CN106566779A - Recombinant yeast strain, construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant yeast strain, a construction method thereof and the application thereof. In the recombinant yeast strain, gal1, gal7, gal10 and ypl062w genes are knocked out, and the recombinant yeast strain comprises 6 gene segments which are integrated to the genome through yeast homologous recombination. On the above basis, the recombinant yeast strain is further integrated onto one gene segment of a yeast genome, so that a better recombinant yeast is obtained. According to the technical scheme of the invention, the yeast strain is constructed based on gene knock-out, and an optimized host cell for producing lycopene is provided. Meanwhile, functional genes crtE crtB and crtI of a specific source combination and used for the synthesis of lycopene, specific yeast endogenous genes and specific exogenous genes are selected. The above selected genes are integrated to the gene knock-out genome of the yeast strain, and then a brand-new recombinant strain capable of producing high-yield lycopene is obtained.

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 structure side Method and application.

Background technology

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

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 are 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, the Host Strains for being adopted focus 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 raise the key gene in endogenous MEP approach and build heterologous MVA paths in 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 The supply that art research institute Ma Yan and seminar pass through optimization Escherichia coli intracellular NADPH and ATP, and utilize ribosome bind site Library screening obtains the recombination bacillus coli that a plant height produces lycopene, and its yield on fed batch fermentation tank is 3.52g/ L (50.6mg/g DCW), maximum output in the category recombinant bacterial strain that at present disclosure is reported.

Saccharomyces cerevisiae compares Escherichia coli as generally acknowledged safe mode microorganism, and its thalline vitamin, protein contain Amount is high, can eat, medicinal and fodder yeast；Trispore Bruce mould is compared, its growth cycle is shorter and is more easy to culture.Cause This, realizes that high yield of the lycopene in saccharomyces cerevisiae will represent great competitiveness in carotenoid industrialization.2014 That what is reported in year Bahieldin et al. bites summer spore Ou Wenshi in saccharomyces cerevisiae by the expression Jing codon optimizations of ADH2 promoters The yield of lycopene only 3.3mg/gDCW of crtE, crtB and the crtI in bacterium source.2015, Zhejiang University was in Hong Wei seminars The bifunctional enzyme crtYB in phaffiafhodozyma source is transformed by albumen orthogenesis means so as to lose lycopene cyclase Encoding function, and only retain its coding phytoene synthetase function；Meanwhile, the crtE to phaffiafhodozyma source Evolution is oriented, the catalytic performance of enzyme is improved；Again, by the copy number of adjustment crtE, crtB and crtI, a plant height is obtained The dliploid recombinant Saccharomyces cerevisiae of lycopene is produced, its 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).Chinese patent CN105087406A discloses a kind of restructuring yeast strains and its construction method and application, and it is by the way that yeast strain is knocked out Gal1, gal7, gal10 or gal80 gene, and the gene constructed gene knockout yeast strains of ypl062w, then choose different next Functional gene crtE, crtB and crtI of the synthesis lycopene in source, and specific yeast entogenous gene etc., Jing modularized designs It is integrated on gene knockout yeast strain genome, obtains the recombinant bacterial strain of one plant of brand-new high yield lycopene, lycopene Yield reach 30-45mg/gDCW；However, this synthesizes the highest of lycopene with the utilization recombination bacillus coli reported before this Yield (50.6mg/g DCW) is compared and still have certain gap.

Chinese patent CN105420134A discloses a kind of restructuring yeast strains and its construction method and application, its pass through by Yeast strain knocks out gal1, gal7, gal10 and ypl062w gene, and comprising on Jing yeast homologous recombination and integration to genome 3 genetic fragments, on its basis its also further knock out rox1 genes and be incorporated into 2 gene pieces on Yeast genome Section, obtains more excellent recombination yeast, and the yield of lycopene is higher than 45mg/gDCW, reaches as high as 54.62mg/gDCW, not only excellent Yeast engineering bacteria in patent CN105087406A, and higher than the lycopene maximum output of recombination bacillus coli instantly. But, develop with saccharomyces cerevisiae and realize that the high yield of lycopene still has very big potentiality and space as host strain, it is worth Researcher goes the product lycopene ability for further lifting recombination engineering.

The content of the invention

In view of this, it is an object of the invention to provide a kind of restructuring yeast strains so that the recombinant bacterial strain can be answered In for 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 present invention provides following technical scheme：

A kind of restructuring yeast strains, it is characterised in that the Yeast genome knock out gal1, gal7, gal10, Ypl062w, rox1 gene, and comprising the following genetic fragment on Jing yeast autologous recombination and integrations to its genome：

Yeast trp1 site upstream homologous sequences, CYC1 terminators, Bt crtI, GAL10 promoters, GAL1 promoters, Pa The genetic fragment 1 that crtB, PGK1 terminator, yeast trp1 sites downstream homologous sequences are sequentially spliced；

Yeast leu2 site upstream homologous sequences, LEU2 marks, TDH2 terminators, ACT1 terminators, the HMG-CoA of truncation Reductase gene tHMGR1, GAL10 promoter, GAL1 promoters, TmcrtE, GPM1 terminator, yeast leu2 sites downstreams are same The genetic fragment 2 that source sequence is sequentially spliced；

TDH2 terminators and its upstream homologous sequence, DR-Kl URA3-DR nutritional labelings, CYC1 terminate in genetic fragment 2 ACT1 terminators and gene that downstream homologous sequence is sequentially spliced in son, Bt crtI, GAL7 promoters, genetic fragment 2 Fragment 3；

Yeast his3 site upstream homologous sequences, HIS3 marks, ENO2 terminators, ACT1 terminators, the HMG-CoA of truncation Reductase gene tHMGR1, GAL10 promoter, GAL1 promoters, fusion BTS1-ERG20, FBA1 terminator, yeast The genetic fragment 4 that his3 sites downstream homologous sequences are sequentially spliced；

Yeast YPRCdelta15 site upstream homologous sequences, DR-Kl URA3-DR nutritional labelings, CPS1 terminators, transcription The genetic fragment that factor INO2, GAL1 promoter, saccharomyces cerevisiae YPRCdelta15 sites downstream homologous sequences are sequentially spliced 5；

Yeast YNRCdelta9 site upstream homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 terminator, DR- (the pattern of genetic fragment 6 that Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream homologous sequences are sequentially spliced Figure is shown in Fig. 1)；

Wherein, gal1, gal7, gal10, ypl062w, rox1 gene and homologous recombination gene piece are knocked out in Yeast genome After section 1-5, intermediate strains, restructuring yeast strains Jing autologous recombination and integration gene piece on the basis of intermediate strains are obtained Obtain after section 6.The particular sequence of genetic fragment 1-5 of the present invention, Genetic elements, synthetic method and ideograph can be found in Described in patent CN105420134A.

The present invention carries out the structure of Genetic elements, netic module by using specific yeast entogenous gene and foreign gene Build, and be transferred on the Yeast genome for knocking out gal1, gal7, gal10, ypl062w, rox1 gene, realize recombinant bacterial strain kind The synthesis output increased of Lycopene.

Wherein, in involved yeast amino acid tag, transcription factor, promoter and terminator, including but not limited to CYC1 terminators, GAL10 promoters, GAL1 promoters, PGK1 terminators, ACT1 terminators, GPM1 terminators, LEU2 mark, TDH2 terminators, FBA1 terminators, ENO2 terminators, HIS3 marks, CPS1 terminators, PGK1 promoters, transcription factor INO2, The acquisition of said gene element can be obtained with Yeast genome as template by PCR amplifications；

In the present invention, the upstream and downstream homologous sequence of above-mentioned each Genetic elements and correlation is with Wine brewing yeast strain BY4741 Genome be template, design and synthesize suitable primer, by PCR amplification obtain；And LEU2 upstreams homologous sequence and LEU2 Mark is to expand from PCR on plasmid pRS405 in the lump, and HIS3 upstreams homologous sequence and HIS3 marks are in the lump from plasmid The upper PCR of pRS313 are expanded.

Foreign gene involved in the present invention includes geranyl pyrophosphate (GGPP) synthase gene crtE, phytoene Synthase gene crtB and Phytoene dehydrogenase gene crtI.Wherein the source of crtE is Taxus x media (Taxus X media), it is abbreviated as Tm crtE；The source of crtB is pantoea agglomerans (Pantoea agglomerans), is abbreviated as Pa crtB；The source of crtI is trispore Bruce mould (Blakeslea trispora), is abbreviated as Bt crtI；VHb is from saturating The bright bacterium that quivers (Vitreoscilla stercoraria), said gene is codon optimization and suitably evades conventional restriction Property restriction enzyme site after obtained by artificial synthesized.

Preferably, the genetic fragment 6 such as SEQ ID NO:Shown in 1.

Preferably, bacterial strain of the intermediate strains for deposit number CGMCC No.11748, the bacterial strain was in 2015 11 The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 27th, and records in detail in patent In CN105420134A；The present invention transformed gene fragment 6 can be obtained on the basis of the bacterial strain of deposit number CGMCC No.11748 New restructuring yeast strains.

Preferably, the yeast be saccharomyces cerevisiae, solution fat belong to yeast or Crewe dimension category yeast.In addition, it is also possible to 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 resolved herxheimer-liked reaction path of these bacterial strains.

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

On the basis of above-mentioned each technical scheme, preferably also include Jing yeast autologous recombination and integrations on its genome Following genetic fragment：

Yeast YFLWtau1 site upstream homologous sequences, PGK1 promoters, from gene VHb, ACT1 of Vitreoscilla The base that terminator, DR-Kl URA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream homologous sequences are sequentially spliced Because of fragment 7 (ideograph is shown in Fig. 1).It is further preferred that the genetic fragment 7 such as SEQ ID NO:Shown in 2.On this basis, It is highly preferred that the bacterial strain is the bacterial strain that deposit number is CGMCC No.13013.

Restructuring yeast strains of the present invention can substantial amounts of synthesis lycopene, it is therefore present invention also offers described heavy Application of the group yeast strain in production lycopene and the application in product of the production with lycopene as intermediate product.

Additionally, present invention also offers the construction method of the restructuring yeast strains, including：

Step 1, structure are by under yeast gal7 downstream of gene homologous sequences, DR-Kl URA3-DR nutritional labelings, gal1 genes Trip homologous sequence be sequentially connected with knockout box fragment 1, by ypl062w upstream region of gene homologous sequences, kanMX resistance labels, The knockout box fragment 2 that ypl062w downstream of gene homologous sequences are sequentially connected with, and build by the homologous sequence of yeast rox1 upstream region of gene The knockout box fragment 3 that row, DR-Kl URA3-DR nutritional labelings, rox1 downstream of gene homologous sequences are sequentially connected with, using knockout box Fragment 1-3 knocks out yeast gal1, gal7, gal10, ypl062w and rox1 gene by the restructuring of yeast autologous, obtains gene Yeast is knocked out, it is standby；

By yeast trp1 site upstream homologous sequences, CYC1 terminators, Bt crtI, GAL10 promoters, GAL1 promoters, Pa crtB, PGK1 terminators, yeast trp1 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 1, i.e. trp1 sites Upstream homologous sequence-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1- trp1 sites downstream homologous sequences, it is standby；

By yeast leu2 site upstream homologous sequences, LEU2 marks, TDH2 terminators, ACT1 terminators, truncate HMG- Under CoA reductase gene tHMGR1, GAL10 promoters, GAL1 promoters, Tm crtE, GPM1 terminators, yeast leu2 sites Trip homologous sequence sequentially splices, and obtains genetic fragment 2, i.e. leu2 site upstreams homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1- P_GAL10-P_GAL1-crtE-T_GPM1- leu2 sites downstream homologous sequences, it is standby；

By TDH2 terminators in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labelings, CYC1 ends Only ACT1 terminators and downstream homologous sequence sequentially splices in son, Bt crtI, GAL7 promoters, genetic fragment 2, obtain base Because of fragment 3, i.e. TDH2 terminators and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL7-T_ACT1-ACT1 Terminator and downstream homologous sequence, it is standby；

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 sequences, HIS3 marks, ENO2 terminators, ACT1 terminators, truncation HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoters, fusion BTS1-ERG20, FBA1 terminate Son, yeast his3 sites downstream homologous sequences sequentially splice, and acquisition genetic fragment 4, i.e. his3 site upstreams homologous sequence- HIS3-T_ENO2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1- his3 sites downstream homologous sequences, it is standby；

By yeast YPRCdelta15 site upstream homologous sequences, DR-KlURA3-DR nutritional labelings, CPS1 terminators, turn Record factor INO2, GAL1 promoter, saccharomyces cerevisiae YPRCdelta15 sites downstream homologous sequences sequentially splice, and obtain gene piece Section 5, i.e. YPRCdelta15 site upstreams homologous sequence-DR-KlURA3-DR-T_CPS1-INO2-P_GAL1- YPRCdelta15 sites Downstream homologous sequence, it is standby；

By yeast YNRCdelta9 site upstream homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 terminator, DR-Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 6, i.e., YNRCdelta9 site upstream homologous sequence-P_GAL1-INO2-T_CPS1- DR-Kl URA3-DR-YNRCdelta9 sites downstreams are homologous Sequence, it is standby；

Step 2, genetic fragment 1 is proceeded in the gene knockout yeast, by genetic fragment 1 by Li-acetate method There is restructuring with trp1 sites on gene knockout Yeast genome and be incorporated into genome in trp1 sites upstream and downstream homologous sequence On；

Genetic fragment 2 is proceeded in the gene knockout yeast by Li-acetate method, by leu2 sites in genetic fragment 2 There is restructuring with leu2 sites on gene knockout Yeast genome and be incorporated on genome in upstream and downstream homologous sequence；

Genetic fragment 3 is continued through into Li-acetate method to be transformed in the gene knockout yeast of transgene fragment 2, is passed through TDH2 terminators and its upstream homologous sequence in genetic fragment 3, ACT1 terminators and homologous sequence downstream, with the base integrated The clpp gene of integrator gene fragment 2 is inserted into because of the TDH2 terminators in fragment 2 and the generation restructuring of ACT1 terminators site Except on Yeast genome；

Genetic fragment 4 is proceeded in the gene knockout yeast by Li-acetate method, by his3 sites in genetic fragment 4 There is restructuring with his3 sites on gene knockout Yeast genome and be incorporated on genome in upstream and downstream homologous sequence；

Genetic fragment 5 is transformed in gene knockout yeast by Li-acetate method, by genetic fragment 5 On YPRCdelta15 sites upstream and downstream homologous sequence and gene knockout Yeast genome YPRCdelta15 sites there is restructuring and It is incorporated on genome；

Intermediate strains are obtained after genetic fragment 1-5 is transformed in gene knockout yeast by Li-acetate method；

Genetic fragment 6 is transformed in intermediate strains by Li-acetate method, by YNRCdelta9 sites in genetic fragment 6 There is restructuring and be incorporated on genome in upstream and downstream homologous sequence, obtain with YNRCdelta9 sites on intermediate strains genome Restructuring yeast strains.

Preferably, the construction method also includes：

Genetic fragment 7 is built, and genetic fragment 7 is proceeded to into acquisition restructuring yeast strains；

Specifically, by yeast YFLWtau1 site upstream homologous sequences, PGK1 promoters, from the gene of Vitreoscilla VHb, ACT1 terminator, DR-KlURA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream homologous sequences sequentially splice, Obtain genetic fragment 7, i.e. YFLWtau1 site upstreams homologous sequence-P_PGK1-VHb-T_ACT1- DR-KlURA3-DR-YFLWtau1 positions Point downstream homologous sequence；

Genetic fragment 7 is transformed into into transgene fragment 1-6 or the base of transgene fragment 1-5 by Li-acetate method In because knocking out yeast, by YFLWtau1 sites upstream and downstream homologous sequence in genetic fragment 7 and gene knockout Yeast genome There is restructuring and be incorporated on genome in YFLWtau1 sites.

Gene knockout yeast (including the detailed structure of each knockout box) of the present invention, the concrete structure of genetic fragment 1-5 Method and detailed record situation can refer to the record of patent CN105420134A, and here of the present invention is by the structure of genetic fragment 6 and 7 Build situation and provide as follows：

Amplification yeast YNRCdelta9 site upstream 256bp homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 Terminator, DR-Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream 267bp homologous sequences, sequentially pass through OE- PCR method is stitched together, and genetic fragment 6 of the two ends comprising PmeI restriction enzyme sites is obtained, by fragment obtained above and flat end Carrier pJET1.2 connects, and obtains the integrated plasmid of genetic fragment 6 and is designated as pYNRCdelta9-P_GAL1-INO2-T_CPS1-DR-Kl URA3-DR, PmeI digestion obtains genetic fragment 6, i.e. YNRCdelta9 site upstreams homologous sequence-P_GAL1-INO2-T_CPS1-DR- Kl URA3-DR-YNRCdelta9 sites downstream homologous sequences, nucleotide sequence such as SEQ ID NO:Shown in 1.

Amplification yeast YFLWtau1 site upstream 297bp homologous sequences, PGK1 promoters, from the gene of Vitreoscilla VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream 208bp homologous sequences, it is suitable It is secondary to be stitched together by OE-PCR methods, genetic fragment 7 of the two ends comprising PmeI restriction enzyme sites is obtained, by fragment obtained above It is connected with flat ends vector pJET1.2, obtains the integrated plasmid of genetic fragment 7 and be designated as pYFLWtau1-P_PGK1-VHb-T_ACT1-DR-Kl URA3-DR, PmeI digestion obtains genetic fragment 7, i.e. YFLWtau1 site upstreams homologous sequence-P_PGK1-VHb-T_ACT1-DR- KlURA3-DR-YFLWtau1 sites downstream homologous sequences, nucleotide sequence such as SEQ ID NO:Shown in 2.

Preferably, in construction method, the yeast is saccharomyces cerevisiae, solution fat belongs to yeast or Crewe dimension belongs to yeast.Remove Outside this, it is also possible to algae, mould (such as streptomycete) and bacterium (such as Escherichia coli, bacillus subtilis) as transformation bacterium The herxheimer-liked reaction path that strain has been resolved the Genetic elements that limited of the present invention and module according to these bacterial strains come Restructuring.It is highly preferred that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiaes or BY series saccharomyces cerevisiaes.It is further preferred that The CEN.PK series saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1D.

When restructuring yeast strains of the present invention are used to produce lycopene, yield of lycopene is 60.97mg/gDCW (356.22mg/L) -66.14mg/gDCW (376.78mg/L), yield is significantly higher than recombination yeast in CN105420134A patents The maximum output of the 54.62mg/gDCW (318.13mg/L) of bacterial strain.

According to the related application of bacterial strain of the present invention, present invention also offers a kind of method for producing lycopene, incites somebody to action Bacterial strain of the present invention is inoculated in fermented and cultured in fermentation medium Jing after seed culture medium culture activation, collects after fermented and cultured Somatic cells extract lycopene.

More specifically, after inoculation of the present invention is cultivated in 5mL seed culture mediums, with initial cell concentration OD₆₀₀=0.2 transfers in fresh 25mL seed culture mediums culture to mid log phase, with initial cell concentration OD₆₀₀=0.5 It is inoculated in 50mL fermentation mediums and cultivates, collects thalline cell extraction lycopene after fermented and cultured.

Wherein, preferably, the seed culture medium is 20g/L glucose, 20g/L peptones, 10g/L yeast extracts, 50mg/L uracils, remaining is water.

Preferably, the fermentation medium is 20g/L glucose, 20g/L peptones, 10g/L yeast extracts, 20mg/L Uracil, 10g/L D- (+)-galactolipin, remaining is water.

Preferably, the culture is to cultivate under the conditions of 30 DEG C, 250rpm.

From above technical scheme, the present invention builds gene knockout yeast strain, and to produce lycopene optimization is provided Host cell, choose functional gene crtE, crtB and crtI of the synthesis lycopene of particular source combination, and specific ferment Female endogenous gene and foreign gene etc., Jing modularized designs are integrated on gene knockout yeast strain genome, obtain one plant The recombinant bacterial strain of brand-new high yield lycopene.

Biological deposits explanation

SyBE_Sc0014D018, Classification And Nomenclature：Saccharomyces cerevisiae, Saccharomyces cerevisiae are in September, 2016 China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited within 19th, address is Chaoyang District, Beijing City North Star west The institute 3 of road 1, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.13013.

Description of the drawings

Fig. 1 show the Genetic elements ideograph of genetic fragment 6；Wherein, two ends YNRCdelta9-L, YNRCdelta9-R Yeast YNRCdelta9 sites upstream and downstream homologous sequence is represented respectively；

Fig. 2 show the Genetic elements ideograph of genetic fragment 7；Wherein, two ends YFLWtau1-L, YFLWtau1-R difference Represent yeast YFLWtau1 sites upstream and downstream homologous sequence；

Fig. 3 show SyBE_Sc0014D014 bacterial strains in patent CN105420134A, SyBE_Sc0014D017 of the present invention With the yield of lycopene column diagram of SyBE_Sc0014D018 bacterial strains；

Fig. 4 show the growth of SyBE_Sc0014D018 bacterial strains shake flask fermentation of the present invention and yield of lycopene curve.

Specific embodiment

The invention discloses a kind of restructuring yeast strains and its construction method and application, those skilled in the art can use for reference Present disclosure, is suitably modified technological parameter realization.Specifically, all similar replacements and change are to this area 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, and related personnel substantially can be in without departing from present invention, spirit and scope to described herein Methods and applications be modified or suitably change with combining realizing and apply the technology of the present invention.

Involved in the present invention some plasmid vectors, bacterial strain are commercially available, such as pJET1.2 plasmid vectors purchase From the CloneJET PCR Cloning Kit of Thermo Scientific companies, #K1231；Wine brewing yeast strain CEN.PK2- 1D buys the EUROSCARF from Germany Scientific Research and Development GmbH, and strain number is 30000A and 30000B；Saccharomyces cerevisiae BY4742 is mono- to strike storehouse bacterial strain YPL062W purchases from U.S. Thermo Fisher Scientific companies；Plasmid pRS405, pRS313 and saccharomyces cerevisiae BY4741 are from BioVector China plasmid vector bacterium The Type Tissue Collection purchase of strain cytogene collection-NTCC countries.Above-mentioned plasmid map and sequence can be found in Patent CN105420134A is recorded.

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

Box fragment 1 is knocked out (referring to the SEQ ID NO in patent CN105420134A:7)：1-40bp is gal7 downstream of gene 40bp homologous sequences；41-1615bp is DR-Kl URA3-DR nutritional labeling sequences；1616-1655bp is gal1 downstream of gene 40bp homologous sequences.

Box fragment 2 is knocked out (referring to the SEQ ID NO in patent CN105420134A:8)：1-394bp is ypl062w genes Upstream 394bp homologous sequences；395-1864bp is kanMX resistance sequence labels；1865-2181bp is ypl062w downstream of gene 317bp homologous sequences.

Box fragment 3 is knocked out (referring to the SEQ ID NO in patent CN105420134A:9)：1-40bp is rox1 upstream region of gene 40bp homologous sequences；41-1615bp is DR-Kl URA3-DR nutritional labeling sequences；1616-1655bp is rox1 downstream of gene 40bp homologous sequences.

Genetic fragment 1 is (referring to the SEQ ID NO in patent CN105420134A:1)：1-631bp is trp1 site upstreams 631bp homologous sequences；632-637bp is BamHI restriction enzyme sites；638-640bp is meaningless sequence；641-646bp is HindIII restriction enzyme sites；647-901bp is CYC1 terminator sequences；902-2650bp is Bt crtI sequences；2651-3318bp For GAL10-GAL1 two-way startup subsequences；3319-4248bp is Pa crtB sequences；4249-4523bp is PGK1 terminator sequences Row；4524-4529bp is XhoI restriction enzyme sites；4530-5262bp is the homologous 733bp sequences of trp1 sites downstreams.

Genetic fragment 2 is (referring to the SEQ ID NO in patent CN105420134A:2)：1-561bp is leu2 site upstreams Homologous 561bp sequences；562-1656bp is LEU2 marks；1657-2056bp is TDH2 terminator sequences；2057-2062bp is BamHI restriction enzyme sites；2063-2349bp is ACT1 terminator sequences；2350-3858bp is the HMG-CoA reductase base for truncating Because of tHMGR1；3859-4526bp is GAL10-GAL1 two-way startup subsequences；4527-5708bp is Tm crtE genes；5709- 6108bp is GPM1 terminator sequences；6109-6114bp is XhoI restriction enzyme sites；6115-6698bp is that leu2 sites downstreams are same Source 584bp sequences.

Genetic fragment 3 is (referring to the SEQ ID NO in patent CN105420134A:3)：1-869bp be TDH2 terminators and Its upstream homologous sequence 869bp；870-2444bp is DR-Kl URA3-DR nutritional labeling sequences；2445-2699bp is CYC1 ends Only subsequence；2700-4448bp is Bt crtI sequences；4449-4948bp is GAL7 promoters；4949-5303bp is ACT1 ends Only son and downstream homologous sequence 355bp.

Genetic fragment 4 is (referring to the SEQ ID NO in patent CN105420134A:4)：1-312bp is his3 site upstreams 312bp homologous sequences；313-975bp is HIS3 marks；976-1375bp is ENO2 terminators；1376-1381bp is BamHI enzymes Enzyme site；1382-1668bp is ACT1 terminator sequences；1669-3177bp is the HMG-CoA reductase gene for truncating tHMGR1；3178-3845bp is GAL10-GAL1 two-way startup subsequences；3846-5921bp is fusion BTS1-ERG20； 5922-6121bp is FBA1 terminator sequences；6122-6127bp is PstI restriction enzyme sites；6128-6705bp is under his3 sites Trip 578bp homologous sequences.

Genetic fragment 5 is (referring to the SEQ ID NO in patent CN105420134A:5)：1-606bp is YPRCdelta15 positions Point upstream 606bp homologous sequences；607-2181bp is DR-Kl URA3-DR nutritional labeling sequences；2182-2372bp is CPS1 ends Only subsequence；2373-3287bp is transcription factor INO2 sequence；3288-3744bp is GAL1 promoter sequences；3745- 4103bp is YPRCdelta15 sites downstream 359bp homologous sequences.

(the SEQ ID NO of the present invention of genetic fragment 6:Shown in 1)：1-256bp is that YPRCdelta9 site upstream 256bp are homologous Sequence；257-713bp is GAL1 promoter sequences；714-1634bp is transcription factor INO2 sequence；1635-1825bp is CPS1 Terminator sequence；1826-3373bp is DR-KlURA3-DR nutritional labeling sequences；3374-3640bp is YPRCdelta9 sites Downstream 267bp homologous sequences；

(the SEQ ID NO of the present invention of genetic fragment 7:Shown in 2)：1-297bp is the homologous sequences of YFLWtau1 site upstream 297bp Row；298-1075bp is PGK1 promoter sequences；1076-1516bp is the gene VHb sequences from Vitreoscilla；1517- 1803bp is ACT1 terminator sequences；1804-3351bp is DR-Kl URA3-DR nutritional labeling sequences；3352-3559bp is YFLWtau1 sites downstream 208bp homologous sequences；

URA3 is Kluyveromyces lactis source (Kluyveromyces in DR-Kl URA3-DR nutritional labeling sequences 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 splicings.Meanwhile, SD culture mediums of the present invention are a kind of trainings commonly used in yeast screening assay field Foster base, according to yeast have which gene defect and specially delete on the composition of minimal medium a certain composition or it is several into Divide to realize filtering out aimed strain.

It is of the invention with saccharomyces cerevisiae CEN.PK2-1D as starting strain according to technical solution of the present invention and preferred version, General introduction recombinant bacterial strain building process：

1). box fragment 1 will be knocked out and proceed to CEN.PK2-1D, knock out gal1, gal7, gal10 gene, obtained strains are named as SyBE_Sc0014D003；

2). box fragment 2 will be knocked out and proceed to SyBE_Sc0014D003, knock out ypl062w genes, obtained strains are named as SyBE_Sc0014D004；

3). genetic fragment 1 is proceeded to into SyBE_Sc0014D004, obtained strains are named as SyBE_Sc0014D005；

4). genetic fragment 2 is proceeded to into SyBE_Sc0014D005, obtained strains are named as SyBE_Sc0014D006；

5). genetic fragment 3 is proceeded to into SyBE_Sc0014D006, obtained strains are named as SyBE_Sc0014D007；

6). genetic fragment 4 is proceeded to into SyBE_Sc0014D007, obtained strains are named as SyBE_Sc0014D009；

7). box fragment 3 will be knocked out and proceed to SyBE_Sc0014D009, knock out rox1 genes, obtained strains are named as SyBE_ Sc0014D011；

8). genetic fragment 5 is proceeded to into SyBE_Sc0014D011, it is (middle that obtained strains are named as SyBE_Sc0014D014 Bacterial strain)；

9). genetic fragment 6 is proceeded to into SyBE_Sc0014D014, obtained strains are named as SyBE_Sc0014D017；

10). genetic fragment 7 is proceeded to into SyBE_Sc0014D017, obtained strains are named as SyBE_Sc0014D018；

Wherein, SyBE_Sc0014D014 is deposited in Chinese microorganism strain preservation management committee on November 27th, 2015 Member's meeting common micro-organisms center, deposit number is CGMCC No.11748.

Additionally, can insert one section in any site for not affecting its normal function in the bacterial strain constructed by the present invention being used for Recognize the sequence of the bacterial strain, it functions as " watermark " or " bar code ", prevent build recombinant bacterial strain leak and cannot be fast Victory identification, described recognition sequence can be any suitable sequence of current area, and it does not interfere with bacterial strain normal function, can use Detect after corresponding amplimer amplification.Described recognition sequence is specially：Two ends are tag sequences (amplimer), middle The nucleotide sequence of one or more amino acid in encode C, M, E, P, S, A, H, V, N, W, T, Y, G, I, D, overall size is 180-220bp, the insertion method of recognition sequence adopts conventional method.

With reference to embodiment, the present invention is expanded on further.

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

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

△ gal1, △ gal7, △ gal10 are built first::DR-Kl URA3-DR knock out box, that is, box fragment 1 is knocked out, with matter Grain pWJ1042 is template, design upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arms and DR-Kl URA3-DR The knockout box fragment of nutritional labeling, the homologous recombination machinery using yeast itself is whole by Li-acetate method yeast conversion by the fragment Close on Yeast genome, using SD-URA solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, list after conversion The kilnitamin powder 2g/L of scarce uracil, 2% agar powder) screened, the transformant for obtaining is carried after point pure culture Take Yeast genome and enter performing PCR checking, to verifying that correct recombinant bacterial strain is applied with a little bacterium solution is taken after YPD fluid nutrient medium cultures Cloth 5- fluororotic acids (5-FOA) solid panel (respectively there is the direct repeat DR of 143bp at DR-KlURA3-DR nutritional labelings two ends, Yeast itself can occur homologous recombination and delete URA3 genes and one of DR using this two sections of identical sequences；YPD is trained Foster gene is that without this screening pressure of amino acid nutrient defect, the saccharomycete of such spontaneous deletion URA3 can grow.So Screened with 5-FOA afterwards, because the bacterial strain containing URA3 can make 5-FOA become paired under the enzyme effect of URA3 gene codes The virose material of cell, makes yeast cells to grow on the culture medium containing 5-FOA, and so as to filter out URA genes are deleted Bacterium), genome extracted after point pure culture of picking single bacterium colony enter performing PCR checking screening and deleted by spontaneous restructuring between DR sequences The correct bacterial strain of URA3 genes, i.e. three gene knock-out bacterial strains, are named as SyBE_Sc0014D003.Knock out gal1, gal7, gal10 Bacterial strain will not metabolism D- galactolipins, such that it is able to maintain intracellular inducer galactose concentration constant, realize efficiently induction.

Then, △ ypl062w are built on the basis of three gene knock-out bacterial strain SyBE_Sc0014D003::KanMX is knocked out Box, with the mono- genomes for singly striking bacterial strain YPL062W struck in storehouse of BY4742 as template, design upstream and downstream primer PCR amplification tape base Because of YPL062W upstream and downstream homologous sequence and the knockout box fragment 2 of kanMX resistance labels, the fragment is integrated by yeast conversion To on Yeast genome, bacterial strain (kanMX resistances are struck in the YPD solid panels screening four after conversion using the resistances of G418 containing 200mg/L Label can produce resistance to this antibiotic of Geneticin G418, so as to select successful knockout gene using G418 screens The bacterium of ypl062w), the transformant for obtaining is extracted genome after point pure culture and enters performing PCR checking, to verifying correct restructuring Bacterial strain preserves glycerol stock, and is named as SyBE_Sc0014D004.

Embodiment 2：The structure of genetic fragment 1

Amplification CYC1 terminators, Bt crtI, GAL10 promoters, GAL1 promoters, Pa crtB, PGK1 terminator and suitable It is secondary to be stitched together by OE-PCR methods, obtain fragment T of the two ends comprising HindIII and XhoI restriction enzyme sites_CYC1-crtI- P_GAL10-P_GAL1-crtB-T_PGK1；

Meanwhile, the homologous 631bp sequences of amplification yeast trp1 site upstreams, the homologous 733bp sequences of yeast trp1 sites downstreams And sequentially pass through OE-PCR methods and be stitched together, two ends are obtained comprising SacI and ApaI restriction enzyme sites, and in yeast trp1 sites Fragment comprising HindIII and XhoI restriction enzyme sites between the homologous sequence of upstream and downstream, then by SacI and ApaI restriction enzyme sites Carrier pRS405 is connected into, TRP1 integrated plasmid pRS405-TRP are obtained, by fragment T obtained above_CYC1-crtI-P_GAL10- P_GAL1-crtB-T_PGK1It is attached by HindIII and XhoI restriction enzyme sites with pRS405-TRP plasmids, obtains genetic fragment 1 Integrated plasmid, is designated as pRS405-TRP-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1；

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with SacI and ApaI double enzyme sites respectively, obtain genetic fragment 1, nucleotide sequence ginseng The SEQ ID NO seen in patent CN105420134A:Shown in 1.

Embodiment 3：The structure of genetic fragment 2

By ACT1 terminators, HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoters, the TM that truncate CrtE, GPM1 terminator sequentially passes through OE-PCR methods and is stitched together, and obtains piece of the two ends comprising BamHI and XhoI restriction enzyme sites Section T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1；Meanwhile, the homologous 561bp sequences of yeast leu2 site upstreams, LEU2 are marked Note, TDH2 terminators, the homologous 584bp sequences of yeast leu2 sites downstreams sequentially pass through OE-PCR methods and are stitched together, and obtain two End includes SacI and ApaI restriction enzyme sites, and BamHI is included between TDH2 terminators, yeast leu2 sites downstream homologous sequences With the fragment of XhoI restriction enzyme sites, carrier pRS405 is connected into by SacI and ApaI restriction enzyme sites, obtains LEU2 integrated plasmids pRS405-LEU.By above-mentioned fragment T for obtaining_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1Pass through with pRS405-LEU plasmids BamHI and XhoI restriction enzyme sites are attached, and obtain the integrated plasmid of genetic fragment 2, are designated as pRS405-LEU-T_ACT1-tHMGR1- P_GAL10-P_GAL1-crtE-T_GPM1。

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with SacI and ApaI double enzyme sites respectively, obtain genetic fragment 2, leu2 site upstreams Homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-crtE-T_GPM1- leu2 sites downstream homologous sequences, nucleotides Sequence is referring to the SEQ ID NO in patent CN105420134A:Shown in 2.

Embodiment 4：The structure of genetic fragment 3

TDH2 terminators and its upstream 869bp homologous sequences in amplification gene fragment 2, ACT1 terminators and downstream 355bp homologous sequences, then terminate TDH2 terminators and its upstream homologous sequence, DR-KlURA3-DR nutritional labelings, CYC1 ACT1 terminators and downstream homologous sequence sequentially passes through OE-PCR methods in son, Bt crtI, GAL7 promoters, genetic fragment 2 It is stitched together, obtains genetic fragment 3 of the two ends comprising PmeI restriction enzyme sites, i.e. TDH2 terminators and its upstream homologous sequence-DR- KlURA3-DR-T_CYC1-Bt crtI-P_GAL7- ACT1 terminators and downstream homologous sequence, are connected with flat ends vector pJET1.2 The integrated plasmid of genetic fragment 3 is obtained, pleu-DR-Kl URA3-DR-T are designated as_CYC1-BtcrtI-P_GAL7。

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with PmeI restriction enzyme sites, obtain genetic fragment 3, nucleotide sequence is referring to patent SEQ ID NO in CN105420134A:Shown in 3.

Embodiment 5：The structure of genetic fragment 4

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is carried out into amalgamation and expression, by OE-PCR side The C-terminal of BTS1 is connected to form fusion BTS1- by method with GGGS linker (GGTGGTGGTTCT) with the N-terminal of ERG20 ERG20；T is expanded from embodiment 4 on LEU2 integrated plasmids pRS405-LEU_ACT1-tHMGR1-P_GAL10-P_GAL1Fragment；

By fragment T_ACT1-tHMGR1-P_GAL10-P_GAL1, fusion BTS1-ERG20, FBA1 terminator pass through OE-PCR side Method is stitched together, and obtains fragment T of the two ends comprising BamHI and PstI restriction enzyme sites_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1- ERG20)-T_FBA1；Meanwhile, PCR expands respectively the homologous 312bp sequences of yeast his3 site upstreams, HIS3 marks, ENO2 terminators And the homologous 578bp sequences of yeast his3 sites downstreams, and sequentially spliced by OE-PCR methods, obtain two ends comprising SacI and ApaI restriction enzyme sites, and BamHI and PstI digestions position is included between ENO2 terminators, yeast his3 sites downstream homologous sequences The fragment of point, by SacI and ApaI restriction enzyme sites carrier pRS405 is connected into, and obtains HIS3 integrated plasmid pRS405-HIS.Will Above-mentioned fragment T for arriving_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1With pRS405-HIS plasmids by BamHI and PstI restriction enzyme sites are attached, and obtain the integrated plasmid of genetic fragment 4, are designated as pRS405-HIS-T_ACT1-tHMGR1-P_GAL10- P_GAL1-(BTS1-ERG20)-T_FBA1；

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with SacI and ApaI double digestions, obtain genetic fragment 4, the i.e. homologous sequence of his3 site upstreams Row-HIS3-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1- his3 sites downstream homologous sequences, nucleotides sequence Row are referring to the SEQ ID NO in patent CN105420134A:Shown in 4.

Embodiment 6：The structure of genetic fragment 5

Amplification yeast YPRCdelta15 site upstream 606bp homologous sequences, DR-Kl URA3-DR nutritional labelings, CPS1 ends Only son, transcription factor INO2, GAL1 promoter, saccharomyces cerevisiae YPRCdelta15 sites downstream 359bp homologous sequences are sequentially passed through OE-PCR methods are stitched together, and obtain genetic fragment 5 of the two ends comprising PmeI restriction enzyme sites, i.e. YPRCdelta15 site upstreams Homologous sequence-DR-KlURA3-DR-T_CPS1-INO2-P_GAL1- YPRCdelta15 sites downstream homologous sequences, will be obtained above Fragment is connected with flat ends vector pJET1.2, obtains the integrated plasmid of genetic fragment 5 and is designated as pYPRCdelta15-DR-KlURA3- DR-T_CPS1-INO2-P_GAL1。

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with PmeI restriction enzyme sites, obtain genetic fragment 5, nucleotide sequence is referring to patent SEQ ID NO in CN105420134A:Shown in 5.

Embodiment 7：The structure of genetic fragment 6

Amplification yeast YNRCdelta9 site upstream 256bp homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 Terminator, DR-Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream 267bp homologous sequences, sequentially pass through OE- PCR method is stitched together, and genetic fragment 6 of the two ends comprising PmeI restriction enzyme sites is obtained, by fragment obtained above and flat end Carrier pJET1.2 connects, and obtains the integrated plasmid of genetic fragment 6 and is designated as pYNRCdelta9-P_GAL1-INO2-T_CPS1-DR-Kl URA3-DR；

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with PmeI restriction enzyme sites, obtain genetic fragment 6, nucleotide sequence is referring in the present invention SEQ ID NO:Shown in 1.

Embodiment 8：The structure of genetic fragment 7

Amplification yeast YFLWtau1 site upstream 297bp homologous sequences, PGK1 promoters, from the gene of Vitreoscilla VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream 208bp homologous sequences, it is suitable It is secondary to be stitched together by OE-PCR methods, genetic fragment 7 of the two ends comprising PmeI restriction enzyme sites is obtained, by fragment obtained above It is connected with flat ends vector pJET1.2, obtains the integrated plasmid of genetic fragment 7 and be designated as pYFLWtau1-P_PGK1-VHb-T_ACT1-DR-Kl URA3-DR；

Integrated plasmid is transformed in E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to guarantee that purpose fragment connection is correct and base sequence is not undergone mutation.

After checking is correct, cut with PmeI restriction enzyme sites, obtain genetic fragment 7, nucleotide sequence is referring in the present invention SEQ ID NO:Shown in 2.

Embodiment 9：Genetic fragment 1-2 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Fragment is converted four gene knockout yeast strain SyBE_Sc0014D004 by genetic fragment 1 using Li-acetate method, is passed through There is restructuring with trp1 sites on Yeast genome and be incorporated on genome in TRP1 upstream and downstream homologous sequence.Adopt after conversion SD-TRP solid panels (synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L, the kilnitamin powder 2g/ of single scarce tryptophan L, 2% agar powder) screened, the transformant for obtaining carries out extraction Yeast genome after line point pure culture and enters performing PCR checking, To verifying that correct recombinant bacterial strain preserves glycerol stock and is respectively designated as SyBE_Sc0014D005.

Then, adopt Li-acetate method by fragment transformed yeast bacterial strain SyBE_Sc0014D005 genetic fragment 2, pass through There is restructuring with leu2 sites on Yeast genome and be incorporated on genome in LEU2 upstream and downstream homologous sequence.Adopt after conversion SD-TRP-LEU solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, double scarce tryptophans and leucic mixing Powder of amino acids 2g/L, 2% agar powder) screened, the transformant for obtaining carries out extracting yeast genes after line point pure culture Group enters performing PCR checking, to verifying that correct recombinant bacterial strain preserves glycerol stock and is named as SyBE_Sc0014D006.

Embodiment 10：Genetic fragment 1-3 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Genetic fragment 3 is passed through fragment conversion restructuring yeast strains SyBE_Sc0014D006 using Li-acetate method There is restructuring and be inserted on genome whole before in TDH2 terminators upstream homologous sequence and ACT1 terminators downstream homologous sequence The centre of the genetic fragment 2 of conjunction.SD-URA-TRP-LEU solid panels (synthetic yeast nitrogen source YNB 6.7g/L, Portugal are adopted after conversion Grape sugar 20g/L, lacks the kilnitamin powder 2g/L of tryptophan, leucine and uracil, 2% agar powder) screened, obtain Transformant carry out extracting Yeast genome after line point pure culture and enter performing PCR checking, to verifying correct recombinant bacterial strain YPD A little bacterium solution coating 5- fluororotic acids (5-FOA) solid panel is taken after fluid nutrient medium culture, is carried after picking single bacterium colony point pure culture Take genome enter performing PCR checking screening the correct bacterial strain of URA genes is deleted by spontaneous restructuring between DR sequences, by correct bacterial strain It is respectively designated as SyBE_Sc0014D007.

Embodiment 11：Genetic fragment 1-4 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Genetic fragment 4 is passed through fragment conversion restructuring yeast strains SyBE_Sc0014D007 using Li-acetate method There is restructuring with his3 sites on Yeast genome and be incorporated on genome in HIS3 upstream and downstream homologous sequence.Yeast after conversion Using SD-TRP-LEU-HIS solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lack tryptophan, histidine and Leucic kilnitamin powder 2g/L, 2% agar powder) screened, the transformant for obtaining carries out line point pure culture Yeast genome is extracted afterwards and enters performing PCR checking, to verifying that correct recombinant bacterial strain preserves glycerol stock and is respectively designated as SyBE_ Sc0014D009。

Embodiment 12：The knockout of rox1 genes

△ rox1 are built first::DR-Kl URA3-DR knock out box, that is, box fragment 3 is knocked out, with plasmid pWJ1042 as mould Plate, designs the knockout of upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arms and DR-Kl URA3-DR nutritional labelings Box fragment, recombinant yeast is incorporated into using the homologous recombination machinery of yeast itself by the fragment by Li-acetate method yeast conversion On strain SyBE_Sc0014D009, SD-TRP-LEU-HIS-URA solid panels (synthetic yeast nitrogen source YNB 6.7g/ is adopted after conversion L, glucose 20g/L, lack uracil, leucine, histidine, the kilnitamin powder 2g/L of tryptophan, 2% agar powder) enter Row screening, the transformant for obtaining is extracted Yeast genome after point pure culture and enters performing PCR checking, to verifying correct recombinant bacterium With a little bacterium solution coating 5- fluororotic acids (5-FOA) solid panel is taken after YPD fluid nutrient medium cultures, (DR-Kl URA3-DR seek for strain Respectively there is the direct repeat DR of 143bp at foster label two ends, and yeast itself can occur homologous heavy using this two sections of identical sequences Group and delete URA3 genes and one of DR；YPD culturing genes are this screening pressure of no amino acid nutrient defect, this The saccharomycete of sample spontaneous deletion URA3 can grow.Then screened with 5-FOA, because the bacterial strain containing URA3 is in URA3 5-FOA can be made under the enzyme effect of gene code to become the virose material of paired cell, make yeast cells in the culture containing 5-FOA Can not grow on base, so as to filter out the bacterium for deleting URA genes), extract genome after picking single bacterium colony point pure culture and enter performing PCR Checking screening deletes the correct bacterial strain of URA3 genes by spontaneous restructuring between DR sequences, to verifying that correct recombinant bacterial strain is preserved Glycerol stock is simultaneously respectively designated as SyBE_Sc0014D011.

Embodiment 13：Genetic fragment 1-5 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Genetic fragment 5 is passed through fragment conversion restructuring yeast strains SyBE_Sc0014D011 using Li-acetate method There is restructuring with YPRCdelta15 sites on Yeast genome and be incorporated into base in YPRCdelta15 sites upstream and downstream homologous sequence Because in group.Yeast adopts SD- TRP-LEU-HIS-URA solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose after conversion 20g/L, lacks uracil, leucine, histidine, the kilnitamin powder 2g/L of tryptophan, 2% agar powder) screened, obtain To transformant carry out extracting Yeast genome after line point pure culture and enter performing PCR checking, to verifying that correct recombinant bacterial strain is used A little bacterium solution coating 5- fluororotic acids (5-FOA) solid panel (DR-Kl URA3-DR nutrition marks are taken after YPD fluid nutrient medium cultures Signing two ends respectively has the direct repeat DR of 143bp, and yeast itself can be using this two sections of identical sequences generation homologous recombinations Delete URA3 genes and one of DR；YPD culturing genes are this screening pressure of no amino acid nutrient defect, so certainly Send out and delete the saccharomycete of URA3 and can grow.Then screened with 5-FOA, because the bacterial strain containing URA3 is in URA3 genes 5-FOA can be made under the enzyme effect of coding to become the virose material of paired cell, make yeast cells on the culture medium containing 5-FOA Can not grow, so as to filter out the bacterium for deleting URA genes), genome is extracted after picking single bacterium colony point pure culture and enters performing PCR checking Screening deletes the correct bacterial strain of URA3 genes by spontaneous restructuring between DR sequences, to verifying that correct recombinant bacterial strain preserves glycerine Bacterium is simultaneously respectively designated as SyBE_Sc0014D014.

Embodiment 14：Genetic fragment 1-6 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Genetic fragment 6 is passed through fragment conversion restructuring yeast strains SyBE_Sc0014D014 using Li-acetate method There is restructuring with YPRCdelta15 sites on Yeast genome and be incorporated into base in YNRCdelta9 sites upstream and downstream homologous sequence Because in group.Yeast adopts SD-TRP-LEU-HIS-URA solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose after conversion 20g/L, lacks uracil, leucine, histidine, the kilnitamin powder 2g/L of tryptophan, 2% agar powder) screened, obtain To transformant carry out extracting Yeast genome after line point pure culture and enter performing PCR checking, to verifying that correct recombinant bacterial strain is used A little bacterium solution coating 5- fluororotic acids (5-FOA) solid panel (DR-Kl URA3-DR nutrition marks are taken after YPD fluid nutrient medium cultures Signing two ends respectively has the direct repeat DR of 143bp, and yeast itself can be using this two sections of identical sequences generation homologous recombinations Delete URA3 genes and one of DR；YPD culturing genes are this screening pressure of no amino acid nutrient defect, so certainly Send out and delete the saccharomycete of URA3 and can grow.Then screened with 5-FOA, because the bacterial strain containing URA3 is in URA3 genes 5-FOA can be made under the enzyme effect of coding to become the virose material of paired cell, make yeast cells on the culture medium containing 5-FOA Can not grow, so as to filter out the bacterium for deleting URA genes), genome is extracted after picking single bacterium colony point pure culture and enters performing PCR checking Screening deletes the correct bacterial strain of URA3 genes by spontaneous restructuring between DR sequences, to verifying that correct recombinant bacterial strain preserves glycerine Bacterium is simultaneously respectively designated as SyBE_Sc0014D017.

Embodiment 15：Genetic fragment 1-7 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D

Genetic fragment 7 is passed through fragment conversion restructuring yeast strains SyBE_Sc0014D017 using Li-acetate method There is restructuring with YPRCdelta15 sites on Yeast genome and be incorporated into gene in YFLWtau1 sites upstream and downstream homologous sequence In group.Yeast adopts SD-TRP-LEU-HIS-URA solid panels (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/ after conversion L, lacks uracil, leucine, histidine, the kilnitamin powder 2g/L of tryptophan, 2% agar powder) screened, obtain Transformant carries out extraction Yeast genome after line point pure culture and enters performing PCR checking, to verifying correct recombinant bacterial strain YPD liquid A little bacterium solution coating 5- fluororotic acids (5-FOA) solid panel (DR-Kl URA3-DR nutritional labelings two ends are taken after body medium culture Respectively there is the direct repeat DR of 143bp, yeast itself can occur homologous recombination and delete using this two sections of identical sequences URA3 genes and one of DR；YPD culturing genes are this screening pressure of no amino acid nutrient defect, so spontaneous to delete Except the saccharomycete of URA3 can grow.Then screened with 5-FOA, because the bacterial strain containing URA3 is in URA3 gene codes Enzyme effect under 5-FOA can be made to become the virose material of paired cell, make the yeast cells can not on the culture medium containing 5-FOA Growth, so as to filter out the bacterium for deleting URA genes), genome is extracted after picking single bacterium colony point pure culture and enters performing PCR checking screening The correct bacterial strain of URA3 genes is deleted by spontaneous restructuring between DR sequences, to verifying that correct recombinant bacterial strain preserves glycerol stock simultaneously It is respectively designated as SyBE_Sc0014D018.

Embodiment 16：The shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain

Bacterial strain：SyBE_Sc0014D0014 (bacterial strain in existing patent CN105420134A), SyBE_Sc0014D017, SyBE_Sc0014D018。

Test method：

Seed culture medium：20g/L glucose, 20g/L peptones, 10g/L yeast extracts, 50mg/L uracils；

Fermentation medium：20g/L glucose, 20g/L peptones, 10g/L yeast extracts, 20mg/L uracils, 10g/L D- (+)-galactolipin.

Above-mentioned bacterial strains are inoculated in 5mL seed culture mediums, in 30 DEG C, 250rpm 14-16h is cultivated, it is dense with initial thalline Degree OD₆₀₀=0.2 transfers in fresh 25mL seed culture mediums, cultivates 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 mediums, cultivates under the conditions of 30 DEG C, 250rpm, Sampling monitoring yield of lycopene during fermentation 48h.

Lycopene quantitative approach：The zymotic fluid of two equal portions, 4000g centrifugation 2min collects thallines are taken, and is washed twice.Will A copy of it thalline is placed in 80 DEG C and dries to constant weight, calculating dry cell weight of weighing；Another thalline is extracted to product, concrete side Method is：With 3N HCl re-suspended cells, it 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, and after washing 2 times acetone, and the 5min that is vortexed are added；Acetone phase is finally collected by centrifugation, is used Upper ultraviolet liquid phase detection after 2 μm of membrane filtrations, lycopene Detection wavelength is 471nm.As a result Fig. 3 and Fig. 4 is seen.

Result of the test：From Fig. 3 and Fig. 4, continue to raise transcription on the basis of Saccharomyces Cerevisiae in S yBE_Sc0014D014 Activity factor INO2, obtains bacterial strain SyBE_Sc0014D017, and yield of lycopene is carried from 54.62mg/gDCW (318.13mg/L) Up to 60.97mg/gDCW (356.22mg/L).The hemoglobin gene VHb in Vitreoscilla source is introduced on this basis to add Strong intracellular oxygen conveying, obtains bacterial strain SyBE_Sc0014D018, and its yield of lycopene is significantly improved, and reaches 66.14mg/ GDCW (376.78mg/L), this has belonged to highest in the open report that lycopene is produced currently with recombinant Saccharomyces cerevisiae.

Embodiment 17：The shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain

Method according to embodiment of the present invention 1-15 repeats to build restructuring yeast strains, names respectively and according to embodiment 16 Method detects yield of lycopene, as a result such as following table：

The restructuring yeast strains yield of lycopene of table 1

Strain number (includes fragment 1-6)	Yield of lycopene	Strain number (includes fragment 1-7)	Yield of lycopene
				SyBE_Sc0014D027	60.97mg/gDCW	SyBE_Sc0014D028	65.03mg/gDCW
SyBE_Sc0014D037	59.88mg/gDCW	SyBE_Sc0014D038	64.98mg/gDCW
				SyBE_Sc0014D047	60.04mg/gDCW	SyBE_Sc0014D048	64.34mg/gDCW

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

SEQUENCE LISTING

<110>University Of Tianjin

<120>A kind of restructuring yeast strains and its construction method and application

<130> MP1614119

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 3640

<212> DNA

<213>Artificial sequence

<400> 1

gacattagga cgccagggta gccagtatta ttcaagtcca tagaacagcc tgtatacctt 60

cacttcatta ggtatattct tggttatgcg ttatttaaat cctcatctgc cgctgcttaa 120

aaaaagcagc taaagtgttg cgtaggcact tcgaacaagt agtcagtaat atcacctttt 180

aacatctaat catcaaaaga gacatttttt gggattaatt gtttataaaa gctatgaact 240

taggtctaca gaatgtagta cggattagaa gccgccgagc gggtgacagc cctccgaagg 300

aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc 360

tcgcgccgca ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag 420

aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa tgaacgaatc aaattaacaa 480

ccataggatg ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa 540

gcgatgattt ttgatctatt aacagatata taaatgcaaa aactgcataa ccactttaac 600

taatactttc aacattttcg gtttgtatta cttcttattc aaatgtaata aaagtatcaa 660

caaaaaattg ttaatatacc tctatacttt aacgtcaagg agaaaaaacc ccgtaaacaa 720

tgtcccaagc aactgggaac gaattactgg gtatcctaga tctggataac gatatagact 780

ttgaaactgc ttaccaaatg ctcagcagta acttcgacga ccaaatgtct gcgcacatac 840

atgaaaacac gtttagtgca acttcccctc ctctgttaac acacgagctc ggcataattc 900

ctaacgtagc aaccgtgcaa ccctctcacg tagaaactat acctgccgat aaccaaactc 960

atcatgctcc tttgcatact catgcacact atctaaatca caaccctcat caaccaagca 1020

tgggttttga tcaagcgctt ggtctcaagt tgtctccttc cagttcgggg ttgttgagca 1080

cgaatgaatc gaatgccatt gaacagtttt tagacaatct aatatcacag gatatgatgt 1140

cttccaacgc ttccatgaac tccgaatcac atctacatat aagatcacca aaaaagcagc 1200

ataggtatac cgaattaaat caaagatatc ctgaaacaca tccacacagt aacacagggg 1260

agttacccac aaacacagca gatgtgccaa ctgagttcac cacgagggaa ggacctcatc 1320

agcctatcgg caatgaccac tacaacccgc caccgttttc agtacctgag atacgaatcc 1380

cagactctga tattccagcc aatatcgagg acgaccctgt gaaggtacgg aaatggaaac 1440

acgttcaaat ggagaagata cgaagaataa acaccaaaga agcctttgaa aggctcatta 1500

aatcagtaag gaccccaccg aaggaaaacg ggaaaagaat tcccaagcat attcttttaa 1560

cttgtgtaat gaacgatatc aagtccatta gaagcgcaaa tgaagcacta cagcacatac 1620

tggatgattc ctgagcgcaa tgattgaata gtcaaagatt tttttttttt aatttttttt 1680

ttttaatttt tttttttttt catagaactt tttatttaaa taaatcacgt ctatatatgt 1740

atcagtataa cgtaaaaaaa aaaacaccgt cagttaaaca aaacataaat aaaaaaaaaa 1800

agaagtgtca aatcaagtgt caaatgtgat tctgggtaga agatcggtct gcattggatg 1860

gtgtaacgca tttttttaca cacattactt gcctcgagca tcaaatggtg gttattcgtg 1920

gatctatatc acgtgatttg cttaagaatt gtcgttcatg gtgacacttt tagctttgac 1980

atgattaagt catctcaatt gatgttatct aaagtcattt caactatcta agatgtggtt 2040

gtgattgggc cattttgtga aagccagtac gccagcgtca atacactccc gtcaattagt 2100

tgcaccatgt ccacaaaatc atataccagt agagctgaga ctcatgcaag tccggttgca 2160

tcgaaacttt tacgttaatg gatgaaaaga agaccaattt gtgtgcttct cttgacgttc 2220

gttcgactga tgagctattg aaacttgttg aaacgttggg tccatacatt tgccttttga 2280

aaacacacgt tgatatcttg gagatttcag ttatgagggt actgtcgttc cattgaaagc 2340

attggcagag aaatacaagt tcttgatatt tgaggacaga aaattcgccg atatcggtaa 2400

cacagtcaaa ttacaatata catcgggcgt taccgtatcg cagaatggtc tgatatcacc 2460

aacgcccacg gggttactgg tgctggtatt gttgctggct tgaaacaagg tgcgcaagag 2520

gtcaccaaag aaccaagggg attattgatg cttgctaatt gtcttccaag ggttctctag 2580

cacacggtga atatactaag ggtaccgttg atattgcaaa gagtgataaa gatttcgtta 2640

ttgggttcat tgctcagaac gatatgggag gaagagaaga aggtttgatt ggctaatcat 2700

gaccccaggt gtaggtttag acgacaaagg cgatgcattg ggtcagcagt acagaaccgt 2760

cgacgaagtt gtaagtggtg gatcagatat catcattgtt ggcagaggac tttcgccaag 2820

ggtagagatc ctaaggttga aggtgaaaga tacagaaatg ctggatggga agcgtaccaa 2880

aagagaatca gcgctcccca ttaattatac aggaaactta atagaacaaa tcacatatta 2940

atctaatagc cacctgcatt ggcacggtgc aacactactt caacttcatc ttacaaaaag 3000

atcacgtgat ctgttgtatt gaactgaaaa ttttttgttt gcttctctct ctctctcttt 3060

catttgtgag atttaaaaac cagaaactac atcatcgaat tccagctgac caccatgagc 3120

cgaattccag cacactggcg gccgttacta gtggatccga gctcggtacc aagctgggct 3180

gcaggaattc gtatcaagct tatcgatgtg attctgggta gaagatcggt ctgcattgga 3240

tggtggtaac gcattttttt acacacatta cttgcctcga gcatcaaatg gtggttattc 3300

gtggatctat atcacgtgtt tgcttaagaa ttgtcgttca tggtgacacg ggcccggtac 3360

ccaattcgcc ctaacaagta gtatgagtac acaccaatag atgattaatt ttttccgaca 3420

atcaaatatt gggatcataa acacacataa tcgccatatc cgttaattcg ggtttcaatc 3480

acttcgtttg tctatcgtat cgcagcctag tgaatattta attctttcaa taaaaaaggc 3540

ttaaaatcac catgaaaatc acaagaggaa tctttcaaca agaacaatag tatacaatcc 3600

atgaatgaag gagttcatat ttgactagaa ctatacaaag 3640

<210> 2

<211> 3559

<212> DNA

<213>Artificial sequence

<400> 2

aaagacgtat agtccaaagc aatattcaag gaaactacaa agtgagatta gctacgtaga 60

aataggagaa aaagtataaa tttaaagtta aattcagatc tcaaattctc ttaatatttg 120

aaataaatcc gctgcgtgac aaatcccgtg atgatctcgg aatatttata tgttaccatt 180

tatacttatg gtagaattat actcactaat gaatgtacgg ttactatttg gaacaagtgg 240

tcatcaattg ccatagtaac attttgaatg cttgcatcgt gcatgaatac ataccgctat 300

tttagattcc tgacttcaac tcaagacgca cagatattat aacatctgca taataggcat 360

ttgcaagaat tactcgtgag taaggaaaga gtgaggaact atcgcatacc tgcatttaaa 420

gatgccgatt tgggcgcgaa tcctttattt tggcttcacc ctcatactat tatcagggcc 480

agaaaaagga agtgtttccc tccttcttga attgatgtta ccctcataaa gcacgtggcc 540

tcttatcgag aaagaaatta ccgtcgctcg tgatttgttt gcaaaaagaa caaaactgaa 600

aaaacccaga cacgctcgac ttcctgtctt cctattgatt gcagcttcca atttcgtcac 660

acaacaaggt cctagcgacg gctcacaggt tttgtaacaa gcaatcgaag gttctggaat 720

ggcgggaaag ggtttagtac cacatgctat gatgcccact gtgatctcca gagcaaagtt 780

cgttcgatcg tactgttact ctctctcttt caaacagaat tgtccgaatc gtgtgacaac 840

aacagcctgt tctcacacac tcttttcttc taaccaaggg ggtggtttag tttagtagaa 900

cctcgtgaaa cttacattta catatatata aacttgcata aattggtcaa tgcaagaaat 960

acatatttgg tcttttctaa ttcgtagttt ttcaagttct tagatgcttt ctttttctct 1020

tttttacaga tcatcaagga agtaattatc tactttttac aacaaatata aaacaatgtt 1080

ggaccaacaa accataaata tcataaaagc cacagtacca gtcttgaagg aacacggtgt 1140

aactatcact accacattct ataagaattt gttcgctaag catccagaag ttagaccatt 1200

gtttgatatg ggtagacaag aatctttaga acaaccaaaa gcattggcaa tgactgtttt 1260

ggctgcagct caaaacatcg aaaatttgcc agctatctta ccagcagtta agaaaattgc 1320

tgttaagcat tgtcaagcag gtgttgcagc tgcacattat ccaattgttg gtcaagaatt 1380

gttgggtgct attaaagaag ttttgggtga cgctgcaaca gatgatattt tagatgcttg 1440

gggtaaagca tacggtgtaa tagccgatgt ctttatccaa gttgaagccg atttgtatgc 1500

ccaagccgtt gaatgatctc tgcttttgtg cgcgtatgtt tatgtatgta cctctctctc 1560

tatttctatt tttaaaccac cctctcaata aaataaaaat aataaagtat ttttaaggaa 1620

aagacgtgtt taagcactga ctttatctac tttttgtacg ttttcattga tataatgtgt 1680

tttgtctctc ccttttctac gaaaatttca aaaattgacc aaaaaaagga atatatatac 1740

gaaaaactat tatatttata tatcatagtg ttgataaaaa atgtttatcc attggaccgt 1800

gtagtgattc tgggtagaag atcggtctgc attggatggt gtaacgcatt tttttacaca 1860

cattacttgc ctcgagcatc aaatggtggt tattcgtgga tctatatcac gtgatttgct 1920

taagaattgt cgttcatggt gacactttta gctttgacat gattaagtca tctcaattga 1980

tgttatctaa agtcatttca actatctaag atgtggttgt gattgggcca ttttgtgaaa 2040

gccagtacgc cagcgtcaat acactcccgt caattagttg caccatgtcc acaaaatcat 2100

ataccagtag agctgagact catgcaagtc cggttgcatc gaaactttta cgttaatgga 2160

tgaaaagaag accaatttgt gtgcttctct tgacgttcgt tcgactgatg agctattgaa 2220

acttgttgaa acgttgggtc catacatttg ccttttgaaa acacacgttg atatcttgga 2280

gatttcagtt atgagggtac tgtcgttcca ttgaaagcat tggcagagaa atacaagttc 2340

ttgatatttg aggacagaaa attcgccgat atcggtaaca cagtcaaatt acaatataca 2400

tcgggcgtta ccgtatcgca gaatggtctg atatcaccaa cgcccacggg gttactggtg 2460

ctggtattgt tgctggcttg aaacaaggtg cgcaagaggt caccaaagaa ccaaggggat 2520

tattgatgct tgctaattgt cttccaaggg ttctctagca cacggtgaat atactaaggg 2580

taccgttgat attgcaaaga gtgataaaga tttcgttatt gggttcattg ctcagaacga 2640

tatgggagga agagaagaag gtttgattgg ctaatcatga ccccaggtgt aggtttagac 2700

gacaaaggcg atgcattggg tcagcagtac agaaccgtcg acgaagttgt aagtggtgga 2760

tcagatatca tcattgttgg cagaggactt tcgccaaggg tagagatcct aaggttgaag 2820

gtgaaagata cagaaatgct ggatgggaag cgtaccaaaa gagaatcagc gctccccatt 2880

aattatacag gaaacttaat agaacaaatc acatattaat ctaatagcca cctgcattgg 2940

cacggtgcaa cactacttca acttcatctt acaaaaagat cacgtgatct gttgtattga 3000

actgaaaatt ttttgtttgc ttctctctct ctctctttca tttgtgagat ttaaaaacca 3060

gaaactacat catcgaattc cagctgacca ccatgagccg aattccagca cactggcggc 3120

cgttactagt ggatccgagc tcggtaccaa gctgggctgc aggaattcgt atcaagctta 3180

tcgatgtgat tctgggtaga agatcggtct gcattggatg gtggtaacgc atttttttac 3240

acacattact tgcctcgagc atcaaatggt ggttattcgt ggatctatat cacgtgtttg 3300

cttaagaatt gtcgttcatg gtgacacggg cccggtaccc aattcgccct atcagcgtgt 3360

gttttatact tctcttatat agtataagaa gatccatatt taatcttcat taacactact 3420

tcttaacctc taattaccaa cgggtcaatg ttaggataat tgttggcatt ccattgttat 3480

taaagggaat caaagtatat taaaaattct tcccaaggat gtaataacct aaaaaaggaa 3540

gcccatattt ctatataat 3559

Claims (20)

1. a kind of restructuring yeast strains, it is characterised in that the Yeast genome knock out gal1, gal7, gal10, ypl062w, Rox1 genes, and comprising the following genetic fragment on Jing yeast autologous recombination and integrations to its genome：

Yeast trp1 site upstream homologous sequences, CYC1 terminators, Bt crtI, GAL10 promoters, GAL1 promoters, Pa The genetic fragment 1 that crtB, PGK1 terminator, yeast trp1 sites downstream homologous sequences are sequentially spliced；

Yeast leu2 site upstream homologous sequences, LEU2 marks, TDH2 terminators, ACT1 terminators, the HMG-CoA reduction of truncation Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoters, Tm crtE, GPM1 terminators, the homologous sequence of yeast leu2 sites downstreams The genetic fragment 2 that row are sequentially spliced；

TDH2 terminators and its upstream homologous sequence, DR-KlURA3-DR nutritional labelings, CYC1 terminators, Bt in genetic fragment 2 ACT1 terminators and genetic fragment 3 that downstream homologous sequence is sequentially spliced in crtI, GAL7 promoter, genetic fragment 2；

Yeast his3 site upstream homologous sequences, HIS3 marks, ENO2 terminators, ACT1 terminators, the HMG-CoA reduction of truncation Enzyme gene tHMGR1, GAL10 promoter, GAL1 promoters, fusion BTS1-ERG20, FBA1 terminator, yeast his3 positions The genetic fragment 4 that point downstream homologous sequence is sequentially spliced；

Yeast YPRCdelta15 site upstream homologous sequences, DR-Kl URA3-DR nutritional labelings, CPS1 terminators, transcription factor The genetic fragment 5 that INO2, GAL1 promoter, saccharomyces cerevisiae YPRCdelta15 sites downstream homologous sequences are sequentially spliced；

Yeast YNRCdelta9 site upstream homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 terminator, DR-Kl The genetic fragment 6 that URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream homologous sequences are sequentially spliced；

Wherein, gal1, gal7, gal10, ypl062w, rox1 gene and homologous recombination genetic fragment 1- are knocked out in Yeast genome After 5, intermediate strains, restructuring yeast strains Jing autologous recombination and integration genetic fragment 6 on the basis of intermediate strains are obtained After obtain.

2. bacterial strain according to claim 1, it is characterised in that the genetic fragment 6 such as SEQ ID NO:Shown in 1.

3. bacterial strain according to claim 1, it is characterised in that the intermediate strains are deposit number CGMCC No.11748's Bacterial strain.

4. bacterial strain according to claim 1, it is characterised in that the yeast is that saccharomyces cerevisiae, solution fat belong to yeast or Crewe dimension Category yeast.

5. bacterial strain according to claim 4, it is characterised in that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiaes or BY systems Row saccharomyces cerevisiae.

6. bacterial strain according to claim 5, it is characterised in that the CEN.PK series saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1D。

7. the bacterial strain according to claim 1-6 any one, it is characterised in that also including Jing yeast autologous recombination and integrations Following genetic fragment on its genome：

Yeast YFLWtau1 site upstream homologous sequences, PGK1 promoters, gene VHb, ACT1 from Vitreoscilla terminate The gene piece that son, DR-KlURA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream homologous sequences are sequentially spliced Section 7.

8. bacterial strain according to claim 7, it is characterised in that the genetic fragment 7 such as SEQ ID NO:Shown in 2.

9. bacterial strain according to claim 8, it is characterised in that the bacterial strain is deposit number for the bacterium of CGMCC No.13013 Strain.

10. the bacterial strain according to claim 1-9 any one, it is characterised in that also including recognition sequence, the recognition sequence two Hold as tag sequences, the nucleotides of one or more amino acid in middle C, M, E, P, S, A, H, V, N, W, T, Y, G, I, D for coding Sequence, overall size is 180-220bp.

Bacterial strain described in 11. claim 1-10 any one production lycopene in application and production with lycopene For the application in the product of intermediate product.

The construction method of restructuring yeast strains described in 12. claims 1, it is characterised in that include：

Step 1, structure are same by yeast gal7 downstream of gene homologous sequences, DR-Kl URA3-DR nutritional labelings, gal1 downstream of gene Knockout box fragment 1 that source sequence is sequentially connected with, by ypl062w upstream region of gene homologous sequences, kanMX resistance labels, ypl062w bases Because of the knockout box fragment 2 that downstream homologous sequence is sequentially connected with, and build by yeast rox1 upstream region of gene homologous sequences, DR-Kl The knockout box fragment 3 that URA3-DR nutritional labelings, rox1 downstream of gene homologous sequences are sequentially connected with, is led to using box fragment 1-3 is knocked out Cross the restructuring of yeast autologous and knock out yeast gal1, gal7, gal10, ypl062w and rox1 gene, obtain gene knockout yeast, It is standby；

By yeast trp1 site upstream homologous sequences, CYC1 terminators, Bt crtI, GAL10 promoters, GAL1 promoters, Pa CrtB, PGK1 terminator, yeast trp1 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 1, i.e. trp1 site upstreams Homologous sequence-T_CYC1-crtI-P_GAL10-P_GAL1-crtB-T_PGK1- trp1 sites downstream homologous sequences, it is standby；

By yeast leu2 site upstream homologous sequences, LEU2 marks, TDH2 terminators, ACT1 terminators, truncate HMG-CoA also Nitroreductase gene tHMGR1, GAL10 promoter, GAL1 promoters, Tm crtE, GPM1 terminators, yeast leu2 sites downstreams are homologous Sequence is sequentially spliced, and obtains genetic fragment 2, i.e. leu2 site upstreams homologous sequence-LEU2-T_TDH2-T_ACT1-tHMGR1-P_GAL10- P_GAL1-crtE-T_GPM1- leu2 sites downstream homologous sequences, it is standby；

By TDH2 terminators in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labelings, CYC1 terminators, ACT1 terminators and downstream homologous sequence sequentially splices in Bt crtI, GAL7 promoters, genetic fragment 2, obtain genetic fragment 3, i.e. TDH2 terminators and its upstream homologous sequence-DR-Kl URA3-DR-T_CYC1-Bt crtI-P_GAL7-T_ACT1- ACT1 terminators And homologous sequence downstream, it is standby；

Yeast entogenous FPP synthase gene ERG20 and GGPP synthase gene BTS1 is attached, fusion BTS1- is obtained ERG20, then by yeast his3 site upstream homologous sequences, HIS3 marks, ENO2 terminators, ACT1 terminators, truncate HMG-CoA reductase gene tHMGR1, GAL10 promoter, GAL1 promoters, fusion BTS1-ERG20, FBA1 terminator, Yeast his3 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 4, i.e. his3 site upstreams homologous sequence-HIS3- T_ENO2-T_ACT1-tHMGR1-P_GAL10-P_GAL1-(BTS1-ERG20)-T_FBA1- his3 sites downstream homologous sequences, it is standby；

By yeast YPRCdelta15 site upstream homologous sequences, DR-Kl URA3-DR nutritional labelings, CPS1 terminators, transcription because Sub- INO2, GAL1 promoter, saccharomyces cerevisiae YPRCdelta15 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 5, That is YPRCdelta15 site upstreams homologous sequence-DR-Kl URA3-DR-T_CPS1-INO2-P_GAL1- YPRCdelta15 sites downstreams Homologous sequence, it is standby；

By yeast YNRCdelta9 site upstream homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 terminator, DR-Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream homologous sequences sequentially splice, and obtain genetic fragment 6, i.e., YNRCdelta9 site upstream homologous sequence-P_GAL1-INO2-T_CPS1- DR-Kl URA3-DR-YNRCdelta9 sites downstreams are homologous Sequence, it is standby；

Step 2, genetic fragment 1 is proceeded in the gene knockout yeast by Li-acetate method, by trp1 positions in genetic fragment 1 There is restructuring with trp1 sites on gene knockout Yeast genome and be incorporated on genome in point upstream and downstream homologous sequence；

Genetic fragment 2 is proceeded in the gene knockout yeast by Li-acetate method, by leu2 sites in genetic fragment 2, There is restructuring with leu2 sites on gene knockout Yeast genome and be incorporated on genome in downstream homologous sequence；

Genetic fragment 3 is continued through into Li-acetate method to be transformed in the gene knockout yeast of transgene fragment 2, by gene TDH2 terminators and its upstream homologous sequence in fragment 3, ACT1 terminators and homologous sequence downstream, with the gene piece integrated There is restructuring and be inserted into the gene knockout ferment of integrator gene fragment 2 in the TDH2 terminators and ACT1 terminators site in section 2 On female genome；

Genetic fragment 4 is proceeded in the gene knockout yeast by Li-acetate method, by his3 sites in genetic fragment 4, There is restructuring with his3 sites on gene knockout Yeast genome and be incorporated on genome in downstream homologous sequence；

Genetic fragment 5 is transformed in gene knockout yeast by Li-acetate method, by YPRCdelta15 positions in genetic fragment 5 There is restructuring with YPRCdelta15 sites on gene knockout Yeast genome and be incorporated into genome in point upstream and downstream homologous sequence On；

Intermediate strains are obtained after genetic fragment 1-5 is transformed in gene knockout yeast by Li-acetate method；

Genetic fragment 6 is transformed in intermediate strains by Li-acetate method, by YNRCdelta9 sites in genetic fragment 6, There is restructuring and be incorporated on genome in downstream homologous sequence, recombinated with YNRCdelta9 sites on intermediate strains genome Yeast strain.

13. according to claim 12 construction method, it is characterised in that also include：Genetic fragment 7 is built, and by gene piece Section 7 proceeds to acquisition restructuring yeast strains；

Specifically, by yeast YFLWtau1 site upstream homologous sequences, PGK1 promoters, the gene VHb from Vitreoscilla, ACT1 terminators, DR-Kl URA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream homologous sequences sequentially splice, and obtain Obtain genetic fragment 7, i.e. YFLWtau1 site upstreams homologous sequence-P_PGK1-VHb-T_ACT1- DR-Kl URA3-DR-YFLWtau1 positions Point downstream homologous sequence；

Genetic fragment 7 is transformed into into transgene fragment 1-6 or the clpp gene of transgene fragment 1-5 by Li-acetate method In except yeast, by YFLWtau1 sites upstream and downstream homologous sequence in genetic fragment 7 and gene knockout Yeast genome There is restructuring and be incorporated on genome in YFLWtau1 sites.

14. according to claim 13 construction method, it is characterised in that the concrete construction method of the genetic fragment 7 is as follows：

Amplification yeast YFLWtau1 site upstream 297bp homologous sequences, PGK1 promoters, the gene VHb from Vitreoscilla, ACT1 terminators, DR-Kl URA3-DR nutritional labelings, saccharomyces cerevisiae YFLWtau1 sites downstream 208bp homologous sequences, sequentially lead to Cross OE-PCR methods to be stitched together, obtain genetic fragment 7 of the two ends comprising PmeI restriction enzyme sites, by fragment obtained above with it is flat Ends vector pJET1.2 connects, and obtains the integrated plasmid of genetic fragment 7 and is designated as pYFLWtau1-P_PGK1-VHb-T_ACT1-DR-Kl URA3-DR, PmeI digestion obtains genetic fragment 7, i.e. YFLWtau1 site upstreams homologous sequence-P_PGK1-VHb-T_ACT1-DR- KlURA3-DR-YFLWtau1 sites downstream homologous sequences, nucleotide sequence such as SEQ ID NO:Shown in 2.

15. according to claim 12 construction method, it is characterised in that the concrete construction method of the genetic fragment 6 is as follows：

Amplification yeast YNRCdelta9 site upstream 256bp homologous sequences, GAL1 promoters, transcription factor INO2, CPS1 terminate Son, DR-Kl URA3-DR nutritional labelings, yeast YNRCdelta9 sites downstream 267bp homologous sequences, sequentially pass through OE-PCR side Method is stitched together, and genetic fragment 6 of the two ends comprising PmeI restriction enzyme sites is obtained, by fragment obtained above and flat ends vector PJET1.2 connects, and obtains the integrated plasmid of genetic fragment 6 and is designated as pYNRCdelta9-P_GAL1-INO2-T_CPS1- DR-Kl URA3-DR, PmeI digestions obtain genetic fragment 6, i.e. YNRCdelta9 site upstreams homologous sequence-P_GAL1-INO2-T_CPS1-DR-Kl URA3- DR-YNRCdelta9 sites downstream homologous sequences, nucleotide sequence such as SEQ ID NO:Shown in 1.

A kind of 16. methods for producing lycopene, it is characterised in that by bacterial strain Jing seeds described in claim 1-8 any one Fermented and cultured in fermentation medium is inoculated in after medium culture activation, collects thalline cell extraction tomato red after fermented and cultured Element.

17. according to claim 16 methods described, it is characterised in that by inoculation described in claim 1-8 any one in After cultivating in 5mL seed culture mediums, with initial cell concentration OD₆₀₀=0.2 transfers in fresh 25mL seed culture mediums culture extremely Mid log phase, with initial cell concentration OD₆₀₀=0.5 is inoculated in culture in 50mL fermentation mediums, collects after fermented and cultured Somatic cells extract lycopene.

18. according to the methods described of claim 16 or 17, it is characterised in that the seed culture medium is 20g/L glucose, 20g/ L peptones, 10g/L yeast extracts, 50mg/L uracils, remaining is water.

19. according to the methods described of claim 16 or 17, it is characterised in that the fermentation medium is 20g/L glucose, 20g/ L peptones, 10g/L yeast extracts, 20mg/L uracils, 10g/L D- (+)-galactolipin, remaining is water.

20. according to the methods described of claim 16 or 17, it is characterised in that the culture is to train under the conditions of 30 DEG C, 250rpm Support.