WO2013183961A1 - Gène de biosynthèse de protopanaxatriol et composition pour la promotion de la biosynthèse de protopanaxatriol - Google Patents

Gène de biosynthèse de protopanaxatriol et composition pour la promotion de la biosynthèse de protopanaxatriol Download PDF

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WO2013183961A1
WO2013183961A1 PCT/KR2013/005033 KR2013005033W WO2013183961A1 WO 2013183961 A1 WO2013183961 A1 WO 2013183961A1 KR 2013005033 W KR2013005033 W KR 2013005033W WO 2013183961 A1 WO2013183961 A1 WO 2013183961A1
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protopanaxatriol
gene
biosynthesis
ginseng
yeast
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최용의
한정연
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Industry Academic Cooperation Foundation of KNU
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the present invention relates to a protopanaxatriol biosynthesis gene and a facilitation composition, and more particularly, a CYP716A53v2 protein or a CYP716A53v2 gene encoding the same, which is involved in protopanaxatriol biosynthesis. It relates to a composition for promoting protopanaxatriol biosynthesis.
  • Ginseng saponins are almost triterpenoid-based dammarane saponins. This is a unique saponin that exists only in plants of the genus Panax, and has the pharmacological effect that ginseng is different from other saponin-containing plants. Dozens of ginseng saponins have been identified to date, and these are called ginsenosides as glycosides contained in ginseng.
  • Triterpenoid saponins are secondary metabolites of isoprene compounds and are found in many higher plants. They exhibit a wide range of structural diversity and biological activities among plant species. These molecules also have considerable commercial value and are used as drugs (Hostettmann, KA, Marston, A. (1995) Saponins. Chemistry and Pharmacology of Natural Products.Cambridge University Press, Cambridge; Vogler, BK et al. (1999), Eur. J. Clin. Pharmacol. 55: 567575; Shibata, S. (2001) J. Korean Med. Sci. 16: S28S37).
  • triterpenoid saponins are oleanane ( ⁇ -amyrin), ursane ( ⁇ -amyrin), lupeol or dammarene-type triterpenoids It is a skeleton.
  • Ginsenosides are known to be the major constituents of ginseng roots that exhibit their biological activity. have. P. ginseng roots contain at least 4% ginsenosides by dry weight (Shibata, 2001). Seven damaren-type tetracyclic triterpenes (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) are known as major ginsenoside components, and only ginsenoside Ro is oleic. Anan-type pentacyclic triterpenes found in P. ginseng very few.
  • the damarene-type ginsenosides are panaxadiol (Rb1, Rb2, Rc and Rd) and panaxatriol groups (Rg1, Re, Rf, and Rg2) depending on the aglycone structure. Are divided into two groups. Damarene-type triterpenes are described by Panax (Kushiro, T. et al. (1997), Biol. Pharm. Bull. 20: 292-294) and Gynostemma (Cui, JF et al. (1999), Eur. J. Pharm. Sci. 8 187-191) are well known as major compounds.
  • the first step in dama-type ginsenoside biosynthesis is the cycling of 2,3-oxidosqualene into dammarenediol, which is a dammarenediol synthase.
  • dammarenediol synthase Is catalyzed by (Fig. 1), P. ginseng Two homologous dammarenediol synthase enzymes DDS And PNA end Known (Tansakul, P. et al. (2006), FEBS Lett. 580: 5143-5149 .; Han, J.Y. Et al (2006) Plant Cell Physiol. 47: 1653-1662).
  • Dammarenediol-II is a hydroxylation of the cytochrome P450 (CYP) enzyme (Shibuya, M. et al.
  • CYP and GT are located in the supergene families of the plant genome.
  • CYP plays an important role in oxidation during the biosynthesis of various plant secondary metabolites, lignin, terpenoids, sterols, fatty acids, hormones, pigments, and defense-related phytoalexins (Schuler). , M. (1996) Plant cytochrome P450 monooxygenases.Crit . Rev. Plant Sci. 15: 235-284).
  • P. ginseng two CYP genes are thought to be involved in damarene-type ginsenoside biosynthesis. One of these genes may be involved in dammarenediol hydroxylation at the C-12 position for protopanaxadiol synthesis.
  • This gene is the CYP716A47 gene, which was first discovered by the inventors of the world (Han et al. 2011) and filed for patent application (Korean Patent Application No. 10-2011-0113784, PCT International Application No. PCT / KR2012 / 003246 ).
  • Another gene may be involved in protopanaxadiol hydroxylation at the C-6 position for protopanaxatriol synthesis, and these two compounds may be used for damarene-type ginsenosides. Used as backbones.
  • the present inventors have proved through experiments that CYP716A47 is a protoparnaxadiol synthase that plays a very important role in ginsenoside biosynthesis (Han et al. 2011, Plant and Cell Physiology, 52: 2062-2073). However, no enzyme has been identified for the synthesis of ProtoPanaxatriol from ProtoPanacodiol.
  • CYP716A is among the putative full CYP gene sequences obtained from the EST sequences of the adventitious roots.
  • CYP716A53v2 a family of genes, is a protofaxatriol synthase that plays a very important role in ginsenoside biosynthesis.
  • An object of the present invention is CYP71653v2, which is a promoter for protofanaxadiol biosynthesis CYP716A53v2 gene or encoded therefrom It is to provide a composition for promoting protopanaxatriol biosynthesis containing a protein.
  • Another object of the present invention is to provide a host cell transformed with the composition.
  • Another object of the present invention to provide a transformed plant transformed with the composition.
  • Another object of the present invention is the CYP716A53v2 It is to provide a method for increasing the production of protoparnaxatriol by increasing the expression of the protofanaxatriol biosynthesis promoting gene.
  • the present invention provides a composition for promoting protopanaxatriol biosynthesis comprising a CYP716A53v2 protein or a CYP716A53v2 gene encoding the same.
  • the gene may be composed of the nucleotide sequence of SEQ ID NO: 1
  • the protein may be composed of the amino acid sequence of SEQ ID NO: 2.
  • the composition is a CYP716A53v2 encoding the CYP716A53v2 protein It may include a recombinant vector or plasmid containing the gene.
  • the CYP716A53v2 protein is characterized in that the protopanaxadiol 6-hydroxylase (protopanaxadiol 6-hydroxylase). Therefore, the composition for promoting protopanaxatriol biosynthesis of the present invention may increase the synthesis of protopanaxatriol in protopanaxadiol through activation of the protopanaxatriol.
  • the present invention also provides a host cell which is transformed with the recombinant vector or plasmid capable of synthesizing protopanaxatriol.
  • the host cell may be yeast or E. coli.
  • the present invention also provides a transformed plant transformed with the recombinant vector or plasmid.
  • the present invention also provides a method of increasing the production of the CYP716A53v2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 or CYP716A53v2 protein consisting of the amino acid sequence of SEQ ID NO: 2 to increase the production of protopanaxtriol.
  • the method may comprise overexpressing the CYP716A53v2 gene or protein by transforming the host with a recombinant vector or plasmid.
  • the host may be a plant including yeast, Escherichia coli or Panax ginseng.
  • the present invention can be usefully used in a method for mass-producing protopanaxatriol or increasing the ginseng saponin biosynthesis of the protopanaxanatriol family.
  • Figure 1 shows the biosynthetic pathway of ginsenosides expected in ginseng ( P. ginseng) .
  • Squalene epoxidase converts squalene into 2,3-oxidosquane, which is triterpene aglycones (dammarenediol or beta) by dammarenediol synthetase or beta-amirin synthase. Amirine).
  • Triterpene aglycones are subsequently oxidized and glycosylated to eventually triterpene saponins (ginsenosides).
  • FIG. 2 shows the phylogenies identified from amino acid sequences inferred for P. ginseng CYPs (bold) and other plant CYPs.
  • Gm Glycine max ;
  • At Arabidopsis thaliana ;
  • Gu Glycyrrhiza uralensis ;
  • Mt Medicago truncatula ;
  • Sb Sorghum bicolor ;
  • Vv Vitis vinifera .
  • Bar 0.1 amino acid substitutions / site.
  • Figure 3 is a total ion chromatogram of LC / APCIMS analysis for the CYP716A53v2 product in yeast.
  • A LC chromatogram of yeast cell extract with empty vector as a control.
  • B LC chromatogram of yeast cell extract with pYES2-CYP716A53v2 vector.
  • C Protopananaxtriol standard LC chromatogram.
  • D Protoparanaxadiol standard LC chromatogram.
  • FIG. 4 is an LC / APCIMS spectrum of peaks detected in yeast with CYP716A53v2.
  • A MS spectrum of the peak detected in the ProtoPanaxatriol standard.
  • B MS spectrum of peak detected in yeast with CYP716A53v2.
  • the present invention provides a CYP716A53v2 protein, which is a cytochrome P450 enzyme derived from ginseng involved in protopanaxatriol biosynthesis, and a CYP716A53v2 encoding the same.
  • the present invention relates to the use of the gene, and provides a method of increasing the production of protopanaxatriol using the CYP716A53v2 gene and its protein.
  • the CYP superfamily is a large and diverse group of enzymes. 246 CYP genes have been reported in A. thaliana (Nelson, D. (2006) Plant cytochrome P450s from moss to poplar.Phytochem . Rev. 5: 193-204).
  • Ginseng saponin is called "ginsenoside” in the sense of ginseng glycoside to distinguish it from other plant-based saponins, and these ginsenoids are from sequualene to dammarenediol as shown in FIG. Produced via II and Protopananacodiol. Accordingly, the present invention provides a composition for promoting protopanaxatriol biosynthesis that can significantly increase the synthesis of protopanaxanatriol, which is an intermediate in the ginsenoid biosynthesis process.
  • CYP716A53v2 as a protopanaxatriol synthase with the ability to activate protopanaxadiol 6-hydroxylase using yeast expression analysis. That is, the recombinant CYP716A53v2 expressing yeast was prepared, and after the feeding of the protopanaxadiol to the yeast, it was confirmed that the protopanaxanatriol was produced from the protopanaxadiol. After expression, only this enzyme was extracted and reacted with protopanaxadiol to confirm that protopanaxanatriol was produced.
  • Ginseng P. ginseng
  • panaxadiol Rb1, Rb2, Rc, and Rd
  • panaxatriol Rg1, depending on the aglycone structure
  • Re, Rf, and Rg2 group is divided into two groups.
  • Each ginsenoside has been found to have different pharmacological effects such as anti-stress, anti-diabetic, anti-inflammatory, anti-oxidant, anti-cancer as well as immune system abnormalities (Briskin, DP (2000) Plant Physiol. 124: 50714 Shibata, 2001).
  • the discovery of the genes and enzymes that produce the protopananasporidium according to the present invention can be introduced into yeast, ginseng and other plants to induce the biosynthesis of the protopanaxanatriol and through the metabolic engineering method. It can be an effective way to drastically increase biosynthesis.
  • the present invention provides a CYP716A53v2 protein or CYP716A53v2 encoding the same. It is possible to provide a composition for promoting protopanaxatriol biosynthesis comprising a gene.
  • the range of the CYP716A53v2 protein includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from ginseng and a functional equivalent of the protein.
  • “functional equivalent” means at least 70%, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution or deletion of an amino acid. Preferably it refers to a protein having a sequence homology of 95% or more, and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2.
  • substantially homogeneous physiological activity refers to the activity involved in protopanaxanatriol biosynthesis in plants.
  • the CYP716A53v2 gene according to the present invention includes all genomic DNA encoding the CYP716A53v2 protein.
  • the gene of the present invention that is, the cDNA of CYP716A53v2 may be composed of the nucleotide sequence represented by SEQ ID NO: 1.
  • variants of the base sequence may be included within the scope of the present invention. Specifically, the variant may include a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1, respectively. Can be.
  • % sequence homology to the polynucleotide is identified by comparing the two optimally arranged sequences with the comparison region, wherein a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (additional Or does not include deletion) or addition or deletion (gap).
  • the present invention can provide a composition for promoting protopanaxatriol biosynthesis comprising a recombinant vector or plasmid comprising the CYP716A53v2 gene according to the present invention.
  • the recombinant vector is preferably a recombinant yeast expression vector or a recombinant plant expression vector.
  • recombinant refers to a cell expressing a heterologous nucleic acid, expressing the nucleic acid, or expressing a protein encoded by a peptide, heterologous peptide, or heterologous nucleic acid.
  • the recombinant cell transformed with the vector may express a gene or a gene fragment which is not expressed in the natural form of the cell in one of the sense and antisense forms.
  • recombinant cells may express genes expressed in cells in a natural state, but the genes are modified and reintroduced into cells by artificial means.
  • Vector refers to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. “Delivers” can often be used interchangeably with “vectors.”
  • An "expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.
  • Yeast expression vectors can include promoter genes, genes encoding target proteins from which translational initiation and termination codons have been removed, and the promoter genes are genes selected from the group consisting of GAPDH, PGK, ADH, PHO5, GAL1 and GAL10. Is preferably, but is not limited thereto.
  • the CYP716A53v2 gene of the present invention comprises a nucleic acid sequence encoding a signal peptide, which allows for the export of the expressed protein.
  • the nucleic acid sequence encoding the signal peptide is preferably bound directly to 5 'of the heterologous gene to be expressed.
  • fusion with a protein sequence having a signal sequence at the N-terminus is required to steer the polypeptide into the secretion apparatus.
  • the vector may be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector (YP).
  • the extrachromosomal plasmid vector is divided into an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromeric plasmid (YCp). Furthermore, artificial yeast chromosomes (YACs) are also possible as expression vectors according to the present invention.
  • YEp episomal yeast plasmid
  • YRp replicative yeast plasmid
  • YCp yeast centromeric plasmid
  • YACs artificial yeast chromosomes
  • yeast vectors are yeast replication plasmids that can be propagated and selected in E. coli , containing the origin of replication ori and an antibiotic resistance cassette. Furthermore, they have ARS sequences capable of independent chromosome replication in yeast cells, such as HARS1 from H. polymorpha, and metabolic yeast selection markers such as URA3 or HLEU2.
  • Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells.
  • a suitable host such as Agrobacterium tumerfaciens
  • Another type of Ti-plasmid vector (see EP 0116718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which properly insert hybrid DNA into the genome of the plant.
  • a particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0120516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those that can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc.
  • CaMV double stranded plant viruses
  • gemini viruses single stranded viruses
  • it may be selected from an incomplete plant viral vector.
  • the use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
  • the expression vector will preferably comprise one or more selectable markers.
  • the marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. It is not limited to this.
  • the promoter of the plant expression vector may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter.
  • the term “promoter” refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the possibility of selection.
  • the terminator may use a conventional terminator, and examples thereof include nopaline synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto.
  • NOS nopaline synthase
  • rice ⁇ -amylase RAmy1 A terminator phaseoline terminator
  • agrobacterium tumefaciens ocrobacterium tumefaciens
  • Terminator of the Fine (Octopine) gene etc.
  • the present invention can be provided with a host cell capable of transforming the recombinant vector or plasmid according to the present invention capable of protopananax triol biosynthesis.
  • the host cell may be, but is not limited to, yeast and E. coli.
  • the present invention may provide a transformed yeast transformed with a recombinant yeast vector comprising the CYP716A53v2 gene.
  • the yeast may be a genus selected from Pichia, Hansenula, Candida, Torulopsis, Saccharomyces, Schizosaccharomyces, Kluyveromyces and Yarrowia.
  • the microorganism may belong to a species selected from Hansenula polymorpha, Saccharomyces Cervisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Yarrowia lipolytica.
  • Transformation of yeast can cause the nucleic acid molecule or vector to be introduced into cells by standard methods known to those skilled in the art, preferably by electroporation, chemical transformation, transformation by plasma fusion, or particle bombardment.
  • Current Protocols in Molecular Biology John Wiley & Sons, Edited by: Fred M. Ausubel et al .; Molecular Cloning: A Laboratory Manual (Third Edition), J. Sambrook and D. Russell, 2001, Cold Spring Harbor Laboratory Press ).
  • the present invention can provide a transgenic plant capable of protofa naxatriol biosynthesis by transformation with a recombinant vector or plasmid comprising the CYP716A53v2 gene of the present invention.
  • the plant may include, but is not limited to, tobacco, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gall, watermelon, melon, Cucumber pumpkin, gourd, strawberry, soybeans, green beans, kidney beans and peas can be a dicotyledonous plant characterized in that selected from, but is preferably Arabidopsis.
  • Plant transformation refers to any method of transferring DNA to a plant.
  • transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including dicotyledonous plants as well as monocotyledonous quantum.
  • any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells.
  • Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A.
  • Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EPA 120516 and US Pat. No. 4,940,838.
  • Plant cells used for plant transformation may be any plant cells.
  • Plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.
  • Plant tissue refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue.
  • the plant tissue may be in planta or in an organ culture, tissue culture or cell culture.
  • the present invention can provide a method of increasing the production of the CYP716A53v2 gene or the CYP716A53v2 protein encoded therefrom, thereby increasing the production of protopanaxtriol.
  • the method may also include overexpressing the CYP716A53v2 gene or protein by transforming the host with the recombinant vector or plasmid according to the present invention.
  • the host may be, but is not limited to, yeast, E. coli, plants, especially ginseng.
  • the CYP716A53v2 gene of the present invention promotes the synthesis of protopanaxtriol in protopanaxadiol through the activation of protopanaxadiol 6-hydroxylase, such a protopanaxanatriol It is possible to provide a composition for promoting biosynthesis and a transformed plant which is excessively produced with ProtoPanaxatriol, and also provides a method for increasing the production of ProtoPanaxatriol.
  • DH10B was used as the host strain and pDNR-LIB was used as the cloning vector.
  • CYP716A47 is a Protopanaxadiol synthetic gene (see our patent application No. 10-2011-0113784) in the EST sequence in ginseng MeJA-treated adventitious roots.
  • the full length of the gene CYP716A53v2 was obtained and registered in GenBank, and the access numbers are CYP716A53v2 JX036031 .
  • CYP716A53v2 gene belongs to the CYP716A family, the amino acid sequence has only 49% similarity to the previously known CYP716A47 gene, which is a gene that is completely different from the gene related to protopanaxadiol synthesis.
  • CYP716A12 found in Medicago truncatula plants, is a multifunctional gene that makes oleanolic acid from beta-amyrin ( ⁇ -amyrin and erythrodiol). It is a completely different gene because it has the same sex.
  • CYP72A219, CYP73A100, CYP736A12, CYP82H23, CYP82D47, CYP71D312, CYP71D313, CYP749A22, and CYP716A47 The amino acid sequence deduced from CYP716A53v2 is 44% of the CYP716A47 gene, Medicago truncatula Origin Only 53% homology with that of CYP716A12.
  • ORFs open reading frames
  • PCR was carried out 25 cycles of 94 °C 40 seconds, 55 °C 40 seconds and 72 °C 2 minutes, using Pfu DNA polymerase (Stratagene).
  • Pfu DNA polymerase (Stratagene).
  • the PCR product was cloned into pYES2.1 using the TOPO TA expression kit (Invitrogen). Primer pairs used to isolate cDNAs are as follows:
  • PCR products were cloned into pYES2.1 / V5-His-TOPO vector and Escherichia coli was transformed.
  • the ORFs were then operably conjugated to the GAL1 promoter.
  • the nucleotide sequence of the inserted DNA was confirmed by sequencing.
  • CYP716A53v2 and the empty vectors were expressed in Saccharomyces cerevisiae strain WAT21 carrying Arabidopsis thaliana NADPH-CYP reductase (Urban, P. et al. (1997) J. Biol. Chem. 272, 19176-19186).
  • WAT21 yeast cells were transformed by known modified lithium acetate methods (Gietz, D., St Jean, A., Woods, RA, Schiestl, RH (1992) Improved method for high efficiency transformation of intact yeast cells.Nucleic Acids Res. 20: 1425). Transformed cells were selected with SC-U (uracil deficient SC minimal medium), cultured for 3 days and passaged in YPG medium (Kribii et al., 1997).
  • SC-U uracil deficient SC minimal medium
  • LC-APCIMS analysis was performed on a Surveyor LC system (Thermo Finnigan Co., San Jose, CA, USA). It consists of four solvent pumps, a Rheodyne injector (5 ml loop) and an HTP Pal autosampler (CTC Analytics, Zwingen, Switzerland). The analytical column used YMC pack-pro C18 RS (5 mm, 2.0 ⁇ 150 mm, YMC Co. LTD. Japan) stored at 408 ° C.
  • Water and acetonitrile gradient application time and composition ratio are as follows: 0 min, 80% acetonitrile and 20% water; 30 minutes, 10% acetonitrile and 90% water; 32 min, 5% acetonitrile and 95% water; 34 minutes, 5% acetonitrile and 95% water; 36 minutes, 80% acetonitrile and 20% water; And 45 min, 80% acetonitrile and 20% water, flow rate of 0.2 ml min ⁇ 1 .
  • Finnigan TSQ Quantum Ultra (Thermo Electron Co., San Jose, Calif., USA), a triple quadrupole mass spectrometer, equipped with an atmospheric pressure chemical ionization (APCI) system, was used for detection.
  • the analytical conditions were as follows: positive mode of 5.0 mA discharge current, vaporizer temperature of 320 ° C. and ion-transfer capillary temperature of 320 ° C. Nitrogen was used as sheath (15 psi) and auxiliary gas (10 psi). For HPLC-UV detection, ginsenosides were observed at 202 nm wavelength. The original protopanaxadiol and protopanaxatriol were tested under the same conditions, and both materials were used as the standard for LC / APCIMS analysis.
  • CYP716A53v2 The full length cDNA clone of (JX036031) is 1,560 bp in size, has an open reading frame (ORF) of 470-amino acids, and produces a protein whose molecular weight is predicted to be 55.3 kDa.
  • ORF open reading frame
  • CYP716A53v2 The ORF region of the cDNA was inserted into the pYES2.1 expression vector and expressed under the control of the GAL1 promoter in WAT21 yeast. Yeast extracts were analyzed using total ion chromatograms from liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC / APCIMS).
  • yeast expressing the CYP716A53v2 gene the protofanaxatriol signal was analyzed using MS fragmentation patterns at a retention time of 19.2 minutes (FIG. 4).
  • yeast expressing the CYP716A53v2 gene the LC / APCIMS fragmentation pattern is m / s of 405 [M-3H 2 O + H] + , 423 [M-2H 2 O + H] + , and 441 [MH 2 O + H] + . z ratios were included, which was the same as for the original ProtoPanaxatriol (FIG. 4).

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Abstract

La présente invention concerne un gène CYP716A53v2 d'enzyme de cytochrome P450 issu du ginseng qui est mis en jeu dans la biosynthèse de protopanaxatriol ; et plus particulièrement concerne, entre autre : une composition favorisant la biosynthèse de protopanaxatriol comprenant la protéine CYP716A53v2 issue du ginseng ou un gène CYP716A53v2 codant pour celle-ci ; un vecteur recombinant ou un plasmide recombinant comprenant le gène CYP716A53v2 ; et un procédé pour améliorer la production de protopanaxatriol par l'amélioration de l'expression du gène CYP716A53v2 ou la production directe de protopanaxatriol à partir d'un transformant provenant de l'altération génétique au moyen de la composition. Le gène CYP716A53v2 issu du ginseng selon la présente invention, qui est mis en jeu dans la biosynthèse de protopanaxatriol, peut être utilisé de façon avantageuse dans un procédé de synthèse de protopanaxatriol en volume ou pour améliorer la biosynthèse de saponines de ginseng à base de protopanaxatriol.
PCT/KR2013/005033 2012-06-07 2013-06-07 Gène de biosynthèse de protopanaxatriol et composition pour la promotion de la biosynthèse de protopanaxatriol Ceased WO2013183961A1 (fr)

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EP3441476A1 (fr) * 2017-08-09 2019-02-13 Intelligent Synthetic Biology Center Production accrue de ginsénosides par amélioration de machinerie de pliage de la protéine de levure
CN111118095A (zh) * 2019-12-10 2020-05-08 武汉克鲁金生物科技有限公司 乳酸克鲁维酵母菌水解人参皂苷生产人参皂苷ck提取物的方法
CN115260299A (zh) * 2022-05-12 2022-11-01 成都大学 人参PgWRKY2转录因子及其应用

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CN114507646B (zh) * 2020-11-17 2023-11-10 生合万物(上海)生物科技有限公司 细胞色素p450突变体蛋白及其应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3441476A1 (fr) * 2017-08-09 2019-02-13 Intelligent Synthetic Biology Center Production accrue de ginsénosides par amélioration de machinerie de pliage de la protéine de levure
US11046990B2 (en) 2017-08-09 2021-06-29 Korea Research Institute Of Chemical Technology Increased production of ginsenosides through improvement of protein-folding machinery of yeast
CN111118095A (zh) * 2019-12-10 2020-05-08 武汉克鲁金生物科技有限公司 乳酸克鲁维酵母菌水解人参皂苷生产人参皂苷ck提取物的方法
CN111118095B (zh) * 2019-12-10 2023-10-24 武汉克鲁金生物科技有限公司 乳酸克鲁维酵母菌水解人参皂苷生产人参皂苷ck提取物的方法
CN115260299A (zh) * 2022-05-12 2022-11-01 成都大学 人参PgWRKY2转录因子及其应用

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