CN106566779B - A kind of restructuring yeast strains and its construction method and application - Google Patents

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

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CN106566779B
CN106566779B CN201610969721.2A CN201610969721A CN106566779B CN 106566779 B CN106566779 B CN 106566779B CN 201610969721 A CN201610969721 A CN 201610969721A CN 106566779 B CN106566779 B CN 106566779B
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李霞
陈艳
肖文海
王颖
姚明东
刘宏
元英进
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Tianjin University
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Abstract

The present invention relates to gene engineering technology field, a kind of restructuring yeast strains and its construction method and application are disclosed.The restructuring yeast strains knock out gal1, gal7, gal10, ypl062w and rox1 gene, it and include through 6 genetic fragments on yeast homologous recombination and integration to genome, the present invention is also further integrated into 1 genetic fragment on Yeast genome on its basis, obtains more preferably recombination yeast.The present invention constructs gene knockout yeast strain, the host cell of optimization is provided for production lycopene, choose functional gene crtE, crtB and crtI of the synthesis lycopene of particular source combination, and specific yeast entogenous gene and foreign gene etc., it is integrated on gene knockout yeast strain genome through modularized design, obtains the recombinant bacterial strain of one plant of completely new high yield lycopene.

Description

A kind of restructuring yeast strains and its construction method and application
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of restructuring yeast strains and its building side Method and application.
Background technique
Worldwide, with economic level and to the raising of health demand, safety, trophism and the function of food Property receive more and more attention, therefore functional nutrient chemicals has become development trend, represents the new of contemporary food development Trend has a vast market foreground.Lycopene is a kind of fat-soluble natural food colour, and class is belonged in chemical structure recklessly Radish element, has extremely strong oxidation resistance, is the hot spot that functional food composition is studied in the world in recent years.
The preparation of lycopene relies primarily on plant extract, chemical synthesis and Microbe synthesis, and first two method respectively has it The deficiency of itself, Microbe synthesis is then with low cost, high yield and Product Safety, it is considered to be most promising method.Mesh Before, in the research of Microbe synthesis lycopene, used host strain focuses primarily upon Escherichia coli and saccharomyces cerevisiae. 2011, Yeong-Su Kim et al. introduced three genes of synthesis lycopene in pantoea agglomerans source in Escherichia coli CrtE, crtB and crtI, while raising the key gene in endogenous MEP approach and building the heterologous path MVA intracellular, it is final logical Cross the yield of lycopene that 1.35g/L (32mg/gDCW) is realized in fed batch fermentation optimization.2014, Tianjin industrial bio skill Art research institute Ma Yan and seminar utilize ribosome bind site by optimizing the supply of Escherichia coli NADPH and ATP intracellular Library screening obtains the recombination bacillus coli that a plant height produces lycopene, and the yield on fed batch fermentation tank is 3.52 G/L (50.6 mg/g DCW) belongs to maximum output in the recombinant bacterial strain of open report at present.
Saccharomyces cerevisiae compares Escherichia coli as generally acknowledged safe mode microorganism, its thallus vitamin, protein contains Amount is high, can eat, medicinal and fodder yeast；Compared to trispore Bruce mould, its growth cycle is shorter and is easier to cultivate.Cause This, realizes that high yield of the lycopene in saccharomyces cerevisiae will show great competitiveness in carotenoid industrialization. Being expressed in saccharomyces cerevisiae by ADH2 promoter for Bahieldin in 2014 et al. report bites summer spore Europe through codon optimization The yield of lycopene of crtE, crtB and the crtI in Wen bacterium source only 3.3mg/gDCW.2015, Zhejiang University's Yu Hongwei class The bifunctional enzyme crtYB in phaffiafhodozyma source is transformed by albumen directed evolution means for topic group, it is made to lose tomato red prime ring Change the encoding function of enzyme, and only retains its function of encoding phytoene synthetase；Meanwhile to phaffiafhodozyma source CrtE is oriented evolution, improves the catalytic performance of enzyme；Again, by adjusting the copy number of crtE, crtB and crtI, one is obtained Plant height produces the diploid recombinant Saccharomyces cerevisiae of lycopene, and shaking flask yield reaches 159.56mg/L (23.23mg/gDCW), most Optimized afterwards by fed batch fermentation, 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, by knocking out yeast straingal1、gal7、gal10Orgal80Gene, andypl062wThen gene constructed gene knockout yeast strain is chosen different next The functional gene of the synthesis lycopene in sourcecrtE、crtBWithcrtI, and specific yeast entogenous gene etc., through modularized design It is integrated on gene knockout yeast strain genome, obtains the recombinant bacterial strain of one plant of completely new high yield lycopene, lycopene Yield reach 30-45mg/gDCW；However, this is with the highest for synthesizing lycopene using recombination bacillus coli reported before this Yield (50.6 mg/g DCW), which is compared, still certain gap.
Chinese patent CN105420134A discloses a kind of restructuring yeast strains and its construction method and application, pass through by Yeast strain knocks outgal1、gal7、gal10Andypl062wGene, and include through on yeast homologous recombination and integration to genome 3 genetic fragments, on its basis its also further knock outrox1Gene and it is integrated into 2 gene pieces on Yeast genome Section obtains more preferably 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 it is higher than the lycopene maximum output of recombination bacillus coli instantly. But exploitation realizes that the high yield of lycopene still remains very big potentiality and space using saccharomyces cerevisiae as host strain, is worth Researcher goes the production lycopene ability for further promoting recombination engineering.
Summary of the invention
In view of this, enabling the recombinant bacterial strain to answer the purpose of the present invention is to provide a kind of restructuring yeast strains For in the biosynthesis of lycopene, and high yield is kept, while providing construction method and the application of the recombinant bacterial strain.
For achieving the above object, the invention provides the following technical scheme:
A kind of restructuring yeast strains, which is characterized in that the Yeast genome knocks outgal1、gal7、gal10、ypl062w、rox1Gene, and include through the following genetic fragment on yeast autologous recombination and integration to its genome:
Yeast trp1 site upstream homologous sequence, CYC1 terminator,Bt crtI, GAL10 promoter, GAL1 promoter,Pa crtB, the genetic fragment 1 that is sequentially spliced of PGK1 terminator, the site yeast trp1 downstream homologous sequence；
Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA Reductase genetHMGR1, GAL10 promoter, GAL1 promoter,TmcrtE, GPM1 terminator, the site yeast leu2 downstream it is same The genetic fragment 2 that source sequence is sequentially spliced；
TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 are terminated in genetic fragment 2 Son,Bt crtI, GAL7 promoter, ACT1 terminator and the gene that homologous sequence is sequentially spliced downstream in genetic fragment 2 Segment 3；
Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA Reductase genetHMGR1, GAL10 promoter, GAL1 promoter, fusionBTS1-ERG20, FBA1 terminator, yeast The genetic fragment 4 that the site his3 downstream homologous sequence is sequentially spliced；
Yeast YPRCdelta15 site upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CPS1 terminator, transcription The factorINO2, the genetic fragment that is sequentially spliced of GAL1 promoter, the site saccharomyces cerevisiae YPRCdelta15 downstream homologous sequence 5；
Yeast YNRCdelta9 site upstream homologous sequence, GAL1 promoter, transcription factorINO2, CPS1 terminator, DR- The genetic fragment 6(mode that Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream homologous sequence are sequentially spliced Figure is shown in Fig. 1)；
Wherein, it is knocked out in Yeast genomegal1、gal7、gal10、ypl062w、rox1Gene and homologous recombination gene piece After section 1-5, intermediate strains are obtained, the restructuring yeast strains are on the basis of intermediate strains through autologous recombination and integration gene piece It is obtained after section 6.Particular sequence, Genetic elements, synthetic method and the ideograph of genetic fragment 1-5 of the present invention can be found in It is recorded in patent CN105420134A.
The present invention passes through the structure that Genetic elements, netic module are carried out using specific yeast entogenous gene and foreign gene It builds, and is transferred to knockoutgal1、gal7、gal10、ypl062w、rox1On the Yeast genome of gene, recombinant bacterial strain kind is realized The synthesis output increased of Lycopene.
Wherein, amino acid tag, transcription factor, promoter and the terminator in related yeast, including but not limited to CYC1 terminator, GAL10 promoter, GAL1 promoter, PGK1 terminator, ACT1 terminator, GPM1 terminator, LEU2 label, TDH2 terminator, FBA1 terminator, ENO2 terminator, HIS3 label, CPS1 terminator, PGK1 promoter, transcription factorINO2, The acquisition of said gene element can be obtained using Yeast genome as template by PCR amplification；
In the present invention, above-mentioned each Genetic elements and relevant upstream and downstream homologous sequence are with Wine brewing yeast strain BY4741 Genome be template, design and synthesize suitable primer, obtained by PCR amplification；And the upstream LEU2 homologous sequence and LEU2 Label is to get off together from PCR amplification on plasmid pRS405, and the upstream HIS3 homologous sequence and HIS3 label are together from plasmid The upper PCR amplification of pRS313 is got off.
Foreign gene according to the present invention includes geranyl pyrophosphate (GGPP) synthase genecrtE, phytoene Synthase genecrtBAnd Phytoene dehydrogenase genecrtI.WhereincrtESource be Taxus x media (Taxus x media), it is abbreviated asTm crtE；crtBSource be pantoea agglomerans (Pantoea agglomerans), it is abbreviated asPa crtB；crtISource be trispore Bruce mould (Blakeslea trispora), it is abbreviated asBt crtI；VHbFrom saturating The bright bacterium that quivers (Vitreoscilla stercoraria), said gene is that codon optimizes and suitably evades common limitation Property restriction enzyme site after obtained by artificial synthesized.
Preferably, the genetic fragment 6 is as shown in SEQ ID NO:1.
Preferably, the intermediate strains are the bacterial strain of 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 27th, and is recorded in patent in detail In CN105420134A；The present invention transformed gene segment 6 can obtain on the basis of the bacterial strain of deposit number CGMCC No.11748 New restructuring yeast strains.
Preferably, the yeast is that saccharomyces cerevisiae, solution rouge category yeast or Crewe dimension belong to yeast.It in addition to this, can also be with It is transformation bacterial strain by institute of the present invention with algae, mould (such as streptomycete etc.) and bacterium (such as Escherichia coli, bacillus subtilis etc.) The Genetic elements and module of restriction are recombinated according to the herxheimer-liked reaction path of these bacterial strains being resolved.
It is highly preferred that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY series saccharomyces cerevisiae.Further preferably Ground, the CEN.PK series saccharomyces cerevisiae are saccharomyces cerevisiae CEN.PK2-1D.
It preferably further include through on yeast autologous recombination and integration to its genome on the basis of above-mentioned each technical solution Following genetic fragment:
Yeast YFLWtau1 site upstream homologous sequence, PGK1 promoter, gene VHb, ACT1 from Vitreoscilla The base that terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream homologous sequence are sequentially spliced Because segment 7(ideograph is shown in Fig. 1).It is further preferred that the genetic fragment 7 is as shown in SEQ ID NO: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 largely synthesize lycopene, therefore the present invention also provides described heavy Group yeast strain is in the application in production lycopene and the application in product of the production using lycopene as intermediate product.
In addition, the present invention also provides the construction methods of the restructuring yeast strains, comprising:
Step 1, building are by yeastgal7Downstream of gene homologous sequence, DR-Kl URA3-DR nutritional labeling,gal1Gene The sequentially connected knockout box segment 1 of downstream homologous sequence, byypl062wUpstream region of gene homologous sequence, kanMX resistance label,ypl062wThe sequentially connected knockout box segment 2 of downstream of gene homologous sequence, and building is by yeastrox1The homologous sequence of upstream region of gene Column, DR-Kl URA3-DR nutritional labeling,rox1The sequentially connected knockout box segment 3 of downstream of gene homologous sequence utilizes knockout box Segment 1-3 knocks out yeast by the recombination of yeast autologousgal1、gal7、gal10、ypl062wWithrox1Gene obtains gene Yeast is knocked out, it is spare；
By yeast trp1 site upstream homologous sequence, CYC1 terminator,Bt crtI, GAL10 promoter, GAL1 starting Son,Pa crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence sequentially splice, obtain genetic fragment 1, i.e. trp1 Point upstream homologous sequence-TCYC1-crtI-PGAL10-PGAL1-crtB-TPGK1The site-trp1 downstream homologous sequence, it is spare；
By yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG- CoA reductase genetHMGR1, GAL10 promoter, GAL1 promoter,TmcrtE, GPM1 terminator, the site yeast leu2 downstream Homologous sequence sequentially splices, and obtains genetic fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-TTDH2-TACT1-tHMGR1- PGAL10-PGAL1-crtE-TGPM1The site-leu2 downstream homologous sequence, it is spare；
TDH2 terminator in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 is whole Only sub,Bt crtI, GAL7 promoter, ACT1 terminator and homologous sequence sequentially splices downstream in genetic fragment 2, obtain base Because of segment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-TCYC1-Bt crtI-PGAL7-TACT1-ACT1 Terminator and downstream homologous sequence, it is spare；
By yeast entogenous FPP synthase geneERG20With GGPP synthase geneBTS1It is attached, obtains fusionBTS1-ERG20, then by yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncation HMG-CoA reductase genetHMGR1, GAL10 promoter, GAL1 promoter, fusionBTS1-ERG20, FBA1 terminate Son, the site yeast his3 downstream homologous sequence sequentially splice, and obtain genetic fragment 4, i.e. his3 site upstream homologous sequence- HIS3-TENO2-TACT1-tHMGR1-PGAL10-PGAL1 -(BTS1-ERG20)-TFBA1The site-his3 downstream homologous sequence, it is spare；
By yeast YPRCdelta15 site upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CPS1 terminator, Transcription factorINO2, GAL1 promoter, the site saccharomyces cerevisiae YPRCdelta15 downstream homologous sequence sequentially splice, obtain gene Segment 5, i.e. YPRCdelta15 site upstream homologous sequence-DR-Kl URA3-DR-TCPS1-INO2-PGAL1- YPRCdelta15 Point downstream homologous sequence, it is spare；
By yeast YNRCdelta9 site upstream homologous sequence, GAL1 promoter, transcription factorINO2, CPS1 terminator, DR-Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream homologous sequence sequentially splice, and obtain genetic fragment 6, i.e., YNRCdelta9 site upstream homologous sequence-PGAL1-INO2-TCPS1The site-DR-Kl URA3-DR-YNRCdelta9 downstream is homologous Sequence, it is spare；
Genetic fragment 1 is transferred in the gene knockout yeast by step 2 by Li-acetate method, by genetic fragment 1 The site trp1 occurs recombination and is integrated into genome on the site trp1 upstream and downstream homologous sequence and gene knockout Yeast genome On；
Genetic fragment 2 is transferred in the gene knockout yeast by Li-acetate method, passes through the site leu2 in genetic fragment 2 The site leu2 occurs recombination and is integrated on genome on upstream and downstream homologous sequence and gene knockout Yeast genome；
Genetic fragment 3 is continued through Li-acetate method to be transformed into the gene knockout yeast of transgene segment 2, is passed through TDH2 terminator and its upstream homologous sequence in genetic fragment 3, ACT1 terminator and homologous sequence downstream, with the base integrated Because in segment 2 TDH2 terminator and ACT1 terminator site recombination occurs due to be inserted into the clpp gene of integrator gene segment 2 Except on Yeast genome；
Genetic fragment 4 is transferred in the gene knockout yeast by Li-acetate method, passes through the site his3 in genetic fragment 4 The site his3 occurs recombination and is integrated on genome on upstream and downstream homologous sequence and gene knockout Yeast genome；
Genetic fragment 5 is transformed into gene knockout yeast by Li-acetate method, by genetic fragment 5 On the site YPRCdelta15 upstream and downstream homologous sequence and gene knockout Yeast genome the site YPRCdelta15 occur recombination and It is integrated on genome；
Intermediate strains are obtained after genetic fragment 1-5 is transformed into gene knockout yeast by Li-acetate method；
Genetic fragment 6 is transformed into intermediate strains by Li-acetate method, passes through the site YNRCdelta9 in genetic fragment 6 The site YNRCdelta9 occurs recombination and is integrated on genome on upstream and downstream homologous sequence and intermediate strains genome, obtains Restructuring yeast strains.
Preferably, the construction method further include:
Genetic fragment 7 is constructed, and genetic fragment 7 is transferred to acquisition restructuring yeast strains；
Specifically, by yeast YFLWtau1 site upstream homologous sequence, PGK1 promoter, from the base of Vitreoscilla Because VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream homologous sequence are sequentially spelled It connects, obtains genetic fragment 7, i.e. YFLWtau1 site upstream homologous sequence-PPGK1-VHb-TACT1-DR-Kl URA3-DR- The site YFLWtau1 downstream homologous sequence；
Genetic fragment 7 is transformed into transgene segment 1-6 or the base of transgene segment 1-5 by Li-acetate method Because knocking out in yeast, by the site YFLWtau1 upstream and downstream homologous sequence in genetic fragment 7 and gene knockout Yeast genome The site YFLWtau1 occurs recombination and is integrated on genome.
Gene knockout yeast of the present invention (including each detailed building for knocking out box), the specific building of genetic fragment 1-5 The record that method and in detail record situation can refer to patent CN105420134A, the present invention is herein by the structure of genetic fragment 6 and 7 It is as follows to build situation offer:
Expand yeast YNRCdelta9 site upstream 256bp homologous sequence, GAL1 promoter, transcription factorINO2、CPS1 Terminator, DR-Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream 267bp homologous sequence, sequentially pass through OE- PCR method is stitched together, and obtains both ends and includesPmeThe genetic fragment 6 of I restriction enzyme site, by segment obtained above and flat end Carrier pJET1.2 connection obtains 6 integrated plasmid of genetic fragment and is denoted as pYNRCdelta9-PGAL1-INO2-TCPS1-DR-Kl URA3-DR,PmeI digestion obtains genetic fragment 6, i.e. YNRCdelta9 site upstream homologous sequence-PGAL1-INO2-TCPS1-DR- The site Kl URA3-DR-YNRCdelta9 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:1.
Expand yeast YFLWtau1 site upstream 297bp homologous sequence, PGK1 promoter, the base from Vitreoscilla Because of VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream 208bp homologous sequence, It sequentially passes through OE-PCR method to be stitched together, obtaining both ends includesPmeThe genetic fragment 7 of I restriction enzyme site, by obtained above The pJET1.2 connection of Duan Yuping ends vector obtains 7 integrated plasmid of genetic fragment and is denoted as pYFLWtau1-PPGK1-VHb-TACT1-DR- Kl URA3-DR,PmeI digestion obtains genetic fragment 7, i.e. YFLWtau1 site upstream homologous sequence-PPGK1-VHb-TACT1-DR- The site Kl URA3-DR-YFLWtau1 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:2.
Preferably, the yeast is that saccharomyces cerevisiae, solution rouge category yeast or Crewe dimension belong to yeast in construction method.It removes It can also be transformation bacterium with algae, mould (such as streptomycete) and bacterium (such as Escherichia coli, bacillus subtilis) except this The herxheimer-liked reaction path that Genetic elements defined by the present invention and module have been resolved by strain according to these bacterial strains To recombinate.It is highly preferred that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY series saccharomyces cerevisiae.Further preferably Ground, the CEN.PK series saccharomyces cerevisiae are saccharomyces cerevisiae CEN.PK2-1D.
Restructuring yeast strains of the present invention are for when producing lycopene, yield of lycopene to be 60.97 mg/gDCW (356.22mg/L) -66.14mg/gDCW (376.78mg/L), yield are significantly higher than recombination yeast in CN105420134A patent The maximum output of the 54.62mg/gDCW (318.13mg/L) of bacterial strain.
The related application of the bacterial strain according to the present invention, the present invention also provides a kind of methods for producing lycopene, will Bacterial strain of the present invention is inoculated in fermented and cultured in fermentation medium after seed culture medium culture activation, collects after fermented and cultured Somatic cells extract lycopene.
More specifically, after strain inoculated of the present invention being cultivated in 5mL seed culture medium, with initial cell concentration OD600=0.2 switching is in fresh 25mL seed culture medium culture to mid log phase, with initial cell concentration OD600=0.5 connects Kind is cultivated in 50mL fermentation medium, and somatic cells are collected after fermented and cultured and extract lycopene.
Wherein, preferably, the seed culture medium be 20g/L glucose, 20g/L peptone, 10g/L yeast extract, 50mg/L uracil, remaining is water.
Preferably, the fermentation medium is 20g/L glucose, 20g/L peptone, 10g/L yeast extract, 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 the above technical scheme, the present invention constructs gene knockout yeast strain, provides optimization for production lycopene Host cell, choose particular source combination synthesis lycopene functional genecrtE、crtBWithcrtIAnd specific ferment Female endogenous gene and foreign gene etc. are integrated on gene knockout yeast strain genome through modularized design, obtain one plant The recombinant bacterial strain of completely new high yield lycopene.
Biological deposits explanation
SyBE_Sc0014D018, classification naming: saccharomyces cerevisiae,Saccharomyces cerevisiaeIn in September, 2016 It is deposited within 19th China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is Chaoyang District, Beijing City North Star west The institute 3 of road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.13013.
Detailed description of the invention
Fig. 1 show the Genetic elements ideograph of genetic fragment 6；Wherein, both ends YNRCdelta9-L, YNRCdelta9-R Respectively indicate the site yeast YNRCdelta9 upstream and downstream homologous sequence；
Fig. 2 show the Genetic elements ideograph of genetic fragment 7；Wherein, both ends YFLWtau1-L, YFLWtau1-R distinguishes Indicate the site yeast YFLWtau1 upstream and downstream homologous sequence；
Fig. 3 show SyBE_Sc0014D014 bacterial strain in patent CN105420134A, SyBE_Sc0014D017 of the present invention With the yield of lycopene column diagram of SyBE_Sc0014D018 bacterial strain；
Fig. 4 show growth and the yield of lycopene curve of SyBE_Sc0014D018 bacterial strain shake flask fermentation of the present invention.
Specific embodiment
The invention discloses a kind of restructuring yeast strains and its construction method and application, those skilled in the art can be used for reference Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the invention and application have passed through Preferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to described herein Methods and applications be modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Some plasmid vectors involved in the present invention, bacterial strain are commercially available, as pJET1.2 plasmid vector is bought From CloneJET PCR the Cloning Kit, #K1231 of Thermo Scientific company；Wine brewing yeast strain CEN.PK2- 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 library bacterial strain YPL062W purchase from U.S. Thermo Fisher Scientific company；Plasmid pRS405, pRS313 and saccharomyces cerevisiae BY4741 are from BioVector China plasmid vector bacterium Strain cytogene collection-NTCC country's Type Tissue Collection purchase.Above-mentioned plasmid map and sequence can be found in Patent CN105420134A is recorded.
For each Genetic elements employed in present invention building restructuring yeast strains, such as amino acid tag, label, endogenous Gene, foreign gene etc. are it is known in the art that its particular sequence as known to those skilled in the art.It is of the invention for convenience of understanding, The present invention is illustrated each Genetic elements in each genetic fragment:
Box segment 1(is knocked out referring to the SEQ ID NO:7 in patent CN105420134A): 1-40bp isgal7Downstream of gene 40bp homologous sequence；41-1615bp is DR-Kl URA3-DR nutritional labeling sequence；1616-1655bp isgal1Downstream of gene 40bp homologous sequence.
Box segment 2(is knocked out referring to the SEQ ID NO:8 in patent CN105420134A): 1-394bp isypl062wGene Upstream 394bp homologous sequence；395-1864bp is kanMX resistance sequence label；1865-2181bp isypl062wDownstream of gene 317bp homologous sequence.
Box segment 3(is knocked out referring to the SEQ ID NO:9 in patent CN105420134A): 1-40bp isrox1Upstream region of gene 40bp homologous sequence；41-1615bp is DR-Kl URA3-DR nutritional labeling sequence；1616-1655bp isrox1Downstream of gene 40bp homologous sequence.
Genetic fragment 1(is referring to the SEQ ID NO:1 in patent CN105420134A): 1-631bp is trp1 site upstream 631bp homologous sequence；632-637bp isBamHI restriction enzyme site；638-640bp is meaningless sequence；641-646bp isHindIII digestion site；647-901bp is CYC1 terminator sequence；902-2650bp isBt crtISequence；2651-3318bp For GAL10-GAL1 two-way startup subsequence；3319-4248bp isPa crtBSequence；4249-4523bp is PGK1 terminator sequence Column；4524-4529bp isXhoI restriction enzyme site；4530-5262bp is the homologous 733bp sequence in the site trp1 downstream.
Genetic fragment 2(is referring to the SEQ ID NO:2 in patent CN105420134A): 1-561bp is leu2 site upstream Homologous 561bp sequence；562-1656bp is LEU2 label；1657-2056bp is TDH2 terminator sequence；2057-2062bp isBamHI restriction enzyme site；2063-2349bp is ACT1 terminator sequence；2350-3858bp is truncated HMG-CoA reductase base CausetHMGR1；3859-4526bp is GAL10-GAL1 two-way startup subsequence；4527-5708bp isTmcrtEGene；5709- 6108bp is GPM1 terminator sequence；6109-6114bp isXhoI restriction enzyme site；6115-6698bp is that the site leu2 downstream is same Source 584bp sequence.
Genetic fragment 3(is referring to the SEQ ID NO:3 in patent CN105420134A): 1-869bp be TDH2 terminator and Its upstream homologous sequence 869bp；870-2444bp is DR-Kl URA3-DR nutritional labeling sequence；2445-2699bp is CYC1 whole Only subsequence；2700-4448bp isBt crtISequence；4449-4948bp is GAL7 promoter；4949-5303bp is ACT1 whole Only son and downstream homologous sequence 355bp.
Genetic fragment 4(is referring to the SEQ ID NO:4 in patent CN105420134A): 1-312bp is his3 site upstream 312bp homologous sequence；313-975bp is HIS3 label；976-1375bp is ENO2 terminator；1376-1381bp isBamHI enzyme Enzyme site；1382-1668bp is ACT1 terminator sequence；1669-3177bp is truncated HMG-CoA reductase genetHMGR1；3178-3845bp is GAL10-GAL1 two-way startup subsequence；3846-5921bp is fusionBTS1-ERG20； 5922-6121bp is FBA1 terminator sequence；6122-6127bp isPstI restriction enzyme site；6128-6705bp is under the site his3 Swim 578bp homologous sequence.
Genetic fragment 5(is referring to the SEQ ID NO:5 in patent CN105420134A): 1-606bp is YPRCdelta15 Point upstream 606bp homologous sequence；607-2181bp is DR-Kl URA3-DR nutritional labeling sequence；2182-2372bp is CPS1 whole Only subsequence；2373-3287bp is transcription factor INO2 sequence；3288-3744bp is GAL1 promoter sequence；3745- 4103bp is the site YPRCdelta15 downstream 359bp homologous sequence.
Shown in genetic fragment 6(SEQ ID NO:1 of the present invention): 1-256bp is that YPRCdelta9 site upstream 256bp is homologous Sequence；257-713bp is GAL1 promoter sequence；714-1634bp is transcription factorINO2Sequence；1635-1825bp is CPS1 Terminator sequence；1826-3373bp is DR-Kl URA3-DR nutritional labeling sequence；3374-3640bp is the site YPRCdelta9 Downstream 267bp homologous sequence；
Shown in genetic fragment 7(SEQ ID NO:2 of the present invention): 1-297bp is the homologous sequence of YFLWtau1 site upstream 297bp Column；298-1075bp is PGK1 promoter sequence；1076-1516bp is the gene VHb sequence from Vitreoscilla；1517- 1803bp is ACT1 terminator sequence；1804-3351 bp is DR-Kl URA3-DR nutritional labeling sequence；3352-3559bp is The site YFLWtau1 downstream 208bp homologous sequence；
URA3 is Kluyveromyces lactis source (Kluyveromyces in DR-Kl URA3-DR nutritional labeling sequence Lactis, Kl).
After the particular sequence for knowing above-mentioned each Genetic elements, those skilled in the art can be according to conventional primer design principle Carry out amplification and OE-PCR splicing.Meanwhile SD culture medium of the present invention is in a kind of commonly training of yeast screening assay field Support base, according to yeast have which gene defect and specially deleted on the composition of minimal medium a certain ingredient or it is several at Divide and filters out aimed strain to realize.
Technical solution and preferred embodiment according to the present invention, the present invention using saccharomyces cerevisiae CEN.PK2-1D as starting strain, Summarize recombinant bacterial strain building process:
1) is transferred to CEN.PK2-1D for box segment 1 is knocked out, and knocks outgal1、gal7、gal10Gene, obtained strains are named as SyBE_Sc0014D003；
2) is transferred to SyBE_Sc0014D003 for box segment 2 is knocked out, and knocks outypl062wGene, obtained strains are named as SyBE_Sc0014D004；
3) genetic fragment 1 is transferred to SyBE_Sc0014D004 by, and obtained strains are named as SyBE_Sc0014D005；
4) genetic fragment 2 is transferred to SyBE_Sc0014D005 by, and obtained strains are named as SyBE_Sc0014D006；
5) genetic fragment 3 is transferred to SyBE_Sc0014D006 by, and obtained strains are named as SyBE_Sc0014D007；
6) genetic fragment 4 is transferred to SyBE_Sc0014D007 by, and obtained strains are named as SyBE_Sc0014D009；
7) is transferred to SyBE_Sc0014D009 for box segment 3 is knocked out, and knocks outrox1Gene, obtained strains are named as SyBE_ Sc0014D011；
8) genetic fragment 5 is transferred to SyBE_Sc0014D011 by, and obtained strains are named as among SyBE_Sc0014D014( Bacterial strain)；
9) genetic fragment 6 is transferred to SyBE_Sc0014D014 by, and obtained strains are named as SyBE_Sc0014D017；
10) genetic fragment 7 is transferred to SyBE_Sc0014D017 by, and obtained strains are named as SyBE_Sc0014D018；
Wherein, SyBE_Sc0014D014 has been deposited in Chinese microorganism strain preservation management committee on November 27th, 2015 Member's meeting common micro-organisms center, deposit number are CGMCC No.11748.
It is used in addition, in bacterial strain constructed by the present invention one section can be inserted into any site for not influencing its normal function The sequence for identifying the bacterial strain functions as " watermark " or " bar code ", prevent building recombinant bacterial strain leak and can not be fast Victory identification, the identification sequence can be any suitable sequence of current area, will not influence bacterial strain normal function, can use It is detected after corresponding amplimer amplification.The identification sequence specifically: both ends are tag sequence (amplimer), intermediate For the nucleotide sequence of one or more amino acid in coding C, M, E, P, S, A, H, V, N, W, T, Y, G, I, D, whole size is 180-220bp identifies the insertion method of sequence using conventional method.
Below with reference to embodiment, the present invention is further explained.
Embodiment 1: the building of gene knock-out bacterial strain
Using saccharomyces cerevisiae CEN.PK2-1D as starting strain, four gene knock-out bacterial strain CEN.PK2-1C are constructed△gal1,△ gal7,△gal10::DR,△ypl062w::kanMX.Detailed process is as follows:
It constructs first△gal1,△gal7,△gal10::DR-Kl URA3-DRBox is knocked out, i.e. knockout box segment 1, with matter Grain pWJ1042 is template, designs upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arm and DR-Kl URA3-DR The knockout box segment of nutritional labeling, it is using the homologous recombination machinery of yeast itself that the segment is whole by Li-acetate method yeast conversion It closes on Yeast genome, using SD-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, list after conversion Lack the kilnitamin powder 2g/L of uracil, 2% agar powder) it is screened, obtained transformant after dividing pure culture by extracting Yeast genome carries out PCR verifying, to taking a little bacterium solution to be coated with after verifying correct recombinant bacterial strain YPD fluid nutrient medium culture (respectively there are the direct repeat DR of 143bp, ferment in DR-Kl URA3-DR nutritional labeling both ends to 5- fluororotic acid (5-FOA) solid panel It is female itself homologous recombination to occur using this two sections identical sequences and deleteURA3Gene and one of DR；YPD culture Gene is no this screening pressure of amino acid nutrient defect, such spontaneous deletionURA3Saccharomycete can grow.Then It is screened with 5-FOA, because containingURA3Bacterial strain existURA35-FOA can be made to become to thin under the enzyme effect of gene coding The virose substance of born of the same parents, grow yeast cells cannot on the culture medium containing 5-FOA, to filter out deletionURAGene Bacterium), picking single colonie divides extraction genome progress PCR verifying screening after pure culture to delete by recombination spontaneous between DR sequenceURA3The correct bacterial strain of gene, i.e. three gene knock-out bacterial strains, are named as SyBE_Sc0014D003.It knocks outgal1、gal7、gal10 Bacterial strain will not be metabolized D- galactolipin, so as to maintain inducer galactose concentration intracellular constant, realize efficiently induction.
Then, it is constructed on the basis of three gene knock-out bacterial strain SyBE_Sc0014D003△ypl062w::kanMXIt knocks out Box, using the mono- genome for singly striking bacterial strain YPL062W struck in library of BY4742 as template, design upstream and downstream primer PCR expands tape base Causeypl062wThe knockout box segment 2 of upstream and downstream homologous sequence and kanMX resistance label integrates the segment by yeast conversion Onto Yeast genome, bacterial strain (kanMX resistance is struck using the YPD solid panel screening four of the resistance of G418 containing 200mg/L after conversion Label can generate resistance to this antibiotic of Geneticin G418, to select successful knockout gene using G418 screenypl062wBacterium), obtained transformant extracts genome and carries out PCR verifying by dividing after pure culture, to verifying correctly recombination Bacterial strain saves glycerol stock, and is named as SyBE_Sc0014D004.
Embodiment 2: the building of genetic fragment 1
Amplification CYC1 terminator,Bt crtI, GAL10 promoter, GAL1 promoter,Pa crtB, PGK1 terminator and suitable Secondary to be stitched together by OE-PCR method, obtaining both ends includesHindIII andXhoThe segment T of I restriction enzyme siteCYC1-crtI- PGAL10-PGAL1-crtB-TPGK1；
Meanwhile expanding the homologous 631bp sequence of yeast trp1 site upstream, the homologous 733bp sequence in the site yeast trp1 downstream And sequentially pass through OE-PCR method and be stitched together, obtaining both ends includesSacI andApaI restriction enzyme site, and in the site yeast trp1 Include between the homologous sequence of upstream and downstreamHindIII andXhoThe segment of I restriction enzyme site, then passes throughSacI andApaI restriction enzyme site It is connected into carrier pRS405, obtains TRP1 integrated plasmid pRS405-TRP, by segment T obtained aboveCYC1-crtI-PGAL10- PGAL1-crtB-TPGK1Pass through with pRS405-TRP plasmidHindIII andXhoI restriction enzyme site is attached, and obtains genetic fragment 1 Integrated plasmid is denoted as pRS405-TRP-TCYC1-crtI-PGAL10-PGAL1-crtB-TPGK1；
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, use respectivelySacI andApaThe cutting of I double enzyme site obtains genetic fragment 1, nucleotide sequence ginseng As shown in the SEQ ID NO:1 in patent CN105420134A.
Embodiment 3: the building of genetic fragment 2
By ACT1 terminator, truncated HMG-CoA reductase genetHMGR1, GAL10 promoter, GAL1 promoter,TMcrtE, GPM1 terminator sequentially pass through OE-PCR method and be stitched together, obtaining both ends includesBamHI andXhoI restriction enzyme site Segment TACT1-tHMGR1-PGAL10-PGAL1-crtE-TGPM1；Meanwhile by the homologous 561bp sequence of yeast leu2 site upstream, LEU2 Label, TDH2 terminator, the homologous 584bp sequence in the site yeast leu2 downstream sequentially pass through OE-PCR method and are stitched together, and obtain Both ends includeSacI andApaI restriction enzyme site, and include between TDH2 terminator, the site yeast leu2 downstream homologous sequenceBamHI andXhoThe segment of I restriction enzyme site, passes throughSacI andApaI restriction enzyme site is connected into carrier pRS405, obtains LEU2 and integrates matter Grain pRS405-LEU.The above-mentioned segment T that will be obtainedACT1-tHMGR1-PGAL10-PGAL1-crtE-TGPM1It is logical with pRS405-LEU plasmid It crossesBamHI andXhoI restriction enzyme site is attached, and is obtained 2 integrated plasmid of genetic fragment, is denoted as pRS405-LEU-TACT1- tHMGR1-PGAL10-PGAL1-crtE-TGPM1。
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, use respectivelySacI andApaThe cutting of I double enzyme site, obtains genetic fragment 2, leu2 site upstream Homologous sequence-LEU2-TTDH2-TACT1-tHMGR1-PGAL10-PGAL1-crtE-TGPM1The site-leu2 downstream homologous sequence, nucleotide Sequence is referring to shown in the SEQ ID NO:2 in patent CN105420134A.
Embodiment 4: the building of genetic fragment 3
TDH2 terminator and its upstream 869bp homologous sequence in amplification gene segment 2, ACT1 terminator and downstream Then 355bp homologous sequence terminates TDH2 terminator and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 Son,Bt crtI, GAL7 promoter, ACT1 terminator and homologous sequence sequentially passes through OE-PCR method downstream in genetic fragment 2 It is stitched together, obtaining both ends includesPmeThe genetic fragment 3 of I restriction enzyme site, i.e. TDH2 terminator and its upstream homologous sequence-DR- Kl URA3-DR-TCYC1-Bt crtI-PGAL7- ACT1 terminator and downstream homologous sequence connect with flat ends vector pJET1.2 It connects to obtain 3 integrated plasmid of genetic fragment, is denoted as pleu-DR-Kl URA3-DR-TCYC1-Bt crtI-PGAL7。
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, usePmeThe cutting of I restriction enzyme site, obtains genetic fragment 3, nucleotide sequence is referring to patent Shown in SEQ ID NO:3 in CN105420134A.
Embodiment 5: the building of genetic fragment 4
By yeast entogenous FPP synthase geneERG20With GGPP synthase geneBTS1Amalgamation and expression is carried out, by the side OE-PCR Method will with GGGS linker (GGTGGTGGTTCT)BTS1C-terminal withERG20N-terminal connect to form fusionBTS1- ERG20；From expanding T on LEU2 integrated plasmid pRS405-LEU in embodiment 4ACT1-tHMGR1-PGAL10-PGAL1Segment；
By segment TACT1-tHMGR1-PGAL10-PGAL1, fusionBTS1-ERG20, FBA1 terminator pass through the side OE-PCR Method is stitched together, and obtains both ends and includesBamHI andPstThe segment T of I restriction enzyme siteACT1-tHMGR1-PGAL10-PGAL1-(BTS1- ERG20)-TFBA1；Meanwhile PCR expands the homologous 312bp sequence of yeast his3 site upstream, HIS3 label, ENO2 terminator respectively And the homologous 578bp sequence in the site yeast his3 downstream, and sequentially spliced by OE-PCR method, obtaining both ends includesSacI andApaI restriction enzyme site, and include between ENO2 terminator, the site yeast his3 downstream homologous sequenceBamHI andPstI digestion position The segment of point, passes throughSacI andApaI restriction enzyme site is connected into carrier pRS405, obtains HIS3 integrated plasmid pRS405-HIS.Will The above-mentioned segment T arrivedACT1-tHMGR1-PGAL10-PGAL1-(BTS1-ERG20)-TFBA1Pass through with pRS405-HIS plasmidBamHI andPstI restriction enzyme site is attached, and is obtained 4 integrated plasmid of genetic fragment, is denoted as pRS405-HIS-TACT1-tHMGR1-PGAL10- PGAL1 -(BTS1-ERG20)-TFBA1；
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, useSacI andApaThe cutting of I double digestion, obtains genetic fragment 4, the i.e. homologous sequence of his3 site upstream Column-HIS3-TACT1-tHMGR1-PGAL10-PGAL1 -(BTS1-ERG20)-TFBA1The site-his3 downstream homologous sequence, nucleotides sequence Column are referring to shown in the SEQ ID NO:4 in patent CN105420134A.
Embodiment 6: the building of genetic fragment 5
It is whole to expand yeast YPRCdelta15 site upstream 606bp homologous sequence, DR-Kl URA3-DR nutritional labeling, CPS1 Only son, transcription factor INO2, GAL1 promoter, the site saccharomyces cerevisiae YPRCdelta15 downstream 359bp homologous sequence sequentially pass through OE-PCR method is stitched together, and obtains both ends and includesPmeThe genetic fragment 5 of I restriction enzyme site, i.e. YPRCdelta15 site upstream Homologous sequence-DR-Kl URA3-DR-TCPS1-INO2-PGAL1The site-YPRCdelta15 downstream homologous sequence, will be obtained above Segment is connect with flat ends vector pJET1.2, is obtained 5 integrated plasmid of genetic fragment and is denoted as pYPRCdelta15-DR-Kl URA3- DR-TCPS1-INO2-PGAL1。
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, usePmeThe cutting of I restriction enzyme site, obtains genetic fragment 5, nucleotide sequence is referring to patent Shown in SEQ ID NO:5 in CN105420134A.
Embodiment 7: the building of genetic fragment 6
Expand yeast YNRCdelta9 site upstream 256bp homologous sequence, GAL1 promoter, transcription factorINO2、CPS1 Terminator, DR-Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream 267bp homologous sequence, sequentially pass through OE- PCR method is stitched together, and obtains both ends and includesPmeThe genetic fragment 6 of I restriction enzyme site, by segment obtained above and flat end Carrier pJET1.2 connection obtains 6 integrated plasmid of genetic fragment and is denoted as pYNRCdelta9-PGAL1-INO2-TCPS1-DR-Kl URA3-DR；
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, usePmeThe cutting of I restriction enzyme site, obtains genetic fragment 6, nucleotide sequence is referring in the present invention Shown in SEQ ID NO:1.
Embodiment 8: the building of genetic fragment 7
Expand yeast YFLWtau1 site upstream 297bp homologous sequence, PGK1 promoter, the gene from Vitreoscilla VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream 208bp homologous sequence, it is suitable Secondary to be stitched together by OE-PCR method, obtaining both ends includesPmeThe genetic fragment 7 of I restriction enzyme site, by segment obtained above It is connect with flat ends vector pJET1.2, obtains 7 integrated plasmid of genetic fragment and be denoted as pYFLWtau1-PPGK1-VHb-TACT1-DR-Kl URA3-DR；
Integrated plasmid is transformed into E. coli competent DH5 α, bacterium colony PCR screening, upgrading grain carry out digestion verification and Sequence verification, to ensure that target fragment connection is correct and base sequence does not mutate.
After verifying is correct, usePmeThe cutting of I restriction enzyme site, obtains genetic fragment 7, nucleotide sequence is referring in the present invention Shown in SEQ ID NO:2.
Embodiment 9: genetic fragment 1-2 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D
Segment is converted four gene knockout yeast strain SyBE_Sc0014D004 using Li-acetate method by genetic fragment 1, is passed through The site trp1 occurs recombination and is integrated on genome on the upstream and downstream TRP1 homologous sequence and Yeast genome.It is used after conversion SD-TRP solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, the kilnitamin powder 2g/ of single scarce tryptophan L, 2% agar powder) it is screened, obtained transformant carries out extracting Yeast genome progress PCR verifying after scribing line divides pure culture, Glycerol stock is saved to the correct recombinant bacterial strain of verifying and is respectively designated as SyBE_Sc0014D005.
Then, it uses Li-acetate method by segment transformed yeast bacterial strain SyBE_Sc0014D005 genetic fragment 2, passes through The site leu2 occurs recombination and is integrated on genome on the upstream and downstream LEU2 homologous sequence and Yeast genome.It is used after conversion SD-TRP-LEU solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, the mixing of double scarce tryptophans and leucine Powder of amino acids 2g/L, 2% agar powder) it is screened, obtained transformant carries out extracting Yeast genome after scribing line divides pure culture PCR verifying is carried out, glycerol stock is saved to the correct recombinant bacterial strain of verifying and is named as SyBE_Sc0014D006.
Embodiment 10: genetic fragment 1-3 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D
The segment is converted into restructuring yeast strains SyBE_Sc0014D006 using Li-acetate method by genetic fragment 3, is passed through TDH2 terminator upstream homologous sequence and ACT1 terminator downstream homologous sequence occur recombination and are inserted on genome whole before The centre of the genetic fragment 2 of conjunction.SD-URA-TRP-LEU solid panel (synthetic yeast nitrogen source YNB 6.7g/L, Portugal are used after conversion Grape sugar 20g/L lacks the kilnitamin powder 2g/L of tryptophan, leucine and uracil, 2% agar powder) it is screened, it obtains Transformant carry out scribing line and divide after pure culture extracting Yeast genome and carrying out PCR verifying, to verifying correct recombinant bacterial strain YPD A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel is taken after fluid nutrient medium culture, picking single colonie mentions after dividing pure culture It takes genome to carry out PCR verifying screening to delete by recombination spontaneous between DR sequenceURAThe correct bacterial strain of gene, 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
The segment is converted into restructuring yeast strains SyBE_Sc0014D007 using Li-acetate method by genetic fragment 4, is passed through The site his3 occurs recombination and is integrated on genome on the upstream and downstream HIS3 homologous sequence and Yeast genome.Yeast after conversion Using SD-TRP-LEU-HIS solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lack tryptophan, histidine and The kilnitamin powder 2g/L of leucine, 2% agar powder) screened, obtained transformant carry out scribing line divide pure culture after It extracts Yeast genome and carries out PCR verifying, glycerol stock is saved to the correct recombinant bacterial strain of verifying and be respectively designated as SyBE_ Sc0014D009。
Embodiment 12:rox1The knockout of gene
It constructs first△rox1::DR-Kl URA3-DRBox is knocked out, i.e. knockout box segment 3, using plasmid pWJ1042 as mould Plate designs the knockout of upstream and downstream primer PCR amplified band gene upstream and downstream 40bp homology arm and DR-Kl URA3-DR nutritional labeling The segment is integrated into recombinant yeast by Li-acetate method yeast conversion using the homologous recombination machinery of yeast itself by box segment On strain SyBE_Sc0014D009, SD-TRP-LEU-HIS-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/ is used after conversion L, glucose 20g/L lack the kilnitamin powder 2g/L of uracil, leucine, histidine, tryptophan, 2% agar powder) it carries out Screening, obtained transformant extracts Yeast genome progress PCR verifying by dividing after pure culture, to the correct recombinant bacterial strain of verifying With taking coating 5- fluororotic acid (5-FOA) solid panel (the DR-Kl URA3-DR nutrition of a little bacterium solution after YPD fluid nutrient medium culture Respectively there is the direct repeat DR of 143bp at label both ends, and using this two sections identical sequences homologous recombination can occur for yeast itself And it deletesURA3Gene and one of DR；YPD culturing gene is no this screening pressure of amino acid nutrient defect, in this way Spontaneous deletionURA3Saccharomycete can grow.Then it is screened with 5-FOA, because containingURA3Bacterial strain existURA3Base Because 5-FOA can be made to become to make yeast cells in the culture medium containing 5-FOA the virose substance of cell under the enzyme effect of coding On cannot grow, to filter out deletionURAThe bacterium of gene), picking single colonie divides extraction genome progress PCR after pure culture to test Card screening is deleted by recombination spontaneous between DR sequenceURA3The correct bacterial strain of gene, to verifying, correct recombinant bacterial strain preservation is sweet Oily bacterium is simultaneously respectively designated as SyBE_Sc0014D011.
Embodiment 13: genetic fragment 1-5 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D
The segment is converted into restructuring yeast strains SyBE_Sc0014D011 using Li-acetate method by genetic fragment 5, is passed through The site YPRCdelta15 occurs recombination and is integrated into base on the site YPRCdelta15 upstream and downstream homologous sequence and Yeast genome Because in group.Yeast uses SD- TRP-LEU-HIS-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose after conversion 20g/L lacks the kilnitamin powder 2g/L of uracil, leucine, histidine, tryptophan, 2% agar powder) it is screened, it obtains To transformant carry out scribing line and divide after pure culture extracting Yeast genome and carrying out PCR verifying, use correct recombinant bacterial strain is verified A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel (DR-Kl URA3-DR nutrition mark is taken after YPD fluid nutrient medium culture Respectively there is a direct repeat DR of 143bp at label both ends, yeast itself can occur homologous recombinations using this two sections identical sequences and It deletesURA3Gene and one of DR；YPD culturing gene is no this screening pressure of amino acid nutrient defect, so certainly Hair deletesURA3Saccharomycete can grow.Then it is screened with 5-FOA, because containingURA3Bacterial strain existURA3Gene 5-FOA can be made to become to make yeast cells on the culture medium containing 5-FOA the virose substance of cell under the enzyme effect of coding It cannot grow, to filter out deletionURAThe bacterium of gene), picking single colonie extracts genome progress PCR verifying after dividing pure culture Screening is deleted by recombination spontaneous between DR sequenceURA3The correct bacterial strain of gene saves glycerol to correct recombinant bacterial strain is verified Bacterium is simultaneously respectively designated as SyBE_Sc0014D014.
Embodiment 14: genetic fragment 1-6 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D
The segment is converted into restructuring yeast strains SyBE_Sc0014D014 using Li-acetate method by genetic fragment 6, is passed through The site YPRCdelta15 occurs recombination and is integrated into base on the site YNRCdelta9 upstream and downstream homologous sequence and Yeast genome Because in group.Yeast uses SD- TRP-LEU-HIS-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose after conversion 20g/L lacks the kilnitamin powder 2g/L of uracil, leucine, histidine, tryptophan, 2% agar powder) it is screened, it obtains To transformant carry out scribing line and divide after pure culture extracting Yeast genome and carrying out PCR verifying, use correct recombinant bacterial strain is verified A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel (DR-Kl URA3-DR nutrition mark is taken after YPD fluid nutrient medium culture Respectively there is a direct repeat DR of 143bp at label both ends, yeast itself can occur homologous recombinations using this two sections identical sequences and It deletesURA3Gene and one of DR；YPD culturing gene is no this screening pressure of amino acid nutrient defect, so certainly Hair deletesURA3Saccharomycete can grow.Then it is screened with 5-FOA, because containingURA3Bacterial strain existURA3Gene 5-FOA can be made to become to make yeast cells on the culture medium containing 5-FOA the virose substance of cell under the enzyme effect of coding It cannot grow, to filter out deletionURAThe bacterium of gene), picking single colonie extracts genome progress PCR verifying after dividing pure culture Screening is deleted by recombination spontaneous between DR sequenceURA3The correct bacterial strain of gene saves glycerol to correct recombinant bacterial strain is verified Bacterium is simultaneously respectively designated as SyBE_Sc0014D017.
Embodiment 15: genetic fragment 1-7 construction and integration recombinant Saccharomyces cerevisiae CEN.PK2-1D
The segment is converted into restructuring yeast strains SyBE_Sc0014D017 using Li-acetate method by genetic fragment 7, is passed through The site YPRCdelta15 occurs recombination and is integrated into gene on the site YFLWtau1 upstream and downstream homologous sequence and Yeast genome In group.Yeast uses SD- TRP-LEU-HIS-URA solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose after conversion 20g/L lacks the kilnitamin powder 2g/L of uracil, leucine, histidine, tryptophan, 2% agar powder) it is screened, it obtains To transformant carry out scribing line and divide after pure culture extracting Yeast genome and carrying out PCR verifying, use correct recombinant bacterial strain is verified A little bacterium solution coating 5- fluororotic acid (5-FOA) solid panel (DR-Kl URA3-DR nutrition mark is taken after YPD fluid nutrient medium culture Respectively there is a direct repeat DR of 143bp at label both ends, yeast itself can occur homologous recombinations using this two sections identical sequences and It deletesURA3Gene and one of DR；YPD culturing gene is no this screening pressure of amino acid nutrient defect, so certainly Hair deletesURA3Saccharomycete can grow.Then it is screened with 5-FOA, because containingURA3Bacterial strain existURA3Gene 5-FOA can be made to become to make yeast cells on the culture medium containing 5-FOA the virose substance of cell under the enzyme effect of coding It cannot grow, to filter out deletionURAThe bacterium of gene), picking single colonie extracts genome progress PCR verifying after dividing pure culture Screening is deleted by recombination spontaneous between DR sequenceURA3The correct bacterial strain of gene saves glycerol to correct recombinant bacterial strain is verified Bacterium is simultaneously respectively designated as SyBE_Sc0014D018.
Embodiment 16: the shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain
Bacterial strain: bacterial strain in the existing patent CN105420134A of SyBE_Sc0014D0014(), SyBE_Sc0014D017, SyBE_Sc0014D018。
Test method:
Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract, 50mg/L uracil；
Fermentation medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract, 20mg/L uracil, 10g/L D- (+)-galactolipin.
Above-mentioned bacterial strains are inoculated in 5mL seed culture medium, cultivate 14-16h in 30 DEG C, 250rpm, it is dense with initial thallus Spend OD600=0.2 switching is cultivated under the conditions of 30 DEG C, 250rpm to mid log phase in fresh 25mL seed culture medium, With initial cell concentration OD600=0.5 is inoculated in respectively in 50mL fermentation medium, cultivates under the conditions of 30 DEG C, 250rpm, fermentation Sampling monitoring yield of lycopene when 48h.
Lycopene quantitative approach: the fermentation liquid of two equal portions is taken, 4000g is centrifuged 2min and collects thallus, and washes twice.It will A copy of it thallus is placed in 80 DEG C, and drying to constant weight, and weighing calculates dry cell weight；Another thallus is extracted to product, specific side Method are as follows: cell is resuspended with 3N HCl, is placed in boiling water bath and boils 2min, immediately after ice bath 3min；By broken cell 12000rpm, 4 DEG C of centrifugation 4min abandon supernatant, acetone are added after washing 2 times, and the 5min that is vortexed；Acetone phase is finally collected by centrifugation, uses Upper ultraviolet liquid phase detection after 2 μm of membrane filtrations, lycopene Detection wavelength is 471nm.As a result see Fig. 3 and Fig. 4.
Test result: by Fig. 3 and Fig. 4 as it can be seen that continuing up-regulation transcription on the basis of Saccharomyces Cerevisiae in S yBE_Sc0014D014 Activity factor INO2, obtains bacterial strain SyBE_Sc0014D017, and yield of lycopene is from 54.62mg/gDCW (318.13mg/L) It improves to 60.97 mg/gDCW (356.22mg/L).The hemoglobin gene VHb in Vitreoscilla source is introduced on this basis To reinforce oxygen conveying intracellular, bacterial strain SyBE_Sc0014D018 is obtained, yield of lycopene significantly improves, and reaches 66.14mg/gDCW (376.78mg/L), this is in disclosing in report currently with recombinant Saccharomyces cerevisiae production lycopene Belong to highest.
Embodiment 17: the shake flask fermentation of recombinant Saccharomyces cerevisiae bacterial strain
It repeats to construct restructuring yeast strains according to the method for 1-15 of the embodiment of the present invention, name respectively and according to embodiment 16 Method detects yield of lycopene, the result is as follows:
1 restructuring yeast strains yield of lycopene of table
Strain number (including segment 1-6) Yield of lycopene Strain number (including segment 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 a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
In order to facilitate describing and quote, the present invention is by 2350-3858bp in the SEQ ID NO:2 of patent CN105420134A And in SEQ ID NO:4 1669-3177bp truncated HMG-CoA reductase genetHMGR1Sequence is according to sequence of the present invention Number is named as SEQ ID NO:3, specific as follows:
t taggatttaa tgcaggtgac ggacccatct ttcaaacgat ttatatcagt ggcgtccaaa ttgttaggtt ttgttggttc agcaggtttc ctgttgtggg tcatatgact ttgaaccaaa tggccggctg ctagggcagc acataaggat aattcacctg ccaagacggc acaggcaact attcttgcta attgacgtgc gttggtacca ggagcggtag catgcgggcc tcttacacct aataagtcca acatggcacc ttgtggttct agaacagtac caccaccgat ggtacctact tcgatggatg gcatggatac ggaaattctc aaatcaccgt ccacttcttt catcaatgtt atacagttgg aactttcaac attttgtgca ggatcttgtc ctaatgccaa gaaaacagct gtcactaaat tagctgcatg tgcgttaaat ccaccaacag acccagccat tgcagatcca accaaattct tagcaatgtt caactcaacc aatgcggaaa catcactttt taacactttt ctgacaacat caccaggaat agtagcttct gcgacgacac tcttaccacg accttcgatc cagttgatgg cagctggttt tttgtcggta cagtagttac cagaaacgga gacaacctcc atatcttccc agccatactc ttctaccatt tgctttaatg agtattcgac acctttagaa atcatattca tacccattgc gtcaccagta gttgttctaa atctcatgaa gagtaaatct cctgctagac aagtttgaat atgttgcaga cgtgcaaatc ttgatgtaga gttaaaagct tttttaattg cgttttgtcc ctcttctgag tctaaccata tcttacaggc accagatctt ttcaaagttg ggaaacggac tactgggcct cttgtcatac catccttagt taaaacagtt gttgcaccac cgccagcatt gattgcctta cagccacgca tggcagaagc taccaaacaa ccctctgtag ttgccattgg tatatgataa gatgtaccat cgataaccaa ggggcctata acaccaacgg gcaaaggcat gtaacctata acattttcac aacaagcgcc aaatacgcgg tcgtagtcat aatttttata tggtaaacga tcagatgcta atacaggagc ttctgccaaa attgaaagag ccttcctacg taccgcaacc gctctcgtag tatcacctaa ttttttctcc aaagcgtaca aaggtaactt accgtgaata accaaggcag cgacctcttt gttcttcaat tgttttgtat ttccactact taataatgct tctaattctt ctaaaggacg tattttctta tccaagcttt caatatcgcg ggaatcatct tcctcactag atgatgaagg tcctgatgag ctcgattgcg cagatgataa acttttgact ttcgatccag aaatgactgt tttattggtt aaaaccat
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 (26)

1. a kind of recombinant Saccharomyces cerevisiae bacterial strain, which is characterized in that the saccharomyces cerevisiae genome knocks outgal1、gal7、gal10、ypl062w、rox1Gene, and include through the following genetic fragment on yeast autologous recombination and integration to its genome:
Yeast trp1 site upstream homologous sequence, CYC1 terminator,Bt crtI, GAL10 promoter, GAL1 promoter,Pa crtB, the genetic fragment 1 that is sequentially spliced of PGK1 terminator, the site yeast trp1 downstream homologous sequence；
Yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme genetHMGR1, GAL10 promoter, GAL1 promoter,TmcrtE, GPM1 terminator, the homologous sequence in the site yeast leu2 downstream Arrange the genetic fragment 2 being sequentially spliced；Truncated HMG-CoA reductase gene described in genetic fragment 2tHMGR1Such as SEQ Shown in ID NO:3；
TDH2 terminator and its upstream homologous sequence in genetic fragment 2, DR-Kl URA3-DR nutritional labeling, CYC1 terminator,Bt crtI, GAL7 promoter, ACT1 terminator and the genetic fragment 3 that homologous sequence is sequentially spliced downstream in genetic fragment 2；
Yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA reduction Enzyme genetHMGR1, GAL10 promoter, GAL1 promoter, fusionBTS1-ERG20, FBA1 terminator, his3, yeast The genetic fragment 4 that point downstream homologous sequence is sequentially spliced；Truncated HMG-CoA reductase gene described in genetic fragment 4tHMGR1As shown in SEQ ID NO:3；
Yeast YPRCdelta15 site upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CPS1 terminator, transcription factorINO2, the genetic fragment 5 that is sequentially spliced of GAL1 promoter, the site saccharomyces cerevisiae YPRCdelta15 downstream homologous sequence；
Yeast YNRCdelta9 site upstream homologous sequence, GAL1 promoter, transcription factorINO2, CPS1 terminator, DR-Kl The genetic fragment 6 that URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream homologous sequence are sequentially spliced；
Wherein, it is knocked out in Yeast genomegal1、gal7、gal10、ypl062w、rox1Gene and homologous recombination genetic fragment 1- After 5, intermediate strains are obtained, the restructuring yeast strains are on the basis of intermediate strains through autologous recombination and integration genetic fragment 6 After obtain.
2. bacterial strain according to claim 1, which is characterized in that the genetic fragment 6 is as shown in SEQ ID NO:1.
3. bacterial strain according to claim 1, which is characterized in that the intermediate strains are deposit number CGMCC No.11748's Bacterial strain.
4. bacterial strain according to claim 1, which is characterized in that the saccharomyces cerevisiae is CEN.PK series saccharomyces cerevisiae or BY system Column saccharomyces cerevisiae.
5. bacterial strain according to claim 4, which is characterized in that the CEN.PK series saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1D。
6. bacterial strain described in -5 any one according to claim 1, which is characterized in that further include through yeast autologous recombination and integration Following genetic fragment on to its genome:
Yeast YFLWtau1 site upstream homologous sequence, PGK1 promoter are terminated from gene VHb, ACT1 of Vitreoscilla The gene piece that son, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream homologous sequence are sequentially spliced Section 7.
7. bacterial strain according to claim 6, which is characterized in that the genetic fragment 7 is as shown in SEQ ID NO:2.
8. bacterial strain according to claim 7, which is characterized in that the bacterial strain is the bacterium that deposit number is CGMCC No.13013 Strain.
9. bacterial strain according to claim 1, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
10. bacterial strain according to claim 2, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
11. bacterial strain according to claim 3, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
12. bacterial strain according to claim 4, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
13. bacterial strain according to claim 5, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
14. bacterial strain according to claim 6, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
15. bacterial strain according to claim 7, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
16. bacterial strain according to claim 8, which is characterized in that further include identification sequence, which, which is inserted in, does not influence Any site of bacterial strain normal function, identification sequence both ends be tag sequence, centre for coding C, M, E, P, S, A, H, V, N, W, T, Y, in G, I, D one or more amino acid nucleotide sequence, whole size is 180-220bp.
17. bacterial strain described in claim 1-16 any one is in the application produced in lycopene and is producing with lycopene For the application in the product of intermediate product.
18. the construction method of recombinant Saccharomyces cerevisiae bacterial strain described in claim 1 characterized by comprising
Step 1, building are by yeastgal7Downstream of gene homologous sequence, DR-Kl URA3-DR nutritional labeling,gal1Downstream of gene is same The sequentially connected knockout box segment 1 of source sequence, byypl062wUpstream region of gene homologous sequence, kanMX resistance label,ypl062wBase Because of the sequentially connected knockout box segment 2 of downstream homologous sequence, and building is by yeastrox1Upstream region of gene homologous sequence, DR-Kl URA3-DR nutritional labeling,rox1The sequentially connected knockout box segment 3 of downstream of gene homologous sequence, it is logical using box segment 1-3 is knocked out It crosses the recombination of yeast autologous and knocks out yeastgal1、gal7、gal10、ypl062wWithrox1Gene obtains gene knockout yeast, It is spare；
By yeast trp1 site upstream homologous sequence, CYC1 terminator,Bt crtI, GAL10 promoter, GAL1 promoter,Pa crtB, PGK1 terminator, the site yeast trp1 downstream homologous sequence sequentially splice, obtain genetic fragment 1, i.e. trp1 site upstream Homologous sequence-TCYC1-crtI-PGAL10-PGAL1-crtB-TPGK1The site-trp1 downstream homologous sequence, it is spare；
Also by yeast leu2 site upstream homologous sequence, LEU2 label, TDH2 terminator, ACT1 terminator, truncated HMG-CoA Nitroreductase genetHMGR1, GAL10 promoter, GAL1 promoter,TmcrtE, GPM1 terminator, the site yeast leu2 downstream it is homologous Sequence is sequentially spliced, and genetic fragment 2, i.e. leu2 site upstream homologous sequence-LEU2-T are obtainedTDH2-TACT1-tHMGR1-PGAL10- PGAL1-crtE-TGPM1The site-leu2 downstream homologous sequence, it is spare；Truncated HMG-CoA reductase base described in genetic fragment 2 CausetHMGR1As shown in SEQ ID NO:3；
By TDH2 terminator in genetic fragment 2 and its upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CYC1 terminator,Bt crtI, GAL7 promoter, ACT1 terminator and homologous sequence sequentially splices downstream in genetic fragment 2, obtain genetic fragment 3, i.e. TDH2 terminator and its upstream homologous sequence-DR-Kl URA3-DR-TCYC1-Bt crtI-PGAL7- ACT1 terminator and its Downstream homologous sequence, it is spare；
By yeast entogenous FPP synthase geneERG20With GGPP synthase geneBTS1It is attached, obtains fusionBTS1- ERG20, then by yeast his3 site upstream homologous sequence, HIS3 label, ENO2 terminator, ACT1 terminator, truncated HMG-CoA reductase genetHMGR1, GAL10 promoter, GAL1 promoter, fusionBTS1-ERG20, FBA1 terminator, The site yeast his3 downstream homologous sequence sequentially splices, and obtains genetic fragment 4, i.e. his3 site upstream homologous sequence-HIS3- TENO2-TACT1-tHMGR1-PGAL10-PGAL1 -(BTS1-ERG20)-TFBA1The site-his3 downstream homologous sequence, it is spare；Gene piece Truncated HMG-CoA reductase gene described in section 4tHMGR1As shown in SEQ ID NO:3；
By yeast YPRCdelta15 site upstream homologous sequence, DR-Kl URA3-DR nutritional labeling, CPS1 terminator, transcription because SonINO2, GAL1 promoter, the site saccharomyces cerevisiae YPRCdelta15 downstream homologous sequence sequentially splice, obtain genetic fragment 5, That is YPRCdelta15 site upstream homologous sequence-DR-Kl URA3-DR-TCPS1-INO2-PGAL1The site-YPRCdelta15 downstream Homologous sequence, it is spare；
By yeast YNRCdelta9 site upstream homologous sequence, GAL1 promoter, transcription factorINO2, CPS1 terminator, DR-Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream homologous sequence sequentially splice, and obtain genetic fragment 6, i.e., YNRCdelta9 site upstream homologous sequence-PGAL1-INO2-TCPS1The site-DR-Kl URA3-DR-YNRCdelta9 downstream is homologous Sequence, it is spare；
Genetic fragment 1 is transferred in the gene knockout yeast by step 2 by Li-acetate method, passes through trp1 in genetic fragment 1 The site trp1 occurs recombination and is integrated on genome on point upstream and downstream homologous sequence and gene knockout Yeast genome；
Genetic fragment 2 is transferred in the gene knockout yeast by Li-acetate method, by the site leu2 in genetic fragment 2, The site leu2 occurs recombination and is integrated on genome on downstream homologous sequence and gene knockout Yeast genome；
Genetic fragment 3 is continued through Li-acetate method to be transformed into the gene knockout yeast of transgene segment 2, passes through gene TDH2 terminator and its upstream homologous sequence in segment 3, ACT1 terminator and homologous sequence downstream, with the gene piece integrated TDH2 terminator and ACT1 terminator site in section 2 occur to recombinate and be inserted into the gene knockout ferment of integrator gene segment 2 On female genome；
Genetic fragment 4 is transferred in the gene knockout yeast by Li-acetate method, by the site his3 in genetic fragment 4, The site his3 occurs recombination and is integrated on genome on downstream homologous sequence and gene knockout Yeast genome；
Genetic fragment 5 is transformed into gene knockout yeast by Li-acetate method, passes through YPRCdelta15 in genetic fragment 5 The site YPRCdelta15 occurs recombination and is integrated into genome on point upstream and downstream homologous sequence and gene knockout Yeast genome On；
Intermediate strains are obtained after genetic fragment 1-5 is transformed into gene knockout yeast by Li-acetate method；
Genetic fragment 6 is transformed into intermediate strains by Li-acetate method, by the site YNRCdelta9 in genetic fragment 6, The site YNRCdelta9 occurs recombination and is integrated on genome on downstream homologous sequence and intermediate strains genome, is recombinated Yeast strain.
19. 8 construction method according to claim 1, which is characterized in that further include: building genetic fragment 7, and by gene piece Section 7 is transferred to acquisition restructuring yeast strains；
Specifically, by yeast YFLWtau1 site upstream homologous sequence, PGK1 promoter, from Vitreoscilla gene VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream homologous sequence sequentially splice, and obtain Obtain genetic fragment 7, i.e. YFLWtau1 site upstream homologous sequence-PPGK1-VHb-TACT1- DR-Kl URA3-DR-YFLWtau1 Point downstream homologous sequence；
Genetic fragment 7 is transformed into transgene segment 1-6 or the clpp gene of transgene segment 1-5 by Li-acetate method Except in yeast, by the site YFLWtau1 upstream and downstream homologous sequence in genetic fragment 7 and gene knockout Yeast genome The site YFLWtau1 occurs recombination and is integrated on genome.
20. 9 construction method according to claim 1, which is characterized in that the specific construction method of the genetic fragment 7 is as follows:
Expand yeast YFLWtau1 site upstream 297bp homologous sequence, PGK1 promoter, from Vitreoscilla gene VHb, ACT1 terminator, DR-Kl URA3-DR nutritional labeling, the site saccharomyces cerevisiae YFLWtau1 downstream 208bp homologous sequence, sequentially lead to It crosses OE-PCR method to be stitched together, obtaining both ends includesPmeThe genetic fragment 7 of I restriction enzyme site by segment obtained above and is put down Ends vector pJET1.2 connection obtains 7 integrated plasmid of genetic fragment and is denoted as pYFLWtau1-PPGK1-VHb-TACT1-DR-Kl URA3-DR,PmeI digestion obtains genetic fragment 7, i.e. YFLWtau1 site upstream homologous sequence-PPGK1-VHb-TACT1-DR-Kl The site URA3-DR-YFLWtau1 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:2.
21. 8 construction method according to claim 1, which is characterized in that the specific construction method of the genetic fragment 6 is as follows:
Expand yeast YNRCdelta9 site upstream 256bp homologous sequence, GAL1 promoter, transcription factorINO2, CPS1 terminate Son, DR-Kl URA3-DR nutritional labeling, the site yeast YNRCdelta9 downstream 267bp homologous sequence, sequentially pass through the side OE-PCR Method is stitched together, and obtains both ends and includesPmeThe genetic fragment 6 of I restriction enzyme site, by segment obtained above and flat ends vector PJET1.2 connection obtains 6 integrated plasmid of genetic fragment and is denoted as pYNRCdelta9-PGAL1-INO2-TCPS1- DR-Kl URA3-DR,PmeI digestion obtains genetic fragment 6, i.e. YNRCdelta9 site upstream homologous sequence-PGAL1-INO2-TCPS1-DR-Kl URA3- The site DR-YNRCdelta9 downstream homologous sequence, nucleotide sequence is as shown in SEQ ID NO:1.
22. a kind of method for producing lycopene, which is characterized in that by bacterial strain described in claim 1-8 any one through seed It is inoculated in fermented and cultured in fermentation medium after culture medium culture activation, collects somatic cells after fermented and cultured and extracts tomato red Element.
23. according to claim 22 the method, which is characterized in that by strain inoculated described in claim 1-8 any one in After being cultivated in 5mL seed culture medium, with initial cell concentration OD600=0.2 transfers in fresh 25mL seed culture medium culture extremely Mid log phase, with initial cell concentration OD600=0.5 is inoculated in 50mL fermentation medium and cultivates, and collects after fermented and cultured Somatic cells extract lycopene.
24. according to claim 22 or 23 the methods, which is characterized in that the seed culture medium is 20g/L glucose, 20g/ L peptone, 10g/L yeast extract, 50mg/L uracil, remaining is water.
25. according to claim 22 or 23 the methods, which is characterized in that the fermentation medium is 20g/L glucose, 20g/ L peptone, 10g/L yeast extract, 20mg/L uracil, 10g/L D- (+)-galactolipin, remaining is water.
26. according to claim 22 or 23 the methods, which is characterized in that the culture is to train under the conditions of 30 DEG C, 250rpm It supports.
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