WO2015167282A1

WO2015167282A1 - A novel method for glycosylation of ginsenoside using a glycosyltransferase derived from panax ginseng

Info

Publication number: WO2015167282A1
Application number: PCT/KR2015/004399
Authority: WO
Inventors: Sun Chang Kim; Gil Tsu Choi; Suk Chae Jung; Myung Keun Park; Woo Hyun Kim; Soo Hwan Lim; Wan Taek Im
Original assignee: Korea Advanced Institute Of Science And Technology; Intelligent Synthetic Biology Center
Priority date: 2014-04-30
Filing date: 2015-04-30
Publication date: 2015-11-05
Also published as: US10174355B2; KR101788608B1; CN106459987B; US20170121750A1; JP6526716B2; JP2017514480A; CN106459987A; EP3137612A1; EP3137612A4; KR20150125902A

Abstract

The present invention relates to a uridine diphosphate (UDP)-glycosyltransferase protein which has glycosylation activity for a hydroxyl group at the C-20 position of a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside, and a method for glycosylation of UDP using the same.

Description

A NOVEL METHOD FOR GLYCOSYLATION OF GINSENOSIDE USING A GLYCOSYLTRANSFERASE DERIVED FROM PANAX GINSENG

The present invention relates to a uridine diphosphate (UDP) glycosyltransferase protein which has glycosylation activity for a hydroxyl group located at the C-20 position of a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside, and a method for UDP glycosylation using the same.

Ginseng is one of the most popular medicinal plants widely used for improving health. The root of ginseng has been consumed as a herbal tea in the traditional medicine and is currently used in a variety of products including candies, instant teas, and tonic drinks. Ginsenosides, which are glycosylated triterpene compounds contained in ginseng, may provide many positive effects on health. In particular, the ginsenosides have been known to have various pharmacological effects such as enhancement of the immune system and revitalization of the body function, and more than 40 different ginsenosides have been identified from the root of ginseng. However, the difficulty in large-scale production of the individual ginsenosides remains as a major obstacle to investigation of the efficacy of each ginsenoside, e.g., its therapeutic effect on specific diseases, and commercial use of the identified ginsenosides.

Ginsenosides are glycosylated dammarene-type tetracyclic triterpenes and can be classified into three different groups based on their aglycone structures: protopanaxadiol (PPD)-type ginsenosides, protopanaxatriol (PPT)-type ginsenosides and oleanolic acid-type ginsenosides. These three groups can be further classified based on the position and number of sugar moieties (aglycones) attached to the C-3, C-6 and C-20 positions of the rings in the chemical structure by a glycosidic bond. PPDs and PPTs have different hydroxylation patterns. While the PPDs have -OH groups at the C-3, C-12 and C-20 positions, the PPTs have -OH groups at the C-3, C-6, C-12 and C-20 positions. The PPDs and PPTs can be glycosylated with glucose or other sugars to be converted into various ginsenosides. The glycosylated PPD-type ginsenosides include ginsenosides Rb₁, Rd, F2, Rg₃, Rh₂, compound K (C-K), Rb₂, Rc, compound MC (C-MC), compound Y (C-Y), etc., and the glycosylated PPT-typeginsenosidesincludeginsenosides Rg₁, Rh₁, F1, Rf, Re, Rg₂, etc.

The biosynthetic pathway of ginsenosides has been only partially identified. The ginsenosides are known to partially share the biosynthetic pathway with other triterpenes until oxidosqualene is synthesized by a series of condensation reactions of isopentenyl diphosphate and dimethylallyl diphosphate (DMADP) by the actions of IPP isomerase (IPI), GPP synthase (GPS), FPP synthase (FPS), squalene synthase (SS), and squalene epoxidase (SE) (Ajikumar et al. Science, 330, 70-74. 2010; Ro et al. Nature, 440, 940-943. 2006; Sun et al. BMC Genomics, 11, 262, 2010). Oxidosqualene is cyclized into dammarenediol-II by dammarenediol-II synthase (DS) which is a triterpene cyclase. Dammarenediol-II has hydroxyl groups at the C-3 and C-20 positions, and is converted to PPD by hydroxylation of the C-12 position by the p450 enzyme protopanaxadiol synthase (PPDS). PPDS can also be converted to PPT by hydroxylation at the C-6 position by another p450 protein, protopanaxatirol synthase (PPTS). PPD can be converted to various PPD-type ginsenosides by glycosylation at the C-3 and/or C-20 position(s), and PPT can be converted to various PPT-type ginsenosides by glycosylation at the C-6 and/or C-20 position(s).

Uridine diphosphate (UDP) glycosyltransferase (UGT) is an enzyme that catalyzes the transfer of a sugar moiety from a UDP-sugar to a wide variety of metabolites such as hormones and secondary metabolites. In general, UGT acts in the final step of the biosynthetic pathway in order to increase the solubility, stability, storage, biological activity or biological availability of metabolites. As recognized by the remarkable diversity of plant metabolites, each plant genome possesses hundreds of different UGTs. For example, a thale cress (Arabidopsis thaliana) plant model contains 107 UGTs that belong to 14 different groups (groups A to N) based on the amino acid sequences. However, although DS, PPDS, and PPTS have been reported as the enzymes involved in ginsenoside biosynthesis, little is known about whether UGT is involved in ginsenoside biosynthesis. Therefore, for production of specific ginsenosides, it is necessary to identify the UGTs, which use ginsenosides as substrates.

Different UGTs exhibit substrate specificity towards both sugar donors and sugar acceptors. For example, UGT78D2 transfers glucose from UDP-glucose to the C-3 position of a flavonol (kaempferol or quercetin) and an anthocyanin (cyanidin) in order to produce flavonol-3-O-glucosides and cyanidin-3-O-glucoside, respectively. It seems that such glycosylation is essential for in-vivo stability and storage of the compounds. On the other hand, UGT89C1 transfers rhanmnose from UDP-rhanmnose to the C-7 position of flavonol-3-O-glucoside in order to produce flavonol-3-O-glucoside-7-O-rhamnoside. Since UGT89C1 does not utilize UDP-glucose and anthocyanin-3-O-glucoside as substrates, it is known to exhibit different specificity towards the UDP-sugar and other acceptors from that of UGT78D2. As described, because different UGTs may have different substrate specificity and regioselectivity, it is necessary to investigate the substrate specificity and regioselectivity of the individual UGTs.

The inventors of the present invention have made a lot of efforts to develop a novel UDP glycosyltransferase having substrate specificity and regioselectivity that can be used for biosynthesis of a particular ginsenoside. As a result, they have identified a novel glycosyltransferase, PgUGT71A1, from Korean ginseng and found out that the PgUGT71A1 can convert the PPD-type ginsenosides PPD, Rh₂ and Rg₃ to the ginsenosides C-K, F2, and Rd, respectively, and can convert PPT to F1. Since the protein has a glycosylation activity for a hydroxyl group at the C-20 position of PPD- and PPT-type ginsenosides, the inventors of the present invention have confirmed that it can be used to produce particular glycosylated ginsenosides and have completed the present invention.

An objective of the present invention is to provide a method for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, comprising treating a UDP glycosyltransferase protein, a transformed cell which is introduced with a vector containing a polynucleotide encoding the protein or a fragment thereof and exhibits activity of the protein, an organism which contains the transformed cell, or a culture of the transformed cell to a PPD- or PPD- type ginsenoside having a hydroxyl group at the C-20 position.

Another objective of the present invention is to provide a composition for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, which comprises one or more selected from the goup consisting of a uridine diphosphate (UDP) glycosyltransferase protein having glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside having the hydroxyl group at the C-20 position, a transformed cell which is introduced with a vector containing a polynucleotide encoding the protein or a fragment thereof, an organism which contains the transformed cell, or a culture of the transformed cell as an active ingredient.

Still another objective of the present invention is to provide a UDP glycosyltransferase protein defined by the amino acid sequence of SEQ ID NO: 1, which has a selective glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside.

Still another objective of the present invention is to provide a polynucleotide which encodes the protein, an expression vector which contains the polynucleotide, a transformed cell which contains the expression vector or a fragment thereof, and an organism which contains the transformed cell.

Still another objective of the present invention is to provide an expression vector which contains polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, and AtCPR proteins, respectively; a transformed cell for producing C-K which contains the expression vector or a fragment thereof; and an organism which contains the transformed cell.

Still another objective of the present invention is to provide an expression vector which contains polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, AtCPR, and PPTS proteins, respectively; a transformed cell for producing F1 which contains the expression vector or a fragment thereof; and an organism which contains the transformed cell.

Still another objective of the present invention is to provide an expression vector which contains polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, AtCPR, and PgUGT74A1 proteins, respectively; a transformed cell for producing F2 which contains the expression vector or a fragment thereof; and an organism which contains the transformed cell.

Still another objective of the present invention is to provide an expression vector which contains polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, AtCPR, PgUGT74A1 and PgUGT94B1 proteins, respectively; a transformed cell for producing Rd which contains the expression vector or a fragment thereof; and an organism which contains the transformed cell.

Since the UDP glycosyltransferase of the present invention is a protein having an activity of selectively transferring a sugar to a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside, it may be usefully used for large-scale production of a ginsenoside having a sugar at the C-20 position, such as C-K, F2, Rd, and F1.

Additionally, in the present invention, the expression vector, and the transformed cell including the expression vector may be usefully used for large-scale production of particular ginsenosides by de novo synthesis of ginsenoside C-K (amount of novel synthesis; 4.5 mg/L), F1, F2, or Rd having a sugar at C-20 position.

Fig. 1 shows the chemical structures of PPD- and PPT-type ginsenosides.

Fig. 2 shows that PgUGT71A1, a UDP glycosyltransferase, has a glycosylation activity of attaching UDP-glucose to a hydroxyl group at the C-20 position of PPD-type and PPT-type ginsenosides. Specifically, a result of analyzing the conversion of PPD to compound K (C-K), Rg₃ to Rd, Rh₂ to F2, and PPT to F1 by PgUGT71A1 through thin-layer chromatography (TLC; top) and high performance liquid chromatography (HPLC; bottom) is shown.

Fig. 3 shows the results of demonstrating the increased expression of the PgUGT71A1 of the present invention in the leaves and roots of ginseng treated with methyl jasmonate (MeJA) via real-time PCR.

Fig. 4 shows the results of demonstrating the increased expression of the PgUGT71A1 of the present invention in the leaf and root of ginseng treated with methyl jasmonate (MeJA) through NGS.

Fig. 5 shows a prepared platform plasmid called pRS306-MET3p-ERG7-CYC1ter(A), and the results of PCR amplification of a cassette from URA3 region to CYC1 terminator using ERG7 knock down-F and ERG7 knock-down-B primers(B).

Fig. 6 shows a biosynthetic pathway of C-K in S. cerevisiae MET3p-ERG7 by introduction of pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, and pRS423-C-K containing PgUGT71A1.

Fig. 7 shows the results of HPLC and LC-MS/MS analyses of demonstrating that the formation of a new peak at 11.97 min, which is identical to the retention time of C-K in HPLC, by the substances produced in a yeast containing PgUGT71A1(A), and the retention time of C-K produced by a yeast, analyzed by LC-MS/MS analysis(B).

Fig. 8 shows a biosynthetic pathway of ginsenoside F1 in S. cerevisiae MET3p-ERG7 by introduction of pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, pRS423-F1 containing PgUGT71A1 and PgPPTS.

Fig. 9 shows the results of HPLC and LC-MS/MS analyses of demonstrating that the formation of a new peak at 6.01 min, which is identical to the retention time of F1 in HPLC, by the substances produced in a yeast containing PgUGT71A1 and PgPPTS(A), and the retention time of F1 produced by a yeast, analyzed by LC-MS/MS analysis(B).

Fig. 10 shows a biosynthetic pathway of ginsenoside F2 in S. cerevisiae MET3p-ERG7 by introduction of pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, pRS423-F2 containing PgUGT74A1 and PgUGT71A1.

Fig. 11 shows the results of HPLC and LC-MS/MS analyses of demonstrating that the formation of a new peak at 7.81 min, which is identical to the retention time of F2 in HPLC, by the substances produced in a yeast containing PgUGT74A1 and PgUGT71A1(A), and the retention time of F2 produced by a yeast, analyzed by LC-MS/MS analysis(B).

Fig. 12 shows a biosynthetic pathway of ginsenoside Rd in S. cerevisiae MET3p-ERG7 by introduction of pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, pRS423-Rd containing PgUGT74A1, PgUGT94B1 and PgUGT71A1.

Fig. 13 shows the results of HPLC and LC-MS/MS analyses of demonstrating that the formation of a new peak at 5.88 min, which is identical to the retention time of Rd in HPLC, by the substances produced in a yeast containing PgUGT74A1, PgUGT94B1, and PgUGT71A1(A), and the retention time of Rd produced by a yeast, analyzed by LC-MS/MS analysis(B).

In an aspect, the present invention provides a method for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, comprising treating a UDP glycosyltransferase protein, a transformed cell which is introduced with a vector containing a polynucleotide encoding the protein or a fragment thereof and exhibits activity of the protein, an organism which contains the transformed cell, or a culture of the transformed cell to a PPD- or PPD- type ginsenoside having a hydroxyl group at the C-20 position.

In the present invention, the term, "uridine diphosphate (UDP) glycosyltransferase" refers to an enzyme that has an activity of transferring a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor, in particular, an enzyme that utilizes a UDP-sugar as a glycosyl donor. In the present invention, the term UDP glycosyltransferase may be used interchangeably with 'UGT'. Since little is known about the ginsenoside UDP glycosyltransferases and since different enzymes having UDP glycosyltransferase activity have different substrate specificity and regioselectivity, it needs to be determined whether the enzyme is a UDP glycosyltransferase which specifically acts on a particular ginsenoside which is a ginseng saponin.

The inventors of the present invention have identified a novel UDP glycosyltransferase derived from ginseng (Panax ginseng C. A. Meyer), which is capable of selectively transferring a sugar moiety to a hydroxyl (-OH) group at the C-20 position of a PPD-type ginsenoside or a PPT-type ginsenoside, for the first time. The UDP glycosyltransferase identified by the inventors of the present invention has an activity of selectively transferring the sugar moiety of UDP-glucose to a hydroxyl group at the C-20 position of a PPD-type ginsenoside or a PPT-type ginsenoside. Accordingly, the UDP glycosyltransferase identified in the present invention can convert the PPD-type ginsenosides PPD, Rh₂ and Rg₃ to the ginsenosides C-K, F2 and Rd, respectively, and can also convert PPT to F1 by transferring a sugar moiety to the hydroxyl group at the C-20 position. A ginsenoside UDP glycosyltransferase having such activity has never been known and has been first identified by the inventors of the present invention.

The UDP glycosyltransferase identified in the present invention is a UDP glycosyltransferase derived from Korean ginseng (Panax ginseng C. A. Meyer) and may be defined by the amino acid sequence of SEQ ID NO: 1. In an exemplary embodiment of the present invention, the UDP glycosyltransferase defined by the amino acid sequence of SEQ ID NO: 1 is designated as 'PgUGT71A1'.

The UDP glycosyltransferase of the present invention may refer to not only a protein having the amino acid sequence of SEQ ID NO: 1 but also a protein having an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more v98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 1 and being capable of substantially transferring a sugar to a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside, without limitation. In addition, if the protein having the sequence similarity has substantially the same or comparable biological activity as UDP glycosyltransferase, a variant of the protein which has a portion of the sequence deleted, modified, substituted or added is included in the scope of the present invention.

The term "similarity" is intended to indicate the degree of similarity to the amino acid sequence of a wild-type protein or a nucleotide sequence that encodes the same, and includes sequences having similarity of the above-described percentage or higher to the amino acid sequence or nucleotide sequence of the present invention. Comparison of the similarity may be conducted with the naked eye or using a commercially readily available comparison program.

Since the UDP glycosyltransferase of the present invention has an activity of selectively transferring a sugar to the C-20 position of a PPD- or PPT-type ginsenoside, specifically to the C-20 position having a hydroxyl group, it may be used to prepare a glycosylated ginsenoside from a PPD- or PPT-type ginsenoside having a hydroxyl group at the C-20 position.

In the present invention, the term "PPD-type ginsenoside" refers to a dammarane-type saponin, which is a PPD having -OH groups at the C-3, C-12 and C-20 positions or a ginsenoside having the -OH groups of the PPD glycosylated. Examples of the PPD-type ginsenoside that can be glycosylated by the glycosyltransferase of the present invention include PPD, Rg₃, Rh₂, etc. having a hydroxyl group at the C-20 position. However, any PPD-type ginsenoside that can be glycosylated by the UDP glycosyltransferase of the present invention may be included without limitation.

In the present invention, the term "PPT-type ginsenoside" refers to a dammarane-type saponin, which is a PPT having -OH groups at the C-3, C-6, C-12 and C-20 positions or a ginsenoside having the -OH groups of the PPT glycosylated. Examples of the PPT-type ginsenoside that can be glycosylated by the glycosyltransferase of the present invention include a PPT having a hydroxyl group at the C-20 position and not having a sugar at the C-6 position. However, any PPT-type ginsenoside that can be glycosylated by the UDP glycosyltransferase of the present invention is included without limitation.

The PPD-type or PPT-type ginsenoside may be an isolated and purified ginsenoside, or a ginsenoside included in a powder or extract of ginseng or red ginseng. That is, a powder or extract of ginseng or red ginseng containing a saponin may be used directly as a ginsenoside to carry out the method of the present invention. Alternatively, a chemically synthesized ginsenoside may be used. Various types of known ginseng may be used in the present invention. Examples include Korean ginseng (Panax ginseng), American ginseng (P. quinquefolius), notoginseng (P. notoginseng), Japanese ginseng (P. japonicus), dwarf ginseng (P. trifolium), Himalayan ginseng (P. pseudoginseng) and Vietnamese ginseng (P. vietnamensis), although not being limited thereto. The chemical structure of the PPD-type or PPT-type ginsenoside is shown in Fig. 1.

In the present invention, the term "glycosylated ginsenoside" refers to a ginsenoside having a monosaccharide or a higher saccharide attached to the hydroxyl group of a non-sugar component (aglycone) that constitutes the ginsenoside. For the purpose of the present invention, the glycosylated ginsenoside includes any glycosylated ginsenoside without limitation as long as it is a ginsenoside glycosylated as a sugar, preferably glucose, is transferred to the C-20 position of the PPD- or PPT-type ginsenoside by the UDP glycosyltransferase of the present invention. Examples include compound K (C-K), F2, Rd or PPT glycosylated by the glycosyltransferase of the present invention from PPD, Rh₂, Rg₃ or F1, respectively, although not being limited thereto.

For preparing a glycosylated ginsenoside by converting a PPD- or PPT-type ginsenoside having a hydroxyl group at the C-20 position, a transformed cell which contains an expression vector containing a polynucleotide encoding the UDP glycosyltransferase or a fragment thereof and exhibits activity of the UDP glycosyltransferase, an organism which contains the transformed cell, or a culture of the transformed cell may be used.

The polynucleotide encoding the UDP glycosyltransferase protein may be preferably a polynucleotide defined by a nucleotide sequence of SEQ ID NO: 2. In addition to the polynucleotide having a nucleotide sequence of SEQ ID NO: 2, any polynucleotide with a nucleotide sequence having a sequence similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, most preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 2 and being capable of substantially encoding a protein having activity of the PgUGT71A1 protein is included, without limitation.

The expression vector containing the polynucleotide of the present invention refers to a nucleic acid construct containing essential regulatory elements which are operably linked to express an inserted nucleic acid, as an expression vector capable of expressing the target protein in a suitable host cell. The desired target protein can be obtained by transforming or transfecting the prepared recombinant vector into the host cell.

The expression vector containing the polynucleotide provided by the present invention includes E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24, and YCp50) and Ti plasmids that may be used in Agrobacterium-mediated transformation, although not particularly limited thereto. Specific examples of phage DNAs include λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, an animal virus such as retrovirus, adenovirus or vaccinia virus, an insect virus such as baculovirus, a double-stranded plant virus (e.g.,, CaMV), a single-stranded virus, or a viral vector derived from geminivirus may be used.

Moreover, as the vector of the present invention, a transcriptional activator protein (e.g., B42)-linked fusion plasmid (e.g., pJG4-5) may be used. In addition, for easier purification of the target protein obtained in the present invention, the plasmid vector may further include other sequences, if necessary. For example, the fusion plasmid may contain a tag such as GST, GFP, His-tag, Myc-tag, etc., although not limited thereto. In an exemplary embodiment of the present invention, a pGEX4T-1 vector which is a GST gene-fused vector may be used to construct the expression vector which contains the polynucleotide encoding the UDP glycosyltransferase protein.

In addition, a fusion protein expressed by a vector containing a fusion sequence may be purified by affinity chromatography. For example, when a glutathione S-transferase is fused, glutathione which is a substrate of the enzyme may be used. And, when hexahistidine is fused, the target protein may be easily recovered by using a Ni-NTA His-bind resin column (Novagen, USA).

In order to insert the polynucleotide of the present invention into the vector, a purified DNA may be cleaved using appropriate restriction enzymes and then inserted into the restriction sites or cloning sites of a suitable vector DNA.

The polynucleotide encoding the UDP glycosyltransferase of the present invention protein may be operably linked to the vector. The vector of the present invention may further contain, in addition to a promoter and the nucleic acid of the present invention, a cis element such as an enhancer, a splicing signal, a poly-A addition signal, a selection marker, a ribosome-binding sequence (SD sequence), etc. As examples of the selection marker, a chloramphenicol resistance gene, a chloramphenicol resistance gene, dihydrofolate reductase, a neomycin resistance gene, etc. may be used. However, the additional elements to be operably linked are not limited to these examples. In the present invention, the term "transformation" refers to introduction of a DNA into a host cell such that the DNA can be replicated as an extra-chromosomal element or by chromosomal integration. That is, transformation refers to a phenomenon of artificial alteration of genes by introducing a foreign DNA into the cell.

The expression vector which contains the polynucleotide encoding the UDP glycosyltransferase of the present invention protein or a fragment of the expression vector may be introduced into a host cell through transformation. As used herein, a fragment of the expression vector refers to a fragment of the expression vector which contains the portion of the polynucleotide that encodes the UDP glycosyltransferase protein such that the activity of the UDP glycosyltransferase protein can be conferred to the host cell. For example, it may be the T-DNA of a Ti plasmid transferred into a host cell in Agrobacterium-mediated transformation, although not limited thereto.

The transformation of the present invention may be performed by any transformation method and can be easily performed according to the method commonly employed in the art. In general, examples of the transformation method include CaCl₂ precipitation, a Hanahan method which is an improved CaCl₂ method using dimethylsulfoxide (DMSO) as a reducing material, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fiber, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate-, lipofectamine- and desiccation/inhibition-mediated transformation, and so forth. However, the method for transforming the vector containing the polynucleotide which encodes the UDP glycosyltransferase of the present invention is not limited to the above examples, and the transformation or transfection methods typically employed in the art may be used without limitation.

In the present invention, the host cell is not particularly limited as long as it is able to express the polynucleotide of the present invention. Specific examples of the host that can be used in the present invention include bacteria belonging to the genus Escherichia such as E. coli, bacteria belonging to the Bacillus genus such as Bacillus subtilis, bacteria belonging to the Pseudomonas genus such as Pseudomonas putida, yeasts such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, animal cells, plant cells and insect cells. Specific examples of the E. coli strain that can be used in the present invention include CL41 (DE3), BL21 or HB101, and specific examples of the Bacillus subtilis that can be used in the present invention include WB700 or LKS87.

The organism that can contain the transformed cell which is introduced with the expression vector encoding the polynucleotide of the present invention or a fragment thereof may be, for example, tobacco, thale cress, potato, ginseng, sesame, citron, daisy, etc., although not particularly limited thereto.

Any promoter can be used as the promoter of the present invention as long as it enables expression of the nucleic acid of the present invention in the host cell. For example, an E. coli- or phage-derived promoter such as a trp promoter, a lac promoter, a PL promoter or a PR promoter, an E. coli-infecting phage-derived promoter such as a T7 promoter, a CaMV35S promoter, a MAS promoter or a histone promoter may be used. Also, an artificially modified promoter such as a tac promoter may be used.

The transformant wherein the expression vector which contains the polynucleotide encoding the UDP glycosyltransferase of the present invention protein is introduced by transformation has a selective glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside, specifically a glycosylation activity of converting PPD to C-K, converting Rh₂ to F2, converting Rg₃ to Rd or converting PPT to F1, although not limited thereto.

In the present invention, the term "culture of the transformed cell" refers to a product obtained by culturing the transformed cell according to a known method of culturing microorganisms. The term culture is used in a broad concept, including a culture containing the transformed cell and one obtained by removing the transformed cell from the culture containing the transformed cell through, e.g., centrifugation.

Since the culture contains the UDP glycosyltransferase of the present invention protein, it has an activity of converting a PPD- or PPT-type ginsenoside to a glycosylated ginsenoside. For example, it may convert PPD to C-K, convert Rh₂ to F2, convert Rg₃ to Rd, and convert PPT to F1.

Since the UDP glycosyltransferase of the present invention protein, a transformed cell which is introduced with an expression vector containing a polynucleotide encoding the protein or a fragment of the expression vector and exhibits activity of the protein, an organism containing the transformed cell, or a culture of the transformed cell may be used to convert a PPD- or PPT-type ginsenoside having a hydroxyl group at the C-20 position to a glycosylated ginsenoside, the method according to the present invention may be usefully used in the fields where ginsenosides with the C-20 position glycosylated are required, particularly where the ginsenosides such as ginsenosides C-K, F2, Rd, F1, etc., are required.

The inventors of the present invention have identified a novel UDP glycosyltransferase which has an activity of selectively transferring a glucose moiety of UDP-glucose to a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside and has an amino acid sequence of SEQ ID NO: 1 from Korean ginseng (Panax ginseng C. A. Meyer) and have named it as PgUGT71A1 (Example 1). In order to investigate the enzymatic activity of the PgUGT71A1 protein, PPD, Rg₃, Rh₂ and PPT, which are representative PPD- or PPT-type ginsenosides having hydroxyl groups at the C-20 position, were reacted with the PgUGT71A1 of the present disclosure and their conversion activity was determined. As a result, the PgUGT71A1 of the present invention converted PPD to C-K, Rg₃ to Rd, Rh₂ to F2, and PPT to F1, suggesting that it has an activity of converting a glucose moiety to a hydroxyl group at the C-20 position of PPD-and PPT-type ginsenosides (Fig. 2). In addition, it was found out that the expression of the protein is enhanced by methyl jasmonate (MeJA), which is known to be expressed in both the leaf and root of ginseng and to enhance the expression of the biosynthetic genes of ginseng (Figs. 3 and 4). These results suggest that the PgUGT71A1 of the present invention is involved in the biosynthesis of ginsenosides, particularly in the biosynthesis of C-K, Rd, F2, F1, etc., and the protein may be used in a process of bioconverting C-K, Rd, F2, F1, etc., through glycosylation at the C-20 position.

In another aspect, the present invention provides a composition for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, which comprises one or more selected from the goup consisting of a uridine diphosphate (UDP) glycosyltransferase protein having glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside having the hydroxyl group at the C-20 position, a transformed cell which is introduced with a vector containing a polynucleotide encoding the protein or a fragment thereof, an organism which contains the transformed cell, or a culture of the transformed cell as an active ingredient.

The UDP glycosyltransferase protein, the transformed cell, the organism, the PPD-type or PPT-type ginsenoside and the glycosylated ginsenoside are the same as described above.

In another aspect, the present invention provides a UDP glycosyltransferase protein defined by the amino acid sequence of SEQ ID NO: 1, which has glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside.

The UDP glycosyltransferase protein and the amino acid sequence of SEQ ID NO: 1 are the same as described above.

The protein may be one that converts PD to C-K, Rh₂ to F2, Rg₃ to Rd, and PPT to F1.

In another aspect, the present invention provides an expression vector, which further includes polynucleotides encoding dammarenediol-Ⅱ synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), and Arabidopsis thaliana cytochrome p450 reductase (AtCPR) proteins, respectively, in addition to the expression vector including the polynucleotide encoding the UDP glycosyltransferase protein; and a transformed cell for producing C-K including the expression vector or a fragment thereof.

The UDP glycosyltransferase protein, the expression vector, and the transformed cell are the same as described above.

The dammarenediol-II synthase (DS), being a triterpenecyclase, refers to an enzyme which cyclizes oxidosqualene into dammarenediol-II. The DS may be defined by an amino acid sequence described iny SEQ ID NO: 29. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the DS variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 29, and has a biological activity substantially the same as or corresponding to that of the DS.

The polynucleotide encoding the DS protein may be preferably a polynucleotide defined by the nucleotide sequence described in SEQ ID NO: 30 or SEQ ID NO: 31 optimized for codon usage of E. coli. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, which encodes a protein having a biological activity substantially the same as that of the DS protein.

The truncated HMG-CoA reductase (tHMGR) is an important enzyme controlling in vivo synthesis of Z10 or cholesterol, and refers to an enzyme that converts HMG-CoA to mevalonate during the process of cholesterol synthesis. The tHMGR may be defined by an amino acid sequence described in SEQ ID NO: 32. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the tHMGR variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 32, which has a biological activity substantially the same as or corresponding to that of the tHMGR.

The polynucleotide encoding the tHMGR protein may be preferably a polynucleotide defined by the amino acid sequence described in SEQ ID NO: 33. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 33, which encodes a protein having a biological activity substantially the same as that of the tHMGR protein.

The protopanaxadiol synthase (PPDS) is a p450 enzyme which converts the C-12 position of dammarenediol-II to PPD by hydroxylation. The PPDS may be defined by an amino acid sequence described in SEQ ID NO: 34. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the PPDS variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 34, which has a biological activity substantially the same as or corresponding to that of the PPDS.

The polynucleotide encoding the PPDS protein may be preferably a polynucleotide defined by the nucleotide sequence described in SEQ ID NO: 35. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 35, which encodes a protein having a biological activity substantially the same as that of the PPDS protein.

The Arabidopsis thaliana cytochrome p450 reductase (AtCPR) may be used as having the same meaning as cytochrome p450 reductase. The AtCPR may be defined by an amino acid sequence described in SEQ ID NO: 36. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the AtCPR vatiant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 36, which has a biological activity substantially the same as or corresponding to that of the AtCPR.

The polynucleotide encoding the AtCPR protein may be preferably a polynucleotide defined by the nucleotide sequence described in SEQ ID NO: 37 or SEQ ID NO: 38 optimized for codon usage of E. coli. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 37 or SEQ ID NO: 38, which encodes a protein having a biological activity substantially the same as that of the AtCPR protein.

In an exemplary embodiment of the present invention, S. cerevisiae C-K strain having a C-K biosynthesis pathway of C-K was constructed by simultaneously introducing pRS424 DS, pRS426 tHMG1, pRS425 PPD containing PPDS and AtCPR, and pRS423 C-K containing PgUGT71A1 into S. cerevisiae MET3p-ERG7 via lithium acetate method (Fig. 6), and the S. cerevisiae C-K strain was cultured via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. Yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using MtOH and purified using Sep-PAK. Purified ginsenosides were analyzed by HPLC and LC-MS/MS. As a result, the yeast harboring PgUGT71A1 produced a new peak at 11.97 min, which is identical to the retention time of C-K in HPLC((A) of Fig. 7), whereas the observed retention time of products in LC-MS/MS detected by MRM was 11.46 min with the transition at 645.4 → 23.2 ((B) of Fig. 7). These results indicate that the PgUGT71A1 enable the de novo production of ginsenosides through yeast fermentation (4.5 mg/L).

Accordingly, the present invention can provide an expression vector, which further includes polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, and AtCPR proteins, respectively, and a transformed cell for producing C-K including the expression vector or a fragment thereof.

The transformed cell may be a yeast, but is not limited thereto.

In another aspect, the present invention provides an expression vector, which further includes polynucleotides encoding dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), and protopanaxatriol synthase (PPTS) proteins, respectively, in addition to the expression vector including the polynucleotide encoding the UDP glycosyltransferase protein; and a transformed cell for producing F1 including the expression vector or a fragment thereof.

The dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), and Arabidopsis thaliana cytochrome p450 reductase (AtCPR) are the same as described above.

The protopanaxatriol synthase (PPTS) is another p450 protein which converts the C-6 position of PPDS to PPT by hydroxylation. The PPTS may be defined by an amino acid sequence described in SEQ ID NO: 39. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the PPTS variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence of SEQ ID NO: 39, which has a biological activity substantially the same as or corresponding to that of the PPTS.

The polynucleotide encoding the PPTS protein may be preferably a polynucleotide defined by the nucleotide sequence described in SEQ ID NO: 40 or SEQ ID NO: 41 optimized for codon usage of yeast. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence of SEQ ID NO: 40 or SEQ ID NO: 41, which encodes a protein having a biological activity substantially the same as that of the PPTS protein.

In an exemplary embodiment of the present invention, S. cerevisiae F1 strain having a biosynthesis pathway of ginsenoside F1 was constructed by simultaneously introducing pRS424 DS, pRS426 tHMG1, pRS425 PPD containing PPDS and AtCPR, pRS423 F1 containing PgUGT71A1 and PgPPTS into S. cerevisiae MET3p-ERG7 via lithium acetate method (Fig. 8), and the S. cerevisiae F1 strain was cultured via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. Additionally, yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then evaporated BtOH. Finally, ginsenoside F1 was re-extracted by MtOH and the extracted ginsenoside was analyzed by HPLC and LC-MS/MS. As a result, the yeast harboring PgUGT71A1 and PgPPTS produced a new peak at 6.01 min, which is identical to the retention time of F1 in HPLC((A) of Fig. 9), whereas the observed retention time of products in LC-MS/MS detected by MRM was 6.13 min with the transition at 661.5 → 203 ((B) of Fig. 9). These results indicate that the PgUGT71A1 and PgPPTS enable the de novo production of ginsenoside F1 through yeast fermentation.

Accordingly, the present invention can provide an expression vector, which further includes polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, AtCPR, and PPTS proteins, respectively, and a transformed cell for producing F1 including the expression vector or a fragment thereof.

The transformed cell may be a yeast, but is not limited thereto.

In another aspect, the present invention provides an expression vector, which further includes polynucleotides encoding dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), and panax ginseng UDP glycosyltransferase 74A1 (PgUGT74A1) proteins, respectively, in addition to the expression vector including the polynucleotide encoding the UDP glycosyltransferase protein; and a transformed cell for producing F2 including the expression vector or a fragment thereof.

Panax ginseng UDP glycosyltransferase 74A1 (PgUGT74A1) is a UDP glycosyltransferase acting specifically on PPD and C-K, which are PPD-type ginsenosides, to cause O-glycosylation of the C-3 position thereby converting PPD and C-K into Rh₂ and F2, respectively. The PgUGT74A1 may be defined by the amino acid sequence described in KR Pat. No. 10-1479615. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the PgUGT74A1 variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence described in KR Pat. No. 10-1479615, which has a biological activity substantially the same as or corresponding to that of the PgUGT74A1.

The polynucleotide encoding the PgUGT74A1 protein may be preferably a polynucleotide defined by the nucleotide sequence described in KR Pat. No. 10-1479615, and any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence described in KR Pat. No. 10-1479615, which encodes a protein having a biological activity substantially the same as that of the PgUGT74A1 protein.

In an exemplary embodiment of the present invention, S. cerevisiae F2 strain having a biosynthesis pathway of ginsenoside F2 was constructed by simultaneously introducing pRS424 DS, pRS426 tHMG1, pRS425 PPD containing PPDS and AtCPR, pRS423 F2 containing PgUGT74A1 and PgUGT71A1 into S. cerevisiae MET3p-ERG7 via lithium acetate method (Fig. 10), and the S. cerevisiae F2 strain was cultured via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. Additionally, yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then evaporated BtOH. Finally, ginsenoside F2 was re-extracted by MtOH and the extracted ginsenoside was analyzed by HPLC and LC-MS/MS. As a result, the yeast harboring PgUGT74A1 and PgUGT71A1 produced a new peak at 7.81 min, which is identical to the retention time of F2 in HPLC((A) of Fig. 11), whereas the observed retention time of products in LC-MS/MS detected by MRM was 7.35 min with the transition at 807.5 → 627.5 ((B) of Fig. 11). These results indicate that the PgUGT74A1 and PgUGT71A1 enable the de novo production of ginsenoside F2 through yeast fermentation.

Accordingly, the present invention can provide an expression vector, which further includes polynucleotides encoding UDP glycosyltransferase proteins of DS, tHMGR, PPDS, AtCPR, and PgUGT74A1 proteins, respectively, and a transformed cell for producing F2 including the expression vector or a fragment thereof.

The transformed cell may be a yeast, but is not limited thereto.

In another aspect, the present invention provides an expression vector, which further includes polynucleotides encoding dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), panax ginseng UDP glycosyltransferase 74A1 (PgUGT74A1), and panax ginseng UDP glycosyltransferase 94B1 (PgUGT94B1) proteins, respectively, in addition to the expression vector including the polynucleotide encoding the UDP glycosyltransferase protein; and a transformed cell for producing Rd including the expression vector or a fragment thereof.

The dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), and panax ginseng UDP glycosyltransferase 74A1 (PgUGT74A1) are the same as described above.

Panax ginseng UDP glycosyltransferase 94B1 (PgUGT94B1) is a UDP glycosyltransferase acting specifically on Rh₂ and F2, which are PPD-type ginsenosides, to cause β-1,2 glycosylation of O-glucoside located at C-3 position thereby converting Rh₂ and F2, which are ginsenosides, into Rg₃ and Rd, respectively. The PgUGT94B1 may be defined by an amino acid sequence in KR Pat. No. 10-1479608. Any protein variants having a deletion, a modification, a substitution, or an addition in part of their amino acid sequences therein may be included without limitation within the scope of the present invention, as long as the PgUGT94B1 variant has an amino acid sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, even much more preferably 98% or higher, and most preferably 99% or higher, to the amino acid sequence described in KR Pat. No. 10-1479608, which has a biological activity substantially the same as or corresponding to that of the PgUGT94B1.

The polynucleotide encoding the PgUGT94B1 protein may be preferably a polynucleotide defined by the nucleotide sequence described in KR Pat. No. 10-1479608. Any polynucleotide variants having a deletion, a modification, a substitution, or an addition in part of their nucleotide sequences therein may be included without limitation within the scope of the present invention, as long as the variant has an nucleotide sequence having similarity of 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even much more preferably 98% or higher, to the nucleotide sequence described in KR Pat. No. 10-1479608, which encodes a protein having a biological activity substantially the same as that of the PgUGT94B1 protein.

In an exemplary embodiment of the present invention, S. cerevisiae Rd strain having a biosynthesis pathway of ginsenoside Rd was constructed by simultaneously introducing pRS424 DS, pRS426 tHMG1, pRS425 PPD containing PPDS and AtCPR, pRS423 Rd containing PgUGT74A1, PgUGT94B1, and PgUGT71A1 into S. cerevisiae MET3p-ERG7 via lithium acetate method (Fig. 12), and the S. cerevisiae Rd strain was cultured via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. Additionally, yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then evaporated BtOH. Finally, ginsenoside Rd was re-extracted by MtOH and the extracted ginsenoside was analyzed by HPLC and LC-MS/MS. As a result, the yeast harboring PgUGT74A1, PgUGT94B1, and PgUGT71A1 produced a new peak at 5.88 min, which is identical to the retention time of Rd in HPLC((A) of Fig. 13), whereas the observed retention time of products in LC-MS/MS detected by MRM was 5.93 min with the transition at 969.5 → 789.5 ((B) of Fig. 13). These results indicate that the PgUGT74A1, PgUGT94B1, and PgUGT71A1 enable the de novo production of ginsenoside Rd through yeast fermentation.

The present invention is described in detail through examples. However, the following examples are for illustrative purpose only, and the scope of the present invention is not limited by the examples.

Example 1: Cloning and purification of ginseng UDP glycosyltransferase PgUGT71A1

From ginseng cDNAs, genes were amplified by PCR using PgUGT71A1-F (5'-AGGCAGGATCCATGAAGTCAGAATTGATATTCTTGCCCGCCCCGGC-3'; SEQ ID NO: 17) and PgUGT71A1-R (5'-AGGCATCTCGAGTCACATAATTTTCTCAAATAGTTTGGCCAATGAAT-3'; SEQ ID NO: 18) primers and polymerases, and the terminals of the genes were digested with the restriction enzymes BamHI and XhoI. Then, the genes were cloned into a pGEX-4T1 vector to construct an expression vector, which was transformed into the E. coli BL21 (DE3)-RIL strain to obtain a PgUGT71A1-expressing strain.

After inducing the strain to express proteins with IPTG, the resulting protein was purified using sepharose-4B resin to obtain the PgUGT71A1 enzyme.

Example 2: In-vitro enzyme assay

A glycosyltransferase assay was conducted in a reaction buffer (10 mM PBS buffer, pH 7) containing the purified PgUGT71A1 (30 ㎍), a ginsenoside compound (5 mM) and UDP-glucose (50 mM). For this assay, 4 different types of ginsenosides, i.e., protopanaxadiol (PPD), protopanaxatriol (PPT), Rh₂ and Rg₃, were caused to react the enzyme of the present disclosure. The structures of the ginsenosides are shown in Fig. 1.

The reaction mixture was incubated at 35 ℃ for 12 hours and the resulting products were analyzed by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC).

The TLC analysis was performed using a mobile phase (acetone:methanol:DDW = 65:35:10 vol/vol) and a 60F254 silica gel plate (Merck, Germany). The resolved product on the TLC plate was detected by spraying 10% (vol/vol) sulfuric acid (H2SO4) and heating at 110℃ for 5 minutes.

The HPLC analysis was performed using an ODS(2) C18 column (Phenomenex, USA). The condition of gradient application of water and acetonitrile was as follows: flow rate = 1 mL/min, 0 minute, 68% water and 32% acetonitrile; 8 minutes, 35% water and 65% acetonitrile; 12 minutes, 0% water and 100% acetonitrile; 20 minutes, 0% water and 100% acetonitrile; 20.1 minutes, 68% water and 32% acetonitrile; and 28 minutes, 68% water and 32% acetonitrile.

The ginsenosides were detected by monitoring at a wavelength of 203 nm using a UV detector (Agilent, USA).

Example 3: RNA isolation and real-time PCR analysis

Total RNA was isolated from the leaf or root of 15-month-old ginseng using the Spectrum Plant Total RNA kit (Sigma-Aldrich). 200 μM methyl jasmonate (MeJA) was sprayed onto the leaves of ginseng every day for a total of 5 days and samples were collected on the 6th day. 1 ㎍ of the total RNA was used for cDNA synthesis.

The expression level of different genes was examined by quantitative RT-PCR using the primer sets listed in Table 1, and the result was normalized to the expression level of tubulin.

Table 1

Gene	Sequence(5'->3')	SEQ ID NO
PgDS	5'-AAATGAAGAAGGTGGTTGGG-3'	3
PgDS	5'-CTCTATGCAGAGGTGTCGGA-3'	4
PgPPDS	5'-GGGAGGATTTGAGGAAGATGAAG-3'	5
PgPPDS	5'-CAGATGCATCTTCCATCCCTTTGG-3'	6
PgPPTS	5'-GAGATTAGTACCTCCTTCTCAAGG-3'	7
PgPPTS	5'-GAATGGCATAGGTCCATCTCCTTC-3'	8
PgUGT74A1	5'-TATCGAACCCGAACGTACAA-3'	9
PgUGT74A1	5'-GTCGAGTTCCAACCACAATG-3'	10
PgUGT94B1	5'-GACAGAGGATTGGTTGTGGA-3'	11
PgUGT94B1	5'-TCAAAGGCTGATCAAGATGC-3'	12
PgUGT71A1	5'-CCTCCGGATGGATAGTATGATCC-3'	13
PgUGT71A1	5'-CATTGCAATCTCCTTCACCTGG-3'	14
PgTubulin	5'-GAAGGCTTTCTTGCATTGGT-3'	15
PgTubulin	5'-CCCAGATCGTCTTCTTCTCC-3'	16

Example 4: NGS analysis

RNA was extracted from the leaf and root of ginseng which had been treated and not treated with MeJA, and a cDNA library was constructed from 1 μg of the total RNA using the TruSeq RNA library kit. Following polyA-selected RNA extraction, RNA fragmentation and random hexamer-primed reverse transcription, 100 nt paired-end sequencing was conducted using Illumina HiSeq2000.

The resulting sequences were subjected to de novo transcriptome assembly using the Trinity (2011-11-26 version) program. The produced contigs (transcripts) were analyzed using Blast (version 2.2.25+). The GO database (released on 20-04-2012) and the NR database of NCBI (download date: 2012/05/07) were used. To determine the expression level of the assembled contigs (transcripts), Bowtie (version 0.12.8) was used as a mapping program. The expression level was expressed as fragments per kilobase of exon per million fragments mapped (FPKM) with a program released by the Trinity group using the RSEM algorithm.

Example 5: Production of ginsenosides in yeast

Example 5-1: Construction of ERG7 down regulation cassette

In order to construct the ERG7 down regulation strain (S. cereivisae MET3p-ERG7), ERG7 down regulation cassette was constructed. For the ERG7 down regulation cassette carrying MET3 promoter, ERG7 gene, and CYC1 terminator, pRS306 plasmid was used as a platform. To suppress ERG7 gene, MET3 promoter (MET3p), which is capable of suppressing transcription level by supply of methionine, was chosen. MET3p was amplified from genomic DNA of S. cerevisiae CEN.PK 2-1D by PCR amplification with the following primer set: MET3p for pRS306-F (SacI) and MET3p for pRS306-B (XbaI) (Table 2).

Table 2

Primer	Sequence(5'->3')	SEQ ID NO
MET3p (pRS306-F(SacI))	5’- AGGCATGAGCTCTTTAGTACTAACAGAGACTTTTGTCACAACTACA TATAAGTGTACAAATA-3’	19
MET3p (pRS306-B(XbaI))	5’- AGGCATTCTAGATGTTAATTATACTTTATTCTTGTTATTATTATACTTTCT TAGTTCCTTTT-3’	20

ERG7 was amplified from genomic DNA of S. cerevisiae CEN.PK 2-1D by PCR amplification with the following primer set: ERG7 for pRS306-F (SpeⅠ) and ERG7 for pRS306-B (XhoⅠ) (Table 3).

Table 3

Primer	Sequence(5'->3')	SEQ ID NO
ERG7 (pRS306-F(SpeⅠ))	5'-AGGCATACTAGTATGACAGAATTTTATTCTGACACAATCGGTCT-3’	21
ERG7 (pRS306-B(XhoⅠ))	5'-AGGCATCTCGAGTTAAAGCGTATGTGTTTCATATGCCCTGCTGT-3’	22

CYC1 terminator was amplified from pRS424-GPD plasmid by PCR amplification with the following primer set: CYC1 terminator for pRS306-F (XhoI) and CYC1 terminator for pRS306-B (KpnI) (Table 4).

Table 4

Primer	Sequence(5'->3')	SEQ ID NO
CYC1ter (pRS306-F(XhoI))	5'-AGGCATCTCGAGTCATGTAATTAGTTATGTCACGCTTACATTCA CGCCCTC-3’	23
CYC1ter (pRS306-B(KpnI))	5'-AGGCATGGTACCGGCCGCAAATTAAAGCCTTCGAGCGTCCCAAAA CC-3’	24

Each fragment was inserted into the pRS306 vector and thereby constructed a platform plasmid called pRS3063p-MET3p-ERG7-CYC1ter ((A) of Fig. 5).

ERG7 down regulation cassette was constructed via PCR using the pRS3063p-MET3p-ERG7-CYC1ter plasmid as a template. The ERG7 down regulation cassette includes URA3 gene and MET3p-ERG6-CYC1ter as markers. The cassette from URA3 region to CYC1 terminator was amplified by PCR with ERG7 knock down-F and ERG7 knock down-B primers(Table 5) which include homologous arms ((B) of Fig. 5).

Table 5

Primer	Sequence(5'->3')	SEQ ID NO
ERG7 knock down-F	5'-GCCCAATAACCTTACCAATAATCGTCGCCCACAAAGAAAGTAC AAAACAGTCAGAGCAGATTGTACTGAGAGTGCACCACGCTTT-3’	25
ERG7 knock down-B	5’- TGCACTAGTTTCTAATTGTTGCAGCCTCTAACAACACTTATAAATAAAACTCGGAATTAACCCTCACTAAAGGGAACAAAAGCTG-3’	26

Example 5-2: Construction of S. cerevisiae MET3p-ERG7 strain and confirmation of ERG7 down regulation

The constructed ERG7 down regulation cassette was integrated into S. cerevisiae CEN.PK 2-1D genome by lithium acetate method (transformation of yeast by lithium acetate, single-stranded carrier DNA, polyethylene glycol method). Transformants were selected on SD-Leu-Trp-His plates. The selected trnasformans were incubated in 5 ml YPD media for genomic DNA isolation for confirmation of cassette integration by PCR amplification with ERG7 knock down confirm-F and ERG7 knock down confirm-B primers (Table 6). After the replacement, yeast was selected on 5-FOA (5-Fluoroorotic acid) plate to recover URA3 marker and the resulting yeast was used for further analysis.

Table 6

Primer	Sequence(5'->3')	SEQ ID NO
ERG7 knock down confirm-F	5’- GCCCAATAACCTTACCAATAATCGTCGCCCACAAAGAAAGTACAAA ACAG-3’	27
ERG7 knock down confirm-B	5’- TGCACTAGTTTCTAATTGTTGCAGCCTCTAACAACACTTATAAATAAA AC-3'	28

For the confirmation of accumulation of 2,3-oxidosqualene, flask cultivation was performed and metabolites were analyzed by HPLC. Seed cultures for flask cultivation were prepared by inoculating 1 mL of frozen cells in 15% (v/v) glycerol into a 250 mL flask containing 50 mL of SD-TRP-LEU-HIS-URA medium. For flask cultivation, seed cultures were grown for 2 days at 30℃ with an OD 600 of 4~7. 20 mL of seed cultures were inoculated into 1 L of batch medium (2% v/v) in 2L flasks. The cultured cells were harvested and refluxed with 20 mL of 20% KOH/50% EtOH solution aq. for 1 hour. After extraction with the same volume of hexane, the extract was evaporated and re-extracted using 1.5 mL of acetone. The extract was analyzed by HPLC analysis. HPLC analysis was performed using an ODS(2) C18 column (Phenomenex, CA, USA) at a flow rate of 1 mL/min as isocratic method with 100% acetonitrile. The metabolites (squalene, 2,3-oxidosqualene) were monitored at a wavelength of 203 nm with a UV-detector (Agilent Technologies, CA, USA).

Example 5-3: De novo synthesis of ginsenoside in S. cerevisiae

De novo synthesis of ginsenoside was examined by culturing S. cerevisiae harboring ginsenoside biosynthetic genes in a bioreactor. A defined medium, described by van Hoek et al., containing methionine was used for the fermentation (van Hoek, de Hulster et al. 2000; Lenihan, Tsuruta et al. 2008; Westfall, Pitera et al. 2012). The batch culture medium contained 20 g/L of glucose, 15 g/L of (NH₄)₂SO₄, 8 g/L of KH₂PO₄, 0.72 g/L of ZnSO₄·7H₂O, 6.15 g/L of MgSO₄·7H₂O, 12 mL/L of a vitamin solution, 0.3 g/L of methionine, and 10 mL/L of a trace metal solution. The trace metal solution for the medium contained 15 g/L of EDTA, 10.2 g/L of ZnSO₄·7H₂O, 0.50 g/L of MnCl₂·4H₂O, 0.5 g/L of anhydrous CuSO₄, 0.86 g/L of CoCl₂·6H₂O, 0.56 g/L of Na₂MoO₄·2H₂O, 3.84 g/L of CaCl₂·2H₂O, and 5.12 g/L of FeSO₄·7H₂O. The vitamin solution contained 0.05 g/L of biotin, 1 g/L of calcium pantothenate, 1 g/L of nicotinic acid, 25 g/L of myoinositol, 1 g/L of thiamine HCL, 1 g/L of pyridoxol HCl, and 0.2 g/L of 4-aminobenzoicacid. The feeding solution contained 578 g/L of glucose, 9 g/L of KH2PO4, 3.5 g/L of K2SO4, 0.28 g/L of Na2SO4, 5.12 g/L of MgSO4?7H2O, and 1 g/L of methionine. Fed-batch fermentation was performed in a 1.5-LBioreactor (Biotron, Korea) at 30°C with air flow of 1 L/min and 400 rpm agitation. The pH-stat feeding strategy was used for fed-batch fermentation by feeding glucose(>pH5.51) and by adding 5% NH₄OH(<pH5.4).

For the analysis of de novo synthesized ginsenosides, 50ml of cells were harvested by centrifugation (10 min, 2898 Xg), resuspended in 20 mL of DDW, and disrupted by sonication (Vibra-cell; Sonics & Materials, CT, USA). Metabolites were extracted with 50% methanol (v/v), purified on a SEP-PAK 18 cartridge, and analyzed via HPLC and LC-MS/MS.

Example 5-4: HPLC analysis and LC-ESI-MS/MS analysis of ginsenosides

HPLC analyses were performed using an ODS(2) C18 column (Phenomenex, CA, USA) at a flow rate of 1 mL/min as follows: 0 min, 68% water and 32% acetonitrile; 8 min, 35% water and 65% acetonitrile; 12 min, 0% water and 100% acetonitrile; 20 min, 0% water and 100% acetonitrile; 20.1 min, 68% water, and 32% acetonitrile; and 28 min, 68% water and 32% acetonitrile. Ginsenosides were monitored at a wavelength of 203 nm with a UV-detector (Agilent Technologies, CA, USA).

Identification of each ginsenoside was performed using an HPLC-MS/MS system composed of an HPLC System (HP1100; Agilent Technologies), a triple-quadrupole tandem mass spectrometer (API-2000; Applied Biosystems, CA, USA) equipped with an autosampler, a heated electrospray ionization source (H-ESI), a triple-stage quadrupole mass analyzer, and Analyst 1.4 software for data acquisition. A reversed-phase column (Fortis H2o C18, 2.1×100 mm, 3-mm pore size; Fortis Technologies Ltd., UK) was used for sample separation. The mobile phase for chromatographic separation consisted of 0.01% acetic acid aqueous water (A), and 0.01% acetic acid aqueous acetonitrile (B). The gradient elution program, with a constant flow rate of 250 μL/min, was as follows: 0 min, 68% A and 32% B; 3 min, 45% A and 55% B; 8 min, 40% A and 60% B; 13 min, 20% A and 80% B; 18 min, 0% A and 100% B; 22 min, 0% A and 100% B; 22.1 min, 68% A and 32% B; 30 min, 68% A and 32% B. The column temperature was set at 25°C. To detect ginsenoside compounds (PPD, Rh₂, Rg₃, C-K, F2, and Rd), the multiple reaction monitoring (MRM) method was used. The transitions were set at m/z 461.1 → 425.5 for PPD, at m/z 645.3 → 23.2 for Rh₂, at m/z 807.4→365.3 for Rg₃, at m/z 645.4 → 23.2 for C-K, m/z 807.5 → 627.5 for F2, and at m/z 969.3 → 789.4 for Rd, respectively. For full-scan MS analyses, spectra were recorded in the m/z range from 400 to 1,000 according to the transition pattern (Kim, Cui et al. 2012). The MS/MS conditions were optimized by introducing a standard solution of analyte via a syringe pump at 10 μL/min. The ES-MS parameters were as follows: ionspray voltage, 4,200V;

ion source gas

1, 20; curtain gas, 20; and collision gas, 2.

Test Example 1: Specific glycosyltransferase activity for hydroxyl group at C-20 position of PPD-type and PPT-type ginsenosides of PgUGT71A1

The substrate specificity and regioselectivity of the PgUGT71A1 identified in Example 1 were investigated as follows.

First, the recombinant PgUGT of Example 1, PgUGT71A1, was incubated with 9 different types of ginsenosides (PPD, Rh₂, Rg₃, C-K, F2, Rd, PPT, F1, and Rh₁) in the presence of UDP-glucose. It was confirmed by thin-layer chromatography (TLC) that reaction occurred for PPD, Rh₂, Rg₃ and PPT.

In order to confirm the result again, 4 different types of ginsenosides (PPD, PPT, Rh₂, and Rg₃) were incubated with PgUGT71A1, and the products converted by the recombinant PgUGT71A1 were analyzed by TLC. The result is shown at the top of Fig. 2. The result was confirmed by comparing the locations of the migrating spots with those of PPD, PPT, Rh₂, Rg₃, C-K, F2, Rd, and F1 used as standard samples.

As a result, it was confirmed that PgUGT71A1 converted PPD to compound K (C-K), Rg₃ to Rd, Rh₂ to F2, and PPT to F1 (Fig. 2, top).

The result was further confirmed by HPLC, as shown at the bottom of Fig. 2.

As in the TLC analysis result, it was confirmed that PgUGT71A1 converted PPD to C-K, Rg₃ to Rd, Rh₂ to F2, and PPT to F1 (Fig. 2, bottom).

Overall, the above results demonstrate that PgUGT71A1 is an enzyme having an activity of transferring UDP-glucose to a hydroxyl group at the C20-position of PPD-type and PPT-type ginsenosides, particularly a PPD-type ginsenoside and a PPT-type ginsenoside not having a sugar at the C-6 position.

Test Example 2: Enhancement of PgUGT71A1 expression by methyl jasmonate (MeJA)

It was investigated whether PgUGT71A1 of the present invention is mainly expressed in the root of ginseng that has been traditionally used for medicinal purposes. Also, the organ-specific expression patterns of PgUGT71A1 of the present invention were examined along with 3 different ginsenoside biosynthetic genes dammarenediol-II synthase (PgDS), protopanaxadiol synthase (PgPPDS), and protopanaxatriol synthase (PgPPTS).

Methyl jasmonate (MeJA) is known to enhance the expression of the biosynthetic genes of ginseng in hairy root cultures. Based on this fact, it was examined whether the expression of PgUGT71A1, i.e., the UDP glycosyltransferase of the present invention, can be increased by MeJA. For this, 15-month-old ginseng grown in a growth chamber under LD conditions was used. MeJA was sprayed onto the leaves of the ginseng every day for a total of 5 days and samples were collected on the 6th day in order to analyze the expression level of the ginsenoside biosynthetic genes.

As a result, all the ginsenoside biosynthetic genes were expressed in both leaves and roots of ginseng, and the expression of the PgUGT71A1 of the present invention was significantly enhanced by the MeJA treatment (Figs. 3 and 4).

Overall, the above results demonstrate that the PgUGT71A1 of the present invention is a protein involved in the biosynthesis of ginseng. In addition, since MeJA enhances the expression of the PgUGT71A1, which is the glycosyltransferase of the present invention, it can be seen that MeJA may be used to enhance the expression of PgUGT71A1.

Test Example 3: PgUGT71A1 enabling the de novo synthesis of C-K in yeast

In order to produce compound K (C-K), pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, and pRS423-C-K containing PgUGT71A1 were introduced into S. cerevisiae MET3p-ERG7, and S. cerevisiae C-K strain having a biosynthetic pathway of C-K was constructed (Fig. 6).

To determine whether ginsenosides C-K could be produced de novo in yeast, S. cerevisiae C-K strain was grown via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. The yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using MtOH and purified using Sep-PAK. The purified ginsenosides were analyzed by HPLC and LC-MS/MS. Yeast harboring PgUGT71A1 produced a new peak at 11.97 min which is identical to the retention time of C-K in HPLC ((A) of Fig. 7). The observed retention time of products in LC-MS/MS detected by MRM was 11.46 min with the transition at 645.4 → 23.2, which is identical to C-K ((B) of Fig. 7). These results indicate that the PgUGT71A1 enables the de novo production of ginsenoside through yeast fermentation (4.5 mg/L).

Test Example 4: PgUGT71A1 and PgPPTS enable the de novo synthesis of ginsenosides F1 in yeast

In order to produce ginsenoside F1, pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, and pRS423-F1 containing PgUGT71A1 and PgPPTS were introduced into S. cerevisiae MET3p-ERG7, and S. cerevisiae F1 strain having a biosynthetic pathway of ginsenoside F1 was constructed (Fig. 8).

To determine whether ginsenosides F1 could be produced de novo in yeast, S. cerevisiae F1 strain was grown via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. Yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then BtOH was evaporated. Finally, ginsenoside F1 was re-extracted using MtOH. Extracted ginsenosides were analyzed by HPLC and LC-MS/MS. Yeast harboring PgUGT71A1 and PgPPTS produced a new peak at 6.01 min which is identical to the retention time of ginsenoside F1 in HPLC ((A) of Fig. 9). The observed retention time of products in LC-MS/MS detected by MRM was 6.13 min with the transition at 661.5 → 203, which is identical to ginsenoside F1 ((B) of Fig. 9). These results indicate that the PgUGT71A1 and PgPPTS enable the de novo production of ginsenoside through yeast fermentation.

Test Example 5: PgUGT74A1 and PgUGT71A1 enable the de novo synthesis of ginsenoside F2 in yeast

In order to produce ginsenoside F2, pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, and pRS423-F2 containing PgUGT74A1 and PgUGT71A1 were introduced into S. cerevisiae MET3p-ERG7, and S. cerevisiae F2 strain having a biosynthetic pathway of ginsenoside F2 was constructed (Fig. 10).

To determine whether ginsenosides F2 could be produced de novo in yeast, S. cerevisiae F2 strain was grown via fed-batch fermentation for 10 d (OD600 80) in the presence of methionine. The yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then BtOH was evaporated. Finally, ginsenoside F2 was re-extracted using MtOH. The extracted ginsenosides were analyzed by HPLC and LC-MS/MS. The yeast harboring PgUGT74A1 and PgUGT71A1 produced a new peak at 7.81 min which is identical to the retention time of ginsenoside F2 in HPLC ((A) of Fig. 11). The observed retention time of products in LC-MS/MS detected by MRM was 7.35 min with the transition at 807.5 → 627.5, which is identical to ginsenoside F2 ((B) of Fig. 11). These results indicate that PgUGT74A1 and PgUGT71A1 enable the de novo production of ginsenoside through yeast fermentation.

Test Example 6: PgUGT74A1, PgUGT94B1 and PgUGT71A1 enable the de novo synthesis of ginsenoside Rd

In order to produce ginsenoside Rd, pRS424-DS, pRS426-tHMG1, pRS425-PPD containing PPDS and AtCPR, and pRS423-Rd containing PgUGT74A1, PgUGT94B1 and PgUGT71A1 were introduced into S. cerevisiae MET3p-ERG7, and S. cerevisiae Rd strain having a biosynthetic pathway of ginsenoside Rd was constructed (Fig. 12).

To determine whether ginsenosides Rd could be produced de novo in yeast, S. cerevisiae Rd strain was grown via fed-batch fermentation for 10 d (OD600

80) in the presence of methionine. The yeast cells were harvested by centrifugation and disrupted by sonication. Ginsenosides were extracted using BtOH and then BtOH was evaporated. Finally, ginsenoside Rd was re-extracted using MtOH. The extracted ginsenosides were analyzed by HPLC and LC-MS/MS. The yeast harboring PgUGT74A1, PgUGT94B1, and PgUGT71A1 produced a new peak at 5.88 min which is identical to the retention time of ginsenoside Rd in HPLC ((A) of Fig. 13). The observed retention time of products in LC-MS/MS detected by MRM was 5.93 min with the transition at 969.5 → 789.5, which is identical to ginsenoside Rd ((B) of Fig. 13). These results indicate that PgUGT74A1, PgUGT94B1 and PgUGT71A1 enable the de novo production of ginsenoside through yeast fermentation.

It will be apparent to those of ordinary skill in the art to which the present invention belongs that various modifications and changes may be made without departing from the scope and spirit of the disclosure. Therefore, it should be understood that the above-described exemplary embodiments are not limitative, but illustrative in all aspects. The scope of the present invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

Claims

A method for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, comprising:

treating a uridine diphosphate (UDP) glycosyltransferase protein, a transformed cell which is introduced with a vector comprising a polynucleotide encoding the protein or a fragment thereof and exhibits activity of the protein, an organism which comprises the transformed cell, or a culture of the transformed cell to a PPD- or PPT-type ginsenoside having a hydroxyl group at the C-20 position.
The method according to claim 1, wherein the PPD- or PPT-type ginsenoside having the hydroxyl group at the C-20 position is one or more selected from the group consisting of PPD, Rh₂, Rg₃, and PPT.
The method according to claim 1, wherein the method comprises one or more conversion step selected from the group consisting of a conversion of PPD to compound K (C-K), a conversion of Rh₂ to F2, a conversion of Rg₃ to Rd, and a conversion of PPT to F1.
The method according to claim 1, wherein the UDP glycosyltransferase protein is defined by the amino acid sequence of SEQ ID NO: 1.
A composition for preparing a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside whose hydroxyl group at the C-20 position is glycosylated, which comprises one or more selected from the group consisting of a uridine diphosphate (UDP) glycosyltransferase protein having glycosylation activity for a hydroxyl group at the C-20 position of a PPD- or PPT-type ginsenoside having the hydroxyl group at the C-20 position, a transformed cell which is introduced with a vector comprising a polynucleotide encoding the protein or a fragment thereof and exhibits activity of the protein, an organism which comprises the transformed cell, or a culture of the transformed cell as an active ingredient.
The composition according to claim 5, wherein the PPD- or PPT-type ginsenoside having a hydroxyl group at the C-20 position is one or more selected from the group consisting of PPD, Rh₂, Rg₃, and PPT.
The composition according to claim 5, wherein the UDP glycosyltransferase protein converts PPD to compound K (C-K), Rh₂ to F2, Rg₃ to Rd, or PPT to F1.
The composition according to claim 5, wherein the UDP glycosyltransferase protein is defined by the amino acid sequence of SEQ ID NO: 1.
An isolated uridine diphosphate (UDP) glycosyltransferase protein (PgUGT71A1) defined by the amino acid sequence of SEQ ID NO: 1.
The uridine diphosphate (UDP) glycosyltransferase protein according to claim 9, wherein the protein has glycosylation activity for a hydroxyl group at the C-20 position of a protopanaxadiol (PPD)- or protopanaxatriol (PPT)-type ginsenoside.
The uridine diphosphate (UDP)-glycosyltransferase protein according to claim 9, wherein the protein converts PPD to compound K (C-K), Rh₂ to F2, Rg₃ to Rd, or PPT to F1.
A polynucleotide encoding the protein according to claim 9.
An expression vector comprising the polynucleotide according to claim 12.
The expression vector according to claim 13, further comprising polynucleotides each of which encodes dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), and Arabidopsis thaliana cytochrome p450 reductase (AtCPR) proteins.
The expression vector according to claim 13, further comprising polynucleotides each of which encodes dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), and UDP-glycosyltransferaseprotopanaxatriol synthase (PPTS) proteins.
The expression vector according to claim 13, further comprising polynucleotides each of which encodes dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), and Panax ginseng UDP-glycosyltransferase 74A1 (PgUGT74A1) proteins.
The expression vector according to claim 13, further comprising polynucleotides each of which encodes dammarenediol-II synthase (DS), truncated HMG-CoA reductase (tHMGR), protopanaxadiol synthase (PPDS), Arabidopsis thaliana cytochrome p450 reductase (AtCPR), Panax ginseng UDP-glycosyltransferase 74A1 (PgUGT74A1), and Panax ginseng UDP-glycosyltransferase 94B1 (PgUGT94B1) proteins.
A transformed cell for producing compound K (C-K) comprising the expression vector according to claim 14 or a fragment thereof.
A transformed cell for producing F1 comprising the expression vector according to claim 15 or a fragment thereof.
A transformed cell for producing F2 comprising the expression vector according to claim 16 or a fragment thereof.
A transformed cell for producing Rd comprising the expression vector according to claim 17 or a fragment thereof.
A transformed cell according to claim 18, wherein the transformed cell is a yeast.
A transformed cell according to claim 19, wherein the transformed cell is a yeast.
A transformed cell according to claim 20, wherein the transformed cell is a yeast.
A transformed cell according to claim 21, wherein the transformed cell is a yeast.