WO2015188742A2 - Groupe de glycosyltransférase et utilisation dudit groupe - Google Patents

Groupe de glycosyltransférase et utilisation dudit groupe Download PDF

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WO2015188742A2
WO2015188742A2 PCT/CN2015/081111 CN2015081111W WO2015188742A2 WO 2015188742 A2 WO2015188742 A2 WO 2015188742A2 CN 2015081111 W CN2015081111 W CN 2015081111W WO 2015188742 A2 WO2015188742 A2 WO 2015188742A2
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ggt29
polypeptide
glycosyl
compound
ginsenoside
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PCT/CN2015/081111
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Chinese (zh)
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WO2015188742A3 (fr
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周志华
严兴
王平平
魏勇军
魏维
许云鹏
李晓东
杨成帅
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids

Definitions

  • the present invention relates to the field of biotechnology and plant biology, and in particular, the present invention relates to a novel set of glycosyltransferases and uses thereof.
  • Ginsenoside is a general term for saponins isolated from ginseng and its genus (such as Panax notoginseng, American ginseng, etc.) and belongs to triterpenoid saponins, which is the main active ingredient in ginseng. At present, at least 60 saponins have been isolated from ginseng, and some of them have been proven to have a wide range of physiological functions and medicinal properties: including anti-tumor, immune regulation, anti-fatigue, heart protection, liver protection and other functions.
  • the ginsenosides Rg3, Rf and Rg2 are all rare ginsenosides, each having a very strong physiological activity as described above.
  • ginsenoside Rg3 has excellent anti-tumor activity, can induce tumor cell apoptosis, and inhibit tumor cell metastasis. It can be combined with radiotherapy and chemotherapy to enhance the effects of radiotherapy and chemotherapy.
  • Ginsenoside Rf has anti-tumor and anti-fatigue effects, can reduce uterine contraction, and has an analgesic effect associated with brain nerve cells.
  • ginsenoside Rg2 has protective effect on the brain of rats with Alzheimer's disease, can enhance the learning and memory ability of rats, and has a repairing effect on myocardial injury. In addition, ginsenoside Rg2 also protects cells from UV damage. The role.
  • the method for producing such a rare ginsenoside is to start from a large amount of ginseng saponins in ginseng, and to perform extraction and purification by selectively hydrolyzing a glycosyl group.
  • the saponins of the genus Panax species or the original ginseng diol saponins are used as raw materials, and are chemically and enzymatically transformed, separated and extracted. Due to the large loss of raw materials in the chemical preparation method, the operation is cumbersome, and there are many by-products, which leads to an increase in cost and difficulty in improving the yield.
  • the acquisition of ginseng total saponins depends on the cultivation of ginseng, the market price of rare ginsenosides produced by conventional methods is high.
  • an in vitro glycosylation method comprising the steps of:
  • glycosyl donor glycosyl group is transferred to the following sites of the tetracyclic triterpenoid in the presence of a glycosyltransferase:
  • glycosyltransferase is a glycosyltransferase as shown in SEQ ID NO.: 61 or a polypeptide derived therefrom.
  • the extended sugar chain comprises a direct extension or a displacement extension.
  • the direct extension is to add a sugar group to the first sugar group at the C-3 and/or C-6 position to extend the sugar chain.
  • the substitution is extended to replace the terminal glycosyl group of the C-3 and/or C-6 sugar chain with a different glycosyl group from the C-3 and/or C-6 position.
  • the sugar chain is extended on the first glycosyl group.
  • the derivative polypeptide is selected from the group consisting of:
  • the amino acid sequence has a homology of SEQ ID NO.: 61 amino acid sequence ⁇ 85% (preferably ⁇ 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and a derivative polypeptide having glycosyltransferase activity;
  • glycosyltransferase activity refers to the ability to transfer a glycosyl donor glycosyl group to the first glycosyl group of the tetracyclic triterpenoid C-3 and/or C-6 to extend the activity of the sugar chain .
  • the derived polypeptide comprises a SEQ ID NO.: 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, The sequence shown in any of 92, 93, 94 or 95.
  • polypeptide selected from the group consisting of:
  • glycosyltransferase activity refers to the ability to transfer a glycosyl donor glycosyl group to the first glycosyl group of the tetracyclic triterpenoid C-3 and/or C-6 to extend the activity of the sugar chain .
  • sequence (c) is a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (a) or (b).
  • the derived polypeptide comprises a SEQ ID NO.: 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, A polypeptide of the amino acid sequence shown in 92, 93, 94 or 95.
  • an isolated polynucleotide having a sequence selected from the group consisting of:
  • the nucleotide sequence is SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87 , 89, or 91.
  • the sequence is as shown in SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
  • the polynucleotide encoding amino acid sequences are SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, respectively.
  • a vector comprising the polynucleotide of the third aspect.
  • the vector comprises an expression vector, a shuttle vector, an integration vector.
  • a fifth aspect of the invention there is provided the use of the isolated polypeptide of the first or second aspect of the invention, which is used to catalyze one or more of the following reactions, or is used to prepare one or more of the following Catalytic preparation for the reaction: transferring a glycosyl group derived from a glycosyl donor to the first glycosyl group at the C-3 position or the C6 position of the tetracyclic triterpenoid to extend the sugar chain; preferably,
  • the glycosyl donor comprises a nucleoside diphosphate selected from the group consisting of UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetyl Glucose, ADP-acetylglucose, TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, GDP-xylose , UDP-xylose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose , TDP-galactose, CDP-galactose, GDP-galactose, U
  • the glycosyl donor comprises a uridine diphosphate (UDP) sugar selected from the group consisting of UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, UDP-rhamnose, or other uridine diphosphate hexose or uridine pentose diphosphate, or a combination thereof.
  • UDP uridine diphosphate
  • the isolated polypeptide is used to catalyze one or more of the following reactions or to prepare a catalytic formulation that catalyzes one or more of the following reactions:
  • R1 is a glycosyl group
  • R2 and R3 are OH or H
  • R4 is a glycosyl group or H
  • R5 is a glycosyl group
  • R5-R1-O is a C3 first glycosyl group-derived glycosyl group, said polypeptide being selected from the group consisting of a polypeptide represented by SEQ ID NO.: 61 or a polypeptide derived therefrom; preferably, selected from the group consisting of 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, 94 or 95 or a polypeptide derived therefrom.
  • R1 is a glucosyl group
  • R2 is H
  • R3 is OH
  • R4 is H
  • the compound of the formula (I) is Rh2.
  • R1 is a glucosyl group
  • R2 is H
  • R3 is OH
  • R4 is a glucosyl group
  • the compound of formula (I) is F2.
  • the substrate (I) compound is Rh2, then the product of formula (II) is Rg3; and the substrate (I) compound is F2, then the product of formula (II) is Rd;
  • the substrate (I) compound is Rh2, and the product of formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)- PPD; the substrate (I) compound is F2, and the product of formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • R1 and R2 are H or a glycosyl group
  • R3 and R4 are a glycosyl group
  • R3-R4-O is the first glycosyl-derived glycosyl group of C6, and the polypeptide is selected from the polypeptide of SEQ ID NO.: 61 or a polypeptide derived therefrom, preferably SEQ ID NO.: 55, 57, 59, 78, 82, 92, 94 or 95 or a polypeptide derived therefrom.
  • the substrate (III) compound is Rh1
  • the product of formula (IV) is Rf
  • the substrate (III) compound is Rh1
  • the product of formula (IV) is Rg2.
  • R1 is a glycosyl group
  • R2 and R3 are OH or H
  • R4 is a glycosyl group or H
  • R5 is a glycosyl group
  • R5-R1-O is a glycosyl group derived from the first glycosyl group of C3
  • R6 is a glycosyl group
  • R6 -R1-O is the first glycosyl-derived glycosyl group of C3, said polypeptide being selected from the polypeptide of SEQ ID NO.: 61 or a polypeptide derived therefrom, preferably SEQ ID NO.: 26, 28, 59, 76 a polypeptide as shown in 84, 86 or 88.
  • R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is H, and the compound of formula (V) is Rg3.
  • R1 is two glucosyl groups
  • R2 is H
  • R3 is OH
  • R4 is a glucosyl group
  • the compound of formula (V) is Rd.
  • the substrate (V) compound is Rg3
  • the product of formula (VI) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)- PPD
  • the substrate (V) compound is Rd
  • the product of formula (VI) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • the glycosyl group is selected from the group consisting of: glucosyl, galacturonic acid, xylose, galactosyl, arabinose, rhamnosyl, and other hexose or pentose base.
  • the compound of the formula (I) or (III) includes, but is not limited to, a dammarane type tetracyclic triterpenoid of the S configuration or the R configuration, a lanolinane type Tetracyclic triterpenoids, apotirucallane type tetracyclic triterpenes, ganthanane type tetracyclic triterpenoids, cycloalkane (cycloaltenane) type tetracyclic triterpenoids, cucurbitane tetracyclic triterpenoids a compound or a decane type tetracyclic triterpenoid.
  • a dammarane type tetracyclic triterpenoid of the S configuration or the R configuration a lanolinane type Tetracyclic triterpenoids, apotirucallane type tetracyclic triterpenes, ganthanane type tetracyclic triterpenoids, cycloalkane (cycl
  • polypeptide is selected from the group consisting of:
  • polynucleotide encoding the nucleotide of the polypeptide is a sequence selected from the group consisting of:
  • (C) a nucleotide sequence as shown in SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91 ;
  • (F) a nucleotide sequence that is complementary (preferably fully complementary) to the nucleotide sequence of any of (A)-(E).
  • sequence of the nucleotide is SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
  • the sequence is as shown in SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
  • the polynucleotide encoding amino acid sequences are SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, respectively.
  • a method of performing a glycosyl transfer catalytic reaction comprising the steps of performing a glycosyl transfer catalytic reaction in the presence of the polypeptide of the second aspect of the invention or a polypeptide derived therefrom.
  • the method further includes the steps of:
  • the method further comprises separately adding the polypeptide and the polypeptide derived therefrom to a catalytic reaction; and/or
  • the polypeptide and its derived polypeptide are simultaneously added to the catalytic reaction.
  • the method further comprises ligating a nucleotide sequence encoding a glycosyltransferase with a key gene in a anabolic pathway of dammar diol and/or protopanaxadiol and/or protosol. / or other glycosyltransferase gene is co-expressed in a host cell to obtain the compound of formula (II), (IV), or (VI).
  • the host cell is a yeast or Escherichia coli.
  • the polypeptide has SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, A polypeptide of the amino acid sequence shown in 92, 93, 94 or 95 and a polypeptide derived therefrom.
  • the nucleotide sequence encoding the polypeptide is SEQ ID NO.: 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91 is shown.
  • the method further comprises: providing an additive for regulating enzyme activity to the reaction system.
  • the additive for regulating enzyme activity is an additive that increases enzyme activity or inhibits enzyme activity.
  • the additive for regulating enzyme activity is selected from the group consisting of Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ . , or Fe 2+ .
  • the additive for regulating enzyme activity is: Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ , Or a substance of Fe 2+ .
  • the glycosyl donor is a nucleoside diphosphate sugar selected from the group consisting of UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetyl Glucose, ADP-acetylglucose, TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, GDP-xylose , UDP-xylose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose , TDP-galactose, CDP-galactose, GDP-galactose,
  • the glycosyl donor is uridine diphosphate, selected from the group consisting of UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-Arabic Sugar, UDP-rhamnose, or other uridine diphosphate hexose or uridine pentose diphosphate, or a combination thereof.
  • the pH of the reaction system is: pH 4.0 to 10.0, preferably pH 5.5 to 9.0.
  • the temperature of the reaction system is from 10 ° C to 105 ° C, preferably from 20 ° C to 50 ° C.
  • the key genes in the darumadiol anabolic pathway include, but are not limited to, the damasenediol synthase gene.
  • the key genes in the proto-ginsengdiol anabolic pathway include, but are not limited to, a dammarenediol synthase gene, a cytochrome P450 CYP716A47 gene, and a reductase gene thereof, or Its combination. .
  • the key genes in the proto-ginsolic triol anabolic pathway include, but are not limited to, a dammarenediol synthase gene, a cytochrome P450 CYP716A47 gene, a reductase gene thereof, and a cell. Pigment P450 CYP716A53V2 gene and its reductase gene, or a combination thereof.
  • the key genes in the ginsenoside Rh2 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene and a reductase gene thereof, and a tetracyclic three ⁇ C-3 position glycosyltransferase gene UGTPg45, or a combination thereof.
  • the key genes in the ginsenoside Rh1 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene, a reductase gene thereof, and a cytochrome.
  • the substrate of the glycosyl catalyzed reaction is a compound of the formula (I) or (III), respectively, and the product is a compound of (II) or (IV), respectively;
  • the compound of formula (I) is ginsenoside Rh2, and the compound of formula (II) is ginsenoside Rg3;
  • the compound of the formula (I) is ginsenoside Rh2, and the compound of the formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD;
  • the compound of formula (I) is ginsenoside F2, and the compound of formula (II) is ginsenoside Rd;
  • the compound of the formula (I) is ginsenoside F2, and the compound of the formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK;
  • the compound of the formula (III) is ginsenoside Rh1, and the compound of the formula (IV) is ginsenoside Rf;
  • the compound of formula (III) is ginsenoside Rh1, and the compound of formula (IV) is ginsenoside Rg2;
  • the compound of the formula (V) is ginsenoside Rg3, and the compound of the formula (VI) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD;
  • the compound of formula (V) is ginsenoside Rd
  • the compound of formula (IV) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • a genetically engineered host cell comprising the vector of the fourth aspect of the invention, or a genome thereof, which integrates the polynucleoside of the third aspect of the invention acid.
  • the glycosyltransferase is a polypeptide as described in the second aspect of the invention or a polypeptide derived therefrom.
  • nucleotide sequence encoding the glycosyltransferase is as described in the third aspect of the invention.
  • the cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.
  • the host cell is a Saccharomyces cerevisiae cell.
  • the host cell is a prokaryotic cell, such as E. coli.
  • the host cell is a ginseng cell.
  • the host cell is not a cell that naturally produces a compound of formula (II), (IV), (VI), (VIII), (II), (IIII).
  • the host cell is not naturally producing a rare ginsenoside Rg3 and/or a rare ginsenoside Rf and/or a rare ginsenoside Rg2, and/or a new ginsenoside 3-O- ⁇ -(D- Cells such as xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD and 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • a rare ginsenoside Rg3 and/or a rare ginsenoside Rf and/or a rare ginsenoside Rg2 and/or a new ginsenoside 3-O- ⁇ -(D- Cells such as xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD and 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosy
  • the key genes in the darumadiol anabolic pathway include, but are not limited to, the damasenediol synthase gene.
  • the host cell contains key genes in the proto-glycol diol anabolic pathway including, but not limited to, the dammarenediol synthase gene, the cytochrome P450 CYP716A47 gene, and its reductase gene. , or a combination thereof.
  • the host cell contains key genes in the original ginseng triol anabolic pathway including, but not limited to, a dammarenediol synthase gene, a cytochrome P450 CYP716A47 gene, and a reductase thereof, P450 CYP716A47 reductase gene and its gene, or a combination thereof. .
  • the key genes in the ginsenoside Rh2 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene and a reductase gene thereof, and a tetracyclic three ⁇ C-3 position glycosyltransferase gene UGTPg45, or a combination thereof.
  • the key genes in the ginsenoside Rh1 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene, a reductase gene thereof, and a cytochrome.
  • the use of the host cell of the eighth aspect is provided 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 formula (II), (IV) Or (VI).
  • the host cell is used to produce a novel saponin 3-O- ⁇ -(D) by glycosylation of ginsenoside Rh2, F2, Rg3, Rd and/or ginsenoside Rh1, Rg1.
  • a method of producing a transgenic plant comprising the steps of: regenerating the genetically engineered host cell of the eighth aspect into a plant, and wherein the genetically engineered host cell is a plant cell.
  • the genetically engineered host cell is a ginseng cell.
  • Figure 1 shows agarose gel electrophoresis patterns of (a) gGT29/gGT29-3 gene and (b) gGT29-4/gGT29-5/gGT29-6 and gGT29-7 gene PCR products.
  • Lane 1 nucleic acid Marker; Lane 2, gGT29/gGT29-3 gene PCR product;
  • Lane 1 Lane 1, gGT29-4/gGT29-5/gGT29-6 gene PCR product;
  • Lane 2 gGT29-7 gene PCR Product; Lane 3, Nucleic Acid Marker.
  • Figure 2 shows SDS-PAGE detection of gGT29 and gGT29-3 expression in Saccharomyces cerevisiae; Lane 1, lysate supernatant of empty vector pYES2 recombinant; Lane 2, lysate supernatant of gGT29-pYES2 yeast recombinant; Lane 3 , lysate supernatant of gGT29-3-pYES2 yeast recombinant.
  • Figure 3 shows Western Blot detection of gGT29 and gGT29-3 expression in Saccharomyces cerevisiae; Lane 1, lysate supernatant of empty vector pYES2 recombinant; Lane 2, lysate supernatant of gGT29-pYES2 yeast recombinant; Lane 3, Lysate supernatant of gGT29-3-pYES2 yeast recombinant.
  • Figure 4 shows TLC detection profiles of glycosyltransferases gGT29 and gGT29-3 catalyzed products of ginsenoside Rh2 and F2; Lane 1, PPD and PPD type saponin mixed standards, Lane 2, gGT29 crude enzyme solution (gGT29-pYES2 yeast recombination) The lysate supernatant of the bacterium catalyzes the formation of Rg3 by Rh2, Lane 3, gGT29 crude enzyme solution catalyzes the Rh2 control, and adds pYES2 empty plasmid yeast recombinant lysate instead of the enzyme solution; Lane 4, gGT29 catalyzes the formation of Rd by F2, Lane 5, gGT29 catalysis F2 control, adding pYES2 empty plasmid yeast recombinant lysate instead of enzyme solution; Lane 6, gGT29-3 crude enzyme solution (gGT29-3-pYES2 yeast lysate supernatant) catalyzes Rh2
  • Figure 5 shows TLC detection of glycotransferase gGT29 and BvUGT73C10 or gGT29 and UGTPg45 in combination with PPD products;
  • gGT29 and BvUGT73C10 combined with catalytic PPD, lane 1, PPD and PPD type saponin mixed standards;
  • lane 2, BvUGT73C10 catalytic PPD Generation of Rh2;
  • Lane 3, gGT29 catalyzes the formation of Rg3 by Rh2;
  • Lane 4, BvUGT73C10 and gGT29 combine to catalyze the formation of Rg3 by PPD;
  • GGT29 and UGTPg45 combined with catalytic PPD, Lane 1, PPD and PPD type saponin mixed standards;
  • Lane 4, UGTPg45 and gGT29 combined with catalytic PPD to generate Rg3.
  • Figure 6 shows that the glycosyltransferases BvUGT73C10 and gGT29 catalyze the TLC detection of 20(R)-PPD and 20(R)-PPD and the combined catalyst 20(R)-PPD, respectively; Lane 1, BvUGT73C10 catalyzes 20(R)- PPD produces 20(R)-Rh2; Lane 2, gGT29 catalyzes the formation of 20(R)-Rh2 by 20(R)-Rg3; Lane 3, BvUGT73C10 and gGT29 combine to catalyze the formation of 20(R)-PP3 by 20(R)-PPD.
  • Figure 7 shows the results of HPLC detection of glycotransferases gGT29 and BvUGT73C10 or gGT29 and UGTPg45 in combination with catalytic PPD products.
  • the first row Rg3, Rh2 and PPD mixed standard samples; the second row, gGT29 and BvUGT73C10 combined catalytic PPD, the third row, gGT29 and UGTPg45 combined catalytic PPD.
  • Figure 8 shows the results of LC/MS detection of the products of glycosyltransferases gGT29 and BvUGT73C10 or gGT29 and UGTPg45 in combination with PPD.
  • the mass spectrum of the standard sample Rg3 and the mass spectrum of the P1 peak (product of gGT29 and BvUGT73C10 in combination with catalytic PPD) and the P2 peak (product product peak of gGT29 and UGTPg45 combined catalytic PPD) are shown in Fig. 7.
  • Figure 9 shows the results of HPLC detection of Rg3 yeast engineering strain A2 cell lysate extract, the first line of samples: original ginseng diol (PPD), dammar diol (DM), ginsenoside Rh2 and Rg3 mixing standards Sample; second row of samples: Rg3 yeast engineering bacteria A2 cell lysate extract.
  • Figure 10 shows the expression of gGT29-4, gGT29-5, gGT29-6, gGT29-7 in recombinant E. coli by SDS-PAGE.
  • Lane 1 gGT29-4-pET28a recombinant E. coli lysed total protein; lane 2, gGT29-4-pET28a recombinant E. coli lysate supernatant; Lane 3, gGT29-5-pET28a recombinant E. coli lysed total protein; Lane 4, gGT29-5-pET28a recombinant E. coli lysate supernatant; Lane 5, gGT29-6-pET28a recombinant E.
  • Figure 11 shows the expression of gGT29-4, gGT29-5, gGT29-6, gGT29-7 in recombinant E. coli by Western Blot.
  • Lane 1 gGT29-4-pET28a recombinant E. coli lysed total protein
  • Lane 2 gGT29-4-pET28a recombinant E. coli lysate supernatant
  • Lane 3 gGT29-5-pET28a recombinant E. coli lysed total protein
  • Lane 4 gGT29-5-pET28a recombinant E. coli lysate supernatant
  • Lane 5 gGT29-6-pET28a recombinant E.
  • Figure 12 shows that the glycosyltransferases gGT29-4, gGT29-5, gGT29-6, gGT29-7 catalyze the TLC detection of the products of Rh2 and F2, respectively.
  • Lane Rh2 indicates the use of saponin Rh2 as a substrate; and lane F2 indicates the use of saponin F2 as a substrate.
  • gGT29-4, gGT29-5, gGT29-6, gGT29-7 indicate that the catalytic reaction was carried out with different enzyme solutions.
  • Figure 13 shows that the glycosyltransferases gGT29-4, gGT29-5, gGT29-6, gGT29-7 catalyze the TLC detection of the product of Rh1, respectively.
  • Lanes 1, 2 and 3 represent the glycosyltransferases gGT29-4, gGT29-5 and gGT29-6 respectively catalyze the product of Rh1, and lane 4 represents the original ginsenotriol-type saponin mixed standard;
  • Lane 1 The product representing Rh1 is catalyzed by the glycosyltransferase gGT29-7, and the lane 2 represents the original ginseng triol type saponin mixed standard.
  • Figure 14 shows the results of the detection of Rh1 by the glycosyltransferase gGT29-7 and its mutant proteins gGT29-7-N343G, gGT29-7-A359P and gGT29-7-N343G/A359P (using both UDP-glucose and UDP-rhamnet) Sugar as a glycosyl donor).
  • Rg1, Rf, Rg2 and Rh1 are mixed with standard samples; the second row, gGT29-7 catalyzes Rh1; the third row, gGT29-7-N343G catalyzes Rh1; the fourth row, gGT29-7-A359P catalyzes Rh1; Five lines, gGT29-7-N343G/A359P catalyze Rh1.
  • Figure 15 shows TLC detection of glycosyltransferases gGT29-3 and gGT29-14 with UDP-xylose as a glycosyl donor, catalyzing the ginsenoside Rh2, Rg3 and Rd products;
  • A lysis of Enterobacteriaceae expressing the empty vector pET28a The supernatant was catalyzed as an enzyme solution;
  • B catalyzed by the supernatant of Enterobacter cloaca expressing pET28a-gGT29-3 as an enzyme solution;
  • C the supernatant of Enterobacter cloaca expressing pET28a-gGT29-14 was used as The enzyme solution is catalyzed.
  • Lanes Rh2, Rg3 and Rd represent the saponins Rh2, Rg3 and Rd as substrates, respectively, and lane M represents the original ginsengdiol-type saponin mixing standard.
  • glycosyltransferase gGT29-7 (SEQ ID NO.: 61) and its derived polypeptides for the first time, such as gGT29 (SEQ ID NO.: 26), gGT29-3 (SEQ ID NO) .: 28), gGT29-4 (SEQ ID NO.: 55), gGT29-5 (SEQ ID NO.: 57), gGT29-6 (SEQ ID NO.: 59), gGT29-8 (SEQ ID NO.: 72), gGT29-9 (SEQ ID NO.: 74), gGT29-10 (SEQ ID NO.: 76), gGT29-11 (SEQ ID NO.: 78), gGT29-12 (SEQ ID NO.: 80) , gGT29-13 (SEQ ID NO.: 82) gGT29-14 (SEQ ID NO.: 84), gGT29-15 (SEQ ID NO.: 86), gGT29-16 (SEQ ID NO.:
  • the glycosyltransferase of the present invention is capable of specifically and efficiently catalyzing the tetracyclic triterpenoid substrate and/or transferring a glycosyl group derived from a glycosyl donor to the C-3 position of a tetracyclic triterpenoid or
  • the first sugar group of C-6 is extended with a sugar chain.
  • ginseng Rh2 can be converted into rare ginsenoside Rg3 with anticancer activity
  • ginsenoside F2 can be converted into ginsenoside Rd
  • ginsenoside Rh1 can be converted into anti-tumor, anti-fatigue saponin Rf
  • ginsenoside Rh1 Transformation of rare ginsenoside Rg2 with neuroprotective effects and UV protection.
  • the invention also provides methods of transformation and catalysis.
  • the glycosyltransferase of the present invention may also be a key enzyme in the anabolic pathway of dammarane diol and/or protopanaxadiol or protopanaxatriol, and a glycosyltransferase of the tetracyclic triterpenoid C-3 or C-6 position.
  • the enzyme is co-expressed in the host cell or used in the preparation of genetically engineered cells of ginsenoside Rh2 and ginsenoside Rh1 for the construction of artificially synthesized ginsenoside Rg3 and Rf.
  • glycosyltransferase of the present invention may also be a key enzyme in the anabolic pathway of dammarane diol and/or protopanaxadiol or protopanaxadiol, and a glycosyltransferase at the C-6 position and a synthetic UDP-mouse.
  • the key enzyme of plum sugar is co-expressed in host cells and used to construct a strain of artificially synthesized ginsenoside Rg2. The present invention has been completed on this basis.
  • active polypeptide refers to glycosyltransferase gGT29-7 (SEQ ID NO.: 61) or a polypeptide derived therefrom.
  • preferred derivative polypeptides include gGT29 (SEQ ID NO.: 26), gGT29-3 (SEQ ID NO.: 28), gGT29-4 (SEQ ID NO.: 55), gGT29-5 (SEQ ID NO.: 57).
  • gGT29-6 SEQ ID NO.: 59
  • gGT29-8 SEQ ID NO.: 72
  • gGT29-9 SEQ ID NO.: 74
  • gGT29-10 SEQ ID NO.: 76
  • gGT29-11 SEQ ID NO.: 78
  • gGT29-12 SEQ ID NO.: 80
  • gGT29-13 SEQ ID NO.: 82
  • gGT29-14 SEQ ID NO.: 84
  • gGT29-15 SEQ ID NO.: 86
  • gGT29-16 SEQ ID NO.: 88
  • gGT29-17 SEQ ID NO.: 90
  • gGT29-18 SEQ ID NO.: 92
  • gGT29-7-N343G SEQ ID NO.: 93
  • gGT29-7-A359P SEQ ID NO.: 94
  • gGT29-7-N343G/A359P SEQ ID NO.: 95
  • the ginsenosides and sapogenins referred to herein are ginsenosides and sapogenins of the C20 position S and/or R configuration.
  • isolated polypeptide means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using amino acid sequences.
  • the active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants).
  • the polypeptide of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the polypeptide.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment).
  • a polypeptide having one or more conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It
  • the active polypeptide of the present invention has glycosyltransferase activity and is capable of catalyzing one or more of the following reactions:
  • R1 is a glycosyl group
  • R2 and R3 are OH or H
  • R4 is a glycosyl group or H
  • R5 is a glycosyl group
  • said polypeptide is selected from the group consisting of SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, 94 or 95 or a polypeptide derived therefrom.
  • R1 is a glucosyl group
  • R2 is H
  • R3 is OH
  • R4 is H
  • the compound of the formula (I) is Rh2.
  • R1 is a glucosyl group
  • R2 is H
  • R3 is OH
  • R4 is a glucosyl group
  • the compound of formula (I) is F2.
  • the substrate (I) compound is Rh2, then the product of formula (II) is Rg3; and the substrate (I) compound is F2, then the product of formula (II) is Rd;
  • the substrate (I) compound is Rh2, and the product of formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)- PPD; the substrate (I) compound is F2, and the product of formula (II) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • R1 and R2 are H or a glycosyl group
  • R3 and R4 are a glycosyl group
  • R3-R4-O is the first glycosyl-derived glycosyl group of C6, and the polypeptide is selected from the group consisting of SEQ ID NO.: 55, 57, 59, 61, 78, 82, 92, 94 or 95 or a polypeptide derived therefrom.
  • the substrate (IIII) compound is Rh1
  • the product of formula (IV) is Rf
  • the substrate (IIII) compound is Rh1
  • the product of formula (IV) is Rg2;
  • R1 is a glycosyl group
  • R2 and R3 are OH or H
  • R4 is a glycosyl group or H
  • R5 is a glycosyl group
  • R5-R1-O is a glycosyl group derived from the first glycosyl group of C3
  • R6 is a glycosyl group
  • R6 -R1-O is the first glycosyl-derived glycosyl group of C3
  • the polypeptide is selected from the group consisting of SEQ ID NO.: 26, 28, 59, 76, 84, 86 or 88 or a polypeptide derived therefrom.
  • R1 is two glucosyl groups, R2 is H, R3 is OH, R4 is H, and the compound of formula (V) is Rg3.
  • R1 is two glucosyl groups
  • R2 is H
  • R3 is OH
  • R4 is a glucosyl group
  • the compound of formula (V) is Rd.
  • the substrate (V) compound is Rg3
  • the product of formula (VI) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)- PPD
  • the substrate (V) compound is Rd
  • the product of formula (VI) is 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • the polypeptide sequence is SEQ ID NO.: 61 or a derivative thereof, preferably a derivative polypeptide 26, 28, 55, 57, 59, 72, 74, 76, 78, 80, 82, 84, 86, 88, a polypeptide as shown in 90, 92, 93, 94 or 95, the term further comprising SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76 having the same function as the polypeptide shown.
  • variations include (but are not limited to): one or more (usually 1-50, Preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually It is an amino acid of 20 or less, preferably 10 or less, more preferably 5 or less.
  • amino acid deletions usually 1-50, Preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually It is an amino acid of 20 or less, preferably 10 or less, more preferably 5 or less.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the proteins of the invention.
  • the invention also provides analogs of the poly
  • the difference between these analogs and the natural polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology. Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the gGT29-7 polypeptide of the present invention or a polypeptide derived therefrom for example, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29
  • the carboxy terminus may also contain one or more polypeptide fragments as a protein tag.
  • the tags can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE, and Ty1. These tags can be used to purify proteins. Table 1 lists some of these tags and their sequences.
  • the gGT29-7 polypeptide or a derivative thereof preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29, may also be used.
  • amino acid amino terminus of the amino acid is added with a signal peptide sequence, such as a pelB signal peptide.
  • the signal peptide can be cleaved off during secretion of the polypeptide from the cell.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 60 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a polypeptide having SEQ ID NO.: 61 or a derivative thereof, preferably SEQ ID NO.: 26, 28, 55, 57, 59, 72, 74.
  • a protein of 76, 78, 80, 82, 84, 86, 88, 90, 92, 93, 94 or 95 but with the coding sequence of SEQ ID NO.: 60 or a derivative thereof, preferably SEQ ID NO.: A nucleic acid sequence having a sequence different from 25, 27, 54, 56, 58, 60, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
  • Polynucleotides of mature polypeptides of 90, 92, 93, 94 or 95 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding) Sequence) and non-coding sequences.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions (or stringent conditions).
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • polypeptide encoded by the hybridizable polynucleotide is SEQ ID NO.: 26, 28, 55, 57, 59, 61, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92
  • the mature polypeptides shown in 93, 94 or 95 have the same biological function and activity.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate encoding gGT29-7 polypeptides or derived polypeptides thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29 -8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29 Polynucleotide of -7-A359P, or gGT29-7-N343G/A359P protein.
  • PCR nucleic acid amplification techniques
  • polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the gGT29-7 polypeptide of the present invention or a polypeptide derived therefrom preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29- 12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G
  • the full-length nucleotide sequence of gGT29-7-A359P or gGT29-7-N343G/A359P or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • RACE method RACE-cDNA end rapid amplification method
  • primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to a vector comprising a polynucleotide of the invention, and a vector of the invention or a gGT29-7 polypeptide or a derivative thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, A host cell produced by genetic engineering of the gGT29-7-A359P or gGT29-7-N343G/A359P protein coding sequence, and a method of producing a polypeptide of the present invention by recombinant techniques.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant gGT29-7 polypeptide or a derivative thereof by conventional recombinant DNA techniques, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29- 7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P polypeptide.
  • gGT29-7 polypeptide of the present invention or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/ A polynucleotide (or variant) of the A359P polypeptide, or a recombinant host vector comprising the polynucleotide, which is transformed or transduced into a suitable host cell;
  • the gGT29-7 polypeptide or a polypeptide derived therefrom is preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29. -12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P polynucleotide sequence It can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell diseases well known in the art. Toxic, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • gGT29-7-containing polypeptide or a derivative thereof preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29- 7-N343G/A359P An expression vector encoding a DNA sequence and a suitable transcription/translation control signal.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • promoters are: lac or trp promoter of E. coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti- Promoters for transcription of viral LTRs and other known controllable genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Usable examples include a 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, a polyoma enhancer on the late side of the replication initiation site, and an adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2.
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include but Not limited to: conventional renaturation treatment, treatment with protein precipitant (salting method), centrifugation, osmotic bacteria, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the active polypeptide or peptidyl transferase gGT29-7 polypeptide or derivative thereof according to the present invention is preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29- 10.
  • N343G/A359P Uses of N343G/A359P include, but are not limited to, the specific and efficient transfer of a glycosyl group from a glycosyl donor to the first glycosyl group at the C-3 and C-6 positions of the tetracyclic triterpenoid To extend the sugar chain.
  • ginsenoside Rh2 can be converted into rare ginsenoside Rg3 with better anticancer activity; ginsenoside F2 can be converted into ginsenoside Rd; ginsenoside Rh1 can be converted into rare ginsenoside Rf having antitumor and anti-fatigue activity; Ginsenoside Rh1 transforms rare ginsenoside Rg2 with neuroprotective effect and UV protection; the glycosyltransferase of the present invention can also synthesize ginsenoside Rh2, Rg3 and Rd into a novel saponin 3-O- ⁇ - (not previously reported) D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD and (3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK.
  • the tetracyclic triterpene compound includes, but is not limited to, a dammarane type, a lanolin type, a ganthanane type, a cycloalkane (cycloaltenane) type in the S configuration or the R configuration, a tetracyclic triterpenoid such as apotirucallane type, cucurbitane or decane type.
  • the present invention provides an industrial catalytic method comprising: using a gGT29-7 polypeptide of the present invention or a derivative thereof, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, under conditions providing a glycosyl donor , gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29
  • a compound of formula (II), (IV) or formula (VI) is obtained from -7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P active polypeptide or peptidyl transferase.
  • the polypeptide used in the (A) reaction is selected from the polypeptide represented by SEQ ID NO.: 61 or a polypeptide derived therefrom, preferably SEQ ID NO.: 26, 28, 55, 57, 59, 72, 74
  • the polypeptide used in the (B) reaction is selected from the group consisting of SEQ ID NO.: 55 , 57, 59, 61, 78, 82, 92, 94 or 95
  • the polypeptide used in the (C) reaction is selected from the polypeptide of SEQ ID NO.: 61 or a polypeptide derived therefrom, preferably SEQ ID NO.: 26, 28, 59, 76, 84, 86 or 88.
  • the glycosyl donor is a nucleoside diphosphate sugar selected from the group consisting of UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetylglucose, ADP-acetylglucose , TDP-acetylglucose, CDP-acetylglucose, GDP-acetylglucose, UDP-xylose, ADP-xylose, TDP-xylose, CDP-xylose, UDP-xylose, GDP-xylose, UDP -galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP - Galactose, GDP-galactose,
  • the glycosyl donor is preferably uridine diphosphate, selected from the group consisting of UDP-glucose, UDP-xylose, UDP-rhamnose, UDP-galacturonic acid, UDP-galactose, UDP-Arabic Sugar, or other uridine diphosphate Acid hexose or uridine pentose diphosphate, or a combination thereof.
  • uridine diphosphate selected from the group consisting of UDP-glucose, UDP-xylose, UDP-rhamnose, UDP-galacturonic acid, UDP-galactose, UDP-Arabic Sugar, or other uridine diphosphate Acid hexose or uridine pentose diphosphate, or a combination thereof.
  • an enzyme active additive (an additive that increases enzyme activity or inhibits enzyme activity) may also be added.
  • the enzyme activity additive may be selected from the group consisting of Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ , or Fe 2+ ; a substance of Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ , or Fe 2+ .
  • the pH conditions of the process are: pH 4.0-10.0, preferably pH 6.0-pH 8.5, more preferably 8.5.
  • the temperature conditions of the process are from 10 ° C to 105 ° C, preferably from 25 ° C to 35 ° C, more preferably 35 ° C.
  • the present invention also provides a composition comprising an effective amount of the active polypeptide or peptidyl transferase gGT29-7 polypeptide of the present invention or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29- 7-N343G, gGT29-7-A359P or gGT29-7-N343G/A359P, and a food or industrially acceptable carrier or excipient.
  • Such carriers include, but are not limited to, water, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • Substances which modulate the glycosyltransferase activity of the present invention may also be added to the composition. Any substance having a function of increasing the activity of the enzyme is available. Preferably, the substance which increases the glycosyltransferase activity of the present invention is selected from the group consisting of mercaptoethanol.
  • many substances can reduce enzyme activity, including but not limited to: Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ , and Fe 2+ ;
  • Substrate can be hydrolyzed to form Ca 2+ , Co 2+ , Mn 2+ , Ba 2+ , Al 3+ , Ni 2+ , Zn 2+ and Fe 2+ .
  • the enzyme can be conveniently applied by a person skilled in the art to function as a transglycosyl group, particularly for the transglycosylation of dammar diol, protopanaxadiol and protopanaxatriol.
  • rare ginsenosides there are also provided two methods for forming rare ginsenosides, one of which comprises: using the gGT29-7 polypeptide of the present invention or a derivative thereof, preferably gGT29, gGT29-3, gGT29- 4.
  • the gGT29-7 polypeptide or a polypeptide derived therefrom is used under the condition of pH 3.5-10.
  • gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G or gGT29-7- A359P or gGT29-7-N343G/A359P enzyme treats the substrate to be transglycosylated.
  • the gGT29-7 polypeptide or a polypeptide derived therefrom is used at a temperature of 30-105 °C.
  • the second method comprises: the gGT29-7 polypeptide of the present invention or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29 -10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, gGT29-7-A359P or gGT29-7 -N343G/A359P gene transfer can be synthesized In the engineered bacteria of saponin Rh2 or Rh1 (for example, yeast or Escherichia coli engineering bacteria), or the gGT29-7 polypeptide or a polypeptide derived therefrom, preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29- 6,
  • the gGT29-7 polypeptide or a polypeptide derived therefrom preferably gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29- 12.
  • gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29-18, gGT29-7-N343G, or gGT29-7-A359P, or gGT29-7-N343G/A359P gene Key enzymes in the anabolic pathway of methenediol and/or protopanaxadiol or protosol ginseng, and glycosyltransferases at the C-6 position of tetracyclic triterpenes and key enzymes for the synthesis of UDP-rhamnose in host cells Expression, applied to the construction of recombinant strains of synthetic ginsenoside R
  • the key genes in the dammar diol anabolic pathway include, but are not limited to, the dammarene diol synthase gene.
  • the key genes in the proto-ginsengdiol anabolic pathway include, but are not limited to, a dammarenediol synthase gene, a cytochrome P450 CYP716A47 gene, and a reductase gene thereof, or combination. Or isozymes of various enzymes and combinations thereof.
  • dammarene diol synthase converts squalene (Saccharomyces cerevisiae self-synthesis) into dammarene diol
  • cytochrome P450 CYP716A47 and its reductase convert dammarene diol into protocanthodiol .
  • the key genes in the proto-ginsolic triol anabolic pathway include, but are not limited to, a dammarenediol synthase gene, a cytochrome P450 CYP716A47 gene, a reductase gene thereof, and a cell. Pigment P450 CYP716A53V2 gene and its reductase gene, or a combination thereof. Or isozymes of various enzymes and combinations thereof.
  • dammarene diol synthase converts squalene (Saccharomyces cerevisiae self-synthesis) into dammarene diol
  • cytochrome P450 CYP716A47 and its reductase convert dammarene diol into protocanthodiol
  • the cytochrome P450 CYP716A53v2 (JII036031) and its reductase further convert the original ginseng diol to the original ginseng triol.
  • the key genes in the ginsenoside Rh2 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene and a reductase gene thereof, and a tetracyclic three ⁇ C-3 position glycosyltransferase UGTPg45 or a combination thereof. Or isozymes of various enzymes and combinations thereof.
  • dammarene diol synthase converts squalene (Saccharomyces cerevisiae self-synthesis) into dammarene diol
  • cytochrome P450 CYP716A47 and its reductase convert dammarene diol into protocanthodiol
  • the glycosyltransferase UGTPg45 can further convert protoglycan diol to Rh2 (Wang et. al, Metabolic Engineering, 2015, 29.97-105).
  • the key genes in the ginsenoside Rh1 anabolic pathway include, but are not limited to, a dammarene diol synthase gene, a cytochrome P450 CYP716A47 gene, a reductase gene thereof, and a cytochrome.
  • dammarene diol synthase converts squalene (Saccharomyces cerevisiae self-synthesis) into dammarane diol
  • cytochrome P450 CYP716A47 and its reductase convert dammarene diol into proto-ginseng diol
  • cytochrome P450 CYP716A53v2 JII036031
  • reductase to further convert proto-ginseng diol into proto-ginstriol
  • glycosyltransferase UGTPg100 The original ginseng triol was further converted to Rh1 (Wei et. al, Molecular Plant, 2015, 15. doi: 10.1016/j.molp. 2015.05.010).
  • the glycosyltransferase of the present invention can specifically and efficiently transfer a glycosyl group derived from a glycosyl donor to the first sugar at the C-3 position and/or the C-6 position of the tetracyclic triterpenoid Extended sugar chain
  • the glycosyltransferase of the present invention is particularly capable of converting ginsenoside Rh2 and Rh1 into rare ginsenoside Rg3 having better anticancer activity, and ginsenoside Rf having antitumor and anti-fatigue effects, respectively, and having neuroprotection
  • ginsenoside Rg2 in the role of UV protection
  • a synthetic route of ginsenosides (Damadiol, Protopanaxadiol, and Protopanaxatriol) or a synthetic route of rare ginsenoside Rh2 or Rh1 was constructed in yeast to achieve a monosaccharide such as glucose. Fermentation with yeast to produce rare ginsenosides Rg3 and Rf.
  • the synthetic route of rare ginsenoside Rh1 and the route of synthesizing UDP-rhamnose were constructed in yeast, and yeast was used to ferment and produce rare ginsenoside Rg2. This not only solves the problem of the source of raw materials for saponin production, but also greatly reduces the production cost of the rare saponins Rg3, Rg2 and Rf.
  • More than 100 cDNA sequences predicted to be glycosyltransferases were extracted from the published expression data of Panax species, and 60 full-length cDNA sequences were cloned and expressed and transglycosylated. 17 of them were analyzed. The expression product has transglycosylation activity on ginsenosides and saponins.
  • Ginseng RNA was extracted and reverse transcribed to obtain cDNA of ginseng.
  • PCR amplification was carried out using this cDNA as a template, using primer pair 1 (SEQ ID NO.: 7, 8); primer pair 2 (SEQ ID NO.: 9, 10); primer pair 3 (SEQ ID NO.: 11) , 12); Primer Pair 5 (SEQ ID NO.: 34, 35); Primer Pair 7 (SEQ ID NO.: 46, 47); Primer Pair 8 (SEQ ID NO.: 62, 63); Primer Pair 9 ( SEQ ID NO.: 64, 65) All obtained amplification products.
  • DNA polymerase uses the high-fidelity KOD DNA polymerase from Biotech Engineering Co., Ltd.
  • the PCR product was detected by agarose gel electrophoresis (Fig. 1). Irradiate in the ultraviolet and cut off the target DNA band. The amplified DNA fragment was then recovered from the agarose gel using an AIIygen Gel EIItraction Kit (AEYGEN). This DNA fragment was ligated with the commercially available cloning vector pMD18-T Vector after the end of A with the rTaq DNA polymerase of Biosciences Co., Ltd., and the ligated product was transformed into a commercially available E. coli EPI300 competent cell, and the transformed large intestine was transformed.
  • a commercially available cloning vector pMD18-T Vector after the end of A with the rTaq DNA polymerase of Biosciences Co., Ltd.
  • the Bacillus solution was coated on an LB plate supplemented with ampicillin 50 ug/mL, IPTG 0.5 mM, II-Gal 25 ⁇ g/mL, and the recombinant clone was further verified by PCR and restriction enzyme digestion.
  • One of the clones was selected to extract the recombinant plasmid and then sequenced.
  • the open reading frame (ORF) was searched using the BESTORF software. By sequence alignment, the ORF encodes a PSPG cassette of the first functional conserved domain of the glycosyltransferase, indicating a glycosyltransferase gene.
  • the gene obtained with primer pair 5 (SEQ ID NO.: 34, 35) has the nucleotide sequence shown by SEQ ID NO.: 25, 27, and is named gGT29 and gGT29-3, respectively. Among them, the corresponding genetic information is shown in Table 2.
  • the nucleotide sequence of the gGT29-7-derived polypeptide having the gene shown in Table 3 was obtained using primer pair 7 (SEQ ID NO.: 62, 63) and designated as gGT29-4, gGT29-5, gGT29-6, gGT29-8. , gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-17 and gGT29-18.
  • the corresponding genetic information is shown in Table 3.
  • the gene obtained with primer pair 8 (SEQ ID NO.: 64, 65) had the nucleotide sequences shown in Table 4 and designated as gGT29-7, gGT29-15 and gGT29-16. Among them, the corresponding genetic information is shown in Table 4.
  • the target genes were amplified using the plasmids gGT29-pMD18T and gGT29-3-pMD18T containing the gGT29 and gGT29-3 genes constructed in Example 1 as templates.
  • the forward primers used for gGT29 were (SEQ ID NO.: 36), and the Kpn I recognition site was added to the 5' end: GGATCC; the reverse primers used were all (SEQ ID NO.: 37), and the 5' end was added with IIhoI.
  • the forward primers used for gGT29-3 were (SEQ ID NO.: 38), and the Kpn I recognition site was added to the 5' end: GGATCC; the reverse primers used were all (SEQ ID NO.: 39), and the 5' end The IIhoI recognition site was added: CTCGAG, and the reverse primer introduced 6-His Tag for Western Blot detection expression and purification.
  • DNA polymerase was selected from Toyobo's high-fidelity DNA polymerase kod, and the PCR program was set up according to the instructions: 94 ° C for 2 min; 94 ° C for 15 s, 58 ° C for 30 s, 68 ° C for 1.5 min, for a total of 30 cycles; 68 ° C for 10 min; 10 ° C Keep warm.
  • the PCR product was detected by agarose gel electrophoresis, and the band of the same size as the target DNA was cut under ultraviolet light.
  • the DNA fragment was then recovered from the agarose gel using AIIYGEN's AIIyPrep DNA Gel EIItraction Kit. Using Takara The DNA fragments recovered by double-cutting of the QuickCut restriction enzymes Kpn I and IIba I were used for 30 min, and the digested products were cleaned and recovered using AIIYGEN's AIIyPrep PCR Cleanup Kit.
  • the digested product was ligated with S. cerevisiae expression plasmid pYES2 (also cut with Kpn I and IIba I and tapped and recovered) using NEB T4 DNA ligase for 2 h at 25 °C. The ligation product was transformed into E.
  • the constructed expression plasmids gGT29-pYES2 and gGT29-3-pYES2 were transformed into Saccharomyces cerevisiae by electroporation and plated on SC-Ura (0.67% yeast amino acid-free basic nitrogen source, 2% glucose). ). Yeast recombinants were verified by colony PCR. Yeast recombinant colonies were picked up in 10 mL of SC-Ura (2% glucose) medium and incubated at 30 ° C for 200 h at 200 rpm.
  • the cells were collected by centrifugation at 3500 g at 4 ° C, the cells were washed twice with sterile deionized water, and the cells were resuspended in induction medium SC-Ura (2% galactose) and inoculated into 50 mL of induction medium to make OD 600. At about 0.4, induction was initiated at 30 ° C at 200 rpm. The cells induced to express for 12 hours were collected by centrifugation at 3500 g at 4 ° C, and the cells were washed twice with sterile deionized water and resuspended in yeast lysis buffer to give an OD 600 between 50 and 100.
  • induction medium SC-Ura 2% galactose
  • the yeast cells were disrupted by shaking with a Fastprep cell disrupter, and the cell debris was removed by centrifugation at 12000 g for 10 min at 4 ° C, and the supernatant of the cell lysate was collected. The appropriate amount of lysate supernatant was subjected to SDS-PAGE electrophoresis. Compared with the pYES2 empty vector recombinant, the gGT29-pYES2 and gGT29-3-pYES2 recombinants showed no significant banding (Fig. 2). The expression was detected by anti-6-His Tag Western Blot.
  • Saccharomyces cerevisiae expressing gGT29 and gGT29-3 showed strong Western Blot signal, indicating that both gGT29 and gGT29-3 were soluble in yeast, and transferred to pYES2 empty vector. The recombinant did not have an anti-6-His Tag Western Blot signal (Figure 3).
  • the recombinant yeast cleavage supernatant expressing gGT29 and gGT29-3 was used as an enzyme solution to catalyze the transglycosylation reaction of ginsenoside Rh2 and F2, and the recombinant yeast cleavage supernatant expressing empty vector was used as a control.
  • the 100 ⁇ L reaction system is shown in Table 3. The reaction was carried out at 35 ° C for 12 h, then 100 ⁇ L of butanol was added to terminate the reaction, and the product was extracted. The product was dried under vacuum and dissolved in methanol.
  • the reaction product was first detected by thin layer chromatography (TLC), and the yeast host cleavage supernatant solution expressing gGT29 and gGT29-3 can be further extended into a glycosyl group at the 3-position glycosyl group of ginsenoside Rh2 and F2 to be converted into ginsenoside. Rg3 and Rd ( Figure 4).
  • the catalytic activity of gGT29 and gGT29-3 is not affected by the glycosylation or hydroxyl configuration of ginsenoside 20, and 20(R)-Rh2 can be converted to 20(R)-Rg3 (Fig. 6).
  • the E. coli host cleavage supernatant expressing BvUGT73C10 (JQ291613) or UGTPg45 (KM401918) and the yeast host cleavage supernatant expressing gGT29 were used as an enzyme solution to co-catalyze the original ginseng diol (PPD).
  • the 100 ⁇ L reaction system is shown in Table 3. 40 ⁇ L of the 73.4 ⁇ L enzyme solution was the large intestine host lysis supernatant of BvUGT73C10, and the remaining 33.4 ⁇ L was the yeast host cleavage supernatant expressing gGT29.
  • the reaction was carried out at 35 ° C for 12 h, then 100 ⁇ L of butanol was added to terminate the reaction, and the product was extracted. The product was dried under vacuum and dissolved in methanol. The reaction product was first detected by thin layer chromatography (TLC) (Fig. 5). It can be seen that the combination of glycosyltransferase BvUGT73C10 and gGT29 or UGTPg45 and gGT29 can convert PPD to Rg3.
  • TLC thin layer chromatography
  • Glycosyltransferase BvUGT73C10 and gGT29 or a combination of 3GT2 and gGT29 can catalyze 20(R)-PPD to form 20(R)-Rg3 ( Figure 6).
  • cytochrome P450 reductase ATR2-1 (NP_849472.2) was integrated into the chromosome trp1 gene locus in the S. cerevisiae BY4742 chromosome (GAL1 promoter, using trp1 original terminator) to construct recombinant yeast A2.
  • Recombinant yeast needs to be supplemented
  • the corresponding amino acid was added (0.01% tryptophan, 0.01% leucine, 0.01% lysine).
  • the recombinant yeast A2 lysate was transferred to a 2 mL EP tube, 1 mL each tube, and an equal volume (1 mL) of n-butanol was added for about 30 min and then centrifuged at 12000 g for 10 min. Pipette the supernatant into a new EP tube. The n-butanol was evaporated to dryness at 45 ° C under vacuum. It was dissolved in 100 ⁇ L of methanol and used for HPLC detection.
  • the cell lysate of recombinant yeast A2 contained dammarene diol, protopanaxadiol (PPD) and ginsenoside active metabolite Rg3 ( Figures 8 and 9) by HPLC and LC-MS analysis.
  • the method of 6.2 is the same as 6.1, except that the glycosyltransferase BvUGT73C10 is used instead of UGTPg45 to obtain recombinant yeast A6.
  • the cell lysate of recombinant yeast A6 also contained dammarene diol, protopanaxadiol (PPD) and ginsenoside active metabolite Rg3 by HPLC analysis.
  • Example 7 Construction of Escherichia coli recombinant expression vector for glycosyltransferase genes gGT29-4, gGT29-5, gGT29-6 and gGT29-7
  • the plasmids gGT29-4-pMD18T, gGT29-5-pMD18T, gGT29-6-pMD18T and gGT29-7-pMD18T containing the gGT29-4, gGT29-5, gGT29-6 and gGT29-7 genes constructed in Example 1 were used as templates. Amplify the target gene.
  • the forward primer used for the gGT29-5 and gGT29-6 genes is shown in SEQ ID NO.: 66, and the sequence homologous to the vector pET28a is added to the 5' end: CTGGTGCCGCGCGGCAGC; the reverse primer used is SEQ ID NO.: As shown at 68, a sequence homologous to the vector pET28a was added to the 5' end: TGCGGCCGCAAGCTTGTC.
  • the forward primer used for the gGT29-4 and gGT29-7 genes is SEQ ID NO.: 67, and the sequence homologous to the vector pET28a is added to the 5' end: CTGGTGCCGCGCGGCAGC; the reverse primer used is SEQ ID NO.: 68, which is 5 The 18-base fragment homologous to the vector pET28a was added to the 'end: TGCGGGCCGAGAGCTTGTC.
  • the gGT29-4, gGT29-5, gGT29-6 and gGT29-7 genes were amplified by a PCR method using the above primers.
  • the amplified gene was selected from NEB's Q5 high-fidelity DNA polymerase.
  • the PCR program was set up according to the instructions: 98 ° C 30 s; 98 ° C 15 s, 58 ° C 30 s, 72 ° C 1 min, a total of 35 cycles; 72 ° C 2 min; 10 ° C insulation .
  • the linearized vector pET28a was obtained using SEQ ID NO.: 69 and SEQ ID NO.: 70 as the forward and reverse primer amplification vectors pET28a, respectively.
  • the linearized vector of amplified pET28a was also selected from NEB's Q5 high-fidelity DNA polymerase.
  • the PCR procedure was set up according to the instructions: 98 ° C for 30 s; 98 ° C for 15 s, 58 ° C for 30 s, 72 ° C for 3 min for 35 cycles; 72 ° C for 2 min. ; 10 ° C insulation.
  • the gGT29-4, gGT29-5, gGT29-6 and gGT29-7 gene PCR products and the linearized vector pET28a were detected by agarose gel electrophoresis, and the bands corresponding to the target DNA size were cut under ultraviolet light. The DNA fragment was then recovered from the agarose gel using Axygen's AxyPrep DNA Gel Extraction Kit.
  • BGclonart Seamless Cloning Kit Instructions of Nuojing Biotechnology Co., Ltd., the linearized pET28a vector fragment recovered, the recovered gGT29-4, gGT29-5, gGT29-6 and gGT29-7 gene fragments and Nuojing Biotechnology
  • the BGclonart seamless cloning reaction solution of the company was mixed in an appropriate ratio for a total of 20 ⁇ l. After mixing, the mixture was incubated at 50 ° C for 30 minutes, and then the mixed reaction solution was transferred to ice. E. coli EPI300 competent cells were transformed with 5 ⁇ l of the reaction solution and plated on LB plates supplemented with 50 ⁇ g/mL kanamycin.
  • E. coli expression vectors gGT29-4-pET28a, gGT29-5-pET28a, gGT29-6-pET28a and gGT29-7-pET28a constructed in Example 19 were transformed into commercially available E. coli BL21.
  • One recombinant was inoculated into LB medium, cultured at 30 ° C, 200 rpm to an OD 600 of about 0.6-0.8, the bacterial solution was cooled to 4 ° C, IPTG was added to a final concentration of 50 ⁇ M, and expression was induced for 15 h at 18 ° C at 200 rpm.
  • the cells were collected by centrifugation at 4 ° C, and the cells were sonicated, and the supernatant of the cell lysate was collected by centrifugation at 12,000 g at 4 ° C, and the sample was taken for SDS-PAGE electrophoresis ( FIG. 10 ).
  • gGT29-4-pET28a, gGT29-5-pET28a, gGT29-6-pET28a, gGT29-7-pET28a and gGT29-7-pET28a recombinant lysates and total protein and supernatant all have distinct protein bands (approximately 50KD), respectively representing sugar Base transferases gGT29-4, gGT29-5, gGT29-6 and gGT29-7. From the results of Western Blot (Fig. 11), it was also confirmed that the target proteins gGT29-4, gGT29-5, gGT29-6 and gGT29-7 achieved soluble expression in the host.
  • the recombinant yeast cleavage supernatant expressing gGT29-4, gGT29-5, gGT29-6 and gGT29-7 was used as an enzyme solution to catalyze the transglycosylation reaction of ginsenoside Rh2 and F2.
  • the 100 ⁇ L reaction system is shown in Table 6. The reaction was carried out at 35 ° C for 12 h, then 100 ⁇ L of butanol was added to terminate the reaction, and the product was extracted. The product was dried under vacuum and dissolved in methanol.
  • the reaction product was detected by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the crude enzyme solution of gGT29-6 can further extend a sugar group on the 3-position glycosyl group of ginsenoside Rh2 and F2 to form ginsenoside Rg3 and Rd, respectively (Fig. 12
  • the crude enzyme solution of gGT29-4, gGT29-5 and gGT29-7 can further extend a glycosyl group at the 3-position glycosyl group of ginsenoside F2 to form saponin Rd, but they cannot catalyze the saponin Rh2 (Fig. 12).
  • the crude enzyme solution of gGT29-4, gGT29-5, gGT29-6 and gGT29-7 can also be extended on the C-6 glycosyl group of the original ginseng triol type saponin Rh1. Glycosyl group formed ginsenoside Rf (Fig. 13) in which gGT29-4, gGT29-5 and gGT29-6 were relatively weak, while gGT29-7 had relatively strong activity (Table 7).
  • Example 10 Glycosyltransferase genes gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-14, gGT29-15, gGT29-16, gGT29-17, gGT29- Construction of 18 E. coli recombinant expression vector, expression in E. coli and identification of products
  • coli expression vector for gGT29-18 (gGT29-8-pET28a, gGT29-9-pET28a, gGT29-10-pET28a, gGT29-11-pET28a, gGT29-12-pET28a, gGT29-13-pET28a, gGT29-14-pET28a , gGT29-15-pET28a, gGT29-16-pET28a, gGT29-17-pET28a, gGT29-18-pET28a), and achieve soluble expression in E. coli.
  • the glycosyltransferase gGT29-7 can be a saccharide-based donor with UDP-glucose, and the C6-O-Glc of ginsenoside Rh1 is catalyzed to extend a glucose to produce ginsenoside Rf.
  • the specificity of the glycosyl donor was altered by site-directed mutagenesis of the glycosyl donor binding region (PSPG) of the glycosyltransferase gGT29-7.
  • Amino acid sequence alignment of rhamnosyltransferase which can be used to catalyze other glycosylation receptors, with gGT29-7, and screening and finally selecting two sites on the gGT29-7PSPG cassette for site-directed mutagenesis (N343G and A359P).
  • the PCR amplification procedure was: 95 ° C for 30 s; 95 ° C for 5 s, 56 ° C for 10 s, and 72 ° C for 1.5 min for a total of 30 cycles; to 10 ° C.
  • the PCR product was digested with DpnI and reacted in a water bath at 37 ° C for 2 h. Then, 3 ⁇ L of the recombinant enzyme EIInase in the kit was used for reconstitution at 37 ° C for 30 min to transform E. coli TOP 10 competent state. The monoclonal was picked and the plasmid was extracted, and the mutation site was verified by sequencing.
  • the obtained plasmid was named pET28a-gGT29-7-N343G.
  • the plasmid pET28a-gGT29-7-N343G was transformed into E. coli BL21 (DE3), and the recombinant strain pET28a-gGT29-7-N343G-BL21 was constructed, and the procedure for inducing expression was the same as in Example 8.
  • the A359 locus of gGT29-7 was subjected to point mutation according to conventional design and synthesis of two primers, and the mutant plasmid pET28a-gGT29-7-A359P and the recombinant strain gGT29-7-N343G-BL21 were constructed, and the procedure was the same as above.
  • the procedure for inducing expression was the same as in Example 8.
  • the single mutant protein gGT29-7-N343G catalyzes the activity of C6-O-Glc of Rh1 to extend a glucose to produce Rf
  • gGT29-7-A359P still retains C6 which catalyzes Rh1.
  • -O-Glc extends the enzyme activity of a glucose.
  • the double mutant protein gGT29-7-N343G/A359P not only retains the wild-type protein catalyzes the C6-O-Glc of Rh1 to extend the activity of a glucose-producing Rf, but also obtains a rhamnose that catalyzes the extension of C6-O-Glc of Rh1.
  • the enzyme activity of Rg2 is generated.
  • the recombinant E. coli supernatant expressing gGT29-10 and gGT29-14 was used as an enzyme solution to catalyze the transglycosylation reaction of ginsenoside Rh2 and F2.
  • the 100 ⁇ L reaction system is shown in Table 6, except that UDP-xylose was used instead of UDP-glucose as a glycosyl donor.
  • the reaction was carried out at 35 ° C for 12 h, then 100 ⁇ L of butanol was added to terminate the reaction, and the product was extracted. The product was dried under vacuum and dissolved in methanol.
  • reaction product was detected by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • gGT29-10 and gGT29-14 were able to extend a xylose in the C-3 glycosyl group of ginsenoside Rh2 to form a new triterpenoid saponin, (3-O- ⁇ ).
  • gGT29-10 and gGT29-14 can replace the second glucose of ginsenoside Rg3C-3 with xylose to form a new triterpenoid saponin ( 3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-PPD); gGT29-10 and gGT29-14 can also replace the second glucose of ginsenoside Rd C-3 with xylose A new trisaponin (3-O- ⁇ -(D-xylopyranosyl)- ⁇ -(D-glucopyranosyl)-CK) (Fig. 15).
  • glycosyltransferases gGT29, gGT29-4, gGT29-5, gGT29-6 and gGT29-7gGT29-8, gGT29-9, gGT29-10, gGT29-11, gGT29-12, gGT29-13, gGT29-15, gGT29-16, gGT29-17 and gGT29-18, with UDP-xylose as a glycosyl donor, catalyze the activities of Rh2, Rg3 and Rd as shown in Table 8.
  • Recombinant yeast A5 was constructed by chromosomal trp1 gene locus in the yeast BY4742 chromosome (GAL1 promoter, using trp1 original terminator). Recombinant yeast requires additional amino acids (0.01% tryptophan, 0.01% leucine, 0.01% lysine).
  • the recombinant yeast A7 lysate was transferred to a 2 mL EP tube, 1 mL per tube, and an equal volume (1 mL) of n-butanol was added for about 30 min and then centrifuged at 12000 g for 10 min. Pipette the supernatant into a new EP tube. The n-butanol was evaporated to dryness at 45 ° C under vacuum. It was dissolved in 100 ⁇ L of methanol and used for HPLC detection.
  • the cell lysate of recombinant yeast A7 contained protopanaxatriol (PPT) and ginsenoside active metabolites Rh1 and Rf by HPLC analysis.
  • PPT protopanaxatriol
  • Rh1 and Rf ginsenoside active metabolites
  • GAL1 promoter using trp1 original terminator
  • UDP-L-rhamnose synthase RHM2 GQ292791.1
  • GAL10 promoter, FBA1 terminator GAL10 promoter, FBA1 terminator
  • the recombinant yeast A8 lysate was transferred to a 2 mL EP tube, 1 mL each tube, and an equal volume (1 mL) of n-butanol was added for about 30 min and then centrifuged at 12000 g for 10 min. Pipette the supernatant into a new EP tube. The n-butanol was evaporated to dryness at 45 ° C under vacuum. It was dissolved in 100 ⁇ L of methanol and used for HPLC detection.
  • the cell lysate of recombinant yeast A8 contained protopanaxatriol (PPT) and ginsenoside active metabolites Rh1 and Rg2 by HPLC analysis.

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Abstract

La présente invention concerne un groupe de glycosyltransférase et l'utilisation dudit groupe. Plus précisément, l'invention concerne l'utilisation de la glycosyltransférase GGT 29-7 et d'un polypeptide dérivé de cette dernière dans la glycosylation catalytique d'un terpénoïde et dans la synthèse de nouvelles saponines. La glycosyltransférase peut transférer spécifiquement et de manière hautement efficace un glycosyl d'un donneur de glycosyl à un premier glycosyl en position C-3 et/ou C-6 d'un triterpénoïde 4-cycles, de manière à étendre la chaîne d'hydrate de carbone. La glycosyltransférase de la présente invention peut également être utilisée dans la construction de ginsénosides synthétisés artificiellement et dans une diversité de nouveaux ginsénosides et de dérivés de ces derniers.
PCT/CN2015/081111 2014-06-09 2015-06-09 Groupe de glycosyltransférase et utilisation dudit groupe WO2015188742A2 (fr)

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CN110872594B (zh) * 2019-12-09 2021-04-06 四川农业大学 黑莓糖基转移酶基因及其应用
CN114807075B (zh) * 2021-01-27 2023-11-21 东北林业大学 糖基转移酶PpUGT73E5及其在重楼皂苷合成中的应用
CN115678952A (zh) * 2021-07-30 2023-02-03 生合万物(苏州)生物科技有限公司 鼠李糖高度专一的糖基转移酶及其应用

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IL128193A (en) * 1999-01-22 2008-03-20 Yeda Res & Dev Rhamnosyl-transferase gene and uses thereof
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CN111892639B (zh) * 2020-07-10 2022-06-28 山东省药学科学院 过山蕨中新的环阿尔廷型皂苷类化合物、制备方法及其应用

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