WO2013183961A1 - Protopanaxatriol biosynthesis gene and promoting composition - Google Patents

Abstract

The present invention relates to a ginseng-derived cytochrome P450 enzyme CYP716A53v2 gene which is involved in protopanaxatriol biosynthesis, and more specifically relates to, inter alia: a protopanaxatriol biosynthesis promoting composition comprising ginseng-derived CYP716A53v2 protein or a CYP716A53v2 gene coding therefor; a recombinant vector or plasmid comprising the CYP716A53v2 gene; and a method for increasing protopanaxatriol production by increasing expression of the CYP716A53v2 gene or the direct production of protopanaxatriol from a transformant resulting from genetic alteration by means of the composition. The ginseng-derived CYP716A53v2 gene according to the present invention, which is involved in protopanaxatriol biosynthesis, can be used advantageously in a method for synthesising protopanaxatriol in volume or increasing the biosynthesis of protopanaxatriol-based ginseng saponins.

Description

ProtoPanaxatriol Biosynthesis Gene and Composition for Promotion

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). The key components of triterpenoid saponins are oleanane (β-amyrin), ursane (α-amyrin), lupeol or dammarene-type triterpenoids It is a skeleton.

Panax ginseng is well known among consumers for its excellent pharmacological effects, especially in cancer, diabetes, and neurodegenerative diseases. 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. Is catalyzed by (Fig. 1),P. ginsengTwo homologous dammarenediol synthase enzymesDDS AndPNAend 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. (2006),FEBS J.273: 948-959) and subsequent glycosylation of glycosyl transferase (GT) (Kushiro, T. et al. (1997),Biol. Pharm. Bull.20: 292-294; Shibuya et al., 2006; Choi, D.W. Et al. (2005),Plant Cell Rep.23: 557-566), and ginsenoside Ro is thought to be synthesized from oleanolic acid, a product of β-amyrin (Shibata, S (1977) Saponins with biological and pharmacological activity.In: H. Wagner and P. Wolff (Eds.), New natural products and plant drugs with pharmacological or therapeutical activity.Springer-Verlag, New York pp. 177-196).

CYP and GT are located in the supergene families of the plant genome. In plants, 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). In 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.

In the present invention, a new gene involved in proto-paroxal triol biosynthesis was identified from proto-paranse diol, which was found to be CYP716A53v2 .

Therefore, the inventors have found that CYP716A is among the putative full CYP gene sequences obtained from the EST sequences of the adventitious roots. Experiments have demonstrated that 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.

In addition, another object of the present invention to provide a transformed plant transformed with the composition.

In addition, 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.

In order to achieve the above object, the present invention provides a composition for promoting protopanaxatriol biosynthesis comprising a CYP716A53v2 protein or a CYP716A53v2 gene encoding the same.

In one embodiment of the present invention, 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.

In one embodiment of the invention, the composition is a CYP716A53v2 encoding the CYP716A53v2 protein It may include a recombinant vector or plasmid containing the gene.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the invention, the method may comprise overexpressing the CYP716A53v2 gene or protein by transforming the host with a recombinant vector or plasmid.

In one embodiment of the present invention, the host may be a plant including yeast, Escherichia coli or Panax ginseng.

It was confirmed that the ginseng-derived CYP716A53v2 gene, which is involved in the protofaxatriol biosynthesis according to the present invention, may increase the protofanaxatriol biosynthesis. Therefore, 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).

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 ; Pg. Panax ginseng; 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.

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).

To successfully find the CYP gene associated with ginsenoside biosynthesis, we have successfully isolated CYP716A53v2 from the EST sequence of MeJA-treated ginseng root root and identified CYP716A53v2 as a novel protopanaxatriol synthase. It was.

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.

The inventors also identified 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.

More than 30 ginsenosides have been identified in Ginseng (P. ginseng) , which are panaxadiol (Rb1, Rb2, Rc, and Rd) and 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).

In addition, recent studies have reported that ginsenoside metabolites have greater biological effects than ginsenosides (Jia, W. et al., (2004) J Clin Oncol ASCO Annual Meeting Proceedings (Post-Meeting Edition). 22: 9663). In addition, it has been reported that protopanaxadiol, a triterpene aglyconate hydrolyzed from various ginsenosides, has apoptosis effects through various signaling pathways in cancer cells and cytotoxicity against multidrug-resistant tumors (Jia et. al. 2004; Popovich and Kitts 2004 Li et al. 2006). Accordingly, some pharmaceutical companies (PanaGin Pharmaceuticals (British Columbia, Canada)) are developing cancer treatments containing Pandimex.

In reality, the synthesis of triterpenes in organic synthesis is difficult, so the compound must be separated from nature or obtained by ginsenoside hydrolysis.

Therefore, 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.

Therefore, 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.

As used herein, "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. As used herein, "substantially homogeneous physiological activity" refers to the activity involved in protopanaxanatriol biosynthesis in plants.

In addition, the CYP716A53v2 gene according to the present invention includes all genomic DNA encoding the CYP716A53v2 protein. Preferably, 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. In addition, 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.

Wherein the “% 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).

In addition, 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. Although not limited thereto, the recombinant vector is preferably a recombinant yeast expression vector or a recombinant plant expression vector.

As used herein, "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. In addition, 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" as used herein 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.

On the other hand, 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. Here, the nucleic acid sequence encoding the signal peptide is preferably bound directly to 5 'of the heterologous gene to be expressed. In the secretion and modification of many eukaryotic cell proteins, 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.

In addition, particularly preferred 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.

Preferred examples of plant expression vectors are 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. 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.

Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those that can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, 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.

In one embodiment of the present invention, 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 α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

In addition, 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.

Specifically, the present invention may provide a transformed yeast transformed with a recombinant yeast vector comprising the CYP716A53v2 gene. Preferably, the yeast may be a genus selected from Pichia, Hansenula, Candida, Torulopsis, Saccharomyces, Schizosaccharomyces, Kluyveromyces and Yarrowia. Particularly preferably, 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 ).

In addition, 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.

Such 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. In principle, 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. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), Agrobacterium tumulopasis (DNA or RNA-coated) particle bombardment (Klein TM et al., 1987, Nature 327, 70), invasion of plants or transformation of mature pollen or vesicles And may be appropriately selected from infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer.

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.

The "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.

Thus, 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.

Since 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.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example 1>

P. ginseng  Isolation of the CYP Gene from the EST Library

<1-1> Plant Materials and EST Library

Total RNA was extracted from adventitious roots of ginseng cultured in vitro. Poly (A) + RNA was extracted using Poly (A) quick mRNA isolation kit (Stratagene). The Creator SMART cDNA library construction kit (Clontech) was used to create the cDNA library. DH10B was used as the host strain and pDNR-LIB was used as the cloning vector.

Single-pass partial sequences were measured using an automated DNA sequencer (ABI Prism 3700, Applied Biosystems). A total of 4,226 cDNA strands (NCBI GenBank dbEST accession Nos. HS076062HS080287) were randomly extracted and sequenced from the 5 'end using an ABI 3730 XL automated DNA sequencer (Applied Biosystems). ESTs results were compared to GenBank and dbEST sequences using the BLASTX algorithm on the NCBI website.

<1-2> Identification and Analysis of CYP716 Family

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 .

Table 1

CYP450 family	Typical EST ID	GenBank Access Number	EST Count	Naming CYPs	Homology to (species name)	Description	Accession No.	E-Value
CYP71	KYERRC001 46-A08	JN604540	7	CYP71D312	CYP71D55 ( Hyoscyamus muticus )	Premnaspirodiene Oxygenase	EF569601	2e -09
	KYERRC001 36-B03	JN604541	5	CYP71D313
	KYERRC001 26-F07		One	CYP71D314
	KYERRC001 18-F12		One	CYP71D315v2
	KYERRC001 17-H02		2	CYP71D315v1
	KYERRC001 10-D12		One	CYP71D316
	KYERRC001 19-G01		One	CYP71D317
	KYERRC001 43-F04		One	CYP71D318v1
	KYERRC001 45-G06		One	CYP71D318v2
CYP72	KYERRC001 39-E08	JN604542	2	CYP72A129	CYP72A57 ( Nicotianat abacum )	Unknown	DQ350360	3e -64
	KYERRC001 42-H03		One	CYP72A220
	KYERRC001 31-H09		One	CYP72A221
CYP73	KYERRC001_19-F10	JN604543	5	CYP73A100	CYP73A5 ( Arabidopsi sthaliana )	Cinnamic acid 4-hydroxylase	D78596	3e -166
CYP82	KYERRC001 42-G11	JN604544	3	CYP82H23	CYP82A1 ( Pisumsativum )	UnknownWound-inducible P450 hydroxylase	AF175278	2e -09
	KYERRC001 19-F02		One	CYP82H24
	KYERRC001 01-H12	JN604545	One	CYP82D47
CYP90	KYERRC001_12-D12		3	CYP90A29	CYP90 A2 ( Pisumsativum )	Putativebrassinosteroid C-23 hydroxylase	AB218762	9e -122
CYP94	KYERRC001_27-A10		One	CYP94A33	CYP94A1 ( Viciasativa )	Fatty acidomega-hydroxylase	081117	0.0
CYP98	KYERRC001 26-B07		One	CYP98A58	CYP98A13 ( Ocimumbasilicum )	p-coumaroyl shikimate 3'-hydroxylase	AY082612	1 x 10 -141
	KYERRC001 08-H05		One	CYP98A59
CYP716	KYERRC001_23-C09	JN604536	7	CYP716A47	CYP716A2 ( Medicagotruncatula )	Oxidation of β-amyrin and erythrodiol at the C-28 position	FN 995 112	6e -169
CYP734	KYERRC001_09-G04		One	CYP734A23	CYP734 A7 (Lycopersiconesculentum)	Brassinosteroid C-26 hydroxylase	AB223042	5e -49
CYP736	KYERRC001_22-F01	JN604539	3	CYP736A12	CYP736B ( Vitisarizonicax Vitisrupestris )	UnknowmDisease Responsive	FJ828518	5e -24
CYP749	KYERRC001_20-C10	JN604538	2	CYP749A20	CYP86A22 ( Petuniaxhybrida )	Fatty acyl-CoA-Hydroxylase	DQ099540	2e -17

(EST sequence homologous to the CYP gene from the root of P. ginseng )

Although the 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. . Meanwhile, CYP716A12, found in Medicago truncatula plants, is a multifunctional gene that makes oleanolic acid from beta-amyrin (β-amyrin and erythrodiol). It is a completely different gene because it has the same sex.

<Example 2>

<2-1> Isolation and Comparison of Cytochrome P450 Protein Sequences

Their nucleotide and expected amino acid sequences were analyzed using the DNASIS program (Hitachi Software Engineering Co.). To analyze the lineage between these gene sequences, amino acid sequences were obtained from EMBL, GenBank and DDBJ sequence data. Multiple sequence alignments were made using the CLUSTAL W program (Thompson, JD et al. (1994) Nucl. Acids Res. 22: 46734680). Phylogenetic analysis of deduced amino acid alignments was performed using a neighbor-joining method using TreeView software (Page, RD (1996) TreeView: an application to display phylogenetic trees on personal computers Comput.Appl.Biosci . 12, 357358). Strength measurement of nodes (nodes) in the tree (tree) was used to bootstrap (bootstrap) analysis of 1000 replicon Kate (replicates) (Felsenstein, J. ( 1985) Confidence limits on phylogenies:. An approach using the bootstrap Evolution 39: 783791).

<2-2> System Analysis Results

P. ginsengWe analyzed the flexible relationship between the nine potential CYP genes (e.g., CYP72A219, CYP73A100, CYP736A12, CYP82H23, CYP82D47, CYP71D312, CYP71D313, CYP749A22, and CYP716A47) of CYP genes (see FIG. 2). , The amino acid sequence deduced from CYP716A53v2 is 44% of the CYP716A47 gene,Medicago truncatulaOrigin Only 53% homology with that of CYP716A12.

<Example 3>

Ectopic Expression of CYP716A53v2 cDNA in WAT21 Yeast

<3-1> Expression of CYP716A53v2 in Yeast

To prepare expression plasmid vectors for yeast, open reading frames (ORFs) were amplified using PCR from the cDNA of CYP716A53v2. PCR was carried out 25 cycles of 94 ℃ 40 seconds, 55 ℃ 40 seconds and 72 ℃ 2 minutes, using 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:

For CYP716A53v2 Synthetase,

5′-ATG GAT CTC TTT ATC TCA TCT CAA-3 ′ (SEQ ID NO: 3) and

5′-TTA AAG CGT ACA AGG TGA TAG ACG-3 ′ (SEQ ID NO: 4)

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).

Culture conditions and methods for induction by galactose and preparation of the triterpene monoalcohol fraction are known, except that protopanaxadiol (50 mg L- ¹ ) is added to the medium as a substrate. Methods were followed (Kushiro, T., Ohno, Y., Shibuya, Y., Ebizuka, Y. (1997) In vitro conversion of 2,3-oxidosqualene into dammarenediol by Panax ginseng microsomes.Biol . Pharm. Bull. 20: 292294). After 1 day of galactose induction, cells were collected by centrifugation at 500 g for 5 minutes and refluxed with 2 ml 20% KOH / 50% EtOH for 5 minutes. After extraction with the same volume of hexane, the extract was analyzed by LC / APCIMS.

<3-2> LC / APCIMS Analysis of Recombinant Yeast

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.

<3-3> Biosynthesis Analysis of Protoparnaxatriol

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. In order to measure the hydroxylation activity of CYP716A53v2 in the production of ProtoPanaxatriol, 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).

Since protopanaxadiol was applied exogenously to yeast, both protopanaxadiol and protopanaxatriol were measured in total ion chromatograms from LC / APCIMS (Fig. 3B). Retention times of the standard ProtoPanaxadiol and ProtoPanaxtriol peaks were 33.6 minutes and 19.2 minutes, respectively (Figures 3C, 3D). Both ProtoPanaxadiol and ProtoPanaxtriol were identified in CYP716A53v2-expressing yeast using LC / APCI-MS (FIG. 3B). In the control yeast extract with an empty vector, the peak of the exogenously added protopanaxadiol was detected, but the protopanaxanatriol signal was not detected (FIG. 3A).

In yeast expressing the CYP716A53v2 gene, the protofanaxatriol signal was analyzed using MS fragmentation patterns at a retention time of 19.2 minutes (FIG. 4). In 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). These results show that CYP716A53v2 is a protopanaxadiol 6-hydroxylase gene and converts protoparanadiol to protopanaxtriol.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> KNU-Industry Cooperation Foundation

<120> Genes for the Biosynthesis of Protopanaxatriol and Composition

         for Promoting and Activating Biosynthesis of Protopanaxatriol

<130> OP13-1014

<150> KR 12/061070

<151> 2012-06-07

<160> 4

<170> KopatentIn 2.0

<210> 1

<211> 1560

<212> DNA

<213> Panax ginseng

<400> 1

gaccactcac caccttcatt ttcatttctg aattttgggc aaaatcacag aattcttgat 60

ccatggatct ctttatctca tctcaactac ttcttctact agtcttttgc ttattcctct 120

tttggaattt caaaccaagt agccaaaaca aactgcctcc gggcaaaaca ggatggccca 180

taattggaga aacactagaa ttcatctcct gtggccaaaa aggcaaccct gaaaagttcg 240

taacacaaag aatgaacaaa tactcccctg atgtcttcac aacatcctta gcaggcgaga 300

aaatggtagt tttctgcggt gcctcgggga acaaattcat tttctccaac gaaaacaagc 360

ttgttgtgtc ctggtggccc cctgccatat ccaaaatcct aactgcaaca ataccttcgg 420

tagagaaaag caaagccttg cggagtctaa ttgttgaatt cttaaaaccc gaagcgctcc 480

acaagtttat ttctgtcatg gatcggacaa cgaggcagca ctttgaagac aaatggaacg 540

ggagtacaga agtgaaagct ttcgctatgt cagagtcgct gacttttgag ttggcctgtt 600

ggctgctctt tagcataaat gatccggtgc aggtgcagaa gctttctcat ctttttgaga 660

aggttaaagc gggattattg tctttacctt taaattttcc gggcacggct tttaaccgtg 720

ggatcaaggc cgccaatctt attagaaaag agctttcggt ggtgataaaa cagaggagaa 780

gtgataaatt acagactcga aaggatcttt tgtcccacgt tatgctttcc aatggcgagg 840

gcgagaaatt tttcagcgaa atggatattg cggacgttgt tcttaattta ctgattgcta 900

gccatgatac cactagcagt gccatgggct ctgtggtcta ctttcttgca gatcatcctc 960

acatctatgc taaagttctc acagaacaaa tggagatcgc aaagtcgaaa ggggcagaag 1020

aacttttgag ctgggaggac ataaagagga tgaagtattc ccgcaatgtt ataaatgaag 1080

ctatgagatt agtacctcct tctcaaggag gttttaaagt agttacaagt aaattcagtt 1140

acgcaaactt catcattccc aaaggatgga agatcttttg gagcgtatac tcgacacata 1200

aagatcccaa atactttaaa aatccagagg agtttgatcc ttcaagattt gaaggagatg 1260

gacctatgcc attcacattt ataccatttg gaggaggacc aaggatgtgc cctgggagtg 1320

agtttgctcg tctggaggta ctaatattca tgcaccattt ggttaccaat tttaagtggg 1380

agaaggtgtt tcccaatgaa aagattattt atactccatt tcccttcccg gagaatggtc 1440

ttcctattcg tctatcacct tgtacgcttt aattttttgg cttttgttaa ggtgaacatt 1500

ttctaatttg tattaatggt gttcatgttt gtgtgatgta ttcaawttat aatgtttcac 1560

                                                                        1560

<210> 2

<211> 469

<212> PRT

<213> Panax ginseng

<400> 2

Met Asp Leu Phe Ile Ser Ser Gln Leu Leu Leu Leu Leu Val Phe Cys

  1 5 10 15

Leu Phe Leu Phe Trp Asn Phe Lys Pro Ser Ser Gln Asn Lys Leu Pro

             20 25 30

Pro Gly Lys Thr Gly Trp Pro Ile Ile Gly Glu Thr Leu Glu Phe Ile

         35 40 45

Ser Cys Gly Gln Lys Gly Asn Pro Glu Lys Phe Val Thr Gln Arg Met

     50 55 60

Asn Lys Tyr Ser Pro Asp Val Phe Thr Thr Ser Leu Ala Gly Glu Lys

 65 70 75 80

Met Val Val Phe Cys Gly Ala Ser Gly Asn Lys Phe Ile Phe Ser Asn

                 85 90 95

Glu Asn Lys Leu Val Val Ser Trp Trp Pro Pro Ala Ile Ser Lys Ile

            100 105 110

Leu Thr Ala Thr Ile Pro Ser Val Glu Lys Ser Lys Ala Leu Arg Ser

        115 120 125

Leu Ile Val Glu Phe Leu Lys Pro Glu Ala Leu His Lys Phe Ile Ser

    130 135 140

Val Met Asp Arg Thr Thr Arg Gln His Phe Glu Asp Lys Trp Asn Gly

145 150 155 160

Ser Thr Glu Val Lys Ala Phe Ala Met Ser Glu Ser Leu Thr Phe Glu

                165 170 175

Leu Ala Cys Trp Leu Leu Phe Ser Ile Asn Asp Pro Val Gln Val Gln

            180 185 190

Lys Leu Ser His Leu Phe Glu Lys Val Lys Ala Gly Leu Leu Ser Leu

        195 200 205

Pro Leu Asn Phe Pro Gly Thr Ala Phe Asn Arg Gly Ile Lys Ala Ala

    210 215 220

Asn Leu Ile Arg Lys Glu Leu Ser Val Val Ile Lys Gln Arg Arg Ser

225 230 235 240

Asp Lys Leu Gln Thr Arg Lys Asp Leu Leu Ser His Val Met Leu Ser

                245 250 255

Asn Gly Glu Gly Glu Lys Phe Phe Ser Glu Met Asp Ile Ala Asp Val

            260 265 270

Val Leu Asn Leu Leu Ile Ala Ser His Asp Thr Thr Ser Ser Ala Met

        275 280 285

Gly Ser Val Val Tyr Phe Leu Ala Asp His Pro His Ile Tyr Ala Lys

    290 295 300

Val Leu Thr Glu Gln Met Glu Ile Ala Lys Ser Lys Gly Ala Glu Glu

305 310 315 320

Leu Leu Ser Trp Glu Asp Ile Lys Arg Met Lys Tyr Ser Arg Asn Val

                325 330 335

Ile Asn Glu Ala Met Arg Leu Val Pro Pro Ser Gln Gly Gly Phe Lys

            340 345 350

Val Val Thr Ser Lys Phe Ser Tyr Ala Asn Phe Ile Ile Pro Lys Gly

        355 360 365

Trp Lys Ile Phe Trp Ser Val Tyr Ser Thr His Lys Asp Pro Lys Tyr

    370 375 380

Phe Lys Asn Pro Glu Glu Phe Asp Pro Ser Arg Phe Glu Gly Asp Gly

385 390 395 400

Pro Met Pro Phe Thr Phe Ile Pro Phe Gly Gly Gly Pro Arg Met Cys

                405 410 415

Pro Gly Ser Glu Phe Ala Arg Leu Glu Val Leu Ile Phe Met His His

            420 425 430

Leu Val Thr Asn Phe Lys Trp Glu Lys Val Phe Pro Asn Glu Lys Ile

        435 440 445

Ile Tyr Thr Pro Phe Pro Phe Pro Glu Asn Gly Leu Pro Ile Arg Leu

    450 455 460

Ser Pro Cys Thr Leu

465

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> CYP716A53v2 forward primer

<400> 3

atggatctct ttatctcatc tcaa 24

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> CYP716A53v2 reverse primer

<400> 4

ttaaagcgta caaggtgata gacg 24

Claims (11)

1. A composition for promoting protopanaxatriol biosynthesis comprising a CYP716A53v2 protein or a CYP716A53v2 gene encoding the same.
2. The method of claim 1,

   The CYP716A53v2 gene is a composition for promoting protopanaxatriol biosynthesis, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
3. The method of claim 1,

   CYP716A53v2 Protein is a composition for promoting protopanaxatriol biosynthesis, characterized in that consisting of the amino acid sequence of SEQ ID NO: 2.
4. The method of claim 1,

   The composition is a composition for promoting protopanaxatriol biosynthesis, characterized in that it comprises a recombinant vector or plasmid containing the CYP716A53v2 gene.
5. The method of claim 1,

   The CYP716A53v2 protein is protopanaxadiol 6-hydroxylase (protopanaxadiol 6-hydroxylase), characterized in that the composition for promoting protopanaxatriol biosynthesis.
6. A host cell transformed with the recombinant vector or plasmid of claim 4.
7. The method of claim 6,

   The host cell is transformed host cell, characterized in that yeast or E. coli.
8. Transgenic plant transformed with the recombinant vector or plasmid of claim 4.
9. A method of increasing the production of CYP716A53v2 protein consisting of the amino acid sequence of SEQ ID NO: 2 or CYP716A53v2 gene encoding the same, thereby increasing the production of protopanaxatriol.
10. The method according to claim 9,

    The method comprises the step of transforming the host with the recombinant vector or plasmid of claim 4, overexpressing the CYP716A53v2 gene or protein.
11. The method of claim 10,

    Said host is yeast, Escherichia coli or plant.

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