efficiency expression method of human interleukin-10 (hIL-10) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003085111A1 (en) * | 2002-04-08 | 2003-10-16 | Kaneka Corporation | Novel promoters |
| CN1544640A (en) * | 2003-11-17 | 2004-11-10 | 中国农业大学 | Expression method for high temperature resistant xylanase and specific expression carrier for same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003085111A1 (en) * | 2002-04-08 | 2003-10-16 | Kaneka Corporation | Novel promoters |
| CN1544640A (en) * | 2003-11-17 | 2004-11-10 | 中国农业大学 | Expression method for high temperature resistant xylanase and specific expression carrier for same |
Non-Patent Citations (6)
| Title |
|---|
| Constitutive expression of recombinant proteins in themethylotrophic yeast Hansenula polymorpha using the PMAIpromoter. Cox, Helen 等人.Yeast,Vol.16 No.13. 2000 |
| Constitutive expression of recombinant proteins in themethylotrophic yeast Hansenula polymorpha using the PMAIpromoter. Cox, Helen 等人.Yeast,Vol.16 No.13. 2000 * |
| 人多形汉逊酵母外源基因表达系统. 陈凤菊等.生物工程学报，第17卷第2期. 2001 |
| 人多形汉逊酵母外源基因表达系统. 陈凤菊等.生物工程学报，第17卷第2期. 2001 * |
| 极端耐热木聚糖酶基因在大肠杆菌和毕赤酶母中的高效表达. 杨梦华等.生物工程学报，第45卷第2期. 2005 |
| 极端耐热木聚糖酶基因在大肠杆菌和毕赤酶母中的高效表达. 杨梦华等.生物工程学报，第45卷第2期. 2005 * |
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