JP7749799B2 - Highly specific glycosyltransferase for rhamnose and its applications - Google Patents

Description

The present invention relates to the fields of biotechnology and plant biology, and specifically to a glycosyltransferase with high specificity for rhamnose and its applications.

Ginsenosides are a collective term for saponins isolated from plants of the Panax genus (such as Korean ginseng, Panax notoginseng, and American ginseng) and Jiaogulan (Gynostemma pentaphyllum). They are a type of triterpene compound. Ginsenosides are also called ginsenosides, notoginsenosides, and gypenosides based on their isolation source. Ginsenosides are the main physiologically active components of these medicinal plants. Approximately 150 types of saponins have been isolated so far. Structurally, ginsenosides are primarily physiologically active small molecules formed after glycosylation of sapogenins. Ginsenosides contain only a limited number of sapogenins, primarily the dammarane-type tetracyclic triterpenes protopanaxadiol and protopanaxatriol, and oleanolic acid. After glycosylation, sapogenins can increase their water solubility, change their intracellular localization, and produce various biological activities. Most protopanaxadiol-type saponins are glycosylated at the hydroxyl groups at the C3 and/or C20 positions, while protopanaxatriol-type saponins are glycosylated at the hydroxyl groups at the C6 and/or C20 positions. Varying types of glycosyl and degrees of glycosylation result in ginsenosides with various molecular structures.

Rhamnosylated ginsenosides possess a wide range of biological activities. For example, Rg2 has one rhamnose molecule extended to the C6-O-Glc of Rh1, and Rg2 is highly effective in treating depression, improving cardiac function, enhancing learning and memory abilities, and preventing Alzheimer's disease. Ginsenoside Re has one rhamnose molecule extended to the C6-O-Glc of Rg1, and may play a role in lowering blood sugar levels and treating diabetes by promoting the secretion of glucagon-like peptide-1 in intestinal tissue.

Ginsenosides are prepared using chemical, enzymatic, and microbial fermentation hydrolysis methods with the total saponins or abundant saponins of ginseng or Panax notoginseng as raw materials. Because wild ginseng resources have essentially been depleted, ginsenoside resources are currently obtained from the artificial cultivation of ginseng and Panax notoginseng. However, artificial cultivation has a long growth cycle (generally taking more than 5-7 years), is geographically limited, is frequently susceptible to damage from pests and diseases, and requires the application of large amounts of pesticides. Therefore, the artificial cultivation of ginseng and Panax notoginseng suffers from significant problems with continuous cropping (the land where ginseng or Panax notoginseng is planted must be left fallow for more than 5-15 years to overcome this problem), and the yield, quality, and safety of ginsenosides all face challenges.

Developments in synthetic biology are bringing new opportunities for the heterologous synthesis of plant-derived natural products. Using yeast as a chassis, metabolic pathway construction and optimization enabled the fermentation synthesis of artemisinic acid or dihydroartemisinic acid from inexpensive monosaccharides, followed by the production of artemisinin through a single-step chemical conversion process, demonstrating the great potential of synthetic biology in the pharmaceutical synthesis of natural products. Heterologous synthesis of ginsenoside monomers was achieved using yeast chassis cells via synthetic biology techniques. The raw material was inexpensive monosaccharides, and the preparation process was a safe and tunable fermentation process, avoiding any external contamination (e.g., pesticides used in the artificial cultivation of the source plants). Preparing ginsenoside monomers through synthetic biology not only offers cost advantages, but also ensures the quality and safety of the final product. Using synthetic biology techniques, we can prepare a variety of high-purity natural and unnatural ginsenoside monomers in sufficient quantities for activity testing and clinical trials, facilitating the development of innovative pharmaceuticals targeting rare ginsenosides.

In recent years, transcriptome and functional genomic studies of Korean ginseng, Panax notoginseng, and American ginseng have significantly advanced the understanding of the synthetic pathways of ginsenoside sapogenins. In 2006, Japanese and Korean scientists each identified a terpene cyclase element (dammarenediol synthase, PgDDS) that converts epoxysqualene to dammarenediol. Between 2011 and 2012, Korean scientists also identified the cytochrome P450 elements CYP716A4 and CYP716A53v2 that oxidize dammarenediol to protopanaxadiol and further oxidize protopanaxadiol to protopanaxatriol.

To artificially synthesize these pharmacologically active ginsenosides using synthetic biology techniques, it is necessary not only to construct a metabolic pathway for sapogenin synthesis but also to identify UDP-glycosyltransferases that catalyze the glycosylation of ginsenosides. The function of UDP-glycosyltransferases is to transfer glycosyl groups from glycosyl donors (nucleoside diphosphate sugars such as UDP-glucose, UDP-rhamnose, UDP-xylose, and UDP-arabinose) to different glycosyl acceptors. Analysis of previously sequenced plant genomes has revealed that they often encode more than 100 different glycosyltransferases. In 2015, Chinese researchers identified a UDP-glycosyltransferase element (UGTPg100) capable of transferring one glucosyl group to the C6 position of protopanaxatriol. Chinese scholars have disclosed in a patent (PCT/CN2015/081111) glycosyltransferases (such as gGT29-7) that can elongate the sugar chain at the C6 position of protopanaxatriol-type saponins. For example, gGT29-7 can catalyze the elongation of one molecule of xylosyl at the C6 position of Rh1 using UDP-Xyl to produce notoginsenoside R2, and can catalyze the elongation of one molecule of glucosyl at the C6 position of Rh1 using UDP-Glc to produce Rf, but cannot basically use UDP-Rha. The gGT29-7 mutant gGT29-7(N343G, A359P) disclosed in the patent (PCT/CN2015/081111) catalyzes the elongation of one rhamnosyl molecule at the C6 position of Rh1 using UDP-Rha to produce Rg2, but its activity is very low, with a conversion rate of only approximately 9%. Furthermore, gGT29-7(N343G, A359P) can perform transglycosylation using UDP-Rha as a donor, as well as UDP-glc as a donor, with higher catalytic efficiency than catalytic reactions using UDP-Rha as a glycosyl donor. Therefore, the activity of gGT29-7(N343G, A359P) catalyzing UDP-Rha is low and nonspecific, resulting in the synthesis of many by-products and failing to meet application needs.

In light of the above background, the inventors screened for glycosyltransferases URT94-1 and URT94-2 from ginseng, which are capable of extending UPD-rhamnose at the C6 position. These glycosyltransferases efficiently catalyze the extension of one rhamnose molecule at the first glycosyl residue at the C-6 position of ginsenoside Rh1, ginsenoside Rg1, or Panax notoginseng R3, specifically using UDP-Rha as the glycosyl donor, to yield ginsenoside Rg2, ginsenoside Re, or Yesanchinoside E, respectively. However, URT94-1 and URT94-2 cannot catalyze the above saponin substrates using UDP-glucose as the glycosyl donor. Therefore, these glycosyltransferases provide highly specific glycosyltransferases for the efficient preparation of saponins such as ginsenoside Rg2, ginsenoside Re, and Yesanchinoside E.

In a first aspect of the present invention, there is provided a method for attaching rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene(s) compound, the method comprising transferring rhamnosyl using a specific glycosyltransferase that is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, or a conservative variant polypeptide thereof.
In another aspect of the present invention, there is provided a use of a specific glycosyltransferase for attaching rhamnosyl (including a catalyst used in this reaction) to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, wherein the specific glycosyltransferase is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14 or a conservative variant polypeptide thereof.

In one or more embodiments, the rhamnosyl is provided by a glycosyl donor; preferably, the glycosyl donor is a glycosyl donor having a rhamnose group; more preferably, the glycosyl donor includes (but is not limited to) uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, cytidine diphosphate (CDP)-rhamnose, and thymidine diphosphate (TDP)-rhamnose.

In one or more embodiments, the tetracyclic triterpene compound is a compound of formula (I), and the compound with a glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (II);

wherein R1 and R2 are H or glycosyl, R3 is monosaccharide glycosyl, and R4 is rhamnosyl; preferably, the glycosyl or monosaccharide glycosyl (R3) is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
Preferably, when R1 is H and R2 and R3 are glucosyl, the compound of formula (I) is ginsenoside Rg1, and the compound of formula (II) is ginsenoside Re; when R1 and R2 are H and R3 is glucosyl, the compound of formula (I) is ginsenoside Rh1, and the compound of formula (II) is ginsenoside Rg2.

In one or more embodiments, the tetracyclic triterpene compound is a compound of formula (III), and the compound with a glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (IV);

wherein R1 is H or glycosyl, R2, R3, R4 are monosaccharide glycosyl, and R5 is rhamnosyl; preferably, the glycosyl (R1) or monosaccharide glycosyl (R2, R3, R4) is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
Preferably, when R1 is H, R2, R3 and R4 are glucosyl, and R5 is rhamnosyl, the compound of formula (III) is notoginsenoside R3, and the compound of formula (IV) is Yesanchinoside E.

In one or more embodiments, the type of group, substrate, or product is as follows:

In one or more embodiments, the type of group, substrate, or product is as follows:

In one or more embodiments, compounds (I) and (III) in the above reaction scheme include, but are not limited to, S- or R-configured dammarane-type tetracyclic triterpene compounds, lanolin-type tetracyclic triterpene compounds, apotirucallane-type tetracyclic triterpenes, tirucallane-type tetracyclic triterpene compounds, cycloartane (cycloartinane)-type tetracyclic triterpene compounds, cucurbitane-type tetracyclic triterpene compounds, or neem-type tetracyclic triterpene compounds.

In one or more embodiments, compound (II) or (IV) in the above reaction scheme includes ginsenoside Rg2, ginsenoside Re, and Yesanchinoside E.

In another aspect of the present invention, there is provided a method for attaching rhamnosyl to a first glycosyl at the C-6 position of a tetracyclic triterpene(s) compound in a cell, the method comprising:
(a) obtaining a recombinant host cell by introducing into a host cell a reactive precursor of a tetracyclic triterpene compound or a construct expressing/forming the reactive precursor, or a specific glycosyltransferase or a construct expressing the reactive precursor, wherein the specific glycosyltransferase is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, or a conservative variant polypeptide thereof; a glycosyl donor having a rhamnose group is present in the host cell, or a glycosyl donor having a rhamnose group (including a construct/precursor capable of forming the glycosyl donor) is introduced;
(b) culturing the recombinant host cell of (a) to produce a tetracyclic triterpene compound having rhamnosyl attached to the first glycosyl at the C-6 position;
Preferably, the reaction precursors of the tetracyclic triterpene compounds include ginsenoside Rg1, ginsenoside Rh1, and notoginsenoside R3; and the corresponding products include ginsenoside Re, ginsenoside Rg2, and Yesanchinoside E;
Preferably, the glycosyl donor includes, but is not limited to, uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, cytidine diphosphate (CDP)-rhamnose, and thymidine diphosphate (TDP)-rhamnose.

In one or more embodiments, the method further includes providing an additive to the reaction system to regulate enzyme activity.

In one or more embodiments, the additive for regulating enzyme activity is an additive that increases or inhibits enzyme activity.

In one or more embodiments, the additive for regulating enzyme activity is selected from Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al 3+ , Ni ²⁺ , Zn ²⁺ , or Fe ²⁺ .

In one or more embodiments, the additive for regulating enzyme activity is a substance capable of generating Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al 3+ , Ni ²⁺ , Zn ²⁺ or Fe ²⁺ .

In one or more embodiments, the pH of the reaction system is 4.0 to 10.0, preferably 5.5 to 9.0.

In one or more embodiments, the temperature of the reaction system is 10°C to 105°C, preferably 20°C to 50°C.

In another aspect of the present invention, there is provided a specific glycosyltransferase, which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14 or a conservative variant polypeptide thereof; preferably, the conservative variant polypeptide is
(1) A polypeptide having the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, which is formed by substituting, deleting, or adding one or more (e.g., 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3) amino acid residues, and which has the function of binding rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound;
(2) A polypeptide having an amino acid sequence that is 50% or more (preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more) identical to a polypeptide having a sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, and that has the function of linking rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound; or (3) A polypeptide formed by adding a tag sequence to the N-terminus or C-terminus of a polypeptide having a sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, or by adding a signal peptide sequence to the N-terminus.
In another aspect of the present invention, there is provided an isolated polynucleotide encoding said specific glycosyltransferase.

In one or more embodiments, the polynucleotide encoding the specific glycosyltransferase comprises a polynucleotide selected from (A) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 13; (B) a nucleotide sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 1, 3, or 13; (E) a nucleotide sequence formed by truncating or adding 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides to the 5' and/or 3' end of the sequence set forth in SEQ ID NO: 1, 3, or 13; (F) a complementary sequence of the nucleotide sequence set forth in any one of (A) to (E); and (G) a 20 to 50 base fragment of the sequence set forth in (A) to (F).

In one or more embodiments, the polynucleotide sequence is selected from any one of SEQ ID NO: 1, 3, or 13, or a complementary sequence thereof.

In another aspect of the present invention, there is provided a nucleic acid construct comprising the polynucleotide or expressing the specific glycosyltransferase; preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

In another aspect of the present invention, a recombinant host cell is provided that expresses the specific glycosyltransferase, contains the polynucleotide, or contains the nucleic acid construct; preferably, the recombinant host cell also contains a reactive precursor of a tetracyclic triterpene compound or a construct that expresses/forms the precursor; preferably, the recombinant host cell also contains a glycosyl donor having a rhamnose group, or a glycosyl donor having a rhamnose group is introduced into the recombinant host cell (including a construct/precursor that can form the glycosyl donor).

In one or more embodiments, the reaction precursors of the tetracyclic triterpene compounds include ginsenoside Rg1, ginsenoside Rh1, and notoginsenoside R3; the corresponding products include ginsenoside Re, ginsenoside Rg2, and yesanchinoside E.

In one or more embodiments, the glycosyl donor includes, but is not limited to, uridine diphosphate (UDP)-rhamnose, uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, cytidine diphosphate (CDP)-rhamnose, and thymidine diphosphate (TDP)-rhamnose.

In one or more embodiments, the host cell is a prokaryotic or eukaryotic cell.
In one or more embodiments, the host cell is a eukaryotic cell such as a yeast cell or a plant cell. In one or more embodiments, the host cell is a Saccharomyces cerevisiae cell. In one or more embodiments, the host cell is a Korean ginseng cell or a Panax notoginseng cell.

In one or more embodiments, the host cell is a prokaryotic cell, such as E. coli.
In one or more embodiments, the host cell is not a cell that naturally produces the product formed after treatment with the specific glycosyltransferase of the invention; e.g., is not a cell that naturally produces the compounds of formula (II) or (IV).

In one or more embodiments, the host cell is not a cell that naturally produces one or more of ginsenoside Rh1, ginsenoside Rg1, notoginsenoside R3, ginsenoside Rg2, ginsenoside Re, and yesanchinoside E.

In one or more embodiments, the host cell also has a characteristic selected from the following:
(a) a mutant expressing a key enzyme in the anabolic pathway of dammarenediol and/or protopanaxadiol saponins and/or protopanaxatriol saponins, and having 50% sequence identity with said enzyme;
(b) a polypeptide expressing a functional fragment comprising the enzyme of (a) or a mutant having 50% sequence identity to the fragment;
(c) a polynucleotide comprising the enzyme according to (a) or the polypeptide according to (b), or a complementary sequence thereof, and/or (d) a nucleic acid construct comprising the coding sequence according to (c).
In one or more embodiments, protopanaxatriol saponins include ginsenoside Rh1, ginsenoside Rg1, notoginsenoside R3, ginsenoside Rg2, ginsenoside Re, and yesanchinoside E.

In one or more embodiments, key genes in the ginsenoside Rh1 assimilation pathway include, but are not limited to, the dammarenediol synthase gene, the cytochrome P450 CYP716A47 gene, the P450 CYP716A47 reductase gene, and the tetracyclic triterpene C6 glycosyltransferase UGTPg100 (Genbank accession number AKQ76388.1), or a combination thereof.

In one or more embodiments, key genes in the ginsenoside Rg1 assimilation pathway include, but are not limited to, the dammarenediol synthase gene, the cytochrome P450 CYP716A47 gene, the P450 CYP716A47 reductase gene, and the tetracyclic triterpene C20 and C6 glycosyltransferases UGTPg1 and UGTPg100 (Genbank accession number AKQ76388.1), or combinations thereof.

In one or more embodiments, key genes in the ginsenoside Rg2 assimilation pathway include (but are not limited to) the dammarenediol synthase gene, the cytochrome P450 CYP716A47 gene, the P450 CYP716A47 reductase gene, the tetracyclic triterpene C6 glycosyltransferase UGTPg100 (Genbank accession number AKQ76388.1), and the glycosyltransferases URT94-1 and URT94-2 that catalyze the elongation of glycosyl groups at the C6 position in the present invention, or a combination thereof.

In one or more embodiments, key genes in the ginsenoside Re assimilation pathway include (but are not limited to) the dammarenediol synthase gene, the cytochrome P450 CYP716A47 gene, the P450 CYP716A47 reductase gene, the tetracyclic triterpene C20 and C6 glycosyltransferases UGTPg1 and UGTPg100 (Genbank accession number AKQ76388.1), and the glycosyltransferases URT94-1 and URT94-2 catalyzing the elongation of the C6 glycosyl group as described herein, or combinations thereof.

In another aspect, the present invention also provides the use of the host cells described in the present invention in the preparation of glycosyltransferases, catalytic reagents, or compounds of formula (II) or (IV).

In another aspect, the present invention also provides a method for producing a glycosyltransferase or a compound of formula (II) or (IV), comprising incubating a host cell described in the present invention.

In another aspect of the present invention, there is also provided the use of the host cells described in the present invention for the preparation of enzyme catalytic reagents, for the production of glycosyltransferases, as catalytic cells, or for the production of compounds of formula (II) or (IV).

In another aspect, the present invention also provides a method for producing a transgenic plant, comprising the step of regenerating a host cell described in the present invention into a plant, wherein the host cell is a plant cell. In one or more embodiments, the host cell is a ginseng cell. In one or more embodiments, the host cell is a Panax notoginseng cell.

In another aspect of the present invention, a kit for glycosyltransferase is provided, which is capable of attaching rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, and which comprises a specific glycosyltransferase that is a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:14, or a conservative variant polypeptide thereof.

In another aspect of the present invention, there is provided a kit for glycosyltransferase, comprising the isolated polynucleotide.

In another aspect of the present invention, there is provided a kit for glycosyltransferase, comprising the nucleic acid construct.

In another aspect of the present invention, there is provided a kit for glycosyltransferase, comprising the recombinant host cell.

In one or more embodiments, the kit also includes a glycosyl donor having a rhamnose group; more preferably, the glycosyl donor includes, but is not limited to, uridine diphosphate (UDP)-rhamnose, guanosine diphosphate (GDP)-rhamnose, adenosine diphosphate (ADP)-rhamnose, cytidine diphosphate (CDP)-rhamnose, and thymidine diphosphate (TDP)-rhamnose.

In one or more embodiments, the kit includes a reaction precursor of a tetracyclic triterpene compound.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred embodiments. To avoid lengthy descriptions, they will not be described in detail here.

FIG. 1 shows the results of DNA agarose gel electrophoresis of the products obtained by amplifying two target bands of glycosyltransferases from a single ginseng plant. 2 shows the expression of glycosyltransferases URT94-1 and URT94-2 in E. coli by Western blot. "1" represents the supernatant of a lysate from an empty vector pET28a E. coli recombinant, "Marker" represents a protein molecular weight standard, "2" represents the supernatant of a lysate from an E. coli recombinant containing glycosyltransferase BL21-URT94-1, "3" represents the supernatant of a lysate from an E. coli recombinant containing glycosyltransferase BL21-URT94-2, "4" represents the supernatant of a lysate from an E. coli recombinant containing glycosyltransferase BL21-gGT29-7, and "5" represents the supernatant of a lysate from an E. coli recombinant containing glycosyltransferase BL21-gGT29-7 (N343G, A359P). 3a shows TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction using protopanaxatriol-type ginsenoside Rh1 as a glycosyl acceptor and UDP-Rha as a glycosyl donor. "1" represents the supernatant of the lysate of the pet28a empty vector recombinant as the enzyme solution, and "2," "3," "4," and "5" represent the supernatants of the lysates of BL21-URT94-1, BL21-URT94-2, BL21-gGT29-7 (N343G, A359P), and BL21-gGT29-7 as the enzyme solutions, respectively. The arrow indicates the migration position of the saponin standard; FIG. 3b shows the HPLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransfer reaction using protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor. 4a shows TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the transglycosylation reaction using protopanaxatriol-type ginsenoside Rg1 as a glycosyl acceptor and UDP-Rha as a glycosyl donor. "1" represents the supernatant of the lysate of the pet28a empty vector recombinant as the enzyme solution, and "2," "3," "4," and "5" represent the supernatants of the lysates of BL21-gGT29-7, BL21-gGT29-7 (N343G, A359P), BL21-URT94-1, and BL21-URT94-2 as the enzyme solutions, respectively. The arrow indicates the migration position of the saponin standard; FIG. 4b shows the HPLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransfer reaction using protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor. 5 shows TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction using protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Glc as the glycosyl donor. "1" represents the supernatant of the lysate of the pet28a empty vector recombinant as the enzyme solution, while "2," "3," "4," and "5" represent the supernatants of the lysates of BL21-gGT29-7, BL21-gGT29-7 (N343G, A359P), BL21-URT94-1, and BL21-URT94-2 as the enzyme solutions, respectively. The arrow indicates the migration position of the saponin standard. 6 shows TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction using protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Glc as the glycosyl donor. "1" represents the supernatant of the lysate of the pet28a empty vector recombinant as the enzyme solution, while "2," "3," "4," and "5" represent the supernatants of the lysates of BL21-gGT29-7, BL21-gGT29-7 (N343G, A359P), BL21-URT94-1, and BL21-URT94-2 as the enzyme solutions, respectively. The arrow indicates the migration position of the saponin standard. FIG. 7 shows a comparison of the catalytic activity of the glycosyltransferase URT94-1m mutant and the wild type. FIG. 8 shows the expression of the glycosyltransferase URT94-1m mutant and wild-type, detected by Western blotting.

As a result of extensive research and screening, the present inventors have provided, for the first time, a specific glycosyltransferase that can catalyze rhamnosylation at a specific position in a substrate and improve catalytic activity. Specifically, the specific glycosyltransferase of the present invention can specifically and efficiently catalyze hydroxyglycosylation of the first glycosyl at the C-6 position of a tetracyclic triterpene compound substrate, thereby extending the rhamnose group.

Definitions As used herein, "isolated polypeptide" or "active polypeptide" means that the polypeptide is essentially free from other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. One of skill in the art can purify the polypeptide using standard protein purification techniques. An essentially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using amino acid sequence.

As used herein, the terms "active polypeptide," "polypeptide of the present invention and derivative polypeptides thereof," "enzyme of the present invention," and "glycosyltransferase" are used interchangeably and include URT94-1 (SEQ ID NO: 2), URT94-2 (SEQ ID NO: 4) polypeptides or derivative polypeptides thereof, and may also refer to mutant glycosyltransferases, including URT94-1m (SEQ ID NO: 14).

As used herein, the term "conservative variant polypeptide" refers to a polypeptide that essentially retains a biological function or activity homologous to the polypeptide. The "conservative variant polypeptide" may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, where the substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substitution at one or more amino acid residues; (iii) a polypeptide formed by fusing the mature polypeptide to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a proteinogenic sequence, or a fusion protein formed with an antigenic IgG fragment). As taught herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

As used herein, the terms "variant" or "mutant" refer to a peptide or polypeptide whose amino acid sequence has been altered by the insertion, deletion, or substitution of one or more amino acids compared to a reference sequence, but which retains at least one biological activity. Mutants described in any embodiment herein include amino acid sequences that share at least 50%, 60%, or 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 97% sequence identity with a reference sequence (e.g., SEQ ID NO: 2, 4, or 14 described herein) and retain the biological activity (e.g., as a glycosyltransferase) of the reference sequence. Sequence identity between two compared sequences can be calculated, for example, using NCBI's BLASTp. Mutants also include amino acid sequences that have one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of the reference sequence while retaining the biological activity of the reference sequence. The number of mutations is usually 1 to 20, for example, 1 to 15, 1 to 10, 1 to 8, 1 to 5, or 1 to 3. The substitutions are preferably conservative substitutions. For example, conservative substitutions with amino acids having close or similar properties are generally known in the art and usually do not alter the function of a protein or polypeptide. "Amino acids with close or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more positions in a polypeptide of the present invention with another amino acid residue from the same side chain class does not substantially affect its activity.

URT94-1m (SEQ ID NO: 14) is a mutant of UR T94-1. The present invention also includes conservative variant polypeptides of UR T94-1m, which are conserved at the amino acid residue corresponding to position 55 of SEQ ID NO: 14.

Active Polypeptide, Gene Encoding It, Vector, and Host The present inventors have mined genome and transcriptome information and combined this with a large amount of research and experimental work to discover a novel glycosyltransferase with specificity that can specifically and efficiently transfer glycosyl to the first glycosyl at C-6 of a tetracyclic triterpene compound substrate, thereby elongating the sugar chain, and the reaction product has high application value in the fields of pharmaceuticals, etc.

The specific glycosyltransferase sequence described in the present invention is preferably the polypeptide shown in SEQ ID NO: 2, 4, or 14. The polypeptide also includes "conservative variant polypeptides" having the sequence of SEQ ID NO: 2, 4, or 14 that have the same function as the polypeptide shown. The present invention also includes fragments, derivatives, and analogs of the polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that essentially retain a biological function or activity homologous to the polypeptide.

In the present invention, the term "conservative variant polypeptide" refers to a polypeptide that essentially retains biological functions or activities homologous to the polypeptide. The "conservative variant polypeptide" may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, where the substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substitution at one or more amino acid residues; (iii) a polypeptide formed by fusing a mature polypeptide to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein-constituting sequence, or a fusion protein formed with an antigen IgG fragment). As taught herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

The "conservative variant polypeptide" includes, but is not limited to, one or more amino acids (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) deleted, inserted, and/or substituted, and one or more amino acids (e.g., 50 or fewer, preferably 20 or 10 or fewer, more preferably 5 or fewer) added or deleted at the C-terminus and/or N-terminus. For example, substitution with amino acids with close or similar properties is known in the art, and typically does not alter the function of the protein. Furthermore, for example, addition of one or more amino acids to the C-terminus and/or N-terminus typically does not alter the function of the protein. The present invention also provides analogs of the above polypeptides. The difference between these analogs and the naturally occurring polypeptide may be in the amino acid sequence, in the form of a modification that does not affect the sequence, or both. These polypeptides include naturally occurring or induced genetic variants. Induced variants can be obtained by a variety of techniques, including, for example, random mutations induced by irradiation or exposure to mutagenic agents, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include those with residues different from naturally occurring L-amino acids (e.g., D-amino acids) and those with non-naturally occurring or synthetic amino acids (e.g., β-, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

The amino or carboxyl terminus of the URT94-1 (SEQ ID NO: 2), URT94-2 (SEQ ID NO: 4), or URT94-lm (SEQ ID NO: 14) polypeptides of the present invention, or their conservative variants, may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-Tag II, AU1, EE, T7, 4A6, ε, B, gE, or Ty1. These tags can be used for protein purification.

When used to produce the specific glycosyltransferase of the present invention or other enzymes (e.g., enzymes used to react in host cells to form substrates for the specific glycosyltransferase of the present invention, or enzymes involved in any step of the product synthesis pathway of the present invention), a signal peptide sequence can also be added to the amino terminus of the polypeptide of the present invention for the purpose of secreting and expressing (e.g., secreting extracellularly) the translated protein. The signal peptide can be cleaved during the process of secreting the polypeptide from the cell.

Active polypeptides of the present invention may be recombinant, natural, or synthetic. Polypeptides of the present invention may be naturally purified products, chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants). Depending on the host used in a recombinant production protocol, polypeptides of the present invention may be glycosylated or non-glycosylated. Polypeptides of the present invention may or may not contain an initial methionine residue.

Polynucleotides encoding the specific glycosyltransferases and other enzymes of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be the coding strand or the non-coding strand. The term "polynucleotide encoding a polypeptide" may refer to a polynucleotide that encodes the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors or polypeptide-encoding sequences of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The present invention relates to nucleic acid constructs comprising the polynucleotides described herein and, operably linked to these sequences, one or more regulatory sequences or sequences necessary for homologous recombination with a genome. The polynucleotides of the present invention may be manipulated in various ways to ensure expression of the polypeptide or protein. The nucleic acid construct may be manipulated prior to insertion into a vector to accommodate differences or requirements in the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are known in the art.

In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a gene knock-in vector. The polynucleotides of the invention may be cloned into various types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence for the protein or polypeptide of the invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Typically, expression of the polynucleotide of the invention is achieved by operably linking the polynucleotide of the invention to a promoter and introducing the construct into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical expression vectors include expression control sequences that can be used to regulate expression of the desired nucleic acid sequence.

A gene knock-in vector is used to integrate the polynucleotide sequence described herein into a desired region of the genome. In addition to the polynucleotide sequence, the gene knock-in vector typically comprises a 5' homology arm and a 3' homology arm, which are necessary for homologous recombination into the genome. In some embodiments, the nucleic acid construct described herein comprises a 5' homology arm, a polynucleotide sequence described herein, and a 3' homology arm. When a gene knock-in vector is used, the polynucleotide sequence can be homologously recombined into the desired location using CRISPR/Cas9 technology. CRISPR/Cas9 technology involves designing a guide RNA for the target gene to guide Cas9 nuclease to modify the genome at the insertion position, increasing the efficiency of homologous recombination of the gene-modified region, and homologously recombining the target fragment contained in the gene knock-in vector into the target site. The steps of the CRISPR/Cas9 technology and the reagents used, such as Cas9 nuclease, are well known in the art.

Nucleic acid constructs can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence may be operatively linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters include the E. coli lac or trp promoter; the lambda phage PL promoter; and eukaryotic promoters, including the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, retroviral LTRs, and other known promoters for expression of regulatable genes in prokaryotic or eukaryotic cells or their viruses. The expression vector may further include a ribosome binding site for translation initiation and a transcription terminator. Additionally, the expression vector preferably contains one or more selectable marker genes for providing a phenotypic trait of the host cell that is used for selection for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for E. coli.

When expressing a polynucleotide of the present invention in higher eukaryotic cells, transcription can be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance gene transcription. Examples include the SV40 enhancer located 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer located on the late side of the replication origin, and adenovirus enhancers.

The present invention also provides host cells for the biosynthesis of a desired product. The host cells may be prokaryotic cells such as, but not limited to, E. coli, yeast, and Streptomyces, and are more preferably E. coli cells. A cellular host is a production tool, and those skilled in the art can achieve biosynthesis as in the present invention by modifying various host cells using any technical means. Host cells and production methods constructed in this way are also intended to be included in the present invention.

The polynucleotide sequences of the present invention can be used to express or produce the polypeptides described herein by conventional recombinant DNA techniques. This generally involves the steps of: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the present invention encoding the specific glycosyltransferase, or an expression vector containing such a polynucleotide; (2) culturing the host cell in a suitable medium; and (3) isolating and purifying the protein from the medium or cells.

A vector containing an appropriate DNA sequence and an appropriate promoter or control sequence, as described above, can be used to transform an appropriate host cell to express the protein. The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells such as fruit fly S2 or Sf9, and animal cells such as CHO, COS, 293, or Bowes melanoma cells. Those skilled in the art will clearly know how to select appropriate vectors, promoters, enhancers, and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. In the above-mentioned methods, the recombinant polypeptide is expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be isolated and purified using various isolation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

Although the present inventors have devoted themselves to the study of glycosyltransferases, their research to date has not yet yielded an enzyme that can efficiently utilize rhamnosyl donors and specifically attach rhamnosyl to the first glycosyl at the C-6 position of tetracyclic triterpene(s). Some existing enzymes are essentially unable to utilize rhamnosyl donors (e.g., UDP-Rha); others have very low activity and cannot fully meet the needs of applications.

In light of the above, the inventors screened ginseng and isolated a specific glycosyltransferase (URT94s) capable of extending rhamnose at the C6 position. The enzyme efficiently catalyzes the extension of one rhamnose molecule at the first glycosyl group at the C-6 position of protopanaxatriol saponins (protopanaxatriol-type saponins/protopanaxatriol-type saponins) known as ginsenoside Rh1, ginsenoside Rg1, and notoginsenoside R3 to yield ginsenoside Rg2, ginsenoside Re, or Yesanchinoside E. This glycosyltransferase is highly specific and provides the efficient preparation of ginsenoside Rg2, ginsenoside Re, or Yesanchinoside E. Preferably, the protopanaxatriol-type saponins include ginsenoside Rh1 and ginsenoside Rg1.

In a specific embodiment of the present invention, the active polypeptide of the present invention has glycosyltransferase activity and can catalyze one or more of the following reactions:

wherein R1 and R2 are H or glycosyl, and R3 and R4 are monosaccharide glycosyl.
In one or more embodiments, the compounds substituted at R1 to R4 are as follows:

That is, when R1 is H and R2 and R3 are glucosyl, the compound of formula (I) is ginsenoside Rg1, and when R4 is rhamnosyl, the compound of formula (II) is notoginsenoside Re; or when R1 and R2 are H and R3 is glucosyl, the compound of formula (I) is ginsenoside Rh1, and when R4 is rhamnosyl, the compound of formula (II) is notoginsenoside Rg2.
As another specific embodiment of the present invention,

wherein R1 is H or glycosyl, and R2, R3, R4, and R5 are monosaccharide glycosyl; the polypeptide is selected from SEQ ID NO: 2, 4, or 14, or a derivative polypeptide thereof.

In one or more embodiments, the compound in which R1 to R5 are substituted is as follows:

That is, when R1 is H and R2, R3, and R4 are glucosyl, the compound of formula (III) is notoginsenoside R3, and when R5 is rhamnosyl, the compound of formula (IV) is Yesanchinoside E.

The present invention also provides a method for constructing a transgenic plant, comprising regenerating a host cell containing a polypeptide or polynucleotide described herein into a plant, wherein the host cell is a plant cell. Methods and reagents for regenerating plant cells are well known in the art.

The glycosyltransferase of the present invention is particularly capable of converting ginsenoside Rh1 to ginsenoside Rg2, which has other activities. The glycosyltransferase of the present invention is particularly capable of converting ginsenoside Rg1 to ginsenoside Re, which has other activities.

The active polypeptide or glycosyltransferase of the present invention can be used to artificially synthesize known ginsenosides, novel ginsenosides, and their derivatives, and can convert Rh1 into active ginsenoside Rg2 and Rg1 into active ginsenoside Re.

The present invention also provides a method for constructing a transgenic plant, comprising transforming a plant with a polynucleotide or nucleic acid construct described herein, and obtaining, by hybridization and screening, a transgenic positive plant comprising the polynucleotide or comprising the nucleic acid construct in plant progeny, which expresses a polypeptide described herein. Methods for transforming plants with nucleic acids, as well as methods for plant hybridization and screening for transgenic positive plants, are well known in the art.

The present invention also provides a kit for biosynthesizing a desired product or an intermediate thereof, comprising a novel specific glycosyltransferase polypeptide set forth in SEQ ID NO: 2, 4, or 14 or a conservative variant thereof; preferably further comprising a glycosyl donor therein; and preferably further comprising a host cell therein. More preferably, the kit also includes instructions describing the biosynthesis method.

The main advantages of the present invention are:
(1) The specific glycosyltransferase of the present invention can specifically and efficiently transfer glycosyl to the first glycosyl at C-6 of a tetracyclic triterpene compound substrate to elongate the sugar chain.
(2) The glycosyltransferase of the present invention can efficiently convert Rh1 to active ginsenoside Rg2; and the glycosyltransferase of the present invention can efficiently convert Rg1 to active ginsenoside Re. Rg2 is active in the prevention and treatment of neurodegenerative diseases, and Re is active in lowering blood sugar levels and treating diabetes. Therefore, the glycosyltransferase of the present invention has broad application value.
(3) High catalytic efficiency: Compared with the glycosyltransferases disclosed in patent PCT/CN2015/081111, URT94-1 and URT94-2 have at least five-fold improved activity in catalyzing the elongation of the sugar chain at the C6 position of Rh1 using UDP-rhamnose as the glycosyl donor.

The present invention will be further described below with reference to specific examples. Note that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, 3rd Edition, edited by J. Sambrook et al., Scientific Press, or conditions recommended by the manufacturer.

Sequence information SEQ ID NO: 1 (URT94-1 nucleic acid)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagca aaaaaactctcaaaacgaaatttttacatatacttttgttccacatctatcaatcttagttccatcaggaaaaaacttgcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctcc ccatctcattcctgatttgatcaaggcccttggtatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccata tcaactgccgccgcctataggtttaaggtggatcctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataat cttgatcaagagttctagagagttcgatgaaaagaatatcgaatactattcccttttgatggacaagaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaaagaatcc tcaacagtttatgtttctttgggagtgagtgctatttgtcagagcctagggatccgagagctggcccatgggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggttttt tgatagggtgaaagatagaggggtgattgtgagttggggccccacaggaaagaatattaggggcatggtggacttggggggatttgtgagt cattgtgggtggggttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcat gctatgtttgtggaggaggtgggcgttggcgtggaggttctaaaagacgagagtggagaatttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaa agaagtggaatgtgtagttgaggagttgaccaaacttttgcaaaaagtatcagaaagtagcagcaggccagggaagcgatgcccctaa

SEQ ID NO: 2 (URT94-1 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKK L AVDDHEAIQLIEFQLTSQTELPPHHHHTTKGLPPHLIPDLIKALGMSGPNVINILNTVNPDLIIYDVFQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDP SIPVPCSRIFLDDTNIRKSPDYDSSSAENSGILDLTFGTAIQSSDIILIKSSREFDEKNIEYYSLLMDKKIVPTGPLVQVNTSVAVHTENEKDDIMEWLSKKE ESSTVYVSFGSECYLSEPRIRELAHGLELSNVNFIWVISFPEGDEEMCNTCIEDVLPEGFLDRVKDRGVIVSWAPQERILGHGGLGGFVSHCGWGSVVEGMSY GVPIIAMPAQYEQPLHAMFVEEVGVGVEVLKDESGEFRRDEIAKAIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKRCP

SEQ ID NO: 3 (URT94-2 nucleic acid)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagca aaaaaactctcaaaacgaaatttttacatatacttttgttccacatctatcaatcttagttccatcaggaaaaaacttgcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctcc ccatctcattcctgatttgatcaaggcccttggtatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccata tcaactgccgccgcctataggtttaaggtggatcctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataat cttgatcaagagttctagagagttcgatgaaaagaatatcgaatactattcccttttgatggacaagaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaaagaatcc tcaacagtttatgtttctttgggagtgagtgctatttgtcagagcctagggatccgagagctggcccatgggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggttttt tgatagggtgaaagatagaggggtgattgtgagttggggccccacaggaaagaatattaggggcatggtggacttggggggatttgtgagt cattgtgggtggggttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcat gctatgtttgtggaggaggtgggcgttggcgtggaggttctaaaagacgagagcggagaatttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaa agaagtggaatgtgtagttgaggagttgaccaaacttttgcaaaaagtatcagaaagtagcagcaggccagggaaggaatgcccctaa

SEQ ID NO: 4 (URT94-2 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKKLAVD DHEAIQLIEFQLTSQTELPPHHHTTKGLPPHLIPDLIKALGMSGPNVINILNTVNPDL IIYDVFQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDPSIPVPCSRIFLDDTNI RKSPDYDSSSAENSGILDLTFGTAIQSSDIILIKSSREFDEKNIEYYSLLMDKKIVPTG PLVQVNTSVAVHTENEKDDIMEWLSKKEESSTVYVSFGSECYLSEPRIRELAHGLELS NVNFIWVISFPEGDEEMCNTCIEDVLPEGFLDRVKDRGVIVSWAPQERILGHGGLGGFV SHCGWGSVVEGMSYGVPIIAMPAQYEQPLHAMFVEEVGVGVEVLKDESGEFRRDEIAK AIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKECP

SEQ ID NO: 5 (primer pair 1-F)
cgcagtacatctaacagaaaaaaga

SEQ ID NO: 6 (primer pair 1-R)
caataatttgaaaaaaaatgaatta

SEQ ID NO: 7 (primer pair 2-F)
cgtgacattaatggtgtcatttat

SEQ ID NO: 8 (primer pair 2-R)
ctttttatatagcttttgctatccct

SEQ ID NO: 9 (URT94-1_Pet28a-F)
ctttaagaaggagagatataccatggataccaatgaaaaaacca

SEQ ID NO: 10 (URT94-1_Pet28a -R)
ctcgagtgcggccgcaagcttggggcatcgcttcccctggccctg

SEQ ID NO: 11 (URT94-2_Pet28a-F)
ctttaagaaggagagatataccatggataccaatgaaaaaaccaga

SEQ ID NO: 12 (URT94-2_Pet28a -R)
ctcgagtgcggccgcaagcttggggcattccttcccctggccctg

SEQ ID NO: 13 (URT94-1m1 nucleic acid)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagca aaaaaactctcaaaacgaaatttttacatatacttttgttccacatctatcaatcttagttccatcaggaaaaaaatggcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctcc ccatctcattcctgatttgatcaaggcccttggtatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccata tcaactgccgccgcctataggtttaaggtggatcctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataat cttgatcaagagttctagagagttcgatgaaaagaatatcgaatactattcccttttgatggacaagaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaaagaatcc tcaacagtttatgtttctttgggagtgagtgctatttgtcagagcctagggatccgagagctggcccatgggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggttttt tgatagggtgaaagatagaggggtgattgtgagttggggccccacaggaaagaatattaggggcatggtggacttggggggatttgtgagt cattgtgggtggggttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcat gctatgtttgtggaggaggtgggcgttggcgtggaggttctaaaagacgagagtggagaatttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaa agaagtggaatgtgtagttgaggagttgaccaaacttttgcaaaaagtatcagaaagtagcagcaggccagggaagcgatgcccctaa

SEQ ID NO: 14 (URT94-1m1 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKK M AVDDHEAIQLIEFQLTSQTELPPHHHHTTKGLPPHLIPDLIKALGMSGPNVINILNTVNPDLIIYDVFQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDP SIPVPCSRIFLDDTNIRKSPDYDSSSAENSGILDLTFGTAIQSSDIILIKSSREFDEKNIEYYSLLMDKKIVPTGPLVQVNTSVAVHTENEKDDIMEWLSKKE ESSTVYVSFGSECYLSEPRIRELAHGLELSNVNFIWVISFPEGDEEMCNTCIEDVLPEGFLDRVKDRGVIVSWAPQERILGHGGLGGFVSHCGWGSVVEGMSY GVPIIAMPAQYEQPLHAMFVEEVGVGVEVLKDESGEFRRDEIAKAIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKRCP

Example 1: Cloning of Ginseng-Derived Glycosyltransferase URT94s As a result of extensive research and screening, the present inventors have cloned and isolated two glycosyltransferases, designated URT94-1 and URT94-2 (URT94s), from a single ginseng plant.

Cloning of URT94s: Ginseng RNA was extracted and reverse-transcribed to obtain ginseng cDNA. This cDNA was used as a template to design two pairs of primers (SEQ ID NO: 5 to SEQ ID NO: 6 amplify URT94-1; SEQ ID NO: 7 to SEQ ID NO: 8 amplify URT94-2) for PCR amplification. The DNA polymerase used was the high-fidelity DNA polymerase PrimeSTAR from Takara Biotechnology Co., Ltd. The PCR product was detected by agarose gel electrophoresis (Figure 1). The target DNA band was excised by UV irradiation. The amplified DNA fragment, DNA, was then recovered from the agarose gel using the AxyPrep DNA Gel Extraction Kit (AXYGEN). This DNA fragment was then ligated into the commercially available cloning vector pMD18T plasmid, after adding A to the termini using Takara Biotechnology Co., Ltd.'s rTaq DNA polymerase, to yield the recombinant plasmids URT94-1-pMD18T and URT94-2-pMD18T. The ligation product was transformed into commercially available Escherichia coli Top10 competent cells, and the transformed E. coli solution was spread onto an LB plate supplemented with 100 μg/mL ampicillin. Recombinant clones were verified by PCR and enzymatic digestion. One clone was individually selected, and the recombinant plasmid was extracted and sequenced. Verification revealed that URT94-1 and URT94-2 are glycosyltransferase genes, and their ORFs encode the PSPG box, a conserved functional domain of glycosyltransferase family 1.

The inventors performed expression and transglycosylation analyses on URT94-1 and URT94-2, respectively. Among them, glycosyltransferases (SEQ ID NO: 2 or 4, respectively) encoded by two nucleic acid sequences (SEQ ID NO: 1 and 3, respectively) were able to catalyze the elongation of one rhamnosyl residue at the C6 position of Rh1 to produce Rg2, and their catalytic activity was at least five-fold improved compared to that of the gGT29-7 mutant gGT29-7 (N343G, A359P) disclosed in a previous patent (PCT/CN2015/081111). Neither mutant was able to catalyze the elongation of one glucosyl residue at the C6 position of Rh1 to produce Rf.

The experimental results showed that ginseng-derived URT94-1 and URT94-2 catalyzed the elongation of one rhamnosyl at the C6 position of Rh1 to produce Rg2 at a conversion rate of over 50%, and both catalyzed the elongation of one rhamnosyl at the C6 position of Rg1 to produce Re at a conversion rate of over 50%. Neither enzyme was able to catalyze the elongation of one glucosyl at the C6 position of Rg1 to produce C20-O-Glc-Rf, demonstrating that they are glycosyltransferases with high specificity for UDP-rhamnose.

Example 2: Construction of recombinant expression plasmid for ginseng glycosyltransferase URT94s gene Using the pMD18T plasmid containing the URT94-1 and URT94-2 genes constructed in Example 1 as an example, the forward primer contained two parts, from the 5' end to the 3' end, containing a 20 bp sequence of the pET28a homologous arm and a 20 bp initiation sequence encoding URT94-1, and the reverse primer contained two parts, from the 5' end to the 3' end, containing a 20 bp sequence of the pET28a homologous arm and a 20 bp termination sequence encoding URT94-1 (SEQ ID NO: 9 to SEQ ID NO: 10, see Table 1). A gene encoding URT94-1 (including the pET28a homologous arm) was obtained by PCR amplification using the above primers. The DNA polymerase used was the high-fidelity DNA polymerase PrimeSTAR from Takara Biotechnology Co., Ltd. The PCR protocol, according to the manufacturer's instructions, was set as follows: 94°C for 2 min, 94°C for 15 s, 57°C for 30 s, 68°C for 1.5 min (a total of 33 cycles), 68°C for 10 min, and incubation at 16°C. The PCR product was detected by agarose gel electrophoresis, and a band of the same size as the target DNA was excised by UV irradiation. The DNA fragment was then recovered from the agarose gel using an AxyPrep DNA Gel Extraction Kit (AXYGEN).

Plasmid pET28a was double-digested with FD restriction endonucleases NcoI and SalI (Thermo Biotechnology). After incubation at 37°C for 50 minutes, the linearized plasmid pET28a was recovered from an agarose gel using an AxyPrep DNA Gel Extraction Kit (AXYGEN). The digested linearized plasmid was homologously recombined with two UGTs, including URT94-1, obtained above, using recombination enzymes (Shanghai Yisheng Biotechnology Co., Ltd.). The ligation product was transformed into E. coli BL21 (DE3) competent cells and plated on LB plates supplemented with 50 μg/mL kanamycin (Kana). Positive transformants were confirmed by colony PCR and sequenced to further verify the successful construction of the recombinant expression plasmid. The positive transformants were designated E. coli BL21-URT94-1 and BL21-URT94-2.

Example 3: Expression of ginseng glycosyltransferase URT94s in E. coli. Correctly sequenced E. coli BL21-URT94-1 and BL21-URT94-2 were each inoculated into 50 mL of LB medium and cultured at 37°C and 200 rpm until the OD600 reached approximately 0.6-0.8. The culture was then cooled to 4°C, and IPTG was added to a final concentration of 200 μM. Expression was induced for 16 hours at 18°C and 120 rpm. The cells were collected by centrifugation at 4°C, disrupted by sonication, and centrifuged at 12,000 g for 10 minutes at 4°C to collect the cell lysate supernatant, yielding a crude enzyme protein solution. A 6xHis tag was added to the C-terminus of proteins URT94-1 and URT94-2 using the 6xHis tag sequence on pET28a. The crude enzyme solutions of both proteins were subjected to Western blotting to detect protein expression. Anti-6xHis tag Western blot (Figure 2) showed a clear band between 45 and 55 kD, indicating that both glycosyltransferases URT94-1 and URT94-2 were expressed in soluble form in E. coli.

Example 4: In vitro transglycosylation activity of glycosyltransferase URT94s using protopanaxatriol-type saponin Rh1 as a substrate and identification of the product. The supernatants of cell lysates from recombinant E. coli BL21-URT94-1 and BL21-URT94-2 from Example 4 were used as crude enzyme solutions to carry out transglycosylation reactions, and the cell lysate of recombinant E. coli transformed with empty vector pET28a was used as a control. Ginseng glycosyltransferases gGT29-7 and gGT29-7 (N343G, A359P) derived from patent PCT/CN2015/081111 were selected as positive controls. In vitro transglycosylation experiments were performed according to the reaction system shown in Table 2, with the reaction mixture left at 35°C overnight.

Reaction results were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), respectively.

As shown in Figures 3a and 3b, when protopanaxatriol-type ginsenoside Rh1 was used as the glycosyl acceptor and UDP-Rha as the glycosyl donor, BL21-URT94-1 and BL21-URT94-2 catalyzed the synthesis of Rg2, and their catalytic efficiency was significantly superior to that of the previously reported glycosyltransferase gGT29-7 (N343G, A359P). Furthermore, the HPLC results were consistent with the TLC results.

Therefore, URT94-1 and URT94-2, like gGT29-7 (N343G, A359P), can catalyze the elongation of one rhamnose molecule at the C6-O-Glc of Rh1 to produce ginsenoside Rg2.

Example 5: In vitro transglycosylation activity of glycosyltransferase URT94s using protopanaxatriol-type saponin Rg1 as a substrate and identification of the product. Transglycosylation reactions were performed using the supernatants of cell lysates from recombinant E. coli BL21-URT94-1 and BL21-URT94-2 (Example 4) as crude enzyme solutions, and cell lysates from recombinant E. coli transformed with empty vector pET28a were used as controls. Ginseng glycosyltransferases gGT29-7 and gGT29-7 (N343G, A359P) derived from patent PCT/CN2015/081111 were selected as positive controls. In vitro transglycosylation experiments were performed according to the reaction system shown in Table 3, with the reaction mixture incubated overnight at 35°C.

Reaction results were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), respectively.

URT94-1 and URT94-2 catalyzed the production of Re using protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor. Their catalytic efficiency was significantly superior to that of the previously described glycosyltransferase gGT29-7 (N343G, A359P) (PCT/CN2015/081111). Furthermore, the HPLC results were consistent with the TLC results, as shown in Figures 4a and 4b.

Therefore, URT94-1 and URT94-2, like gGT29-7 (N343G, A359P), can catalyze the elongation of one rhamnose molecule at the C6-O-Glc of Rg1 to produce ginsenoside Re.

Example 6: In Vitro Glycosyltransferase Activity and Product Identification of URT94s, a Glycosyltransferase Using Protopanaxatriol-Type Saponins Rh1/Rg1 as Substrates and UDP-Glc as Glycosyl Donor. Transglycosylation reactions were performed using the supernatants of cell lysates from recombinant E. coli BL21-URT94-1 and BL21-URT94-2 (Example 4) as crude enzyme solutions, and cell lysates from recombinant E. coli transformed with empty vector pET28a were used as controls. Ginseng glycosyltransferases gGT29-7 and gGT29-7(N343G, A359P), derived from patent PCT/CN2015/081111, were selected as positive controls. In vitro transglycosylation experiments were performed according to the reaction system shown in Table 3, with the reaction mixture incubated overnight at 35°C. The reaction results were detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively.

When protopanaxatriol-type ginsenoside Rh1 was used as the glycosyl acceptor and UDP-Glc as the glycosyl donor, URT94-1 and URT94-2 were unable to catalyze the synthesis to produce Rf, and the HPLC results were consistent with the TLC results. Therefore, unlike gGT29-7 and gGT29-7 (N343G, A359P), the glycosyltransferases URT94-1 and URT94-2 of the present invention are unable to catalyze the elongation of one glucose molecule at C6-O-Glc of Rh1 to produce ginsenoside Rf, as shown in Figure 5.

When protopanaxatriol-type ginsenoside Rg1 was used as the glycosyl acceptor and UDP-Glc as the glycosyl donor, URT94-1 and URT94-2 were unable to catalyze the synthesis to produce C20-O-Glc-Rf, and the HPLC results were consistent with the TLC results. Therefore, unlike gGT29-7 and gGT29-7(N343G, A359P), the glycosyltransferases URT94-1 and URT94-2 of the present invention were unable to catalyze the elongation of one glucose molecule at C6-O-Glc of Rg1 to produce ginsenoside C20-O-Glc-Rf, as shown in Figure 6. URT94-1 and URT94-2 were shown to be glycosyltransferases with high specificity for UDP-rhamnose.

Example 7: Comparison of the Efficiency of URT94s in Catalyzing the Elongation of One Rhamnose Molecule at C6. The glycosyltransferase gGT29-7 derived from patent PCT/CN2015/081111 can elongate one glucose molecule at C6, while gGT29-7(N343G, A359P) can elongate one glucose molecule at C6 and also one rhamnose molecule at C6. The glycosyltransferases gGT29-7 and gGT29-7(N343G, A359P) of the present invention, and the glycosyltransferases URT94-1 and URT94-2 of the present invention were expressed according to the method of Example 4, and crude enzyme solutions were prepared. Using UDP-Rha as the glycosyl donor and Rh1 and/or Rg1 as the glycosyl acceptor, the enzyme-catalyzed reaction was carried out according to Example 5, reacted at 35°C for 1 hour, and the product was quantified by HPLC. The catalytic efficiency was calculated according to the following formula:
Conversion efficiency (%) = amount of product/(amount of substrate + amount of product)

As shown in Table 4, compared to the glycosyltransferases gGT29-7 and gGT29-7 (N343G, A359P) disclosed in patent PCT/CN2015/081111, URT94-1 and URT94-2 have improved activity in catalyzing the elongation of the glycan at the C6 position of Rh1 and/or Rg1 using UDP-rhamnose as the glycosyl donor.

Therefore, unlike previous glycosyltransferases, URT94-1 and URT94-2 of the present invention can specifically and efficiently add rhamnosyl to the first glycosyl at C-6 of a tetracyclic triterpene compound substrate, thereby elongating the sugar chain.

Example 8: Mutant proteins of highly efficient rhamnosyltransferase URT94-1 To further improve the catalytic activity of rhamnosyltransferase, the present inventors constructed a library of mutants for URT94-1 using random mutagenesis.

(1) Error-prone PCR
Using the rhamnosyltransferase URT94-1 gene sequence (SEQ ID NO: 1) as a template, error-prone PCR was performed with primers URT94-1_Pet28a-F (5'-ctttaagaaggagatataccatggataccaatgaaaaaacca-3' (SEQ ID NO: 9)) and URT94-1_Pet28a-R (5'-ctcgagtgcggccgcaagcttggggcatcgcttcccctggcctg-3' (SEQ ID NO: 10)). The error-prone PCR was performed using Stratagene's GeneMorph II Random Mutagenesis Kit. The PCR procedure was 95°C for 2 min, 95°C for 10 s, 55°C for 15 s, and 72°C for 2 min (a total of 28 cycles), and 72°C for 10 min, then reduced to 10°C. The template amount was 50 ng. The PCR product was recovered after agarose gel electrophoresis, and the error-prone PCR product of rhamnosyltransferase URT94-1 was obtained.

(2) Enzyme Expression The PCR product was ligated into pET28a plasmid (One-Step Cloning Kit, purchased from Shanghai Yisheng), and the ligation product was transformed into laboratory-prepared E. coli BL21 competent cells. The transformed E. coli solution was spread on an LB plate supplemented with 100 μg/mL kanamycin, and recombinant clones were verified by PCR. Several clones were individually selected, and recombinant plasmids were extracted and sequenced.

(3) Measurement of Enzyme Activity and Screening Several strains of Escherichia coli expressing the ginseng glycosyltransferase URT94-1 mutant obtained in step (2) were selected and each was inoculated into 50 mL of LB liquid medium and cultured at 37°C and 200 rpm until the OD600 reached 0.6 to 0.8. The medium was then induced with 0.2 mM IPTG and cultured at 16°C and 110 rpm for 18 hours. After recovering the bacterial cells at low temperature, the bacterial cells were reselected using 2 mL of 50 mM tris-HCl (pH 8.0) and disrupted using a cell disrupter to obtain a crude protein enzyme solution.

By comparing enzyme activity using the enzyme activity measurement reaction system shown in Table 3 and analyzing and screening numerous mutants, the inventors isolated one mutant exhibiting particularly high enzyme activity, designated URT94-1m1 (nucleic acid sequence as shown in SEQ ID NO: 13, protein sequence as shown in SEQ ID NO: 14). This mutant corresponds to wild-type URT94-1, but has a mutation from L to M at position 55 (L55M). The comparative activity of rhamnosyltransferase URT94-1 and its mutant URT94-1m1 is shown in Table 5.

According to Table 5, the catalytic activity of this mutant was significantly higher than that of URT94-1, with the efficiency of catalyzing Rh1 to synthesize Rg2 increasing to 92%, and the efficiency of catalyzing Rg1 to produce Re reaching 99%. See Table 5 and Figure 7 for details.

The inventors detected protein expression using Western blot analysis, and the results are shown in Figure 8, demonstrating that the mutant UR94-1m1 can be efficiently expressed.

All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, after reading the above content of the present invention, those skilled in the art may make various variations and modifications to the present invention, and it is understood that these equivalents are also within the scope of the claims of the present invention.

This application claims priority to an application filed on July 30, 2021, bearing Chinese application number 202110871374.0.

Claims (38)

A method for attaching rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, comprising transferring rhamnosyl using a specific glycosyltransferase that is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, or a conservative variant polypeptide having at least 95% sequence identity to said amino acid sequence. 2. The method of claim 1, wherein the rhamnosyl is provided by a glycosyl donor. The method of claim 2, wherein the glycosyl donor is a glycosyl donor having a rhamnose group. 3. The method of claim 2, wherein the glycosyl donor comprises uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, thymidine diphosphate-rhamnose, or a combination thereof. The tetracyclic triterpene compound is a compound of formula (I), and the compound with glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (II);
2. The method of claim 1, wherein R1 and R2 are H or glycosyl, R3 is a monosaccharide glycosyl, and R4 is rhamnosyl. 6. The method of claim 5, wherein the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl or rhamnosyl. The method of claim 5, characterized in that when R1 is H and R2 and R3 are glucosyl, the compound of formula (I) is ginsenoside Rg1 and the compound of formula (II) is ginsenoside Re; when R1 and R2 are H and R3 is glucosyl, the compound of formula (I) is ginsenoside Rh1 and the compound of formula (II) is ginsenoside Rg2. The tetracyclic triterpene compound is a compound of formula (III), and the compound with glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (IV);
The method according to claim 1, wherein R1 is H or glycosyl, R2, R3, and R4 are monosaccharide glycosyl, and R5 is rhamnosyl. 9. The method of claim 8, wherein the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl or rhamnosyl. The method according to claim 8, characterized in that when R1 is H, R2, R3 and R4 are glucosyl, and R5 is rhamnosyl, the compound of formula (III) is notoginsenoside R3 and the compound of formula (IV) is Yesanchinoside E. 1. Use of a specific glycosyltransferase for attaching rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, wherein the specific glycosyltransferase is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide having at least 95% sequence identity to said amino acid sequence . 12. The application according to claim 11, characterized in that the rhamnosyl is provided by a glycosyl donor. The application according to claim 12, characterized in that the glycosyl donor is a glycosyl donor having a rhamnose group. 13. The application of claim 12, wherein the glycosyl donor comprises uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, thymidine diphosphate-rhamnose, or a combination thereof. The tetracyclic triterpene compound is a compound of formula (I), and the compound with glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (II);
The use according to claim 11 , wherein R1 and R2 are H or glycosyl, R3 is monosaccharide glycosyl, and R4 is rhamnosyl. 16. The application according to claim 15, characterized in that the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl or rhamnosyl. The application described in claim 15, characterized in that when R1 is H and R2 and R3 are glucosyl, the compound of formula (I) is ginsenoside Rg1 and the compound of formula (II) is ginsenoside Re; when R1 and R2 are H and R3 is glucosyl, the compound of formula (I) is ginsenoside Rh1 and the compound of formula (II) is ginsenoside Rg2. The tetracyclic triterpene compound is a compound of formula (III), and the compound with glycosyl bonded to the glycosyl at the C-6 position is a compound of formula (IV);
The use of claim 11 , wherein R1 is H or glycosyl, R2, R3, R4 are monosaccharide glycosyl, and R5 is rhamnosyl. 19. The application according to claim 18, characterized in that the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl or rhamnosyl. The application described in claim 18, characterized in that when R1 is H, R2, R3 and R4 are glucosyl, and R5 is rhamnosyl, the compound of formula (III) is notoginsenoside R3 and the compound of formula (IV) is Yesanchinoside E. (a) obtaining a recombinant host cell by introducing into the host cell a reaction precursor of a tetracyclic triterpene compound or a construct for expressing/forming the precursor, or a specific glycosyltransferase or a construct for expressing the specific glycosyltransferase; the specific glycosyltransferase is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:
a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide having at least 95% sequence identity to said amino acid sequence ; wherein a glycosyl donor having a rhamnose group is present in said host cell or a glycosyl donor having a rhamnose group is introduced into said host cell;
(b) a method for attaching rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound in the cell, comprising culturing the recombinant host cell of (a) to obtain a product of the tetracyclic triterpene compound in which rhamnosyl is attached to the first glycosyl at the C-6 position. The method according to claim 21, characterized in that the reaction precursors of the tetracyclic triterpene compounds include ginsenoside Rg1, ginsenoside Rh1, and notoginsenoside R3 ; and the corresponding products include ginsenoside Re, ginsenoside Rg2, and Yesanchinoside E. 22. The method of claim 21, wherein the glycosyl donor comprises uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, thymidine diphosphate-rhamnose, or a combination thereof. A specific glycosyltransferase, which is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide having at least 95% sequence identity to said amino acid sequence . The conservative variant polypeptide is
(1) A polypeptide having the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, formed by the substitution, deletion, or addition of one or more amino acid residues, and having the function of binding rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound;
(2) A polypeptide having an amino acid sequence that is 95% or more identical to the polypeptide of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, and that has the function of linking rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound; or
(3) The specific glycosyltransferase according to claim 24, characterized in that it comprises a polypeptide formed by adding a tag sequence to the N-terminus or C-terminus of a polypeptide having a sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 14, or by adding a signal peptide sequence to the N-terminus thereof. 25. An isolated polynucleotide encoding the specific glycosyltransferase of claim 24 . A nucleic acid construct that expresses a specific glycosyltransferase, comprising the polynucleotide of claim 24 . A nucleic acid construct that expresses the specific glycosyltransferase of claim 24. The nucleic acid construct according to claim 27 or 28, characterized in that the nucleic acid construct is an expression vector or a homologous recombination vector. 29. A recombinant host cell that expresses the specific glycosyltransferase of claim 24 , or that contains the polynucleotide of claim 26 , or that contains the nucleic acid construct of claim 27 or 28 . The recombinant host cell of claim 30, characterized in that the recombinant host cell contains a reactive precursor of a tetracyclic triterpene compound or a construct that expresses/forms the same. 31. The recombinant host cell of claim 30, wherein a glycosyl donor having a rhamnose group is also present in the recombinant host cell or a glycosyl donor having a rhamnose group is introduced. The recombinant host cell of claim 31, characterized in that the reaction precursors of the tetracyclic triterpene compounds include ginsenoside Rg1, ginsenoside Rh1, and notoginsenoside R3; and the corresponding products include ginsenoside Re, ginsenoside Rg2, and Yesanchinoside E. 33. The recombinant host cell of claim 32, wherein the glycosyl donor comprises uridine diphosphate-rhamnose, uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, thymidine diphosphate-rhamnose, or a combination thereof. A polypeptide capable of binding rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, or a conservative variant polypeptide having at least 95% sequence identity with said amino acid sequence .
29. A kit for glycosyltransferase, comprising: the specific glycosyltransferase of claim 24 ; or the isolated polynucleotide of claim 26 ; or the nucleic acid construct of claim 27 or 28 . 36. A kit for transglycosylation according to claim 35, characterized in that it comprises a glycosyl donor having a rhamnose group. 37. The kit for transglycosylation according to claim 36, wherein the glycosyl donor comprises uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, or thymidine diphosphate-rhamnose. 36. A kit for transglycosylation according to claim 35, characterized in that it contains a reaction precursor of a tetracyclic triterpene compound.