CN105087406A

CN105087406A - Recombinant yeast strain as well as construction method and application thereof

Info

Publication number: CN105087406A
Application number: CN201510437222.4A
Authority: CN
Inventors: 肖文海; 陈艳; 李霞; 元英进
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2015-07-22
Filing date: 2015-07-22
Publication date: 2015-11-25
Anticipated expiration: 2035-07-22
Also published as: CN105087406B

Abstract

The invention relates to the technical field of genetic engineering, and discloses a recombinant yeast strain as well as a construction method and an application thereof. The recombinant yeast strain has the knockout gene gal1, gal7, gal10 or gal80, and the knockout gene ypl062w, and contains 2-4 gene segments integrated to the genome through the yeast homologous recombination. According to the invention, the yeast strain with the knockout genes is established, an optimized host cell is provided for producing lycopene, and functional genes crtE, crtB and crt I with different sources for synthetizing lycopene, and the specific yeast endogenous genes are selected, and integrated to the genome of the yeast strain with the knockout gene through the modular design, and thus a brand-new recombinant strain with high yield of lycopene is obtained.

Description

A kind of restructuring yeast strains and construction process thereof and application

Technical field

The present invention relates to gene engineering technology field, relate to a kind of restructuring yeast strains and construction process thereof and application in particular.

Background technology

Worldwide, along with economic level and the raising to health demand, the security of food, trophicity and functionally receive increasing concern, therefore functional nutrient chemical becomes development trend, represent the new trend of contemporary food development, there are wide market outlook.Lyeopene is a kind of fat-soluble natural food colour, and chemical structure belongs to carotenoid, possesses extremely strong resistance of oxidation, is the focus of functional food composition research in the world in recent years.

The preparation of Lyeopene mainly relies on plant extract, chemosynthesis and Microbe synthesis, and first two method respectively has himself deficiency, and Microbe synthesis then with low cost, high yield and Product Safety, is considered to the most promising method.At present, in the research of Microbe synthesis Lyeopene, the Host Strains adopted mainly concentrates on intestinal bacteria and yeast saccharomyces cerevisiae.2011, the people such as Yeong-SuKim introduce synthesis Lyeopene three genes crtE, crtB and the crtI in pantoea agglomerans source in intestinal bacteria, raise the key gene in endogenous MEP approach simultaneously and in born of the same parents, build allos MVA path, realizing the yield of lycopene of 1.35g/L (32mg/gDCW) eventually through fed-batch fermentation optimization.2014, Tianjin Institute of Industrial Biotechnology Ma Yan and seminar are by optimizing the supply of NADPH and ATP in Escherichia coli cell, and utilize ribosome bind site library screening to obtain the recombination bacillus coli of a plant height product Lyeopene, its output on fed-batch fermentation tank is 3.52g/L (50.6mg/gDCW), belongs to production peak in the recombinant bacterial strain of open report at present.

Yeast saccharomyces cerevisiae is as the safe mode microorganism of generally acknowledging, compare intestinal bacteria, its thalline VITAMIN, protein content are high, can eat, medicinal and fodder yeast; Compare trispore Bruce mould, its growth cycle is shorter and more easily cultivate.Therefore, realize the high yield of Lyeopene in yeast saccharomyces cerevisiae and will represent great competitive power in carotenoid industrialization.The yield of lycopene only 3.3mg/gDCW passing through crtE, crtB and crtI that ADH2 promoter expression is originated through codon optimized uredo erwinia phage in yeast saccharomyces cerevisiae of people's reports such as Bahieldin in 2014.2015, the bifunctional enzyme crtYB of Zhejiang University in Hong Wei seminar by albumen orthogenesis means transformation phaffiafhodozyma source, made it lose the encoding function of lycopene cyclase, and only retains the function of its coding phytoene synthetase; Meanwhile, orthogenesis is carried out to the crtE in phaffiafhodozyma source, improves the catalytic performance of enzyme; Again, by adjusting the copy number of crtE, crtB and crtI, obtain the diploid recombinant Saccharomyces cerevisiae that a plant height produces Lyeopene, its shaking flask output reaches 159.56mg/L (23.23mg/gDCW), finally by fed-batch fermentation optimization, the output of Lyeopene reaches 1.61g/L (24.41mg/gDCW), and the recombinant Saccharomyces cerevisiae that utilizes belonging to open report at present synthesizes the production peak of Lyeopene.But this still has larger gap compared with the production peak (50.6mg/gDCW) utilizing recombination bacillus coli to synthesize Lyeopene reported before this.Therefore, develop with yeast saccharomyces cerevisiae is that the high yield that host strain realizes Lyeopene still exists very large potentiality and space.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of restructuring yeast strains, described recombinant bacterial strain can be applied in the biosynthesizing of Lyeopene, and keep high yield, construction process and the application of described recombinant bacterial strain is provided simultaneously.

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

A kind of restructuring yeast strains, described Yeast genome knocks out gal1, gal7, gal10 and ypl062w gene or gal80 and ypl062w gene, and to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 1 (mode chart is shown in Fig. 1) that yeast trp1 site upstream homologous sequence, CYC1 terminator, BtcrtI, GAL10 promotor, GAL1 promotor, PacrtB, PGK1 terminator, yeast trp1 sites downstream homologous sequence are spliced in turn;

The gene fragment 2 (mode chart is shown in Fig. 2) that crtE, GPM1 terminator in HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast leu2 site upstream homologous sequence, LEU2 mark, TDH2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media source, yeast leu2 sites downstream homologous sequence are spliced in turn.

The structure of the present invention by utilizing specific yeast entogenous gene and foreign gene to carry out Genetic elements, netic module, and be transferred to and knock out gal1, gal7, gal10 and ypl062w gene or knock out on the Yeast genome of gal80 and ypl062w gene, realize the synthesis output increased of recombinant bacterial strain Lyeopene.

Wherein, involved yeast entogenous gene comprises 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) the reductase gene tHMGR1 of brachymemma, is template, is obtained by pcr amplification by Yeast genome;

Simultaneously, the invention still further relates to the amino acid tag, promotor and the terminator that adopt in yeast, comprise CYC1 terminator, GAL10 promotor, GAL1 promotor, PGK1 terminator, ACT1 terminator, GPM1 terminator, LEU2 mark, TDH2 terminator, FBA1 terminator, ENO2 terminator, HIS3 mark, the acquisition of said gene element can Yeast genome be also template, is obtained by pcr amplification;

In the present invention, above-mentioned each Genetic elements and relevant upstream and downstream homologous sequence for template with the genome of Wine brewing yeast strain BY4741, are designed and synthesized suitable primer, are obtained by pcr amplification; And LEU2 upstream homologous sequence and LEU2 mark get off from pcr amplification plasmid pRS405 in the lump, HIS3 upstream homologous sequence and HIS3 mark get off from pcr amplification plasmid pRS313 in the lump.

Foreign gene involved in the present invention comprises geranyl pyrophosphate (GGPP) synthase gene crtE, phytoene synthase gene crtB and Phytoene dehydrogenase gene crtI.Wherein the source of crtE comprises pantoea agglomerans (Pantoeaagglomerans), trispore Bruce mould (Blakesleatrispora), Taxus x media (Taxusxmedia), sulfolobus acidocaldarius (Sulfolobusacidocaldarius) and luminous ancient green-ball bacterium (Archaeoglobusfulgidus), is abbreviated as PacrtE, BtcrtE, TmcrtE, SacrtE, AfcrtE successively; The source of crtB comprises pantoea agglomerans (Pantoeaagglomerans) and aquatic secondary coccus (Agrobacteriumaurantiacum), is abbreviated as PacrtB, AacrtB successively; The source of crtI comprises pantoea agglomerans (Pantoeaagglomerans), aquatic secondary coccus (Agrobacteriumaurantiacum) and trispore Bruce mould (Blakesleatrispora), is abbreviated as PacrtI, AacrtI, BtcrtI successively.Said gene is after codon optimization also suitably evades conventional restriction enzyme site and is obtained by synthetic.

As preferably, described bacterial strain also to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 3 (mode chart is shown in Fig. 3) that in gene fragment 2, in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2, ACT1 terminator and downstream homologous sequence thereof are spliced in turn.Further preferably, described gene fragment 3 is as shown in SEQIDNO:5.

More preferably, described bacterial strain also to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 4 (mode chart is shown in Fig. 4) that HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast his3 site upstream homologous sequence, HIS3 mark, ENO2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, fusion gene BTS1-ERG20, FBA1 terminator, yeast his3 sites downstream homologous sequence are spliced in turn.Further preferably, described gene fragment 4 is as shown in SEQIDNO:6.

Most preferably, described restructuring yeast strains for starting strain with yeast saccharomyces cerevisiae CEN.PK2-1D, comprises the crtE in Taxus x media source, knocks out gal1, gal7, gal10 and ypl062w gene, deposit number is CGMCCNo.10754, is designated as SyBE_Sc0014D019 in the present invention.Side by side, described restructuring yeast strains for starting strain with yeast saccharomyces cerevisiae CEN.PK2-1C, comprises the crtE in Taxus x media source, knocks out gal80 and ypl062w gene.

As preferably, described gene fragment 1 as shown in SEQIDNO:1, gene fragment 2 is as shown in SEQIDNO:2-4 any one;

Wherein, the gene fragment 2 of luminous ancient green-ball bacterium source crtE gene is comprised as shown in SEQIDNO:2; Comprise the gene fragment 2 of trispore Bruce mould source crtE gene as shown in SEQIDNO:3; Comprise the gene fragment 2 of Taxus x media source crtE gene as shown in SEQIDNO:4.

As preferably, described yeast is yeast saccharomyces cerevisiae, separate fat belongs to yeast or Crewe dimension belongs to yeast.In addition, also can with algae, mould (as streptomycete etc.) and bacterium (as intestinal bacteria, subtilis etc.) for transformation bacterial strain by the present invention the Genetic elements that limits and module recombinate according to the resolved herxheimer-liked reaction path of these bacterial strains.

More preferably, described yeast saccharomyces cerevisiae is CEN.PK series yeast saccharomyces cerevisiae or BY series yeast saccharomyces cerevisiae.Further preferably, described CEN.PK series yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae CEN.PK2-1C or yeast saccharomyces cerevisiae CEN.PK2-1D.

Restructuring yeast strains of the present invention can be a large amount of synthesis Lyeopene, therefore present invention also offers described restructuring yeast strains in the application of producing in Lyeopene and producing with Lyeopene the application in the product being intermediate product.

In addition, present invention also offers the construction process of described restructuring yeast strains, comprising:

Step 1, build by yeast gal7 downstream of gene homologous sequence, DR-KlURA3-DR nutritional labeling, what gal1 downstream of gene homologous sequence connected in turn knock out box fragment 1 or by yeast gal80 upstream region of gene homologous sequence, DR-KlURA3-DR nutritional labeling, what gal80 downstream of gene homologous sequence connected in turn knocks out box fragment 2, and by ypl062w upstream region of gene homologous sequence, kanMX resistance label, what ypl062w downstream of gene homologous sequence connected in turn knocks out box fragment 3, utilization knock out box fragment 1 and 3 or utilize knock out box fragment 2 and 3 by yeast autologous restructuring knock out yeast gal1, gal7, gal10 and ypl062w gene or knock out yeast gal80 and ypl062w gene, obtain gene knockout yeast, for subsequent use,

Yeast trp1 site upstream homologous sequence, CYC1 terminator, BtcrtI, GAL10 promotor, GAL1 promotor, PacrtB, PGK1 terminator, yeast trp1 sites downstream homologous sequence are spliced in turn, obtain gene fragment 1, i.e. trp1 site upstream homologous sequence-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1-trp1 sites downstream homologous sequence, for subsequent use;

CrtE, GPM1 terminator of being originated in HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast leu2 site upstream homologous sequence, LEU2 mark, TDH2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media, yeast leu2 sites downstream homologous sequence splice in turn, obtain gene fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T _tDH2-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1-leu2 sites downstream homologous sequence, for subsequent use;

Step 2, gene fragment 1 is proceeded in described gene knockout yeast by Li-acetate method, occur to recombinate by trp1 site on trp1 site upstream and downstream homologous sequence in gene fragment 1 and gene knockout Yeast genome and be incorporated on genome;

Gene fragment 2 is proceeded in described gene knockout yeast by Li-acetate method, occurs to recombinate by leu2 site on leu2 site upstream and downstream homologous sequence in gene fragment 2 and gene knockout Yeast genome and be incorporated on genome, obtain restructuring yeast strains.

As preferably, described construction process also comprises:

Build gene fragment 3, and after transgene fragment 1 and 2, gene fragment 3 transgene is knocked out in yeast and obtains restructuring yeast strains;

Be specially, ACT1 terminator and downstream homologous sequence thereof in TDH2 terminator in gene fragment 2 and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2 are spliced in turn, obtain gene fragment 3, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-T _aCT1-ACT1 terminator and downstream homologous sequence thereof, for subsequent use;

Gene fragment 3 being continued through Li-acetate method is transformed in the gene knockout yeast of transgene fragment 2, by TDH2 terminator in gene fragment 3 and upstream homologous sequence, ACT1 terminator and downstream homologous sequence thereof, occur to recombinate with the TDH2 terminator in the gene fragment 2 integrated and ACT1 terminator site and be inserted on the gene knockout Yeast genome of integrator gene fragment 2.

Further preferably, described construction process also comprises:

Build gene fragment 4, and after transgene fragment 1,2 and 3, gene fragment 4 transgene is knocked out in yeast and obtains restructuring yeast strains;

Be specially, yeast entogenous FPP synthase gene ERG20 is connected with GGPP synthase gene BTS1, obtain fusion gene BTS1-ERG20, then yeast his3 site upstream homologous sequence, HIS3 mark, ENO2 terminator, ACT1 terminator, HMG-CoA reductase gene tHMGR1, GAL10 promotor of brachymemma, GAL1 promotor, fusion gene BTS1-ERG20, FBA1 terminator, yeast his3 sites downstream homologous sequence are spliced in turn, obtain gene fragment 4, i.e. his3 site upstream homologous sequence-HIS3-T _eNO2-T _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1-his3 sites downstream homologous sequence;

Gene fragment 4 is proceeded in described gene knockout yeast by Li-acetate method, occurs to recombinate by his3 site on his3 site upstream and downstream homologous sequence in gene fragment 4 and gene knockout Yeast genome and be incorporated on genome.

As preferably, the concrete construction process of described gene knockout yeast is:

With plasmid pWJ1042 (complete genome sequence is shown in shown in SEQIDNO:7) for template, in design, downstream primer (relates to upstream and downstream primer for DR-KlURA3-DR nutritional labeling in plasmid pWJ1042, and upper, downstream primer 5 ' end respectively adds 40bp homologous sequence) pcr amplification is by yeast gal7 downstream of gene 40bp homologous sequence, DR-KlURA3-DR nutritional labeling, what gal1 downstream of gene 40bp homologous sequence connected in turn knocks out in box fragment 1 or design, downstream primer pcr amplification is by yeast gal80 upstream region of gene 40bp homologous sequence, DR-KlURA3-DR nutritional labeling, what gal80 downstream of gene 40bp homologous sequence connected in turn knocks out box fragment 2, proceed in yeast by Li-acetate method, utilization knocks out gal7 downstream of gene 40bp homologous sequence in box fragment 1, gal1 downstream of gene 40bp homologous sequence or utilization knock out gal80 upstream region of gene 40bp homologous sequence in box fragment 2, gal80 downstream of gene 40bp homologous sequence, with the gal7 that Yeast genome is connected in turn, gal10, there is restructuring or recombinate with gal80 gene in gal1 tri-genes, DR-KlURA3-DR nutritional labeling is substituted gal1, gal7, gal10 tri-genes or replacement gal80 gene integration are on genome, complete gal1, gal7, knocking out (Fig. 5 and Fig. 6 be shown in by schematic diagram) of gal10 gene or gal80 gene, then correct bacterial strain is filtered out by SD-URA solid medium, correct bacterial strain YPD liquid nutrient medium is got a little bacterium liquid and is coated on 5-fluororotic acid solid board and again screens to reclaim KlURA3 label after cultivating, obtain transition gene knockout yeast,

The genome singly striking bacterial strain YPL062W in storehouse is struck for template so that yeast saccharomyces cerevisiae BY4742 is mono-, in design, downstream primer pcr amplification is by ypl062w upstream region of gene 394bp homologous sequence, kanMX resistance label, what ypl062w downstream of gene 317bp homologous sequence connected in turn knocks out box fragment 3, proceed in transition gene knockout yeast by Li-acetate method, utilization to knock out in box fragment 3 on ypl062w gene, downstream homologous sequence, recombinate with the ypl062w gene on transition gene knockout Yeast genome, kanMX resistance label is substituted ypl062w gene integration on genome, complete knocking out (Fig. 7 is shown in by schematic diagram) of ypl062w gene, then correct bacterial strain is filtered out by the YPD solid board containing G418 resistance, obtain gene knockout yeast.

As preferably, the concrete construction process of described gene fragment 1 is:

CYC1 terminator, BtcrtI, GAL10 promotor, GAL1 promotor, PacrtB, PGK1 terminator are sequentially passed through OE-PCR method and be stitched together, the fragment T that two ends comprise HindIII and XhoI restriction enzyme site must be arrived _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1;

Simultaneously, yeast trp1 site upstream homology 631bp sequence, yeast trp1 sites downstream homology 733bp sequence are sequentially passed through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between the homologous sequence of upstream and downstream, yeast trp1 site, comprise the fragment of HindIII and XhoI restriction enzyme site, then (complete genome sequence is shown in shown in SEQIDNO:8 to be connected into carrier pRS405 by SacI and ApaI restriction enzyme site, plasmid map is shown in Fig. 8), obtain TRP1 integrated plasmid pRS405-TRP, by fragment T obtained above _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1be connected by HindIII and XhoI restriction enzyme site with pRS405-TRP plasmid, obtain gene fragment 1 integrated plasmid, be designated as pRS405-TRP-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1, SacI and ApaI double digestion obtains gene fragment 1, i.e. trp1 site upstream homologous sequence-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1-trp1 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:1.

As preferably, the concrete construction process of described gene fragment 2 is:

CrtE, GPM1 terminator originated in HMG-CoA reductase gene tHMGR1, GAL10 promotor of ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media is sequentially passed through OE-PCR method be stitched together, the fragment T that two ends comprise BamHI and XhoI restriction enzyme site must be arrived _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1;

Simultaneously, yeast leu2 site upstream homology 561bp sequence, LEU2 mark, TDH2 terminator, yeast leu2 sites downstream homology 584bp sequence are sequentially passed through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between TDH2 terminator, yeast leu2 sites downstream homologous sequence, comprise the fragment of BamHI and XhoI restriction enzyme site, then carrier pRS405 is connected into by SacI and ApaI restriction enzyme site, obtain LEU2 integrated plasmid pRS405-LEU, by fragment T obtained above _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1be connected by BamHI and XhoI restriction enzyme site with pRS405-LEU plasmid, obtain gene fragment 2 integrated plasmid, be designated as pRS405-LEU-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1, SacI and ApaI double digestion obtains gene fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T _tDH2-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1-leu2 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:2-4.Wherein, the gene fragment 2 of luminous ancient green-ball bacterium source crtE gene is comprised as shown in SEQIDNO:2; Comprise the gene fragment 2 of trispore Bruce mould source crtE gene as shown in SEQIDNO:3; Comprise the gene fragment 2 of Taxus x media source crtE gene as shown in SEQIDNO:4.

As preferably, the concrete construction process of described gene fragment 3 is:

TDH2 terminator and upstream 869bp homologous sequence, ACT1 terminator and downstream 355bp homologous sequence thereof in amplification gene fragment 2, then ACT1 terminator and downstream homologous sequence thereof in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2 are sequentially passed through OE-PCR method to be stitched together, obtain the gene fragment 3 that two ends comprise PmeI restriction enzyme site, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-ACT1 terminator and downstream homologous sequence thereof, be connected fragment obtained above with flat ends vector pJET1.2 (plasmid map is shown in Fig. 9), obtains gene fragment 3 integrated plasmid and be designated as pleu-DR-KlURA3-DR-T _cYC1-BtcrtI-P _gAL3, PmeI enzyme is cut and is obtained gene fragment 3, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-ACT1 terminator and downstream homologous sequence thereof, nucleotide sequence is as shown in SEQIDNO:5.

As preferably, the concrete construction process of described gene fragment 4 is:

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is carried out amalgamation and expression, the C of BTS1 is held to hold with the N of ERG20 by OE-PCR method GGGSlinker and be connected to form fusion gene BTS1-ERG20;

HMG-CoA reductase gene tHMGR1, GAL10 promotor of ACT1 terminator, brachymemma, GAL1 promotor are sequentially passed through OE-PCR method and be stitched together, obtains fragment T _aCT1-tHMGR1-P _gAL10-P _gAL1;

By fragment T _aCT1-tHMGR1-P _gAL10-P _gAL1, fusion gene BTS1-ERG20, FBA1 terminator is stitched together by OE-PCR method, must arrive the fragment T that two ends comprise BamHI and PstI restriction enzyme site _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1;

PCR increases yeast his3 site upstream homology 312bp sequence, HIS3 mark, ENO2 terminator and yeast his3 sites downstream homology 578bp sequence respectively, and spliced in turn by OE-PCR method, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between ENO2 terminator, yeast his3 sites downstream homologous sequence, comprise the fragment of BamHI and PstI restriction enzyme site, then carrier pRS405 is connected into by SacI and ApaI restriction enzyme site, obtain HIS3 integrated plasmid pRS405-HIS, by fragment T obtained above _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1be connected by BamHI and PstI restriction enzyme site with pRS405-HIS plasmid, obtain gene fragment 4 integrated plasmid, be designated as pRS405-HIS-T _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1, SacI and ApaI double digestion obtains gene fragment 4, i.e. his3 site upstream homologous sequence-HIS-T _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1-his3 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:6.

As preferably, in construction process, described yeast is yeast saccharomyces cerevisiae, solution fat belongs to yeast or Crewe dimension belongs to yeast.In addition, also can with algae, mould (as streptomycete etc.) and bacterium (as intestinal bacteria, subtilis etc.) for transformation bacterial strain by the present invention the herxheimer-liked reaction path that has been resolved according to these bacterial strains of the Genetic elements that limits and module recombinate.More preferably, described yeast saccharomyces cerevisiae is CEN.PK series yeast saccharomyces cerevisiae or BY series yeast saccharomyces cerevisiae.Further preferably, described CEN.PK series yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae CEN.PK2-1C or yeast saccharomyces cerevisiae CEN.PK2-1D.

In addition, present invention also offers a kind of diploid restructuring yeast strains, obtained by restructuring yeast strains CEN.PK2-1C and recombinant Saccharomyces cerevisiae CEN.PK2-1D mating;

The genome of described restructuring yeast strains CEN.PK2-1C and restructuring yeast strains CEN.PK2-1D all knocks out gal1, gal7, gal10 and ypl062w gene, and to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 1 that yeast trp1 site upstream homologous sequence, CYC1 terminator, BtcrtI, GAL10 promotor, GAL1 promotor, PacrtB, PGK1 terminator, yeast trp1 sites downstream homologous sequence are spliced in turn;

The gene fragment 2 that crtE, GPM1 terminator in HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast leu2 site upstream homologous sequence, LEU2 mark, TDH2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media source, yeast leu2 sites downstream homologous sequence are spliced in turn;

The gene fragment 3 that in gene fragment 2, in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2, ACT1 terminator and downstream homologous sequence thereof are spliced in turn;

The gene fragment 4 that HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast his3 site upstream homologous sequence, HIS3 mark, ENO2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, fusion gene BTS1-ERG20, FBA1 terminator, yeast his3 sites downstream homologous sequence are spliced in turn.

Mating in diploid restructuring yeast strains of the present invention is the method that this area routine obtains diploid yeast bacterial strain, generally cultivate in the medium two haploid yeast bacterial strain mixing are mating and form diploid strains, and described gene fragment 1-4 is with reference to aforesaid restriction and preferred version.

When restructuring yeast strains of the present invention is for the production of Lyeopene, yield of lycopene between 30-45mg/gDCW, far beyond the production peak of the 24.41mg/gDCW of current restructuring yeast strains.Meanwhile, the yield of lycopene of diploid restructuring yeast strains of the present invention higher than 26mg/gDCW, also higher than existing highest level.

According to the related application of bacterial strain of the present invention, present invention also offers a kind of method of producing Lyeopene, restructuring yeast strains of the present invention is inoculated in fermention medium after seed culture medium activation and cultivates, collect somatic cells after cultivating and extract Lyeopene.

Wherein, described seed culture medium is preferably 40g/L glucose, 20g/L peptone, 10g/L yeast leaching powder, and all the other are water.

Described ferment substratum is 40g/L glucose, 20g/L peptone, 10g/L yeast leaching powder, and 10g/LD-semi-lactosi, all the other are water.

Described cultivation be preferably 30 DEG C, cultivate under 250rpm condition.

From above technical scheme, the present invention builds gene knockout yeast strain, the host cell of optimization is provided for producing Lyeopene, choose functional gene crtE, crtB and crtI of the synthesis Lyeopene of different sources, and specific yeast entogenous gene etc., be integrated on gene knockout yeast strain genome through modular design, obtain the recombinant bacterial strain of the brand-new high yield Lyeopene of a strain.

Biological deposits explanation

SyBE_Sc0014D019, Classification And Nomenclature: yeast saccharomyces cerevisiae, Saccharomycescerevisiae is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 28th, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.10754.

Accompanying drawing explanation

Figure 1 shows that the Genetic elements mode chart of gene fragment 1; Wherein, two ends TRP1-L, TRP1-R represents upstream and downstream, yeast trp1 site homologous sequence respectively;

Figure 2 shows that the Genetic elements mode chart of gene fragment 2; Wherein, two ends LEU2-L, LEU2-R represents upstream and downstream, yeast leu2 site homologous sequence respectively;

Figure 3 shows that the Genetic elements mode chart of gene fragment 3; Wherein, two ends T _tDH2-L, T _aCT1-R to represent in gene fragment 2 TDH2 terminator and upstream homologous sequence thereof and ACT1 terminator and downstream homologous sequence thereof respectively;

Figure 4 shows that the Genetic elements mode chart of gene fragment 4; Wherein, two ends HIS3-L, HIS3-R represents upstream and downstream, yeast his3 site homologous sequence respectively;

Figure 5 shows that and knock out the schematic diagram that box fragment 1 knocks out gal1, gal7, gal10 gene;

Figure 6 shows that and knock out the schematic diagram that box fragment 2 knocks out gal80 gene;

Figure 7 shows that and knock out the schematic diagram that box fragment 3 knocks out ypl062w gene;

Figure 8 shows that plasmid pRS405 collection of illustrative plates;

Figure 9 shows that plasmid pJET1.2 collection of illustrative plates;

Figure 10 shows that plasmid pRS313 collection of illustrative plates;

Figure 11 shows that the yield of lycopene column diagram of the restructuring yeast strains proceeding to the combination of different foreign gene;

Figure 12 shows that the yield of lycopene column diagram of the restructuring yeast strains built for starting strain with CEN.PK2-1C and CEN.PK2-1D;

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

Embodiment

The invention discloses a kind of restructuring yeast strains and construction process thereof and application, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.The method of the invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.

Some involved in the present invention plasmid vectors, bacterial strain are all commercially available, as pJET1.2 plasmid vector buys the CloneJETPCRCloningKit from ThermoScientific company, and #K1231; Wine brewing yeast strain CEN.PK2-1C and CEN.PK2-1D buys the EUROSCARF from German ScientificResearchandDevelopmentGmbH, and strain number is 30000A and 30000B; The mono-storehouse bacterial strain YPL062W that strikes of yeast saccharomyces cerevisiae BY4742 buys from ThermoFisherScientific company of the U.S.; Plasmid pRS405, pRS313 (plasmid map is shown in Figure 10) and yeast saccharomyces cerevisiae BY4741 buy from plasmid vector strain cell gene preservation center-NTCC country of BioVector China Type Tissue Collection.

The present invention is built to each Genetic elements adopted in restructuring yeast strains, as amino acid tag, label, native gene, foreign gene etc. are known in this field, those skilled in the art know its concrete sequence.Understand the present invention for convenience, the present invention is described each Genetic elements in each gene fragment:

Knock out box fragment 1 (shown in SEQIDNO:9): 1-40bp is gal7 downstream of gene 40bp homologous sequence; 41-1615bp is DR-KlURA3-DR nutritional labeling sequence; 1616-1655bp is gal1 downstream of gene 40bp homologous sequence.

Knock out box fragment 2 (shown in SEQIDNO:10): 1-40bp is gal80 upstream region of gene 40bp homologous sequence; 41-1615bp is DR-KlURA3-DR nutritional labeling sequence; 1616-1655bp is gal80 downstream of gene 40bp homologous sequence.

Knock out box fragment 3 (shown in SEQIDNO: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.

Gene fragment 1 (shown in SEQIDNO: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 BtcrtI sequence; 2651-3318bp is GAL10-GAL1 two-way startup subsequence; 3319-4248bp is PacrtB sequence; 4249-4523bp is PGK1 terminator sequence; 4524-4529bp is XhoI restriction enzyme site; 4530-5262bp is trp1 sites downstream homology 733bp sequence.

Comprise the gene fragment 2 (shown in SEQIDNO:2) of luminous ancient green-ball bacterium source crtE gene: 1-561bp is leu2 site upstream homology 561bp sequence; 562-1656bp is LEU2 mark; 1657-2056bp is TDH2 terminator sequence; 2057-2062bp is BamHI restriction enzyme site; 2063-2349bp is ACT1 terminator sequence; 2350-3858bp is the HMG-CoA reductase gene tHMGR1 of brachymemma; 3859-4526bp is GAL10-GAL1 two-way startup subsequence; 4527-5480bp is luminous ancient green-ball bacterium source crtE gene; 5481-5880bp is GPM1 terminator sequence; 5881-5886bp is XhoI restriction enzyme site; 5887-6470bp is leu2 sites downstream homology 584bp sequence.

Comprise the gene fragment 2 (shown in SEQIDNO:3) of trispore Bruce mould source crtE gene: 1-561bp is leu2 site upstream homology 561bp sequence; 562-1656bp is LEU2 mark; 1657-2056bp is TDH2 terminator sequence; 2057-2062bp is BamHI restriction enzyme site; 2063-2349bp is ACT1 terminator sequence; 2350-3858bp is the HMG-CoA reductase gene tHMGR1 of brachymemma; 3859-4526bp is GAL10-GAL1 two-way startup subsequence; 4527-5489bp is trispore Bruce mould source crtE gene; 5490-5889bp is GPM1 terminator sequence; 5890-5895bp is XhoI restriction enzyme site; 5896-6479bp is leu2 sites downstream homology 584bp sequence.

Comprise the gene fragment 2 (shown in SEQIDNO:4) of Taxus x media source crtE gene: 1-561bp is leu2 site upstream homology 561bp sequence; 562-1656bp is LEU2 mark; 1657-2056bp is TDH2 terminator sequence; 2057-2062bp is BamHI restriction enzyme site; 2063-2349bp is ACT1 terminator sequence; 2350-3858bp is the HMG-CoA reductase gene tHMGR1 of brachymemma; 3859-4526bp is GAL10-GAL1 two-way startup subsequence; 4527-5708bp is Taxus x media source crtE gene; 5709-6108bp is GPM1 terminator sequence; 6109-6114bp is XhoI restriction enzyme site; 6115-6698bp is leu2 sites downstream homology 584bp sequence.

Gene fragment 3 (shown in SEQIDNO:5): 1-869bp is TDH2 terminator and upstream homologous sequence 869bp thereof; 870-2444bp is DR-KlURA3-DR nutritional labeling sequence; 2445-2699bp is CYC1 terminator sequence; 2700-4448bp is BtcrtI sequence; 4449-5108bp is GAL3 promotor; 5109-5463bp is ACT1 terminator and downstream homologous sequence 355bp thereof.

Gene fragment 4 (shown in SEQIDNO:6): 1-312bp is his3 site upstream 312bp homologous sequence; 313-975bp is HIS3 mark; 976-1375bp is ENO2 terminator; 1376-1381bp is BamHI restriction enzyme site; 1382-1668bp is ACT1 terminator sequence; 1669-3177bp is the HMG-CoA reductase gene tHMGR1 of brachymemma; 3178-3845bp is GAL10-GAL1 two-way startup subsequence; 3846-5921bp is fusion gene BTS1-ERG20; 5922-6121bp is FBA1 terminator sequence; 6122-6127bp is PstI restriction enzyme site; 6128-6705bp is his3 sites downstream 578bp homologous sequence.

In DR-KlURA3-DR nutritional labeling sequence, URA3 is Kluyveromyces lactis source (Kluyveromyceslactis, Kl).

After the concrete sequence knowing above-mentioned each Genetic elements, those skilled in the art conveniently design of primers principle can carry out amplification and OE-PCR splicing.Meanwhile, SD substratum of the present invention is a kind of substratum conventional in yeast screening assay field, has which genetic flaw and on the composition of minimum medium, specially delete that the realization of assigning to of a certain composition or several one-tenth filters out aimed strain according to yeast.

Below in conjunction with embodiment, set forth the present invention further.

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

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

First △ gal1 is built, △ gal7, △ gal10::DR-KlURA3-DR knocks out box, namely box fragment 1 is knocked out, with plasmid pWJ1042 for template, in design, on downstream primer pcr amplification carries gene, downstream 40bp homology arm and DR-KlURA3-DR nutritional labeling knock out box fragment, the homologous recombination machinery of yeast self is utilized to be incorporated on Yeast genome by this fragment by Li-acetate method yeast conversion, SD-URA solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, single kilnitamin powder 2g/L lacking uridylic, 2% agar powder) screen, the transformant obtained carries out PCR checking by extracting Yeast genome after point pure culture, the recombinant bacterial strain YPD liquid nutrient medium correct to checking gets a little bacterium liquid coating 5-fluororotic acid (5-FOA) solid board after cultivating (respectively there is the direct repeat DR of 143bp at DR-KlURA3-DR nutritional labeling two ends, yeast self can utilize these two sections identical sequence generation homologous recombination and delete URA3 gene and one of them DR, YPD culturing gene is not for having this screening pressure of amino acid nutrient defect, and the yeast of such spontaneous deletion URA3 can grow.Then screen with 5-FOA, because the bacterial strain containing URA3 can make 5-FOA become the virose material of cell under the enzyme effect of URA3 genes encoding, yeast cell can not grown containing on the substratum of 5-FOA, thus filter out the bacterium of deleting URA gene), extract genome after picking list bacterium colony divides pure culture and carry out URA3 gene is deleted in PCR checking screening correct bacterial strain by restructuring spontaneous between DR sequence, this correct Strain Designation is SyBE_Sc0014C011 by i.e. three gene knock-out bacterial strains.The bacterial strain knocking out gal1, gal7, gal10 can not metabolism D-semi-lactosi, thus it is constant to maintain inductor galactose concentration in born of the same parents, realizes efficiently inducing.

Then, the basis of three gene knock-out bacterial strain SyBE_Sc0014C011 builds △ ypl062w::kanMX and knocks out box, the genome singly striking bacterial strain YPL062W in storehouse is struck for template so that BY4742 is mono-, in design, on downstream primer pcr amplification carries gene YPL062W, downstream homologous sequence and kanMX resistance label knock out box fragment 3, this fragment is incorporated on Yeast genome by yeast conversion, (kanMX resistance label can produce resistance to this microbiotic of Geneticin G418 to adopt the YPD solid board screening four containing 200mg/LG418 resistance to strike bacterial strain after transforming, thus utilize G418 plate to filter out the bacterium of successful knockout gene ypl062w), the transformant obtained carries out PCR checking by extracting genome after point pure culture, the recombinant bacterial strain correct to checking preserves glycerol stock, and by its called after SyBE_Sc0014C012.

In the manner described above with yeast saccharomyces cerevisiae CEN.PK2-1D for starting strain, build four gene knock-out bacterial strain CEN.PK2-1D △ gal1, △ gal7, △ gal10::DR, △ ypl062w::kanMX, the transformant obtained carries out PCR checking by extracting genome after point pure culture, and the recombinant bacterial strain correct to checking preserves glycerol stock, and by its called after SyBE_Sc0014D004.

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

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

First build △ gal80::DR-KlURA3-DR and knock out box, namely box fragment 2 is knocked out, with plasmid pWJ1042 for template, in design, on downstream primer pcr amplification carries gene, downstream 40bp homology arm and DR-KlURA3-DR nutritional labeling knock out box fragment, the homologous recombination machinery of yeast self is utilized to be incorporated on Yeast genome by this fragment by Li-acetate method yeast conversion, SD-URA solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, single kilnitamin powder 2g/L lacking uridylic, 2% agar powder) screen, the transformant obtained carries out PCR checking by extracting Yeast genome after point pure culture, the recombinant bacterial strain YPD liquid nutrient medium correct to checking gets a little bacterium liquid coating 5-fluororotic acid (5-FOA) solid board after cultivating (respectively there is the direct repeat DR of 143bp at DR-KlURA3-DR nutritional labeling two ends, yeast self can utilize these two sections identical sequence generation homologous recombination and delete URA3 gene and one of them DR, YPD culturing gene is not for having this screening pressure of amino acid nutrient defect, and the yeast of such spontaneous deletion URA3 can grow.Then screen with 5-FOA, because the bacterial strain containing URA3 can make 5-FOA become the virose material of cell under the enzyme effect of URA3 genes encoding, yeast cell can not grown containing on the substratum of 5-FOA, thus filter out the bacterium of deleting URA gene), extract genome after picking list bacterium colony divides pure culture and carry out URA3 gene is deleted in PCR checking screening correct bacterial strain by restructuring spontaneous between DR sequence, this correct Strain Designation is SyBE_Sc0014D001 by i.e. single-gene knock-out bacterial strain.

Then, the basis of single-gene knock-out bacterial strain SyBE_Sc0014D001 builds △ ypl062w::kanMX and knocks out box, the genome singly striking bacterial strain YPL062W in storehouse is struck for template so that BY4742 is mono-, in design, on downstream primer pcr amplification carries gene YPL062W, downstream homologous sequence and kanMX resistance label knock out box fragment 3, this fragment is incorporated on Yeast genome by yeast conversion, (kanMX resistance label can produce resistance to this microbiotic of Geneticin G418 to adopt the YPD solid board containing 200mg/LG418 resistance to screen dual-gene knock-out bacterial strain after transforming, thus utilize G418 plate to filter out the bacterium of successful knockout gene ypl062w), the transformant obtained carries out PCR checking by extracting genome after point pure culture, the recombinant bacterial strain correct to checking preserves glycerol stock, and by its called after SyBE_Sc0014D002.

Embodiment 3: the structure of gene fragment 1

Amplification CYC1 terminator, BtcrtI, GAL10 promotor, GAL1 promotor, PacrtB, PGK1 terminator sequentially pass through OE-PCR method and be stitched together, must arrive the fragment T that two ends comprise HindIII and XhoI restriction enzyme site _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1;

Simultaneously, amplification yeast trp1 site upstream homology 631bp sequence, yeast trp1 sites downstream homology 733bp sequence sequentially pass through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between the homologous sequence of upstream and downstream, yeast trp1 site, comprise the fragment of HindIII and XhoI restriction enzyme site, then (complete genome sequence is shown in shown in SEQIDNO:8 to be connected into carrier pRS405 by SacI and ApaI restriction enzyme site, plasmid map is shown in Fig. 8), obtain TRP1 integrated plasmid pRS405-TRP, by fragment T obtained above _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1be connected by HindIII and XhoI restriction enzyme site with pRS405-TRP plasmid, obtain gene fragment 1 integrated plasmid, be designated as pRS405-TRP-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1;

Integrated plasmid is transformed in E. coli competent DH5 α, and bacterium colony PCR screens, and upgrading grain carries out digestion verification and sequence verification, to guarantee that object fragment connects correctly and base sequence is not undergone mutation.

After checking is correct, respectively with the cutting of SacI and ApaI double enzyme site, obtain gene fragment 1, nucleotide sequence is as shown in SEQIDNO:1.

Embodiment 4: the structure of gene fragment 2

CrtE, GPM1 terminator originated in HMG-CoA reductase gene tHMGR1, GAL10 promotor of ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media is sequentially passed through OE-PCR method be stitched together, the fragment T that two ends comprise BamHI and XhoI restriction enzyme site must be arrived _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1; Simultaneously, yeast leu2 site upstream homology 561bp sequence, LEU2 mark, TDH2 terminator, yeast leu2 sites downstream homology 584bp sequence are sequentially passed through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between TDH2 terminator, yeast leu2 sites downstream homologous sequence, comprise the fragment of BamHI and XhoI restriction enzyme site, be connected into carrier pRS405 by SacI and ApaI restriction enzyme site, obtain LEU2 integrated plasmid pRS405-LEU.By the above-mentioned fragment T obtained _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1be connected by BamHI with XhoI restriction enzyme site with pRS405-LEU plasmid, obtain gene fragment 2 integrated plasmid, be designated as pRS405-LEU-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1.

After checking is correct, respectively with the cutting of SacI and ApaI double enzyme site, obtain gene fragment 2, leu2 site upstream homologous sequence-LEU2-T _tDH2-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1-leu2 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:2-4.Wherein, the gene fragment 2 of luminous ancient green-ball bacterium source crtE gene is comprised as shown in SEQIDNO:2; Comprise the gene fragment 2 of trispore Bruce mould source crtE gene as shown in SEQIDNO:3; Comprise the gene fragment 2 of Taxus x media source crtE gene as shown in SEQIDNO:4.

Embodiment 5: the structure of gene fragment 3

TDH2 terminator and upstream 869bp homologous sequence, ACT1 terminator and downstream 355bp homologous sequence thereof in amplification gene fragment 2, then ACT1 terminator and downstream homologous sequence thereof in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2 are sequentially passed through OE-PCR method to be stitched together, obtain the gene fragment 3 that two ends comprise PmeI restriction enzyme site, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-ACT1 terminator and downstream homologous sequence thereof, be connected with flat ends vector pJET1.2 and obtain gene fragment 3 integrated plasmid, be designated as pleu-DR-KlURA3-DR-T _cYC1-BtcrtI-P _gAL3.

After checking is correct, with the cutting of PmeI restriction enzyme site, obtain gene fragment 3, nucleotide sequence is as shown in SEQIDNO:5.

Embodiment 6: the structure of gene fragment 4

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is carried out amalgamation and expression, the C of BTS1 is held to hold with the N of ERG20 by OE-PCR method GGGSlinker (GGTGGTGGTTCT) and be connected to form fusion gene BTS1-ERG20; From embodiment 4, LEU2 integrated plasmid pRS405-LEU increases T _aCT1-tHMGR1-P _gAL10-P _gAL1fragment;

By fragment T _aCT1-tHMGR1-P _gAL10-P _gAL1, fusion gene BTS1-ERG20, FBA1 terminator is stitched together by OE-PCR method, must arrive the fragment T that two ends comprise BamHI and PstI restriction enzyme site _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1; Simultaneously, PCR increases yeast his3 site upstream homology 312bp sequence, HIS3 mark, ENO2 terminator and yeast his3 sites downstream homology 578bp sequence respectively, and spliced in turn by OE-PCR method, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between ENO2 terminator, yeast his3 sites downstream homologous sequence, comprise the fragment of BamHI and PstI restriction enzyme site, be connected into carrier pRS405 by SacI and ApaI restriction enzyme site, obtain HIS3 integrated plasmid pRS405-HIS.By the above-mentioned fragment T obtained _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1be connected by BamHI with PstI restriction enzyme site with pRS405-HIS plasmid, obtain gene fragment 4 integrated plasmid, be designated as pRS405-HIS-T _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1;

After checking is correct, with the cutting of SacI and ApaI double digestion, obtain gene fragment 4, i.e. his3 site upstream homologous sequence-HIS3-T _aCT1-tHMGR1-P _gAL10-P _gAL1-(BTS1-ERG20)-T _fBA1-his3 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:6.

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

Gene fragment 1 adopts Li-acetate method fragment to be transformed four gene knockout yeast strain SyBE_Sc0014C012, occurs to recombinate and be incorporated on genome by trp1 site on TRP1 upstream and downstream homologous sequence and Yeast genome.SD-TRP solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, single kilnitamin powder 2g/L lacking tryptophane, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, and the recombinant bacterial strain correct to checking preserves glycerol stock and difference called after SyBE_Sc0014C015.

Then, adopt Li-acetate method by fragment transformed yeast strain SyBE_Sc0014C015 the gene fragment 2 of the crtE containing 3 kinds of different sourcess, occur to recombinate by leu2 site on LEU2 upstream and downstream homologous sequence and Yeast genome and be incorporated on genome.SD-TRP-LEU solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, two scarce tryptophane and leucic kilnitamin powder 2g/L, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, and the recombinant bacterial strain correct to checking preserves glycerol stock and difference called after SyBE_Sc0014C029, SyBE_Sc0014C032, SyBE_Sc0014C035.Wherein:

SyBE_Sc0014C029:SyBE_Sc0014C015,LEU2::T _ACT1-tHMGR-P _GAL10-P _GAL1-AfcrtE-T _GPM1；

SyBE_Sc0014C032:SyBE_Sc0014C015,LEU2::T _ACT1-tHMGR-P _GAL10-P _GAL1-BtcrtE-T _GPM1；

SyBE_Sc0014C035:SyBE_Sc0014C015,LEU2::T _ACT1-tHMGR-P _GAL10-P _GAL1-TmcrtE-T _GPM1。

In a manner mentioned above, the gene fragment 2 of gene fragment 1 and the crtE containing Taxus x media source is proceeded to four gene knockout yeast strain SyBE_Sc0014D004, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, and the recombinant bacterial strain correct to checking preserves glycerol stock called after SyBE_Sc0014D006.

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

Adopt Li-acetate method that this fragment is transformed restructuring yeast strains SyBE_Sc0014C035 and SyBE_Sc0014D006 respectively gene fragment 3, the centre of the gene fragment 2 integrated before restructuring occurring by TDH2 terminator upstream homologous sequence and ACT1 terminator downstream homologous sequence and is inserted on genome.SD-URA-TRP-LEU solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, lack tryptophane, the kilnitamin powder 2g/L of leucine and uridylic, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, the recombinant bacterial strain YPD liquid nutrient medium correct to checking gets a little bacterium liquid coating 5-fluororotic acid (5-FOA) solid board after cultivating, extract genome after picking list bacterium colony divides pure culture and carry out URA gene is deleted in PCR checking screening correct bacterial strain by restructuring spontaneous between DR sequence, by correct bacterial strain called after SyBE_Sc0014C037 and SyBE_Sc0014D008 respectively.

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

Adopt Li-acetate method that this fragment is transformed restructuring yeast strains SyBE_Sc0014C037 and SyBE_Sc0014D008 respectively gene fragment 4, occur to recombinate by his3 site on HIS3 upstream and downstream homologous sequence and Yeast genome and be incorporated on genome.After transforming, yeast adopts SD-TRP-LEU-HIS solid board (synthetic yeast nitrogenous source YNB6.7g/L, glucose 20g/L, lack tryptophane, Histidine and leucic kilnitamin powder 2g/L, the agar powder of 2%) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, and the recombinant bacterial strain correct to checking preserves glycerol stock and difference called after SyBE_Sc0014C040 and SyBE_Sc0014D019.

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

Gene fragment 1 adopts Li-acetate method fragment to be transformed and dual-genely knocks out yeast strain SyBE_Sc0014D002, occurs to recombinate and be incorporated on genome by trp1 site on TRP1 upstream and downstream homologous sequence and Yeast genome.SD-TRP solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, single kilnitamin powder 2g/L lacking tryptophane, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking.

Then, the gene fragment 2 of the crtE originated containing Taxus x media adopted Li-acetate method fragment to be transformed in the yeast strain incorporating gene fragment 1, occur to recombinate by leu2 site on LEU2 upstream and downstream homologous sequence and Yeast genome and be incorporated on genome.SD-TRP-LEU solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, two scarce tryptophane and leucic kilnitamin powder 2g/L, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking.

Adopting Li-acetate method to be transformed into gene fragment 3 incorporates in the yeast strain of gene fragment 2, the centre of the gene fragment 2 integrated before restructuring being occurred by TDH2 terminator upstream homologous sequence and ACT1 terminator downstream homologous sequence and is inserted on genome.SD-URA-TRP-LEU solid board (synthetic yeast nitrogenous source YNB6.7g/L is adopted after transforming, glucose 20g/L, lack tryptophane, the kilnitamin powder 2g/L of leucine and uridylic, 2% agar powder) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, the recombinant bacterial strain YPD liquid nutrient medium correct to checking gets a little bacterium liquid coating 5-fluororotic acid (5-FOA) solid board after cultivating, extract genome after picking list bacterium colony divides pure culture and carry out URA gene is deleted in PCR checking screening correct bacterial strain by restructuring spontaneous between DR sequence.

Adopting Li-acetate method to be transformed into gene fragment 4 incorporates in the yeast strain of gene fragment 3, occurs to recombinate and be incorporated on genome by his3 site on HIS3 upstream and downstream homologous sequence and Yeast genome.After transforming, yeast adopts SD-TRP-LEU-HIS solid board (synthetic yeast nitrogenous source YNB6.7g/L, glucose 20g/L, lack tryptophane, Histidine and leucic kilnitamin powder 2g/L, the agar powder of 2%) screen, the transformant obtained extracts Yeast genome after carrying out line point pure culture and carries out PCR checking, and the recombinant bacterial strain correct to checking preserves glycerol stock called after SyBE_Sc0014D022.

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

Take a morsel SyBE_Sc0014C040 and SyBE_Sc0014D019 (prepared by embodiment 9) the bacterium liquid mixing growing to logarithmic phase on YPD liquid nutrient medium, the mixed bacterium that therefrom takes a morsel drop one drops on YPD flat board, after 30 DEG C of cultivation 12h, line divides pure, picking list bacterium colony extracts Yeast genome after cultivating and verifies mating type by PCR, and the diploid recombinant bacterial strain correct to checking preserves glycerol stock and called after SyBE_Sc0014CD01.

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

Test materials of the present invention: SyBE_Sc0014C029, SyBE_Sc0014C032, SyBE_Sc0014C035 in embodiment 7.

Simultaneous test material: other the 12 kinds of bacterial strains built according to the method for embodiment 7, difference is only that integrated gene fragment 1 is different, specific as follows with the external source assortment of genes in gene fragment 2:

SyBE_Sc0014C021：AacrtI+AacrtB+PacrtE

SyBE_Sc0014C022：PacrtI+PacrtB+PacrtE

SyBE_Sc0014C023：BtcrtI+PacrtB+PacrtE

SyBE_Sc0014C024：AacrtI+AacrtB+SacrtE

SyBE_Sc0014C025：PacrtI+PacrtB+SacrtE

SyBE_Sc0014C026：BtcrtI+PacrtB+SacrtE

SyBE_Sc0014C027：AacrtI+AacrtB+AfcrtE

SyBE_Sc0014C028：BtcrtI+PacrtB+AfcrtE

SyBE_Sc0014C029：BtcrtI+PacrtB+AfcrtE

SyBE_Sc0014C030：AacrtI+AacrtB+BtcrtE

SyBE_Sc0014C031：BtcrtI+PacrtB+BtcrtE

SyBE_Sc0014C032：BtcrtI+PacrtB+BtcrtE

SyBE_Sc0014C033：AacrtI+AacrtB+TmcrtE

SyBE_Sc0014C034：BtcrtI+PacrtB+TmcrtE

SyBE_Sc0014C035：BtcrtI+PacrtB+TmcrtE

Test method:

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

Fermention medium: 40g/L glucose, 20g/L peptone, 10g/L yeast leaching powder, 10g/LD-semi-lactosi.

Above-mentioned bacterial strains is inoculated in 5mL seed culture medium, 30 DEG C, 250rpm cultivates 14-16h, with initial cell concentration OD ₆₀₀=0.2 transfers in fresh 25mL seed culture medium, in 30 DEG C, be cultured to mid log phase, with initial cell concentration OD under 250rpm condition ₆₀₀=0.5 is inoculated in 50mL fermention medium respectively, in 30 DEG C, cultivate under 250rpm condition, the cell density (OD600) in monitoring fermenting process and yield of lycopene.

Lyeopene quantivative approach: the fermented liquid getting two equal portions, the centrifugal 2min of 4000g collects thalline, and washes twice.A copy of it thalline is placed in 80 DEG C to dry to constant weight, calculating dry cell weight of weighing; Another part of thalline extracts in order to product, and concrete grammar is: use 3NHCl re-suspended cell, is placed in boiling water bath and boils 2min, then ice bath 3min immediately; The cell 12000rpm of fragmentation, 4 DEG C of centrifugal 4min are abandoned supernatant, after washing 2 times, adds acetone, and vortex 5min; Last collected by centrifugation acetone phase, by ultraviolet Liquid Detection upper after 2 μm of membrane filtrations, Lyeopene determined wavelength is 471nm.

Test-results: by the yield of lycopene of Figure 11 bacterial strain SyBE_Sc0014C021-SyBE_Sc0014C035, the output of fermentation 60h, PacrtB+BtcrtI combination is taken second place higher than AacrtB+AacrtI, PacrtB+PacrtI far away.And the impact of crtE on yield of lycopene of comparing different sources under corresponding PacrtB+BtcrtI combination is visible, the unit cell output of SacrtE is minimum, PacrtE takes second place, and the unit cell yield of lycopene of AfcrtE, BtcrtE and TmcrtE all reaches more than 30mg/gDCW, this has belonged to the highest in the open report utilizing recombinant Saccharomyces cerevisiae production Lyeopene at present, wherein the yield of lycopene of Taxus x media source crtE, pantoea agglomerans source crtB and the crtI combination of trispore Bruce mould source is the highest, reaches 37.25 ± 0.52mg/gDCW.The above results not only shows the output of the Lyeopene of the restructuring yeast strains constructed by the present invention, is significantly higher than the current production peak reported; And the bacterial strain that builds can significantly improve the output of Lyeopene when showing only in gene fragment 1 and 2 of the present invention foreign gene combination.

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

Test materials: 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-results: as seen from Figure 12, no matter take CEN.PK2-1C as starting strain or be that starting strain builds restructuring yeast strains with CEN.PK2-1D, the recombinant bacterial strain of corresponding transgene fragment 1-2,1-3,1-4, the output of its Lyeopene is all higher than 35mg/gDCW; This wherein bacterial strain SyBE_Sc0014D019 yield of lycopene up to 44.76 ± 0.86mg/gDCW.

As seen from Figure 13, no matter monoploid restructuring yeast strains SyBE_Sc0014D019, SyBE_Sc0014C040, or amphiploid restructuring yeast strains SyBE_Sc0014CD01, the output of Lyeopene is also all higher than 35mg/gDCW;

The above results shows, the output of the Lyeopene of the restructuring yeast strains constructed by the present invention, is all significantly higher than the current production peak reported.

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

Test materials: SyBE_Sc0014D022 and SyBE_Sc0014D019;

Test method: with embodiment 12;

Test-results: although knock out the gene constructed restructuring yeast strains SyBE_Sc0014D022 output of gal80 and ypl062w far below the restructuring yeast strains SyBE_Sc0014D019 knocking out gal1, gal10, gal7 and ypl062w gene, but the output of its Lyeopene is 26.48 ± 0.50mg/gDCW, still higher than production peak 24.41mg/gDCW instantly.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

1. a restructuring yeast strains, is characterized in that, described Yeast genome knocks out gal1, gal7, gal10 and ypl062w gene or gal80 and ypl062w gene, and to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 2 that crtE, GPM1 terminator in HMG-CoA reductase gene tHMGR1, GAL10 promotor of yeast leu2 site upstream homologous sequence, LEU2 mark, TDH2 terminator, ACT1 terminator, brachymemma, GAL1 promotor, luminous ancient green-ball bacterium source or trispore Bruce mould source or Taxus x media source, yeast leu2 sites downstream homologous sequence are spliced in turn.

2. bacterial strain according to claim 1, is characterized in that, also to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

The gene fragment 3 that in gene fragment 2, in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2, ACT1 terminator and downstream homologous sequence thereof are spliced in turn.

3. bacterial strain according to claim 2, it is characterized in that, described gene fragment 3 is as shown in SEQIDNO:5.

4. bacterial strain according to claim 2, is characterized in that, also to comprise through yeast autologous recombination and integration to the following gene fragment on its genome:

5. bacterial strain according to claim 4, it is characterized in that, described gene fragment 4 is as shown in SEQIDNO:6.

6. bacterial strain according to claim 4, it is characterized in that, described restructuring yeast strains for starting strain, comprises the crtE in Taxus x media source with yeast saccharomyces cerevisiae CEN.PK2-1D, knock out gal1, gal7, gal10 and ypl062w gene, deposit number is CGMCCNo.10754.

7. bacterial strain according to claim 4, is characterized in that, described restructuring yeast strains for starting strain with yeast saccharomyces cerevisiae CEN.PK2-1C, comprises the crtE in Taxus x media source, knocks out gal80 and ypl062w gene.

8. bacterial strain according to claim 1, is characterized in that, described gene fragment 1 as shown in SEQIDNO:1, gene fragment 2 is as shown in SEQIDNO:2-4 any one;

9. bacterial strain according to claim 1, is characterized in that, described yeast is yeast saccharomyces cerevisiae, separate fat belongs to yeast or Crewe dimension belongs to yeast.

10. bacterial strain according to claim 9, is characterized in that, described yeast saccharomyces cerevisiae is CEN.PK series yeast saccharomyces cerevisiae or BY series yeast saccharomyces cerevisiae.

11. bacterial strains according to claim 10, is characterized in that, described CEN.PK series yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae CEN.PK2-1C or yeast saccharomyces cerevisiae CEN.PK2-1D.

Bacterial strain described in 12. claim 1-11 any one is in the application of producing in Lyeopene and producing with Lyeopene the application in the product being intermediate product.

Described in 13. claims 1, the construction process of restructuring yeast strains, is characterized in that, comprising:

14., according to construction process described in claim 13, is characterized in that, also comprise:

15., according to construction process described in claim 14, is characterized in that, also comprise:

16., according to construction process described in claim 13, is characterized in that, the concrete construction process of described gene knockout yeast is:

With plasmid pWJ1042 for template, in design, downstream primer pcr amplification is by yeast gal7 downstream of gene 40bp homologous sequence, DR-KlURA3-DR nutritional labeling, what gal1 downstream of gene 40bp homologous sequence connected in turn knocks out in box fragment 1 or design, downstream primer pcr amplification is by yeast gal80 upstream region of gene 40bp homologous sequence, DR-KlURA3-DR nutritional labeling, what gal80 downstream of gene 40bp homologous sequence connected in turn knocks out box fragment 2, proceeds in yeast by Li-acetate method, utilizes and knocks out gal7 downstream of gene 40bp homologous sequence in box fragment 1, gal1 downstream of gene 40bp homologous sequence or utilization knock out gal80 upstream region of gene 40bp homologous sequence in box fragment 2, gal80 downstream of gene 40bp homologous sequence, with the gal7 that Yeast genome is connected in turn, gal10, there is restructuring or recombinate with gal80 gene in gal1 tri-genes, DR-KlURA3-DR nutritional labeling is substituted gal1, gal7, gal10 tri-genes or replacement gal80 gene integration, on genome, complete gal1, gal7, knocking out of gal10 gene or gal80 gene, then correct bacterial strain is filtered out by SD-URA solid medium, correct bacterial strain YPD liquid nutrient medium is got a little bacterium liquid and is coated on 5-fluororotic acid solid board and again screens to reclaim KlURA3 label after cultivating, obtain transition gene knockout yeast,

The genome singly striking bacterial strain YPL062W in storehouse is struck for template so that yeast saccharomyces cerevisiae BY4742 is mono-, in design, downstream primer pcr amplification is by ypl062w upstream region of gene 394bp homologous sequence, kanMX resistance label, what ypl062w downstream of gene 317bp homologous sequence connected in turn knocks out box fragment 3, proceed in transition gene knockout yeast by Li-acetate method, utilization to knock out in box fragment 3 on ypl062w gene, downstream homologous sequence, recombinate with the ypl062w gene on transition gene knockout Yeast genome, kanMX resistance label is substituted ypl062w gene integration on genome, complete knocking out of ypl062w gene, then correct bacterial strain is filtered out by the YPD solid board containing G418 resistance, obtain gene knockout yeast.

17., according to construction process described in claim 13, is characterized in that, the concrete construction process of described gene fragment 1 is:

Simultaneously, yeast trp1 site upstream homology 631bp sequence, yeast trp1 sites downstream homology 733bp sequence are sequentially passed through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between the homologous sequence of upstream and downstream, yeast trp1 site, comprise the fragment of HindIII and XhoI restriction enzyme site, then carrier pRS405 is connected into by SacI and ApaI restriction enzyme site, obtain TRP1 integrated plasmid pRS405-TRP, by fragment T obtained above _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1be connected by HindIII and XhoI restriction enzyme site with pRS405-TRP plasmid, obtain gene fragment 1 integrated plasmid, be designated as pRS405-TRP-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1, SacI and ApaI double digestion obtains gene fragment 1, i.e. trp1 site upstream homologous sequence-T _cYC1-crtI-P _gAL10-P _gAL1-crtB-T _pGK1-trp1 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:1.

18., according to construction process described in claim 13, is characterized in that, the concrete construction process of described gene fragment 2 is:

Simultaneously, yeast leu2 site upstream homology 561bp sequence, LEU2 mark, TDH2 terminator, yeast leu2 sites downstream homology 584bp sequence are sequentially passed through OE-PCR method and be stitched together, two ends must be arrived and comprise SacI and ApaI restriction enzyme site, and between TDH2 terminator, yeast leu2 sites downstream homologous sequence, comprise the fragment of BamHI and XhoI restriction enzyme site, then carrier pRS405 is connected into by SacI and ApaI restriction enzyme site, obtain LEU2 integrated plasmid pRS405-LEU, by fragment T obtained above _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1be connected by BamHI and XhoI restriction enzyme site with pRS405-LEU plasmid, obtain gene fragment 2 integrated plasmid, be designated as pRS405-LEU-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1, SacI and ApaI double digestion obtains gene fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T _tDH2-T _aCT1-tHMGR1-P _gAL10-P _gAL1-crtE-T _gPM1-leu2 sites downstream homologous sequence, nucleotide sequence is as shown in SEQIDNO:2-4.

19., according to construction process described in claim 13, is characterized in that, the concrete construction process of described gene fragment 3 is:

TDH2 terminator and upstream 869bp homologous sequence, ACT1 terminator and downstream 355bp homologous sequence thereof in amplification gene fragment 2, then ACT1 terminator and downstream homologous sequence thereof in TDH2 terminator and upstream homologous sequence thereof, DR-KlURA3-DR nutritional labeling, CYC1 terminator, BtcrtI, GAL3 promotor, gene fragment 2 are sequentially passed through OE-PCR method to be stitched together, obtain the gene fragment 3 that two ends comprise PmeI restriction enzyme site, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-ACT1 terminator and downstream homologous sequence thereof, be connected fragment obtained above with flat ends vector pJET1.2, obtains gene fragment 3 integrated plasmid and be designated as pleu-DR-KlURA3-DR-T _cYC1-BtcrtI-P _gAL3, PmeI enzyme is cut and is obtained gene fragment 3, i.e. TDH2 terminator and upstream homologous sequence-DR-KlURA3-DR-T thereof _cYC1-BtcrtI-P _gAL3-ACT1 terminator and downstream homologous sequence thereof, nucleotide sequence is as shown in SEQIDNO:5.

20., according to construction process described in claim 13, is characterized in that, the concrete construction process of described gene fragment 4 is:

21. 1 kinds of diploid restructuring yeast strains, is characterized in that, are obtained by restructuring yeast strains CEN.PK2-1C and recombinant Saccharomyces cerevisiae CEN.PK2-1D mating;