WO2015199043A1 - Caroténoïde synthase et son utilisation - Google Patents
Caroténoïde synthase et son utilisation Download PDFInfo
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- WO2015199043A1 WO2015199043A1 PCT/JP2015/067927 JP2015067927W WO2015199043A1 WO 2015199043 A1 WO2015199043 A1 WO 2015199043A1 JP 2015067927 W JP2015067927 W JP 2015067927W WO 2015199043 A1 WO2015199043 A1 WO 2015199043A1
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- 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
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- 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
-
- 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
Definitions
- the present invention relates to a novel carotenoid synthase and use thereof.
- Carotenoids typified by astaxanthin and carotene are widely used in foods, cosmetics, pharmaceuticals and feeds.
- Non-Patent Documents 1 to 14 a gene involved in carotenoid biosynthesis is identified (for example, see Non-Patent Documents 1 to 14), a transformant into which the identified gene is introduced is prepared, and the carotenoid is produced by the transformant. Attempts have been made to produce (see, for example, Patent Documents 1 to 3).
- Patent Document 1 discloses a technique for producing carotenoids using a transformant into which a gene involved in carotenoid biosynthesis of Brevibacterium flavum has been introduced.
- Patent Document 2 discloses a gene encoding phytoene synthase, a gene encoding phytoene desaturase, a gene encoding ⁇ -carotene desaturase, Also disclosed is a technique for producing carotenoids using a transformant into which at least one gene selected from the group consisting of genes encoding lycopene cyclase (Lycopene cyclase) has been introduced.
- Patent Document 3 discloses a gene encoding phytoene desaturase, a gene encoding lycopene cyclase, a gene encoding ⁇ -carotene hydroxylase, and a gene encoding lycopene cyclase.
- a technique for producing carotenoids using a transformant introduced with a gene encoding ⁇ -carotene-C (4) -oxygenase ( ⁇ -carotene C (4) oxygenase) is disclosed.
- JP 2001-149077 A released June 5, 2001
- JP 2002-537841 A published on November 12, 2002
- JP 2006-75097 A published March 23, 2006
- the conventional techniques as described above produce carotenoids by a multi-stage enzymatic reaction using a transformant that artificially expresses many types of enzymes simultaneously.
- carotenoid productivity (for example, production amount) is greatly influenced by the enzyme with the slowest reaction rate and / or the enzyme with the least amount of expression among many types of enzymes. .
- the conventional techniques as described above use transformants in which many types of enzymes are artificially expressed at the same time. Therefore, among these enzymes, enzymes with very slow reaction rates, As a result, the enzyme reaction stage through the enzyme becomes the rate-determining stage in the carotenoid biosynthesis system, resulting in a decrease in carotenoid productivity. ing.
- the present invention has been made in view of the above conventional problems, and an object thereof is to provide a novel carotenoid synthase capable of improving the productivity of carotenoid and use thereof.
- the protein of the present invention is characterized by including a phytoene desaturase domain, a phytoene synthase domain, and a lycopene cyclase domain.
- the phytoene desaturase domain, the phytoene synthase domain, and the lycopene cyclase domain are preferably arranged in this order from the amino terminal side of the protein.
- the protein of the present invention preferably contains at least part of the protein represented by the following (a) or (b). That means (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, or (B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having an activity of generating ⁇ -carotene from geranylgeranyl pyrophosphate.
- the gene of the present invention is characterized by encoding the protein of the present invention in order to solve the above problems.
- the gene of the present invention preferably contains at least a part of the following polynucleotide (c) or (d). That means (C) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or 4, or (D) hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 4 and produces ⁇ -carotene from geranylgeranyl pyrophosphate A polynucleotide encoding a protein having activity.
- the vector of the present invention is characterized in that the gene of the present invention is inserted in order to solve the above problems.
- the transformant of the present invention is characterized in that the vector of the present invention is introduced in order to solve the above problems.
- the method for producing ⁇ -carotene of the present invention is characterized by including a step of culturing the transformant of the present invention in order to solve the above problems.
- the transformant is preferably a transformant capable of producing geranylgeranyl pyrophosphate.
- the method for producing astaxanthin of the present invention includes a step of culturing the transformant of the present invention in order to solve the above problems, and the transformant is a transformant capable of producing astaxanthin from ⁇ -carotene. It is characterized by.
- the transformant is preferably further introduced with a CrtS gene.
- the transformant is preferably further introduced with a CrtR gene.
- the present invention can catalyze a multi-step reaction from the generation of geranylgeranyl pyrophosphate to ⁇ -carotene with a single enzyme, the productivity of carotenoid can be improved.
- the multi-step reaction from the formation of ⁇ -carotene from geranylgeranyl pyrophosphate is catalyzed by a single enzyme, and the multi-step reaction proceeds continuously.
- accumulation of intermediates in the transformation can be suppressed. That is, in the present invention, the amount of geranylgeranyl pyrophosphate used for the production of substances other than ⁇ -carotene can be reduced. Therefore, the present invention has an effect that the production amount of carotenoid can be increased.
- the present invention has an effect that the production process of the transformant, more specifically, the production process of carotenoid can be simplified.
- a transformant that simultaneously expresses a plurality of enzymes as in the prior art tends to change its trait and it is difficult to maintain a desired trait for a long period of time. As a result, it is difficult for the prior art to stably produce carotenoids.
- a transformant expressing one enzyme may be used, and such a transformant is relatively easy to maintain a desired trait for a long period of time. Therefore, the present invention has an effect that carotenoid can be produced stably.
- the protein of the present embodiment is a protein including a phytoene desaturase domain, a phytoene synthase domain, and a lycopene cyclase domain.
- the term “protein” is used interchangeably with “polypeptide”, and a polymer of amino acids is intended.
- phytoene desaturase domain means a domain having the activity of phytoene desaturase, in other words, a domain having high amino acid sequence homology with phytoene desaturase.
- activity of phytoene desaturase means the activity of converting phytoene to lycopene.
- the activity of phytoene desaturase can be confirmed by bringing a desired domain into contact with phytoene. Specifically, if lycopene is generated when a desired domain is brought into contact with phytoene, the domain can be determined to be a phytoene desaturase domain.
- a specific method for testing that lycopene is produced from phytoene is not particularly limited, and a known method can be used as appropriate.
- the presence of lycopene in a mixed solution of a desired domain and phytoene may be detected by various types of chromatography (for example, thin layer chromatography).
- Whether or not the domain has high amino acid sequence homology with phytoene desaturase can be confirmed, for example, by comparing the amino acid sequence of the desired domain with the amino acid sequence of a known phytoene desaturase. Can do.
- phytoene desaturases include, for example, Fulvivirga, Haloquadratum, Arabidopsis, Ectocarpus, or Xanthophyllomyces phytoene desaturase (for example, Accession No. ELR70603, CAJA20735A, AJA5735C, CAJA2A7A7, CAJAA2 .
- the amino acid sequence of the desired domain and the amino acid sequence of a known phytoene desaturase are 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85 % Or more, more preferably 90% or more, and most preferably 95% or more, the domain can be determined to be a phytoene desaturase domain.
- the homology of amino acid sequences can be determined by a known method. Specifically, GENETYX-WIN (manufactured by Genetics Co., Ltd.) is used in accordance with the GENETYX-WIN manual, for example, a homology search (homology search) between a specific amino acid sequence and an amino acid sequence to be compared is performed, and the same The homology can be calculated as the percentage (%) of amino acids.
- GENETYX-WIN manufactured by Genetics Co., Ltd.
- the homology can be calculated as the percentage (%) of amino acids.
- the homology can be calculated as the ratio (%) of the number of identical amino acids to the total number of amino acids of the longer amino acid sequence of the amino acid sequences to be compared.
- the domain having high amino acid sequence homology with phytoene desaturase is one or several (for example, within 20, 19, within 18, within the amino acid sequence of the well-known phytoene desaturase, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less
- phytoene synthase domain means a domain having the activity of phytoene synthase, in other words, a domain having high amino acid sequence homology with phytoene synthase.
- the activity of phytoene synthase intends the activity of converting geranylgeranyl pyrophosphate into phytoene.
- the activity of phytoene synthase can be confirmed by contacting a desired domain with geranylgeranyl pyrophosphate. Specifically, if phytoene is produced when a desired domain is contacted with geranylgeranyl pyrophosphate, the domain can be determined to be a phytoene synthase domain.
- the specific method for testing that phytoene is produced from geranylgeranyl pyrophosphate is not particularly limited, and a known method can be used as appropriate.
- the presence of phytoene in a mixed solution of a desired domain and geranylgeranyl pyrophosphate may be detected by various types of chromatography (for example, high performance liquid chromatography).
- Whether or not a domain has high amino acid sequence homology with phytoene synthase can be confirmed, for example, by comparing the amino acid sequence of a desired domain with the amino acid sequence of a known phytoene synthase.
- phytoene synthases include, for example, phytoene synthases of Fulvivirga, Haloquadratum, Arabidopsis, Ectocarpus, or Xanthophyllomyces. (For example, Accession No. ELR70639, CAJ52967, AED92400, CBJ31337 or AAO47570).
- the amino acid sequence of the desired domain and the amino acid sequence of a known phytoene synthase are 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more. More preferably, if the homology is 90% or more, most preferably 95% or more, the domain can be determined to be a phytoene synthase domain.
- the homology of the amino acid sequence can be determined in the same manner as in the “phytoene desaturase domain”.
- the domain having high amino acid sequence homology with phytoene synthase is one or several in the amino acid sequence of the well-known phytoene synthase described above (for example, 20 or less, 19 or less, 18 or less, 17 or less, 16 pieces, 15 pieces, 14 pieces, 13 pieces, 12 pieces, 11 pieces, 10 pieces, 9 pieces, 8 pieces, 7 pieces, 6 pieces, 5 pieces, 4 pieces Or a domain consisting of an amino acid sequence in which 3 amino acids are deleted, substituted or added.
- lycopene cyclase domain means a domain having lycopene cyclase activity, in other words, a domain having a high amino acid sequence homology with lycopene cyclase.
- the activity of lycopene cyclase is intended to convert lycopene to ⁇ -carotene.
- lycopene cyclase The activity of lycopene cyclase can be confirmed by bringing a desired domain into contact with lycopene. Specifically, if ⁇ -carotene is produced when a desired domain is brought into contact with lycopene, the domain can be determined to be a lycopene cyclase domain.
- the specific method for testing the production of ⁇ -carotene from lycopene is not particularly limited, and a known method can be used as appropriate.
- the presence of ⁇ -carotene in the mixed solution of the desired domain and lycopene may be detected by various types of chromatography (eg, thin layer chromatography).
- a domain having high amino acid sequence homology with lycopene cyclase can be confirmed, for example, by comparing the amino acid sequence of the desired domain with the amino acid sequence of a known lycopene cyclase. .
- lycopene cyclase examples include fulvivirga, Haloquadratum, Arabidopsis, Ectocarpus, or Xanthophyllomyces lycopene cyclase. (For example, Accession No. ELR70634, CAJ51148, AEE74875, CBN78004 or AAO47570).
- the amino acid sequence of the desired domain and the amino acid sequence of a known lycopene cyclase are 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85%. As described above, if the homology is 90% or more, most preferably 95% or more, the domain can be determined to be a lycopene cyclase domain. The homology of the amino acid sequence can be determined in the same manner as in the “phytoene desaturase domain”.
- one or several domains (for example, 20 or less, 19 or less, 18 or less, 17) of the amino acid sequence of the well-known lycopene cyclizing enzyme described above have high homology with lycopene cyclase. Up to 16, up to 15, up to 14, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, It may be a domain consisting of an amino acid sequence in which 4 amino acids, 3 amino acids, or 2 amino acids are deleted, substituted or added.
- the positions of the phytoene desaturase domain, phytoene synthase domain, and lycopene cyclase domain in the protein of the present embodiment are not particularly limited, but from the amino terminal side of the protein, the phytoene desaturase domain, They can be arranged in the order of the phytoene synthase domain and the lycopene cyclase domain.
- phytoene synthase functions first, then phytoene desaturase functions, and finally lycopene cyclase functions.
- the order of the phytoene desaturase domain, the phytoene synthase domain, and the lycopene cyclase domain from the amino terminal side of the protein. Can be arranged.
- the order of the arrangement is a unique configuration of the present invention that cannot be conceived from conventional technical common sense.
- the protein of the present embodiment may be a protein consisting of the protein represented by the following (a) or (b), or a protein containing the protein as at least a part: (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, or (B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having an activity of generating ⁇ -carotene from geranylgeranyl pyrophosphate.
- the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 means that geranylgeranyl pyrophosphate to ⁇ -carotene found in the genus Aurantiochytrium genus KH105 is a diploid organism. Is a novel carotenoid synthase capable of catalyzing a multi-step reaction until it is produced.
- a protein is a protein having the activity of generating ⁇ -carotene from geranylgeranyl pyrophosphate, whether ⁇ -carotene is generated when the protein is contacted with geranylgeranyl pyrophosphate. You can test it. That is, if ⁇ -carotene is produced when the protein is contacted with geranylgeranyl pyrophosphate, the protein can be determined to be a protein having an activity of producing ⁇ -carotene from geranylgeranyl pyrophosphate.
- the specific determination method is not particularly limited, and a known method can be used as appropriate.
- the extract of the solution or cell is separated by various chromatography (for example, thin layer chromatography), and ⁇ -The presence or absence of carotene may be confirmed based on color or absorbance.
- an organic solvent for example, an acetone / methanol mixed solution
- acetone / methanol mixed solution may be appropriately selected and used.
- amino acid in which one or several amino acids are deleted, substituted or added the position where the deletion, substitution or addition occurs is not particularly limited.
- the number of amino acids intended by “one or several amino acids” is not particularly limited, but is within 20 pieces, within 19 pieces, within 18 pieces, within 17 pieces, within 16 pieces, within 15 pieces, within 14 pieces, Less than 13, Less than 12, Less than 11, Less than 10, Less than 9, Less than 8, Less than 7, Less than 6, Less than 5, Less than 4, Less than 3, Less than 2, or It can be a single amino acid.
- the amino acid substitution is preferably a conservative substitution.
- the conservative substitution means that a specific amino acid is substituted with another amino acid having the same chemical properties and / or structure as the amino acid.
- Chemical properties include, for example, hydrophobicity (hydrophobic and hydrophilic), charge (neutral, acidic and basic).
- Examples of the structure include an aromatic ring, an aliphatic hydrocarbon group, and a carboxyl group that exist as a side chain or a functional group of the side chain.
- conservative substitutions include, for example, substitution of serine and threonine, substitution of lysine and arginine, and substitution of phenylalanine and tryptophanamino.
- substitutions include, for example, substitution of serine and threonine, substitution of lysine and arginine, and substitution of phenylalanine and tryptophanamino.
- the present invention is not limited to these substitutions.
- the protein of the present embodiment may be a protein containing at least a part of the protein represented by the following (e) or a protein comprising the protein represented by the following (e): (E) 50% or more of the amino acid sequence represented by SEQ ID NO: 1 or 2, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably Proteins with homology of 90% or more, most preferably 95% or more.
- the homology of amino acid sequences can be determined by a known method. Specifically, GENETYX-WIN (manufactured by Genetics Co., Ltd.) is used in accordance with the GENETYX-WIN manual, for example, a homology search (homology search) between a specific amino acid sequence and an amino acid sequence to be compared is performed, and the same The homology can be calculated as the percentage (%) of amino acids.
- GENETYX-WIN manufactured by Genetics Co., Ltd.
- the homology can be calculated as the percentage (%) of amino acids.
- the homology can be calculated as the ratio (%) of the number of identical amino acids to the total number of amino acids of the longer amino acid sequence of the amino acid sequences to be compared.
- the protein of the present embodiment may contain a configuration other than the above-described phytoene desaturase domain, phytoene synthase domain, and lycopene cyclase domain as necessary.
- Examples of such a configuration include a linker that connects domains, a tag (for example, His tag, Flag tag, HA tag, Myc tag, etc.), and a protein (for example, GST protein, fluorescent protein, etc.).
- a linker that connects domains
- a tag for example, His tag, Flag tag, HA tag, Myc tag, etc.
- a protein for example, GST protein, fluorescent protein, etc.
- the linker may be a linker made of a polypeptide (for example, a linker made of 1 to 20 or 1 to 10 amino acids), or a well-known cross-linking agent capable of linking proteins. May be. If the linker is provided, the relative positions of the domains can be appropriately adjusted, so that a more active protein and / or a more heat-stable protein can be realized.
- a linker made of a polypeptide for example, a linker made of 1 to 20 or 1 to 10 amino acids
- a well-known cross-linking agent capable of linking proteins May be. If the linker is provided, the relative positions of the domains can be appropriately adjusted, so that a more active protein and / or a more heat-stable protein can be realized.
- the gene of the present embodiment is [1. It is a gene encoding the protein of the present invention explained in the column of [Protein].
- the term “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule”, and is intended to be a polymer of nucleotides.
- the codon encoding each amino acid constituting the protein is not limited, and may be a desired codon.
- the gene of the present embodiment may be in the form of DNA (for example, cDNA or genomic DNA) or RNA (for example, mRNA). Furthermore, when it is in the form of DNA, it may be double-stranded DNA or single-stranded DNA. Furthermore, the single-stranded DNA and RNA may be a sense strand encoding a protein, or may be an antisense strand to the sense strand.
- the gene of the present embodiment may be a gene comprising the following polynucleotide (c) or (d), or a gene containing the gene as at least a part thereof. That is, (c) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or 4, or (D) hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3 or 4 and produces ⁇ -carotene from geranylgeranyl pyrophosphate A polynucleotide encoding a protein having activity.
- the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 4 is a gene obtained from geranylgeranyl pyrophosphate found in an Aurantiochytrium genus KH105 strain until the production of ⁇ -carotene. It is a polynucleotide encoding a novel carotenoid synthase capable of catalyzing the step reaction alone.
- stringent conditions refers to hybridization solutions (50% formamide, 5 ⁇ SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7). .6) Incubate overnight at 42 ° C. in 5 ⁇ Denhart solution, 10% dextran sulfate, and 20 ⁇ g / ml denatured sheared salmon sperm DNA, then in 0.1 ⁇ SSC at about 65 ° C.
- the washing conditions at high stringency can be changed as appropriate. For example, when using mammalian-derived DNA, 0.1% SDS containing 0.1% SDS is used.
- washing at 65 ° C. in 5 ⁇ SSC (preferably 15 minutes ⁇ 2 times) is preferred, and EWhen using E. coli-derived DNA, washing at 68 ° C. in 0.1 ⁇ SSC containing 0.1% SDS (preferably 15 minutes ⁇ 2 times) is preferable, and when using RNA, 0.1% Washing at 68 ° C. in 0.1 ⁇ SSC containing SDS (preferably twice for 15 minutes) is preferable, and when using oligonucleotide, in 0.1 ⁇ SSC containing 0.1% SDS Washing at the hybridization temperature (preferably 15 minutes ⁇ 2 times) is preferred.
- the above hybridization was performed by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989).
- the gene according to the present embodiment includes a configuration other than the region encoding the phytoene desaturase domain, the region encoding the phytoene synthase domain, and the region encoding the lycopene cyclase domain as necessary. You may go out.
- a polynucleotide encoding a linker that connects domains for example, a linker consisting of 1 to 20 or 1 to 10 amino acids
- a tag for example, a His tag, a Flag tag
- Examples include polynucleotides encoding HA tags, Myc tags, and the like, and polynucleotides encoding proteins (eg, GST protein, fluorescent protein, etc.).
- the linker is provided, the relative positions of the domains can be appropriately adjusted, so that a more active protein and / or a more highly heat-stable protein can be realized.
- the vector of the present embodiment is described in [2. It is a vector containing the gene of the present invention described in the section “Gene”. In other words, the vector of the present embodiment is [2. It is a vector into which the gene of the present invention described in the section “Gene” is inserted.
- the specific type of the vector is not particularly limited, and the gene of the present invention is expressed in a desired host (for example, bacteria (for example, E. coli, Bacillus subtilis), yeast, filamentous fungi, fungi, animal cells, plant cells). Any vector can be used. Therefore, for known expression vectors [2. Vectors into which the gene of the present invention described in the section “Gene” is inserted are included in the vector of the present invention.
- the vector only needs to contain an expression control region (for example, promoter, terminator, and / or replication origin) according to the type of host to be introduced.
- an expression control region for example, promoter, terminator, and / or replication origin
- the host is a bacterium, for example, a trc promoter, a tac promoter, or a lac promoter can be used as the promoter.
- the promoter can be, for example, a GAL1 promoter, glyceraldehyde, or the like.
- a triphosphate dehydrogenase promoter, a PHO5 promoter or the like can be used.
- the host is a filamentous fungus, for example, an amylase promoter or a trpC promoter can be used as the promoter, and the host is an animal.
- the SV40 early promoter or the SV40 late promoter can be used as the promoter.
- the present invention is not limited to these configurations.
- the expression control region may be a promoter capable of inducing expression (eg, tac promoter, GAL1 promoter, etc.).
- the expression of the protein of the present invention can be induced at a specific timing in the host life cycle (in other words, a specific timing at which a specific metabolic pathway functions in the host). That is, in this configuration, A) a specific enzyme that is not originally contacted in the host is contacted with ⁇ -carotene, and / or B) the amount of phytoene, ⁇ -carotene, and lycopene in the host is determined. Can be reduced. As a result, production of a new carotenoid and / or improvement in production efficiency of a specific carotenoid can be realized.
- the vector of this embodiment can be produced according to a conventional method. Transformation of the host with the vector of the present embodiment can also be performed according to conventional techniques.
- the transformant of the present embodiment is [3.
- the transformant of the present embodiment is [3.
- the host is not particularly limited, and a desired host (for example, bacteria (for example, Escherichia coli, Bacillus subtilis), yeast, filamentous fungi, fungi, animal cells, plant cells) can be used as appropriate.
- the host include, for example, a host in which geranylgeranyl pyrophosphate can be present in cells (for example, the following (1) to (3)), geranylgeranyl pyrophosphate can be present in cells, and Examples include hosts capable of producing astaxanthin from ⁇ -carotene (for example, (4) to (9) below).
- a given host (6) Artificially imparted the ability to produce geranylgeranyl pyrophosphate (in other words, biosynthetic pathway) and originally produced the ability to produce astaxanthin from ⁇ -carotene (in other words, biosynthetic pathway).
- geranylgeranyl pyrophosphate is present in the host.
- the transformant when the transformant is prepared by introducing the vector of the present invention into such a host, the transformant contains the protein of the present invention expressed by the vector and geranylgeranyl pyrophosphate which is a substrate of the protein. Will come into contact. As a result, ⁇ -carotene can be produced from geranylgeranyl pyrophosphate.
- the transformant can be obtained by introducing the vector of the present invention into such a host. As a result, the transformant can produce astaxanthin from ⁇ -carotene produced by the protein of the present invention.
- a method for imparting to a host the ability to produce geranylgeranyl pyrophosphate is not particularly limited.
- a gene encoding at least one enzyme that catalyzes a biosynthesis reaction of geranylgeranyl pyrophosphate can be expressed. Examples thereof include a method for introduction into a host.
- Examples of such a gene include a gene encoding an enzyme that generates geranyl pyrophosphate from isopentenyl pyrophosphate (for example, a GDPS gene (Accession No. Y17376)), and an enzyme that generates farnesyl pyrophosphate from geranyl pyrophosphate.
- Genes encoding eg, FDPS gene (Accession No. AY063112)
- genes encoding enzymes that produce geranylgeranyl pyrophosphate from farnesyl pyrophosphate eg, BTS1 gene (Accession No. U31632), and CrtE gene ( Accession No. DQ016502)).
- a gene encoding an enzyme that generates geranylgeranyl pyrophosphate from farnesyl pyrophosphate may be introduced into the host, or an enzyme that generates geranylgeranyl pyrophosphate from farnesyl pyrophosphate to the host.
- the gene encoding and the gene encoding the enzyme that generates farnesyl pyrophosphate from geranyl pyrophosphate may be introduced, or the gene encoding the enzyme generating geranylgeranyl pyrophosphate from farnesyl pyrophosphate to the host and geranyl pyroline
- a gene encoding an enzyme that generates farnesyl pyrophosphate from an acid and a gene encoding an enzyme that generates geranyl pyrophosphate from isopentenyl pyrophosphate may be introduced.
- a method for imparting to a host the ability to produce astaxanthin from ⁇ -carotene is not particularly limited.
- it encodes at least one enzyme that catalyzes a biosynthetic reaction for producing astaxanthin from ⁇ -carotene.
- Examples thereof include a method for introducing a gene into a host so that the gene can be expressed.
- enzymes examples include genes encoding enzymes that produce astaxanthin from ⁇ -carotene (for example, CrtS gene (Accession No. DQ202402) and CrtR gene (Accession No. EU884134)), ⁇ -carotene Genes encoding enzymes that produce canthaxanthin (for example, CrtO gene (Accession No. DQ257290) and CrtW gene (Accession No. EF031051)) and genes encoding enzymes that produce astaxanthin from canthaxanthin (for example, CrtZ gene (Accession No. EF031052)).
- CrtS gene Accession No. DQ202402
- CrtR gene accesion No. EU884134
- ⁇ -carotene Genes encoding enzymes that produce canthaxanthin (for example, CrtO gene (Accession No. DQ257290) and CrtW gene (Accession No. EF031051)) and genes
- a gene encoding an enzyme that generates astaxanthin from ⁇ -carotene may be introduced into the host, or a gene encoding an enzyme that generates canthaxanthin from ⁇ -carotene to the host. And a gene encoding an enzyme that produces astaxanthin from canthaxanthin may be introduced.
- hosts include various microorganisms that have been confirmed to be carotenoid-produced (for example, the genus Hematotococcus, Monoraphidium, Phaffia (Xanthophyllomyces), Paracoccus, Brevundimonas, Gordoniaco (ccord)) , Nostoc genus and Gloeobacter genus), plants (eg oysters, oranges, carrots, tomatoes and tobacco).
- the present invention is not limited to these hosts.
- the method for producing ⁇ -carotene of the present embodiment includes a step of culturing the transformant of the present invention.
- Specific culture methods for example, culture medium, culture time, culture temperature, etc. are not limited, and may be appropriately selected according to the host used for production of the transformant.
- the transformant is preferably a transformant capable of producing geranylgeranyl pyrophosphate.
- “transformants capable of producing geranylgeranyl pyrophosphate” include [4.
- the hosts described in (1) to (9) described in the section “Transformant” are added to [3. Examples thereof include a transformant prepared by introducing the vector of the present invention described in the “Vector” column.
- geranylgeranyl pyrophosphate is present in the cell.
- the transformant when the transformant is prepared by introducing the vector of the present invention into such a host, the transformant contains the protein of the present invention expressed by the vector and geranylgeranyl pyrophosphate which is a substrate of the protein. Will come into contact. As a result, ⁇ -carotene can be produced from geranylgeranyl pyrophosphate.
- the method for producing ⁇ -carotene of the present embodiment may include a step other than the step of culturing the transformant.
- Examples of the step include a step of purifying ⁇ -carotene.
- ⁇ -carotene may be purified by various chromatography.
- the method for producing astaxanthin according to the present embodiment includes the step of culturing the transformant of the present invention, and the transformant is a transformant capable of producing astaxanthin from ⁇ -carotene.
- Specific culture methods for example, culture medium, culture time, culture temperature, etc. are not limited, and may be appropriately selected according to the host used for production of the transformant.
- a transformant is prepared by introducing the vector of the present invention into such a host.
- the transformant can produce astaxanthin from ⁇ -carotene produced by the protein of the present invention.
- the method for producing astaxanthin according to the present embodiment may include a step other than the step of culturing the transformant.
- Examples of the step include a step of purifying astaxanthin.
- astaxanthin may be purified by various chromatography.
- KH105 strain was cultured in GPY medium (3% glucose, 0.6% polypeptone, 0.2% yeast extract, 2% sea salts) for 4 days, and the cells were collected by centrifugation. After the collected cells are washed with distilled water, the cells are lysed in buffer (0.25 M Tris-HCl, pH 8.2, 0.1 M EDTA (pH 8.0), 2% SDS, 0.1 M NaCl). It was suspended in.
- the upper layer separated by the above centrifugation is collected in a new test tube, an equal amount of a phenol / chloroform / isoamyl alcohol mixed solution (25: 24: 1, v / v) is added, and after gently tumbling for 20 minutes, The solution was centrifuged at 8000 rpm for 15 minutes at room temperature.
- a phenol / chloroform / isoamyl alcohol mixed solution 25: 24: 1, v / v
- the upper layer separated by the above centrifugation is collected in a new test tube, an equal volume of chloroform / isoamyl alcohol mixed solution (24: 1, v / v) is added, and the solution is gently tumbled for 20 minutes. And centrifuged at room temperature for 15 minutes.
- the upper layer separated by the above centrifugation was collected in a new test tube, 2.5 times the amount of chilled ethanol was added, and the mixture was inverted and stirred, and then the solution was centrifuged at 15000 rpm for 15 minutes.
- TE buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
- RNase A was added so as to be / mL, and incubation was performed at 37 ° C. for 1 hour.
- the upper layer separated by the above centrifugation is collected in a new test tube, an equal volume of chloroform / isoamyl alcohol mixed solution (24: 1, v / v) is added, and after inversion stirring, the solution is 8000 rpm, 15 minutes, Centrifuge at room temperature.
- the upper layer separated by the above centrifugation was collected in a new test tube, 2.5 times the amount of chilled ethanol was added, and the mixture was inverted and stirred, and then the solution was centrifuged at 15000 rpm for 15 minutes.
- the CrtIBY gene was amplified and isolated as follows by PCR using the genomic DNA of the KH105 strain as a template.
- a PCR reaction solution containing 150 ng of genomic DNA, 1 unit / 50 ⁇ L of KOD FX neo (manufactured by Toyobo), 0.4 mM of dNTP, 0.3 ⁇ M of primer 1 and 0.3 ⁇ M of primer 2 was prepared.
- Primer 1 5'-CAGTGTGCTGGAATTATGGCGCGCAGGGCGTCG-3 '(SEQ ID NO: 5)
- Primer 2 5'-GATATCTGCAGAATTTCAGGCATTCTTGTACAGCGGGAGC-3 '(SEQ ID NO: 6)
- the PCR reaction solution was heated at 94 ° C. for 2 minutes, followed by a reaction cycle comprising a denaturation reaction at 98 ° C. for 10 seconds, an annealing reaction at 70 ° C. for 30 seconds, and an extension reaction at 68 ° C. for 2 minutes. Was repeated 30 times.
- the PCR reaction solution was separated by agarose gel electrophoresis, and the amplified CrtIBY gene was purified from the agarose gel according to a conventional method.
- each of the DNA base sequences of the two types of genes is shown in SEQ ID NOs: 3 and 4 (hereinafter also referred to as CrtIBY gene). Moreover, each of the amino acid sequences of the proteins encoded by the two types of genes is shown in SEQ ID NOs: 1 and 2.
- the total length of the DNA encoding the protein was 3807 bp (including one stop codon), and encoded a polypeptide consisting of 1268 amino acids.
- a protein having high homology to the polypeptide was searched from a database.
- the polypeptide was derived from various organisms such as phytoene synthase (CrtB), phytoene desaturase. (CrtI) and lycopene cyclase (CrtY) were found to have domains showing high homology.
- the above two types of polypeptides are, in order from the amino-terminal side, a domain having high homology with phytoene desaturase (CrtI) and a high homology with phytoene synthase (CrtB). And a domain exhibiting high homology with lycopene cyclase (CrtY).
- Fig. 2 shows a molecular phylogenetic tree created based on homology with proteins derived from various organisms.
- the molecular phylogenetic tree was created using a clustalw and a saveaw program. The specific creation procedure was in accordance with the manual attached to the program. As shown in FIG. 2, it has been clarified that proteins derived from any organisms are located in positions close to evolutionary phylogeny.
- Saccharomyces cerevisiae INVSc1 (MATa his3D1 leu2 trp1-289 ura3-52 / MAT ⁇ his3D1 leu2 trp1-289 ura3-52) was used as an expression host.
- the expression of the CrtIBY gene was performed by S. Yeast expression vector capable of autonomous replication in cerevisiae, having URA3 as a selection marker, GAL1 promoter and CYC1 terminator capable of inducing expression in galactose as a gene expression control region, and ampicillin resistance gene as a selection marker in E. coli pYES2 (Life Technologies) was used.
- PYES2 was digested with restriction enzyme Eco RI, the digest was subjected to agarose electrophoresis, and linearized pYES2 was purified from an agarose gel by a conventional method.
- Linearized pYES2 100 ng of the PCR product containing the full length of the CrtIBY gene obtained in the previous section, and 5 ⁇ L of 5 ⁇ In-fusion HD enzyme premix (Clontech (Laboratories, Inc.) were mixed, and the mixture was Incubated at 50 ° C. for 15 minutes.
- E. coli competent cells Stella TM Competent Cells (F-, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, ⁇ 80d lacZ ⁇ m15, ⁇ (lacZYA-argF) U169, ⁇ 50 ⁇ L of a solution containing (mrr-hsdRMS-mcrBC), ⁇ mcrA, ⁇ -) (Clontech Laboratories, Inc.) was added, and the cell suspension was ice-cooled for 30 minutes and then heat shocked at 42 ° C. for 45 seconds. And immediately left on ice for 1 minute.
- the bacterial cell suspension was applied to LB plate medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 50 ⁇ g / mL ampicillin, and cultured at 37 ° C. for 12 hours.
- LB plate medium 1% tryptone, 0.5% yeast extract, 1% NaCl, 2% agar
- Colonies that appeared on the LB plate medium were inoculated into 5 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing 50 ⁇ g / mL ampicillin and cultured at 37 ° C., 300 rpm for 12 hours.
- the plasmid was extracted from the obtained cells in accordance with the protocol of PowerPrep Express Plasmid Miniprep System (OriGene).
- the obtained plasmid was digested with restriction enzyme Xho I, and the digest was confirmed by agarose gel electrophoresis. As a result, it was judged that the target plasmid was obtained because it showed an expected band pattern.
- An Escherichia coli strain having the target plasmid was inoculated into 50 mL of LB medium containing 50 ⁇ g / mL ampicillin and cultured at 37 ° C. and 160 rpm for 12 hours. From the obtained cells, NucleoBond (registered trademark) Xtra Midi Plus (The target plasmid, that is, the expression plasmid pYES2-CrtIBY was obtained according to the protocol of Clontech (Laboratories, Inc.).
- Yeast cells were transformed with each of the above-described pYES2-CrtIBY and pYES2 serving as a negative control.
- S. Cerevisiae INVSc1 was cultured in 5 mL of YPD medium (2% polypeptone, 1% yeast extract, 2% D-glucose) at 28 ° C. and 300 rpm for 12 hours. 1 mL of the culture solution was dispensed into a microtube, and the culture solution was centrifuged at 1500 g for 5 minutes at room temperature.
- YPD medium 2% polypeptone, 1% yeast extract, 2% D-glucose
- plasmid pYES2-CrtIBY or pYES2
- the suspension after the incubation was added to SD plate medium (0.67% without uracil). Yeast nitrogen base w / o amino acids, 0.19% yeast synthetic drop out medium w / o uracil, 2% D-glucose, 2% agar) and cultured at 28 ° C.
- Transformants that appeared on the SD plate medium were treated with 3 mL of SCT w / o ura medium (0.67% yeast nitrogen base w / o amino acids, 0.19% yeast synthetic dropout medium w / o uracil, 4% D -Raffinose) and inoculated at 28 ° C. and 300 rpm for 15 hours.
- SCT w / o ura medium 0.67% yeast nitrogen base w / o amino acids, 0.19% yeast synthetic dropout medium w / o uracil, 4% D -Raffinose
- the carotenoid pigment was extracted from the transformant in which the expression of the CrtIBY gene was induced as follows.
- the supernatant separated by the above centrifugation is removed, and the precipitated cells are collected.
- the cells are suspended in 1 mL of distilled water, and then the suspension is transferred to a screw-mouth test tube at 2000 g for 5 minutes. Centrifuge at 4 ° C.
- the supernatant separated by the above centrifugation is removed and the precipitated cells are collected, and 1 mL of 3N HCl is added to the cells and suspended vigorously, and then the suspension is allowed to stand still in a boiling water bath for 3 minutes. I put it.
- the suspension after standing was centrifuged at 2000 g for 5 minutes at 4 ° C., the supernatant was removed and the precipitated cells were collected, and 1 mL of distilled water was added to the cells and suspended.
- the suspension was centrifuged at 2000 g for 5 minutes at 4 ° C., the supernatant was removed and the precipitated cells were collected, and 1 mL of an acetone / methanol mixture (7: 3, v / v) was added and vigorously suspended.
- the suspension is centrifuged at 2000 g for 5 minutes at 4 ° C., and the resulting supernatant is transferred to a new screw-mouth test tube.
- the supernatant is evaporated to dryness with a heat block at 60 ° C. under a nitrogen stream. Solidified.
- the dried product was dissolved in 50 ⁇ L of acetone and transferred to a vial, and the dissolved product was used as a dye sample for each transformant.
- the dye composition of the obtained dye sample was confirmed by thin-layer chromatography (TLC).
- Acetone / hexane mixture (3: 7, v / v) was used as a developing solvent, and 20 ⁇ L of each dye sample was developed in the dark on TLC Silica gel 60 F 254 (Merck).
- 10 ⁇ L each of 1 mg / mL ⁇ -carotene and astaxanthin was similarly developed.
- lane 1 shows ⁇ -carotene as a comparative control
- lane 2 shows astaxanthin as a comparative control
- Lane 3 shows the test results of the yeast into which pYES2 was introduced, and production of ⁇ -carotene was not observed in the yeast.
- lane 4 shows the test results of the yeast introduced with pYES2-CrtIBY, and production of ⁇ -carotene was observed in the yeast.
- FIG. 4 shows the biosynthetic pathway and metabolic pathway of ⁇ -carotene, and genes encoding enzymes involved in the biosynthetic pathway and metabolic pathway.
- Cerevisiae is a route originally owned by S. cerevisiae. This is a route that cerevisiae does not originally have.
- IPP indicates isopentenyl pyrophosphate
- GPP indicates geranyl pyrophosphate
- FPP indicates farnesyl pyrophosphate
- GGPP indicates geranylgeranyl pyrophosphate.
- Cerevisiae can biosynthesize geranylgeranyl pyrophosphate (GGPP), which is a substrate of phytoene synthase (CrtB), but cannot produce phytoene because it does not have phytoene synthase (CrtB).
- GGPP phytoene synthase
- S. Cerevisiae does not have phytoene desaturase / lycopene synthase (CrtI) and lycopene cyclase (CrtY), and therefore cannot produce ⁇ -carotene.
- the CrtIBY gene encodes an enzyme having an activity to produce ⁇ -carotene using GGPP as a substrate.
- a BTS1 gene (derived from S. cerevisiae INVSc1 geneome) (amino acid sequence: SEQ ID NO: 11, nucleotide sequence: SEQ ID NO: 12) encoding an enzyme responsible for the production of GGPP was further introduced. , Investigated its effect. The following tests were performed according to the method described in “Ukibe et al. (2009) Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance. Appl. Environ. Microbiol. 75: 7205-7211.
- pHV1 The yeast constitutive expression plasmid pHV1, which can autonomously replicate in cerevisiae and has HIS3 as a selection marker, was used. More specifically, pHV-BTS1-crtS was constructed in which the BTS1 gene and the CrtS gene were inserted in tandem downstream of the ADH1 promoter in pHV1.
- Transformation into yeast cells was performed by the method described above.
- SD w / o ura his plate medium (0.67% yeast nitrogen base w / o amino acids, 0.19% yeast synthetic dropout medium w / ouracil, histidine, leucine, tryptophan, 2% D-glucose, 76 mg / L L-tryptophan, 76 mg / L L-leucine, 2% agar).
- the obtained transformants were respectively SCT w / o ura, his medium (0.67% yeast nitrogen base w / o amino acid, 0.19% yeast synthetic dropout medium w / ouracile, histidine, histidine.
- 4% D-raffinose, 76 mg / L L-tryptophan, 76 mg / L L-leucine was cultured in the same manner as described above, and the pigment composition was examined.
- lane 5 shows the test results when a CrtS gene and a BTS1 gene were further expressed in yeast in addition to the CrtIBY gene.
- the yeast expressing the CrtIBY gene, the BTS1 gene and the CrtS gene significantly increased the amount of ⁇ -carotene produced, and further, astaxanthin Production was barely observable. This indicates that the amount of GGPP serving as a substrate for the enzyme encoded by the CrtIBY gene was increased by the action of the BTS1 gene, and that astaxanthin was generated by the action of the CrtS gene.
- the plasmid pAD4 for yeast constitutive expression that can replicate autonomously in cerevisiae and has LEU2 as a selection marker was used. More specifically, pAD-crtR in which the CrtR gene was inserted downstream of the ADH1 promoter in pAD4 was constructed.
- Transformation into yeast cells was performed by the method described above, and pAD-crtR was also introduced into yeast into which only pYES2 and pHV-BTS1-crtS were introduced as a negative control.
- SD w / o ura, his, leu plate medium 0.67% yeast nitrogen base w / o amino acids, 0.19% yeast synthetic dropout medium w / ouracile Histidine, leucine, tryptophan, 2% D-glucose, 76 mg / L L-tryptophan, 2% agar).
- the resulting transformants were respectively SCT w / o ura, his, leu medium (0.67% yeast nitrogen base w / o amino acids, 0.19% yeast synthetic dropout medium w / o uracilin, histidine. , Tryptophan, 4% D-raffinose, 76 mg / L L-tryptophan) and cultured in the same manner as described above to examine the pigment composition.
- the present invention can be used in the field of producing carotenoids. More specifically, the present invention can be used in the field of producing foods, cosmetics, pharmaceuticals, samples and the like.
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Abstract
L'invention concerne une nouvelle caroténoïde synthase grâce à laquelle il est possible d'améliorer la productivité des caroténoïdes, et son utilisation. La protéine de la présente invention comprend un domaine phytoène désaturase, un domaine phytoène synthase et un domaine lycopène cyclase.
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JP2014133368A JP2016010347A (ja) | 2014-06-27 | 2014-06-27 | カロテノイド合成酵素およびその利用 |
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CN109593738A (zh) * | 2019-01-16 | 2019-04-09 | 淮阴工学院 | 秋葵类胡萝卜素生物合成和抗逆性相关蛋白AePDS及其编码基因与应用 |
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JP6722209B2 (ja) * | 2017-01-24 | 2020-07-15 | 長瀬産業株式会社 | カロテノイド組成物、カロテノイド組成物の製造方法、カロテノイド組成物を生産する微生物 |
Citations (3)
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JP2005532047A (ja) * | 2002-05-14 | 2005-10-27 | マーテック・バイオサイエンシーズ・コーポレーション | カロチンシンターゼ遺伝子およびその用途 |
JP2006075097A (ja) * | 2004-09-10 | 2006-03-23 | Tadashi Matsunaga | カロテノイド生産能を有する形質転換体及びカロテノイドの生産方法 |
JP2007516697A (ja) * | 2003-06-12 | 2007-06-28 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | カロテノイド化合物をコードする遺伝子 |
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JP2005532047A (ja) * | 2002-05-14 | 2005-10-27 | マーテック・バイオサイエンシーズ・コーポレーション | カロチンシンターゼ遺伝子およびその用途 |
JP2007516697A (ja) * | 2003-06-12 | 2007-06-28 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | カロテノイド化合物をコードする遺伝子 |
JP2006075097A (ja) * | 2004-09-10 | 2006-03-23 | Tadashi Matsunaga | カロテノイド生産能を有する形質転換体及びカロテノイドの生産方法 |
Non-Patent Citations (8)
Cited By (2)
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CN109593738A (zh) * | 2019-01-16 | 2019-04-09 | 淮阴工学院 | 秋葵类胡萝卜素生物合成和抗逆性相关蛋白AePDS及其编码基因与应用 |
CN109593738B (zh) * | 2019-01-16 | 2022-03-29 | 淮阴工学院 | 秋葵类胡萝卜素生物合成和抗逆性相关蛋白AePDS及其编码基因与应用 |
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