CN110499272A - Beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway - Google Patents

Beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway Download PDF

Info

Publication number
CN110499272A
CN110499272A CN201910665228.5A CN201910665228A CN110499272A CN 110499272 A CN110499272 A CN 110499272A CN 201910665228 A CN201910665228 A CN 201910665228A CN 110499272 A CN110499272 A CN 110499272A
Authority
CN
China
Prior art keywords
ala
leu
gly
val
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910665228.5A
Other languages
Chinese (zh)
Other versions
CN110499272B (en
Inventor
姜建国
陈浩宏
谢红
梁志聪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Priority to CN201910665228.5A priority Critical patent/CN110499272B/en
Publication of CN110499272A publication Critical patent/CN110499272A/en
Application granted granted Critical
Publication of CN110499272B publication Critical patent/CN110499272B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
    • C12Y103/05005—15-Cis-phytoene desaturase (1.3.5.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
    • C12Y103/05006—9,9'-Di-cis-zeta-carotene desaturase (1.3.5.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01029—Geranylgeranyl diphosphate synthase (2.5.1.29)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01032—15-Cis-phytoene synthase (2.5.1.32)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01012—Zeta-carotene isomerase (5.2.1.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01013—Prolycopene isomerase (5.2.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y505/00—Intramolecular lyases (5.5)
    • C12Y505/01—Intramolecular lyases (5.5.1)
    • C12Y505/01019—Lycopene beta-cyclase (5.5.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention discloses a kind of beta carotene high production bacteria based on Du Shi algae metabolic pathway and its construction method and application, is related to genetic engineering bacterium field.The present invention realizes all of the gene of the carotenoid approach from Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) engineering bacteria for constructing high yield beta carotene for the first time.When Du Shi algae is Du Shi Pasteur algae, the yield of the beta carotene of the beta carotene high production bacteria is 2.7mg/g (dry cell weight)；When Du Shi algae is Dunaliella salina, the yield of the beta carotene of the beta carotene high production bacteria is 3.3mg/g (dry cell weight).The promoter for the carrier that the present invention uses all is T7 promoter, includes lacZ controlling element, high can be expressed in the e. coli bl21 (DE3) of IPTG effect, this is conducive to a large amount of accumulation of beta carotene, can be used for practical application production.

Description

Beta carotene high production bacteria and its construction method based on Du Shi algae metabolic pathway With application
Technical field
The present invention relates to genetic engineering bacterium fields, are related to the building clone system of Carotenoid in Plants metabolic pathway carrier System, in particular to a kind of beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway；Tool Body is related to a kind of Du Shi algae (such as Du Shi Pasteur algae (Dunaliella bardawil), Dunaliella salina (Dunaliella Saline yak base Mang ox base pyrophosphate synthetase (Ggps), phytoene synthetase (Psy), octahydro tomato in)) Phytoene dehydrogenase enzyme (Pds), 15- be cis--sigma carotene isomerase (Ziso), sigma carotene dehydrogenase (Zds), carotenoids Plain isomerase (Crtiso), lycopene beta cyclase (Lycb) gene cDNA and construct the engineering of high yield beta carotene Bacterium.
Background technique
Carotenoid molecule is the terpenoid containing 8 isoprene units, is widely present as organic pigment In the chloroplaset and chromoplast of plant and in some other photosynthetic tissues, such as algae, in part bacterium and fungi. Under given conditions, carotenoid content can reach the 14% of dry cell weight in Du Shi frustule.Its high-content may be with enzyme Transformation efficiency have very big relationship.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of based on Du Shi algae generation Thank to the beta carotene high production bacteria of approach.
Another object of the present invention is to provide the above-mentioned beta carotene high production bacterias based on Du Shi algae metabolic pathway Construction method.
A further object of the present invention is to provide the above-mentioned beta carotene high production bacterias based on Du Shi algae metabolic pathway Application.
The purpose of the invention is achieved by the following technical solution:
A kind of beta carotene high production bacteria based on Du Shi algae metabolic pathway is based on Du Shi Pasteur algae When (Dunaliella bardawil) metabolic pathway, the beta carotene high production bacteria contains coding GenBank: Yak base Mang ox base pyrophosphate synthetase (Ggps) gene, the coding SEQ ID of amino acid sequence shown in APW83740.1 Phytoene synthetase (Psy) gene of amino acid sequence shown in NO:2 encodes ammonia shown in GenBank:ADD52599.1 The 15- of amino acid sequence shown in phytoene dehydrogenase (Pds) gene of base acid sequence, coding SEQ ID NO:4 is cis-- The sigma carotene dehydrogenase of amino acid sequence shown in sigma carotene isomerase (Ziso) gene, coding SEQ ID NO:6 (Zds) gene, coding SEQ ID NO:8 shown in amino acid sequence carotenoid isomerase (Crtiso) gene and coding Lycopene beta cyclase (Lycb) gene of amino acid sequence shown in GenBank:ANY98896.1；
When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the beta carotene high production bacteria contains There are yak base Mang ox base pyrophosphate synthetase (Ggps) gene, the coding of amino acid sequence shown in coding SEQ ID NO:10 Phytoene synthetase (Psy) gene, the coding GenBank of amino acid sequence shown in GenBank:AAB51287.1: Phytoene dehydrogenase (Pds) gene of amino acid sequence shown in CAA75094.1 encodes ammonia shown in SEQ ID NO:12 The 15- of base acid sequence is cis--sigma carotene isomerase (Ziso) gene, amino acid sequence shown in coding SEQ ID NO:14 The carotenoid isomerase of amino acid sequence shown in sigma carotene dehydrogenase (Zds) gene, coding SEQ ID NO:16 (Crtiso) lycopene beta cyclase (Lycb) base of gene and amino acid sequence shown in coding GenBank:ADX41685.1 Cause；
Preferably, when being based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, GenBank is encoded: The nucleotide sequence of yak base Mang ox base pyrophosphate synthetase (Ggps) gene of amino acid sequence shown in APW83740.1 For the nucleotide sequence as shown in GenBank:KX231795.1；Wherein, GenBank:APW83740.1 and GenBank: Source Dunaliella salina record in KX231795.1 is wrong, and practical source should be Dunaliella bardawil；
Encode the nucleotides sequence of phytoene synthetase (Psy) gene of amino acid sequence shown in SEQ ID NO:2 It is classified as the nucleotide sequence as shown in SEQ ID NO:1；
Encode the core of phytoene dehydrogenase (Pds) gene of amino acid sequence shown in GenBank:ADD52599.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:GQ923693.1；Wherein, GenBank:ADD52599.1 with Source Dunaliella salina record in GenBank:GQ923693.1 is wrong, and practical source should be Dunaliella bardawil；
The 15- for encoding amino acid sequence shown in SEQ ID NO:4 is cis--core of sigma carotene isomerase (Ziso) gene Nucleotide sequence is the nucleotide sequence as shown in SEQ ID NO:3；
Encode the nucleotide sequence of sigma carotene dehydrogenase (Zds) gene of amino acid sequence shown in SEQ ID NO:6 For the nucleotide sequence as shown in SEQ ID NO:5；
Encode the nucleotides sequence of carotenoid isomerase (Crtiso) gene of amino acid sequence shown in SEQ ID NO:8 It is classified as the nucleotide sequence as shown in SEQ ID NO:7；
Encode the core of lycopene beta cyclase (Lycb) gene of amino acid sequence shown in GenBank:ANY98896.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:KX218392.1；Wherein, GenBank:ANY98896.1 with Source Dunaliella salina record in GenBank:KX218392.1 is wrong, and practical source should be Dunaliella bardawil；
Preferably, it when being based on Dunaliella salina (Dunaliella saline) metabolic pathway, encodes shown in SEQ ID NO:10 The nucleotides sequence of yak base Mang ox base pyrophosphate synthetase (Ggps) gene of amino acid sequence is classified as such as SEQ ID NO:9 Shown in nucleotide sequence；
Encode the core of phytoene synthetase (Psy) gene of amino acid sequence shown in GenBank:AAB51287.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:U91900.1；Wherein, the source in GenBank:U91900.1 Dunaliella bardawil record is wrong, and practical source should be Dunaliella saline；
Encode the core of phytoene dehydrogenase (Pds) gene of amino acid sequence shown in GenBank:CAA75094.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:Y14807.1；Wherein, the source in GenBank:Y14807.1 Dunaliella bardawil record is wrong, and practical source should be Dunaliella saline；
The 15- for encoding amino acid sequence shown in SEQ ID NO:12 is cis--sigma carotene isomerase (Ziso) gene Nucleotides sequence is classified as the nucleotide sequence as shown in SEQ ID NO:11；
Encode the nucleotide sequence of sigma carotene dehydrogenase (Zds) gene of amino acid sequence shown in SEQ ID NO:14 For the nucleotide sequence as shown in SEQ ID NO:13；
Encode the nucleotide of carotenoid isomerase (Crtiso) gene of amino acid sequence shown in SEQ ID NO:16 Sequence is the nucleotide sequence as shown in SEQ ID NO:15；
Encode the core of lycopene beta cyclase (Lycb) gene of amino acid sequence shown in GenBank:ADX41685.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:HQ728089.1.
The construction method of the beta carotene high production bacteria based on Du Shi algae metabolic pathway, including walk as follows It is rapid:
(1) yak base Mang ox base pyrophosphate synthetase is cloned into from Du Shi algae using related gene engineering means (Ggps), phytoene synthetase (Psy), phytoene dehydrogenase (Pds), 15- it is cis--sigma carotene isomery Enzyme (Ziso), sigma carotene dehydrogenase (Zds), carotenoid isomerase (Crtiso), lycopene beta cyclase (Lycb)；
(2) by Ggps and Psy building on pACYduet-1 carrier, chlorampenicol resistant obtains recombinant vector pACYduet- ggps-psy；By Pds, Zds and Lycb building on pCDFduet-1 carrier, streptomycin resistance obtains recombinant vector pCDFduet-pds-zds-lycb；By Ziso and Crtiso building on pETduet-1 carrier, ammonia benzyl resistance obtains recombination and carries Body pETduet-ziso-crtiso；
(3) then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), base is obtained In the beta carotene high production bacteria of Du Shi algae metabolic pathway.
Preferably, the Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil) or Dunaliella salina (Dunaliella saline)。
Preferably, when construction of recombinant vector, rbs (ribosomes combination is respectively connected in the N-terminal (5 ' end) of 7 target gene Site) sequence, alternatively, being respectively connected with T7 promoter (T7_promoter) sequence and rbs (ribosome bind site) sequence.
The beta carotene high production bacteria based on Du Shi algae metabolic pathway answering in production beta carotene With.
When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the beta carotene high production bacteria Beta carotene yield be 2.7mg/g (dry cell weight).
When Du Shi algae is Dunaliella salina (Dunaliella saline), the β-of the beta carotene high production bacteria The yield of carrotene is 3.3mg/g (dry cell weight).
The present invention has the following advantages and effects with respect to the prior art:
The present invention is realized for the first time all of the carotenoids for coming from Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) The gene of plain approach constructs engineering bacteria.And Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) is known production beta carotene One of highest plant.The promoter for the carrier that the present invention uses all is T7 promoter, includes lacZ controlling element, in IPTG It high can be expressed in the e. coli bl21 (DE3) of effect, this is conducive to a large amount of accumulation of beta carotene, can be used for actually answering With production.
Detailed description of the invention
Fig. 1 is pACYduet-Dbggps-Dbpsy of the present invention (A) and pCDFduet-Dbpds-Dbzds-Dblycb (B) structure Build figure.
Fig. 2 is pETduet-Dbziso-Dbcrtiso structure figures of the present invention.
Fig. 3 is that the present invention is based on the beta carotene high production bacterias of Du Shi algae metabolic pathway and Bacillus coli communis BL21 (DE3) color compares；Wherein, A is the beta carotene high production bacteria based on Du Shi Pasteur's algae metabolic pathway；B is based on Du The beta carotene high production bacteria of family name's salt algae metabolic pathway.
Fig. 4 is beta carotene liquid phase figure；Wherein, A is embodiment 1；B is embodiment 2.
Fig. 5 is pACYduet1-Dsggps-Dspsy structure figures of the present invention.
Fig. 6 is pCDFduet1-Dspds-Dszds-Dslycb structure figures of the present invention.
Fig. 7 is pETduet1-Dsziso-Dscrtiso structure figures of the present invention.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels And material.
Embodiment 1
1. preparing linearized vector
(1) NcoI and HindIII (being purchased from Thermo Fisher Scientific) double digestion plasmid pACYduet-1 are used, PCDFduet-1 and pETduet-1 (being purchased from EMD Biosciences).37 DEG C of water-bath 3h.Endonuclease reaction system is as follows:
10 × Buffer, 5 μ L, each 1 μ L of two kinds of enzymes, 10 μ L of plasmid, Add dH2O To 50μL。
(2) 1% agarose gel electrophoresis detect digestion products.
(3) after digestion, using PCR Purification Kit linearized vector.
2.PCR expands target fragment
(1) Chinese Academy of Sciences aquatile research institute fresh water algae (is purchased from Du Shi Pasteur algae (Dunaliella bardawil) Library, number FACHB-847) reverse transcription cDNA be template, use the PrimeSTAR HS DNA Polymerase of Takara (high fidelity enzyme is purchased from Takara), carries out PCR reaction.
(2) 1% agarose gel electrophoresis detect PCR product.
(3) purpose piece is recycled with the plastic recovery kit E.Z.N.A.TM Gel Extraction Kit of OMEGA company Section, purified pcr product.Sequencing result such as GenBank:KX231795.1, SEQ ID NO:1, GenBank:GQ923693.1, SEQ ID NO:3,5,7, shown in GenBank:KX218392.1, the amino acid sequence such as GenBank of corresponding coding: APW83740.1, SEQ ID NO:2, GenBank:ADD52599.1, SEQ ID NO:4,6,8, GenBank:ANY98896.1 It is shown.
3.In-Fusion clone
With NcoI and HindIII double digestion plasmid pACYduet-1 and insertion primer Pac_dbggps_F and Pac_ The Dbggps that dbggps_R is expanded, obtains pACYduet-Dbggps；NdeI and EcoRV double digestion pACYduet- is used again The Dbpsy that Dbggps and insertion are expanded with primer Pac_dbpsy_F and Pac_dbpsy_R, obtains pACYduet- Dbggps-Dbpsy.Primer sequence is as shown in table 1.
With NcoI and HindIII double digestion plasmid pCDFduet-1 and insertion primer Pcd_dbpds_F and Pcd_ The Dbpds that dbpds_R is expanded, obtains pCDFduet-Dbpds；NdeI and EcoRV double digestion pCDFduet-Dbpds is used again And the Dbzds that insertion is expanded with primer Pcd_dbzds_F and Pcd_dbzds_R, obtain pCDFduet-Dbpds-Dbzds； It with AatII and KpnI double digestion pCDFduet-Dbpds-Dbzds and is inserted into again with primer Pcd_dblycb_F and Pcd_ The Dblycb that dblycb_R is expanded, obtains pCDFduet-Dbpds-Dbzds-Dblycb.Primer sequence is as shown in table 1.
With NcoI and HindIII double digestion plasmid pETduet-1 and insertion primer Pet_dbziso_F and Pet_ The Dbziso that dbziso_R is expanded, obtains pETduet-Dbziso；NdeI and EcoRV double digestion pETduet- is used again The Dbcrtiso that Dbziso and insertion are expanded with primer Pet_dbcrtiso_F and Pet_dbcrtiso_R, obtains pETduet-Dbziso-Dbcrtiso.Primer sequence is as shown in table 1.
(1) In-Fusion (purchased from Takara) cloning reaction system is established:
5X In-Fusion HD Enzyme Premix 2μL
Linearized vector 1μL
The PCR fragment of purifying 2μL
dH2O 5μL
Total 10μL
(2) 50 DEG C of incubation 15min, are subsequently placed on ice.
(3) connection product converts bacillus coli DH 5 alpha respectively, is coated on containing chloramphenicol (CmR or CamR), streptomysin (Sm) or on the corresponding plate of ampicillin (Ap or Amp).It is inverted 12~16h of culture respectively in 37 DEG C of biochemical cultivation cases. Notice that incubation time is unsuitable too long, otherwise resistance fails, and grows satellite colony, causes difficulty to screening positive clone.
The primer of 1 construction recombination plasmid of table
4. the extraction of plasmid
Referring to the specification of the E.Z.N.A.TM Plasmid Mini Kit kit of OMEGA company, recombinant plasmid is extracted PACYduet-Dbggps-Dbpsy (Figure 1A), pCDFduet-Dbpds-Dbzds-Dblycb (Figure 1B), pETduet-Dbziso- Dbcrtiso (Fig. 2).
(1) 10000g, 1min centrifugation, collect thallus.(2) 250 μ L Solution I are added, are vortexed more than ten seconds, mix. (3) 250 μ L Solution II are added, turns upside down 4~6 times, is placed at room temperature for 2min.(4) 350 μ L Solution III are added, It gently shakes up, 10000g, 10min centrifugation.(5) turn supernatant to Hibind column, 10000g, 1min centrifugation.(6) filtrate is discarded, is added Enter 500 μ L Buffer HB, 10000g, 1min centrifugations.(7) filtrate is discarded, 700 μ L DNA Wash Buffer are added, 10000g, 1min centrifugation.(8) it repeats to wash primary (step (7)).(9) 10000g, blank pipe are centrifuged 2min.(10) collecting pipe is discarded HiBind column sleeve is managed into clean 1.5mL EP, 50~100 μ L aqua sterilisas of addition (60 DEG C of preheatings) eluted dna, standing 1~ After 2min, 10000g, 1min centrifugation.
5. converting e. coli bl21 (DE3)
By recombinant plasmid pACYduet-Dbggps-Dbpsy, pCDFduet-Dbpds-Dbzds-Dblycb of extraction and PETduet-Dbziso-Dbcrtiso cotransformation is coated on to e. coli bl21 (DE3) containing CmR, Sm and Ap antibiotic On plate, 12~16h of culture is inverted in 37 DEG C of biochemical cultivation cases.Purpose bacterial strain is obtained by screening, that is, is based on Du Shi Pasteur The beta carotene high production bacteria of algae metabolic pathway, as shown in Figure 3A, white are BL21 (DE3) control strain, khaki For high yield beta carotene bacterial strain.
6. the culture of transformant
In the sterilized LB liquid medium of 50mL (chloramphenicol, ampicillin, strepto- is added in purpose strain inoculated Plain resistance), the shaken cultivation 6h at 200rpm, 37 DEG C.IPTG is added to be induced, cultivates 6 hours.
7. extracting the carotenoid of transformant
(1) 24mL bacterium solution is centrifuged under 4 DEG C, 8000rpm 2min, abandons supernatant.(2) it is suspended again large intestine with deionized water Bacilli-cell, 4 DEG C, 8000rpm is centrifuged 2min, abandons supernatant.(3) step (2) are repeated.(4) plus 6mL acetone is heavy to Escherichia coli It forms sediment, vortex oscillation dispersion.(5) 55 DEG C of water-bath 20min, it is primary every 5min oscillation.(6) by mixture in 4 DEG C, under 8000rpm It is centrifuged 20min, collects supernatant.(7) 6mL, -20 DEG C of preservations are settled to acetone.
The carotenoid composition of 8.HPLC qualitative analysis transformant
(1) mobile phase is subjected to suction filtration processing with 0.22 μm of aperture filter membrane, be subsequently ultrasonicated in 60min removal liquid Bubble.
(2) it by Carotenoids Extractss and beta carotene standard solution, is filtered with the polycarbonate in 0.2 μm of aperture Film is filtered processing.
(3) chromatographic column (C30YMC carotenoid column, 5 μm, 250*4.6mm) temperature is adjusted at 25 DEG C, with The flow velocity of 1.0mL/min is rinsed with methanol to balance (about 2h).
(4) be changed to mobile phase methyl tertiary butyl ether(MTBE) (A phase)/methanol (B phase) (gradient elution: 0min, 90%A phase and 10%B phase；10min, 60%A phase and 40%B phase；20min 50%A phase and 50%B phase；25min, 10%A phase and 90%B phase； 29.5min, 90%A phase and 10%B phase), it carries out washing column with the flow velocity of 1mL/min, until balance.
(5) the beta carotene standard items that concentration is 100 μ g/mL are stored into liquid and is diluted to 0.625 μ g/mL, 1.25 μ g/ The working solution of the various concentrations such as mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL and 20 μ g/mL is measured under 473nm wavelength with HPLC instrument The peak area of working solution draws standard curve according to result.
(6) 20 μ L of loading, detects 40min at wavelength 473nm.
As a result as shown in Figure 4 A, show the characteristic peak of beta carotene occur in 16.2min or so, this and standard items Appearance time is consistent, according further to the peak area, in conjunction with obtained calibration curve equation: y=34.791x+59.973 (x For peak area, y is the content of beta carotene), further according to the dry weight of cell, the yield that can calculate beta carotene is 2.7mg/g (dry cell weight).
Embodiment 2
1. preparing linearized vector
(1) Asc I and Sal I (being purchased from Thermo Fisher Scientific) double digestion plasmid pACYduet-1 is used, PCDFduet-1 and pETduet-1 (being purchased from EMD Biosciences).37 DEG C of water-bath 3h.Endonuclease reaction system is as follows:
10 × Buffer, 5 μ L, each 1 μ L of two kinds of enzymes, 10 μ L of plasmid, Add dH2O To 50μL。
(2) 1% agarose gel electrophoresis detect digestion products.
(3) after digestion, using PCR Purification Kit linearized vector.
2.PCR expands target fragment
(1) Culture Collection of Algae and (is purchased from Dunaliella salina (Dunaliella saline) Protozoa, Britain；Number CCAP19/18) reverse transcription cDNA be template, use the PrimeSTAR HS DNA of Takara Polymerase (high fidelity enzyme is purchased from Takara), carries out PCR reaction.
(2) 1% agarose gel electrophoresis detect PCR product.
(3) purpose piece is recycled with the plastic recovery kit E.Z.N.A.TM Gel Extraction Kit of OMEGA company Section, purified pcr product.Sequencing result such as SEQ ID NO:9, GenBank:U91900.1, GenBank:Y14807.1, SEQ ID NO:11,13,15, shown in GenBank:HQ728089.1, amino acid sequence such as SEQ ID NO:10 of corresponding coding, GenBank:AAB51287.1, GenBank:CAA75094.1, SEQ ID NO:12,14,16, GenBank:ADX41685.1 institute Show.
3.In-Fusion clone
With Asc I and Sal I double digestion plasmid pACYduet-1 and insertion primer Pac_dsggps_F and Pac_ The Dsggps that dsggps_R is expanded, obtains pACYduet1-Dsggps；NdeI and EcoRV double digestion pACYduet1- is used again The Dspsy that Dsggps and insertion are expanded with primer Pac_dspsy_F and Pac_dspsy_R, obtains pACYduet1- Dsggps-Dspsy.Primer sequence is as shown in table 2.
With Asc I and Sal I double digestion plasmid pCDFduet-1 and insertion primer Pcd_dspds_F and Pcd_dspds_ The Dspds that R is expanded, obtains pCDFduet1-Dspds；It with NdeI and EcoRV double digestion pCDFduet1-Dspds and inserts again Enter the Dszds expanded with primer Pcd_dszds_F and Pcd_dszds_R, obtains pCDFduet1-Dspds-Dszds；Again With AatII and KpnI double digestion pCDFduet1-Dspds-Dszds and insertion primer Pcd_dslycb_F and Pcd_dslycb_ The Dslycb that R is expanded, obtains pCDFduet1-Dspds-Dszds-Dslycb.Primer sequence is as shown in table 2.
With Asc I and Sal I double digestion plasmid pETduet-1 and insertion primer Pet_dsziso_F and Pet_ The Dsziso that dsziso_R is expanded, obtains pETduet1-Dsziso；NdeI and EcoRV double digestion pETduet1- is used again The Dscrtiso that Dsziso and insertion are expanded with primer Pet_dscrtiso_F and Pet_dscrtiso_R, obtains pETduet1-Dsziso-Dscrtiso.Primer sequence is as shown in table 2.
(1) In-Fusion (purchased from Takara) cloning reaction system is established:
5X In-Fusion HD Enzyme Premix 2μL
Linearized vector 1μL
The PCR fragment of purifying 2μL
dH2O 5μL
Total 10μL
(2) 50 DEG C of incubation 15min, are subsequently placed on ice.
(3) connection product converts bacillus coli DH 5 alpha respectively, is coated on containing chloramphenicol (CmR or CamR), streptomysin (Sm) or on the corresponding plate of ampicillin (Ap or Amp).In 37 DEG C of biochemical cultivation cases respectively be inverted culture 12~ 16h.Notice that incubation time is unsuitable too long, otherwise resistance fails, and grows satellite colony, causes difficulty to screening positive clone.
The primer of 2 construction recombination plasmid of table
4. the extraction of plasmid
Referring to the specification of the E.Z.N.A.TM Plasmid Mini Kit kit of OMEGA company, recombinant plasmid is extracted PACYduet1-Dsggps-Dspsy (Fig. 5), pCDFduet1-Dspds-Dszds-Dslycb (Fig. 6), pETduet1- Dsziso-Dscrtiso (Fig. 7).
(1) 10000g, 1min centrifugation, collect thallus.(2) 250 μ L Solution I are added, are vortexed more than ten seconds, mix. (3) 250 μ L Solution II are added, turns upside down 4~6 times, is placed at room temperature for 2min.(4) 350 μ L Solution III are added, It gently shakes up, 10000g, 10min centrifugation.(5) turn supernatant to Hibind column, 10000g, 1min centrifugation.(6) filtrate is discarded, is added Enter 500 μ L Buffer HB, 10000g, 1min centrifugations.(7) filtrate is discarded, 700 μ L DNA Wash Buffer are added, 10000g, 1min centrifugation.(8) it repeats to wash primary (step (7)).(9) 10000g, blank pipe are centrifuged 2min.(10) collecting pipe is discarded HiBind column sleeve is managed into clean 1.5mL EP, 50~100 μ L aqua sterilisas of addition (60 DEG C of preheatings) eluted dna, standing 1~ After 2min, 10000g, 1min centrifugation.
5. converting e. coli bl21 (DE3)
By recombinant plasmid pACYduet1-Dsggps-Dspsy, pCDFduet1-Dspds-Dszds-Dslycb of extraction and PETduet1-Dsziso-Dscrtiso cotransformation is coated on to e. coli bl21 (DE3) containing CamR, Sm and Amp antibiosis On the plate of element, 12~16h of culture is inverted in 37 DEG C of biochemical cultivation cases.Purpose bacterial strain is obtained by screening, that is, is based on Du Shi The beta carotene high production bacteria of salt algae metabolic pathway, as shown in Figure 3B, white are BL21 (DE3) control strain, khaki It is high yield beta carotene bacterial strain.
6. the culture of transformant
In the sterilized LB liquid medium of 50mL (chloramphenicol, ampicillin, strepto- is added in purpose strain inoculated Plain resistance), the shaken cultivation 6h at 200rpm, 37 DEG C.IPTG is added to be induced, cultivates 6 hours.
7. extracting the carotenoid of transformant
(1) 24mL bacterium solution is centrifuged under 4 DEG C, 8000rpm 2min, abandons supernatant.(2) it is suspended again large intestine with deionized water Bacilli-cell, 4 DEG C, 8000rpm is centrifuged 2min, abandons supernatant.(3) step (2) are repeated.(4) plus 6mL acetone is heavy to Escherichia coli It forms sediment, vortex oscillation dispersion.(5) 55 DEG C of water-bath 20min, it is primary every 5min oscillation.(6) by mixture in 4 DEG C, under 8000rpm It is centrifuged 20min, collects supernatant.(7) 6mL, -20 DEG C of preservations are settled to acetone.
The carotenoid composition of 8.HPLC qualitative analysis transformant
(1) mobile phase is subjected to suction filtration processing with 0.22 μm of aperture filter membrane, be subsequently ultrasonicated in 60min removal liquid Bubble.
(2) it by Carotenoids Extractss and beta carotene standard solution, is filtered with the polycarbonate in 0.2 μm of aperture Film is filtered processing.
(3) chromatographic column (C30YMC carotenoid column, 5 μm, 250*4.6mm) temperature is adjusted at 25 DEG C, with The flow velocity of 1.0mL/min is rinsed with methanol to balance (about 2h).
(4) be changed to mobile phase methyl tertiary butyl ether(MTBE) (A phase)/methanol (B phase) (gradient elution: 0min, 90%A phase and 10%B phase；10min, 60%A phase and 40%B phase；20min 50%A phase and 50%B phase；25min, 10%A phase and 90%B phase； 29.5min, 90%A phase and 10%B phase), it carries out washing column with the flow velocity of 1mL/min, until balance.
(5) standard curve of beta carotene is referring to 1 step 8 of embodiment (5).
(6) 20 μ L of loading, detects 40min at wavelength 473nm.
As a result as shown in Figure 4 B, show the characteristic peak of beta carotene occur in 16.2min or so, this and standard items Appearance time is consistent, according further to the peak area, in conjunction with obtained calibration curve equation: y=34.791x+59.973 (x For peak area, y is the content of beta carotene), further according to the dry weight of cell, the yield that can calculate beta carotene is 3.3mg/g (dry cell weight).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>South China Science & Engineering University
<120>beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway
<160> 16
<170> SIPOSequenceListing 1.0
<211> 1305
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Psy
<400> 1
atgacgctgt caatgttgga cgcgcgaagg atggcacagc gaacagcaac ttcctcctcc 60
tcctctccta gcatcatata tgccccatcg cccataagca atcgcagcgg caggcgcgca 120
gcagcgaatc acggcatcag gaatggtagt cgcagagcag caggccggat gggactctgc 180
agcactgtgc aagtgaactg cacgctcgcg atgccccagc ccaaccacgg ccagaagatg 240
cgattgcagc agcagcagca gcaacaactg cagcagcagc agcaacagca actatcggga 300
aagcaggtgg aggagcaggc gatgctgcag tgcataaaga ccgctcagtc agtgcccccc 360
tccaccggac tcctcaatcc tcgcggcctg cgatggcagg gcagcagctt ggaagcagcg 420
tacgagcgat gtggggcggt gtgcagcgag tacgccaaga ccttctacct cggtactcag 480
ctcatgacac cagtgcaggc caggtgcatc tgggccatct atgtgtggtg ccgccgcaca 540
gatgagctgg tggatggccc caatgcatca aagatcacgc ctcaggccct agacagatgg 600
gaggagcgcc ttgaaagcat gttccaaggc aagccctatg atgtgctgga cgcggcgctc 660
acagacacca tctccaaatt ccctctggag gtgcaaccct tcagagacat gatcgagggc 720
atgcgaatgg acctcttcaa gtcgcggtat cacacctttg atgagctgta cgagtactgc 780
tatcgtgtgg cgggcacagt ggggctgatg accatgccag tgatggggat tgatcccaac 840
tacaagggtc caattgacaa ggtctacaag gccgcccttg cgctgggtac ggcaaaccag 900
ctcaccaaca ttctgcgaga tgtgggagag gacatcagag agcgtgaccg tatctacttg 960
cccctggatg agctcaagca gttcggcatc tccgaagagg aggtaaaagc aggtatccac 1020
aagccatcgc aaggcaaggt ggatgagcgg tggcgagcgt tcatgaagtt ccagatcaag 1080
cgtgcgcgag agtacttcca ggaagcagag gatggggtag actacttgga cgtgaaggcg 1140
cggtggccag tgtggtcagc gctgatcctt taccgccaaa tcttggatgt cattgagaag 1200
aatgactacg acaacttctc catgcgcgca tacgtgccta agtccaagaa gtttgcatcg 1260
ttgccgatgg ccttgttccg ggccttggtg cccaagaaca aataa 1305
<211> 434
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Psy
<400> 2
Met Thr Leu Ser Met Leu Asp Ala Arg Arg Met Ala Gln Arg Thr Ala
1 5 10 15
Thr Ser Ser Ser Ser Ser Pro Ser Ile Ile Tyr Ala Pro Ser Pro Ile
20 25 30
Ser Asn Arg Ser Gly Arg Arg Ala Ala Ala Asn His Gly Ile Arg Asn
35 40 45
Gly Ser Arg Arg Ala Ala Gly Arg Met Gly Leu Cys Ser Thr Val Gln
50 55 60
Val Asn Cys Thr Leu Ala Met Pro Gln Pro Asn His Gly Gln Lys Met
65 70 75 80
Arg Leu Gln Gln Gln Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln
85 90 95
Gln Leu Ser Gly Lys Gln Val Glu Glu Gln Ala Met Leu Gln Cys Ile
100 105 110
Lys Thr Ala Gln Ser Val Pro Pro Ser Thr Gly Leu Leu Asn Pro Arg
115 120 125
Gly Leu Arg Trp Gln Gly Ser Ser Leu Glu Ala Ala Tyr Glu Arg Cys
130 135 140
Gly Ala Val Cys Ser Glu Tyr Ala Lys Thr Phe Tyr Leu Gly Thr Gln
145 150 155 160
Leu Met Thr Pro Val Gln Ala Arg Cys Ile Trp Ala Ile Tyr Val Trp
165 170 175
Cys Arg Arg Thr Asp Glu Leu Val Asp Gly Pro Asn Ala Ser Lys Ile
180 185 190
Thr Pro Gln Ala Leu Asp Arg Trp Glu Glu Arg Leu Glu Ser Met Phe
195 200 205
Gln Gly Lys Pro Tyr Asp Val Leu Asp Ala Ala Leu Thr Asp Thr Ile
210 215 220
Ser Lys Phe Pro Leu Glu Val Gln Pro Phe Arg Asp Met Ile Glu Gly
225 230 235 240
Met Arg Met Asp Leu Phe Lys Ser Arg Tyr His Thr Phe Asp Glu Leu
245 250 255
Tyr Glu Tyr Cys Tyr Arg Val Ala Gly Thr Val Gly Leu Met Thr Met
260 265 270
Pro Val Met Gly Ile Asp Pro Asn Tyr Lys Gly Pro Ile Asp Lys Val
275 280 285
Tyr Lys Ala Ala Leu Ala Leu Gly Thr Ala Asn Gln Leu Thr Asn Ile
290 295 300
Leu Arg Asp Val Gly Glu Asp Ile Arg Glu Arg Asp Arg Ile Tyr Leu
305 310 315 320
Pro Leu Asp Glu Leu Lys Gln Phe Gly Ile Ser Glu Glu Glu Val Lys
325 330 335
Ala Gly Ile His Lys Pro Ser Gln Gly Lys Val Asp Glu Arg Trp Arg
340 345 350
Ala Phe Met Lys Phe Gln Ile Lys Arg Ala Arg Glu Tyr Phe Gln Glu
355 360 365
Ala Glu Asp Gly Val Asp Tyr Leu Asp Val Lys Ala Arg Trp Pro Val
370 375 380
Trp Ser Ala Leu Ile Leu Tyr Arg Gln Ile Leu Asp Val Ile Glu Lys
385 390 395 400
Asn Asp Tyr Asp Asn Phe Ser Met Arg Ala Tyr Val Pro Lys Ser Lys
405 410 415
Lys Phe Ala Ser Leu Pro Met Ala Leu Phe Arg Ala Leu Val Pro Lys
420 425 430
Asn Lys
<211> 1104
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Ziso
<400> 3
atggcgagct tgtgtagagc tgcccttggg caggccagtg cgaaggggct gagtggctta 60
caaacttcct ccaagcctct catctccaag agccctcttg tagcaagacc accatgcatc 120
agaattagcg aacgctgccc agtcttagaa aattccgtat ggagcagaag acgagcggag 180
gtgtgtgtgc gggctgctag cgacgaggag tcgccccgac ctgcggggct tgttggcgag 240
gatgcggcag ccttcgacgt ttcccagcag agtaccaagt cctgggcgct gtttactggg 300
cttctgactg gcgtgctggg cctcatttac ctggtatgga tccagccggg agcaggtctg 360
gcagatgact tcttggggtt ggtcgagggc ttcagcaaca acaacccgga ggcaacaatc 420
ctccttatcc tctttgtgtt tgctgttgta cacagtgggc tggcaggcct gcgccccaaa 480
ggggagcagc tgatcggcgc acgagcatac cgggtgattt ttgcccttgt cagcctgccc 540
ttggccatcg tggccatcgt gtacttcatc aaccatagat acgatggcat gcccttatgg 600
gatctcaggg gtgtgacggg agtgcatgag ctggtgtggc tcctcaactt tgtgtccttc 660
tacttccttt acccttccac ctttaacatc cttgaggtgg ctgcagtgga cgagcccaag 720
ctgcatatgt gggaaacggg aatcatgcga atcactcgtc acccacaaat ggtagggcag 780
ctaatatggt gcgcagcaca cacgttgtgg atcgggaaca gcttcatgct cgtgacctca 840
gcaggcctca tggctcatca tctctttggt tgctggcatg gtgaccggcg gttgtctgcc 900
aagtatggcg aggcctttga aatcgtcaag gcgcggacaa gtacctttcc attgcaagct 960
atttgggagg gccgccaggt cttgccagct gattactaca aggagttcct gcgggcgccc 1020
tacttcgctg tgactgcctt caccctgggc gcctacttcg cacaccccat catgcagtct 1080
gccagcttct atttgagatg gtag 1104
<211> 367
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Ziso
<400> 4
Met Ala Ser Leu Cys Arg Ala Ala Leu Gly Gln Ala Ser Ala Lys Gly
1 5 10 15
Leu Ser Gly Leu Gln Thr Ser Ser Lys Pro Leu Ile Ser Lys Ser Pro
20 25 30
Leu Val Ala Arg Pro Pro Cys Ile Arg Ile Ser Glu Arg Cys Pro Val
35 40 45
Leu Glu Asn Ser Val Trp Ser Arg Arg Arg Ala Glu Val Cys Val Arg
50 55 60
Ala Ala Ser Asp Glu Glu Ser Pro Arg Pro Ala Gly Leu Val Gly Glu
65 70 75 80
Asp Ala Ala Ala Phe Asp Val Ser Gln Gln Ser Thr Lys Ser Trp Ala
85 90 95
Leu Phe Thr Gly Leu Leu Thr Gly Val Leu Gly Leu Ile Tyr Leu Val
100 105 110
Trp Ile Gln Pro Gly Ala Gly Leu Ala Asp Asp Phe Leu Gly Leu Val
115 120 125
Glu Gly Phe Ser Asn Asn Asn Pro Glu Ala Thr Ile Leu Leu Ile Leu
130 135 140
Phe Val Phe Ala Val Val His Ser Gly Leu Ala Gly Leu Arg Pro Lys
145 150 155 160
Gly Glu Gln Leu Ile Gly Ala Arg Ala Tyr Arg Val Ile Phe Ala Leu
165 170 175
Val Ser Leu Pro Leu Ala Ile Val Ala Ile Val Tyr Phe Ile Asn His
180 185 190
Arg Tyr Asp Gly Met Pro Leu Trp Asp Leu Arg Gly Val Thr Gly Val
195 200 205
His Glu Leu Val Trp Leu Leu Asn Phe Val Ser Phe Tyr Phe Leu Tyr
210 215 220
Pro Ser Thr Phe Asn Ile Leu Glu Val Ala Ala Val Asp Glu Pro Lys
225 230 235 240
Leu His Met Trp Glu Thr Gly Ile Met Arg Ile Thr Arg His Pro Gln
245 250 255
Met Val Gly Gln Leu Ile Trp Cys Ala Ala His Thr Leu Trp Ile Gly
260 265 270
Asn Ser Phe Met Leu Val Thr Ser Ala Gly Leu Met Ala His His Leu
275 280 285
Phe Gly Cys Trp His Gly Asp Arg Arg Leu Ser Ala Lys Tyr Gly Glu
290 295 300
Ala Phe Glu Ile Val Lys Ala Arg Thr Ser Thr Phe Pro Leu Gln Ala
305 310 315 320
Ile Trp Glu Gly Arg Gln Val Leu Pro Ala Asp Tyr Tyr Lys Glu Phe
325 330 335
Leu Arg Ala Pro Tyr Phe Ala Val Thr Ala Phe Thr Leu Gly Ala Tyr
340 345 350
Phe Ala His Pro Ile Met Gln Ser Ala Ser Phe Tyr Leu Arg Trp
355 360 365
<211> 1749
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Zds
<400> 5
atgttggggc tgcagagcaa ggaatcgcaa ctgtgcacca caaatgtgcc agccaggcgt 60
ggctatgcgc agtgcaccag tacccgcacc cgcagacgga cgcgctgcac cacccaagcc 120
attgccaccc cccctgctcc tccaaagacc acacccaggg agtggaccac ccaggatgtg 180
agcaaggtgg ccttgaagga tgtgcctttg aagtccttgt acccggatga gcctgcccct 240
ccaaagccag gtgcccccaa gatgcgtgtg gccattgtgg gcagtggact ggctggcctg 300
tcgacagcag tggagctgct agaccaaggg catgaggtgg acatctatga ccagcgcagc 360
tgggttggag gcaaggtggc ctcatggcaa gacaaggatg gcaaccacat tgagatgggc 420
ttgcacgtct tcttcggctg ctaccacaac cttttccgtc tgatggccaa gtgtggagta 480
ctggagaact tgctggtgaa ggagcatact cacacctttt gcaacaatga tggggatgtc 540
agggagcttg acttccgttt tgaggtcgga ggacagaaaa tcggggctcc cttccatggc 600
ctgaaagcct tcttcacaac cccccagctt tctgtgggag acaaggcagc caatgcgctg 660
gcgctgggca ccagccctat tgtgcgctcc ttgatagacc cagagggggg catgagtgat 720
gtgcgaaacc tggacaacat cagcttctgg gactggttca agagccatgg cgggtctgag 780
caatccatga agcgcatgtg ggatcccatt gcgtatgctt tgggtttctt ggactgcaaa 840
gacatcagtg cgcgctgcat gctgaccatc ttccagttct ttgccaccaa gaccgacgcc 900
tccgtcctgc gcatgctgaa cggatctcca gctgagaggc tcctgaagcc catcaccgac 960
tacattgagg ccaagggagg ccgcatccac ttgcgtcagg gttgcaagga ggttctattt 1020
gaggatggcc ctgacggcaa gcctgtagtg accggcatgt gcatgggccg ggatggccaa 1080
gttgtgaagg ctgatgccta tgttgcagcc ctggacgtcc ctggggcgaa gcagctcttg 1140
ccgcaggcat ggagaaagta cccccagttt gacaaaatct acaagctcaa tggcgtgcct 1200
gtgatcactg tacagctgcg ctacaatggt tgggtgacgg agatgcagga cccagagaag 1260
gtcaagcagc tgacccagcc ccaaggcatc aacaacctgt tgtacagccc tgatgcgttc 1320
ttctcctgct tcgccgacct tgcccttgtg agccctgtgg agtacttcca tgagggcaag 1380
ggctccctca tgcaagtcgt gatcacgcct gctgccccgt acatgccatg gaccaacgag 1440
gccattgctg aggaggctga ccggcaggtc cgccagctct tccccagcgc ccgtaagctg 1500
gacatgatct ggcatagtgt tgtgaagatt ggccagtcct tgtaccagga ggccccaggc 1560
atggaccctt acaggcccga gcaggccacg cctgtgccca acttcttcct ggcaggcagc 1620
tacaccaagc aagactacat cgactcaatg gagggcgcca ccttgtcagg tcggcagtgt 1680
gctggtgaga ttatgaaggc tgtgcccttg atccaaagcc tgtcaaaggc gccacttccg 1740
agcatgtaa 1749
<211> 582
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Zds
<400> 6
Met Leu Gly Leu Gln Ser Lys Glu Ser Gln Leu Cys Thr Thr Asn Val
1 5 10 15
Pro Ala Arg Arg Gly Tyr Ala Gln Cys Thr Ser Thr Arg Thr Arg Arg
20 25 30
Arg Thr Arg Cys Thr Thr Gln Ala Ile Ala Thr Pro Pro Ala Pro Pro
35 40 45
Lys Thr Thr Pro Arg Glu Trp Thr Thr Gln Asp Val Ser Lys Val Ala
50 55 60
Leu Lys Asp Val Pro Leu Lys Ser Leu Tyr Pro Asp Glu Pro Ala Pro
65 70 75 80
Pro Lys Pro Gly Ala Pro Lys Met Arg Val Ala Ile Val Gly Ser Gly
85 90 95
Leu Ala Gly Leu Ser Thr Ala Val Glu Leu Leu Asp Gln Gly His Glu
100 105 110
Val Asp Ile Tyr Asp Gln Arg Ser Trp Val Gly Gly Lys Val Ala Ser
115 120 125
Trp Gln Asp Lys Asp Gly Asn His Ile Glu Met Gly Leu His Val Phe
130 135 140
Phe Gly Cys Tyr His Asn Leu Phe Arg Leu Met Ala Lys Cys Gly Val
145 150 155 160
Leu Glu Asn Leu Leu Val Lys Glu His Thr His Thr Phe Cys Asn Asn
165 170 175
Asp Gly Asp Val Arg Glu Leu Asp Phe Arg Phe Glu Val Gly Gly Gln
180 185 190
Lys Ile Gly Ala Pro Phe His Gly Leu Lys Ala Phe Phe Thr Thr Pro
195 200 205
Gln Leu Ser Val Gly Asp Lys Ala Ala Asn Ala Leu Ala Leu Gly Thr
210 215 220
Ser Pro Ile Val Arg Ser Leu Ile Asp Pro Glu Gly Gly Met Ser Asp
225 230 235 240
Val Arg Asn Leu Asp Asn Ile Ser Phe Trp Asp Trp Phe Lys Ser His
245 250 255
Gly Gly Ser Glu Gln Ser Met Lys Arg Met Trp Asp Pro Ile Ala Tyr
260 265 270
Ala Leu Gly Phe Leu Asp Cys Lys Asp Ile Ser Ala Arg Cys Met Leu
275 280 285
Thr Ile Phe Gln Phe Phe Ala Thr Lys Thr Asp Ala Ser Val Leu Arg
290 295 300
Met Leu Asn Gly Ser Pro Ala Glu Arg Leu Leu Lys Pro Ile Thr Asp
305 310 315 320
Tyr Ile Glu Ala Lys Gly Gly Arg Ile His Leu Arg Gln Gly Cys Lys
325 330 335
Glu Val Leu Phe Glu Asp Gly Pro Asp Gly Lys Pro Val Val Thr Gly
340 345 350
Met Cys Met Gly Arg Asp Gly Gln Val Val Lys Ala Asp Ala Tyr Val
355 360 365
Ala Ala Leu Asp Val Pro Gly Ala Lys Gln Leu Leu Pro Gln Ala Trp
370 375 380
Arg Lys Tyr Pro Gln Phe Asp Lys Ile Tyr Lys Leu Asn Gly Val Pro
385 390 395 400
Val Ile Thr Val Gln Leu Arg Tyr Asn Gly Trp Val Thr Glu Met Gln
405 410 415
Asp Pro Glu Lys Val Lys Gln Leu Thr Gln Pro Gln Gly Ile Asn Asn
420 425 430
Leu Leu Tyr Ser Pro Asp Ala Phe Phe Ser Cys Phe Ala Asp Leu Ala
435 440 445
Leu Val Ser Pro Val Glu Tyr Phe His Glu Gly Lys Gly Ser Leu Met
450 455 460
Gln Val Val Ile Thr Pro Ala Ala Pro Tyr Met Pro Trp Thr Asn Glu
465 470 475 480
Ala Ile Ala Glu Glu Ala Asp Arg Gln Val Arg Gln Leu Phe Pro Ser
485 490 495
Ala Arg Lys Leu Asp Met Ile Trp His Ser Val Val Lys Ile Gly Gln
500 505 510
Ser Leu Tyr Gln Glu Ala Pro Gly Met Asp Pro Tyr Arg Pro Glu Gln
515 520 525
Ala Thr Pro Val Pro Asn Phe Phe Leu Ala Gly Ser Tyr Thr Lys Gln
530 535 540
Asp Tyr Ile Asp Ser Met Glu Gly Ala Thr Leu Ser Gly Arg Gln Cys
545 550 555 560
Ala Gly Glu Ile Met Lys Ala Val Pro Leu Ile Gln Ser Leu Ser Lys
565 570 575
Ala Pro Leu Pro Ser Met
580
<211> 1905
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Crtiso
<400> 7
atgtttgcaa cgaatggttg tccaaggggg ctgcaagcag ggaaggccag tggctcgcag 60
gcaggaaaga gagttgtcgg actccgtgat tctggcctgg accatgtcat caggcatgtt 120
ccacacccta gcacctccat acccgctagc agcagcagga ccactcaggg caccagtaat 180
agcgcccgcc caggcatcaa cagcagccgc agacaacagc ggttgcagcg ctgcacagcc 240
gctgcgtccg caacggctta tcctcctgca gaggtggtga aggctctacc aacagatgcc 300
ccaacagatg tggagtacga tgccgttatt gtggggagtg gcatgggggg actggcaact 360
gcctcccagc tggcagcaaa gggagcccgt gtggttgtcc tggaaaagta cctcatccct 420
ggaggcagtg cagggcactt caagagagag ggctacacct ttgatgtcgg ctcctcgatg 480
atgtttggct ttggagacaa gggaaccacc aacctgctga cccgctgcct agccgccctg 540
gacaagaaaa ttgagactgt gcctgacccg acccaggtgt actaccattt gcctgcaacc 600
aaagctcacc ccaatgggct ggacgtccag gtctggagaa agtacgagga cttcgtgtca 660
gagctgtgcg ctcaattccc tcacgagagc aatggcatca aggcattcta tgacgagtgc 720
tggaagatct tcaatgctct caacaccttg gagctcaaga gcttggaaga gatccggtac 780
ctgctgcgag agtttgcaaa gcatcccatc gagtgcctga cgcttgcggc atatgccact 840
tccaacgtcg cggacatctc gcgcaaatac atcaaggacc ctgagctcct gcgcttcatt 900
gacctggagt gctacatctg gtccactgtg cctgctgagc tgacgcccat gatgaacgct 960
ggcatggtgt tctgtgacag gcattatggt ggcatcaact accccatagg tggagtgggt 1020
cgcattccgg aagtgctagc agaaggcttg acagagcgtg gcagtcatgt agtctacaag 1080
gccaatgtga agcgcatcct tacggaaaag caaggcgagg agatgaaggc agtaggggtg 1140
gagctggcag atggacgtgt gtttaggggc aagagcatta tttccaatgc caccaggtgg 1200
gacacctttg agtccatgat cggggatgac aacctgccac ccagtgaaaa gctgttcagg 1260
gagcggtaca agaaagcacc ctcatttttc tccatgcaca tgggcgtgga agccagtgtg 1320
tttgagggtt caggagagac agtggactgc caccatgtga ttgtagacga ctggtcaaag 1380
ctggaagatg cctatggaac actttttgtg tccatgccct ccctgctaga cccatccctg 1440
gccccgcccg gcaagcacat tgtccatgca ttcactccag actggataga ctcctggcag 1500
ggcctgtctg tacaagatta cgaggctaag aaggaggaag tttctgcgca gctcattgac 1560
cgcctcgatg ctgtgtttcc tggactgaag caaggggtgg tcttcaagga ggtggggacg 1620
ccacgcacgc accgtcgctt tttgaaccgg acttcaggca cgtacggccc catcccttca 1680
aggccacccc tgggcatgtt gtccatgcct ttcaacagga ctgctgtgca agggctttat 1740
tgtgtgggag actcaacttt tccaggccaa ggagtgaatg cagtcgtctt ctccggcttt 1800
ggctgtgccc atcgagtggc ttgcgacatt gggcttgagc ccacctggcc tgccctggac 1860
aagcctttta acaagttcct cgagtatgtg cgggacagct cataa 1905
<211> 634
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Du Shi Pasteur algae (Dunaliella bardawil): Crtiso
<400> 8
Met Phe Ala Thr Asn Gly Cys Pro Arg Gly Leu Gln Ala Gly Lys Ala
1 5 10 15
Ser Gly Ser Gln Ala Gly Lys Arg Val Val Gly Leu Arg Asp Ser Gly
20 25 30
Leu Asp His Val Ile Arg His Val Pro His Pro Ser Thr Ser Ile Pro
35 40 45
Ala Ser Ser Ser Arg Thr Thr Gln Gly Thr Ser Asn Ser Ala Arg Pro
50 55 60
Gly Ile Asn Ser Ser Arg Arg Gln Gln Arg Leu Gln Arg Cys Thr Ala
65 70 75 80
Ala Ala Ser Ala Thr Ala Tyr Pro Pro Ala Glu Val Val Lys Ala Leu
85 90 95
Pro Thr Asp Ala Pro Thr Asp Val Glu Tyr Asp Ala Val Ile Val Gly
100 105 110
Ser Gly Met Gly Gly Leu Ala Thr Ala Ser Gln Leu Ala Ala Lys Gly
115 120 125
Ala Arg Val Val Val Leu Glu Lys Tyr Leu Ile Pro Gly Gly Ser Ala
130 135 140
Gly His Phe Lys Arg Glu Gly Tyr Thr Phe Asp Val Gly Ser Ser Met
145 150 155 160
Met Phe Gly Phe Gly Asp Lys Gly Thr Thr Asn Leu Leu Thr Arg Cys
165 170 175
Leu Ala Ala Leu Asp Lys Lys Ile Glu Thr Val Pro Asp Pro Thr Gln
180 185 190
Val Tyr Tyr His Leu Pro Ala Thr Lys Ala His Pro Asn Gly Leu Asp
195 200 205
Val Gln Val Trp Arg Lys Tyr Glu Asp Phe Val Ser Glu Leu Cys Ala
210 215 220
Gln Phe Pro His Glu Ser Asn Gly Ile Lys Ala Phe Tyr Asp Glu Cys
225 230 235 240
Trp Lys Ile Phe Asn Ala Leu Asn Thr Leu Glu Leu Lys Ser Leu Glu
245 250 255
Glu Ile Arg Tyr Leu Leu Arg Glu Phe Ala Lys His Pro Ile Glu Cys
260 265 270
Leu Thr Leu Ala Ala Tyr Ala Thr Ser Asn Val Ala Asp Ile Ser Arg
275 280 285
Lys Tyr Ile Lys Asp Pro Glu Leu Leu Arg Phe Ile Asp Leu Glu Cys
290 295 300
Tyr Ile Trp Ser Thr Val Pro Ala Glu Leu Thr Pro Met Met Asn Ala
305 310 315 320
Gly Met Val Phe Cys Asp Arg His Tyr Gly Gly Ile Asn Tyr Pro Ile
325 330 335
Gly Gly Val Gly Arg Ile Pro Glu Val Leu Ala Glu Gly Leu Thr Glu
340 345 350
Arg Gly Ser His Val Val Tyr Lys Ala Asn Val Lys Arg Ile Leu Thr
355 360 365
Glu Lys Gln Gly Glu Glu Met Lys Ala Val Gly Val Glu Leu Ala Asp
370 375 380
Gly Arg Val Phe Arg Gly Lys Ser Ile Ile Ser Asn Ala Thr Arg Trp
385 390 395 400
Asp Thr Phe Glu Ser Met Ile Gly Asp Asp Asn Leu Pro Pro Ser Glu
405 410 415
Lys Leu Phe Arg Glu Arg Tyr Lys Lys Ala Pro Ser Phe Phe Ser Met
420 425 430
His Met Gly Val Glu Ala Ser Val Phe Glu Gly Ser Gly Glu Thr Val
435 440 445
Asp Cys His His Val Ile Val Asp Asp Trp Ser Lys Leu Glu Asp Ala
450 455 460
Tyr Gly Thr Leu Phe Val Ser Met Pro Ser Leu Leu Asp Pro Ser Leu
465 470 475 480
Ala Pro Pro Gly Lys His Ile Val His Ala Phe Thr Pro Asp Trp Ile
485 490 495
Asp Ser Trp Gln Gly Leu Ser Val Gln Asp Tyr Glu Ala Lys Lys Glu
500 505 510
Glu Val Ser Ala Gln Leu Ile Asp Arg Leu Asp Ala Val Phe Pro Gly
515 520 525
Leu Lys Gln Gly Val Val Phe Lys Glu Val Gly Thr Pro Arg Thr His
530 535 540
Arg Arg Phe Leu Asn Arg Thr Ser Gly Thr Tyr Gly Pro Ile Pro Ser
545 550 555 560
Arg Pro Pro Leu Gly Met Leu Ser Met Pro Phe Asn Arg Thr Ala Val
565 570 575
Gln Gly Leu Tyr Cys Val Gly Asp Ser Thr Phe Pro Gly Gln Gly Val
580 585 590
Asn Ala Val Val Phe Ser Gly Phe Gly Cys Ala His Arg Val Ala Cys
595 600 605
Asp Ile Gly Leu Glu Pro Thr Trp Pro Ala Leu Asp Lys Pro Phe Asn
610 615 620
Lys Phe Leu Glu Tyr Val Arg Asp Ser Ser
625 630
<211> 1077
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Ggps
<400> 9
atggccgccc atcagatgca gctctttagt agccaacgga tgtgctcaac gtcctcaagg 60
agcatgaggc cagcagtctg cagccggccg caagtgccac gaattcgccc tgcaaacgtt 120
agacaaggtc gccatcaggc cttcaggaca atggccattg caactgcaga cgaggcccag 180
cagtccactt cttcctttga cttcaagagc tatatgaagg agcgtgcagt catggtgaat 240
gatgcgttgg acaaggccct gccgcagcgc tatccagagg tgctgctaga gtctatgagg 300
tactcactcc tagctggtgg caagcgcgtg cgcccatgcc tgaccttggc tgcctgcgaa 360
tgcgttggag gggacattgc gcacgcaatg cccactgcct gtgcaatgga ggtggttcac 420
accatgagcc tgatccacga cgacctaccc tccatggaca atgatgattt ccgcagaggg 480
tctcccacca accacaagaa atacggagag gacattgcca ttcttgccgg agacgccctg 540
ctttctttcg ccttcgagca cgtcgcgcgc gccaccactg gcacctcgcc tgagcgcgtg 600
ttgcgtgtga tcattgagct gggcaaggct gtgggtgcag atgggctaac aggaggacag 660
gttgtggaca tcaagagcga aaaccaggag gtgggcctgg aagttctgca gtacatccat 720
gagcacaaga cagcggccct gctagaggca gctgtggtgt gtggcgcgct ggtgggcggc 780
gcggatgatg tgacagtgga gaagatgcgc aagtttgcac tcaacatcgg ccttgcattc 840
caggtggtgg acgacatcct ggattgtacc cagaccacag agcagctggg caagactgca 900
ggcaaggaca tgggcgtgaa caagaccaca taccccaaac tgcttggcct ggagaagagt 960
aagcagaccg cggaggacct gatcacagag gccatccagc agctagatgg cttccccccg 1020
gagaagcgcg ccccccttgt agctctggcc aagtacattg gctaccgtca aaactaa 1077
<211> 358
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Ggps
<400> 10
Met Ala Ala His Gln Met Gln Leu Phe Ser Ser Gln Arg Met Cys Ser
1 5 10 15
Thr Ser Ser Arg Ser Met Arg Pro Ala Val Cys Ser Arg Pro Gln Val
20 25 30
Pro Arg Ile Arg Pro Ala Asn Val Arg Gln Gly Arg His Gln Ala Phe
35 40 45
Arg Thr Met Ala Ile Ala Thr Ala Asp Glu Ala Gln Gln Ser Thr Ser
50 55 60
Ser Phe Asp Phe Lys Ser Tyr Met Lys Glu Arg Ala Val Met Val Asn
65 70 75 80
Asp Ala Leu Asp Lys Ala Leu Pro Gln Arg Tyr Pro Glu Val Leu Leu
85 90 95
Glu Ser Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg Pro
100 105 110
Cys Leu Thr Leu Ala Ala Cys Glu Cys Val Gly Gly Asp Ile Ala His
115 120 125
Ala Met Pro Thr Ala Cys Ala Met Glu Val Val His Thr Met Ser Leu
130 135 140
Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Phe Arg Arg Gly
145 150 155 160
Ser Pro Thr Asn His Lys Lys Tyr Gly Glu Asp Ile Ala Ile Leu Ala
165 170 175
Gly Asp Ala Leu Leu Ser Phe Ala Phe Glu His Val Ala Arg Ala Thr
180 185 190
Thr Gly Thr Ser Pro Glu Arg Val Leu Arg Val Ile Ile Glu Leu Gly
195 200 205
Lys Ala Val Gly Ala Asp Gly Leu Thr Gly Gly Gln Val Val Asp Ile
210 215 220
Lys Ser Glu Asn Gln Glu Val Gly Leu Glu Val Leu Gln Tyr Ile His
225 230 235 240
Glu His Lys Thr Ala Ala Leu Leu Glu Ala Ala Val Val Cys Gly Ala
245 250 255
Leu Val Gly Gly Ala Asp Asp Val Thr Val Glu Lys Met Arg Lys Phe
260 265 270
Ala Leu Asn Ile Gly Leu Ala Phe Gln Val Val Asp Asp Ile Leu Asp
275 280 285
Cys Thr Gln Thr Thr Glu Gln Leu Gly Lys Thr Ala Gly Lys Asp Met
290 295 300
Gly Val Asn Lys Thr Thr Tyr Pro Lys Leu Leu Gly Leu Glu Lys Ser
305 310 315 320
Lys Gln Thr Ala Glu Asp Leu Ile Thr Glu Ala Ile Gln Gln Leu Asp
325 330 335
Gly Phe Pro Pro Glu Lys Arg Ala Pro Leu Val Ala Leu Ala Lys Tyr
340 345 350
Ile Gly Tyr Arg Gln Asn
355
<211> 1122
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Ziso
<400> 11
atggcaagct tgtgtgcagg tcgggccctt gggctggctg gtcaggggct gactggctca 60
catgcctcct ctcgaggcct cccagccaag cctctcgtct ccaggagccc gatccttgcc 120
aagacgccac catgcatcag aaatagggat ctccagcaag ccttaggaat ttccgtaccc 180
cacagaagac gatcggaagt gtgtgtgcgg gctgccagcg aggaggagtc gtccccacct 240
gcagggcttg tgggcgagga tgcggctgcc ttcgacgttt ctcaacaaag caccaagtct 300
tgggcgatat tcactgggct tctgactggc gtgctgggcc tcatttacct ggtttggatc 360
cagccgggag cagggctggc agatgacttc ctgagcactg tcgagagctt cagcaacaac 420
aaccccgagg caacaatcct cctcatcctg tttgtgtttg cggtcgcgca cagtgggcta 480
gcagccctgc gcccaaaagg cgagcagctg atcggtgctc gagcattccg cgtgattttt 540
gcccttgtca gcctgcccct ggccatcgtg gcggtggtgt atttcatcaa ccacagatat 600
gatggcatac ccttatggga tctcaggggt gtgacgggag tgcatgagct ggtgtggacc 660
ctcaacttca tttccttcta cttcctgtac ccgtccacct tcaacatcct tgaggtggct 720
gcagtggatg agcccaagct ccacatgtgg gaaaccggca tcatgcgaat cactcgtcac 780
ccacaaatgg tagggcaggc catctggtgt gcagcgcaca cgctgtggat cgggaacagc 840
ttcatgctgg tgacttcggc aggcctcatg gctcaccacc ttttcgggtg ctggcatggt 900
gacaagcgcc tgtcaaacaa gtatggcgag gcctttgaaa tcgtgaagac gcgcaccagc 960
actttcccga tgcaggctgt ttgggagggc cgccagaaat tgccagctga cttctacaag 1020
gagttcctgc gagcccccta ctttgctgtg actgcgttca ccctgggcgc ctattatgca 1080
caccccatca tgcagtctgc tagcttctac ctggggtggt ga 1122
<211> 373
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Ziso
<400> 12
Met Ala Ser Leu Cys Ala Gly Arg Ala Leu Gly Leu Ala Gly Gln Gly
1 5 10 15
Leu Thr Gly Ser His Ala Ser Ser Arg Gly Leu Pro Ala Lys Pro Leu
20 25 30
Val Ser Arg Ser Pro Ile Leu Ala Lys Thr Pro Pro Cys Ile Arg Asn
35 40 45
Arg Asp Leu Gln Gln Ala Leu Gly Ile Ser Val Pro His Arg Arg Arg
50 55 60
Ser Glu Val Cys Val Arg Ala Ala Ser Glu Glu Glu Ser Ser Pro Pro
65 70 75 80
Ala Gly Leu Val Gly Glu Asp Ala Ala Ala Phe Asp Val Ser Gln Gln
85 90 95
Ser Thr Lys Ser Trp Ala Ile Phe Thr Gly Leu Leu Thr Gly Val Leu
100 105 110
Gly Leu Ile Tyr Leu Val Trp Ile Gln Pro Gly Ala Gly Leu Ala Asp
115 120 125
Asp Phe Leu Ser Thr Val Glu Ser Phe Ser Asn Asn Asn Pro Glu Ala
130 135 140
Thr Ile Leu Leu Ile Leu Phe Val Phe Ala Val Ala His Ser Gly Leu
145 150 155 160
Ala Ala Leu Arg Pro Lys Gly Glu Gln Leu Ile Gly Ala Arg Ala Phe
165 170 175
Arg Val Ile Phe Ala Leu Val Ser Leu Pro Leu Ala Ile Val Ala Val
180 185 190
Val Tyr Phe Ile Asn His Arg Tyr Asp Gly Ile Pro Leu Trp Asp Leu
195 200 205
Arg Gly Val Thr Gly Val His Glu Leu Val Trp Thr Leu Asn Phe Ile
210 215 220
Ser Phe Tyr Phe Leu Tyr Pro Ser Thr Phe Asn Ile Leu Glu Val Ala
225 230 235 240
Ala Val Asp Glu Pro Lys Leu His Met Trp Glu Thr Gly Ile Met Arg
245 250 255
Ile Thr Arg His Pro Gln Met Val Gly Gln Ala Ile Trp Cys Ala Ala
260 265 270
His Thr Leu Trp Ile Gly Asn Ser Phe Met Leu Val Thr Ser Ala Gly
275 280 285
Leu Met Ala His His Leu Phe Gly Cys Trp His Gly Asp Lys Arg Leu
290 295 300
Ser Asn Lys Tyr Gly Glu Ala Phe Glu Ile Val Lys Thr Arg Thr Ser
305 310 315 320
Thr Phe Pro Met Gln Ala Val Trp Glu Gly Arg Gln Lys Leu Pro Ala
325 330 335
Asp Phe Tyr Lys Glu Phe Leu Arg Ala Pro Tyr Phe Ala Val Thr Ala
340 345 350
Phe Thr Leu Gly Ala Tyr Tyr Ala His Pro Ile Met Gln Ser Ala Ser
355 360 365
Phe Tyr Leu Gly Trp
370
<211> 1752
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Zds
<400> 13
atgctgggac tgcacggtaa ggatgccctc caggctggcc gcaccaacca gggcccgcca 60
gtcaggacta gctttgtgca gtctacccgt accaggagaa ggacgcgctg caccacccat 120
gccatcgctg cgccccctgc acctccaaag accacaccca aggagtggac cacccaggat 180
cttagcaagg tggccctgaa agacaggcct ctgaagtccc tgtacccgga tgagcccgcc 240
ccaccaaagc caggtgatcc caagctacgc gtggccatcg tgggcagtgg actggctgga 300
ctgtccacag cagtggagct gctagaccag gggcatgagg tggacatcta tgaccagcga 360
ccctttctag gaggcaaagt ggcttcatgg gtagacaagg atggcaacca catagagatg 420
ggcttgcatg tcttcttcgg gtgctaccac aaccttttcc gactgatggc caagtgcggt 480
gtactggaaa acttgctggt caaggatcac acccacacct tttgcaacgc tgatggggac 540
gtcagggagc ttgacttccg ctttgaagct ggaggacaga agattggggc cccattccat 600
ggcctgaaag ccttctttac cacccctcag ctcactgtgg ccgacaaggc acaaaatgca 660
ttggcactgg gcacgagccc tatcgtgcgc gccttgatag atcctgaggg gggtatgcaa 720
gatgtgagga acctagacaa cattagcttc tgggactggt tcaagagcca tggcggatca 780
gagctctcca tgaagcgtat gtgggacccc attgcctacg ctttgggctt cttggactgc 840
aaggacatca gtgcgcgctg catgttgaca atcttccaat tttttgccac caagaccgac 900
gcttctgtcc tccgcatgct taacggatct cctggggaga ggctgttgaa gcccatcgtt 960
aactacattg agtccaaggg tggccgcatc cacctgcgcc agggttgcaa ggaagtactc 1020
tacgaggatg gccctgacgg cacacctgtg gtgactggca tgcgcatggg ccgggatggc 1080
caaattgtga aagctgatgc ctatgtggct gccttggatg ttcctggggc caagcagctc 1140
ttaccgcagg cgtggaggaa gtaccctcaa tttgacaaca tctacagcct gattggcgtg 1200
cctgtgatca ccgttcagct gcgatacaat ggatgggtga ccgagatgca ggacccagag 1260
aaggtcaagc agctgaccca gccccaaggc atcaacaact tgctatacag ccctgatgcg 1320
ttctactcct gctttgctga cctcgctctt gtgagccctg tggagtactt caaggagggc 1380
caggggtcgc tcatgcaagt cgtgattaca ccagcggccc cctacatgcc atgggaaaac 1440
aaggccattg ctgaggaggc tgaccgccag acccgccggc tgttccccag cgcgcgcaac 1500
ttggacatga tctggcacag tgttgtgaag atcggccagt ccctgtacca ggaggcccca 1560
ggcatggacc ctttcaggcc tgagcaggcc acgcctgtgc ccaacttctt cttggcaggc 1620
agttacacca agcaagacta catcgactcc atggagggtg ccaccctgtc aggccgacaa 1680
tgtgctggcg aggtcgtgaa agccgtgccc cggatccaga gcctatcggt ggcgccactg 1740
gcaagcattt ga 1752
<211> 583
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Zds
<400> 14
Met Leu Gly Leu His Gly Lys Asp Ala Leu Gln Ala Gly Arg Thr Asn
1 5 10 15
Gln Gly Pro Pro Val Arg Thr Ser Phe Val Gln Ser Thr Arg Thr Arg
20 25 30
Arg Arg Thr Arg Cys Thr Thr His Ala Ile Ala Ala Pro Pro Ala Pro
35 40 45
Pro Lys Thr Thr Pro Lys Glu Trp Thr Thr Gln Asp Leu Ser Lys Val
50 55 60
Ala Leu Lys Asp Arg Pro Leu Lys Ser Leu Tyr Pro Asp Glu Pro Ala
65 70 75 80
Pro Pro Lys Pro Gly Asp Pro Lys Leu Arg Val Ala Ile Val Gly Ser
85 90 95
Gly Leu Ala Gly Leu Ser Thr Ala Val Glu Leu Leu Asp Gln Gly His
100 105 110
Glu Val Asp Ile Tyr Asp Gln Arg Pro Phe Leu Gly Gly Lys Val Ala
115 120 125
Ser Trp Val Asp Lys Asp Gly Asn His Ile Glu Met Gly Leu His Val
130 135 140
Phe Phe Gly Cys Tyr His Asn Leu Phe Arg Leu Met Ala Lys Cys Gly
145 150 155 160
Val Leu Glu Asn Leu Leu Val Lys Asp His Thr His Thr Phe Cys Asn
165 170 175
Ala Asp Gly Asp Val Arg Glu Leu Asp Phe Arg Phe Glu Ala Gly Gly
180 185 190
Gln Lys Ile Gly Ala Pro Phe His Gly Leu Lys Ala Phe Phe Thr Thr
195 200 205
Pro Gln Leu Thr Val Ala Asp Lys Ala Gln Asn Ala Leu Ala Leu Gly
210 215 220
Thr Ser Pro Ile Val Arg Ala Leu Ile Asp Pro Glu Gly Gly Met Gln
225 230 235 240
Asp Val Arg Asn Leu Asp Asn Ile Ser Phe Trp Asp Trp Phe Lys Ser
245 250 255
His Gly Gly Ser Glu Leu Ser Met Lys Arg Met Trp Asp Pro Ile Ala
260 265 270
Tyr Ala Leu Gly Phe Leu Asp Cys Lys Asp Ile Ser Ala Arg Cys Met
275 280 285
Leu Thr Ile Phe Gln Phe Phe Ala Thr Lys Thr Asp Ala Ser Val Leu
290 295 300
Arg Met Leu Asn Gly Ser Pro Gly Glu Arg Leu Leu Lys Pro Ile Val
305 310 315 320
Asn Tyr Ile Glu Ser Lys Gly Gly Arg Ile His Leu Arg Gln Gly Cys
325 330 335
Lys Glu Val Leu Tyr Glu Asp Gly Pro Asp Gly Thr Pro Val Val Thr
340 345 350
Gly Met Arg Met Gly Arg Asp Gly Gln Ile Val Lys Ala Asp Ala Tyr
355 360 365
Val Ala Ala Leu Asp Val Pro Gly Ala Lys Gln Leu Leu Pro Gln Ala
370 375 380
Trp Arg Lys Tyr Pro Gln Phe Asp Asn Ile Tyr Ser Leu Ile Gly Val
385 390 395 400
Pro Val Ile Thr Val Gln Leu Arg Tyr Asn Gly Trp Val Thr Glu Met
405 410 415
Gln Asp Pro Glu Lys Val Lys Gln Leu Thr Gln Pro Gln Gly Ile Asn
420 425 430
Asn Leu Leu Tyr Ser Pro Asp Ala Phe Tyr Ser Cys Phe Ala Asp Leu
435 440 445
Ala Leu Val Ser Pro Val Glu Tyr Phe Lys Glu Gly Gln Gly Ser Leu
450 455 460
Met Gln Val Val Ile Thr Pro Ala Ala Pro Tyr Met Pro Trp Glu Asn
465 470 475 480
Lys Ala Ile Ala Glu Glu Ala Asp Arg Gln Thr Arg Arg Leu Phe Pro
485 490 495
Ser Ala Arg Asn Leu Asp Met Ile Trp His Ser Val Val Lys Ile Gly
500 505 510
Gln Ser Leu Tyr Gln Glu Ala Pro Gly Met Asp Pro Phe Arg Pro Glu
515 520 525
Gln Ala Thr Pro Val Pro Asn Phe Phe Leu Ala Gly Ser Tyr Thr Lys
530 535 540
Gln Asp Tyr Ile Asp Ser Met Glu Gly Ala Thr Leu Ser Gly Arg Gln
545 550 555 560
Cys Ala Gly Glu Val Val Lys Ala Val Pro Arg Ile Gln Ser Leu Ser
565 570 575
Val Ala Pro Leu Ala Ser Ile
580
<211> 1887
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Crtiso
<400> 15
atgtttgcaa tcactgggcc atcagggata acaagtggct ctcaggcagg aaggggggtc 60
gcaagtctcc gcgtgtccgg tctggaccaa gtcatcaggc atgttcctca cccttccacc 120
tctacacctg ccagcaccag ctggccctct cggaccccca atagtgatgc ccgtccaagc 180
gcattcagca gcaaaaggca gcagcgattg cagcggacaa ccactgcagc caccacaatg 240
gcttaccccc ctgcagaggt tgtgaaggct cttccaacag atgccccgac tgatgtggag 300
tatgatgccg tgattgtggg gagtggcatg gggggattgg caactgcctc ccagctggcg 360
gcaaaaggcg cccgcgtggt tgtcctggaa aagtatctca tccctggcgg cagcgcaggg 420
cactacaggc gtgagggcta cacctttgat gttggctcct ccatgatgtt tggctttgga 480
gacaagggaa ccaccaacct gctgacccgc tgcctagccg ccctcggcaa aaagatcgag 540
acagtaccgg accaaaccca ggtgttctac cacctgcctg catctgaagc ccaccccaac 600
gggctgaacg tccaggtgtg gaggaagtac gaggattttg tggccgagct ttgcgaccgt 660
ttcccccacg agagcaacgg catcaaagca ttctacgatg aaaactggag gatcttcaac 720
tctctcaaca ccttggagct caagagcttg gaggaaatcc ggtacctgct gggagagttt 780
gttaagcacc ccatcgcgtg cctgacgctt gcctcgtacg ccactacgaa cgttgcggac 840
atctcgcgca agtacatcaa ggatcccgag ctcctgcgct tcatcgacct cgagtgcttc 900
atctggtcca ccgtgtcagc tgaattgacg cctatgatga acgccggcat ggtgttttgt 960
gatcggcact atggcggcat caattacccg aagggtggag tgggccgcat tccggagttg 1020
ttagcagaag gcttgacgga gcgcggcagc catgtggtct acaaggccaa tgtgaagcgc 1080
atcctcacag aaaagcaagg caacgagaca aaggcagtgg gcgtggaact tgcggatgga 1140
cgcgtgtaca ggggcaagag catcgtctcc aatgccacaa ggtgggacac ctttgagtcc 1200
atgatcggcg aggaccagct gccgcccagt gaacaggcgt tcagggagcg ttacaagaag 1260
gcgccctcgt ttttcaccat gcacttgggc gttgaagcga gtgtgtttga gggcaaagga 1320
gaagtggact gccaccatgt gatcgtgaat gactggtcaa agctagaaga tgcatacggc 1380
acactctttg tgtccatgcc ttccctactg gacccatccc tggctccgcc tggcaagcac 1440
attgtacatg cattcactcc agactggatt gataactggc agggcctgtc tgtgcaagat 1500
tacgaggcca agaaggagga agtttcggca cagttcattg accgcctgga tgctgtgttt 1560
cctgggctta agcagggcgt ggtcttcaag gaggttggga caccacgcac gcaccgtcgc 1620
tttttgaacc ggaatgctgg cacatacggt cccatccctt caaggcgccc tctgggcatg 1680
ctgtccatgc ctctcaacag gactgctgtg caagggcttt attgcgcggg agactcaact 1740
ttcccgggcc aaggagtgaa tgcagttgtc ttctctggct ttggctgcgc ccatcgtgtg 1800
gcttgcgaca ttggactgga gcccacttgg cctgccttgg acaagccctt ccacaagttc 1860
ctcgactacg tgcgcgacaa ctcatag 1887
<211> 628
<212> PRT
<213>artificial sequence (Artificial Sequence)
<220>
<223>Dunaliella salina (Dunaliella saline): Crtiso
<400> 16
Met Phe Ala Ile Thr Gly Pro Ser Gly Ile Thr Ser Gly Ser Gln Ala
1 5 10 15
Gly Arg Gly Val Ala Ser Leu Arg Val Ser Gly Leu Asp Gln Val Ile
20 25 30
Arg His Val Pro His Pro Ser Thr Ser Thr Pro Ala Ser Thr Ser Trp
35 40 45
Pro Ser Arg Thr Pro Asn Ser Asp Ala Arg Pro Ser Ala Phe Ser Ser
50 55 60
Lys Arg Gln Gln Arg Leu Gln Arg Thr Thr Thr Ala Ala Thr Thr Met
65 70 75 80
Ala Tyr Pro Pro Ala Glu Val Val Lys Ala Leu Pro Thr Asp Ala Pro
85 90 95
Thr Asp Val Glu Tyr Asp Ala Val Ile Val Gly Ser Gly Met Gly Gly
100 105 110
Leu Ala Thr Ala Ser Gln Leu Ala Ala Lys Gly Ala Arg Val Val Val
115 120 125
Leu Glu Lys Tyr Leu Ile Pro Gly Gly Ser Ala Gly His Tyr Arg Arg
130 135 140
Glu Gly Tyr Thr Phe Asp Val Gly Ser Ser Met Met Phe Gly Phe Gly
145 150 155 160
Asp Lys Gly Thr Thr Asn Leu Leu Thr Arg Cys Leu Ala Ala Leu Gly
165 170 175
Lys Lys Ile Glu Thr Val Pro Asp Gln Thr Gln Val Phe Tyr His Leu
180 185 190
Pro Ala Ser Glu Ala His Pro Asn Gly Leu Asn Val Gln Val Trp Arg
195 200 205
Lys Tyr Glu Asp Phe Val Ala Glu Leu Cys Asp Arg Phe Pro His Glu
210 215 220
Ser Asn Gly Ile Lys Ala Phe Tyr Asp Glu Asn Trp Arg Ile Phe Asn
225 230 235 240
Ser Leu Asn Thr Leu Glu Leu Lys Ser Leu Glu Glu Ile Arg Tyr Leu
245 250 255
Leu Gly Glu Phe Val Lys His Pro Ile Ala Cys Leu Thr Leu Ala Ser
260 265 270
Tyr Ala Thr Thr Asn Val Ala Asp Ile Ser Arg Lys Tyr Ile Lys Asp
275 280 285
Pro Glu Leu Leu Arg Phe Ile Asp Leu Glu Cys Phe Ile Trp Ser Thr
290 295 300
Val Ser Ala Glu Leu Thr Pro Met Met Asn Ala Gly Met Val Phe Cys
305 310 315 320
Asp Arg His Tyr Gly Gly Ile Asn Tyr Pro Lys Gly Gly Val Gly Arg
325 330 335
Ile Pro Glu Leu Leu Ala Glu Gly Leu Thr Glu Arg Gly Ser His Val
340 345 350
Val Tyr Lys Ala Asn Val Lys Arg Ile Leu Thr Glu Lys Gln Gly Asn
355 360 365
Glu Thr Lys Ala Val Gly Val Glu Leu Ala Asp Gly Arg Val Tyr Arg
370 375 380
Gly Lys Ser Ile Val Ser Asn Ala Thr Arg Trp Asp Thr Phe Glu Ser
385 390 395 400
Met Ile Gly Glu Asp Gln Leu Pro Pro Ser Glu Gln Ala Phe Arg Glu
405 410 415
Arg Tyr Lys Lys Ala Pro Ser Phe Phe Thr Met His Leu Gly Val Glu
420 425 430
Ala Ser Val Phe Glu Gly Lys Gly Glu Val Asp Cys His His Val Ile
435 440 445
Val Asn Asp Trp Ser Lys Leu Glu Asp Ala Tyr Gly Thr Leu Phe Val
450 455 460
Ser Met Pro Ser Leu Leu Asp Pro Ser Leu Ala Pro Pro Gly Lys His
465 470 475 480
Ile Val His Ala Phe Thr Pro Asp Trp Ile Asp Asn Trp Gln Gly Leu
485 490 495
Ser Val Gln Asp Tyr Glu Ala Lys Lys Glu Glu Val Ser Ala Gln Phe
500 505 510
Ile Asp Arg Leu Asp Ala Val Phe Pro Gly Leu Lys Gln Gly Val Val
515 520 525
Phe Lys Glu Val Gly Thr Pro Arg Thr His Arg Arg Phe Leu Asn Arg
530 535 540
Asn Ala Gly Thr Tyr Gly Pro Ile Pro Ser Arg Arg Pro Leu Gly Met
545 550 555 560
Leu Ser Met Pro Leu Asn Arg Thr Ala Val Gln Gly Leu Tyr Cys Ala
565 570 575
Gly Asp Ser Thr Phe Pro Gly Gln Gly Val Asn Ala Val Val Phe Ser
580 585 590
Gly Phe Gly Cys Ala His Arg Val Ala Cys Asp Ile Gly Leu Glu Pro
595 600 605
Thr Trp Pro Ala Leu Asp Lys Pro Phe His Lys Phe Leu Asp Tyr Val
610 615 620
Arg Asp Asn Ser
625

Claims (8)

1. a kind of beta carotene high production bacteria based on Du Shi algae metabolic pathway, it is characterised in that:
When based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, the beta carotene high production bacteria contains Have coding GenBank:APW83740.1 shown in amino acid sequence yak base Mang ox base pyrophosphate synthetase Ggps gene, Encode phytoene synthetase Psy gene, the coding GenBank of amino acid sequence shown in SEQ ID NO:2: The phytoene dehydrogenase Pds gene of amino acid sequence shown in ADD52599.1 encodes amino acid shown in SEQ ID NO:4 The 15- of sequence is cis--sigma carotene isomerase Ziso gene, amino acid sequence shown in coding SEQ ID NO:6 ζ-Hu Luo Bu Su dehydrogenase Zds gene, coding SEQ ID NO:8 shown in amino acid sequence carotenoid isomerase Crtiso gene and Encode the lycopene beta cyclase Lycb gene of amino acid sequence shown in GenBank:ANY98896.1；
When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the beta carotene high production bacteria contains volume Yak base Mang ox base pyrophosphate synthetase Ggps gene, the coding of amino acid sequence shown in code SEQ ID NO:10 Phytoene synthetase Psy gene, the coding GenBank of amino acid sequence shown in GenBank:AAB51287.1: The phytoene dehydrogenase Pds gene of amino acid sequence shown in CAA75094.1 encodes amino shown in SEQ ID NO:12 The 15- of acid sequence is cis--sigma carotene isomerase Ziso gene, amino acid sequence shown in coding SEQ ID NO:14 ζ-Hu The carotenoid isomerase Crtiso base of amino acid sequence shown in radish element dehydrogenase Zds gene, coding SEQ ID NO:16 Cause and the lycopene beta cyclase Lycb gene for encoding amino acid sequence shown in GenBank:ADX41685.1.
2. the beta carotene high production bacteria according to claim 1 based on Du Shi algae metabolic pathway, it is characterised in that:
When based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, encode shown in GenBank:APW83740.1 The nucleotides sequence of the yak base Mang ox base pyrophosphate synthetase Ggps gene of amino acid sequence is classified as such as GenBank: Nucleotide sequence shown in KX231795.1；
Coding SEQ ID NO:2 shown in amino acid sequence phytoene synthetase Psy gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:1；
Encode the nucleotides sequence of the phytoene dehydrogenase Pds gene of amino acid sequence shown in GenBank:ADD52599.1 It is classified as the nucleotide sequence as shown in GenBank:GQ923693.1；
The 15- for encoding amino acid sequence shown in SEQ ID NO:4 is cis--nucleotides sequence of sigma carotene isomerase Ziso gene It is classified as the nucleotide sequence as shown in SEQ ID NO:3；
The nucleotides sequence of the sigma carotene dehydrogenase Zds gene of amino acid sequence shown in coding SEQ ID NO:6 is classified as such as SEQ Nucleotide sequence shown in ID NO:5；
The nucleotides sequence of carotenoid isomerase (Crtiso) gene of amino acid sequence shown in coding SEQ ID NO:8 is classified as The nucleotide sequence as shown in SEQ ID NO:7；
Encode the nucleotides sequence of the lycopene beta cyclase Lycb gene of amino acid sequence shown in GenBank:ANY98896.1 It is classified as the nucleotide sequence as shown in GenBank:KX218392.1.
3. the beta carotene high production bacteria according to claim 1 or 2 based on Du Shi algae metabolic pathway, feature exist In:
When based on Dunaliella salina (Dunaliella saline) metabolic pathway, amino acid sequence shown in SEQ ID NO:10 is encoded The nucleotides sequence of yak base Mang ox base pyrophosphate synthetase Ggps gene be classified as the nucleotide as shown in SEQ ID NO:9 Sequence；
Encode the nucleotides sequence of the phytoene synthetase Psy gene of amino acid sequence shown in GenBank:AAB51287.1 It is classified as the nucleotide sequence as shown in GenBank:U91900.1；
Encode the nucleotides sequence of the phytoene dehydrogenase Pds gene of amino acid sequence shown in GenBank:CAA75094.1 It is classified as the nucleotide sequence as shown in GenBank:Y14807.1；
The 15- for encoding amino acid sequence shown in SEQ ID NO:12 is cis--nucleotide of sigma carotene isomerase Ziso gene Sequence is the nucleotide sequence as shown in SEQ ID NO:11；
Coding SEQ ID NO:14 shown in amino acid sequence sigma carotene dehydrogenase Zds gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:13；
The nucleotides sequence of the carotenoid isomerase Crtiso gene of amino acid sequence shown in coding SEQ ID NO:16 is classified as The nucleotide sequence as shown in SEQ ID NO:15；
Encode the nucleotides sequence of the lycopene beta cyclase Lycb gene of amino acid sequence shown in GenBank:ADX41685.1 It is classified as the nucleotide sequence as shown in GenBank:HQ728089.1.
4. the building of the described in any item beta carotene high production bacterias based on Du Shi algae metabolic pathway of claims 1 to 3 Method, which comprises the steps of:
(1) be cloned into from Du Shi algae using related gene engineering means yak base Mang ox base pyrophosphate synthetase Ggps, Phytoene synthetase Psy, phytoene dehydrogenase Pds, 15- be cis--sigma carotene isomerase Ziso, ζ-recklessly Radish element dehydrogenase Zds, carotenoid isomerase Crtiso, lycopene beta cyclase Lycb；
(2) by Ggps and Psy building on pACYduet-1 carrier, chlorampenicol resistant obtains recombinant vector pACYduet- ggps-psy；By Pds, Zds and Lycb building on pCDFduet-1 carrier, streptomycin resistance obtains recombinant vector pCDFduet-pds-zds-lycb；By Ziso and Crtiso building on pETduet-1 carrier, ammonia benzyl resistance obtains recombination and carries Body pETduet-ziso-crtiso；
(3) it then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), obtains based on Du The beta carotene high production bacteria of family name's algae metabolic pathway；
The Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil) or Dunaliella salina (Dunaliella saline)。
5. the construction method of the beta carotene high production bacteria according to claim 4 based on Du Shi algae metabolic pathway, It is characterized by:
When construction of recombinant vector, it is respectively connected with ribosome bind site rbs sequence at 5 ' ends of 7 target gene, alternatively, connecting It is connected to T7_promoter sequence and ribosome bind site rbs sequence.
6. the described in any item beta carotene high production bacterias based on Du Shi algae metabolic pathway of claims 1 to 3 are producing Application in beta carotene.
7. application according to claim 6, is characterized in that:
When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the β-of the beta carotene high production bacteria The yield of carrotene is 2.7mg/g dry cell weight.
8. application according to claim 6, is characterized in that:
When Du Shi algae is Dunaliella salina (Dunaliella saline), the β-Hu Luo of the beta carotene high production bacteria The yield of Bu Su is 3.3mg/g dry cell weight.
CN201910665228.5A 2019-07-23 2019-07-23 Beta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof Active CN110499272B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910665228.5A CN110499272B (en) 2019-07-23 2019-07-23 Beta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910665228.5A CN110499272B (en) 2019-07-23 2019-07-23 Beta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof

Publications (2)

Publication Number Publication Date
CN110499272A true CN110499272A (en) 2019-11-26
CN110499272B CN110499272B (en) 2021-03-30

Family

ID=68586358

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910665228.5A Active CN110499272B (en) 2019-07-23 2019-07-23 Beta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof

Country Status (1)

Country Link
CN (1) CN110499272B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111909850A (en) * 2020-07-09 2020-11-10 华南理工大学 Astaxanthin-producing engineering bacteria based on Dunaliella salina metabolic pathway and CBFD and HBFD of Adonis amurensis, construction and application thereof
CN112029782A (en) * 2020-09-11 2020-12-04 深圳大学 Beta-carotene hydroxylase, gene and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1670212A (en) * 2005-02-22 2005-09-21 华南理工大学 Gene for coding phytoene dehydrogenase of Dunaliella salina
CN102154330A (en) * 2011-01-14 2011-08-17 华南理工大学 Beta-carotene engineering bacteria based on Dunaliella bardawil metabolic pathway and construction method thereof
CN103154250A (en) * 2010-03-09 2013-06-12 耶路撒冷希伯来大学伊森姆研究发展有限公司 Organism with altered carotenoid content and method of producing same
CN106434356A (en) * 2016-12-12 2017-02-22 国家开发投资公司 Microalga culture method
CN106987550A (en) * 2017-05-18 2017-07-28 陕西师范大学 A kind of recombinant bacterium for producing bata-carotene and its construction method and application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1670212A (en) * 2005-02-22 2005-09-21 华南理工大学 Gene for coding phytoene dehydrogenase of Dunaliella salina
CN103154250A (en) * 2010-03-09 2013-06-12 耶路撒冷希伯来大学伊森姆研究发展有限公司 Organism with altered carotenoid content and method of producing same
CN102154330A (en) * 2011-01-14 2011-08-17 华南理工大学 Beta-carotene engineering bacteria based on Dunaliella bardawil metabolic pathway and construction method thereof
CN106434356A (en) * 2016-12-12 2017-02-22 国家开发投资公司 Microalga culture method
CN106987550A (en) * 2017-05-18 2017-07-28 陕西师范大学 A kind of recombinant bacterium for producing bata-carotene and its construction method and application

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
TAN.C.P: "EU328287.1", 《GENBANK》 *
劳永民: "巴氏杜氏藻类胡萝卜素合成关键酶基因启动子的克隆及其盐调控分子机制研究", 《中国博士学位论文全文数据库基础科学辑》 *
叶志伟: "杜氏藻类胡萝卜素合成关键酶基因的分子克隆及外源表达系统的构建", 《中国博士学位论文全文数据库》 *
梁明华: "巴氏杜氏藻类胡萝卜素合成与降解相关基因的功能鉴定及盐胁迫响应机制研究", 《中国博士学位论文全文数据库基础科学辑》 *
梁颖婕: "巴氏杜氏藻类胡萝卜素异构酶基因的分子克隆及外源表达系统的构建", 《中国优秀硕士学位论文全文数据库基础科学辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111909850A (en) * 2020-07-09 2020-11-10 华南理工大学 Astaxanthin-producing engineering bacteria based on Dunaliella salina metabolic pathway and CBFD and HBFD of Adonis amurensis, construction and application thereof
CN111909850B (en) * 2020-07-09 2022-07-26 华南理工大学 Astaxanthin-producing engineering bacteria based on Dunaliella salina metabolic pathway and CBFD and HBFD of Adonis amurensis, construction and application thereof
CN112029782A (en) * 2020-09-11 2020-12-04 深圳大学 Beta-carotene hydroxylase, gene and application thereof

Also Published As

Publication number Publication date
CN110499272B (en) 2021-03-30

Similar Documents

Publication Publication Date Title
Gebhardt et al. Molecular evolution of flavonoid dioxygenases in the family Apiaceae
CN109777815B (en) HMG-CoA synthetase gene RKHMGCS and application thereof
CN109666683B (en) Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof
CN107586763B (en) Carbonyl reductase mutant, vector, engineering bacterium and application thereof
JP2005503103A (en) Production of carotenoids from a single carbon substrate
CN109536518A (en) A kind of Phytoene dehydrogenase gene RKcrtI and its application
CN110747206B (en) 3-hydroxy-3-methylglutaryl coenzyme A reductase gene RKHMGR and application thereof
CN110499272A (en) Beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway
CN109486794B (en) Chitinase mutant with improved enzyme activity
CN111454854B (en) Rhodosporidium toruloides gene engineering strain for producing astaxanthin
CN106282137A (en) The preparation method and applications of one Carotenoids 9,10 &#39; dioxygenase
CN115011616A (en) Acetaldehyde dehydrogenase gene RKALDH and application thereof
CN112795547B (en) Reverse transcriptase mutant with high reverse transcription efficiency
CN114369147B (en) Application of BFNE gene in tomato plant type improvement and biological yield improvement
CN114672525B (en) Biosynthesis method and application of N-acetyl-5-methoxy tryptamine
CN110540983B (en) Zeta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof
CN109913380A (en) Recombinant yarrowia lipolytica for producing (-) - α -bisabolol and construction method and application thereof
WO2021073220A1 (en) Gene dicx4 having herbicide dicamba degradation function and use thereof
Shang et al. Identification of interacting proteins of transcription factor DpAP2 related to carotenoid biosynthesis from marine microalga Dunaliella parva
CN112746062A (en) Protein related to biosynthesis of perilla terpene substances as well as encoding gene and application thereof
CN110511919A (en) Lycopene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway
CN110511874A (en) Neurosporene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway
Ye et al. Analysis of an essential carotenogenic enzyme: ζ-carotene desaturase from unicellular alga Dunaliella salina
Li et al. Characteristics of a new carotenoid cleavage dioxygenase NtCCD10 derived from Nicotiana tabacum
CN114989274B (en) Phaeodactylum tricornutum Myb transcription factor PtMYB3 gene and encoding protein and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant