JP2024528104A - Highly specific glycosyltransferase for rhamnose and its application - Google Patents

Abstract

The present invention provides a glycosyltransferase with high specificity for rhamnose and applications thereof. The present invention discloses for the first time a specific glycosyltransferase capable of catalyzing rhamnosylation at a specific position of a substrate and having high catalytic activity. Specifically, the specific glycosyltransferase of the present invention can specifically and efficiently catalyze glycosylation at the first glycosyl at the C-6 position of a tetracyclic triterpene compound substrate to extend the rhamnose group. The present invention also provides a mutant of the specific glycosyltransferase. The specific glycosyltransferase of the present invention has excellent specificity and high efficiency and can be applied to the construction of artificially synthesized ginsenosides, various kinds of novel ginsenosides and their derivatives, and has high application value in the fields of medicine, etc.
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Description

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

Ginsenosides are a general term for saponins isolated from plants of the Panax genus (such as Korean ginseng, Panax notoginseng, and American ginseng) and Jiaogulan, and are a type of triterpene compound. Based on their isolation source, ginsenosides are also called ginsenosides, notoginsenosides, and gypenosides. Ginsenosides are the main bioactive components of these medicinal plants. Currently, about 150 types of saponins have been isolated. From the structural point of view, ginsenosides are bioactive small molecules mainly formed after glycosylation of sapogenins. There are only a limited number of sapogenins in ginsenosides, mainly the dammarane-type tetracyclic triterpenes protopanaxadiol and protopanaxatriol, and oleanolic acid. After glycosylation, sapogenins can increase water solubility, change intracellular localization, and produce various biological activities. Most protopanaxadiol-type saponins are glycosylated at the hydroxyl groups at the C3 and/or C20 positions, whereas protopanaxatriol-type saponins are glycosylated at the hydroxyl groups at the C6 and/or C20 positions. Different types of glycosyl and degrees of glycosylation produce ginsenosides with various molecular structures.

Rhamnosylated ginsenosides have a wide range of biological activities. For example, Rg2 is a compound in which one molecule of rhamnose is extended to the C6-O-Glc of Rh1, and Rg2 has excellent effects in treating depression, improving cardiac function, enhancing learning and memory abilities, and preventing Alzheimer's disease. Ginsenoside Re is a compound in which one molecule of rhamnose is 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 by chemical, enzyme and microbial fermentation hydrolysis using total saponin or abundant saponin from ginseng or Sanchi ginseng as raw materials. Wild ginseng resources have been basically exhausted, so ginsenoside resources are currently obtained from the artificial cultivation of ginseng and Sanchi ginseng. However, artificial cultivation has a long growth cycle (generally more than 5 to 7 years), is geographically restricted, is often damaged by pests and diseases, and requires the spraying of large amounts of pesticides. Therefore, the artificial cultivation of ginseng and Sanchi ginseng has significant problems with continuous cropping (the plantation of ginseng or Sanchi ginseng needs to be left fallow for more than 5 to 15 years to overcome the problems of continuous cropping), and the yield, quality and safety of ginsenosides all face challenges.

The development of synthetic biology brings new opportunities for the heterologous synthesis of plant-derived natural products. By using yeast as a chassis and constructing and optimizing metabolic pathways, it is possible to synthesize artemisinic acid or dihydroartemisinic acid from inexpensive monosaccharides by fermentation, and then produce artemisinin by a one-step chemical conversion method, demonstrating the great potential of synthetic biology in the pharmaceutical synthesis of natural products. Heterologous synthesis of ginsenoside monomers is achieved using yeast chassis cells through synthetic biology techniques. The raw material is an inexpensive monosaccharide, and the preparation process is a safe and adjustable fermentation process, so any external pollution (e.g., pesticides used in the artificial cultivation of raw material plants) can be avoided. Preparing ginsenoside monomers through synthetic biology technology not only has cost advantages, but also ensures the quality and safety of the final product. Using synthetic biology technology to prepare a variety of high-purity natural and non-natural ginsenoside monomers in sufficient quantities for activity measurement and clinical trials, promoting the development of innovative medicines for rare ginsenosides.

In recent years, the analysis of the synthesis pathway of ginsenoside sapogenin has progressed greatly through transcriptomic and functional genomic studies of Korean ginseng, Panax notoginseng and American ginseng. In 2006, Japanese and Korean scientists respectively identified terpene cyclase elements (dammarenediol synthase, PgDDS) that convert epoxysqualene to dammarenediol. In 2011-2012, Korean scientists also identified cytochrome P450 elements CYP716A4 and CYP716A53v2 that oxidize dammarenediol to protopanaxadiol and further oxidize 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 the glycosyl of glycosyl donors (nucleoside diphosphate sugars such as UDP-glucose, UDP-rhamnose, UDP-xylose, UDP-arabinose, etc.) to different glycosyl acceptors. From the analysis of plant genomes sequenced so far, plant genomes often encode more than 100 different glycosyltransferases. In 2015, Chinese scholars identified a UDP-glycosyltransferase element (UGTPg100) that can transfer one glucosyl to the C6 position of protopanaxatriol. Chinese scholars have disclosed in a patent (PCT/CN2015/081111) glycosyltransferases (such as gGT29-7) that can carry out glycan elongation 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 basically cannot use UDP-Rha. The mutant gGT29-7 (N343G, A359P) of gGT29-7 disclosed in the patent (PCT/CN2015/081111) catalyzes the elongation of one molecule of rhamnosyl at the C6 position of Rh1 using UDP-Rha to produce Rg2, but its activity is very low, with a conversion rate of only about 9%. And gGT29-7 (N343G, A359P) can perform transglycosylation using UDP-Rha as a donor in addition to performing transglycosylation, and the catalytic efficiency is higher than that of the catalytic reaction using UDP-Rha as a glycosyl donor. Therefore, the activity of gGT29-7 (N343G, A359P) catalyzing UDP-Rha is low and not specific, so many by-products are synthesized and the application needs cannot be met.

In light of the above background, the present inventors have screened for glycosyltransferases URT94-1 and URT94-2 capable of extending UDP-rhamnose at the C6 position from ginseng, which can efficiently catalyze the extension of one molecule of rhamnose at the first glycosyl at the C-6 position of ginsenoside Rh1, ginsenoside Rg1, or Sanchi ginseng R3 using UDP-Rha as a glycosyl donor to obtain ginsenoside Rg2, ginsenoside Re, or Yesanchinoside E, respectively. However, URT94-1 and URT94-2 cannot catalyze the above saponin substrates using UDP-glucose as a glycosyl donor. Thus, 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 a first glycosyl at the C-6 position of a tetracyclic triterpene compound, comprising transferring the rhamnosyl with 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.
In another aspect of the present invention, there is provided an application of a specific glycosyltransferase for coupling rhamnosyl (including a catalyst for this reaction) to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, said specific glycosyltransferase being 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 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, said 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 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 types of groups, substrates or products are as follows:

In one or more embodiments, the types of groups, substrates or products are as follows:

In one or more embodiments, compounds (I) and (III) in the reaction formula include, but are not limited to, S- or R-configured dammarane-type tetracyclic triterpene compounds, lanolin-type tetracyclic triterpene compounds, apotirucallane-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 reaction scheme includes ginsenoside Rg2, ginsenoside Re, and Yesanchinoside E.

In another aspect of the 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 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 specific glycosyltransferase into a host cell; 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; 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; 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, thymidine diphosphate (TDP)-rhamnose.

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

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

In one or more embodiments, the additive for regulating enzyme activity is selected from Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al3+, 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⁺ , Al3+, Ni ²⁺ , Zn ²⁺ or Fe ²⁺ .

In one or more embodiments, the pH of the reaction system is pH 4.0 to 10.0, preferably pH 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 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, said conservative variant polypeptide is
(1) A polypeptide having a sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14, which is formed by the substitution, deletion or addition of one or more (e.g., 1 to 20, preferably 1 to 10, more preferably 1 to 5, 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 identical (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) to a polypeptide having a sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:14, and having the function of binding 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) a nucleotide sequence shown in SEQ ID NO: 1, 3, or 13, (B) a nucleotide sequence having at least 95% identity to the sequence shown 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' end and/or 3' end of the sequence shown in SEQ ID NO: 1, 3, or 13, (F) a complementary sequence of any one of the nucleotide sequences shown in (A) to (E), and (G) a 20 to 50 base fragment of the sequence shown 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, a nucleic acid construct is provided that contains the polynucleotide or expresses the specific glycosyltransferase; preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

In another aspect of the invention, a recombinant host cell is provided which expresses said specific glycosyltransferase or contains said polynucleotide or contains said nucleic acid construct; preferably, said recombinant host cell also contains a reactive precursor of a tetracyclic triterpene compound or a construct expressing/forming same; preferably, said recombinant host cell also contains a glycosyl donor having a rhamnose group or a glycosyl donor having a rhamnose group is introduced (including a construct/precursor capable of forming said 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 following treatment with the specific glycosyltransferase of the invention; e.g., is not a cell that naturally produces the compound of formula (II), (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 according to (a) or a mutant having 50% sequence identity to said 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 a 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 anabolic pathway of ginsenoside Rh1 include (but are not limited to) dammarenediol synthase gene, cytochrome P450 CYP716A47 gene, P450 CYP716A47 reductase gene, and tetracyclic triterpene C6 glycosyltransferase UGTPg100 (Genbank accession number AKQ76388.1), or combinations 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 reductase gene of P450 CYP716A47, and the glycosyltransferases UGTPg1 and UGTPg100 at the C20 and C6 positions of tetracyclic triterpenes (Genbank accession number AKQ76388.1), or combinations thereof.

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

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

In another aspect of the present invention, there is also provided the use of a host cell according to the present invention in the preparation of a glycosyltransferase, a catalytic reagent, or a compound of formula (II) or (IV).

In another aspect of the present invention, there is also provided a method for producing a glycosyltransferase or a compound of formula (II) or (IV), comprising incubating a host cell according to the present invention.

In another aspect of the invention, there is also provided the use of a host cell according to the invention for the preparation of an enzyme catalytic reagent, or for the production of a glycosyltransferase, or as a catalytic cell, or for the production of a compound of formula (II) or (IV).

In another aspect of the present invention, there is also provided a method for producing a transgenic plant, comprising the step of regenerating a host cell according to 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 glycosyltransfer is provided, which is capable of binding rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, and includes a specific glycosyltransferase that 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 another aspect of the present invention, a kit for glycosylation is provided, comprising the isolated polynucleotide.

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

In another aspect of the present invention, a kit for glycosylation is provided, 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 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. In order 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 glycosyltransferase 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 lysate supernatant of empty vector pET28a E. coli recombinant, Marker represents the molecular weight standard of protein, "2" represents the lysate supernatant of glycosyltransferase BL21-URT94-1 E. coli recombinant, "3" represents the lysate supernatant of glycosyltransferase BL21-URT94-2 E. coli recombinant, "4" represents the lysate supernatant of glycosyltransferase BL21-gGT29-7 E. coli recombinant, and "5" represents the lysate supernatant of glycosyltransferase BL21-gGT29-7 (N343G, A359P) E. coli recombinant. 3a shows the TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction with protopanaxatriol-type ginsenoside Rh1 as a glycosyl acceptor and UDP-Rha as a glycosyl donor. "1" represents the supernatant of the lysate of pet28a empty vector recombinant as an 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 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 glycosyl transfer reaction with protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor. 4a shows the TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction with protopanaxatriol-type ginsenoside Rg1 as a glycosyl acceptor and UDP-Rha as a glycosyl donor. "1" represents the supernatant of the lysate of pet28a empty vector recombinant as an 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 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 glycosyl transfer reaction using protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Rha as the glycosyl donor. 5 shows the TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction with protopanaxatriol-type ginsenoside Rh1 as the glycosyl acceptor and UDP-Glc as the glycosyl donor. "1" represents the supernatant of the lysate of 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. 6 shows the TLC spectra of glycosyltransferases URT94-1 and URT94-2, which catalyze the glycosyltransferase reaction with protopanaxatriol-type ginsenoside Rg1 as the glycosyl acceptor and UDP-Glc as the glycosyl donor. "1" represents the supernatant of the lysate of 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. 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 the wild type as detected by Western blotting.

As a result of intensive research and screening, the present inventors have provided, for the first time, a specific glycosyltransferase that can catalyze rhamnosylation at a specific position of 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 to extend the rhamnose group.

DEFINITIONS As used herein, an "isolated polypeptide" or an "active polypeptide" means that the polypeptide is essentially free of 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 invention and derivative polypeptides thereof", "enzyme of the 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, while they can 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, which 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 or a protein-constituting sequence, or a fusion protein formed with an antigen IgG fragment). As taught herein, these fragments, derivatives and analogs are within the knowledge of those skilled in the art.

As used herein, the term "variant" or "mutant" refers 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 have at least 50%, 60% or 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, 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. The sequence identity between the two sequences being compared can be calculated, for example, using BLASTp from NCBI. Mutants also include amino acid sequences that have one or more mutations (insertion, deletion, or substitution) 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 substitution is preferably a conservative substitution. For example, conservative substitution with amino acids having close or similar properties in the art usually does not change 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more sites in a polypeptide of the 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 URT94-1. In the present invention, a conservative variant polypeptide of URT94-1m may also be included, and this conservative variant polypeptide is conserved at the amino acid residue corresponding to position 55 of SEQ ID NO: 14.

Active Polypeptide, Gene Encoding the Same, 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 to elongate the glycan, and the reaction product has high application value in the fields of pharmaceuticals, etc.

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

In the present invention, the "conservative variant polypeptide" refers to a polypeptide that basically 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) are substituted, the substituted amino acid residues may or may not be encoded by the genetic code, (ii) a polypeptide having a substituent 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 or a protein-constituting sequence, or a fusion protein formed with an antigen IgG fragment). As taught herein, these fragments, derivatives and analogs are within the knowledge of those skilled in the art.

The "conservative variant polypeptide" includes, but is not limited to, one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids deleted, inserted and/or substituted, and one or more (e.g., 50 or less, preferably 20 or 10 or less, more preferably 5 or less) amino acids added or deleted at the C-terminus and/or N-terminus. For example, in the present field, substitution with amino acids having close or similar properties usually does not change the function of the protein. Also, for example, addition of one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. The present invention also provides analogs of the polypeptide. The difference between these analogs and the natural polypeptide may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced mutants can be obtained by a variety of techniques, including random mutations induced by radiation or exposure to mutagenic agents, or by site-directed mutagenesis or other known molecular biology techniques. Analogs further include analogs having residues different from natural L-amino acids (e.g., D-amino acids), and analogs having 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 URT94-1m (SEQ ID NO:14) or conservative variant thereof polypeptide of the present invention may also include one or more polypeptide fragments as a protein tag. Any suitable tag may be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags may be used for protein purification.

When used to produce a specific glycosyltransferase of the invention or other enzymes (e.g., enzymes used to react in a host cell to form a substrate for a specific glycosyltransferase of the invention, enzymes involved in any step of a product synthesis pathway of the invention), a signal peptide sequence can also be added to the amino terminus of the polypeptide of the 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.

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

The polynucleotides encoding the specific glycosyltransferases and other enzymes of the present invention may be in DNA or RNA form. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The term "polynucleotide encoding a polypeptide" may be 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 coding 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 one or more regulatory sequences or sequences required for homologous recombination of a genome operably linked to these sequences. The polynucleotides of the present invention may be manipulated in various ways to ensure expression of said polypeptide or protein. The nucleic acid construct may be manipulated according to differences or requirements of the expression vector before it is inserted into the vector. Techniques for modifying polynucleotide sequences by 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, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide coding sequences for the proteins or polypeptides of the invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Typically, expression of the polynucleotides of the invention is achieved by operably linking the polynucleotides 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. Exemplary 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 a genome. In addition to the polynucleotide sequence, the gene knock-in vector can typically include a 5' homology arm and a 3' homology arm required for homologous recombination of the genome. In some embodiments, the nucleic acid construct of the present specification includes 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 a desired position using CRISPR/Cas9 technology. The CRISPR/Cas9 technology involves designing a guide RNA for a target gene to guide Cas9 nuclease to modify the genome at the insertion position, increasing the efficiency of homologous recombination of the gene modification 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.

The construction of the nucleic acid construct can be carried out by methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to a suitable promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters include the lac or trp promoters of E. coli; 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 of controllable genes expressed in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site and a transcription terminator that are utilized for translation initiation. Additionally, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait of the host cell that is used for selection of 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 the polynucleotide of the present invention in higher eukaryotic cells, transcription is enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 base pairs, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer, 100-270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The present invention also provides a host cell for the biosynthesis of a product of interest. The host cell may be a prokaryotic cell, such as, but not limited to, E. coli, yeast, and Streptomyces, and is more preferably an E. coli cell. The cell host is a production tool, and a person skilled in the art can modify various host cells by any technical means to realize the biosynthesis of the present invention, and the host cell and the production method thus constructed are also 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. In general, the method includes 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 E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells such as fruit fly S2 or Sf9, animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc. A person skilled in the art will clearly know how to select the appropriate vector, promoter, enhancer, and host cell.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. The recombinant polypeptide in the above method is expressed intracellularly or at the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be isolated and purified by various isolation methods, taking advantage of 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 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 been devoted to the study of glycosyltransferases, in the research so far, no enzyme has been obtained that can efficiently utilize rhamnosyl donors and specifically bind rhamnosyl to the first glycosyl at the C-6 position of tetracyclic triterpene(s) compounds. Some of the existing enzymes basically cannot utilize rhamnosyl donors (e.g. UDP-Rha); some have very low activity and cannot fully meet the needs of applications.

Under the above background, the present inventors have screened from Korean ginseng and obtained a specific glycosyltransferase (URT94s) capable of extending rhamnose at the C6 position, which can efficiently catalyze the extension of one molecule of rhamnose in the first glycosyl at the C-6 position of protopanaxatriol saponins (protopanaxatriol-type saponins/protopanaxatriol-type saponins) such as ginsenoside Rh1, ginsenoside Rg1, and notoginsenoside R3, to obtain ginsenoside Rg2, ginsenoside Re, or Yesanchinoside E. The glycosyltransferase is a highly specific glycosyltransferase provided for efficiently preparing 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 compound in which R1-R4 are substituted is 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 comprising 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 can convert ginsenoside Rh1, in particular, to ginsenoside Rg2, which has other activities. The glycosyltransferase of the present invention can convert ginsenoside Rg1, in particular, to ginsenoside Re, which has other activities.

The active polypeptide or glycosyltransferase of the present invention can be used to artificially synthesize known ginsenosides, new ginsenosides and their derivatives, and can convert Rh1 to active ginsenoside Rg2 and Rg1 to 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, screening, in progeny of the plant, a transgenic positive plant comprising said polynucleotide or comprising said nucleic acid construct, 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 intermediate thereof, comprising a novel specific glycosyltransferase or conservative variant polypeptide thereof as set forth in SEQ ID NO: 2, 4 or 14; preferably further comprising a glycosyl donor therein; and preferably further comprising a host cell therein. More preferably, the kit also includes instructions describing the biosynthetic 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; the glycosyltransferase of the present invention can efficiently convert Rg1 to active ginsenoside Re. Rg2 is active in preventing and treating neurodegenerative diseases, and Re is active in lowering blood sugar levels and treating diabetes. Therefore, the glycosyltransferase of the present invention has a wide range of application value.
(3) High catalytic efficiency: Compared with the glycosyltransferases disclosed in patent PCT/CN2015/081111, URT94-1 and URT94-2 have at least 5-fold improved activity in catalyzing the elongation of the glycan 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 only for the purpose of illustrating the present invention 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., Science Press, or follow conditions recommended by the manufacturer.

Sequence information SEQ ID NO: 1 (URT94-1 nucleic acid)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagcaaaaaaactctcaaaacgaaatttttacatatact tttgttccacatctatcaatcttagttccatcaggaaaaaacttgcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctccccatctcattcctgatttgatcaaggcccttgg tatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccatatcaactgccgccgcctataggtttaaggtggatc ctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataatcttgatcaagagttctagagagttcgatgaaaa gaatatcgaatactattcccttttgatggacaagaaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaagaatcctcaacagttatgtttcttttgggagtgagtgct atttgtcagagcctaggatccgagagctggcccatggggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggtttcttgatagggtgaaagatagaggggtgattgtgag ttgggccccacaggaaagaatattagggcatggtggacttgggggatttgtgagt cattgtgggtgggtttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcatgctatgtttgtggaggggtgggcgttggcgtgg aggttctaaaagacgagagtggagaattttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaagaagaagtggaatgtgtagttgaggagttgaccaa actttgcaaaaagtatcagaaagtagcagcaggccaggggaagcgatgcccctaa

SEQ ID NO:2 (URT94-1 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKK VFQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDPSIPVPCSRIFLDDTNIRKSPDYD VHTENEKDDIMEWLSKKE ESSTVYVSFGSECYLSEPRIRELAHGLELSNVNFIWVISFPEGDEEMCNTCIEDVLPEGFLDRVKDRGVIVSWAPQERILGHGGLGGFVSHCGWGSVVEGMSYGVPIIAMPAQYEQPLHAM FVEEVGVGVEVLKDESGEFRRDEIAKAIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKRCP

SEQ ID NO: 3 (URT94-2 nucleic acid)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagcaaaaaaactctcaaaacgaaatttttacatatact tttgttccacatctatcaatcttagttccatcaggaaaaaacttgcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctccccatctcattcctgatttgatcaaggcccttgg tatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccatatcaactgccgccgcctataggtttaaggtggatc ctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataatcttgatcaagagttctagagagttcgatgaaaa gaatatcgaatactattcccttttgatggacaagaaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaagaatcctcaacagttatgtttcttttgggagtgagtgct atttgtcagagcctaggatccgagagctggcccatggggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggttttcttgatagggtgaaagatagagaggggtgattgtgag ttgggccccacaggaaagaatattagggcatggtggacttgggggatttgtgagt cattgtgggtgggtttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcatgctatgtttgtggaggaggtgggcgttggcgtgg aggttctaaaagacgagagcggagaatttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaagaagaagtggaatgtgtagttgaggagttgaccaa actttgcaaaaagtatcagaaagtagcagcaggccaggggaaggaatgcccctaa

SEQ ID NO: 4 (URT94-2 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKKLAVDDHEAIQLIEFQLTSQTELPPHHHTTKGLPPHLIPDLIKALGMSGPNVINILNTTVNPDLIIYDV FQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDPSIPVPCSRIFLDDTNIRKSPDYDSSSAENSGILDLTFGTAIQSSDIILIKSSREFDEKNIEYYSLLMDKKIVPTG PL V WGSVVEGMSYGVPIIAMPAQYEQPLHAMFVEEVGVGVEVLKDESGEFRRDEIAKAIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKECP

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)
ctcgagtgcggccgcaagcttggggcatcgcttccccctggccctg

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)
atggataccaatgaaaaaaccagaataaaagttgtaatgttaccatggctggcatatggtcacatatcaccctatctagagctagcaaaaaaactctcaaaacgaaatttttacatatact tttgttccacatctatcaatcttagttccatcaggaaaaaaatggcagttgatg atcacgaggcaatacagctgatagaattccagttaacttcacaaaccgagctgccgccgcaccatcacacaaccaaaggtctccctccccatctcattcctgatttgatcaaggcccttgg tatgtccggcccccaacgtcatcaacattctaaatacagtaaaccctgatttaatc atctacgatgtcttccagttatgggtgcctgcatttgcagcctctcttcaaatcccagctgtccatttccaagtagtcggagccatatcaactgccgccgcctataggtttaaggtggatc ctagtataccggttccttgttcaagaatctttctggatgacaccaacataagga aaagccccgattatgattcatcttcagcagaaaatagtggtattcttgaccttacatttggtacagctatacaatcgtcagatataatcttgatcaagagttctagagagttcgatgaaaa gaatatcgaatactattcccttttgatggacaagaaagattgtgcctacgggtcca cttgtacaagtcaacacatctgtggctgtccataccgaaaatgagaaggacgatataatggagtggctaagcaagaaagaagaatcctcaacagttatgtttcttttgggagtgagtgct atttgtcagagcctaggatccgagagctggcccatggggctagagcttagcaatg taaatttcatatgggttattagttttccagaggagagatgaggaaatgtgtaatacttgtattgaagatgtattaccggaaggtttcttgatagggtgaaagatagaggggtgattgtgag ttgggccccacaggaaagaatattagggcatggtggacttgggggatttgtgagt cattgtgggtgggtttctgtagtggaaggcatgagctatggagttccaataattgccatgcccgcgcaatatgaacagcctttgcatgctatgtttgtggaggggtgggcgttggcgtgg aggttctaaaagacgagagtggagaattttaggagggatgaaatagcaaaagcta taaaaaaggttgtggtggagaaaaatggagagaaggtgtgaggaagaaggcaagagagatgggcaaggcaataaaaaagagaggagaagaagaagaagtggaatgtgtagttgaggagttgaccaa actttgcaaaaagtatcagaaagtagcagcaggccaggggaagcgatgcccctaa

SEQ ID NO: 14 (URT94-1m1 protein)
MDTNEKTRIKVVMLPWLAYGHISPYLELAKKLSKRNFYIYFCSTSINLSSIRKK VFQLWVPAFAASLQIPAVHFQVVGAISTAAAYRFKVDPSIPVPCSRIFLDDTNIRKSPDYD VHTENEKDDIMEWLSKKE ESSTVYVSFGSECYLSEPRIRELAHGLELSNVNFIWVISFPEGDEEMCNTCIEDVLPEGFLDRVKDRGVIVSWAPQERILGHGGLGGFVSHCGWGSVVEGMSYGVPIIAMPAQYEQPLHAM FVEEVGVGVEVLKDESGEFRRDEIAKAIKKVVVEKNGEGVRKKAREMGKAIKKRGEEEVECVVEELTKLCKKYQKVAAGQGKRCP

Example 1: Cloning of Ginseng-Derived Glycosyltransferase URT94s As a result of intensive research and screening, the present inventors have cloned and obtained two glycosyltransferases, named 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. Using this cDNA as a template, two pairs of primers (SEQ ID NO:5-SEQ ID NO:6 amplify URT94-1; SEQ ID NO:7-SEQ ID NO:8 amplify URT94-2) were designed and amplified by PCR. 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. Next, the amplified DNA fragment DNA was recovered from the agarose gel using AxyPrep DNA Gel Extraction Kit (AXYGEN). The DNA fragment was added with A at the end using rTaq DNA polymerase from Takara Biotechnology Co., Ltd., and then ligated to the commercially available cloning vector pMD18T plasmid to obtain recombinant plasmids URT94-1-pMD18T and URT94-2-pMD18T. The ligation product was transformed into commercially available E. coli Top10 competent cells, and the transformed E. coli solution was spread on an LB plate containing 100 ug/mL ampicillin, and the recombinant clones were verified by PCR and enzymatic digestion. One of the clones was separately selected to extract the recombinant plasmid and determine the sequence. The 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 present inventors performed expression analysis and glycosyltransferase analysis for 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 at the C6 position of Rh1 to produce Rg2, and their catalytic activity was at least 5-fold improved compared with that of the mutant gGT29-7 (N343G, A359P) of gGT29-7 disclosed in a previous patent (PCT/CN2015/081111), and both were unable to catalyze the elongation of one glucosyl at the C6 position of Rh1 to produce Rf.

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

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

The plasmid pET28a was double digested with FD restriction endonucleases NcoI and SalI (Thermo), and after 50 min at 37°C, the linear plasmid pET28a was recovered from the agarose gel using AxyPrep DNA Gel Extraction Kit (AXYGEN). The enzyme-digested linear plasmid was homologously recombined with two UGTs such as URT94-1 obtained above using recombination enzymes (Shanghai Yisheng Biological Technology Co., Ltd.), and the ligation product was transformed into E. coli BL21 (DE3) competent cells and spread on LB plates supplemented with 50 μg/mL kanamycin (Kana). The positive transformants were confirmed by colony PCR and sequenced to further verify whether the recombinant expression plasmid was successfully constructed. The positive transformants were called 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 inoculated into 50 mL LB medium and cultured at 37°C and 200 rpm until OD600 reached about 0.6-0.8. The bacterial solution was cooled to 4°C, IPTG was added to a final concentration of 200 μM, and expression was induced for 16 h at 18°C and 120 rpm. The cells were collected by centrifugation at 4°C, disrupted by ultrasonic treatment, and centrifuged at 12000g, 10 min, and 4°C to collect the supernatant of the cell lysate, and a crude protein enzyme solution was obtained. 6×His tag was added to the C-terminus of each of the proteins URT94-1 and URT94-2 by the 6×His tag sequence on pET28a. The expression of both proteins was detected by Western blotting using crude enzyme solutions. 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 glycosyltransferase activity of URT94s using protopanaxatriol-type saponin Rh1 as a substrate and identification of the product. The supernatant of the cell lysate of recombinant E. coli BL21-URT94-1 and BL21-URT94-2 in Example 4 was used as a crude enzyme solution to carry out glycosyltransferase reaction, and the cell lysate of recombinant E. coli transformed with empty vector pET28a was used as a control. Ginseng glycosyltransferase gGT29-7 and gGT29-7 (N343G, A359P) derived from patent PCT/CN2015/081111 were selected as positive controls. In vitro glycosyltransferase test was carried out according to the reaction system shown in Table 2, and the reaction was carried out at 35°C overnight.

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

As shown in Figure 3a and b, when protopanaxatriol-type ginsenoside Rh1 was used as the glycosyl acceptor and UDP-Rha was used as the glycosyl donor, BL21-URT94-1 and BL21-URT94-2 catalyzed it to produce Rg2, and their catalytic efficiency was obviously superior to that of the previously disclosed glycosyltransferase gGT29-7 (N343G, A359P). Moreover, 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 glycosyltransferase activity of URT94s using protopanaxatriol-type saponin Rg1 as a substrate and identification of the product. The supernatant of the cell lysate of recombinant E. coli BL21-URT94-1 and BL21-URT94-2 in Example 4 was used as a crude enzyme solution to carry out glycosyltransferase reaction, and the cell lysate of recombinant E. coli transformed with empty vector pET28a was used as a control. Ginseng glycosyltransferase gGT29-7 and gGT29-7 (N343G, A359P) derived from patent PCT/CN2015/081111 were selected as positive controls. In vitro glycosyltransferase test was carried out according to the reaction system shown in Table 3, and the reaction was carried out at 35°C overnight.

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

When protopanaxatriol-type ginsenoside Rg1 was used as the glycosyl acceptor and UDP-Rha as the glycosyl donor, URT94-1 and URT94-2 catalyzed it to produce Re, and their catalytic efficiency was obviously superior to that of the previously disclosed glycosyltransferase gGT29-7 (N343G, A359P) (PCT/CN2015/081111). Moreover, the HPLC results were consistent with the TLC results, as shown in Figure 4a and b.

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 of URT94s using protopanaxatriol-type saponin Rh1/Rg1 as substrate and UDP-Glc as glycosyl donor and identification of product. The supernatant of the cell lysate of recombinant E. coli BL21-URT94-1 and BL21-URT94-2 in Example 4 was used as a crude enzyme solution to carry out glycosyltransferase reaction, and the cell lysate of recombinant E. coli transformed with empty vector pET28a was used as a control. Ginseng glycosyltransferase gGT29-7 and gGT29-7 (N343G, A359P) derived from patent PCT/CN2015/081111 were selected as positive controls. In vitro glycosyltransferase test was carried out according to the reaction system shown in Table 3, and the reaction was carried out 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 was used as the glycosyl donor, URT94-1 and URT94-2 could not catalyze it 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 cannot 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 was used as the glycosyl donor, URT94-1 and URT94-2 were unable to catalyze the synthesis of C20-O-Glc-Rf, and the HPLC results were consistent with the TLC results. Thus, 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 FIG. 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 extension of one molecule of rhamnose at C6 Glycosyltransferase gGT29-7 derived from patent PCT/CN2015/081111 can extend one molecule of glucose to C6, and gGT29-7(N343G, A359P) can extend one molecule of glucose to C6 and can also extend one molecule of rhamnose to C6. Glycosyltransferase gGT29-7, gGT29-7(N343G, A359P) of the present invention, and glycosyltransferase URT94-1, URT94-2 of the present invention were expressed according to the method of Example 4 to prepare a crude enzyme solution. UDP-Rha was used as a glycosyl donor, and Rh1 and/or Rg1 were used as a glycosyl acceptor, and the enzyme catalysis 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 a glycosyl donor.

Therefore, unlike conventional 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 to extend the glycan 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 carried out using 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 carried out 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, 72° C. for 2 min, total of 28 cycles, 72° C. for 10 min, then reduced to 10° C., and the amount of template used was 50 ng. The PCR product was recovered after agarose gel electrophoresis to obtain the error-prone PCR product of rhamnosyltransferase URT94-1.

(2) Expression of enzyme The PCR product was ligated into pET28a plasmid (One-Step Cloning Kit, purchased from Shanghai Yisheng), and the ligation product was transformed into E. coli BL21 competent cells prepared in the laboratory. The transformed E. coli solution was spread on an LB plate supplemented with 100ug/mL kanamycin, and the recombinant clones were verified by PCR. Several clones were selected separately to extract the recombinant plasmids and determine the sequences.

(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, 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, induced with 0.2 mM IPTG, and cultured at 16°C and 110 rpm for 18 hours. After recovering the cells at low temperature, the cells were reselected using 2 mL of 50 mM tris-HCl pH 8.0, and the cells were disrupted using a cell disrupter to obtain a crude protein enzyme solution.

By comparing the enzyme activities using the enzyme activity measurement reaction system in Table 3 and analyzing and screening a large number of mutants, the inventors obtained one mutant that exhibited particularly high enzyme activity, which was named URT94-1m1 (nucleic acid sequence as shown in SEQ ID NO: 13, protein sequence as shown in SEQ ID NO: 14), which corresponds to wild-type URT94-1 and has a mutation at position 55 from L to M (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 is significantly higher than that of URT94-1, the efficiency of catalyzing Rh1 to synthesize Rg2 increases to 92%, and the efficiency of catalyzing Rg1 to produce Re reaches 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 URT94-1m1 can be efficiently expressed.

All references related to the present invention are incorporated herein by reference as if each reference were incorporated individually. Furthermore, after reading the above content of the present invention, a person skilled in the art may make various variations and modifications to the present invention, and it is understood that equivalents of these variations are also included 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 (15)

A method for attaching rhamnosyl to a 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 shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:14, or a conservative variant polypeptide thereof. The method according to claim 1, characterized in that 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 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);
wherein R1 and R2 are H or glycosyl, R3 is monosaccharide glycosyl, and R4 is rhamnosyl; preferably, said glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
The method according to claim 1, characterized in that, preferably, when R1 is H, R2 and R3 are glucosyl, the compound of formula (I) is ginsenoside Rg1 and the compound of formula (II) is ginsenoside Re; and 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);
wherein R1 is H or glycosyl, R2, R3, R4 are monosaccharide glycosyl, and R5 is rhamnosyl; preferably, the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
The method according to claim 1, 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. Application of a specific glycosyltransferase for binding rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound, the specific glycosyltransferase being 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. The application according to claim 5, characterized in that 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 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);
wherein R1 and R2 are H or glycosyl, R3 is monosaccharide glycosyl, and R4 is rhamnosyl; preferably, said glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
The application according to claim 5, characterized in that when R1 is H, 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);
wherein R1 is H or glycosyl, R2, R3, R4 are monosaccharide glycosyl, and R5 is rhamnosyl; preferably, the glycosyl or monosaccharide glycosyl is selected from glucosyl, xylosyl, arabinosyl, or rhamnosyl;
Preferably, the compound of formula (III) is Notoginsenoside R3, and the compound of formula (IV) is Yesanchinoside E, when R1 is H, R2, R3 and R4 are glucosyl, and R5 is rhamnosyl. (a) obtaining a recombinant host cell by introducing a reaction precursor of a tetracyclic triterpene compound or a construct expressing/forming the precursor, or a specific glycosyltransferase or a construct expressing the precursor into a host cell; said 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; a glycosyl donor having a rhamnose group is present in said host cell or a glycosyl donor having a rhamnose group is introduced;
(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 a tetracyclic triterpene compound having rhamnosyl attached to the first glycosyl at the C-6 position. 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;
10. The method of claim 9, 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, comprising 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, formed by 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 50% 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 having the function of linking rhamnosyl to the first glycosyl at the C-6 position of a tetracyclic triterpene compound; or (3) a specific glycosyltransferase comprising 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. An isolated polynucleotide encoding the specific glycosyltransferase of claim 11. A nucleic acid construct comprising the polynucleotide of claim 11 or expressing the specific glycosyltransferase of claim 11; preferably, the nucleic acid construct is an expression vector or a homologous recombination vector. A recombinant host cell expressing a specific glycosyltransferase according to claim 11 or comprising a polynucleotide according to claim 12 or comprising a nucleic acid construct according to claim 13; preferably said recombinant host cell also comprising a reactive precursor of a tetracyclic triterpene compound or a construct expressing/forming same; preferably said recombinant host cell also comprises a glycosyl donor having a rhamnose group or a glycosyl donor having a rhamnose group has been introduced;
Preferably, 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;
Preferably, 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, the 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 thereof.
The specific glycosyltransferase of claim 7; or the isolated polynucleotide of claim 12; or the nucleic acid construct of claim 13; or the recombinant host cell of claim 14;
Preferably, it also comprises a glycosyl donor having a rhamnose group; more preferably, said glycosyl donor comprises uridine diphosphate-rhamnose, guanosine diphosphate-rhamnose, adenosine diphosphate-rhamnose, cytidine diphosphate-rhamnose, thymidine diphosphate-rhamnose;
Preferably, the kit for transglycosylation also comprises a reaction precursor of a tetracyclic triterpene compound.