CN103865818A - Construction method of genetically engineered bacterium for producing astaxanthin - Google Patents
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Abstract
The invention discloses a construction method of genetically engineered bacterium for producing astaxanthin, which is characterized by comprising the following steps: A)constructing a gene expression module, wherein the gene expression module comprises relative gene for producing astaxanthin, a promoter at the upstream of relative gene for producing astaxanthin and a terminator at the downstream of the relative gene for producing astaxanthin; and B)performing cotransformation of the gene expression module to saccharomyces cerevisiae. The method has the advantages of simple, rapid and high efficiency performance, multi-grade clone is avoided, restriction enzyme cutting site is not required for depending on, multiple fragments enable cotransformation, the homologous recombination efficiency is high, and the engineering bacteria construction time can be obviously shortened.
Description
Technical field
The invention belongs to microorganism fermentation field, specifically, is the construction process that produces astaxanthin genetic engineering bacterium about a kind of.
Background technology
Up to now, report 600 multiple types carotene, carried out derived bacterium, algae, yeast and plant have concurrently, and astaxanthin is one of them.The chemical full name of astaxanthin is 3,3 '-dihydroxyl-4,4 '-diketo-β, and β '-carotene, structural formula is:
Astaxanthin, as the most effective natural colorant at present, has a wide range of applications in food, textile industry.In addition extremely effectively antioxidant of astaxanthin or one, can Cell protection avoids the damage of free radical, and owing to having good anticancer, enhancing body immunizing power and the anti-ageing vital role of waiting for a long time, and extremely medicine, health care and cosmetic industry are paid close attention to.
The method of producing at present astaxanthin mainly contains chemical synthesis and microbe fermentation method.The eighties in last century, F.Hoffmann-La Roche company completed first astaxanthin chemosynthesis, and successful commercialization, but chemical synthesis process is comparatively complicated, equipment requirements is higher simultaneously, and cost is higher, and has the shortcoming that product structure is single compared with natural astaxanthin, the astaxanthin that causes chemosynthesis to be produced suffers the vigilant of human consumer day by day, and starts to be subject to the legislation restriction of Countries.The source of Production of Astaxanthin from Fermentation by Microorganisms is mainly Haematocoocus Pluvialls and phaffiafhodozyma at present, zymotechnique maturation, and all successfully realize suitability for industrialized production, the content astaxanthin of Haematocoocus Pluvialls can be up to 2.0% of dry cell weight, and phaffiafhodozyma produce astaxanthin proportion in carotenoid can be up to 85%.Compared with chemical synthesis, microbe fermentation method is because its technique is simple, environmental protection, and industrial production cost is low, good product quality and be more subject to the favor of each production company.But Haematocoocus Pluvialls and phaffiafhodozyma have all limited its further development and production optimization to a certain extent because of its bacterial strain self-characteristic, and finding better production of astaxanthin bacterial strain becomes pursuing a goal of more and more investigators.
Pattern bacterium has been proved to be good genetic engineering bacterium host as intestinal bacteria, yeast saccharomyces cerevisiae etc.At present, investigator identified and comprised Haematocoocus Pluvialls, and phaffiafhodozyma is in the chemical activators approach of interior multiple-microorganism.
The homologous recombination efficiency of yeast is far away higher than bacterium or other higher eucaryote, yeast is as a kind of engineering bacteria service platform, be widely used in the heterogenous expression of bio-chemical pathway in approach engineering and metabolic engineering field, Chromosome recombination obtains engineering bacteria simultaneously, there is inheritance stability, the advantage of avoiding foreign gene to lose.
Ken etc. use yeast saccharomyces cerevisiae as Host Strains, structure comprises astaxanthin expresses multiple plasmids of required gene, then import Host Strains and produce astaxanthin (Ken Ukibe, Keisuke Hashida, Nobuyuki Yoshida, and Hiroshi Takagi.Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance.APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2009:7205-7211).But the method for this astaxanthin construction of genetic engineering need to be expressed astaxanthin relevant gene integration on yeast karyomit(e) successively, relates to multistage clone, and depend on suitable restriction enzyme site selection, complex operation, the cycle is long.
Summary of the invention
This research is the carotenogenesis approach based on phaffiafhodozyma (Xanthophyllomyces dendrorhous), and derive from the genes involved of Brevundimonas sp. and Nostoc punctiforme chemical activators, taking yeast saccharomyces cerevisiae as Host Strains, build product astaxanthin genetic engineering bacterium.
The present invention aims to provide a kind of construction process that produces astaxanthin genetic engineering bacterium.
The construction process of product astaxanthin genetic engineering bacterium of the present invention, comprises the following steps:
A) build genetic expression module, described genetic expression module comprises the terminator in the gene downstream that the promotor of gene that production of astaxanthin is relevant, upstream region of gene that described production of astaxanthin is relevant is relevant with described production of astaxanthin;
B) by described genetic expression module cotransformation to yeast saccharomyces cerevisiae.
According to the present invention, described steps A) described in genetic expression module also comprise the promotor of resistant gene, described resistant gene upstream and the terminator in described resistant gene downstream.
According to the present invention, described steps A) described in build genetic expression module and comprise the following steps:
A1) obtain the promoter fragment of the gene that described production of astaxanthin is relevant, described resistant gene fragment, upstream region of gene that described production of astaxanthin is relevant, the promoter fragment of described resistant gene upstream, the terminator fragment in gene downstream and the terminator fragment in described resistant gene downstream that described production of astaxanthin is relevant by pcr amplification;
A2) taking the promoter fragment of the relevant gene of described production of astaxanthin, described resistant gene fragment, upstream region of gene that described production of astaxanthin is relevant, the promoter fragment of described resistant gene upstream, the terminator fragment in gene downstream that described production of astaxanthin is relevant and the terminator fragment in described resistant gene downstream as template, obtain genetic expression module by overlapping pcr amplification.
According to a preferred embodiment of the invention, described steps A) described in the relevant gene of production of astaxanthin be crtE, crtYB, crtI, crtZ and crtW.
According to a preferred embodiment of the invention, the GenBank accession number of described crtE is DQ016502.1, the GenBank accession number of described crtYB is AY177204.1, the GenBank accession number of described crtI is AY177424.1, the GenBank accession number of described crtZ is AB181388.1, and the GenBank accession number of described crtW is CP001037.1.
According to a preferred embodiment of the invention, described steps A) described in promotor be selected from ADH1p, TEF1p, TPI1p, PGK1p, FBA1p and PDC1p.
According to a preferred embodiment of the invention, described promotor derives from yeast saccharomyces cerevisiae.
According to a preferred embodiment of the invention, described steps A) described in terminator be selected from TKL1t, ADH1t, ENO2t, TDH2t, CYC1t and TPI1t.
According to a preferred embodiment of the invention, described terminator derives from yeast saccharomyces cerevisiae.
According to a preferred embodiment of the invention, described resistant gene is ble gene.
According to a preferred embodiment of the invention, described resistant gene derives from pGAPZ α A.
Beneficial effect of the present invention: exogenous genetic fragment is proceeded in yeast cell simultaneously, utilize yeast self born of the same parents endogenous substance system one step that each fragment is assembled, and further Homologous integration arrives the chromosome position of design, there is simple and quick efficient advantage, avoided multistage clone, also do not need to rely on restriction enzyme site, multi-disc section transforms jointly, homologous recombination efficiency is high, within 1 ~ 2 week, can obtain engineering strain, and this good Yeast gene engineering method can obviously shorten engineering bacteria and build the time.The fermentation yield of the astaxanthin of the genetic engineering bacterium obtaining according to method of the present invention can reach about 600 μ g/gDCW, has good prospects for commercial application.
Brief description of the drawings
Fig. 1 is the chemical activators approach of engineering bacteria of the present invention, wherein, S.c represents yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), X.d represents phaffiafhodozyma (Xanthophyllomyces dendrorhous), BTS1, and crtE represents four Isoprenoid synthetic enzyme, crtI represents eight blue or green Lyeopene desaturases, crtYB represents eight blue or green Lyeopene synthase/lycopene cyclases, and crtZ represents β-carotene hydroxylase, and crtW represents β-carotene ketolase.
Fig. 2 is the integration assembling sequence schematic diagram of each gene module on karyomit(e).
Fig. 3 is the PCR checking electrophoresis detection figure of engineering bacteria of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting the scope of the invention.
Experimental program design of the present invention is as follows:
Purchased from EUROSCARF, number Y10000, bacterial strain information: MAT α at yeast saccharomyces cerevisiae BY4742(, his3 △ 1, leu2 △ 0, lys2 △ 0, ura3 △ 0) middle 5 gene crtE(GenBank:DQ016502.1 that express chemical activators approach, derive from Xanthophyllomyces dendrorhous ATCC24202), crtYB(GenBank:AY177204.1, derive from Xanthophyllomyces dendrorhous ATCC24202), crtI(GenBank:AY177424.1, derive from Xanthophyllomyces dendrorhous ATCC24202), crtZ(GenBank:AB181388.1, derive from Brevundimonas sp.SD212) and crtW(GenBank:CP001037.1, derive from Nostoc punctiforme PCC73102), and express ble gene (Zeocin resistant gene) as selection markers simultaneously, select yeast chromosomal δ site as integration site, the integration assembling sequence of each gene module on karyomit(e) as shown in Figure 1.Each promotor and gene are numbered respectively, as shown in table 1.
Table 1, each promotor and gene numbering
| Promotor numbering | Promotor title | Gene numbering | Gene |
| A | TEF1p | ||
| 1 | CrtE | ||
| B | TPI1p | 2 | CrtYB |
| C | PGK1p | 3 | CrtI |
| D | FBA1p | 4 | CrtZ |
| E | PDC1p | 5 | CrtW |
Utilize overlapping PCR(overlap PCR) method build following genetic expression module: delta53 '-ADH1p-ble-TKL1t, TEFlp-CrtE-ADH1t, TPI1p-CrtYB-ENO2t, PGK1p-CrtI-TDH2t, FBA1p-crtZ-TPI1t, PDC1p-crtW-CYC1t-delta5 5 ', the i.e. combination of A1B2C3D4E5.
Wherein, approach builds needs the information of little DNA fragmentation of pcr amplification as shown in table 2.
Table 2, little DNA fragmentation
According to the technical requirements of overlapping PCR, use the Vector NTI10.0 design PCR primer sequence above each small segment that is used for increasing, primer sequence is as shown in table 3.
Table 3, primer sequence
embodiment 2, genetic engineering bacterium structure
2.1, plasmid pYES2-CrtE, the structure of pYES2-CrtYB, pYES2-CrtI, pYES2-CrtZ and pSH62-CrtW
Reference literature (Ken Ukibe, Keisuke Hashida, Nobuyuki Yoshida, and Hiroshi Takagi.Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance.APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2009:7205-7211, Verwaal R, Wang J, Meijnen JP et al.High-level production of Beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.Appl Environ Microbiol, 2007, method 73:4342-4350), obtain crtE, crtYB and crtI gene, and be inserted into respectively pYES2.0(purchased from Invitrogen company) in, obtain plasmid pYES2-CrtE, pYES2-CrtYB and pYES2-CrtI, be transformed into E.coli bacterial strain DH5 α, preserve plasmid.
Entrust Synesis Company (Nanjing Genscript Biotechnology Co., Ltd.) to build the plasmid pYES2-CrtZ and the pSH62-CrtW that comprise CrtZ and CrtW gene fragment.First, adopt the method for chemical synthesising DNA sequence to obtain CrtZ and CrtW gene fragment, wherein, CrtZ sequence is identical with the crtZ gene order (GenBank:AB181388.1) of 10188bp～10673bp in Brevundimonas sp.SD212 carotenoid gene cluster, and CrtW sequence is identical with the genomic orf148 sequence of Nostoc punctiforme PCC73102 (GenBank:CP001037.1).Then synthetic gene C rtZ is inserted on plasmid vector pYES2.0 and obtains pYES2-CrtZ, synthetic gene C rtW is inserted into plasmid vector pSH62(purchased from EUROSCARF) on obtain pSH62-CrtW.
2.2, the extraction of template DNA
The extracting method of yeast saccharomyces cerevisiae BY4742 chromosomal DNA is as follows: the full single bacterium colony of picking is connected to 3mlYPD liquid nutrient medium test tube (20ml shaking flask), 30 DEG C, 220rpm/min, the about 18h of incubated overnight left and right, collect bacterium liquid, use genome extraction agent box (purchased from Axygen company) to extract genome.
The extracting method of plasmid is as follows: the DH5 α bacterium that inoculation contains corresponding plasmid is in 3ml LB liquid nutrient medium (20ml shaking flask), wherein containing penbritin final concentration is 100 μ g/ml, 37 DEG C, 220rpm/min, the about 15h of incubated overnight left and right, is used plasmid to bring up again test kit (purchased from Axygen company) extracting plasmid pGAPZ α A(purchased from Invitrogen company), pYES2-CrtE, pYES2-CrtYB, pYES2-CrtI, pYES2-crtZ and pSH62-crtW.
2.3, the pcr amplification of little DNA fragmentation
Each little DNA fragmentation amplification is all used high-fidelity KOD-Neo-Plus enzyme (spinning company purchased from TOYOBO Japan), according to 50 μ l KOD-PCR system preparation pcr amplification reaction liquid (table 4).
The pcr amplification reaction liquid preparation system of table 4, little DNA fragmentation
Above-mentioned PCR program 1 is for the δ 53 ' that increases, δ 55 ', TEF1p, TPI1p, PGK1p, TKL1t, ADH1t, ENO2t, TDH2t, ble, FBA1p, PDC1p, CYC1t, TPI1t, CrtZ, CrtW, crtE fragment.
PCR program 2 is as follows:
Above-mentioned PCR program 2 is for amplifying ADH 1p, crtYB, crtI fragment.
Use the agarose gel electrophoresis of 1% concentration to separate PCR product, rubber tapping, is used gel to reclaim test kit (purchased from Axygen company) purifying and reclaims fragment, and makes the heavy molten concentration of each fragment keep quite, saving backup.
The structure of 2.3, genetic expression module
Taking in 2.1 obtain each little DNA fragmentation as template, adopt Overlap PCR to build each genetic expression module delta53 '-ADH1p-ble-TKL1t, TEF1p-CrtE-ADH1t, TPI1p-CrtYB-ENO2t, PGK1p-CrtI-TDH2t, FBA1p-crtZ-TPI1t, PDClp-crtW-CYC1t-delta5 5 '.Use high-fidelity KOD-Neo-Plus enzyme (spinning company purchased from TOYOBO Japan), according to 50 μ l KOD-PCR system preparation pcr amplification reaction liquid (table 5).
The pcr amplification reaction liquid preparation system of table 5, genetic expression module
Overlap PCR program is as follows:
Overlap PCR product is used to the agarose gel electrophoresis of 1% concentration, rubber tapping, is used gel to reclaim test kit (purchased from Axygen company) purifying and reclaims fragment, saves backup and transforms.
2.3, genetic expression module cotransformation yeast saccharomyces cerevisiae BY4742
Adopt lithium acetate conversion method by genetic expression module delta53 '-ADHlp-ble-TKL1t, TEFlp-CrtE-ADH1t, TPI1p-CrtYB-ENO2t, PGKlp-CrtI-TDH2t, FBA1p-crtZ-TPI1t, PDC1p-crtW-CYC1t-delta5 5 ' cotransformation yeast saccharomyces cerevisiae BY4742.
Working method is as follows: picking list bacterium colony connects YPD test tube, in 30 DEG C, and 230rpm/min overnight incubation, the fresh 25ml YPD substratum (250ml shaking flask) of transferring, 30 DEG C, 230rpm/min shaking culture, treats bacterium liquid OD
600reach at 0.8 ~ 1.0 o'clock, 4 DEG C, 4000rpm, the centrifugal collection thalline of 5min, outwells supernatant, washs respectively thalline with ice bath sterilized water and 100m M LiAc, then uses the resuspended thalline of 100mM LiAc to cell concn approximately 2 × 10
9individual/mL, makes transformed competence colibacillus cell.Get 50 μ l competent cells, 4 DEG C, 13000rpm, 30s is centrifugal, and collection thalline is abandoned supernatant, adds successively the PEG6000 of 240 μ l 50%, the LiAc of 36 μ l 1M, 50 μ l strand milt DNA(are by the milt DNA boiling water boiling 5min depositing in advance, single stranded), the aseptic ddH of 24 μ l
2o and the plasmid DNA that is concentrated to 10 μ l left and right, vortex mixes, then 30 DEG C of water-bath 30min, 42 DEG C of water-bath 25min, 13000rpm, the centrifugal collection thalline of 30s, with rifle head sucking-off supernatant, add 1mlYPD, resuspended thalline, in 30 DEG C, more than 230rpm recovery 6h, 13000rpm after recovery, 30s is centrifugal, the resuspended thalline of 100 μ l sterilized water, it is that the microbiotic Zeocin(of 200 μ g/ml is purchased from Invitrogen company that coating contains final concentration) YPD solid medium, cultivate 3 ~ 4 days in 30 DEG C.
2.4, qualification
Select the darker clone's bacterium colony of color, be inoculated in 3ml YPD liquid nutrient medium test tube, 30 DEG C, 220rpm/min, the about 18h of incubated overnight left and right, gets bacterium liquid, use genome extraction agent box (purchased from Axygen company) to extract genome, to L1 as shown in Figure 2, L2, L3, L4, L5, L6 fragment is carried out PCR checking, result as shown in Figure 3, obtains a strain PCR and verifies the genotype positive colony that six bands all become clear.Above-mentioned construction process can repeat, and this is apparent for the person of ordinary skill of the art.
3.1, fermentation
Seed culture method is as follows:
Single bacterium colony of the positive colony that screening obtains from culture plate picking embodiment 2, access 25ml YPD substratum (250ml shaking flask), 30 DEG C, 220rpm/min, cultivates 48h.
Fermentation culture method is as follows:
By seed liquor, according in 4% inoculative proportion access 25ml YPD substratum (250ml shaking flask), 30 DEG C, 220rpm/min, cultivates 72h.
3.2, the extraction of astaxanthin
The extracting method of astaxanthin is as follows:
Fermented liquid is proceeded to the centrifuge tube of having weighed, 4 DEG C, 4000r/min, 5min is centrifugal, abandons supernatant, and sterilized water washs once, abandons supernatant, weighs.Every gram of wet thallus, correspondence adds 17.5ml to be preheated to the DMSO of 55 DEG C, mixes, in 55 DEG C of water-bath 12min; Add 25ml acetone again, mix, in 45 DEG C of water-bath lixiviate 15min, 4 DEG C, the centrifugal 10min of 4000r/min, gets supernatant.
3.3, the detection of astaxanthin
The detection method of astaxanthin is as follows:
To extract sample and adopt analysis mode Agilent 1100 liquid phase systems to analyze content astaxanthin, condition is as follows:
Analytical column: chromatographic column Diamonsil C18 post, 200mm × 4.6mm, 5 μ m
Column temperature: 30 DEG C;
Detect wavelength: 472nm;
Sample size: 5 μ l;
Analysis time: 13min;
Condition of gradient elution is as shown in table 6.
Table 6, condition of gradient elution
3.4, the determination of yield of astaxanthin
Through 10 batches of fermentations, the astaxanthin yield of the genetic engineering bacterium building by above step on average can reach 602 μ g/gDCW.
The detection of structure, fermentation and the astaxanthin of the genetic engineering bacterium of embodiment 4 ~ 7, five kind of different promoters and the assortment of genes
Ensure that the combination of resistant gene module is constant, other gene orders are constant, and ensure the promotor difference of each gene, build other four kinds of promotors and gene according to the genetic engineering bacterium of different order permutation and combination according to the method described in embodiment 1 ~ 2, wherein, four kinds of promotors and the assortment of genes put in order as follows: B1C2D3E4A5, C1D2E3A4B5, D1E2A3B4C5, E1A2B3C4D5.
The output of the genetic engineering bacterium of above-mentioned four kinds of combinations being fermented, extracts and measure astaxanthin according to the method for embodiment 3, result is as shown in table 7.
The output (5 batches of mean value) of the astaxanthin of the genetic engineering bacterium of table 7, five kinds of combination structures
Claims (11)
1. a construction process that produces astaxanthin genetic engineering bacterium, is characterized in that, comprises the following steps:
A) build genetic expression module, described genetic expression module comprises the terminator in the gene downstream that the promotor of gene that production of astaxanthin is relevant, upstream region of gene that described production of astaxanthin is relevant is relevant with described production of astaxanthin;
B) by described genetic expression module cotransformation to yeast saccharomyces cerevisiae.
2. the method for claim 1, is characterized in that, described steps A) described in genetic expression module also comprise the promotor of resistant gene, described resistant gene upstream and the terminator in described resistant gene downstream.
3. method as claimed in claim 1 or 2, is characterized in that, described steps A) described in build genetic expression module and comprise the following steps:
A1) obtain the promoter fragment of the gene that described production of astaxanthin is relevant, described resistant gene fragment, upstream region of gene that described production of astaxanthin is relevant, the promoter fragment of described resistant gene upstream, the terminator fragment in gene downstream and the terminator fragment in described resistant gene downstream that described production of astaxanthin is relevant by pcr amplification;
A2) taking the promoter fragment of the relevant gene of described production of astaxanthin, described resistant gene fragment, upstream region of gene that described production of astaxanthin is relevant, the promoter fragment of described resistant gene upstream, the terminator fragment in gene downstream that described production of astaxanthin is relevant and the terminator fragment in described resistant gene downstream as template, obtain genetic expression module by overlapping pcr amplification.
4. the method as described in claim 1 or 3, is characterized in that, described steps A) described in the relevant gene of production of astaxanthin be crtE, crtYB, crtI, crtZ and crtW.
5. method as claimed in claim 4, it is characterized in that, the GenBank accession number of described crtE is DQ016502.1, the GenBank accession number of described crtYB is AY177204.1, the GenBank accession number of described crtI is AY177424.1, the GenBank accession number of described crtZ is AB181388.1, and the GenBank accession number of described crtW is CP001037.1.
6. the method as described in claim 1 or 2 or 3, is characterized in that described steps A) described in promotor be selected from ADH1p, TEF1p, TPI1p, PGK1p, FBA1p and PDC1p.
7. method as claimed in claim 6, is characterized in that, described promotor derives from yeast saccharomyces cerevisiae.
8. the method as described in claim 1 or 2 or 3, is characterized in that described steps A) described in terminator be selected from TKL1t, ADH1t, ENO2t, TDH2t, CYC1t and TPI1t.
9. method as claimed in claim 8, is characterized in that, described terminator derives from yeast saccharomyces cerevisiae.
10. method as claimed in claim 2 or claim 3, is characterized in that, described resistant gene is ble gene.
11. methods as claimed in claim 10, is characterized in that, described resistant gene derives from pGAPZ α A.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107129995A (en) * | 2014-09-03 | 2017-09-05 | 武汉生物技术研究院 | A kind of construction method for producing astaxanthin genetic engineering bacterium |
| CN108998465A (en) * | 2018-06-25 | 2018-12-14 | 浙江皇冠科技有限公司 | The preparation method of recombinant microorganism source natural astaxanthin genetic engineering bacterium and its product |
| CN109609519A (en) * | 2018-10-31 | 2019-04-12 | 昆明理工大学 | A kind of gene RKcrtYB and its application |
| CN109971664A (en) * | 2019-04-23 | 2019-07-05 | 天津大学 | The bacterial strain of high-yield astaxanthin and its application |
| CN111454854A (en) * | 2020-05-02 | 2020-07-28 | 昆明理工大学 | Rhodosporidium toruloides gene engineering strain for producing astaxanthin |
| CN111635907A (en) * | 2020-07-02 | 2020-09-08 | 浙江华睿生物技术有限公司 | Method for constructing astaxanthin-producing strain |
| CN111979132A (en) * | 2020-08-20 | 2020-11-24 | 宜昌东阳光生化制药有限公司 | Fermentation medium for high-yield astaxanthin and application thereof |
| CN113699052A (en) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN113699053A (en) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN114317307A (en) * | 2021-12-30 | 2022-04-12 | 广州智特奇生物科技股份有限公司 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof |
| CN114574516A (en) * | 2022-01-18 | 2022-06-03 | 浙江大学杭州国际科创中心 | CRISPR/Cas 9-based yeast genome stable integration method |
| CN115807026A (en) * | 2022-08-01 | 2023-03-17 | 深圳大学 | Construction method and application of synthetic path of astaxanthin in chlamydomonas reinhardtii |
| CN114317307B (en) * | 2021-12-30 | 2024-05-10 | 广州智特奇生物科技股份有限公司 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof |
-
2012
- 2012-12-07 CN CN201210526173.8A patent/CN103865818A/en active Pending
Non-Patent Citations (7)
| Title |
|---|
| JAE HYUNG LEE ET AL.: "Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis", 《GENE》 * |
| JAE HYUNG LEE: "Functional expression of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis", 《BIOTECHNOL LETT》 * |
| MAURICIO NIKLITSCHEK: "Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous", 《BIOL RES》 * |
| RENÉ VERWAAL ET AL.: "High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 彭炳银等: "不同启动子控制下木酮糖激酶的差异表达及其对酿酒酵母木糖代谢的影响", 《微生物学报》 * |
| 葛菁萍等: "木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达", 《微生物学报》 * |
| 谭兆涛: "葡萄酒酿酒酵母基因工程菌构建的研究进展", 《食品与发酵科技》 * |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107129995A (en) * | 2014-09-03 | 2017-09-05 | 武汉生物技术研究院 | A kind of construction method for producing astaxanthin genetic engineering bacterium |
| CN107142250A (en) * | 2014-09-03 | 2017-09-08 | 武汉生物技术研究院 | β-the Cartoene hydroxylase and its encoding gene of Sphingol single-cell with its production astaxanthin in application |
| CN107142250B (en) * | 2014-09-03 | 2019-09-10 | 武汉生物技术研究院 | β-the Cartoene hydroxylase and its encoding gene of Sphingol single-cell and its application in production astaxanthin |
| CN108998465A (en) * | 2018-06-25 | 2018-12-14 | 浙江皇冠科技有限公司 | The preparation method of recombinant microorganism source natural astaxanthin genetic engineering bacterium and its product |
| CN109609519A (en) * | 2018-10-31 | 2019-04-12 | 昆明理工大学 | A kind of gene RKcrtYB and its application |
| CN109971664A (en) * | 2019-04-23 | 2019-07-05 | 天津大学 | The bacterial strain of high-yield astaxanthin and its application |
| CN111454854A (en) * | 2020-05-02 | 2020-07-28 | 昆明理工大学 | Rhodosporidium toruloides gene engineering strain for producing astaxanthin |
| CN111454854B (en) * | 2020-05-02 | 2022-05-06 | 昆明理工大学 | Rhodosporidium toruloides gene engineering strain for producing astaxanthin |
| CN113699053A (en) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN113699052A (en) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN113699053B (en) * | 2020-05-20 | 2023-08-11 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN113699052B (en) * | 2020-05-20 | 2023-08-11 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN111635907A (en) * | 2020-07-02 | 2020-09-08 | 浙江华睿生物技术有限公司 | Method for constructing astaxanthin-producing strain |
| CN111979132A (en) * | 2020-08-20 | 2020-11-24 | 宜昌东阳光生化制药有限公司 | Fermentation medium for high-yield astaxanthin and application thereof |
| CN111979132B (en) * | 2020-08-20 | 2022-07-26 | 宜昌东阳光生化制药有限公司 | Fermentation medium for high-yield astaxanthin and application thereof |
| CN114317307A (en) * | 2021-12-30 | 2022-04-12 | 广州智特奇生物科技股份有限公司 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof |
| CN114317307B (en) * | 2021-12-30 | 2024-05-10 | 广州智特奇生物科技股份有限公司 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof |
| CN114574516A (en) * | 2022-01-18 | 2022-06-03 | 浙江大学杭州国际科创中心 | CRISPR/Cas 9-based yeast genome stable integration method |
| CN114574516B (en) * | 2022-01-18 | 2023-10-27 | 浙江大学杭州国际科创中心 | CRISPR/Cas 9-based yeast genome stable integration method |
| CN115807026A (en) * | 2022-08-01 | 2023-03-17 | 深圳大学 | Construction method and application of synthetic path of astaxanthin in chlamydomonas reinhardtii |
| CN115807026B (en) * | 2022-08-01 | 2023-09-01 | 深圳大学 | Construction method and application of astaxanthin synthesis path in Chlamydomonas reinhardtii |
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