CN105087406B - A kind of restructuring yeast strains and its construction method and application - Google Patents

Abstract

The present invention relates to gene engineering technology field, a kind of restructuring yeast strains and its construction method and application are disclosed.The restructuring yeast strains knock out gal1, gal7, gal10 or gal80 gene and ypl062w gene, and include through 2-4 genetic fragment on yeast homologous recombination and integration to genome.The present invention constructs gene knockout yeast strain, the host cell of optimization is provided for production lycopene, choose functional gene crtE, crtB and crtI of the synthesis lycopene of separate sources, and specific yeast entogenous gene etc., it is integrated on gene knockout yeast strain genome through modularized design, obtains the recombinant bacterial strain of one plant of completely new high yield lycopene.

Description

A kind of restructuring yeast strains and its construction method and application

Technical field

The present invention relates to gene engineering technology field, more particularly to a kind of restructuring yeast strains and its building side Method and application.

Background technique

Worldwide, with economic level and to the raising of health demand, safety, trophism and the function of food Property receive more and more attention, therefore functional nutrient chemicals has become development trend, represents the new of contemporary food development Trend has a vast market foreground.Lycopene is a kind of fat-soluble natural food colour, and class is belonged in chemical structure recklessly Radish element, has extremely strong oxidation resistance, is the hot spot that functional food composition is studied in the world in recent years.

The preparation of lycopene relies primarily on plant extract, chemical synthesis and Microbe synthesis, and first two method respectively has it The deficiency of itself, Microbe synthesis is then with low cost, high yield and Product Safety, it is considered to be most promising method.Mesh Before, in the research of Microbe synthesis lycopene, used host strain focuses primarily upon Escherichia coli and saccharomyces cerevisiae. 2011, Yeong-Su Kim et al. introduced three genes of synthesis lycopene in pantoea agglomerans source in Escherichia coli CrtE, crtB and crtI, while raising the key gene in endogenous MEP approach and building the heterologous path MVA intracellular, it is final logical Cross the yield of lycopene that 1.35g/L (32mg/gDCW) is realized in fed batch fermentation optimization.2014, Tianjin industrial bio skill Art research institute Ma Yan and seminar utilize ribosome bind site by optimizing the supply of Escherichia coli NADPH and ATP intracellular Library screening obtains the recombination bacillus coli that a plant height produces lycopene, and the yield on fed batch fermentation tank is 3.52g/ L (50.6mg/g DCW) belongs to maximum output in the recombinant bacterial strain of open report at present.

Saccharomyces cerevisiae compares Escherichia coli as generally acknowledged safe mode microorganism, its thallus vitamin, protein contains Amount is high, can eat, medicinal and fodder yeast；Compared to trispore Bruce mould, its growth cycle is shorter and is easier to cultivate.Cause This, realizes that high yield of the lycopene in saccharomyces cerevisiae will show great competitiveness in carotenoid industrialization.2014 Being expressed in saccharomyces cerevisiae by ADH2 promoter for year Bahieldin et al. report bites summer spore Ou Wenshi through codon optimization The yield of lycopene of crtE, crtB and the crtI in bacterium source only 3.3mg/gDCW.2015, Yu Hongwei seminar, Zhejiang University The bifunctional enzyme crtYB in phaffiafhodozyma source is transformed by albumen directed evolution means, it is made to lose lycopene cyclase Encoding function, and only retain its encode phytoene synthetase function；Meanwhile to the crtE in phaffiafhodozyma source It is oriented evolution, improves the catalytic performance of enzyme；Again, by adjusting the copy number of crtE, crtB and crtI, a plant height is obtained The diploid recombinant Saccharomyces cerevisiae of lycopene is produced, shaking flask yield reaches 159.56mg/L (23.23mg/gDCW), finally leads to Fed batch fermentation optimization is crossed, the yield of lycopene reaches 1.61g/L (24.41mg/gDCW), belongs to the benefit of open report at present With the maximum output of recombinant Saccharomyces cerevisiae synthesis lycopene.However, this is synthesized with what is reported before this using recombination bacillus coli The maximum output (50.6mg/g DCW) of lycopene, which is compared, still biggish gap.Therefore, exploitation is host with saccharomyces cerevisiae Bacterial strain realizes that the high yield of lycopene still remains very big potentiality and space.

Summary of the invention

In view of this, enabling the recombinant bacterial strain to answer the purpose of the present invention is to provide a kind of restructuring yeast strains For in the biosynthesis of lycopene, and high yield is kept, while providing construction method and the application of the recombinant bacterial strain.

For achieving the above object, the invention provides the following technical scheme:

A kind of restructuring yeast strains, the Yeast genome knock out gal1, gal7, gal10 and ypl062w gene or Gal80 and ypl062w gene, and include through the following genetic fragment on yeast autologous recombination and integration to its genome:

Yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa The genetic fragment 1 that crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence are sequentially spliced (ideograph is shown in Fig. 1)；

Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA Reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, shine ancient green-ball bacterium source or trispore Bruce mould source Or the base that crtE, GPM1 terminator, the site the yeast leu2 downstream homologous sequence in Taxus x media source are sequentially spliced Because of segment 2 (ideograph is shown in Fig. 2).

The present invention passes through the structure that Genetic elements, netic module are carried out using specific yeast entogenous gene and foreign gene It builds, and is transferred to the yeast genes for knocking out gal1, gal7, gal10 and ypl062w gene or knocking out gal80 and ypl062w gene In group, the synthesis output increased of recombinant bacterial strain lycopene is realized.

Wherein, related yeast entogenous gene includes truncated 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG- CoA) reductase gene tHMGR1 can be template by Yeast genome, be obtained by PCR amplification；

Meanwhile the invention further relates to terminated using amino acid tag, promoter and the terminator in yeast, including CYC1 Son, GAL10 promoter, GAL1 promoter, PGK1 terminator, ACT1 terminator, GPM1 terminator, LEU2 label, TDH2 are terminated The acquisition of son, FBA1 terminator, ENO2 terminator, HIS3 label, said gene element can also lead to using Yeast genome as template Cross PCR amplification acquisition；

In the present invention, above-mentioned each Genetic elements and relevant upstream and downstream homologous sequence are with Wine brewing yeast strain BY4741 Genome be template, design and synthesize suitable primer, obtained by PCR amplification；And the upstream LEU2 homologous sequence and LEU2 Label is to get off together from PCR amplification on plasmid pRS405, and the upstream HIS3 homologous sequence and HIS3 label are together from plasmid The upper PCR amplification of pRS313 is got off.

Foreign gene according to the present invention includes geranyl pyrophosphate (GGPP) synthase gene crtE, phytoene Synthase gene crtB and Phytoene dehydrogenase gene crtI.Wherein the source of crtE includes pantoea agglomerans (Pantoea Agglomerans), trispore Bruce mould (Blakeslea trispora), Taxus x media (Taxus x media), Sulfolobus acidocaldarius (Sulfolobus acidocaldarius) and the ancient green-ball bacterium (Archaeoglobus that shines Fulgidus), successively it is abbreviated as Pa crtE, Bt crtE, Tm crtE, Sa crtE, Af crtE；The source of crtB includes into The general bacterium (Pantoea agglomerans) of group and aquatic secondary coccus (Agrobacterium aurantiacum), are successively abbreviated as Pa crtB,Aa crtB；The source of crtI includes pantoea agglomerans (Pantoea agglomerans), aquatic secondary coccus (Agrobacterium aurantiacum) and trispore Bruce mould (Blakeslea trispora), is successively abbreviated as Pa crtI,Aa crtI,Bt crtI.Said gene is after codon optimizes and suitably evades common restriction enzyme site It is obtained by artificial synthesized.

Preferably, the bacterial strain further includes through the following gene piece on yeast autologous recombination and integration to its genome Section:

TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 are terminated in genetic fragment 2 Son, Bt crtI, GAL3 promoter, ACT1 terminator and the gene that homologous sequence is sequentially spliced downstream in genetic fragment 2 Segment 3 (ideograph is shown in Fig. 3).It is further preferred that the genetic fragment 3 is as shown in SEQ ID NO:5.

It is highly preferred that the bacterial strain further includes through the following gene piece on yeast autologous recombination and integration to its genome Section:

Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA Reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 terminator, yeast The genetic fragment 4 that the site his3 downstream homologous sequence is sequentially spliced (ideograph is shown in Fig. 4).It is further preferred that the base Because segment 4 is as shown in SEQ ID NO:6.

Most preferably, the restructuring yeast strains are sub- red comprising graceful ground using saccharomyces cerevisiae CEN.PK2-1D as starting strain The crtE in beans China fir source knocks out gal1, gal7, gal10 and ypl062w gene, and deposit number is CGMCC No.10754, at this SyBE_Sc0014D019 is denoted as in invention.Side by side, the restructuring yeast strains are bacterium germination with saccharomyces cerevisiae CEN.PK2-1C Strain, the crtE comprising Taxus x media source knock out gal80 and ypl062w gene.

Preferably, the genetic fragment 1 is as shown in SEQ ID NO:1, genetic fragment 2 such as SEQ ID NO:2-4 Shown in meaning one；

Wherein, the genetic fragment 2 comprising the ancient green-ball bacterium source crtE gene that shines is as shown in SEQ ID NO:2；Include three The genetic fragment 2 of spore cloth Laplace mould source crtE gene is as shown in SEQ ID NO:3；Include Taxus x media source crtE The genetic fragment 2 of gene is as shown in SEQ ID NO:4.

Preferably, the yeast is that saccharomyces cerevisiae, solution rouge category yeast or Crewe dimension belong to yeast.It in addition to this, can also be with It is transformation bacterial strain by institute of the present invention with algae, mould (such as streptomycete etc.) and bacterium (such as Escherichia coli, bacillus subtilis etc.) The Genetic elements and module of restriction are recombinated according to the herxheimer-liked reaction path of these bacterial strains being resolved.

It is highly preferred that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY series saccharomyces cerevisiae.Further preferably Ground, the CEN.PK series saccharomyces cerevisiae are saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D.

Restructuring yeast strains of the present invention can largely synthesize lycopene, therefore the present invention also provides described heavy Group yeast strain is in the application in production lycopene and the application in product of the production using lycopene as intermediate product.

In addition, the present invention also provides the construction methods of the restructuring yeast strains, comprising:

Step 1, building are by under yeast gal7 downstream of gene homologous sequence, DR-Kl URA3-DR nutritional labeling, gal1 gene It swims the sequentially connected knockout box segment 1 of homologous sequence or is sought by yeast gal80 upstream region of gene homologous sequence, DR-Kl URA3-DR Label, the sequentially connected knockout box segment 2 of gal80 downstream of gene homologous sequence are supported, and by the homologous sequence of ypl062w upstream region of gene Column, kanMX resistance label, the sequentially connected knockout box segment 3 of ypl062w downstream of gene homologous sequence utilize knockout box segment 1 Yeast gal1, gal7, gal10 and ypl062w are knocked out by the recombination of yeast autologous with 3 or using box segment 2 and 3 is knocked out Gene knocks out yeast gal80 and ypl062w gene, obtains gene knockout yeast, spare；

By yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence sequentially splice, and obtain genetic fragment 1, the i.e. site trp1 Upstream homologous sequence-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1The site-trp1 downstream homologous sequence, it is spare；

By yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG- CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, luminous ancient green-ball bacterium source or trispore Bruce mould come CrtE, GPM1 terminator in source or Taxus x media source, the site yeast leu2 downstream homologous sequence sequentially splice, and obtain base Because of segment 2, i.e. leu2 site upstream homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1-leu2 Site downstream homologous sequence, it is spare；

Genetic fragment 1 is transferred in the gene knockout yeast by step 2 by Li-acetate method, by genetic fragment 1 The site trp1 occurs recombination and is integrated into genome on the site trp1 upstream and downstream homologous sequence and gene knockout Yeast genome On；

Genetic fragment 2 is transferred in the gene knockout yeast by Li-acetate method, passes through the site leu2 in genetic fragment 2 The site leu2 occurs recombination and is integrated on genome on upstream and downstream homologous sequence and gene knockout Yeast genome, is weighed Group yeast strain.

Preferably, the construction method further include:

Genetic fragment 3 is constructed, and after transgene segment 1 and 2,3 transgene of genetic fragment is knocked out in yeast and is obtained Obtain restructuring yeast strains；

Specifically, by TDH2 terminator in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 terminator, Bt crtI, GAL3 promoter, ACT1 terminator and homologous sequence sequentially splices downstream in genetic fragment 2, Obtain genetic fragment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3- T_ACT1- ACT1 terminator and downstream homologous sequence, it is spare；

Genetic fragment 3 is continued through Li-acetate method to be transformed into the gene knockout yeast of transgene segment 2, is passed through TDH2 terminator and its upstream homologous sequence in genetic fragment 3, ACT1 terminator and homologous sequence downstream, with the base integrated Because in segment 2 TDH2 terminator and ACT1 terminator site recombination occurs due to be inserted into the clpp gene of integrator gene segment 2 Except on Yeast genome.

It is further preferred that the construction method further include:

Genetic fragment 4 is constructed, and after transgene segment 1,2 and 3,4 transgene of genetic fragment is knocked out in yeast Obtain restructuring yeast strains；

Specifically, yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is attached, merged Gene BTS1-ERG20, then by yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, Truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 Terminator, the site yeast his3 downstream homologous sequence sequentially splice, and obtain genetic fragment 4, i.e. his3 site upstream homologous sequence- HIS3-T_ENO2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1The site-his3 downstream homologous sequence；

Genetic fragment 4 is transferred in the gene knockout yeast by Li-acetate method, passes through the site his3 in genetic fragment 4 The site his3 occurs recombination and is integrated on genome on upstream and downstream homologous sequence and gene knockout Yeast genome.

Preferably, the specific construction method of gene knockout yeast are as follows:

With plasmid pWJ1042 (complete genome sequence is as shown in SEQ ID NO:7) for template, design upstream and downstream primer (is directed to DR-Kl URA3-DR nutritional labeling is related to upstream and downstream primer in plasmid pWJ1042, and respectively adds at the end of upstream and downstream primer 5 ' 40bp homologous sequence) PCR amplification by yeast gal7 downstream of gene 40bp homologous sequence, DR-Kl URA3-DR nutritional labeling, The sequentially connected knockout box segment 1 of gal1 downstream of gene 40bp homologous sequence or design upstream and downstream primer PCR amplification are by yeast Gal80 upstream region of gene 40bp homologous sequence, DR-Kl URA3-DR nutritional labeling, gal80 downstream of gene 40bp homologous sequence are sequentially The knockout box segment 2 of connection, is transferred in yeast by Li-acetate method, and using knocking out, gal7 downstream of gene 40bp in box segment 1 is same Source sequence, gal1 downstream of gene 40bp homologous sequence utilize the homologous sequence of gal80 upstream region of gene 40bp in knockout box segment 2 Column, gal80 downstream of gene 40bp homologous sequence, with tri- genes of gal7, gal10, gal1 sequentially connected on Yeast genome Recombination occurs or is recombinated with gal80 gene, DR-Kl URA3-DR nutritional labeling is substituted gal1, gal7, gal10 tri- Gene replaces on gal80 gene integration to genome, and the knockout for completing gal1, gal7, gal10 gene or gal80 gene (is shown Intention is shown in Fig. 5 and Fig. 6), correct bacterial strain, correct bacterial strain YPD Liquid Culture are then filtered out by SD-URA solid medium It takes a little bacterium solution to be coated on 5- fluororotic acid solid panel after base culture to be screened again to recycle Kl URA3 label, obtains transition base Because knocking out yeast；

Using the mono- genome for singly striking bacterial strain YPL062W struck in library of saccharomyces cerevisiae BY4742 as template, design upstream and downstream are drawn Object PCR amplification is same by ypl062w upstream region of gene 394bp homologous sequence, kanMX resistance label, ypl062w downstream of gene 317bp The sequentially connected knockout box segment 3 of source sequence, is transferred in transition gene knockout yeast by Li-acetate method, utilizes knockout box segment Weight occurs for the ypl062w gene in 3 on ypl062w gene upstream and downstream homologous sequence, with transition gene knockout Yeast genome Group substitutes kanMX resistance label on ypl062w gene integration to genome, completes the knockout (schematic diagram of ypl062w gene See Fig. 7), correct bacterial strain is then filtered out by the YPD solid panel of the resistance containing G418, obtains gene knockout yeast.

Preferably, the specific construction method of the genetic fragment 1 are as follows:

CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator are sequentially led to It crosses OE-PCR method to be stitched together, obtains the segment T that both ends include HindIII and XhoI restriction enzyme site_CYC1-crtI-P_GAL10- P_GAL1-crtB-T_PGK1；

Meanwhile it is the homologous 631bp sequence of yeast trp1 site upstream, the homologous 733bp sequence in the site yeast trp1 downstream is suitable Secondary to be stitched together by OE-PCR method, obtaining both ends includes SacI and ApaI restriction enzyme site, and upper and lower in the site yeast trp1 The segment between homologous sequence comprising HindIII and XhoI restriction enzyme site is swum, is then connected by SacI and ApaI restriction enzyme site Carrier pRS405 (for complete genome sequence as shown in SEQ ID NO:8, plasmid map is shown in Fig. 8), obtains TRP1 integrated plasmid pRS405- TRP, by segment T obtained above_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1With pRS405-TRP plasmid by HindIII and XhoI restriction enzyme site is attached, and is obtained 1 integrated plasmid of genetic fragment, is denoted as pRS405-TRP-T_CYC1-crtI-P_GAL10- P_GAL1-crtB-T_PGK1, SacI and ApaI double digestion acquisition genetic fragment 1, i.e. trp1 site upstream homologous sequence-T_CYC1-crtI- P_GAL10-P_GAL1-crtB-T_PGK1The site-trp1 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:1.

Preferably, the specific construction method of the genetic fragment 2 are as follows:

By ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, hair CrtE, GPM1 terminator in light Gu green-ball bacterium source or trispore Bruce mould source or Taxus x media source sequentially pass through OE-PCR method is stitched together, and obtains the segment T that both ends include BamHI and XhoI restriction enzyme site_ACT1-tHMGR1-P_GAL10- P_GAL1-crtE-T_GPM1；

Meanwhile by the homologous 561bp sequence of yeast leu2 site upstream, LEU2 label, TDH2 terminator, the site yeast leu2 The homologous 584bp sequence in downstream sequentially passes through OE-PCR method and is stitched together, and obtaining both ends includes SacI and ApaI restriction enzyme site, and Comprising the segment of BamHI and XhoI restriction enzyme site between TDH2 terminator, the site yeast leu2 downstream homologous sequence, then lead to It crosses SacI and ApaI restriction enzyme site and is connected into carrier pRS405, obtain LEU2 integrated plasmid pRS405-LEU, by obtained above Section T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1Connected with pRS405-LEU plasmid by BamHI and XhoI restriction enzyme site It connects, obtains 2 integrated plasmid of genetic fragment, be denoted as pRS405-LEU-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1, SacI and ApaI double digestion obtains genetic fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10- P_GAL1-crtE-T_GPM1The site-leu2 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:2-4.Wherein, comprising hair The genetic fragment 2 of light Gu green-ball bacterium source crtE gene is as shown in SEQ ID NO:2；Include trispore Bruce mould source crtE The genetic fragment 2 of gene is as shown in SEQ ID NO:3；Genetic fragment 2 such as SEQ comprising Taxus x media source crtE gene Shown in ID NO:4.

Preferably, the specific construction method of the genetic fragment 3 are as follows:

TDH2 terminator and its upstream 869bp homologous sequence in amplification gene segment 2, ACT1 terminator and downstream Then 355bp homologous sequence terminates TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 Son, Bt crtI, GAL3 promoter, ACT1 terminator and homologous sequence sequentially passes through OE-PCR method downstream in genetic fragment 2 It is stitched together, obtains the genetic fragment 3 that both ends include PmeI restriction enzyme site, i.e. TDH2 terminator and its upstream homologous sequence-DR- Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3- ACT1 terminator and downstream homologous sequence, by segment obtained above and flat end Carrier pJET1.2 (plasmid map is shown in Fig. 9) connection is held, 3 integrated plasmid of genetic fragment is obtained and is denoted as pleu-DR-Kl URA3-DR- T_CYC1-Bt crtI-P_GAL3, PmeI digestion acquisition genetic fragment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3- ACT1 terminator and downstream homologous sequence, nucleotide sequence such as SEQ ID NO:5 institute Show.

Preferably, the specific construction method of the genetic fragment 4 are as follows:

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is subjected to amalgamation and expression, by the side OE-PCR Method connects the N-terminal of the C-terminal of BTS1 and ERG20 to form fusion BTS1-ERG20 with GGGS linker；

Sequentially by ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter It is stitched together by OE-PCR method, obtains segment T_ACT1-tHMGR1-P_GAL10-P_GAL1；

By segment T_ACT1-tHMGR1-P_GAL10-P_GAL1, fusion BTS1-ERG20, FBA1 terminator pass through the side OE-PCR Method is stitched together, and obtains the segment T that both ends include BamHI and PstI restriction enzyme site_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1- ERG20)-T_FBA1；

PCR expands the homologous 312bp sequence of yeast his3 site upstream, HIS3 label, ENO2 terminator and yeast respectively The homologous 578bp sequence in the site his3 downstream, and sequentially spliced by OE-PCR method, obtaining both ends includes SacI and ApaI digestion Site, and include the piece of BamHI and PstI restriction enzyme site between ENO2 terminator, the site yeast his3 downstream homologous sequence Section, is then connected into carrier pRS405 by SacI and ApaI restriction enzyme site, obtains HIS3 integrated plasmid pRS405-HIS, will be above-mentioned Obtained segment T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1With pRS405-HIS plasmid by BamHI and PstI restriction enzyme site is attached, and is obtained 4 integrated plasmid of genetic fragment, is denoted as pRS405-HIS-T_ACT1-tHMGR1-P_GAL10- P_GAL1-(BTS1-ERG20)-T_FBA1, SacI and ApaI double digestion acquisition genetic fragment 4, i.e. his3 site upstream homologous sequence- HIS-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1The site-his3 downstream homologous sequence, nucleotide sequence is such as Shown in SEQ ID NO:6.

Preferably, the yeast is that saccharomyces cerevisiae, solution rouge category yeast or Crewe dimension belong to yeast in construction method.It removes It can also be transformation bacterium with algae, mould (such as streptomycete) and bacterium (such as Escherichia coli, bacillus subtilis) except this Strain by Genetic elements defined by the present invention and module according to the herxheimer-liked reaction path that these bacterial strains have been resolved come Recombination.It is highly preferred that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY series saccharomyces cerevisiae.It is further preferred that The CEN.PK series saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D.

In addition, the present invention also provides a kind of diploid restructuring yeast strains, by restructuring yeast strains CEN.PK2-1C and Recombinant Saccharomyces cerevisiae CEN.PK2-1D mating obtains；

The genome of the restructuring yeast strains CEN.PK2-1C and restructuring yeast strains CEN.PK2-1D knock out gal1, Gal7, gal10 and ypl062w gene, and include through the following gene piece on yeast autologous recombination and integration to its genome Section:

Yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa The genetic fragment 1 that crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence are sequentially spliced；

Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA Reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, shine ancient green-ball bacterium source or trispore Bruce mould source Or the base that crtE, GPM1 terminator, the site the yeast leu2 downstream homologous sequence in Taxus x media source are sequentially spliced Because of segment 2；

TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 are terminated in genetic fragment 2 Son, Bt crtI, GAL3 promoter, ACT1 terminator and the gene that homologous sequence is sequentially spliced downstream in genetic fragment 2 Segment 3；

Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA Reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 terminator, yeast The genetic fragment 4 that the site his3 downstream homologous sequence is sequentially spliced.

Mating in diploid restructuring yeast strains of the present invention is that this field routinely obtains diploid yeast bacterial strain Cultivate in the medium two haploid yeast bacterial strains are generally mixed and as mate and form diploid strains by method, and The genetic fragment 1-4 is referring to restriction above-mentioned and preferred embodiment.

Restructuring yeast strains of the present invention are for when producing lycopene, yield of lycopene to be between 30-45mg/ GDCW, far beyond the maximum output of the 24.41mg/gDCW of current restructuring yeast strains.Meanwhile diploid of the present invention The yield of lycopene of restructuring yeast strains is higher than 26mg/gDCW, also above existing highest level.

The related application of the bacterial strain according to the present invention, the present invention also provides a kind of methods for producing lycopene, will Restructuring yeast strains of the present invention are inoculated in fermentation medium after seed culture medium activates and cultivate, and thallus is collected after culture Cell extraction lycopene.

Wherein, the seed culture medium is preferably 40g/L glucose, 20g/L peptone, 10g/L yeast extract, remaining is Water.

The ferment culture medium be 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L D- galactolipin, Yu Weishui.

The culture is preferably cultivated under the conditions of 30 DEG C, 250rpm.

From the above technical scheme, the present invention constructs gene knockout yeast strain, provides optimization for production lycopene Host cell, choose functional gene crtE, crtB and crtI of the synthesis lycopene of separate sources, and in specific yeast Source gene etc. is integrated on gene knockout yeast strain genome through modularized design, obtains one plant of completely new high yield tomato red The recombinant bacterial strain of element.

Biological deposits explanation

SyBE_Sc0014D019, classification naming: saccharomyces cerevisiae, Saccharomyces cerevisiae is in April, 2015 It is deposited within 28th China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is Chaoyang District, Beijing City North Star west The institute 3 of road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.10754.

Detailed description of the invention

Fig. 1 show the Genetic elements ideograph of genetic fragment 1；Wherein, both ends TRP1-L, TRP1-R respectively indicates yeast The site trp1 upstream and downstream homologous sequence；

Fig. 2 show the Genetic elements ideograph of genetic fragment 2；Wherein, both ends LEU2-L, LEU2-R respectively indicates yeast The site leu2 upstream and downstream homologous sequence；

Fig. 3 show the Genetic elements ideograph of genetic fragment 3；Wherein, both ends T_TDH2-L、T_ACT1- R respectively indicates gene TDH2 terminator and its upstream homologous sequence and ACT1 terminator and homologous sequence downstream in segment 2；

Fig. 4 show the Genetic elements ideograph of genetic fragment 4；Wherein, both ends HIS3-L, HIS3-R respectively indicates yeast The site his3 upstream and downstream homologous sequence；

Fig. 5, which is shown, knocks out the schematic diagram that box segment 1 knocks out gal1, gal7, gal10 gene；

Fig. 6, which is shown, knocks out the schematic diagram that box segment 2 knocks out gal80 gene；

Fig. 7, which is shown, knocks out the schematic diagram that box segment 3 knocks out ypl062w gene；

Fig. 8 show plasmid pRS405 map；

Fig. 9 show plasmid pJET1.2 map；

Figure 10 show plasmid pRS313 map；

Figure 11 show the yield of lycopene column diagram for being transferred to the restructuring yeast strains of different foreign gene combinations；

Figure 12 is shown using the tomato of the CEN.PK2-1C and CEN.PK2-1D restructuring yeast strains constructed as starting strain Red pigment yield column diagram；

Figure 13 show the yield of lycopene column diagram of monoploid restructuring yeast strains and amphiploid restructuring yeast strains.

Specific embodiment

The invention discloses a kind of restructuring yeast strains and its construction method and application, those skilled in the art can be used for reference Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the invention and application have passed through Preferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to described herein Methods and applications be modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

Some plasmid vectors involved in the present invention, bacterial strain are commercially available, as pJET1.2 plasmid vector is bought From CloneJET PCR the Cloning Kit, #K1231 of Thermo Scientific company；Wine brewing yeast strain CEN.PK2- 1C and CEN.PK2-1D buys the EUROSCARF from Germany Scientific Research and Development GmbH, Strain number is 30000A and 30000B；Saccharomyces cerevisiae BY4742 is mono- to strike library bacterial strain YPL062W purchase from U.S. Thermo Fisher Scientific company；Plasmid pRS405, pRS313 (plasmid map is shown in Figure 10) and saccharomyces cerevisiae BY4741 be from The purchase of BioVector China plasmid vector strain cell gene collection-NTCC country's Type Tissue Collection.

For each Genetic elements employed in present invention building restructuring yeast strains, such as amino acid tag, label, endogenous Gene, foreign gene etc. are it is known in the art that its particular sequence as known to those skilled in the art.It is of the invention for convenience of understanding, The present invention is illustrated each Genetic elements in each genetic fragment:

Knockout box segment 1 (shown in SEQ ID NO:9): 1-40bp is gal7 downstream of gene 40bp homologous sequence；41- 1615bp is DR-Kl URA3-DR nutritional labeling sequence；1616-1655bp is gal1 downstream of gene 40bp homologous sequence.

Knockout box segment 2 (shown in SEQ ID NO:10): 1-40bp is gal80 upstream region of gene 40bp homologous sequence；41- 1615bp is DR-Kl URA3-DR nutritional labeling sequence；1616-1655bp is gal80 downstream of gene 40bp homologous sequence.

Knockout box segment 3 (shown in SEQ ID NO:11): 1-394bp is ypl062w upstream region of gene 394bp homologous sequence； 395-1864bp is kanMX resistance sequence label；1865-2181bp is ypl062w downstream of gene 317bp homologous sequence.

Genetic fragment 1 (shown in SEQ ID NO:1): 1-631bp is trp1 site upstream 631bp homologous sequence；632- 637bp is BamHI restriction enzyme site；638-640bp is meaningless sequence；641-646bp is HindIII restriction enzyme site；647- 901bp is CYC1 terminator sequence；902-2650bp is Bt crtI sequence；2651-3318bp is GAL10-GAL1 two-way startup Subsequence；3319-4248bp is Pa crtB sequence；4249-4523bp is PGK1 terminator sequence；4524-4529bp is XhoI Restriction enzyme site；4530-5262bp is the homologous 733bp sequence in the site trp1 downstream.

Genetic fragment 2 (shown in SEQ ID NO:2) comprising the ancient green-ball bacterium source crtE gene that shines: 1-561bp is The homologous 561bp sequence of leu2 site upstream；562-1656bp is LEU2 label；1657-2056bp is TDH2 terminator sequence； 2057-2062bp is BamHI restriction enzyme site；2063-2349bp is ACT1 terminator sequence；2350-3858bp is truncated HMG-CoA reductase gene tHMGR1；3859-4526bp is GAL10-GAL1 two-way startup subsequence；4527-5480bp is hair Light Gu green-ball bacterium source crtE gene；5481-5880bp is GPM1 terminator sequence；5881-5886bp is XhoI restriction enzyme site； 5887-6470bp is the homologous 584bp sequence in the site leu2 downstream.

Genetic fragment 2 (shown in SEQ ID NO:3) comprising trispore Bruce mould source crtE gene: 1-561bp is The homologous 561bp sequence of leu2 site upstream；562-1656bp is LEU2 label；1657-2056bp is TDH2 terminator sequence； 2057-2062bp is BamHI restriction enzyme site；2063-2349bp is ACT1 terminator sequence；2350-3858bp is truncated HMG-CoA reductase gene tHMGR1；3859-4526bp is GAL10-GAL1 two-way startup subsequence；4527-5489bp is three Spore cloth Laplace mould source crtE gene；5490-5889bp is GPM1 terminator sequence；5890-5895bp is XhoI digestion position Point；5896-6479bp is the homologous 584bp sequence in the site leu2 downstream.

Genetic fragment 2 (shown in SEQ ID NO:4) comprising Taxus x media source crtE gene: 1-561bp is The homologous 561bp sequence of leu2 site upstream；562-1656bp is LEU2 label；1657-2056bp is TDH2 terminator sequence； 2057-2062bp is BamHI restriction enzyme site；2063-2349bp is ACT1 terminator sequence；2350-3858bp is truncated HMG-CoA reductase gene tHMGR1；3859-4526bp is GAL10-GAL1 two-way startup subsequence；4527-5708bp is graceful Ground Asia Chinese yew source crtE gene；5709-6108bp is GPM1 terminator sequence；6109-6114bp is XhoI restriction enzyme site； 6115-6698bp is the homologous 584bp sequence in the site leu2 downstream.

Genetic fragment 3 (shown in SEQ ID NO:5): 1-869bp is TDH2 terminator and its upstream homologous sequence 869bp； 870-2444bp is DR-Kl URA3-DR nutritional labeling sequence；2445-2699bp is CYC1 terminator sequence；2700-4448bp For Bt crtI sequence；4449-5108bp is GAL3 promoter；5109-5463bp homologous sequence for ACT1 terminator and downstream 355bp。

Genetic fragment 4 (shown in SEQ ID NO:6): 1-312bp is his3 site upstream 312bp homologous sequence；313- 975bp is HIS3 label；976-1375bp is ENO2 terminator；1376-1381bp is BamHI restriction enzyme site；1382-1668bp For ACT1 terminator sequence；1669-3177bp is truncated HMG-CoA reductase gene tHMGR1；3178-3845bp is GAL10-GAL1 two-way startup subsequence；3846-5921bp is fusion BTS1-ERG20；5922-6121bp is FBA1 whole Only subsequence；6122-6127bp is PstI restriction enzyme site；6128-6705bp is the site his3 downstream 578bp homologous sequence.

URA3 is Kluyveromyces lactis source (Kluyveromyces in DR-Kl URA3-DR nutritional labeling sequence Lactis, Kl).

After the particular sequence for knowing above-mentioned each Genetic elements, those skilled in the art can be according to conventional primer design principle Carry out amplification and OE-PCR splicing.Meanwhile SD culture medium of the present invention is in a kind of commonly training of yeast screening assay field Support base, according to yeast have which gene defect and specially deleted on the composition of minimal medium a certain ingredient or it is several at Divide and filters out aimed strain to realize.

Below with reference to embodiment, the present invention is further explained.

Embodiment 1: the building of gene knock-out bacterial strain

Using saccharomyces cerevisiae CEN.PK2-1C as starting strain, four gene knock-out bacterial strain CEN.PK2-1C △ gal1, △ are constructed gal7,△gal10::DR,△ypl062w::kanMX.Detailed process is as follows:

△ gal1 is constructed first, and △ gal7, △ gal10::DR-Kl URA3-DR knocks out box, i.e. knockout box segment 1, with matter Grain pWJ1042 is template, designs upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arm and DR-Kl URA3-DR The knockout box segment of nutritional labeling, it is using the homologous recombination machinery of yeast itself that the segment is whole by Li-acetate method yeast conversion It closes on Yeast genome, using SD-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, list after conversion Lack the kilnitamin powder 2g/L of uracil, 2% agar powder) it is screened, obtained transformant after dividing pure culture by mentioning Yeast genome is taken to carry out PCR verifying, to taking a little bacterium solution to apply after verifying correct recombinant bacterial strain YPD fluid nutrient medium culture Cloth 5- fluororotic acid (5-FOA) solid panel (respectively there is the direct repeat DR of 143bp at DR-Kl URA3-DR nutritional labeling both ends, Yeast itself can occur homologous recombination using this two sections identical sequences and delete URA3 gene and one of DR；YPD training Supporting gene is no this screening pressure of amino acid nutrient defect, and the saccharomycete of such spontaneous deletion URA3 can be grown.So It is screened afterwards with 5-FOA, because under the enzyme effect that URA3 gene encodes 5-FOA can become pair the bacterial strain containing URA3 The virose substance of cell, grow yeast cells cannot on the culture medium containing 5-FOA, delete URA gene to filter out Bacterium), picking single colonie divides after pure culture to extract genome and carry out PCR verifying screening is deleted by recombination spontaneous between DR sequence The correct Strain Designation is SyBE_Sc0014C011 by the correct bacterial strain of URA3 gene, i.e. three gene knock-out bacterial strains.It knocks out The bacterial strain of gal1, gal7, gal10 will not be metabolized D- galactolipin, so as to maintain inducer galactose concentration intracellular constant, Realize efficiently induction.

Then, △ ypl062w::kanMX is constructed on the basis of three gene knock-out bacterial strain SyBE_Sc0014C011 to knock out Box, using the mono- genome for singly striking bacterial strain YPL062W struck in library of BY4742 as template, design upstream and downstream primer PCR expands tape base Because of the knockout box segment 3 of the upstream and downstream YPL062W homologous sequence and kanMX resistance label, which is integrated by yeast conversion Onto Yeast genome, bacterial strain (kanMX resistance is struck using the YPD solid panel screening four of the resistance of G418 containing 200mg/L after conversion Label can generate resistance to this antibiotic of Geneticin G418, to select successful knockout gene using G418 screen The bacterium of ypl062w), obtained transformant extracts genome progress PCR verifying by dividing after pure culture, to verifying correctly recombination Bacterial strain saves glycerol stock, and is named as SyBE_Sc0014C012.

In the manner described above using saccharomyces cerevisiae CEN.PK2-1D as starting strain, four gene knock-out bacterial strain CEN.PK2- are constructed 1D △ gal1, △ gal7, △ gal10::DR, △ ypl062w::kanMX, obtained transformant is by extracting base after dividing pure culture Because of a group progress PCR verifying, glycerol stock is saved to correct recombinant bacterial strain is verified, and be named as SyBE_Sc0014D004.

Embodiment 2: the building of gene knock-out bacterial strain

Using saccharomyces cerevisiae CEN.PK2-1C as starting strain, dual-gene knock-out bacterial strain CEN.PK2-1C △ gal80: is constructed: DR,△ypl062w::kanMX.Detailed process is as follows:

Building △ gal80::DR-Kl URA3-DR first knocks out box, i.e. knockout box segment 2, using plasmid pWJ1042 as mould Plate designs the knockout of upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arm and DR-Kl URA3-DR nutritional labeling The segment is integrated into Yeast genome by Li-acetate method yeast conversion using the homologous recombination machinery of yeast itself by box segment On, using SD-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, the mixing of single scarce uracil after conversion Powder of amino acids 2g/L, 2% agar powder) screened, obtained transformant by divide after pure culture extract Yeast genome into Row PCR verifying, to taking a little bacterium solution to be coated with 5- fluororotic acid after verifying correct recombinant bacterial strain YPD fluid nutrient medium culture (respectively there is the direct repeat DR of 143bp at DR-Kl URA3-DR nutritional labeling both ends to (5-FOA) solid panel, and yeast itself can benefit Homologous recombination occurs with this two sections identical sequences and deletes URA3 gene and one of DR；YPD culturing gene is not have The saccharomycete of amino acid nutrient defect this screening pressure, such spontaneous deletion URA3 can be grown.Then it is carried out with 5-FOA Screening, because the bacterial strain containing URA3 can make 5-FOA become virose to cell under the enzyme effect that URA3 gene encodes Substance grow yeast cells cannot on the culture medium containing 5-FOA, to filter out the bacterium for deleting URA gene), picking list Bacterium colony, which divides after pure culture to extract genome and carry out PCR verifying screening, is deleting URA3 gene just by recombination spontaneous between DR sequence The correct Strain Designation is SyBE_Sc0014D001 by true bacterial strain, i.e. single-gene knock-out bacterial strain.

Then, △ ypl062w::kanMX is constructed on the basis of single-gene knock-out bacterial strain SyBE_Sc0014D001 to knock out Box, using the mono- genome for singly striking bacterial strain YPL062W struck in library of BY4742 as template, design upstream and downstream primer PCR expands tape base Because of the knockout box segment 3 of the upstream and downstream YPL062W homologous sequence and kanMX resistance label, which is integrated by yeast conversion Onto Yeast genome, dual-gene knock-out bacterial strain is screened using the YPD solid panel of the resistance of G418 containing 200mg/L after conversion (kanMX resistance label can generate resistance to this antibiotic of Geneticin G418, to select successful knockout using G418 screen The bacterium of gene ypl062w), obtained transformant extracts genome after pure culture and carries out PCR verifying by dividing, correct to verifying Recombinant bacterial strain save glycerol stock, and be named as SyBE_Sc0014D002.

Embodiment 3: the building of genetic fragment 1

Expand CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator and suitable It is secondary to be stitched together by OE-PCR method, obtain the segment T that both ends include HindIII and XhoI restriction enzyme site_CYC1-crtI- P_GAL10-P_GAL1-crtB-T_PGK1；

Meanwhile expanding the homologous 631bp sequence of yeast trp1 site upstream, the homologous 733bp sequence in the site yeast trp1 downstream And sequentially pass through OE-PCR method and be stitched together, obtaining both ends includes SacI and ApaI restriction enzyme site, and in the site yeast trp1 Include the segment of HindIII and XhoI restriction enzyme site between the homologous sequence of upstream and downstream, then passes through SacI and ApaI restriction enzyme site It is connected into carrier pRS405 (for complete genome sequence as shown in SEQ ID NO:8, plasmid map is shown in Fig. 8), obtains TRP1 integrated plasmid PRS405-TRP, by segment T obtained above_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1Pass through with pRS405-TRP plasmid HindIII and XhoI restriction enzyme site is attached, and is obtained 1 integrated plasmid of genetic fragment, is denoted as pRS405-TRP-T_CYC1-crtI- P_GAL10-P_GAL1-crtB-T_PGK1；

Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.

It after verifying is correct, is cut respectively with SacI and ApaI double enzyme site, obtains genetic fragment 1, nucleotide sequence is such as Shown in SEQ ID NO:1.

Embodiment 4: the building of genetic fragment 2

By ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, hair CrtE, GPM1 terminator in light Gu green-ball bacterium source or trispore Bruce mould source or Taxus x media source sequentially pass through OE-PCR method is stitched together, and obtains the segment T that both ends include BamHI and XhoI restriction enzyme site_ACT1-tHMGR1-P_GAL10- P_GAL1-crtE-T_GPM1；Meanwhile the homologous 561bp sequence of yeast leu2 site upstream, LEU2 being marked, TDH2 terminator, yeast The homologous 584bp sequence in the site leu2 downstream sequentially passes through OE-PCR method and is stitched together, and obtaining both ends includes SacI and ApaI enzyme Enzyme site, and include the piece of BamHI and XhoI restriction enzyme site between TDH2 terminator, the site yeast leu2 downstream homologous sequence Section, is connected into carrier pRS405 by SacI and ApaI restriction enzyme site, obtains LEU2 integrated plasmid pRS405-LEU.It is upper by what is obtained State segment T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1With pRS405-LEU plasmid by BamHI and XhoI restriction enzyme site into Row connection, obtains 2 integrated plasmid of genetic fragment, is denoted as pRS405-LEU-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1。

Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.

It after verifying is correct, is cut respectively with SacI and ApaI double enzyme site, obtains genetic fragment 2, leu2 site upstream Homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1The site-leu2 downstream homologous sequence, nucleotide Sequence is as shown in SEQ ID NO:2-4.Wherein, genetic fragment 2 such as SEQ ID comprising the ancient green-ball bacterium source crtE gene that shines Shown in NO:2；Genetic fragment 2 comprising trispore Bruce mould source crtE gene is as shown in SEQ ID NO:3；Comprising gracefully The genetic fragment 2 of sub- Chinese yew source crtE gene is as shown in SEQ ID NO:4.

Embodiment 5: the building of genetic fragment 3

TDH2 terminator and its upstream 869bp homologous sequence in amplification gene segment 2, ACT1 terminator and downstream Then 355bp homologous sequence terminates TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 Son, Bt crtI, GAL3 promoter, ACT1 terminator and homologous sequence sequentially passes through OE-PCR method downstream in genetic fragment 2 It is stitched together, obtains the genetic fragment 3 that both ends include PmeI restriction enzyme site, i.e. TDH2 terminator and its upstream homologous sequence-DR- Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3- ACT1 terminator and downstream homologous sequence connect with flat ends vector pJET1.2 It connects to obtain 3 integrated plasmid of genetic fragment, is denoted as pleu-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3。

Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.

It after verifying is correct, is cut with PmeI restriction enzyme site, obtains genetic fragment 3, nucleotide sequence such as SEQ ID NO:5 institute Show.

Embodiment 6: the building of genetic fragment 4

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is subjected to amalgamation and expression, by the side OE-PCR Method connects the N-terminal of the C-terminal of BTS1 and ERG20 to form fusion BTS1- with GGGS linker (GGTGGTGGTTCT) ERG20；From expanding T on LEU2 integrated plasmid pRS405-LEU in embodiment 4_ACT1-tHMGR1-P_GAL10-P_GAL1Segment；

By segment T_ACT1-tHMGR1-P_GAL10-P_GAL1, fusion BTS1-ERG20, FBA1 terminator pass through the side OE-PCR Method is stitched together, and obtains the segment T that both ends include BamHI and PstI restriction enzyme site_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1- ERG20)-T_FBA1；Meanwhile PCR expands the homologous 312bp sequence of yeast his3 site upstream, HIS3 label, ENO2 terminator respectively And the homologous 578bp sequence in the site yeast his3 downstream, and sequentially spliced by OE-PCR method, obtain both ends include SacI and ApaI restriction enzyme site, and include BamHI and PstI digestion position between ENO2 terminator, the site yeast his3 downstream homologous sequence The segment of point, is connected into carrier pRS405 by SacI and ApaI restriction enzyme site, obtains HIS3 integrated plasmid pRS405-HIS.Will The above-mentioned segment T arrived_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1With pRS405-HIS plasmid by BamHI and PstI restriction enzyme site is attached, and is obtained 4 integrated plasmid of genetic fragment, is denoted as pRS405-HIS-T_ACT1-tHMGR1-P_GAL10- P_GAL1-(BTS1-ERG20)-T_FBA1；

Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.

It after verifying is correct, is cut with SacI and ApaI double digestion, obtains genetic fragment 4, the i.e. homologous sequence of his3 site upstream Column-HIS3-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1The site-his3 downstream homologous sequence, nucleotides sequence Column are as shown in SEQ ID NO:6.

Embodiment 7: genetic fragment 1-2 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1C and recombinant Saccharomyces cerevisiae CEN.PK2-1D

Segment is converted four gene knockout yeast strain SyBE_Sc0014C012 using Li-acetate method by genetic fragment 1, is passed through The site trp1 occurs recombination and is integrated on genome on the upstream and downstream TRP1 homologous sequence and Yeast genome.It is used after conversion SD-TRP solid panel (synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L, the kilnitamin powder 2g/ of single scarce tryptophan L, 2% agar powder) it is screened, obtained transformant carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture, Glycerol stock is saved to the correct recombinant bacterial strain of verifying and is respectively designated as SyBE_Sc0014C015.

Then, use Li-acetate method by segment transformed yeast bacterial strain the genetic fragment 2 of the crtE containing 3 kinds of separate sources SyBE_Sc0014C015 occurs recombination with the site leu2 on Yeast genome by the upstream and downstream LEU2 homologous sequence and is integrated into On genome.SD-TRP-LEU solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, double scarce colors are used after conversion The kilnitamin powder 2g/L of propylhomoserin and leucine, 2% agar powder) it is screened, it is pure that obtained transformant carries out scribing line point Yeast genome is extracted after culture and carries out PCR verifying, and glycerol stock is saved to the correct recombinant bacterial strain of verifying and is respectively designated as SyBE_Sc0014C029,SyBE_Sc0014C032,SyBE_Sc0014C035.Wherein:

SyBE_Sc0014C029:SyBE_Sc0014C015,LEU2::T_ACT1-tHMGR-P_GAL10-P_GAL1-A f crtE- T_GPM1；

SyBE_Sc0014C032:SyBE_Sc0014C015,LEU2::T_ACT1-tHMGR-P_GAL10-P_GAL1-B t crtE- T_GPM1；

SyBE_Sc0014C035:SyBE_Sc0014C015,LEU2::T_ACT1-tHMGR-P_GAL10-P_GAL1-T m crtE- T_GPM1。

In a manner mentioned above, the genetic fragment 2 of genetic fragment 1 and the crtE containing Taxus x media source is transferred to Four gene knockout yeast strain SyBE_Sc0014D004, obtained transformant carry out extracting yeast genes after scribing line divides pure culture Group carries out PCR verifying, is named as SyBE_Sc0014D006 to correct recombinant bacterial strain preservation glycerol stock is verified.

Embodiment 8: genetic fragment 1-3 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1C and recombinant Saccharomyces cerevisiae CEN.PK2-1D

By genetic fragment 3 using Li-acetate method by the segment convert respectively restructuring yeast strains SyBE_Sc0014C035 and By TDH2 terminator upstream homologous sequence and ACT1 terminator downstream homologous sequence recombination occurs for SyBE_Sc0014D006 The centre for the genetic fragment 2 integrated before being inserted on genome.Using SD-URA-TRP-LEU solid panel (synthesis ferment after conversion Female nitrogen source YNB 6.7g/L, glucose 20g/L, lack the kilnitamin powder 2g/L of tryptophan, leucine and uracil, and 2% Agar powder) it is screened, obtained transformant carries out scribing line and divides after pure culture extracting Yeast genome and carrying out PCR verifying, to testing A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel is taken after demonstrate,proving the YPD fluid nutrient medium culture of correct recombinant bacterial strain, is chosen Genome progress PCR verifying screening is extracted after taking single colonie to divide pure culture, and URA gene is deleted by recombination spontaneous between DR sequence Correct bacterial strain, correct bacterial strain is respectively designated as SyBE_Sc0014C037 and SyBE_Sc0014D008.

Embodiment 9: genetic fragment 1-4 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1C and recombinant Saccharomyces cerevisiae CEN.PK2-1D

By genetic fragment 4 using Li-acetate method by the segment convert respectively restructuring yeast strains SyBE_Sc0014C037 and SyBE_Sc0014D008 occurs recombination with the site his3 on Yeast genome by the upstream and downstream HIS3 homologous sequence and is integrated into On genome.Yeast uses SD-TRP-LEU-HIS solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/ after conversion L lacks the kilnitamin powder 2g/L of tryptophan, histidine and leucine, 2% agar powder) it is screened, obtained conversion Son carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture, saves glycerol stock to correct recombinant bacterial strain is verified And it is respectively designated as SyBE_Sc0014C040 and SyBE_Sc0014D019.

Embodiment 10: genetic fragment 1-4 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Segment is converted dual-gene knockout yeast strain SyBE_Sc0014D002 using Li-acetate method by genetic fragment 1, is passed through The site trp1 occurs recombination and is integrated on genome on the upstream and downstream TRP1 homologous sequence and Yeast genome.It is used after conversion SD-TRP solid panel (synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L, the kilnitamin powder 2g/ of single scarce tryptophan L, 2% agar powder) it is screened, obtained transformant carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture.

Then, segment is transformed by the genetic fragment 2 of the crtE containing Taxus x media source using Li-acetate method In the yeast strain for incorporating genetic fragment 1, occurred by the site leu2 on the upstream and downstream LEU2 homologous sequence and Yeast genome It recombinates and is integrated on genome.SD-TRP-LEU solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose are used after conversion The kilnitamin powder 2g/L of 20g/L, double scarce tryptophans and leucine, 2% agar powder) it is screened, obtained transformant It carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture.

Genetic fragment 3 is transformed into the yeast strain for incorporating genetic fragment 2 using Li-acetate method, is terminated by TDH2 Sub- upstream homologous sequence and ACT1 terminator downstream homologous sequence occur recombination and are inserted into the gene integrated before on genome The centre of segment 2.SD-URA-TRP-LEU solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/ are used after conversion L lacks the kilnitamin powder 2g/L of tryptophan, leucine and uracil, 2% agar powder) it is screened, obtained transformant It carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture, to the correct recombinant bacterial strain YPD Liquid Culture of verifying A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel is taken after base culture, picking single colonie extracts genome after dividing pure culture Carry out the correct bacterial strain that URA gene is deleted in PCR verifying screening by recombination spontaneous between DR sequence.

Genetic fragment 4 is transformed into the yeast strain for incorporating genetic fragment 3 using Li-acetate method, by HIS3, The site his3 occurs recombination and is integrated on genome on downstream homologous sequence and Yeast genome.Yeast uses SD- after conversion (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L lack tryptophan, histidine and leucine to TRP-LEU-HIS solid panel Kilnitamin powder 2g/L, 2% agar powder) screened, obtained transformant carry out scribing line divide pure culture after extract Yeast genome carries out PCR verifying, is named as SyBE_Sc0014D022 to correct recombinant bacterial strain preservation glycerol stock is verified.

Embodiment 11: the building of diploid restructuring yeast strains of the present invention

Take the SyBE_Sc0014C040 and SyBE_Sc0014D019 for growing to logarithmic phase on YPD fluid nutrient medium on a small quantity The mixing of (preparation of embodiment 9) bacterium solution therefrom takes a small amount of drop of Mixed Microbes drop one on YPD plate, scribing line point after 30 DEG C of culture 12h It is pure, Yeast genome is extracted after picking single colonie culture by PCR and verifies mating type, to the correct diploid recombinant bacterial strain of verifying It saves glycerol stock and is named as SyBE_Sc0014CD01.

Embodiment 12: the shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain

Test material of the present invention: SyBE_Sc0014C029, SyBE_Sc0014C032, SyBE_ in embodiment 7 Sc0014C035。

Comparative test material: other the 12 kinds of bacterial strains constructed according to the method for embodiment 7, difference are only that integrated base It is specific as follows because segment 1 is different with the external source assortment of genes in genetic fragment 2:

SyBE_Sc0014C021:Aa crtI+Aa crtB+Pa crtE

SyBE_Sc0014C022:Pa crtI+Pa crtB+Pa crtE

SyBE_Sc0014C023:Bt crtI+Pa crtB+Pa crtE

SyBE_Sc0014C024:Aa crtI+Aa crtB+Sa crtE

SyBE_Sc0014C025:Pa crtI+Pa crtB+Sa crtE

SyBE_Sc0014C026:Bt crtI+Pa crtB+Sa crtE

SyBE_Sc0014C027:Aa crtI+Aa crtB+Af crtE

SyBE_Sc0014C028:Bt crtI+Pa crtB+Af crtE

SyBE_Sc0014C029:Bt crtI+Pa crtB+Af crtE

SyBE_Sc0014C030:Aa crtI+Aa crtB+Bt crtE

SyBE_Sc0014C031:Bt crtI+Pa crtB+Bt crtE

SyBE_Sc0014C032:Bt crtI+Pa crtB+Bt crtE

SyBE_Sc0014C033:Aa crtI+Aa crtB+Tm crtE

SyBE_Sc0014C034:Bt crtI+Pa crtB+Tm crtE

SyBE_Sc0014C035:Bt crtI+Pa crtB+Tm crtE

Test method:

Seed culture medium: 40g/L glucose, 20g/L peptone, 10g/L yeast extract；

Fermentation medium: 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L D- galactolipin.

Above-mentioned bacterial strains are inoculated in 5mL seed culture medium, cultivate 14-16h in 30 DEG C, 250rpm, it is dense with initial thallus Spend OD₆₀₀=0.2 switching is in fresh 25mL seed culture medium, and culture is into logarithmic growth under the conditions of 30 DEG C, 250rpm Phase, with initial cell concentration OD₆₀₀=0.5 is inoculated in respectively in 50mL fermentation medium, cultivates under the conditions of 30 DEG C, 250rpm, Monitor the cell density (OD600) and yield of lycopene in fermentation process.

Lycopene quantitative approach: the fermentation liquid of two equal portions is taken, 4000g is centrifuged 2min and collects thallus, and washes twice.It will A copy of it thallus is placed in 80 DEG C, and drying to constant weight, and weighing calculates dry cell weight；Another thallus is extracted to product, specific side Method are as follows: cell is resuspended with 3N HCl, is placed in boiling water bath and boils 2min, immediately after ice bath 3min；By broken cell 12000rpm, 4 DEG C of centrifugation 4min abandon supernatant, acetone are added after washing 2 times, and the 5min that is vortexed；Acetone phase is finally collected by centrifugation, uses Upper ultraviolet liquid phase detection after 2 μm of membrane filtrations, lycopene Detection wavelength is 471nm.

Test result: from the point of view of the yield of lycopene by Figure 11 bacterial strain SyBE_Sc0014C021-SyBE_Sc0014C035, Ferment 60h, and the yield of Pa crtB+Bt crtI combination is significantly larger than Aa crtB+Aa crtI, Pa crtB+Pa crtI and takes second place. And influence of the crtE of the lower relatively separate sources of Pa crtB+Bt crtI combination to yield of lycopene is corresponded to as it can be seen that Sa crtE Unit cell yield it is minimum, Pa crtE takes second place, and the unit cell lycopene of Af crtE, Bt crtE and Tm crtE produce Amount reaches 30mg/gDCW or more, this has belonged to most in the open report currently with recombinant Saccharomyces cerevisiae production lycopene Height, wherein Taxus x media source crtE, pantoea agglomerans source crtB and trispore Bruce mould source crtI are combined Yield of lycopene highest, reaches 37.25 ± 0.52mg/gDCW.The above results not only show recombination ferment constructed by the present invention The yield of the lycopene of mother strains is significantly higher than current reported maximum output；And it also indicates that only in base of the present invention Because the bacterial strain constructed in the case of the foreign gene combination in segment 1 and 2 can significantly improve the yield of lycopene.

Embodiment 13: the shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain

Test material: SyBE_Sc0014C035, SyBE_Sc0014C037, SyBE_Sc0014C040, SyBE_ Sc0014D006, SyBE_Sc0014D008, SyBE_Sc0014D019 and diploid recombinant bacterial strain SyBE_Sc0014CD01；

Test method: with embodiment 12；

Test result: being still either with CEN.PK2-1D by starting strain of CEN.PK2-1C as seen from Figure 12 Send out strain construction restructuring yeast strains, the recombinant bacterial strain of corresponding transgene segment 1-2,1-3,1-4, the yield of lycopene It is above 35mg/gDCW；Bacterial strain SyBE_Sc0014D019 yield of lycopene is up to 44.76 ± 0.86mg/gDCW among these.

As seen from Figure 13, no matter monoploid restructuring yeast strains SyBE_Sc0014D019, SyBE_Sc0014C040, still Amphiploid restructuring yeast strains SyBE_Sc0014CD01, the yield of lycopene are also above 35mg/gDCW；

The above results show the yield of the lycopene of restructuring yeast strains constructed by the present invention, are all remarkably higher than and work as Preceding reported maximum output.

Embodiment 14: the shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain

Test material: SyBE_Sc0014D022 and SyBE_Sc0014D019；

Test method: with embodiment 12；

Test result: although knocking out the gene constructed restructuring yeast strains SyBE_Sc0014D022 of gal80 and ypl062w Yield is far below the restructuring yeast strains SyBE_Sc0014D019 for knocking out gal1, gal10, gal7 and ypl062w gene, but its The yield of lycopene is 26.48 ± 0.50mg/gDCW, is still higher than maximum output 24.41mg/gDCW instantly.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (20)

1. a kind of recombinant Saccharomyces cerevisiae bacterial strain, which is characterized in that the saccharomyces cerevisiae genome knock out gal1, gal7, gal10 and Ypl062w gene or gal80 and ypl062w gene, and include through on yeast autologous recombination and integration to its genome Following genetic fragment:

Yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa The genetic fragment 1 that crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence are sequentially spliced；

Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoter, shine ancient green-ball bacterium source or trispore Bruce mould source or graceful The gene piece that crtE, GPM1 terminator, the site the yeast leu2 downstream homologous sequence in ground Asia Chinese yew source are sequentially spliced Section 2；The truncated HMG-CoA reductase gene tHMGR1 sequence is as shown in 2350-3858bp in SEQ ID NO:2.

2. bacterial strain according to claim 1, which is characterized in that further include through yeast autologous recombination and integration to its genome On following genetic fragment:

TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 terminator, Bt in genetic fragment 2 ACT1 terminator and the genetic fragment 3 that homologous sequence is sequentially spliced downstream in crtI, GAL3 promoter, genetic fragment 2.

3. bacterial strain according to claim 2, which is characterized in that the genetic fragment 3 is as shown in SEQ ID NO:5.

4. bacterial strain according to claim 2, which is characterized in that further include through yeast autologous recombination and integration to its genome On following genetic fragment:

Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 terminator, his3, yeast The genetic fragment 4 that point downstream homologous sequence is sequentially spliced；The truncated HMG-CoA reductase gene tHMGR1 sequence is such as In SEQ ID NO:6 shown in 1669-3177bp.

5. bacterial strain according to claim 4, which is characterized in that the genetic fragment 4 is as shown in SEQ ID NO:6.

6. bacterial strain according to claim 4, which is characterized in that the restructuring yeast strains are with saccharomyces cerevisiae CEN.PK2-1D Starting strain, the crtE comprising Taxus x media source knock out gal1, gal7, gal10 and ypl062w gene, deposit number For CGMCC No.10754.

7. bacterial strain according to claim 4, which is characterized in that the restructuring yeast strains are with saccharomyces cerevisiae CEN.PK2-1C Starting strain, the crtE comprising Taxus x media source knock out gal80 and ypl062w gene.

8. bacterial strain according to claim 1, which is characterized in that the genetic fragment 1 is as shown in SEQ ID NO:1, gene piece Section 2 is as shown in SEQ ID NO:2-4 any one；

Wherein, the genetic fragment 2 comprising the ancient green-ball bacterium source crtE gene that shines is as shown in SEQ ID NO:2；Include three spore cloth The genetic fragment 2 of Laplace mould source crtE gene is as shown in SEQ ID NO:3；Include Taxus x media source crtE gene Genetic fragment 2 as shown in SEQ ID NO:4.

9. bacterial strain according to claim 1, which is characterized in that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY system Column saccharomyces cerevisiae.

10. bacterial strain according to claim 9, which is characterized in that the CEN.PK series saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D.

11. bacterial strain described in claim 1-10 any one is in the application produced in lycopene and is producing with lycopene For the application in the product of intermediate product.

12. the construction method of recombinant Saccharomyces cerevisiae bacterial strain described in claim 1 characterized by comprising

Step 1, building are same by yeast gal7 downstream of gene homologous sequence, DR-Kl URA3-DR nutritional labeling, gal1 downstream of gene The sequentially connected knockout box segment 1 of source sequence or by yeast gal80 upstream region of gene homologous sequence, DR-Kl URA3-DR nutrition mark Label, the sequentially connected knockout box segment 2 of gal80 downstream of gene homologous sequence, and by ypl062w upstream region of gene homologous sequence, The sequentially connected knockout box segment 3 of kanMX resistance label, ypl062w downstream of gene homologous sequence utilizes knockout box segment 1 and 3 Or yeast gal1, gal7, gal10 and ypl062w base are knocked out by the recombination of yeast autologous using box segment 2 and 3 is knocked out Cause knocks out yeast gal80 and ypl062w gene, obtains gene knockout yeast, spare；

By yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa CrtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence sequentially splice, and obtain genetic fragment 1, i.e. trp1 site upstream Homologous sequence-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1The site-trp1 downstream homologous sequence, it is spare；

Also by yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA Nitroreductase gene tHMGR1, GAL10 promoter, GAL1 promoter, shine ancient green-ball bacterium source or trispore Bruce mould source or CrtE, GPM1 terminator, the site the yeast leu2 downstream homologous sequence in Taxus x media source sequentially splice, and obtain gene piece Section 2, i.e. leu2 site upstream homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1The site-leu2 Downstream homologous sequence, it is spare；2350- in the truncated HMG-CoA reductase gene tHMGR1 sequence such as SEQ ID NO:2 Shown in 3858bp；

Genetic fragment 1 is transferred in the gene knockout yeast by step 2 by Li-acetate method, passes through trp1 in genetic fragment 1 The site trp1 occurs recombination and is integrated on genome on point upstream and downstream homologous sequence and gene knockout Yeast genome；

Genetic fragment 2 is transferred in the gene knockout yeast by Li-acetate method, by the site leu2 in genetic fragment 2, The site leu2 occurs recombination and is integrated on genome on downstream homologous sequence and gene knockout Yeast genome, obtains recombination ferment Mother strains.

13. construction method according to claim 12, which is characterized in that further include:

Genetic fragment 3 is constructed, and after transgene segment 1 and 2,3 transgene of genetic fragment is knocked out in yeast and is weighed Group yeast strain；

Specifically, by TDH2 terminator in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 Terminator, Bt crtI, GAL3 promoter, ACT1 terminator and homologous sequence sequentially splices downstream in genetic fragment 2, obtain Genetic fragment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3-T_ACT1- ACT1 terminator and downstream homologous sequence, it is spare；

Genetic fragment 3 is continued through Li-acetate method to be transformed into the gene knockout yeast of transgene segment 2, passes through gene TDH2 terminator and its upstream homologous sequence in segment 3, ACT1 terminator and homologous sequence downstream, with the gene piece integrated TDH2 terminator and ACT1 terminator site in section 2 occur to recombinate and be inserted into the gene knockout ferment of integrator gene segment 2 On female genome.

14. 3 construction method according to claim 1, which is characterized in that further include:

Genetic fragment 4 is constructed, and after transgene segment 1,2 and 3,4 transgene of genetic fragment is knocked out in yeast and is obtained Restructuring yeast strains；

Specifically, yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is attached, fusion is obtained BTS1-ERG20, then by yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncation HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 terminate Son, the site yeast his3 downstream homologous sequence sequentially splice, and obtain genetic fragment 4, i.e. his3 site upstream homologous sequence- HIS3-T_ENO2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1The site-his3 downstream homologous sequence；Described section Short HMG-CoA reductase gene tHMGR1 sequence is as shown in 1669-3177bp in SEQ ID NO:6；

Genetic fragment 4 is transferred in the gene knockout yeast by Li-acetate method, by the site his3 in genetic fragment 4, The site his3 occurs recombination and is integrated on genome on downstream homologous sequence and gene knockout Yeast genome.

15. construction method according to claim 12, which is characterized in that the specific construction method of gene knockout yeast are as follows:

Using plasmid pWJ1042 as template, design upstream and downstream primer PCR amplification by yeast gal7 downstream of gene 40bp homologous sequence, DR-Kl URA3-DR nutritional labeling, the sequentially connected knockout box segment 1 of gal1 downstream of gene 40bp homologous sequence or design Upstream and downstream primer PCR is expanded by yeast gal80 upstream region of gene 40bp homologous sequence, DR-Kl URA3-DR nutritional labeling, gal80 The sequentially connected knockout box segment 2 of downstream of gene 40bp homologous sequence, is transferred in yeast by Li-acetate method, utilizes knockout box piece Gal7 downstream of gene 40bp homologous sequence, gal1 downstream of gene 40bp homologous sequence or utilization knock out in box segment 2 in section 1 Gal80 upstream region of gene 40bp homologous sequence, gal80 downstream of gene 40bp homologous sequence, and it is sequentially connected on Yeast genome Tri- genes of gal7, gal10, gal1 occur recombination or recombinate with gal80 gene, by DR-Kl URA3-DR nutritional labeling It substitutes tri- genes of gal1, gal7, gal10 or replaces on gal80 gene integration to genome, complete gal1, gal7, gal10 Then the knockout of gene or gal80 gene filters out correct bacterial strain, correct bacterial strain YPD liquid by SD-URA solid medium It takes a little bacterium solution to be coated on 5- fluororotic acid solid panel after body culture medium culture to be screened again to recycle Kl URA3 label, obtain Transition gene knockout yeast；

Using the mono- genome for singly striking bacterial strain YPL062W struck in library of saccharomyces cerevisiae BY4742 as template, upstream and downstream primer is designed PCR amplification is homologous by ypl062w upstream region of gene 394bp homologous sequence, kanMX resistance label, ypl062w downstream of gene 317bp The sequentially connected knockout box segment 3 of sequence, is transferred in transition gene knockout yeast by Li-acetate method, utilizes knockout box segment 3 Middle ypl062w gene upstream and downstream homologous sequence, recombinates with the ypl062w gene on transition gene knockout Yeast genome, KanMX resistance label is substituted on ypl062w gene integration to genome, the knockout of ypl062w gene is completed, then by containing The YPD solid panel of G418 resistance filters out correct bacterial strain, obtains gene knockout yeast.

16. construction method according to claim 12, which is characterized in that the specific construction method of the genetic fragment 1 are as follows:

CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator are sequentially passed through into OE- PCR method is stitched together, and obtains the segment T that both ends include HindIII and XhoI restriction enzyme site_CYC1-crtI-P_GAL10-P_GAL1- crtB-T_PGK1；

Meanwhile the homologous 631bp sequence of yeast trp1 site upstream, the homologous 733bp sequence in the site yeast trp1 downstream sequentially being led to It crosses OE-PCR method to be stitched together, obtaining both ends includes SacI and ApaI restriction enzyme site, and same in the site yeast trp1 upstream and downstream Include the segment of HindIII and XhoI restriction enzyme site between source sequence, carrier is then connected by SacI and ApaI restriction enzyme site PRS405 obtains TRP1 integrated plasmid pRS405-TRP, by segment T obtained above_CYC1-crtI-P_GAL10-P_GAL1-crtB- T_PGK1It is attached with pRS405-TRP plasmid by HindIII and XhoI restriction enzyme site, obtains 1 integrated plasmid of genetic fragment, It is denoted as pRS405-TRP-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1, SacI and ApaI double digestion acquisition genetic fragment 1, i.e., Trp1 site upstream homologous sequence-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1The site-trp1 downstream homologous sequence, nucleotide Sequence is as shown in SEQ ID NO:1.

17. construction method according to claim 12, which is characterized in that the specific construction method of the genetic fragment 2 are as follows:

By ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter, shine Gu CrtE, GPM1 terminator in green-ball bacterium source or trispore Bruce mould source or Taxus x media source sequentially pass through OE- PCR method is stitched together, and obtains the segment T that both ends include BamHI and XhoI restriction enzyme site_ACT1-tHMGR1-P_GAL10-P_GAL1- crtE-T_GPM1；

Meanwhile by the homologous 561bp sequence of yeast leu2 site upstream, LEU2 label, TDH2 terminator, the site yeast leu2 downstream Homologous 584bp sequence sequentially passes through OE-PCR method and is stitched together, and obtaining both ends includes SacI and ApaI restriction enzyme site, and Include the segment of BamHI and XhoI restriction enzyme site between TDH2 terminator, the site yeast leu2 downstream homologous sequence, then passes through SacI and ApaI restriction enzyme site is connected into carrier pRS405, obtains LEU2 integrated plasmid pRS405-LEU, by segment obtained above T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1Connected with pRS405-LEU plasmid by BamHI and XhoI restriction enzyme site It connects, obtains 2 integrated plasmid of genetic fragment, be denoted as pRS405-LEU-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1, SacI and ApaI double digestion obtains genetic fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10- P_GAL1-crtE-T_GPM1The site-leu2 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:2-4.

18. 3 construction method according to claim 1, which is characterized in that the specific construction method of the genetic fragment 3 are as follows:

TDH2 terminator and its upstream 869bp homologous sequence, ACT1 terminator and 355bp is same downstream in amplification gene segment 2 Source sequence, then by TDH2 terminator and its upstream homologous sequence, DR-KlURA3-DR nutritional labeling, CYC1 terminator, Bt ACT1 terminator and homologous sequence sequentially passes through OE-PCR method and splices downstream in crtI, GAL3 promoter, genetic fragment 2 Come, obtains the genetic fragment 3 that both ends include PmeI restriction enzyme site, i.e. TDH2 terminator and its upstream homologous sequence-DR- KlURA3-DR-T_CYC1-Bt crtI-P_GAL3- ACT1 terminator and downstream homologous sequence, by segment obtained above and flat end Carrier pJET1.2 connection is held, 3 integrated plasmid of genetic fragment is obtained and is denoted as pleu-DR-KlURA3-DR-T_CYC1-Bt crtI- P_GAL3, PmeI digestion acquisition genetic fragment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL3- ACT1 terminator and downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:5.

19. 4 construction method according to claim 1, which is characterized in that the specific construction method of the genetic fragment 4 are as follows:

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is subjected to amalgamation and expression, is used by OE-PCR method GGGS linker connects the N-terminal of the C-terminal of BTS1 and ERG20 to form fusion BTS1-ERG20；

ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoter are sequentially passed through OE-PCR method is stitched together, and obtains segment T_ACT1-tHMGR1-P_GAL10-P_GAL1；

By segment T_ACT1-tHMGR1-P_GAL10-P_GAL1, fusion BTS1-ERG20, FBA1 terminator spelled by OE-PCR method It picks up and, obtain the segment T that both ends include BamHI and PstI restriction enzyme site_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1- ERG20)-T_FBA1；

PCR expands the homologous 312bp sequence of yeast his3 site upstream, HIS3 label, ENO2 terminator and yeast his3 respectively The homologous 578bp sequence in site downstream, and sequentially spliced by OE-PCR method, obtaining both ends includes SacI and ApaI digestion position Point, and between ENO2 terminator, the site yeast his3 downstream homologous sequence include BamHI and PstI restriction enzyme site segment, Then carrier pRS405 is connected by SacI and ApaI restriction enzyme site, obtains HIS3 integrated plasmid pRS405-HIS, obtained above-mentioned The segment T arrived_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1Pass through BamHI and PstI with pRS405-HIS plasmid Restriction enzyme site is attached, and is obtained 4 integrated plasmid of genetic fragment, is denoted as pRS405-HIS-T_ACT1-tHMGR1-P_GAL10-P_GAL1- (BTS1-ERG20)-T_FBA1, SacI and ApaI double digestion acquisition genetic fragment 4, i.e. his3 site upstream homologous sequence-HIS- T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1The site-his3 downstream homologous sequence, nucleotide sequence such as SEQ Shown in ID NO:6.

20. a kind of diploid recombinant Saccharomyces cerevisiae bacterial strain, which is characterized in that by recombinant Saccharomyces cerevisiae bacterial strain CEN.PK2-1C and again Group saccharomyces cerevisiae CEN.PK2-1D mating obtains；

The genome of the recombinant Saccharomyces cerevisiae bacterial strain CEN.PK2-1C and recombinant Saccharomyces cerevisiae bacterial strain CEN.PK2-1D knock out Gal1, gal7, gal10 and ypl062w gene, and include through the following base on yeast autologous recombination and integration to its genome Because of segment:

Yeast trp1 site upstream homologous sequence, CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa The genetic fragment 1 that crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence are sequentially spliced；

Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoter, shine ancient green-ball bacterium source or trispore Bruce mould source or graceful The gene piece that crtE, GPM1 terminator, the site the yeast leu2 downstream homologous sequence in ground Asia Chinese yew source are sequentially spliced Section 2；The truncated HMG-CoA reductase gene tHMGR1 sequence is as shown in 2350-3858bp in SEQ ID NO:2；

TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 terminator, Bt in genetic fragment 2 ACT1 terminator and the genetic fragment 3 that homologous sequence is sequentially spliced downstream in crtI, GAL3 promoter, genetic fragment 2；

Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoter, fusion BTS1-ERG20, FBA1 terminator, his3, yeast The genetic fragment 4 that point downstream homologous sequence is sequentially spliced；The truncated HMG-CoA reductase gene tHMGR1 sequence is such as In SEQ ID NO:6 shown in 1669-3177bp.