CN108660085A

CN108660085A - One plant height produces nucleic acid saccharomyces cerevisiae engineered yeast and its construction method and application

Info

Publication number: CN108660085A
Application number: CN201810537366.0A
Authority: CN
Inventors: 郭学武; 倪晓丰; 王东旭; 肖冬光; 陈叶福; 赵宾
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2018-10-16
Anticipated expiration: 2038-05-30
Also published as: CN108660085B

Abstract

The present invention provides the fermentation verifications of a kind of Wine brewing yeast strain of high yield nucleic acid and its construction method and application, including gene overexpression and selection and breeding bacterial strain.Using Pep1 genes as purpose gene, the yeast strain of selection and breeding is overexpressed saccharomyces cerevisiae (Saccharomyces cerevisiae) vacuole protein sorting receptor Pep1, compared with parent strain, constructed saccharomyces cerevisiae (Saccharomyces cerevisiae) basic fermenting property of bacterial strain is unaffected, after shake flask fermentation, for selection and breeding bacterial strain nucleic acid content up to 14.70%, parent strain nucleic acid content is 9.32%, and the selection and breeding bacterial strain parent strain that compares improves 57.72%.The Wine brewing yeast strain of selection and breeding significantly improves nucleic acid content, is widely applied in yeast and nucleic acid industry.

Description

One plant height produces nucleic acid saccharomyces cerevisiae engineered yeast and its construction method and application

Technical field

The invention belongs to technical field of bioengineering, are related to the breeding of industrial microorganism, especially a kind of high yield nucleic acid Saccharomyces cerevisiae engineered yeast.

Background technology

Nucleic acid yeast industry is China's new industry.Ribonucleic acid (abbreviation nucleic acid, RNA) be widely used in food industry, On medical industry and agricultural production.In terms of food industry, nucleic acid is the exploitation essential raw material of novel foodstuff flavouring.Wind The condiments such as the second generation monosodium glutamate (strength monosodium glutamate) in the row world, third generation monosodium glutamate (flavor monosodium glutamate), special delicious sauce, are all core Acid and its result of derivative application.On medical industry, nucleic acid is the diseases such as manufacture treatment coronary heart disease, tumour, myocardial infarction The raw material of drug.Agriculturally, the growth that nucleic acid and its derivative are widely used as crops (such as rice, melon and fruit, beans) promotees Into substance.

Saccharomyces cerevisiae intracellular RNA includes mainly：MRNA, rRNA, transfer RNA and non-coding RNA.Wherein ribose Body RNA is a kind of RNA that wherein content is most, accounts for 82% or so of RNA total amounts, and wherein relative molecular mass maximum one Class RNA.So it is a kind of effective breeding high-nucleic acid saccharomyces cerevisiae to increase the yield of intracellular rRNA or generating rate Method.

The considerations of recently as food-safe property, more and more researchs concentrate on breeding high-nucleic acid saccharomyces cerevisiae Work aspect rather than Candida etc. are not belonging to the bacterial strain of GRAS (Generally recognized as safe).

Yang Yi is suitable, fourth Yue, Cao Jing, and fermentation by saccharomyces cerevisiae is waited to prepare research [J] Pharmaceutical Biotechnologies of RNA, and 2013 (4):310-313. utilizes potassium chloride sensibility, by DES mutagenesises, screens an Accharomyces cerevisiae, rna content improves 25%.

Article Chuwattanakul V, Kim Y H, Sugiyama M, et al.Construction of a Saccharomyces cerevisiae strain with a high level of RNA.[J].Journal of Bioscience&Bioengineering,2011,112(1):1, by knocking out RRN10 genes in saccharomyces cerevisiae, then passes through EMS mutagenesis screenings obtain back mutation strain, finally by the integrant expression RRN10 genes in mutant strain, obtain high yield nucleic acid Yeast strain makes nucleic acid content improve 30%.

Article Peterson M R, Emr S D.The class C Vps complex functions at multiple stages of the vacuolar transport pathway.[J].Traffic,2001,2(7):476- 486, once disclosed the function of vacuole protein sorting receptor PEP1 (also referred to as VPS10 or VPT1), it is indicated that it is in protein point Very crucial effect is played during secreting.It relates generally to soluble CPY albumen from the organelle before vacuole (such as in core Body) to the transport between vacuole.Article Marcusson, Eric G, Horazdovsky, et al.The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene [J].Cell,1994,77(4):It is pointed out in 579：The method of subcellular fractionation is fixed by PEP1 (also referred to as VPS10 or VPT1) gene Positioned at the golgiosome later stage, for the protein sorting from endosome to golgiosome.PEP1 albumen is in saccharomyces cerevisiae Cytoplasmic tail structural domain executes more wheel sortings by the iterative cycles from golgiosome to endosome：Point of protein fragments CPY It selects signal PEP1 albumen to interact with p2PCY, forms receptor --- ligand complexes, which is packaged into vesicle, PEP1 protein delivery p2PCY, then receptor PEP1 return to golgiosome continue a new round sorting, CPY precursors continuation transported It is defeated to arrive vacuole, until its activity is formed.When PEP1 is lacked, although CPY protein transports the mistake to golgiosome from endoplasmic reticulum Journey is continuous, but due to lacking cytoplasmic tail structure receptor, CPY precursor substances are secreted into endoplasmic reticulum, and It cannot be transported to vacuole, and then its activity cannot be activated, to influence the physiological activity of cell.

The art is directed to the research of vacuole protein sorting receptor and focuses on the functional right of the albumen substantially at present In the influence of cellular physiological activity.

Invention content

The problem of nucleic acid yeast bacterial strain strain performance is low present invention aim to address producing, nucleic acid low output, provides one plant The saccharomyces cerevisiae of high yield nucleic acid.

In order to solve the above technical problems, technical solution of the present invention is as follows：

One plant height produces nucleic acid saccharomyces cerevisiae engineered yeast, is to pass through genetic engineering means using S. cervisiae as starting strain Vacuole protein sorting acceptor gene PEP1 (also referred to as VPS10 or VPT1) is overexpressed to obtain, the PEP1 gene orders such as SEQ Shown in ID NO.1.

The Gene ID of the PEP1 genes are：852264.

Further, the high yield nucleic acid saccharomyces cerevisiae engineered yeast be by build the sorting of strong promoter and vacuole protein by The junction fragment of body gene PEP1 and riddled basins, by the method for homologous recombination, by junction fragment with non-turn of rDNA Record interval region 1 (NTS1) is integrated into as insertion point in the gene of host strain saccharomyces cerevisiae, to obtain recombination engineering.

Further, the rDNA is saccharomyces cerevisiae 25s rDNA (Gene ID:9164935)、5.8s rDNA(Gene ID:9164934)、18s rDNA(Gene ID:9164923) sequence, rDNA nucleic acid sequences are shown in nucleic acid sequence table SEQ ID NO.2。

Preferably, the starting strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) W303a.

Preferably, the strong promoter is strong promoter PGK1.

Preferably, the riddled basins are Kan genetic fragments.

Preferably, the host strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) W303a.

Another object of the present invention is to provide the construction method of above-mentioned high yield nucleic acid saccharomyces cerevisiae engineered yeast, specific steps It is as follows：

(1) strong promoter PGK1 is connected on vector plasmid Yep352, obtains recombinant plasmid Yep352-PGK1；

(2) using the total DNA genome of saccharomyces cerevisiae W303a as template, PCR amplification obtains PEP1 genetic fragments, by PEP1 Genetic fragment is connected on recombinant plasmid Yep352-PGK1, obtains recombinant plasmid Yep352-PGK1-PEP1；

(3) it is template through PCR amplification using recombinant plasmid Yep352-PGK1-PEP1, obtains strong promoter PGK1 and PEP1 and connect Tab segments；

(4) insertion point rDNA nontranscribed spacers are obtained as template PCR using the Wine brewing yeast strain W303a genomes that set out The upstream homology arm and downstream homology arm in domain 1；

(5) segment and selection markers Kan segments that are obtained in step (3) (4) are transformed by lithium acetate transformation method It sets out in Wine brewing yeast strain, middle and upper reaches homology arm, Kan segments, PGK1 and PEP1 junction fragments, downstream homology arm sequence connect It connects, and screens and obtain the engineering bacteria of multicopy expression PEP1 genes.

Into one, in the step (1), the upstream homology arm is rDNA-A segments, and the downstream homology arm is rDNA- B segments.

Preferably, the preparation method of the Kan segments with selection markers is：It is obtained by template PCR of plasmid PUC6 Riddled basins Kan segments.

It is a further object of the present invention to provide above-mentioned high yield nucleic acid saccharomyces cerevisiae engineered yeast is applied in production nucleic acid Purposes.

Preferably, the fermentation process of the high yield nucleic acid saccharomyces cerevisiae engineered yeast is as follows：

Seed liquor culture：By bacterium mud from inclined-plane one ring of picking, saccharomyces cerevisiae engineered yeast is inoculated into YPD culture medium test tubes In, 28-30 DEG C, cultivate 10-12h under the conditions of 180-190rpm, obtain seed liquor；

Fermented and cultured：Seed liquor is inoculated according to inoculum concentration 3-5% (percent by volume) in YPD fluid nutrient mediums, 28- 30 DEG C, fermented and cultured 4h under the conditions of 180-190rpm.

Above, the integration site is as follows with reference to article：

Sun H,Zang X,Liu Y,et al.Expression of a chimeric human/salmon calcitonin gene i ntegrated into the Saccharomyces cerevisiae,genome using rDNA sequences as reco mbination sites[J].Applied Microbiology&Biotechnology, 2015,99(23):10097-106.

Advantageous effect：

1 and up to the present, in existing open source literature be also not directed to albumen sorting receptor PEP1 and nucleic acid high yield pass The problems such as system, the two whether there is relevance.And high yield nucleic acid saccharomyces cerevisiae engineered yeast provided by the invention can keep good Under the premise of good fermenting property, multicopy is overexpressed vacuole protein and sorts acceptor gene PEP1, surprisingly obtains and significantly improves core The technique effect of acid yield.The low problem of production nucleic acid yeast bacterial strain strain performance is not only overcome, growth performance is significantly improved With fermentative activity, while the yield of nucleic acid is significantly improved.

2, the saccharomyces cerevisiae nucleic acid production quantity that selection and breeding of the present invention obtain significantly improves.After being verified by shake flask fermentation, mistake Expression PEP1 bacterial strain nucleic acid contents have reached 14.70%, and starting strain W303a nucleic acid contents are 9.32%, are improved 57.72%.

Description of the drawings

Fig. 1 is target fragment and construction of recombinant plasmid verification electrophoretogram；Wherein, it is marker to scheme M in (a), and 1 is PGK Promoter electrophoretic band；It is marker to scheme M in (b), and 1 verifies band for the PCR of plasmid Yep352-PGK；Scheme in (c), M is Marker, the 1 PEP1 genes obtained for PCR amplification；(d) figure, M marker, 1 is that segment PEP1 is connected to Yep352- PCR verification electrophoretograms (recombinant plasmid Yep352-PGK1-PEP1) on PGK1；

The electrophoretogram of Fig. 2 target fragments, wherein M swimming lanes are 10000bp DNA marker, and 1 swimming lane is rDNA-B segment electricity Swimming band, 1084bp；2 swimming lanes are Kan fragment electrophoretic bands, 1613bp；3 swimming lanes are promoter and PEP1 junction fragment electrophoresis strips Band, 6512bp；4 swimming lanes are rDNA-A fragment electrophoretic bands, 1462bp；

Fig. 3 is target fragment and Yeast genome homologous recombination schematic diagram, wherein (1) show the Wine brewing yeast strain that sets out Haploid genome；

Fig. 4 is structure successful saccharomyces cerevisiae engineered yeast verification electrophoretogram, and wherein M swimming lanes are 5000bp DNA maker, 1 Swimming lane is the PCR product for verifying primer M1-U/M1-D, and 2 swimming lanes are the PCR product for verifying primer M2-U/M2-D；

Fig. 5 is the growth curve for being overexpressed PEP1 bacterial strains and starting strain.

Specific implementation mode

The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this Under the premise of invention spirit and scope, various changes or change to material component and dosage progress in these embodiments It belongs to the scope of protection of the present invention.

Saccharomyces cerevisiae used in the present invention is the saccharomyces cerevisiae engineered yeast that any source may be used.

(strong promoter PGK1 as described below includes that PGK1 starts to 1 Yep352-PGK-PEP1 plasmid constructions of embodiment Sub (PGK1p) and PGK1 terminators (PGK1t) segment)

Using the plasmid containing PGK1 as template, (such as with application No. is the plasmids of 201410277435.0 patent disclosures PUC-PGK1 is template) use primer PGK1-U (sequence shown in SEQ ID NO.17) and PGK1-D (shown in SEQ ID NO.18 Sequence) PCR amplification is carried out, PGK1 promoters and terminator junction fragment (1771bp) are obtained, restriction enzyme Kpn I are used Digested plasmid Yep352, using recombinase, and using the 15-20bp homology arms of primer both ends design, connection obtains Yep352- PGK1 recombinant plasmids.Saccharomyces cerevisiae W303a genomes are template, use primer P-U (sequence shown in SEQ ID NO.19) and P-D (sequence shown in SEQ ID NO.20) PCR amplification obtains PEP1 (sequence shown in SEQ ID NO.1) segment, 4740bp.Use limit Property restriction endonuclease Xho I digestion Yep352-PGK1 recombinant plasmids processed using recombinase, and utilize the 15-20bp of primer both ends design Homology arm, connection obtains Yep352-PGK1-PEP1 recombinant plasmids, and (wherein, the restriction enzyme site of Xho I enzymes is in PGK1p and PGK1t Between).

Utilize verification primer PGKY-U (sequence shown in SEQ ID NO.21) and PGKY-D (sequences shown in SEQ ID NO.22 Row) strong promoter PGK1 is verified；Utilize verification primer PY-U (sequence shown in SEQ ID NO.23) and PY-D (SEQ Sequence shown in ID NO.24) PEP1 segments are verified.

Such as attached drawing 1, target fragment and construction of recombinant plasmid verify electrophoretogram, and wherein M swimming lanes are 5000bp DNA maker.1 swimming lane is strong promoter PGK1 electrophoretic bands in figure (a), 1771bp, and size is correct；It is plasmid to scheme (b) the 1st swimming lane The PCR of Yep352-PGK1 verifies band, and 2823bp, stripe size is correct, and strong promoter PGK1 connections are correct；Scheme in (c), 1 swimming Road is the PEP1 genes that PCR amplification obtains, and length 4740bp is known, stripe size is correct by figure, and band is single；(d) figure is Segment PEP1 is connected to the PCR on Yep352-PGK1 and verifies electrophoretogram, stripe size 1309bp, as seen from the figure, PEP1 connect It connects correct.

Whole process the primer is shown in Table 1.

PCR primer during 1 carrier construction of table

The structure of 2 high yield nucleic acid saccharomyces cerevisiae engineered yeast of embodiment

(strong promoter PGK1 as described below includes PGK1 promoters (PGK1p) and PGK1 terminators (PGK1t) piece Section)

The main building process of bacterial strain is following (such as attached drawing 2,3)：

1) acquisition of target fragment

First using saccharomyces cerevisiae W303a genomes as template, use primer rDNA-AU (sequence shown in SEQ ID NO.5) With rDNA-AD (sequence shown in SEQ ID NO.6), PCR amplification obtains upstream homology arm rDNA-A segments (SEQ ID NO.3 institutes Show sequence), 1462bp；Using saccharomyces cerevisiae BY23 genomes as template, primer rDNA-BU (sequences shown in SEQ ID NO.11 are used Row) and rDNA-BD (sequence shown in SEQ ID NO.12), PCR amplification obtain downstream homology arm rDNA-B segments (SEQ ID Sequence shown in NO.4), 1084bp；Using plasmid PUC6 as template, using primer Kan-U (sequence shown in SEQ ID NO.7) and Kan-D (sequence shown in SEQ ID NO.8), PCR obtains Kan segments, 1613bp；With the recombinant plasmid prepared by embodiment 1 YEP352-PGK1-PEP1 is template, uses primer PEP1-U (sequence shown in SEQ ID NO.9) and PEP1-D (SEQ ID Sequence shown in NO.10), PCR obtains strong promoter PGK1 and PEP1 junction fragment, 6512bp.

Whole process the primer is shown in Table 2.

PCR primer in 2 engineering bacteria building process of table

Such as attached drawing 2, the electrophoretogram of target fragment, wherein M swimming lanes are 10000bp DNA marker, and 1 swimming lane is rDNA-B Fragment electrophoretic band, 1084bp, size are correct；2 swimming lanes are Kan fragment electrophoretic bands, 1613bp, and size is correct；3 swimming lanes are to open Mover and PEP1 junction fragment electrophoretic bands, 6512bp, size are correct；4 swimming lanes are rDNA-A fragment electrophoretic bands, 1462bp, Size is correct；

2) multicopy is overexpressed the structure of PEP1 saccharomyces cerevisiae engineered yeasts

4 segments obtained in step (1) are transformed into saccharomyces cerevisiae engineered yeast W303a with lithium acetate transformation method In, middle and upper reaches homology arm rDNA-A, Kan segment, PGK1p-PEP1-PGK1t junction fragments, downstream homology arm rDNA-B sequences Connection obtains multicopy and is overexpressed PEP1 saccharomyces cerevisiae engineered yeasts (such as attached drawing 3).

Multicopy is overexpressed the verification (such as Fig. 4) of PEP1 saccharomyces cerevisiae engineered yeasts：

Two groups of primers are separately designed, as：M1-U (sequence shown in SEQ ID NO.13), M1-D (SEQ ID NO.14 institutes Show sequence), M2-U (sequence shown in SEQ ID NO.15), M2-D (sequence shown in SEQ ID NO.16), to grow preferably single times Plasmid is template in body transformant, carries out PCR amplification, verifies recon.Obtained PCR product is carried out to 0.8% fine jade respectively Sepharose electrophoresis.A 3930bp bands are respectively obtained, a 3000bp bands illustrate that successfully saccharomyces cerevisiae list is arrived in recombination to segment In times body genome, and recombinable site is also correct.

If attached drawing 4 is that the successful saccharomyces cerevisiae engineered yeast of structure verifies electrophoretogram, wherein M swimming lanes are 5000bp DNA Maker, 1 swimming lane are the PCR product for verifying primer M1-U/M1-D, are the specific bands of a size 3930bp, size with It is expected that consistent；2 swimming lanes are the PCR product for verifying primer M2-U/M2-D, through 0.8% agarose gel electrophoresis, it can be seen that one The specific band of size 3000bp, size with it is expected consistent, illustrate that successfully saccharomyces cerevisiae monoploid base is arrived in recombination to segment Because in group, and recombinable site is also correct；

Embodiment 3 is overexpressed the experiment of PEP1 bacterial strain shake flask fermentations

1. being overexpressed the comparison of PEP1 bacterial strains and starting strain growth performance

Experimental group is to be overexpressed the saccharomyces cerevisiae W303a of PEP1, and control group is saccharomyces cerevisiae W303a original strains.

1) growth curve measures：

Seed liquor culture：Using oese the examination of YPD culture mediums is inoculated by bacterium mud from one ring of picking on a little inclined-planes Guan Zhong, 30 DEG C, cultivate 12h under the conditions of 180rpm.

Fermented and cultured：Seed liquor is inoculated in 100ml Yepd fluid nutrient mediums, per every other hour or two hours (according to Growing state determines) sampling 2ml.Bacterium solution is taken out into 1ml, certain multiple is diluted to it to OD₆₀₀Value in 0.4-0.6 ranges, Its absorbance is measured at wavelength 600nm.According to its absorbance value and sample time corresponding relationship, make growth curve.

As a result as shown in Fig. 5, compared with starting strain W303a, it is overexpressed the growth performance of PEP1 bacterial strains and is not affected by Any influence, and in identical initial OD values, the fermentation for being overexpressed bacterial strain terminates time (arrival OD₆₀₀=1 time) it is 4 Hour, and the fermentation of starting strain terminates time (arrival OD₆₀₀=1 time) it is 6 hours, bacterial strain is overexpressed compared with starting strain Thus about 2 hours ahead of time illustrate that the fermentative activity of the engineering bacteria of the present invention significantly improves.

2. extraction and the measurement of intracellular total serum IgE：

(the following method for measuring nucleic acid comes from document

Chuwattanakul V,Kim Y H,Sugiyama M,et al.Construction of a Saccharomyces cer evisiae strain with a high level of RNA.[J].Journal of Bioscience&Bioengineering,2 011,112(1):1.)

1) dry weight curve：Using oese the examination of YPD culture mediums is inoculated by bacterium mud from one ring of picking on a little inclined-planes Guan Zhong, 30 DEG C, cultivate 12h under the conditions of 180rpm.Primary seed solution is inoculated in 100ml Yepd fluid nutrient mediums, every 2 Absorbance value and dry weight at its 600nm is measured by sampling within a hour, with dry weight (y) to OD₆₀₀(x) make dry weight curve y=0.382x+ 0.832。

2) strain fermentation culture

Primary seed solution：Using oese, bacterium mud is inoculated into YPD cultures from one ring of picking on a little inclined-planes respectively In base test tube, 30 DEG C, cultivate 12h under the conditions of 180rpm；

Primary seed solution is inoculated in 50ml YEPD fluid nutrient mediums, makes its initial OD₆₀₀It is identical, 30 DEG C, 180rpm items Shaking flask culture is to OD under part₂₆₀=1.0 (middle exponential growths) are divided into three pipes, measure the OD of often pipe₆₀₀, centrifuge, wash, receive Collect thalline；Thalline is resuspended with 0.5mol/L perchloric acid, 70 DEG C of water-bath 20min, and supernatant is collected by centrifugation, and dilutes 50 times, is measured dilute Release liquid 260nm OD values, and as follows calculate intracellular total RNA content (%).

(OD₂₆₀*2500)/(12.224*OD₆₀₀+26.624)

3 are the results are shown in Table, list 3 shows：In shake flask fermentation experiment, control group：The nucleic acid content of original strain W303a reaches To 9.32% (mass percent) of dry cell weight, and experimental group：The nucleic acid content for the overexpression bacterial strain that the present invention obtains reaches The 14.70% of dry cell weight, the parent strain that compares improve 57.72%.This illustrates that the bacterial strain that the present invention obtains can be one The amount for determining to improve yeast strain intracellular nucleic acid in degree provides theoretical foundation for the selection and breeding of high yield nucleic acid yeast bacterial strain.

The nucleic acid content of 3 bacterial strain of table

Note：Shown data are respectively the average value of every group of three parallel test results

Although having been presented for some embodiments of the present invention herein, it will be appreciated by those of skill in the art that Without departing from the spirit of the invention, the embodiment of the present invention can be changed.Above-described embodiment is exemplary, It should not be using the embodiments herein as the restriction of interest field of the present invention.

Sequence table

<110>University Of Science and Technology Of Tianjin

<120>One plant height produces nucleic acid saccharomyces cerevisiae engineered yeast and its construction method and application

<130>On March 2nd, 2018

<141> 2018-05-30

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<170> SIPOSequenceListing 1.0

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agctttgatg attccaacac tttaattaga aaacaagaca cctccgttac tataagcttt 180

gacgatggtg aaacatggga aaaagttgaa ggcattgaag gcgaaatcac ttggatatac 240

atcgacccct tcaatagaca tgacagagcc gttgcaacgg caatgaatgg gtcgtacctt 300

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agtatttcac ctcgtgaatg ctatatagaa acccatccct tgaataagaa ctattttctt 420

gcaaagtgca actattgtga gaaaacagaa gtaaacaatg acaatgaaga gaattcgggg 480

gacgaagagg gacaatttga aatatttaat attacacgtt gcacagacaa ggtttttgca 540

agtaatgatg gtggaaaatc cttttctgag atcaagtctt ctctagaaag gaacgaaaat 600

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agtagtgata cctcaataat ctgtcttttt caaaatatgc agcttattat ggatgagttt 720

agttctcctt acaccgaaag taaattggtc ctaactacgg actggggcaa atcactaaaa 780

gaatttgacc aatttaaaga taaggtcgtc aatggttaca ggatattgaa atctcacatg 840

gttgtcttaa cccagggcga cagatataat gatatgtctt ccatggatgt gtgggtatca 900

aatgatctgt caaactttaa aatggcttac atgcctactc agttaaggca ttctatgcaa 960

ggagaaatat atgaggacgc tatgggaaga attatcttgc ccatgagtag ggaaagaagt 1020

gatcaagagg aggataaggg catcgtgtct gaaattttaa tttccgactc acaagggtta 1080

aaattttccc ccatcccatg gaccgcaaat gaggtgtttg gttatattaa tttttatcaa 1140

cctacttact tgaaaggaac gatgattgcc tcactttacc ctctatctag gcgtcgtaac 1200

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actccaaccg ccggaattct aatgacaaca ggtattgttg gcgatggtag tgtcttcgac 1440

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acttggaccg aatatcagct agaaactact atctacccaa atgaagtaat gaatacaacg 1680

cccgacggat ctggagctaa atttattcta aatgggttta ctttggcgca tatggatggt 1740

acaacgaatt tcatctatgc aattgatttt tcaacagcct ttaatgataa gacatgcgaa 1800

gaaaatgatt tcgaggattg gaatttagct gaggggaagt gtgtcaatgg agtcaagtac 1860

aagatcagaa gaagaaaaca ggacgctcag tgcttggtga agaaagtttt tgaagactta 1920

caattatttg agactgcttg tgacaagtgt accgaggctg attacgaatg cgcgtttgaa 1980

tttgttaggg acgcgaccgg gaaatgcgta ccagactaca acctaatcgt tctctctgac 2040

gtatgtgata agacaaagaa aaaaactgtg cctgtaaaac cattgcaact agttaaaggt 2100

gataaatgta aaaaaccaat gacagtcaaa tcagtggata tttcgtgtga gggagttcca 2160

aagaagggaa cgaatgataa agaaatagtg gttacagaaa acaaatttga tttcaagatt 2220

caattctatc aatactttga cacagtcacc gacgaatccc tcctcatgat caattcaaga 2280

ggagaagctt atatatctca tgatggtgga caaacaataa aaaggttcga cagtaatggt 2340

gaaacaatta ttgaagttgt gtttaatcca tactacaatt cttcagctta tctgtttggt 2400

tccaaaggta gcattttctc tacccatgat aggggatact cttttatgac tgctaaattg 2460

cccgaggcta ggcagttagg tatgccatta gactttaacg ctaaggcaca ggatacattt 2520

atctattatg gtggtaagaa ttgtgagtca atcttaagtc cggaatgtca tgcggtagca 2580

tacctgacca atgatggggg cgaaacgttt acggaaatgc ttgataatgc aattcattgt 2640

gagtttgcgg gctcactttt caaatatccg tcaaatgagg atatggttat gtgtcaagtg 2700

aaggaaaagt cttcgcagac aagaagctta gtttcttcta ctgatttttt ccaggatgat 2760

aaaaataccg tctttgaaaa tattatcggc tacttatcca ctggtggcta tatcatcgtt 2820

gctgttcctc atgagaacaa cgaattgaga gcatacgtaa ctatcgatgg tactgagttt 2880

gccgaggcaa aattcccata tgatgaagat gttgggaagc aagaggcatt cactatatta 2940

gagtctgaga aaggatcgat attcttacat ttagcaacaa acttagtacc aggacgcgat 3000

tttggcaatc ttttgaaatc caactcaaat ggtacttctt ttgtcacgtt ggagcatgcc 3060

gttaatagaa acacattcgg ctatgttgac tttgaaaaaa ttcaaggtct cgaaggcatt 3120

attctcacca acatcgtttc aaatagtgac aaggtcgccg agaataaaga agacaaacaa 3180

ttgaagacga agatcacctt taatgaaggt tcagattgga actttttgaa acctccgaag 3240

agggattcag aaggaaaaaa gttttcttgc agctccaaat cactggatga gtgttcattg 3300

cacttacatg gctatactga acgtaaggat attagagata catattcttc cggttctgca 3360

ttaggaatga tgttcggcgt tggcaacgtt ggtcctaacc ttttaccata taaagaatgt 3420

tccaccttct tcaccaccga tggtggcgaa acgtgggctg aagttaagaa gactcctcac 3480

caatgggaat acggtgacca cggtgggatt ttagttttag ttcctgaaaa ctcagaaact 3540

gattctattt cctattctac cgattttggt aaaacatgga aagattataa attctgcgct 3600

gataaggttt tagtaaagga tataaccact gttcccaggg attctgcttt gagatttttg 3660

ctgtttggag aggcagcaga tattggaggc agctcattta gaacgtacac aattgatttt 3720

agaaacatct tcgaaagaca atgtgatttc gacatcactg gtaaggaaag cgcagattat 3780

aaatactctc ctctgggttc caaaagcaat tgcctatttg gtcaccaaac cgagttttta 3840

cgtaaaaccg atgaaaattg ttttattggg aatattccac tttctgaatt ttcaagaaat 3900

atcaaaaact gttcttgtac aagacaagat ttcgagtgtg attacaactt ttacaaagct 3960

aacgatggta cttgtaaatt agtcaaagga ctaagcccag caaatgctgc agacgtttgt 4020

aaaaaagagc cagatttaat cgaatatttt gaatcgtcag gctacagaaa gatccctcta 4080

tcaacctgtg agggtggcct gaaattggat gctccctcat caccacatgc ttgcccagga 4140

aaagaaaaag aattcaagga aaagtactca gtaagtgccg gtccctttgc atttattttc 4200

atttcaattc ttttaataat tttctttgcc gcatggtttg tatatgacag aggtatcaga 4260

agaaatgggg gatttgcaag gtttggagaa attaggctag gtgacgatgg tttaatagaa 4320

aacaataata ctgacagagt tgtcaataac attgtgaaat caggatttta cgttttctca 4380

aatatcgggt ctcttttaca gcacacaaaa actaatatag cgcatgctat ctccaaaatt 4440

agaggaaggt ttggaaacag aacaggtcca agctactcat ccctgatcca tgatcaattt 4500

ttggatgaag cagatgacct gcttgctggc cacgatgaag acgccaatga cttatccagt 4560

ttcatggatc agggtagtaa ttttgaaatc gaagaagatg atgttccaac acttgaagaa 4620

gagcatacat catatacaga tcaacctacg accaccgatg ttccagatac attaccagaa 4680

ggaaatgagg aaaacatcga caggcctgat tctacagcgc catctaacga aaaccagtag 4740

<210> 2

<211> 2250

<212> DNA

<213> Saccharomyces cerevisiae rDNA

<400> 2

ggaacctcta atcattggct ttacctcata aaactgatac gagcttctgc tatcctgagg 60

gaaacttcgg caggaaccag ctactagatg gttcgattag tctttcgccc ctatacccaa 120

attcgacgat cgatttgcac gtcagaaccg ctacgagcct ccaccagagt ttcctctggc 180

ttcaccctat tcaggcatag ttcaccatct ttcgggtccc aacagctatg ctcttactca 240

aatccatccg aagacatcag gatcggtcga ttgtgcacct cttgcgaggc cccaacctac 300

gttcactttc attacgcgta tgggttttac acccaaacac tcgcatagac gttagactcc 360

ttggtccgtg tttcaagacg ggcggcatat aaccattatg ccagcatcct tgacttacgt 420

cgcagtcctc agtcccagct ggcagtattc ccacaggcta taatacttac cgaggcaagc 480

tacattccta tggatttatc ctgccaccaa aactgatgct ggcccagtga aatgcgagat 540

tcccctaccc acaaggagca gagggcacaa aacaccatgt ctgatcaaat gcccttccct 600

ttcaacaatt tcacgtactt tttcactctc ttttcaaagt tcttttcatc tttccatcac 660

tgtacttgtt cgctatcggt ctctcgccaa tatttagctt tagatggaat ttaccaccca 720

cttagagctg cattcccaaa caactcgact cttcgaaggc actttacaaa gaaccgcact 780

cctcgccaca cgggattctc accctctatg acgtcctgtt ccaaggaaca tagacaagga 840

acggccccaa agttgccctc tccaaattac aactcgggca ccgaaggtac cagatttcaa 900

atttgagctt ttgccgcttc actcgccgtt actaaggcaa tcccggttgg tttcttttcc 960

tccgcttatt gatatgctta agttcagcgg gtactcctac ctgatttgag gtcaaacttt 1020

aagaacattg ttcgcctaga cgctctcttc ttatcgataa cgttccaata cgctcagtat 1080

aaaaaagatt agccgcagtt ggtaaaacct aaaacgaccg tacttgcatt atacctcaag 1140

cacgcagaga aacctctctt tggaaaaaaa aaacatccaa tgaaaaggcc agcaatttca 1200

agttaactcc aaagagtatc actcactacc aaacagaatg tttgagaagg aaatgacgct 1260

caaacaggca tgccccctgg aataccaagg ggcgcaatgt gcgttcaaag attcgatgat 1320

tcacggaatt ctgcaattca cattacgtat cgcatttcgc tgcgttcttc atcgatgcga 1380

gaaccaagag atccgttgtt gaaagttttt aatattttaa aattcccagt tacgaaaatt 1440

cttgtttttg acaaaaattt aatgaataaa taaaattgtt tgtgtttgtt acctctgggc 1500

cccgattgct cgaatgccca aagaaaaagt tgcaaagata tgaaaactcc acagtgtgtt 1560

gtattgaaac ggttttaatt gtcctataac aaaagcacag aaatctctca ccgtttggaa 1620

tagcaagaaa gaaacttaca agcctagcaa gaccgcgcac ttaagcgcag gcccggctgg 1680

actctccatc tcttgtcttc ttgcccagta aaagctctca tgctcttgcc aaaacaaaaa 1740

aaatccattt tcaaaattat taaatttctt taatgatcct tccgcaggtt cacctacgga 1800

aaccttgtta cgacttttag ttcctctaaa tgaccaagtt tgtccaaatt ctccgctctg 1860

agatggagtt gcccccttct ctaagcagat cctgaggcct cactaagcca ttcaatcggt 1920

actagcgacg ggcggtgtgt acaaagggca gggacgtaat caacgcaagc tgatgacttg 1980

cgcttactag gaattcctcg ttgaagagca ataattacaa tgctctatcc ccagcacgac 2040

ggagtttcac aagattacca agacctctcg gccaaggtta gactcgctgg ctccgtcagt 2100

gtagcgcgcg tgcggcccag aacgtctaag ggcatcacag acctgttatt gcctcaaact 2160

tccatcggct tgaaaccgat agtccctcta agaagtggat aaccagcaaa tgctagcacc 2220

actatttagt aggttaaggt ctcgttcgtt 2250

<210> 3

<211> 1462

<212> DNA

<213>Saccharomyces cerevisiae upstream homology arm rDNA-A ()

<400> 3

gttaactata ggaaatgagc ttttctcaat tctctaaact tatacaagca ctcatgtttg 60

ccgctctgat ggtgcggaaa aaactgctcc atgaagcaaa ctgtccgggc aaatcctttc 120

acgctcggga agctttgtga aagcccttct ctttcaaccc atctttgcaa cgaaaaaaaa 180

aaaaaaaata aaaaataaaa agaccaaata gtaaatagta acttacatac attagtaaat 240

ggtacactct tacacactat catcctcatc gtatattata atagatatat acaatacatg 300

tttttacccg gatcatagaa ttcttaagac aaataaaatt tatagagact tgttcagtct 360

acttctctct aaactaggcc ccggctcctg ccagtaccca cttagaaaga aataaaaaac 420

aaatcagaca acaaaggctt aatctcagca gatcgtaaca acaaggctac tctactgctt 480

acaatacccc gttgtacatc taagtcgtat acaaatgatt tatccccacg caaaatgaca 540

ttgcaattcg ccagcaagca cccaaggcct ttccgccaag tgcaccgttg ctagcctgct 600

atggttcagc gacgccacaa ggacgcctta ttcgtatcca tctatattgt gtggagcaaa 660

gaaatcaccg cgttctagca tggattctga cttagaggcg ttcagccata atccagcgga 720

tggtagcttc gcggcaatgc ctgatcagac agccgcaaaa accaattatc cgaatgaact 780

gttcctctcg tactaagttc aattactatt gcggtaacat tcatcagtag ggtaaaacta 840

acctgtctca cgacggtcta aacccagctc acgttcccta ttagtgggtg aacaatccaa 900

cgcttaccga attctgcttc ggtatgatag gaagagccga catcgaagaa tcaaaaagca 960

atgtcgctat gaacgcttga ctgccacaag ccagttatcc ctgtggtaac ttttctggca 1020

cctctagcct caaattccga gggactaaag gatcgatagg ccacactttc atggtttgta 1080

ttcacactga aaatcaaaat caagggggct tttacccttt tgttctactg gagatttctg 1140

ttctccatga gcccccctta ggacatctgc gttatcgttt aacagatgtg ccgccccagc 1200

caaactcccc acctgacaat gtcttcaacc cggatcagcc ccgaatggga ccttgaatgc 1260

tagaacgtgg aaaatgaatt ccagctccgc ttcattgaat aagtaaagaa actataaagg 1320

tagtggtatt tcactggcgc cgaagctccc acttattcta caccctctat gtctcttcac 1380

aatgtcaaac tagagtcaag ctcaacaggg tcttctttcc ccgctgattc tgccaagccc 1440

gttcccttgg ctgtggtttc gc 1462

<210> 4

<211> 1084

<212> DNA

<213>Saccharomyces cerevisiae downstream homology arm rDNA-B ()

<400> 4

acctaccgac caactttcat gttctgtttc gacctacctc ttgtaaatga caaatcacct 60

ttttcatcgt atgcacctta ttctccacat cacaatgcac tattgctttt gctttttcac 120

ctgtcatatc ctattgctat tagatgaaat ataataaaaa ttgtcctcca cccataacac 180

ctctcactcc cacctactga acatgtctgg accctgccct catatcacct gcgtttccgt 240

taaactatcg gttgcggcca tatctaccag aaagcaccgt ttcccgtccg atcaactgta 300

gttaagctgg taagagcctg accgagtagt gtagtgggtg accatacgcg aaactcaggt 360

gctgcaatct ttatttcttt tttttttttt tttttttttt ttttttctag tttcttggct 420

tcctatgcta aatcccataa ctaacctacc attcgattca gaaaaattcg cactatccag 480

ctgcactctt cttctgaaga gttaagcact ccattatgct cattgggttg ctactacttg 540

atatgtacaa acaatattct cctccgatat tcctacaaaa aaaaaaaaaa aaacactccg 600

gttttgttct cttccctcca tttccctctc ttctacggtt aatactttcc tcttcgtctt 660

tttctacacc ctcgtttagt tgcttcttat tccttcccgc tttcctgcac taacattttg 720

ccgcattaca ctatatgatc gtagtacatc ttacaactcc gcataccgcg tcgccgcgtc 780

gccgcgtcgc caaaaattta cttcgccaac cattccatat ctgttaagta tacatgtata 840

tattgcactg gctattcatc ttgcactttt cctctttctt cttcccagta gcctcatcct 900

tttacgctgc ctctctggaa cttgccatca tcattcccta gaaactgcca tttacttaaa 960

aaaaaaaaaa aaaaaaaaat gtccccactg ttcactgttc actgttcact tgtctcttac 1020

atctttcttg gtaaaatcgt agttcgtagt attttttttc atatcaaagg catgtcctgt 1080

taac 1084

<210> 5

<211> 26

<212> DNA

<213>Artificial sequence ()

<400> 5

gttaactata ggaaatgagc ttttct 26

<210> 6

<211> 44

<212> DNA

<213>Artificial sequence ()

<400> 6

ctgcagcgta cgaagcttca gctggcgaaa ccacagccaa ggga 44

<210> 7

<211> 44

<212> DNA

<213>Artificial sequence ()

<400> 7

tcccttggct gtggtttcgc cagctgaagc ttcgtacgct gcag 44

<210> 8

<211> 45

<212> DNA

<213>Artificial sequence ()

<400> 8

gttttggata gatcagttag agcataggcc actagtggat ctgat 45

<210> 9

<211> 45

<212> DNA

<213>Artificial sequence ()

<400> 9

atcagatcca ctagtggcct atgctctaac tgatctatcc aaaac 45

<210> 10

<211> 45

<212> DNA

<213>Artificial sequence ()

<400> 10

catgaaagtt ggtcggtagg ttaacgaacg cagaattttc gagtt 45

<210> 11

<211> 46

<212> DNA

<213>Artificial sequence ()

<400> 11

aactcgaaaa ttctgcgttc gttaacctac cgaccaactt tcatgt 46

<210> 12

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 12

accccaccac actcctac 18

<210> 13

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 13

accccaccac actcctac 18

<210> 14

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 14

acgctcgtca tcaaaatc 18

<210> 15

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 15

tcacctgcgt ttccgtta 18

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 16

ccctgggagg agttatct 18

<210> 17

<211> 44

<212> DNA

<213>Artificial sequence ()

<400> 17

acgaattcga gctcggtacc tctaactgat ctatccaaaa ctga 44

<210> 18

<211> 39

<212> DNA

<213>Artificial sequence ()

<400> 18

tcgacggatc cccgggtacc taacgaacgc agaattttc 39

<210> 19

<211> 40

<212> DNA

<213>Artificial sequence ()

<400> 19

ggaattccag atctcctcga gatgatatta cttcattttg 40

<210> 20

<211> 39

<212> DNA

<213>Artificial sequence ()

<400> 20

atctatcgca gatccctcga gctactggtt ttcgttaga 39

<210> 21

<211> 19

<212> DNA

<213>Artificial sequence ()

<400> 21

tctaactgat ctatccaaa 19

<210> 22

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 22

aggaacgtgc tgctactc 18

<210> 23

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 23

tttgtgctct tatgggac 18

<210> 24

<211> 18

<212> DNA

<213>Artificial sequence ()

<400> 24

attcttcggc attagtta 18

Claims

It is using S. cervisiae as starting strain 1. a plant height produces nucleic acid saccharomyces cerevisiae engineered yeast, it is characterised in that：Pass through gene Engineering means are overexpressed vacuole protein sorting acceptor gene PEP1 and obtain, and the PEP1 gene orders are as shown in SEQ ID NO.1.
2. a plant height as described in claim 1 produces nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that the high yield nucleic acid wine brewing Yeast engineering bacteria is the connection by building strong promoter and vacuole protein sorting acceptor gene PEP1 and riddled basins Junction fragment is integrated into host by segment by the method for homologous recombination using rDNA nontranscribed spacers region 1 as insertion point In the gene of bacterium saccharomyces cerevisiae, to obtain recombination engineering.
3. a plant height as claimed in claim 1 or 2 produces nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that the starting strain For saccharomyces cerevisiae W303a.
4. a plant height as claimed in claim 2 produces nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that the strong promoter is strong Promoter PGK1.
5. a plant height as claimed in claim 2 produces nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that the riddled basins Segment is Kan genetic fragments.
6. a plant height as claimed in claim 2 produces nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that the host strain is wine brewing Yeast W303a.
7. a kind of construction method of high yield nucleic acid saccharomyces cerevisiae engineered yeast, which is characterized in that specific as follows：

(1) strong promoter PGK1 is connected on vector plasmid Yep352, obtains recombinant plasmid Yep352-PGK1；

(2) using the total DNA genome of saccharomyces cerevisiae W303a as template, PCR amplification obtains PEP1 genetic fragments, by PEP1 genes Segment is connected on recombinant plasmid Yep352-PGK1, obtains recombinant plasmid Yep352-PGK1-PEP1；

(3) it is template through PCR amplification using recombinant plasmid Yep352-PGK1-PEP1, obtains strong promoter PGK1 and PEP1 connection sheet Section；

(4) insertion point rDNA nontranscribed spacers region 1 is obtained as template PCR using the Wine brewing yeast strain W303a genomes that set out Upstream homology arm and downstream homology arm；

(5) segment and selection markers Kan segments that are obtained in step (3) (4) are transformed by lithium acetate transformation method and are set out In Wine brewing yeast strain, middle and upper reaches homology arm, Kan segments, PGK1 and PEP1 junction fragments, downstream homology arm are linked in sequence, And it screens and obtains the engineering bacteria of multicopy expression PEP1 genes.
8. any high yield nucleic acid saccharomyces cerevisiae engineered yeasts of claim 1-6 are applied to the purposes in production nucleic acid.
9. the high yield nucleic acid saccharomyces cerevisiae engineered yeast as claimed in claim 8 is applied to the purposes in production nucleic acid, feature exists In：

The fermentation process of the high yield nucleic acid saccharomyces cerevisiae engineered yeast is as follows：Seed liquor culture：By bacterium mud from picking on a little inclined-planes S. cervisiae is inoculated into YPD culture medium test tubes by one ring, 28-30 DEG C, cultivate 10-12h under the conditions of 180-190rpm；

Fermented and cultured：Seed liquor is inoculated according to inoculum concentration 3-5% in YPD fluid nutrient mediums, fermented and cultured 4h.