CN105063080A - Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain - Google Patents
Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain Download PDFInfo
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Abstract
The invention provides a signal peptide-free recombinant vector for exogenous gene expression in a Kluyveromyces marxianus nutritional deficient strain as well as a preparation method and application thereof. The recombinant vector sequentially comprises an ampicillin resistance gene, a PKD1 vector, an inulase promoter, multiple cloning sites, an inulase terminator, a nutritional gene promoter and a nutritional gene open reading frame. The signal peptide-free recombinant vector and the preparation method thereof, constructed by the invention, can be used for constructing transformants to realize exogenous gene expression.
Description
Technical field
The present invention relates to a kind of recombinant expression vector used in auxotrophic strain, particularly relate to a kind of recombinant expression vector not containing signal peptide.
Background technology
Yeast is single celled eukaryote, and it has characteristic and the Eukaryotic albumen synthesis system of processing of microorganism concurrently.Therefore, it is widely used in and expresses multiple external source eukaryotic protein.The yeast expression system of current main flow comprises pichia spp and Saccharomyces Serevisiae Expression System.But the protein expression of pichia spp needs, with methanol induction, not to be suitable for the production of food protein.The easy producing and ethanol of yeast saccharomyces cerevisiae, stand density is low, and expression level is on the low side.For this series of problems, the yeast expression system that security is high and output is high of Development of Novel has very high industrial value.
Kluyveromyces marxianus (Kluyveromycesmarxianus) have passed GRAS and the QPS safety certification of US and European, himself is not only considered to safe microorganism strains, also be considered to the safe microorganisms bacterial strain that can be used for preparing food grade recombinant protein (enzyme), be a kind of yeast of the food grade approved by European Union simultaneously.It has nutritional requirement extremely simple, grow the features such as vigorous, biomass large, growth temperature wide accommodation.Meanwhile, it also has the feature of efficient secretion albumen.Therefore, Kluyveromyces marxianus has high expression and eats the great potential with feed protein.
Expression system is made up of Host Strains and expression vector.For other yeast expression system of aliment security level, Host Strains selects nutrient defect type mark usually.
Summary of the invention
The invention provides a kind of recombinant vectors carrying out exogenous gene expression in auxotrophic strain.
The present invention first aspect is to provide a kind of recombinant vectors carrying out exogenous gene expression in kluyveromyces marxianus auxotrophic strain not containing signal peptide, comprises ammonia benzyl resistant gene, PKD1 carrier, inulinase promotor, multiple clone site, inulinase terminator, vegetative gene promotor, vegetative gene open reading frame (ORF) in order.
Wherein, described vegetative gene is preferably the vegetative gene of Marx's Crewe dimension bacterial strain, as any one or a few in URA3 gene, HIS3 gene, ADE2 gene, is more preferably URA3 gene.
Wherein, described multiple clone site can be the sequence comprising one or more sites restriction enzyme site, described restriction enzyme site is as any one or a few in SmaI, XmaI, SpeI, NotI, be more preferably in SpeI, NotI and SmaI and/or XmaI any one or a few, as SmaI/XmaI, SpeI, NotI.
Wherein, described multiple clone site length is preferably 15-25bp, is more preferably 18-23bp, is more preferably 19-21bp.
In one preferred embodiment of the invention, the DNA sequence dna of described recombinant vectors is as shown in SEQIDNo.1.
The present invention second aspect is to provide a kind of method building above-mentioned recombinant vectors and comprises:
Amplification pUC19 plasmid, PKD1 carrier, obtain the fragment I that pUC19 and PKD1 connects;
Amplification Kluyveromyces marxianus genome, obtains the fragment II comprising vegetative gene promotor or ORF;
Amplification Kluyveromyces marxianus genome, obtains the fragment III comprising inulinase gene promoter, inulinase signal peptide and part multiple clone site;
Amplification Kluyveromyces marxianus genome, obtains the fragment IV comprising inulinase terminator and part multiple clone site,
Fragment I, II, III, IV are connected, obtains the first recombinant vectors.
Use site-directed mutagenesis kit, described first recombinant vectors is suddenlyd change, the recombinant vectors not containing signal peptide described in acquisition.
Wherein, described site-directed mutagenesis kit is preferably QuikChangeII test kit.
Wherein, described sudden change the primer is preferably:
5’-ATAAGTGACACATTTAATTTTTTTTTTGTTAGATACTAGTCCCGGGGCGGCCGCTTAAGGCCGCAAGCTT-3’；
5’-AAGCTTGCGGCCTTAAGCGGCCGCCCCGGGACTAGTATCTAACAAAAAAAAAATTAAATGTGTCACTTAT-3’。
Wherein, the primer of amplification pUC19 plasmid is preferably:
Forward primer:
5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'；
Reverse primer:
5'-TACAATTTTATGGTGCACTCTCAGTACAATCTGCT-3'。
Wherein, the primer of amplification PKD1 plasmid is preferably:
Forward primer:
5'-CCAAGCTTGCATGCATCACTAATGAAAAGCATACGACGCC-3'；
Reverse primer:
5'-AATTCGAGCTCGGTACCCCTCCGTTGCAGCGTGCGAATCGGCACGG-3'。
Wherein, in an advantageous embodiment, the fragment I method obtaining pUC19 and PKD1 connection is as follows: amplification pUC19 plasmid obtains A fragment, the pcYGW of amplification Gateway systemic vectors obtains B fragment, amplification PKD1 carrier obtains C fragment, A, B are connected with C fragment, increase connecting the plasmid obtained, obtaining the fragment I comprising pUC19 and PKD1.
Wherein, the pcYGW the primer that increases is preferably:
Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3';
Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'.
Wherein, the primer of amplification plasmid is preferably:
Forward primer: 5'-GATCCGCTTAAGAGGGGTACCGAGCTCGAATT-3';
Reverse primer:
5'-ACCTTTTCGGATCGGCATGCCGTTGCAGCGTGCGAATCGGCACGG-3'。
Wherein, amplification obtains the primer of fragment II preferably:
Forward primer: 5'-CTAGGATCGGTCGAATTCTGATTGGAAAGACCATT-3';
Reverse primer: 5'-GTACCCCTCTTAAGCGGATCTGCCTACTCTCTTCA-3'.
Wherein, amplification obtains the primer of fragment III preferably:
Forward primer: 5'-GCATGCCGATCCGAAAAGGTAAACAGACACAAAAAC-3';
Reverse primer:
5'-GTCCCGGGGTCACCGTCTCTCTTGTAATTGATCACTGAAG-3'。
Wherein, amplification obtains the primer of fragment IV preferably:
Forward primer:
5'-ACGGTGACCCCGGGACTAGTGCGGCCGCTTAAGGCCGCAAGCTTTGATCTG-3'；
Reverse primer: 5'-CAGAATTCGACCGATCCTAGAATGTTGGTCAGATGTG-3'.
Third aspect of the present invention is to provide a kind of transformant, is inserted into by foreign gene in the multiple clone site of described recombinant vectors, obtains the plasmid containing foreign gene, is proceeded in vegetative gene deficient strain by described plasmid, obtain described transformant.
Wherein, described vegetative gene deficient strain is preferably vegetative gene defective type Marx Crewe dimension bacterial strain.
Described vegetative gene defect preferably comprise in URA3 gene, HIS3 gene, ADE2 genetic flaw any one or a few, be more preferably URA3 genetic flaw.
The recombinant vectors not containing signal peptide that the present invention builds, can be used for building transformant, realizes the expression of foreign gene.
Accompanying drawing explanation
Fig. 1 is constructed recombinant vectors schematic diagram in the embodiment of the present invention;
Embodiment
Step 1, amplification pUC19 plasmid
Forward primer:
5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'
Reverse primer: 5'-TACAATTTTATGGTGCACTCTCAGTACAATCTGCT-3'
Utilize described primer amplification pUC19 plasmid.Pcr amplification reaction carries out according to the working instructions of the PhantaSuperFidelityDNAPolymerase of Vazyme company, and annealing temperature is 58 DEG C, and the extension time is 3 minutes, 30 circulations.PCR primer called after A fragment.
Step 2, the pcYGW of amplification Gateway systemic vectors
Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3'
Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'
Utilize the pcYGW of described primer amplification Gateway systemic vectors.Pcr amplification conditional synchronization rapid 1, except the extension time is 1 minute.PCR primer called after B fragment.
Step 3, amplification PKD1 carrier
Forward primer: 5'-CCAAGCTTGCATGCATCACTAATGAAAAGCATACGACGCC-3' reverse primer:
5'-AATTCGAGCTCGGTACCCCTCCGTTGCAGCGTGCGAATCGGCACGG-3'
Utilize described primer amplification PKD1 carrier.Pcr amplification conditional synchronization rapid 1, except the extension time is 5 minutes.PCR primer called after C fragment.
Step 4, junction fragment A, B, C
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, Segment A, B, C are connected, transformation of E. coli, the plasmid called after PUC19-PKD1 of acquisition.
Step 5, amplification PUC19-PKD1 plasmid
Forward primer: 5'-GATCCGCTTAAGAGGGGTACCGAGCTCGAATT-3'
Reverse primer:
5'-ACCTTTTCGGATCGGCATGCCGTTGCAGCGTGCGAATCGGCACGG-3'
Utilize described primer amplification PUC19-PKD1 plasmid.Pcr amplification conditional synchronization rapid 1, except the extension time is 8 minutes.PCR primer called after D fragment.Part PUC19 sequence and PKD1 sequence is included in D fragment.
Step 6, amplification URA3 gene promoter and ORF fragment
Forward primer: 5'-CTAGGATCGGTCGAATTCTGATTGGAAAGACCATT-3'
Reverse primer: 5'-GTACCCCTCTTAAGCGGATCTGCCTACTCTCTTCA-3'
Extract the specification sheets operation of test kit (DP307) according to Yeast genome, utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1.5 minutes.PCR primer called after E fragment.Promotor and the ORF of URA3 gene is contained in E fragment.
Step 7, the promotor of amplification inulinase gene and signal peptide fragment
Forward primer: 5'-GCATGCCGATCCGAAAAGGTAAACAGACACAAAAAC-3'
Reverse primer: 5'-GTCCCGGGGTCACCGTCTCTCTTGTAATTGATCACTGAAG-3'
Utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1.5 minutes.PCR primer called after F fragment.The promotor of inulinase gene, signal peptide and part multiple clone site is contained in F fragment.
Step 8, the terminator fragment of amplification inulinase gene
Forward primer:
5'-ACGGTGACCCCGGGACTAGTGCGGCCGCTTAAGGCCGCAAGCTTTGATCTG-3'
Reverse primer: 5'-CAGAATTCGACCGATCCTAGAATGTTGGTCAGATGTG-3'
Utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1 minute.PCR primer called after G fragment.Terminator and the part multiple clone site of inulinase gene is contained in G fragment.
Step 9, connects D, E, F, G fragment
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, fragment D, E, F, G are connected, transformation of E. coli, the plasmid called after PUKDN112 of acquisition.
Step 10, sudden change PUKDN112 plasmid
Operate according to the specification sheets of the QuikChangeII test kit of Agilent company, PUKDN112 plasmid is suddenlyd change.The primer used that suddenlys change is:
5’-ATAAGTGACACATTTAATTTTTTTTTTGTTAGATACTAGTCCCGGGGCGGCCGCTTAAGGCCGCAAGCTT-3’；
5’-AAGCTTGCGGCCTTAAGCGGCCGCCCCGGGACTAGTATCTAACAAAAAAAAAATTAAATGTGTCACTTAT-3’。
Plasmid after sudden change is PUKDN115.By Jie Li company, PUKDN115 is checked order, determine the sequence of multiple clone site.Sequencing primer is 5'-TCCCTTGAATCGTGTTTGCCAGTT-3'.
With reference to Fig. 1, sequencing result is as follows:
Total length: 11394bp
1031-1171bp:beta-lactamase (ammonia benzyl resistant gene)
3112-7870:PKD1 carrier sequence
7879-9094: inulinase promotor
9095-9114: multiple clone site, comprises SpeI, SmaI/XmaI, NotI tri-sites.
9119-9983: inulinase terminator
9984-10569:KmURA3 promotor
10570-11373：KmURA3ORF
PUKD115 complete sequence is as shown in SEQIDNo.1.
Purposes:
Foreign gene is inserted in the multiple clone site of PUKDN115.Utilize primer 5'-TCCCTTGAATCGTGTTTGCCAGTT-3' to check order, determine that whether gene order is correct.
Transform and adopt LiAc method, carry out operating (WorldJournalofMicrobiology & Biotechnology16:653-654,2000) according to reference.Proceed in the bacterial strain of the Kluyveromycesmarxianus of URA3 transgenation by the PUKDN115 plasmid containing foreign gene, on the substratum lacking Uracil, screening obtains URA3
+transformant.URA3
+transformant can be used for the expression of foreign gene, and expression and the method detected are carried out (ApplMicrobiolBiotechnol (2005) 67:364 – 369) according to reference.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (9)
1. the recombinant vectors carrying out exogenous gene expression in kluyveromyces marxianus auxotrophic strain not containing signal peptide, it is characterized in that, comprise ammonia benzyl resistant gene, PKD1 carrier, inulinase promotor, multiple clone site, inulinase terminator, vegetative gene promotor, vegetative gene open reading frame in order.
2. recombinant vectors according to claim 1, is characterized in that, described vegetative gene is Marx's Crewe dimension bacterium URA3 gene.
3. recombinant vectors according to claim 1, is characterized in that, the DNA sequence dna of described recombinant vectors is SEQIDNo.1.
4. recombinant vectors according to claim 1, is characterized in that, described multiple clone site be selected from restriction enzyme site SmaI, XmaI, SpeI, NotI any one or a few.
5. build a method for recombinant vectors described in claim 1, it is characterized in that, comprising:
Amplification pUC19 plasmid, PKD1 carrier, obtain the fragment I that pUC19 and PKD1 connects;
Amplification Kluyveromyces marxianus genome, obtains the fragment II comprising vegetative gene promotor or ORF;
Amplification Kluyveromyces marxianus genome, obtains the fragment III comprising inulinase gene promoter, inulinase signal peptide and part multiple clone site;
Amplification Kluyveromyces marxianus genome, obtains the fragment IV comprising inulinase terminator and part multiple clone site,
Fragment I, II, III, IV are connected, obtains the first recombinant vectors.
Use site-directed mutagenesis kit, described first recombinant vectors is suddenlyd change, the recombinant vectors not containing signal peptide described in acquisition.
6. method according to claim 5, is characterized in that, described site-directed mutagenesis kit is QuikChangeII test kit.
7. method according to claim 5, is characterized in that, sudden change the primer is:
5’-ATAAGTGACACATTTAATTTTTTTTTTGTTAGATACTAGTCCCGGGGCGGCCGCTTAAGGCCGCAAGCTT-3’；
5’-AAGCTTGCGGCCTTAAGCGGCCGCCCCGGGACTAGTATCTAACAAAAAAAAAATTAAATGTGTCACTTAT-3’。
8. method according to claim 5, is characterized in that, the fragment I method obtaining pUC19 and PKD1 connection is as follows:
Amplification pUC19 plasmid obtains A fragment, and the pcYGW of amplification Gateway systemic vectors obtains B fragment,
Amplification PKD1 carrier obtains C fragment, A, B is connected with C fragment, increasing, obtaining the fragment I comprising pUC19 and PKD1 by connecting the plasmid obtained.
9. a transformant, is characterized in that, is inserted into by foreign gene in the multiple clone site of recombinant vectors described in claim 1, obtains the plasmid containing foreign gene, is proceeded in vegetative gene deficient strain by described plasmid, obtain described transformant.
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| PCT/CN2016/094868 WO2017036294A1 (en) | 2015-08-28 | 2016-08-12 | Kluyveromyces marxianus and use thereof |
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Cited By (5)
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| WO2017036294A1 (en) * | 2015-08-28 | 2017-03-09 | 复旦大学 | Kluyveromyces marxianus and use thereof |
| CN108410870A (en) * | 2018-02-22 | 2018-08-17 | 复旦大学 | K marxianus promoter, secreting signal peptide and its preparation and application |
| CN108486105A (en) * | 2018-02-22 | 2018-09-04 | 复旦大学 | A kind of K marxianus promoter and the preparation method and application thereof |
| CN115976094A (en) * | 2022-12-15 | 2023-04-18 | 浙江大学杭州国际科创中心 | Genetic engineering bacterium for improving endogenous enzyme secretion and construction method and application thereof |
| CN116042691A (en) * | 2022-09-07 | 2023-05-02 | 四川大学华西第二医院 | Recombinant vector for expressing exogenous gene in Kluyveromyces marxianus strain |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017036294A1 (en) * | 2015-08-28 | 2017-03-09 | 复旦大学 | Kluyveromyces marxianus and use thereof |
| CN108410870A (en) * | 2018-02-22 | 2018-08-17 | 复旦大学 | K marxianus promoter, secreting signal peptide and its preparation and application |
| CN108486105A (en) * | 2018-02-22 | 2018-09-04 | 复旦大学 | A kind of K marxianus promoter and the preparation method and application thereof |
| CN108486105B (en) * | 2018-02-22 | 2021-10-26 | 复旦大学 | Kluyveromyces marxianus promoter as well as preparation method and application thereof |
| CN108410870B (en) * | 2018-02-22 | 2021-11-19 | 复旦大学 | Kluyveromyces marxianus promoter, secretion signal peptide, preparation and application thereof |
| CN116042691A (en) * | 2022-09-07 | 2023-05-02 | 四川大学华西第二医院 | Recombinant vector for expressing exogenous gene in Kluyveromyces marxianus strain |
| CN115976094A (en) * | 2022-12-15 | 2023-04-18 | 浙江大学杭州国际科创中心 | Genetic engineering bacterium for improving endogenous enzyme secretion and construction method and application thereof |
| CN115976094B (en) * | 2022-12-15 | 2024-02-20 | 浙江大学杭州国际科创中心 | Genetically engineered bacterium for improving secretion of endogenous enzyme and construction method and application thereof |
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Application publication date: 20151118 |