CN108866020A - Glycosyl transferase, mutant and its application - Google Patents

Abstract

The present invention relates to glycosyltransferases, mutants and applications thereof. Specifically, an in vitro glycosylation method is provided, comprising the steps of: in the presence of a glycosyltransferase, transferring the glycosyl of the glycosyl donor to the C-3 hydroxyl group of a tetracyclic triterpenoid; thereby forming Glycosylated tetracyclic triterpenoids; wherein, the glycosyltransferase is the glycosyltransferase shown in SEQ ID NO.4 or its derivative polypeptide; or the sugar shown in SEQ ID NO.21 base transferase or its derivative polypeptide.

Description

Glycosyltransferases, mutants and applications thereof

technical field

The invention relates to the fields of biotechnology and plant biology, in particular, the invention relates to a glycosyltransferase for the synthesis of ginsenoside Rh2, a mutant of the glycosyltransferase and applications thereof.

Background technique

Ginsenoside is the main active substance in Panax genus Araliaceae (such as ginseng, Panax notoginseng, American ginseng, etc.), and some ginsenosides have also been found in Cucurbitaceae Panax notoginseng in recent years. At present, scientists at home and abroad have isolated at least 100 saponins from plants such as ginseng and Panax notoginseng, and ginsenosides belong to triterpenoid saponins. Some ginsenosides have been proved to have a wide range of physiological functions and medicinal value: including anti-tumor, immune regulation, anti-fatigue, heart protection, liver protection and other functions. A variety of saponins have been used clinically. For example, Shenyi Capsules, a drug mainly composed of ginsenoside Rg3 monomer, can improve the symptoms of Qi deficiency in tumor patients and improve the immune function of the body. Jinxing Capsule, which is mainly composed of ginsenoside Rh2 monomer, is a health care medicine used to improve the body's immunity and enhance disease resistance.

From a structural point of view, ginsenosides are biologically active small molecules formed by glycosylation of saponins. There are only a limited number of saponins in ginsenosides, mainly dammarane-type protopanaxadiol and protopanaxatriol, and oleanane-type balsam. In addition to the differences in sapogenins, the structural differences between ginsenosides are mainly reflected in the different glycosylation modifications of sapogenins. The sugar chains of ginsenosides are generally bound to the C3, C6, or C20 hydroxyl groups of saponins, and the sugar groups can be glucose, rhamnose, xylose, and arabinose.

Different glycosyl binding sites, sugar chain composition and length make ginsenosides have great differences in physiological functions and medicinal value. For example, ginsenosides Rb1, Rd, and Rc are all saponins with protopanaxadiol as saponin, and the difference between them is only the difference in sugar modification, but there are many differences in their physiological functions. Rb1 has the function of stabilizing the central neuron system, while the function of Rc is to inhibit the function of the central neuron system. The physiological functions of Rb1 are very extensive, while Rd has only a few very limited functions.

Rare ginsenosides refer to saponins whose content is extremely low in ginseng. Ginsenoside Rh2 (3-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol) belongs to protopanaxadiol saponin, and a glucose group is attached to the C-3 hydroxyl group of saponin. The content of ginsenoside Rh2 is only about one ten-thousandth of the dry weight of ginseng. However, ginsenoside Rh2 has good anti-tumor activity and is one of the most important anti-tumor active ingredients in ginseng. It can inhibit the growth of tumor cells and induce tumors. Apoptosis, anti-tumor metastasis. Studies have shown that ginsenoside Rh2 can inhibit the proliferation of lung cancer cells 3LL (mice), Morris liver cancer cells (rats), B-16melanoma cells (mice), and HeLa cells (human). Clinically, ginsenoside Rh2 can be combined with radiotherapy or chemotherapy to enhance the effect of radiotherapy and chemotherapy. In addition, ginsenoside Rh2 also has the functions of anti-allergy, improving the body's immunity, and inhibiting the inflammation produced by NO and PGE.

The function of glycosyltransferases is to transfer sugars from sugar donors (nucleoside diphosphate sugars, such as UDP-glucose) to different sugar acceptors. According to the different amino acid sequences, there are currently 94 families of glycosyltransferases. More than a hundred different glycosyltransferases have been found in the plant genomes that have been sequenced so far. Glycosyl acceptors for these glycosyltransferases include sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules. The glycosyltransferase involved in the glycosylation of saponins in ginseng is to transfer the glycosyl on the glycosyl donor to the C3, C6 or C20 hydroxyl of saponin or aglycon, thus forming a glycoside with different medicinal value. saponins.

At present, there is still a lack of an effective method for producing rare ginsenoside Rh2 and ginsenoside F2 in this field, so it is urgent to develop a variety of specific and efficient glycosyltransferases.

Contents of the invention

The object of the present invention is to provide a class of glycosyltransferase and its application for synthesizing rare ginsenoside Rh2 and ginsenoside F2.

The first aspect of the present invention provides an isolated polypeptide, the amino acid sequence of the isolated polypeptide corresponding to the amino acid residue at position 222 of the amino acid sequence shown in SEQ ID NO: 19 is non-Gln and/or at The amino acid residue corresponding to the 322nd position of the amino acid sequence shown in SEQ ID NO: 19 is not Ala.

In another preference, the isolated polypeptide:

i). has the amino acid sequence shown in SEQ ID NO: 19 and the amino acid residue at position 222 is non-Gln and/or the amino acid residue at position 322 is non-Ala, or

ii). Having the sequence defined in i) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 An isolated polypeptide derived from i) that is a sequence formed by substitution, deletion or addition of amino acid residues and substantially has the function of the isolated polypeptide defined in i).

In another preferred example, the isolated polypeptide has the sequence defined in i) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably An isolated polypeptide derived from i), a sequence formed by the addition of 1-3, most preferably 1 amino acid residue, and substantially having the function of the isolated polypeptide defined in i).

In another preferred example, the amino acid sequence of the isolated polypeptide corresponds to at least one of the following amino acids at the amino acid residue at position 222 of the amino acid sequence shown in SEQ ID NO: 19: His, Asn, Gln, Lys and Arg.

In another preferred example, the amino acid residue corresponding to the 222nd position of the amino acid sequence shown in SEQ ID NO: 19 in the amino acid sequence of the isolated polypeptide is His.

In another preferred example, the amino acid sequence of the isolated polypeptide corresponding to the 322nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 19 is selected from at least one of the following amino acids: Val, Ile, Leu, Met and Phe.

In another preferred example, the amino acid residue corresponding to the 322nd position of the amino acid sequence shown in SEQ ID NO: 19 in the amino acid sequence of the isolated polypeptide is Val.

In another preferred example, the amino acid sequence of the isolated polypeptide corresponds to the amino acid residue at position 222 of the amino acid sequence shown in SEQ ID NO: 19 is His, and the amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO: 19 is His. The amino acid residue at position 322 is Val.

In another preference, the isolated polypeptide:

iii). The amino acid sequence shown in SEQ ID NO: 19 and the amino acid residue at position 222 is non-Gln and/or the amino acid residue at position 322 is non-Ala, or

iv). Having a sequence defined by iii) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 An isolated polypeptide derived from iii) that is a sequence formed by substitution, deletion or addition of amino acid residues and substantially has the function of the isolated polypeptide as defined in iii).

In another preferred example, the isolated polypeptide has the sequence defined in iii) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably An isolated polypeptide derived from iii), a sequence formed by the addition of 1-3, most preferably 1 amino acid residue, and substantially having the function of the isolated polypeptide defined in iii).

In another preferred example, the amino acid residue of the amino acid sequence of the isolated polypeptide at position 222 of the amino acid sequence shown in SEQ ID NO: 19 is selected from at least one of the following amino acids: His, Asn, Gln, Lys and Arg.

In another preferred example, the amino acid residue at position 222 of the amino acid sequence of the isolated polypeptide shown in SEQ ID NO: 19 is His.

In another preferred example, the amino acid residue of the amino acid sequence of the isolated polypeptide at position 322 of the amino acid sequence shown in SEQ ID NO: 19 is selected from at least one of the following amino acids: Val, Ile, Leu, Met and Phe.

In another preferred example, the amino acid residue at position 322 of the amino acid sequence of the isolated polypeptide shown in SEQ ID NO: 19 is Val.

In another preferred example, the amino acid residue of the isolated polypeptide at position 222 of the amino acid sequence shown in SEQ ID NO: 19 is His, and the amino acid residue at position 322 of the amino acid sequence shown in SEQ ID NO: 19 is His. The amino acid residue is Val.

In another preferred embodiment, the isolated polypeptide is a glycosyltransferase.

In another preferred example, the glycosyltransferase is derived from plants of the genus Panax.

In another preferred example, the glycosyltransferase is derived from ginseng, American ginseng and/or Panax notoginseng.

In another preferred example, the polypeptide is selected from the following group:

(a) a polypeptide having the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21;

(b) passing one or several amino acid residues to the polypeptide of the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21, preferably 1-20, more preferably 1-15, more preferably 1-10 A derivative polypeptide formed by substitution, deletion or addition of one, more preferably 1-3, most preferably 1 amino acid residue, or after adding a signal peptide sequence, and having glycosyltransferase activity;

(c) a derivative polypeptide containing the polypeptide sequence described in (a) or (b) in its sequence;

(d) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21 is ≥85% (preferably ≥90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), and a derivative polypeptide having glycosyltransferase activity.

In another preferred example, the sequence (c) is a fusion protein formed by adding a tag sequence, signal sequence or secretion signal sequence to (a) or (b).

In another preferred example, the glycosyltransferase activity refers to the activity that can transfer the sugar group of the sugar group donor to the C-3 hydroxyl group of the tetracyclic triterpenoid compound.

In another preferred embodiment, the glycosyltransferase can increase the yield of ginsenoside Rh2 and/or ginsenoside F2.

In another preference, in the artificially constructed strain, the glycosyltransferase can increase the yield of ginsenoside Rh2; preferably, by 5-150%; more preferably, by 10-100%; more preferably , increased by 20-80%; most preferably, increased by 28-70%.

In another preferred example, the artificially constructed strain is selected from the group consisting of Saccharomyces cerevisiae strains, Escherichia coli strains, Pichia pastoris strains, Schizosaccharomyces strains, and Kluyveromyces strains.

The second aspect of the present invention provides an isolated polynucleotide, said polynucleotide being a sequence selected from the group consisting of:

(A) a nucleotide sequence encoding the polypeptide described in the first aspect of the present invention;

(B) a nucleotide sequence encoding a polypeptide as shown in SEQ ID NO.:4 or SEQ ID NO.:21 or a derivative thereof;

(C) a nucleotide sequence as shown in SEQ ID NO.:3 or SEQ ID NO.:22;

(D) Homology with the sequence shown in SEQ ID NO.:3 or SEQ ID NO.:22 ≥90% (preferably ≥91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence;

(E) Truncating or adding 1-60 (preferably 1-30, more Preferably, a nucleotide sequence formed by 1-10) nucleotides;

(F) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A)-(E).

The third aspect of the present invention provides a vector containing the polynucleotide described in the second aspect of the present invention.

In another preferred example, the vectors include expression vectors, shuttle vectors, and integration vectors.

The fourth aspect of the present invention provides the use of the isolated polypeptide described in the first aspect of the present invention, which is used to catalyze the following reactions, or is used to prepare catalytic preparations that catalyze the following reactions:

(i) Transfer of a glycosyl from a glycosyl donor to the C-3 hydroxyl group of a tetracyclic triterpenoid.

In another preferred example, the glycosyl donor includes nucleoside diphosphate sugars selected from the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-acetyl Glucose, ADP-Acetyl Glucose, TDP-Acetyl Glucose, CDP-Acetyl Glucose, GDP-Acetyl Glucose, 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, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-mouse Litose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other hexose nucleoside diphosphates or pentose nucleoside diphosphates, or combinations thereof.

In another preferred example, the glycosyl donor includes uridine diphosphate (UDP) sugars selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, UDP-rhamnose, or other uridine diphosphate hexoses or uridine diphosphate pentoses, or combinations thereof.

In another preferred embodiment, the isolated polypeptide is used to catalyze the following reactions or is used to prepare catalytic preparations that catalyze the following reactions:

Wherein, R1 is H or OH; R2 is H or OH; R3 is H or glycosyl; R4 is glycosyl.

In another preferred embodiment, the isolated polypeptide is used to catalyze the following reactions or is used to prepare catalytic preparations that catalyze the following reactions:

The polypeptide is selected from the polypeptides shown in SEQ ID NO.:4 or SEQ ID NO.:21 or derivatives thereof.

In another preferred embodiment, the sugar group is selected from: glucose group, galacturonic acid group, xylosyl group, galactosyl group, arabinosyl group, rhamnosyl group, and other hexosyl or pentose groups base.

In another preferred example, the reaction products of the reactions (A) and or (B) include (but not limited to): S-configuration or R-configuration dammarane-type tetracyclic triterpenoids, lanolin type tetracyclic triterpenoids, apotirucallane type tetracyclic triterpenoids, cuminane type tetracyclic triterpenoids, cycloartane (cycloaltinane) type tetracyclic triterpenoids, cucurbitane tetracyclic triterpenoids compounds, or neem-type tetracyclic triterpenoids.

The fifth aspect of the present invention provides an in vitro glycosylation method, comprising the steps of:

In the presence of a glycosyltransferase, the glycosyl of the glycosyl donor is transferred to the C-3 hydroxyl group of the tetracyclic triterpenoid; thereby forming a glycosylated tetracyclic triterpenoid;

Wherein, the glycosyltransferase is the polypeptide described in the first aspect of the present invention or a derivative polypeptide thereof.

In another preferred example, the derivative polypeptide is selected from:

The polypeptide of the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21 is formed by substituting, deleting or adding one or several amino acid residues, or is formed after adding a signal peptide sequence, and has a derivative polypeptide with glycosyltransferase activity; or

Amino acid sequence and SEQ ID NO.: 4 or SEQ ID NO.: 21 amino acid sequence homology ≥ 85% (preferably ≥ 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%), and a derivative polypeptide with glycosyltransferase activity;

Wherein, the glycosyltransferase activity refers to the activity that can transfer the glycosyl of the glycosyl donor to the C-3 hydroxyl group of the tetracyclic triterpenoid compound.

The sixth aspect of the present invention provides a method for catalyzing a glycosyl reaction, comprising the step of: performing the catalyzed reaction of a glycosyl in the presence of the polypeptide described in the first aspect of the present invention or its derivative polypeptide.

In another preferred example, the method also includes the steps of:

The compound of formula (I) is converted into said compound of formula (II) in the presence of a glycosyl donor and said polypeptide of the first aspect of the invention or a derivative thereof.

In another preferred example, the compound of formula (I) is protopanaxadiol PPD, and the compound of formula (II) is ginsenoside Rh2; or

The compound of formula (I) is Compound K, and the compound of formula (II) is ginsenoside F2.

In another preferred embodiment, the method further includes adding the polypeptide and its derivative polypeptide to the catalytic reaction respectively; and/or

The polypeptide and its derived polypeptide are simultaneously added to the catalytic reaction.

In another preferred example, the method also includes combining the nucleotide sequence encoding glycosyltransferase with key genes and/or other glycosyltransferases in the synthetic metabolic pathway of dammarindiol and/or protopanaxadiol The enzyme gene is co-expressed in the host cell, thereby obtaining the compound of formula (II).

In another preferred example, the compound of formula (II) is ginsenoside Rh2 or ginsenoside F2.

In another preferred example, the host cell is yeast or Escherichia coli.

In another preferred example, the method further includes: providing an additive for regulating enzyme activity into the reaction system.

In another preferred example, the additive for regulating enzyme activity is: an additive for increasing enzyme activity or inhibiting enzyme activity.

In another preferred example, 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⁺ .

In another preferred example, the additives used to regulate enzyme activity are: capable of generating Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al ³⁺ , Ni ²⁺ , Zn ²⁺ , or Fe ²⁺ species.

In another preferred example, 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-Acetyl Glucose, TDP-Acetyl Glucose, CDP-Acetyl Glucose, GDP-Acetyl Glucose, 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, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-mouse Litose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other hexose nucleoside diphosphates or pentose nucleoside diphosphates, or combinations thereof.

In another preferred example, the glycosyl donor is uridine diphosphate sugar, selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-galacturonic acid, UDP-galactose, UDP-arabino Sugar, UDP-rhamnose, or other uridine diphosphate hexoses or uridine diphosphate pentoses, or combinations thereof.

In another preferred embodiment, the pH of the reaction system is: pH 4.0-10.0, preferably pH 5.5-9.0.

In another preferred example, the temperature of the reaction system is: 10°C-105°C, preferably 20°C-50°C.

In another preferred example, the key genes in the synthetic metabolic pathway of dammarenediol include (but not limited to): dammarenediol synthase gene.

In another preferred example, the key genes in the synthetic metabolic pathway of protopanaxadiol include (but not limited to): dammarenediol synthase gene, cytochrome P450 gene CYP716A47 of protopanaxadiol synthesis and other The reductase gene, or a combination thereof.

In another preferred embodiment, the substrate of the sugar-catalyzed reaction is the compound of formula (I), and the product is the compound of formula (II).

In another preferred example, the compound of formula (I) is protopanaxadiol PPD, and the compound of formula (II) is ginsenoside Rh2; or

The compound of formula (I) is Compound K, and the compound of formula (II) is ginsenoside F2.

The seventh aspect of the present invention provides a genetically engineered host cell containing the vector described in the third aspect of the present invention, or the vector described in the second aspect of the present invention is integrated into its genome. polynucleotide.

In another preferred embodiment, the glycosyltransferase is the polypeptide described in the first aspect of the present invention or a derivative thereof.

In another preferred example, the nucleotide sequence encoding the glycosyltransferase is as described in the second aspect of the present invention.

In another preferred example, the cells are prokaryotic cells or eukaryotic cells.

In another preferred embodiment, the host cells are eukaryotic cells, such as yeast cells or plant cells.

In another preferred example, the host cell is a Saccharomyces cerevisiae cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

In another preferred example, the host cells are ginseng cells.

In another preferred embodiment, the host cell is not a cell that naturally produces the compound of formula (II).

In another preferred example, the host cell is not a cell that naturally produces ginsenoside Rh2 or ginsenoside F2.

In another preferred example, the key genes in the synthetic metabolic pathway of dammarenediol include (but not limited to): dammarenediol synthase gene.

In another preferred example, the host cell contains key genes in the synthetic metabolic pathway of protopanaxadiol, including (but not limited to): dammarenediol synthase gene, cytochrome P450 gene synthesized by protopanaxadiol CYP716A47 and its reductase gene, or a combination thereof.

The eighth aspect of the present invention provides the use of the host cell described in the seventh aspect of the present invention for preparing enzyme catalytic reagents, or producing glycosyltransferases, or as catalytic cells, or producing glycosylated tetracyclic Triterpenes.

In another preferred example, the tetracyclic triterpenoid compound is a compound of formula (II).

In another preferred embodiment, the host cell is used to produce the compound of formula (II) through glycosylation of the compound of formula (I).

In another preferred embodiment, the host cell is used to produce ginsenoside Rh2 and/or ginsenoside F2 through glycosylation of protopanaxadiol PPD and/or Compound K.

A ninth aspect of the present invention provides a method for producing a transgenic plant, comprising the steps of: regenerating the genetically engineered host cell described in the seventh aspect of the present invention into a plant, and the genetically engineered host cell for plant cells.

In another preferred example, the genetically engineered host cells are selected from: ginseng cells, American ginseng cells, Panax notoginseng cells, and tobacco cells.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 Agarose gel electrophoresis diagram of glycosyltransferase gene Pn50.

Figure 2 TLC analysis diagram of glycosyltransferase gene Pn50 catalyzed ginsenosides.

Fig. 3 is an HPLC analysis chart of rare ginsenoside Rh2 produced by a recombinant Saccharomyces cerevisiae strain constructed by using Pn50.

Detailed ways

After extensive and in-depth research, the present inventors provided for the first time the mutant 8E7 (SEQ ID NO.: 21) of notoginseng glycosyltransferase Pn50 (SEQ ID NO.: 4) and ginseng-derived glycosyltransferase UGTPg45 in terpene Application in the catalysis of glycosylation of compounds and the synthesis of new saponins.

Specifically, the glycosyltransferase of the present invention can specifically and efficiently catalyze the tetracyclic triterpenoid substrate) and/or transfer the glycosyl from the glycosyl donor to the C-3 position of the tetracyclic triterpenoid on the hydroxyl group. In particular, it can efficiently convert protopanaxadiol PPD into rare ginsenoside Rh2 with anti-tumor activity, and convert rare ginsenoside Compound K (with a glycosyl modification at C20 of PPD) into product ginsenoside F2. Unexpectedly, the recombinant Saccharomyces cerevisiae strain ZWBY04RS-Pn50 constructed by introducing the glycosyltransferase gene Pn50 derived from Panax notoginseng into the S. Compared with the strain ZWBY04RS-UGTPg45 of the glycosyltransferase gene UGTPg45, the yield of ginsenoside Rh2 of the strain ZWBY04RS-Pn50 increased by 28% (45.55/35.66-1=28%); the mutant gene 8E7 obtained by transforming UGTPg45 by random mutation Compared with the strain ZWBY04RS-UGTPg45 constructed by using UGTPg45, the Rh2 production of the artificially synthesized rare ginsenoside Rh2 strain ZWBY04RS-8E7 was increased by 70% (60.48/35.66-1=70%).

The invention also provides conversion and catalytic methods. The glycosyltransferase of the present invention can also be synthesized with dammarenediol and/or key enzymes in the protopanaxadiol synthetic metabolic pathway (such as the cytochrome P450 gene of dammarenediol synthase gene PgDDS, protopanaxadiol synthesis) CYP716A47 and its reductase gene (PgCPR1) are co-expressed in host cells, or applied to genetically engineered cells for preparing ginsenoside Rh2, and used to construct strains for artificially synthesizing rare ginsenoside Rh2.

In addition, the glycosyltransferase of the present invention can also be co-expressed with key enzymes in the synthetic metabolic pathway of dammarenediol and/or protopanaxadiol in host cells, and applied to construct strains for artificially synthesizing rare ginsenoside Rh2. The present invention has been accomplished on this basis.

definition

As used herein, the terms "active polypeptide", "polypeptide of the present invention and derivative polypeptide thereof", "enzyme of the present invention", "glycosyltransferase" or "glycosyltransferase of the present invention" are used interchangeably, and has the meaning commonly understood by those of ordinary skill in the art. The glycosyltransferase of the present invention has the activity of transferring the glycosyl of the glycosyl donor to the C-3 hydroxyl group of the tetracyclic triterpenoid compound.

In a preferred example of the present invention, the amino acid sequence of the glycosyltransferase of the present invention corresponds to the amino acid residue at position 222 of the amino acid sequence shown in SEQ ID NO: 19 as non-Gln and/or at the amino acid residue corresponding to SEQ ID NO The amino acid residue at position 322 of the amino acid sequence shown in :19 is not Ala.

In a preferred example of the present invention, the glycosyltransferase of the present invention:

i). has the amino acid sequence shown in SEQ ID NO: 19 and the amino acid residue at position 222 is non-Gln and/or the amino acid residue at position 322 is non-Ala, or

ii). Having the sequence defined in i) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 An isolated polypeptide derived from i) that is a sequence formed by substitution, deletion or addition of amino acid residues and substantially has the function of the isolated polypeptide defined in i).

In a preferred embodiment of the present invention, the glycosyltransferase of the present invention has the sequence defined in i) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10 , more preferably 1-3, most preferably 1 amino acid residue addition sequence, and the isolated polypeptide derived from i) substantially having the function of the isolated polypeptide defined in i).

In a preferred example of the present invention, the amino acid sequence of the glycosyltransferase of the present invention corresponds to the 222nd amino acid residue of the amino acid sequence shown in SEQ ID NO: 19 and is selected from at least one of the following amino acids: His, Asn , Gln, Lys and Arg.

In a preferred example of the present invention, the amino acid residue corresponding to the 222nd position of the amino acid sequence shown in SEQ ID NO:19 in the amino acid sequence of the glycosyltransferase of the present invention is His.

In a preferred example of the present invention, the amino acid sequence of the glycosyltransferase of the present invention corresponds to at least one of the following amino acids at the amino acid residue at position 322 of the amino acid sequence shown in SEQ ID NO: 19: Val, Ile , Leu, Met and Phe.

In a preferred example of the present invention, the amino acid residue corresponding to the 322nd position of the amino acid sequence shown in SEQ ID NO: 19 in the amino acid sequence of the glycosyltransferase of the present invention is Val.

In a preferred example of the present invention, the amino acid sequence of the glycosyltransferase of the present invention corresponds to the 222nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 19 is His, and in the amino acid residue corresponding to SEQ ID NO: 19 The amino acid residue at position 322 in the amino acid sequence is Val.

In a preferred embodiment of the present invention, the glycosyltransferase of the present invention is selected from the following group:

(a) a polypeptide having the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21;

(b) passing one or several amino acid residues to the polypeptide of the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21, preferably 1-20, more preferably 1-15, more preferably 1-10 A derivative polypeptide formed by substitution, deletion or addition of one, more preferably 1-3, most preferably 1 amino acid residue, or after adding a signal peptide sequence, and having glycosyltransferase activity;

(c) a derivative polypeptide containing the polypeptide sequence described in (a) or (b) in its sequence;

(d) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.:4 or SEQ ID NO.:21 is ≥85% (preferably ≥90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), and a derivative polypeptide having glycosyltransferase activity.

In a specific embodiment, the glycosyltransferase activity of the present invention refers to the activity of transferring the glycosyl group of the glycosyl donor to the C-3 hydroxyl group of the tetracyclic triterpenoid compound.

In a preferred example of the present invention, a new glycosyltransferase gene Pn50 cloned from Panax notoginseng can catalyze the C3 glycosylation of various dammarane-type ginsenosides by using this new glycosyltransferase.

Through the analysis of the Panax notoginseng transcriptome data, a full-length glycosyltransferase gene sequence was spliced from it, and named Pn50. It was cloned into the cloning vector PMDT-18T, and then the expression primers were designed and constructed into the Escherichia coli expression vector pET28a to induce expression in Escherichia coli. The obtained protein can catalyze the C3 hydroxyl glycosylation of protohuman diol and Compound K.

The substitution of the notoginseng glycosyltransferase gene Pn50 for the UGTPg45 derived from ginseng can greatly increase the production of Rh2 (increased by 28%).

In another preferred embodiment of the present invention, a glycosyltransferase mutant protein 8E7 is provided. The glycosyltransferase mutant protein is a mutant protein of the ginseng-derived wild-type gene UGTPg45, and the replacement of the ginseng-derived wild-type gene UGTPg45 by the mutant glycosyltransferase gene 8E7 can greatly increase Rh2 production (70% increase).

It is not difficult for those skilled in the art to know that changing a few amino acid residues in some regions of the polypeptide, such as non-essential regions, will not basically change the biological activity, for example, the sequence obtained by properly replacing some amino acids will not affect its activity ( See Watson et al., Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224). Accordingly, one of ordinary skill in the art is able to perform such substitutions and ensure that the resulting molecule still possesses the desired biological activity.

Therefore, the polypeptide of the present invention may be non-Gln at the amino acid residue corresponding to the 222nd position of the amino acid sequence shown in SEQ ID NO:19 and/or at the amino acid corresponding to the 322nd position of the amino acid sequence shown in SEQ ID NO:19 On the basis of residues other than Ala, further mutations are carried out to still have the function and activity of the glycosyltransferase of the present invention. For example, the glycosyltransferase of the present invention (a) has an amino acid sequence as shown in SEQ ID NO: 21; or (b) comprises the sequence defined in (a) through one or more amino acid residues, preferably 1-20, More preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably a sequence formed by substitution, deletion or addition of 1 amino acid residue, and basically has the polypeptide function defined in (a) A polypeptide derived from (a).

In the present invention, the glycosyltransferase of the present invention includes at most 20, preferably at most 10, and more preferably at most 3 compared with the amino acid sequence of the glycosyltransferase shown in SEQ ID NO: 21 , more preferably at most 2, most preferably at most 1 amino acid is replaced by an amino acid with similar or similar properties to form a mutant. Mutants of these conservative variations can be generated, for example, by making amino acid substitutions as shown in the table below.

initial residue Representative Substituted Residues Preferred Substitution Residues Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The present invention also provides polynucleotides encoding the polypeptides of the present invention. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

Therefore, as used herein, "comprising", "having" or "comprising" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of Consists of ", "essentially composed of" and "consisting of" belong to the sub-concepts of "contain", "have" or "include".

Corresponding to the amino acid residue at position 222/322 of the amino acid sequence shown in SEQ ID NO:19

Those of ordinary skill in the art know that various mutations, such as substitutions, additions or deletions, can be made to some amino acid residues in the amino acid sequence of a certain protein, but the obtained mutants can still have the function or activity of the original protein. Therefore, those of ordinary skill in the art can make certain changes to the amino acid sequence specifically disclosed in the present invention to obtain a mutant that still has the desired activity, then the 222nd/ The amino acid residue corresponding to the amino acid residue at position 322 may not be position 222/322, but the mutant thus obtained should still fall within the protection scope of the present invention.

The term "corresponding to" used herein has a meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to the specified position in the other sequence. Therefore, in terms of "corresponding to the amino acid residue at position 222/322 of the amino acid sequence shown in SEQ ID NO: 19", if a 6-His tag is added to one end of the amino acid sequence shown in SEQ ID NO: 19, Then the 222nd/322th position corresponding to the amino acid sequence shown in SEQ ID NO:19 in the resulting mutant may be the 228th/328th position; and if a few amino acid residues in the amino acid sequence shown in SEQ ID NO:19 are deleted base, then the 222nd/322th position corresponding to the amino acid sequence shown in SEQID NO: 19 in the resulting mutant may be the 220th/320th position, and so on. For another example, if a sequence with 400 amino acid residues has higher homology or sequence identity with the 20th-420th amino acid sequence shown in SEQ ID NO: 19, then the resulting mutant corresponding to SEQ ID The 222nd/322nd position of the amino acid sequence shown in NO:19 may be the 202nd/302nd position.

In a specific embodiment, the homology or sequence identity may be more than 80%, preferably more than 90%, more preferably 95%-98%, most preferably more than 99%.

Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, A.M. Ed., Oxford University Press, New York, 1988; Biological Computing: Information Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds., HumanaPress , New Jersey, 1994; Sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer (Sequence Analysis Primer), Gribskov, M. and Devereux, J. Ed. M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method of determining identity is to obtain the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well known Smith Waterman algorithm can also be used to determine identity.

Unless otherwise specified, the ginsenosides and saponins mentioned herein refer to ginsenosides and saponins in the S and/or R configuration at the C20 position.

As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the polypeptides using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the polypeptide can also be further analyzed by amino acid sequence.

The active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the present invention may be purified from nature, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of said polypeptides. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the polypeptide.

The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides 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 in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The active polypeptide of the present invention has glycosyltransferase activity, and can catalyze one or more of the following reactions:

The polypeptide sequence is SEQ ID NO.:4, SEQ ID NO.:21 or derivative polypeptides thereof, and the term also includes SEQ ID NO.:4, SEQ ID NO.:21 having the same function as the indicated polypeptide variants of the sequence. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the proteins of the invention. The invention also provides analogs of said polypeptides. The difference between these analogs and the natural polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

The amino terminus or carboxyl terminus of the Pn50, 8E7 polypeptide or derivative polypeptides of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins. Table 1 lists some of these tags and their sequences.

Table 1

Label number of residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-TagII 8 WSHPQFEK C-myc 10 WQKLISEEDL GST 220 6 LVPRGS behind

In order to secrete and express the translated protein (such as secreted outside the cell), a signal peptide sequence, such as pelB signal peptide, can also be added to the amino acid terminal of the Pn50, 8E7 polypeptide or its derivative polypeptide. The signal peptide can be cleaved during the secretion of the polypeptide from the cell.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the 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.:3 or SEQ ID NO.:22 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to encoding a polypeptide having the amino acid sequence shown in SEQ ID NO.: 4 or SEQ ID NO.: 21, or a derivative thereof, but with SEQ ID NO.: 4 or the coding sequence of SEQ ID NO.:21 or its derivative polypeptide, preferably a nucleic acid sequence that differs from the sequence shown in SEQ ID NO.:3 or SEQ ID NO.:22.

The polynucleotide encoding the mature polypeptide of the polypeptide shown in SEQ ID NO.:4 or SEQ ID NO.:21 or its derivative polypeptide includes: the coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; The coding sequence (and optionally additional coding sequences) and non-coding sequences of the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present 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. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.:4 or SEQ ID NO.:21.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding Pn50, 8E7 polypeptides or derivatives thereof.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The Pn50, 8E7 polypeptide or its derivative polypeptide nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the Pn50, 8E7 polypeptide or its derivative polypeptide, and the production of the polypeptide of the present invention by recombinant techniques Methods.

By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant Pn50, 8E7 polypeptide or derivative polypeptide. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the Pn50, 8E7 polypeptide or its derivative polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

In the present invention, polynucleotide sequences encoding Pn50, 8E7 polypeptides or derivative polypeptides can be inserted into recombinant expression vectors. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of Pn50, 8E7 polypeptides or derivative polypeptides and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of 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.

In addition, the expression vector preferably contains 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.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may 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. Representative examples are: Escherichia coli, Streptomyces spp; 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.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

application

The use of the active polypeptide or glycosyltransferase Pn50, 8E7 polypeptide or its derivative polypeptide involved in the present invention includes (but not limited to): specifically and efficiently transferring the glycosyl from the glycosyl donor to the tetracyclic triterpenoid On the C-3 hydroxyl group. In particular, the compound of formula (I) can be converted into the compound of formula (II), for example, protopanaxadiol PPD can be converted into rare ginsenoside Rh2 with better antitumor activity; Compound K can be converted into ginsenoside F2.

The tetracyclic triterpene compounds include (but are not limited to): S-configuration or R-configuration dammarane type, lanolin type, cuminane type, cycloartane (cycloartean) type, Apotirucallane type, cucurbitane, neem type and other tetracyclic triterpenoids.

The invention provides an industrial catalysis method, comprising: obtaining ginsenoside Rh2 and ginsenoside F2 by using the Pn50, 8E7 polypeptide or derivative polypeptide of the invention under the condition of providing glycosyl donors. Specifically, the polypeptide used in the (A) reaction is selected from the polypeptides shown in SEQ ID NO.:4 or SEQ ID NO.:21 or derivative polypeptides thereof; the polypeptide used in the (B) reaction is SEQ ID NO.: The polypeptide shown in 4 or its derivative polypeptide.

In a preferred example of the present invention, a method for synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae is provided by using the glycosyltransferase Pn50 of Panax notoginseng and the glycosyltransferase mutant protein 8E7.

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-Acetyl Glucose, CDP-Acetyl Glucose, GDP-Acetyl Glucose, 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, UDP-Arabinose, ADP-Arabinose, TDP-Arabinose, CDP-Arabinose, GDP-Arabinose, UDP-Rhamnose, ADP-Rhamnose, TDP-Rhamnose Sugar, CDP-rhamnose, GDP-rhamnose, or other hexose nucleoside diphosphates or pentose nucleoside diphosphates, or combinations thereof.

The glycosyl donor is preferably uridine diphosphate sugar, selected from the group consisting of: UDP-glucose, UDP-xylose, UDP-rhamnose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose sugar, or other hexose uridine diphosphate or pentose uridine diphosphate, or a combination thereof.

In the method, an enzyme activity additive (an enzyme activity-enhancing or enzyme-inhibiting additive) may also be added. The additive of the enzyme activity can be selected from the following group: Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al ³⁺ , Ni ²⁺ , Zn ²⁺ , or Fe ²⁺ ; or can generate Substances of Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al ³⁺ , Ni ²⁺ , Zn ²⁺ , or Fe ²⁺ .

The pH condition of the method is: pH4.0-10.0, preferably pH6.0-pH8.5, more preferably 8.5.

The temperature condition of the method is: 10°C-105°C, preferably 25°C-35°C, more preferably 35°C.

The present invention also provides a composition, which contains an effective amount of the active polypeptide or glycosyltransferase Pn50, 8E7 polypeptide or derivative polypeptides of the present invention, and a food-acceptable or industrially acceptable carrier or excipient. Such carriers include, but are not limited to, water, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.

Substances that regulate the activity of the glycosyltransferase of the present invention can also be added to the composition. Any substance having a function of increasing enzyme activity is usable. Preferably, the substance improving the activity of the glycosyltransferase of the present invention is selected from mercaptoethanol. In addition, 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⁺ ; or when added to Substrates can be hydrolyzed to form Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Ba ²⁺ , Al ³⁺ , Ni ²⁺ , Zn ²⁺ and Fe ²⁺ substances.

After obtaining the Pn50, 8E7 polypeptide or its derivative polypeptide of the present invention, those skilled in the art can conveniently use the enzyme to play the role of transglycosylation, especially the transglycosylation of dammarindiol and protopanaxadiol effect. As a preferred mode of the present invention, two methods for forming rare ginsenosides are also provided, one of which includes: treating the substrate to be transglycosylated with the Pn50, 8E7 polypeptide or derivative polypeptide described in the present invention, said The substrates include tetracyclic triterpenoids such as dammarene diol, protopanaxadiol and its derivatives. Preferably, under the condition of pH 3.5-10, the substrate to be transglycosylated is treated with the Pn50, 8E7 polypeptide or its derivative peptidase. Preferably, the substrate to be transglycosylated is treated with the Pn50, 8E7 polypeptide or its derivative peptidase at a temperature of 30-105°C.

The second method comprises: transferring the Pn50, 8E7 polypeptide or its derivative polypeptide gene according to the present invention into engineering bacteria capable of synthesizing protopanaxadiol PPD (for example, yeast or Escherichia coli engineering bacteria), or, Pn50, The 8E7 polypeptide or its derivative polypeptide gene is co-expressed in host cells (such as yeast cells or Escherichia coli) with key genes in the synthetic metabolic pathway of dammarene diol, protopanaxadiol PPD and optionally other glycosyltransferase genes, Obtain recombinant bacteria that directly produce rare ginsenoside Rh2 and/or ginsenoside F2. Alternatively, the key enzymes and optionally other glycosyltransferases and synthetic UDP- The key enzyme of rhamnose is co-expressed in host cells, and it is applied to construct recombinant strains for artificially synthesizing rare ginsenoside Rh2 and ginsenoside F2.

The key genes in the synthetic metabolic pathway of dammarenediol include (but not limited to): dammarenediol synthase gene.

In another preferred example, the key genes in the synthetic metabolic pathway of protopanaxadiol include (but not limited to): dammarenediol synthase gene PgDDS, cytochrome P450 gene CYP716A47 synthesized by protopanaxadiol and Its reductase gene, or a combination thereof. Or isoenzymes and combinations thereof of the above various enzymes. Among them, dammarenediol synthase converts epoxysqualene (synthesized by Saccharomyces cerevisiae) into dammarenediol, and cytochrome P450CYP716A47 and its reductase convert dammarenediol into protopanaxadiol PPD . (Han et.al, plant&cell physiology, 2011, 52.2062-73)

Main advantage of the present invention:

(1) The method of the present invention utilizing Saccharomyces cerevisiae to produce ginsenoside Rh2 has the advantages of low cost, short period and stable quality compared to the traditional method relying on the extraction of Panax genus plants and the hydrolysis of glycosyl groups;

(2) The glycosyltransferase Pn50 obtained from Panax notoginseng for the first time in the present invention can catalyze PPD and Rh2, and catalyze the synthesis of F2 from CK. Efficient synthesis of rare ginsenoside Rh2;

(3) The present invention obtains the mutant gene 8E7 by random mutation of the wild-type glycosyltransferase UGTPg45 in ginseng, and introducing it into the PPD-producing strain can significantly increase the rare ginsenoside Rh2 compared with the wild-type glycosyltransferase UGTPg45 in ginseng. synthetic efficiency.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Through the following specific implementation methods, the specific implementation process of the present invention can be further understood.

Example 1. Cloning of the notoginseng glycosyltransferase gene Pn50

Two primers such as Pn50 cloning primer F, SEQ ID NO: 1 (ATGGAGAGAGAAATGTTGAGCA) and Pn50 cloning primer R, SEQ ID NO: 2 (TCAGGAGGAAACAAGCTTTGAA) were synthesized. Using the cDNA obtained by reverse transcription of the RNA extracted from Panax notoginseng as a template, PCR was performed using the above primers. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. The PCR amplification program was as follows: 94°C for 2min; 94°C for 15s, 58°C for 30s, 68°C for 2min, a total of 35 cycles; 68°C for 10min to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 1.

Under UV irradiation, the target DNA band is excised. Then, Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the DNA fragment of the amplified glycosyltransferase gene. The recovered PCR product was cloned into a PMDT vector using the PMD18-T cloning kit of Takara Bioengineering (Dalian) Co., Ltd. (Takara), and the constructed vector was named PMDT-Pn50. The gene sequence of Pn50 was obtained by sequencing.

The Pn50 gene has the nucleotide sequence of SEQ ID NO:3. From the 1st-1368th nucleotide of the 5' end of SEQ ID NO:3 is the open reading frame (Open Reading Frame, ORF) of Pn50, from the 1st-3rd nucleotide of the 5' end of SEQ ID NO:3 It is the start codon ATG of the Pn50 gene, and the 1366-1368 nucleotides from the 5' end of SEQ ID NO:3 are the stop codon TGA of the Pn50 gene. The glycosyltransferase Pn50 gene encodes a protein Pn50 containing 455 amino acids, which has the amino acid residue sequence of SEQ ID NO: 4. The theoretical molecular weight of the protein is predicted to be 51.1 kDa by software, and the isoelectric point pI is 5.10. The 332-375th amino acid from the amino terminal of SEQ ID NO:4 is the conserved functional domain of glycosyltransferase PSPG.

Pn50 nucleotide sequence SEQ ID NO:3

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGCCCAATGATGCAATTCGTCAAGCGTTTAGCCTGGAAAGGCGTGCGAATCACGATAGTTCTTCCGGCTGAGATTCGAGATTCTATGCAAATAAACAACTCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGATGATGAGATGCCATACAGCATGCGGGCTTATATGGGAGTTGTAAAGCTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAAAACAAATGGCTACCCTGTTAATTTGCTAGTGGTCGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGGGGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGAAGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAAATGATTTGCCAATTATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGATCTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCAATGGCCAATTACATCCATCGGACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGACATTAATTTCTACAAGGCAGACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAAGCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACACAGCAAATATCACTTCATATGGGTTGTTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGCAGAGGAAAAAGGCCTAATAGTGAGTTGGTGCCCTC AACTCGAAGTTTTGTCACATAAATCTATAGGTAGTTTTATGACTCATTGTGGTTGGAACTCGACGGTTGAGGCATTGAGTTTGGGCGTGCCAATGGTGGCAGTGCCACAACAGTTTGATCAGCCTGTTAATGCCAAGTATATCGTGGATGTATGGCGAATTGGGGTTCAGGTTCCGATTGGTGAAAATGGGGTTCTTTTGAGGGGAGAAGTTGCTAACTGTATAAAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGTGGAGGCAATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

Pn50 amino acid sequence SEQ ID NO:4

MEREMLSKTHIMFIPFPAQGHMSPMMQFVKRLAWKGVRITIVLPAEIRDSMQINNSLINTECISFDFDKDDEMPYSMRAYMGVVKLKVTNKLSDLLEKQKTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNARLGKLKIPPEEGLTSVSLPSIPLLGRNDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSQWPITSIGPLIPSMYLDKQLPNDKDNDINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKYHFIWVVIESERTKLSSDFLAEAEEKGLIVSWCPQLEVLSHKSIGSFMTHCGWNSTVEALSLGVPMVAVPQQFDQPVNAKYIVDVWRIGVQVPIGENGVLLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKLVSS

Example 2. Expression of the notoginseng glycosyltransferase gene Pn50 in Escherichia coli

Two primers such as the sequence Pn50 expression primer F SEQ ID NO: 5 (GGATCCATGGAGAGAGAAATGTTGAGCA) and Pn50 expression primer R SEQ ID NO: 6 (CTCGAGTCAGGAGGAAACAAGCTTTGAA) were synthesized. Two restriction sites, BamH I and Xho I, were respectively added to the synthesized primers F/R, and PCR was carried out using cDNA extracted from plants as a template. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. The PCR amplification program was as follows: 94°C for 2min; 94°C for 15s, 58°C for 30s, 68°C for 2min, a total of 35 cycles; 68°C for 10min to 10°C. PCR products were detected by agarose gel electrophoresis. Under UV irradiation, the target DNA band is excised. Then, Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the DNA fragment of the amplified glycosyltransferase gene. The recovered two PCR products were digested with BamH I and Xho I and then ligated with pET28a that was also digested with BamH I and Xho I respectively, and the ligation product was transformed into Escherichia coli EPI300 competent cells, and the transformed Escherichia coli bacteria were The solution was spread on the LB plate added with 50ug/mL kanamycin, and the recombinant clone was further verified by PCR and enzyme digestion. Select one of the clones to extract the recombinant plasmid and perform sequencing verification. After the sequencing verification is correct, transform the recombinant plasmid into Escherichia coli BL21 (DE3) to induce expression. The induction expression method is: pick a single clone from the plate and inoculate it into the Inoculate 1% of mycin in the LB test tube overnight, inoculate it into a 50ml Erlenmeyer flask with shaking at 37°C until the OD600 is 0.6-0.7, add IPTG with a final concentration of 0.1mM for induction, and induce at 18°C for 16h. 12000g, 3min to collect the bacteria, add 10ml PBS buffer to resuspend each gram of wet weight of the bacteria, lyse the bacteria, centrifuge and take the supernatant as the crude enzyme solution.

Example 3. Notoginseng glycosyltransferase gene Pn50 catalyzes different substrate reactions and detection of its products

Configure the glycosyltransferase Pn50 catalytic reaction system (100 μL) as follows:

The reaction was carried out in a water bath at 37°C for 2h. After the reaction was completed, an equal volume of n-butanol was added for extraction, and the n-butanol phase was taken, concentrated in vacuo, and the reaction product was dissolved in 10 μL of methanol, and the results were detected by TLC or HPLC. As can be seen from the results in Figure 2, the glycosyltransferase Pn50 used in the present invention can catalyze protopanaxadiol PPD to form a new product (reaction is shown in formula A), and its migration position and Rh2 migration on the TLC plate The positions are consistent, which proves that this new product is ginsenoside Rh2. In addition, Pn50 can also catalyze compound K, and the generated product is speculated to be ginsenoside F2 according to the migration position on the TLC plate and the regional specificity of Pn50 (Figure 2).

Example 4. Using the glycosyltransferase gene Pn50 derived from Panax notoginseng to synthesize rare ginsenoside Rh2 in Saccharomyces cerevisiae

(1) The glycosyltransferase gene Pn50 derived from Panax notoginseng can catalyze the C3 hydroxyl glycosylation of protopanaxadiol to synthesize rare ginsenoside Rh2. In order to realize the synthesis of rare ginsenoside Rh2 in Saccharomyces cerevisiae, the present invention first constructed a Saccharomyces cerevisiae chassis cells producing protopanaxadiol. The dammarenediol synthase gene PgDDS, the cytochrome P450 gene CYP716A47 and its reductase gene PgCPR1 synthesized by protopanaxadiol were introduced into wild-type Saccharomyces cerevisiae, and the 2,3 - Epoxy squalene can synthesize protopanaxadiol. A S. cerevisiae strain ZWBY04RS with high protopanaxadiol production was obtained by optimizing the artificially constructed protopanaxadiol synthesis pathway, including optimizing the rate-limiting steps of synthesis and optimizing the supply of precursors.

(2) Synthesize 12 primers such as the sequence of SEQ ID NO:7-18, obtain the promoter expressed by the glycosyltransferase, terminator, ORF, screening markers, upstream and downstream homology arm fragments with the PCR method, and the PCR method is the same as Example 1. After mixing 100 ng of the above PCR fragment and the ORF of UGTPg45, the recombinant Saccharomyces cerevisiae strain ZWBY04RS was transformed using the conventional LiAc/ssDNA transformation method of Saccharomyces cerevisiae to obtain the recombinant Saccharomyces cerevisiae strain ZWBY04RS-UGTPg45. Similarly, 100 ng of the above PCR fragment and the ORF of Pn50 were mixed, and the recombinant Saccharomyces cerevisiae strain ZWBY04RS was transformed using the conventional LiAc/ssDNA transformation method of Saccharomyces cerevisiae to obtain the recombinant Saccharomyces cerevisiae strain ZWBY04RS-Pn50.

Promoter primer F SEQ_ID_NO.7

TAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGAGATT

Promoter primer R SEQ_ID_NO.8

ACTGTCAAGGAGGGTATTCTGGGCCTCCATGTCGCTGCTATATAACAGTTGAAATTTGGATAAGAACAT

ORF Primer F SEQ_ID_NO.9

GCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAGAATTACAATAGT

ORF Primer R SEQ_ID_NO.10

TCTGGTGAGGATTTACGGTATGATCATGCTGGTAAGCTTCGTTA

Terminator primer F SEQ_ID_NO.11

GAAAAGAAGATAATATTTTTTATATAATTATATTAAATCTCAGGAGGAAACAAGCTTTGAA

Terminator primer R SEQ_ID_NO.12

GCATAGCAATCTAATCTAAGTTTTTAATTACAAAATGGAGAGAGAAATGTTGAGCAAAAC

Screening marker primer F SEQ_ID_NO.13

CGCGTTGAGAAGATGTTTCTTATCCAAATTTCAACTGTTATATAGCAGCGA

Screening marker primer R SEQ_ID_NO.14

TTACTTCTTGCAGACATCAGACATACTATTGTAATTCTCGACACTGGATGGCGGCGTTA

Upstream homology arm primer F SEQ_ID_NO.15

GGCTGTCGCCATTCAAGAGCAGATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC

Upstream homology arm primer R SEQ_ID_NO.16

CATAATGTGAGTTTTGCTCAACATTTCTCTCTCCATTTTGTAATTAAAACTTAGATTAG

Downstream homology arm primer F SEQ_ID_NO.17

CCCAAAAGCTAAGAGTCCCATTTTTATTC

Downstream homology arm primer R SEQ_ID_NO.18

GAGTAGAAACATTTTGAAGCTATCTGCTCTTGAATGGCGACAGCTATTGCCCCAGTGT

(3) Prepare solid medium: Prepare medium: 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose) (glucose), 2% agar powder.

Configure liquid medium: configure medium: 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose) (glucose).

Pick the recombinant Saccharomyces cerevisiae ZWBY04RS-UGTPg45 and ZWBY04RS-Pn50 that were streaked on the solid medium plate, and shake them in test tubes containing 5mL liquid medium for overnight culture (30°C, 250rpm, 16h); centrifuge to collect the cells, transfer Put it into a 50mL Erlenmeyer flask with 10mL of liquid medium, adjust the OD600 to 0.05, culture at 30°C with shaking at 250rpm for 4 days to obtain the fermentation product. In this method, a parallel experiment is set up for each strain of recombinant yeast.

Extraction and detection of protopanaxadiol and rare ginsenoside Rh2: draw 100 μL of fermentation broth from 10 mL of fermentation broth, shake and lyse the yeast with Fastprep, add an equal volume of n-butanol for extraction, and then distill n-butanol under vacuum conditions Dry. After dissolved in 100 μL of methanol, the yield of the target product was detected by HPLC. The HPLC results are shown in FIG. 3 .

The recombinant Saccharomyces cerevisiae strain ZWBY04RS-UGTPg45 constructed by introducing the glycosyltransferase gene UGTPg45 derived from ginseng into the Saccharomyces cerevisiae strain producing protopanaxadiol can synthesize rare ginsenoside Rh2, and its yield is 35.66mg/L. The recombinant Saccharomyces cerevisiae strain ZWBY04RS-Pn50 constructed by introducing the glycosyltransferase gene Pn50 derived from Panax notoginseng into the Saccharomyces cerevisiae strain producing protopanaxadiol can synthesize rare ginsenoside Rh2, and its yield is 45.55mg/L.

Example 5. Construction of ginseng-derived glycosyltransferase UGTPg45 random mutation library

以质粒UGTPg45-pMD18T为模板，使用GeneMorph II Random Mutagenesis Kit(Agilent Technology)，UGTPg45随机突变引物F SEQ ID NO:23(Gcatagcaatctaatctaagttttaattacaaaatggagagagaaatgttgagcaaaac)和UGTPg45随机突变引物R SEQ ID NO:24(Gaaaagaagataatatttttatataattatattaatctcaggaggaaacaagctttgaa)为引物进行易Incorrect PCR, the program is as follows: pre-denaturation at 95°C for 2min; denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 3min and 15s, 30 cycles; final extension at 72°C for 10min. According to the kit instructions, 1ug, 1.5ug, 2ug templates were added to explore the mutation rate. The PCR product was recovered by tapping the rubber, and was connected to the vector pMD18T with Taq enzyme plus A, and transformed into Escherichia coli TOP10. Randomly pick 10 positive clones for sequencing, and determine that the mutation rate is 1-2 bases/gene and the corresponding template dosage is 1.5ug. Subsequent experiments use this condition to carry out error-prone PCR to construct a random mutation library of UGTPg45.

Using primer SEQ ID NO:25 (fragment 7 primer F) (Acactggggcaataggctgtcgccattcaagagcagatagcttcaaaatgtttctactc) and SEQ ID NO:26 (fragment 7 primer R) (Cataatgtgagttttgctcaacatttctctctccattttgtaattaaaacttagattag), using Saccharomyces cerevisiae genomic DNA as NO template PCR to obtain fragment ID 7, using primer SEQ: 27 (Fragment 8 Primer F) (Ttgatgagttcatttcaaagcttgtttcctcctgagattaatataattatataaaaata) and SEQ ID NO: 28 (Fragment 8 Primer R) (Actgtcaaggagggtattctgggcctccatgtcgctgctatataacagttgaaatttgg) obtained Saccharomyces cerevisiae genomic DNA as template PCR to obtain Fragment F fragment 8, using primer 9 (SEQ ID NO: 2) (Cccaaagctaagagtcccattttattc)和SEQ ID NO:30(片段9引物R)(Gaagagtaaaaaaggagtagaaacattttgaagctatctgctcttgaatggcgacagcc)，SEQ ID NO:31(片段10引物F)(Tgtcgattcgatactaacgccgccatccagtgtcgagaattacaatagtatgtctgatg)和SEQID NO:32(片段10引物R)(Tctggtgaggatttacggtatg)以酿酒酵母Fragments 9 and 10 were obtained by PCR using genomic DNA as a template. Fragment 11 was obtained by PCR using primers SEQ ID NO: 33 (Primer F for Fragment 11) (Aagatgttcttatccaaatttcaactgttatatagcagcgacatggaggcccagaatac) and SEQ ID NO: 34 (Primer R for Fragment 11) (tacttcttgcagacatcagacatactattgtaattctcgacactggatggcggcgttag) using plasmid PLKAN as a template. The above fragments 7-11 were mixed in equimolar ratio, transformed into PPD high-yield strain ZWBY04RS, coated with YPD plate (adding 100ug/ml G418), cultured at 30°C for 2 days, and the yeast transformant expressing UGTPg45 mutant gene grew out. Similarly, yeast transformants were constructed by transforming wild-type UGTPg45 as a control.

Add 600ul of YPD medium (100ug/ml G418) to each well of a 96-well plate, pick a single yeast clone into the medium, and culture at 280rpm at 30°C for 1 day. Transfer 6 ul of the culture to a new 96-well plate containing 600 ul of YPD medium, and culture at 30°C with shaking at 280 rpm for 3 days. Add 600ul of n-butanol to each hole, cover the rubber cover and seal it with tape, and extract by rotation for 3h. Centrifuge at 4000rpm for 10min, pipette 150ul of n-butanol phase into a new 96-well plate, and perform product determination by HPLC.

The Rh2 production of the rare ginsenoside Rh2 strain ZWBY04RS-8E7 constructed using the mutant gene 8E7 was 70% higher than that of the strain ZWBY04RS-UGTPg45 constructed using UGTPg45, reaching 60.48mg/L.

The wild-type gene UGTPg45 has the nucleotide sequence of SEQ ID NO:20. The 1st-1374th nucleotide from the 5' end of SEQ ID NO:20 is the open reading frame of UGTPg45, and the 1st-3rd nucleotide from the 5' end of SEQ ID NO:20 is the initiation codon of UGTPg45 gene Sub ATG, the 1371-1374 nucleotides from the 5' end of SEQ ID NO: 20 is the stop codon TGA of UGTPg45 gene. The glycosyltransferase UGTPg45 gene encodes a protein UGTPg45 containing 457 amino acids, which has the amino acid residue sequence of SEQ ID NO: 19. The theoretical molecular weight of the protein is predicted to be 51.1 kDa by software, and the isoelectric point pI is 5.10. The 332-375th amino acid from the amino terminal of SEQ ID NO: 19 is the conserved functional domain of glycosyltransferase PSPG.

UGTPg45 amino acid sequence SEQ_ID_NO.19

MEREMLSKTHIMFIPFPAQGHMSPMMQFAKRLAWKGLRITIVLPAQIRDFMQITNPLINTECISFDFDKDDGMPYSMQAYMGVVKLKVTNKLSDLLEKQRTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNARLGKLKIPPEEGLTSVSLPSIPLLGRDDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSQWPITSIGPLIPSMYLDKQLPNDKDNGINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKYHFIWVVIESERTKLSSDFLAEAEAEEKGLIVSWCPQLQVLSHKSIGSFMTHCGWNSTVEALSLGVPMVALPQQFDQPANAKYIVDVWQIGVRVPIGEEGVVLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKLVSS

UGTPg45 nucleotide sequence SEQ_ID_NO.20

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGCCCAATGATGCAATTCGCCAAGCGTTTAGCCTGGAAAGGCCTGCGAATCACGATAGTTCTTCCGGCTCAAATTCGAGATTTCATGCAAATAACCAACCCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGACGATGGGATGCCATACAGCATGCAGGCTTATATGGGAGTTGTAAAACTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAGAACAAATGGCTACCCTGTTAATTTGCTAGTGGTTGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGGGGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGAAGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAGATGATTTGCCAATTATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGATCTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCAATGGCCAATTACATCCATCGGACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGGCATTAATTTCTACAAGGCAGACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAAGCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACATAGCAAATATCACTTCATATGGGTTGTTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGCAGAGGCAGAGGAAAAAGGCCTAATAGTGAGTTGGT GCCCTCAACTCCAAGTTTTGTCACATAAATCTATAGGGAGTTTTATGACTCATTGTGGTTGGAACTCGACGGTTGAGGCATTGAGTTTGGGCGTGCCAATGGTGGCACTGCCACAACAGTTTGATCAGCCTGCTAATGCCAAGTATATCGTGGATGTATGGCAAATTGGGGTTCGGGTTCCGATTGGTGAAGAGGGGGTTGTTTTGAGGGGAGAAGTTGCTAACTGTATAAAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGTGGAGGCAATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

The mutant gene 8E7 has the nucleotide sequence of SEQ ID NO:22. The 1-1374 nucleotides from the 5' end of SEQ ID NO:22 are the open reading frame of 8E7, and the 1-3 nucleotides from the 5' end of SEQ ID NO:22 are the initiation codon of the 8E7 gene Sub ATG, the 1371-1374th nucleotides from the 5' end of SEQ ID NO:22 is the stop codon TGA of the 8E7 gene. The glycosyltransferase 8E7 gene encodes a protein 8E7 containing 457 amino acids, and has the amino acid residue sequence of SEQ ID NO:21.

8E7 amino acid sequence SEQ_ID_NO.21

MEREMLSKTHIMFIPFPAQGHMSPMMQFAKRLAWKGLRITIVLPAQIRDFMQITNPLINTECISFDFDKDDGMPYSMQAYMGVVKLKVTNKLSDLLEKQRTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNARLGKLKIPPEEGLTSVSLPSIPLLGRDDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSHWPITSIGPLIPSMYLDKQLPNDKDNGINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKYHFIWVVIESERTKLSSDFLAEVEAEEKGLIVSWCPQLQVLSHKSIGSFMTHCGWNSTVEALSLGVPMVALPQQFDQPANAKYIVDVWQIGVRVPIGEEGVVLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKLVSS

8E7 nucleotide sequence SEQ_ID_NO.22

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGCCCAATGATGCAATTCGCCAAGCGTTTAGCCTGGAAAGGCCTGCGAATCACGATAGTTCTTCCGGCTCAAATTCGAGATTTCATGCAAATAACCAACCCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGACGATGGGATGCCATACAGCATGCAGGCTTATATGGGAGTTGTAAAACTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAGAACAAATGGCTACCCTGTTAATTTGCTAGTGGTTGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGGGGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGAAGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAGATGATTTGCCAATTATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGATCTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCATTGGCCAATTACATCCATCGGACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGGCATTAATTTCTACAAGGCAGACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAAGCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACATAGCAAATATCACTTCATATGGGTTGTTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGTAGAGGCAGAGGAAAAAGGCCTAATAGTGAGTTGGT GCCCTCAACTCCAAGTTTTGTCACATAAATCTATAGGGAGTTTTATGACTCATTGTGGTTGGAACTCGACGGTTGAGGCATTGAGTTTGGGCGTGCCAATGGTGGCACTGCCACAACAGTTTGATCAGCCTGCTAATGCCAAGTATATCGTGGATGTATGGCAAATTGGGGTTCGGGTTCCGATTGGTGAAGAGGGGGTTGTTTTGAGGGGAGAAGTTGCTAACTGTATAAAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGTGGAGGCAATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

The above results show that replacing the glycosyltransferase gene UGTPg45 derived from ginseng with the glycosyltransferase gene Pn50 derived from Panax notoginseng or the mutant gene 8E7 obtained by genetically modifying the wild-type glycosyltransferase can greatly increase the rare ginsenoside The synthesis efficiency and yield of Rh2 have significant beneficial effects.

discuss

At present, through the transcriptome analysis of ginseng, American ginseng and Panax notoginseng, researchers have discovered a large number of glycosyltransferase candidate genes, but only a very small number of glycosyltransferases have been verified to participate in the synthesis of ginsenosides. The glycosyltransferase involved in the synthesis of ginsenosides in Panax notoginseng has not been reported so far. Since Panax notoginseng also synthesizes the same ginsenosides, the discovery of glycosyltransferases from Panax notoginseng can enable us to better understand the synthetic pathways of ginsenosides in these two types of plants, and on the other hand, it can provide insights into the synthetic biology of ginsenosides. Richer components are of great significance.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Glycosyltransferases, mutants and applications thereof

<130> P2017-0476

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 22

<212>DNA

<213> Artificial sequence

<400> 1

atggagagag aaatgttgag ca 22

<210> 2

<211> 22

<212>DNA

<213> Artificial sequence

<400> 2

tcaggaggaa acaagctttg aa 22

<210> 3

<211> 1368

<212>DNA

<213> Artificial sequence

<400> 3

atggagagag aaatgttgag caaaactcac attatgttca tcccattccc agctcaaggc 60

cacatgagcc caatgatgca attcgtcaag cgtttagcct ggaaaggcgt gcgaatcacg 120

atagttcttc cggctgagat tcgagattct atgcaaataa acaactcatt gatcaacact 180

gagtgcatct cctttgattt tgataaagat gatgagatgc catacagcat gcgggcttat 240

atgggagttg taaagctcaa ggtcacaaat aaactgagtg acctactcga gaagcaaaaa 300

acaaatggct accctgttaa tttgctagtg gtcgattcat tatatccatc tcgggtagaa 360

atgtgccacc aacttggggt aaaaggagct ccatttttca ctcactcttg tgctgttggt 420

gccatttatt ataatgctcg cttagggaaa ttgaagatac ctcctgagga agggttgact 480

tctgtttcat tgccttcaat tccattgttg gggagaaatg atttgccaat tattaggact 540

ggcacctttc ctgatctctt tgagcatttg gggaatcagt tttcagatct tgataaagcg 600

gattggatct ttttcaatac ttttgataag cttgaaaatg aggaagcaaa atggctatct 660

agccaatggc caattacatc catcggacca ttaatccctt caatgtactt agacaaacaa 720

ttaccaaatg acaaagacaa tgacattaat ttctacaagg cagacgtcgg atcgtgcatc 780

aagtggctag acgccaaaga ccctggctcg gtagtctacg cctcattcgg gagcgtgaag 840

cacaacctcg gcgatgacta catggacgaa gtagcatggg gcttgttaca cagcaaatat 900

cacttcatat gggttgttat agaatccgaa cgtacaaagc tctctagcga tttcttggca 960

gaggcagagg aaaaaggcct aatagtgagt tggtgccctc aactcgaagt tttgtcacat 1020

aaatctatag gtagttttat gactcattgt ggttggaact cgacggttga ggcattgagt 1080

ttgggcgtgc caatggtggc agtgccacaa cagtttgatc agcctgttaa tgccaagtat 1140

atcgtggatg tatggcgaat tggggttcag gttccgattg gtgaaaatgg ggttcttttg 1200

aggggagaag ttgctaactg tataaaggat gttatggagg gggaaatagg ggatgagctt 1260

agagggaatg ctttgaaatg gaaggggttg gctgtggagg caatggagaa aggggggtagc 1320

tctgataaga atattgatga gttcatttca aagcttgttt cctcctga 1368

<210> 4

<211> 455

<212> PRT

<213> Artificial sequence

<400> 4

Met Glu Arg Glu Met Leu Ser Lys Thr His Ile Met Phe Ile Pro Phe

1 5 10 15

Pro Ala Gln Gly His Met Ser Pro Met Met Gln Phe Val Lys Arg Leu

20 25 30

Ala Trp Lys Gly Val Arg Ile Thr Ile Val Leu Pro Ala Glu Ile Arg

35 40 45

Asp Ser Met Gln Ile Asn Asn Ser Leu Ile Asn Thr Glu Cys Ile Ser

50 55 60

Phe Asp Phe Asp Lys Asp Asp Glu Met Pro Tyr Ser Met Arg Ala Tyr

65 70 75 80

Met Gly Val Val Lys Leu Lys Val Thr Asn Lys Leu Ser Asp Leu Leu

85 90 95

Glu Lys Gln Lys Thr Asn Gly Tyr Pro Val Asn Leu Leu Val Val Asp

100 105 110

Ser Leu Tyr Pro Ser Arg Val Glu Met Cys His Gln Leu Gly Val Lys

115 120 125

Gly Ala Pro Phe Phe Thr His Ser Cys Ala Val Gly Ala Ile Tyr Tyr

130 135 140

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

145 150 155 160

Ser Val Ser Leu Pro Ser Ile Pro Leu Leu Gly Arg Asn Asp Leu Pro

165 170 175

Ile Ile Arg Thr Gly Thr Phe Pro Asp Leu Phe Glu His Leu Gly Asn

180 185 190

Gln Phe Ser Asp Leu Asp Lys Ala Asp Trp Ile Phe Phe Asn Thr Phe

195 200 205

Asp Lys Leu Glu Asn Glu Glu Ala Lys Trp Leu Ser Ser Gln Trp Pro

210 215 220

Ile Thr Ser Ile Gly Pro Leu Ile Pro Ser Met Tyr Leu Asp Lys Gln

225 230 235 240

Leu Pro Asn Asp Lys Asp Asn Asp Ile Asn Phe Tyr Lys Ala Asp Val

245 250 255

Gly Ser Cys Ile Lys Trp Leu Asp Ala Lys Asp Pro Gly Ser Val Val

260 265 270

Tyr Ala Ser Phe Gly Ser Val Lys His Asn Leu Gly Asp Asp Tyr Met

275 280 285

Asp Glu Val Ala Trp Gly Leu Leu His Ser Lys Tyr His Phe Ile Trp

290 295 300

Val Val Ile Glu Ser Glu Arg Thr Lys Leu Ser Ser Asp Phe Leu Ala

305 310 315 320

Glu Ala Glu Glu Lys Gly Leu Ile Val Ser Trp Cys Pro Gln Leu Glu

325 330 335

Val Leu Ser His Lys Ser Ile Gly Ser Phe Met Thr His Cys Gly Trp

340 345 350

Asn Ser Thr Val Glu Ala Leu Ser Leu Gly Val Pro Met Val Ala Val

355 360 365

Pro Gln Gln Phe Asp Gln Pro Val Asn Ala Lys Tyr Ile Val Asp Val

370 375 380

Trp Arg Ile Gly Val Gln Val Pro Ile Gly Glu Asn Gly Val Leu Leu

385 390 395 400

Arg Gly Glu Val Ala Asn Cys Ile Lys Asp Val Met Glu Gly Glu Ile

405 410 415

Gly Asp Glu Leu Arg Gly Asn Ala Leu Lys Trp Lys Gly Leu Ala Val

420 425 430

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

435 440 445

Ile Ser Lys Leu Val Ser Ser

450 455

<210> 5

<211> 28

<212>DNA

<213> Artificial sequence

<400> 5

ggatccatgg agagagaaat gttgagca 28

<210> 6

<211> 28

<212>DNA

<213> Artificial sequence

<400> 6

ctcgagtcag gaggaaacaa gctttgaa 28

<210> 7

<211> 56

<212>DNA

<213> Artificial sequence

<400> 7

tagctctgat aagaatattg atgagttcat ttcaaagctt gtttcctcct gagatt 56

<210> 8

<211> 69

<212>DNA

<213> Artificial sequence

<400> 8

actgtcaagg agggtattct gggcctccat gtcgctgcta tataacagtt gaaatttgga 60

taagaacat 69

<210> 9

<211> 59

<212>DNA

<213> Artificial sequence

<400> 9

gctatactgc tgtcgattcg atactaacgc cgccatccag tgtcgagaat tacaatagt 59

<210> 10

<211> 44

<212>DNA

<213> Artificial sequence

<400> 10

tctggtgagg atttacggta tgatcatgct ggtaagcttc gtta 44

<210> 11

<211> 59

<212>DNA

<213> Artificial sequence

<400> 11

gaaaagaaga taatattttt atataattat attaatctca ggaggaaaca agctttgaa 59

<210> 12

<211> 59

<212>DNA

<213> Artificial sequence

<400> 12

gcatagcaat ctaatctaag ttttaattac aaaatggaga gagaaatgtt gagcaaaac 59

<210> 13

<211> 50

<212>DNA

<213> Artificial sequence

<400> 13

cgcgttgaga agatgttctt atccaaattt caactgttat atagcagcga 50

<210> 14

<211> 59

<212>DNA

<213> Artificial sequence

<400> 14

ttacttcttg cagacatcag acatactatt gtaattctcg acactggatg gcggcgtta 59

<210> 15

<211> 59

<212>DNA

<213> Artificial sequence

<400> 15

ggctgtcgcc attcaagagc agatagcttc aaaatgtttc tactcctttt ttactcttc 59

<210> 16

<211> 59

<212>DNA

<213> Artificial sequence

<400> 16

cataatgtga gttttgctca aatttctct ctccatttg taattaaaac ttagattag 59

<210> 17

<211> 27

<212>DNA

<213> Artificial sequence

<400> 17

cccaaagcta agagtcccat tttattc 27

<210> 18

<211> 59

<212>DNA

<213> Artificial sequence

<400> 18

gagtagaaac attttgaagc tatctgctct tgaatggcga cagcctattg ccccagtgt59

<210> 19

<211> 457

<212> PRT

<213> Artificial sequence

<400> 19

Met Glu Arg Glu Met Leu Ser Lys Thr His Ile Met Phe Ile Pro Phe

1 5 10 15

Pro Ala Gln Gly His Met Ser Pro Met Met Gln Phe Ala Lys Arg Leu

20 25 30

Ala Trp Lys Gly Leu Arg Ile Thr Ile Val Leu Pro Ala Gln Ile Arg

35 40 45

Asp Phe Met Gln Ile Thr Asn Pro Leu Ile Asn Thr Glu Cys Ile Ser

50 55 60

Phe Asp Phe Asp Lys Asp Asp Gly Met Pro Tyr Ser Met Gln Ala Tyr

65 70 75 80

Met Gly Val Val Lys Leu Lys Val Thr Asn Lys Leu Ser Asp Leu Leu

85 90 95

Glu Lys Gln Arg Thr Asn Gly Tyr Pro Val Asn Leu Leu Val Val Asp

100 105 110

Ser Leu Tyr Pro Ser Arg Val Glu Met Cys His Gln Leu Gly Val Lys

115 120 125

Gly Ala Pro Phe Phe Thr His Ser Cys Ala Val Gly Ala Ile Tyr Tyr

130 135 140

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

145 150 155 160

Ser Val Ser Leu Pro Ser Ile Pro Leu Leu Gly Arg Asp Asp Leu Pro

165 170 175

Ile Ile Arg Thr Gly Thr Phe Pro Asp Leu Phe Glu His Leu Gly Asn

180 185 190

Gln Phe Ser Asp Leu Asp Lys Ala Asp Trp Ile Phe Phe Asn Thr Phe

195 200 205

Asp Lys Leu Glu Asn Glu Glu Ala Lys Trp Leu Ser Ser Gln Trp Pro

210 215 220

Ile Thr Ser Ile Gly Pro Leu Ile Pro Ser Met Tyr Leu Asp Lys Gln

225 230 235 240

Leu Pro Asn Asp Lys Asp Asn Gly Ile Asn Phe Tyr Lys Ala Asp Val

245 250 255

Gly Ser Cys Ile Lys Trp Leu Asp Ala Lys Asp Pro Gly Ser Val Val

260 265 270

Tyr Ala Ser Phe Gly Ser Val Lys His Asn Leu Gly Asp Asp Tyr Met

275 280 285

Asp Glu Val Ala Trp Gly Leu Leu His Ser Lys Tyr His Phe Ile Trp

290 295 300

Val Val Ile Glu Ser Glu Arg Thr Lys Leu Ser Ser Asp Phe Leu Ala

305 310 315 320

Glu Ala Glu Ala Glu Glu Lys Gly Leu Ile Val Ser Trp Cys Pro Gln

325 330 335

Leu Gln Val Leu Ser His Lys Ser Ile Gly Ser Phe Met Thr His Cys

340 345 350

Gly Trp Asn Ser Thr Val Glu Ala Leu Ser Leu Gly Val Pro Met Val

355 360 365

Ala Leu Pro Gln Gln Phe Asp Gln Pro Ala Asn Ala Lys Tyr Ile Val

370 375 380

Asp Val Trp Gln Ile Gly Val Arg Val Pro Ile Gly Glu Glu Gly Val

385 390 395 400

Val Leu Arg Gly Glu Val Ala Asn Cys Ile Lys Asp Val Met Glu Gly

405 410 415

Glu Ile Gly Asp Glu Leu Arg Gly Asn Ala Leu Lys Trp Lys Gly Leu

420 425 430

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

435 440 445

Glu Phe Ile Ser Lys Leu Val Ser Ser

450 455

<210> 20

<211> 1374

<212>DNA

<213> Artificial sequence

<400> 20

atggagagag aaatgttgag caaaactcac attatgttca tcccattccc agctcaaggc 60

cacatgagcc caatgatgca attcgccaag cgtttagcct ggaaaggcct gcgaatcacg 120

atagttcttc cggctcaaat tcgagatttc atgcaaataa ccaacccatt gatcaacact 180

gagtgcatct cctttgattt tgataaagac gatgggatgc catacagcat gcaggcttat 240

atgggagttg taaaactcaa ggtcacaaat aaactgagtg acctactcga gaagcaaaga 300

acaaatggct accctgttaa tttgctagtg gttgattcat tatatccatc tcgggtagaa 360

atgtgccacc aacttggggt aaaaggagct ccatttttca ctcactcttg tgctgttggt 420

gccatttatt ataatgctcg cttagggaaa ttgaagatac ctcctgagga agggttgact 480

tctgtttcat tgccttcaat tccattgttg gggagagatg atttgccaat tattaggact 540

ggcacctttc ctgatctctt tgagcatttg gggaatcagt tttcagatct tgataaagcg 600

gattggatct ttttcaatac ttttgataag cttgaaaatg aggaagcaaa atggctatct 660

agccaatggc caattacatc catcggacca ttaatccctt caatgtactt agacaaacaa 720

ttaccaaatg acaaagacaa tggcattaat ttctacaagg cagacgtcgg atcgtgcatc 780

aagtggctag acgccaaaga ccctggctcg gtagtctacg cctcattcgg gagcgtgaag 840

cacaacctcg gcgatgacta catggacgaa gtagcatggg gcttgttaca tagcaaatat 900

cacttcatat gggttgttat agaatccgaa cgtacaaagc tctctagcga tttcttggca 960

gaggcagagg cagaggaaaa aggcctaata gtgagttggt gccctcaact ccaagttttg 1020

tcacataaat ctataggggag ttttatgact cattgtggtt ggaactcgac ggttgaggca 1080

ttgagtttgg gcgtgccaat ggtggcactg ccacaacagt ttgatcagcc tgctaatgcc 1140

aagtatatcg tggatgtatg gcaaattggg gttcgggttc cgattggtga agagggggtt 1200

gttttgaggg gagaagttgc taactgtata aaggatgtta tggaggggga aataggggat 1260

gagcttagag ggaatgcttt gaaatggaag gggttggctg tggaggcaat ggagaaaggg 1320

ggtagctctg ataagaatat tgatgagttc atttcaaagc ttgtttcctc ctga 1374

<210> 21

<211> 457

<212> PRT

<213> Artificial sequence

<400> 21

Met Glu Arg Glu Met Leu Ser Lys Thr His Ile Met Phe Ile Pro Phe

1 5 10 15

Pro Ala Gln Gly His Met Ser Pro Met Met Gln Phe Ala Lys Arg Leu

20 25 30

Ala Trp Lys Gly Leu Arg Ile Thr Ile Val Leu Pro Ala Gln Ile Arg

35 40 45

Asp Phe Met Gln Ile Thr Asn Pro Leu Ile Asn Thr Glu Cys Ile Ser

50 55 60

Phe Asp Phe Asp Lys Asp Asp Gly Met Pro Tyr Ser Met Gln Ala Tyr

65 70 75 80

Met Gly Val Val Lys Leu Lys Val Thr Asn Lys Leu Ser Asp Leu Leu

85 90 95

Glu Lys Gln Arg Thr Asn Gly Tyr Pro Val Asn Leu Leu Val Val Asp

100 105 110

Ser Leu Tyr Pro Ser Arg Val Glu Met Cys His Gln Leu Gly Val Lys

115 120 125

Gly Ala Pro Phe Phe Thr His Ser Cys Ala Val Gly Ala Ile Tyr Tyr

130 135 140

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

145 150 155 160

Ser Val Ser Leu Pro Ser Ile Pro Leu Leu Gly Arg Asp Asp Leu Pro

165 170 175

Ile Ile Arg Thr Gly Thr Phe Pro Asp Leu Phe Glu His Leu Gly Asn

180 185 190

Gln Phe Ser Asp Leu Asp Lys Ala Asp Trp Ile Phe Phe Asn Thr Phe

195 200 205

Asp Lys Leu Glu Asn Glu Glu Ala Lys Trp Leu Ser Ser His Trp Pro

210 215 220

Ile Thr Ser Ile Gly Pro Leu Ile Pro Ser Met Tyr Leu Asp Lys Gln

225 230 235 240

Leu Pro Asn Asp Lys Asp Asn Gly Ile Asn Phe Tyr Lys Ala Asp Val

245 250 255

Gly Ser Cys Ile Lys Trp Leu Asp Ala Lys Asp Pro Gly Ser Val Val

260 265 270

Tyr Ala Ser Phe Gly Ser Val Lys His Asn Leu Gly Asp Asp Tyr Met

275 280 285

Asp Glu Val Ala Trp Gly Leu Leu His Ser Lys Tyr His Phe Ile Trp

290 295 300

Val Val Ile Glu Ser Glu Arg Thr Lys Leu Ser Ser Asp Phe Leu Ala

305 310 315 320

Glu Val Glu Ala Glu Glu Lys Gly Leu Ile Val Ser Trp Cys Pro Gln

325 330 335

Leu Gln Val Leu Ser His Lys Ser Ile Gly Ser Phe Met Thr His Cys

340 345 350

Gly Trp Asn Ser Thr Val Glu Ala Leu Ser Leu Gly Val Pro Met Val

355 360 365

Ala Leu Pro Gln Gln Phe Asp Gln Pro Ala Asn Ala Lys Tyr Ile Val

370 375 380

Asp Val Trp Gln Ile Gly Val Arg Val Pro Ile Gly Glu Glu Gly Val

385 390 395 400

Val Leu Arg Gly Glu Val Ala Asn Cys Ile Lys Asp Val Met Glu Gly

405 410 415

Glu Ile Gly Asp Glu Leu Arg Gly Asn Ala Leu Lys Trp Lys Gly Leu

420 425 430

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

435 440 445

Glu Phe Ile Ser Lys Leu Val Ser Ser

450 455

<210> 22

<211> 1374

<212>DNA

<213> Artificial sequence

<400> 22

atggagagag aaatgttgag caaaactcac attatgttca tcccattccc agctcaaggc 60

cacatgagcc caatgatgca attcgccaag cgtttagcct ggaaaggcct gcgaatcacg 120

atagttcttc cggctcaaat tcgagatttc atgcaaataa ccaacccatt gatcaacact 180

gagtgcatct cctttgattt tgataaagac gatgggatgc catacagcat gcaggcttat 240

atgggagttg taaaactcaa ggtcacaaat aaactgagtg acctactcga gaagcaaaga 300

acaaatggct accctgttaa tttgctagtg gttgattcat tatatccatc tcgggtagaa 360

atgtgccacc aacttggggt aaaaggagct ccatttttca ctcactcttg tgctgttggt 420

gccatttatt ataatgctcg cttagggaaa ttgaagatac ctcctgagga agggttgact 480

tctgtttcat tgccttcaat tccattgttg gggagagatg atttgccaat tattaggact 540

ggcacctttc ctgatctctt tgagcatttg gggaatcagt tttcagatct tgataaagcg 600

gattggatct ttttcaatac ttttgataag cttgaaaatg aggaagcaaa atggctatct 660

agccattggc caattacatc catcggacca ttaatccctt caatgtactt agacaaacaa 720

ttaccaaatg acaaagacaa tggcattaat ttctacaagg cagacgtcgg atcgtgcatc 780

aagtggctag acgccaaaga ccctggctcg gtagtctacg cctcattcgg gagcgtgaag 840

cacaacctcg gcgatgacta catggacgaa gtagcatggg gcttgttaca tagcaaatat 900

cacttcatat gggttgttat agaatccgaa cgtacaaagc tctctagcga tttcttggca 960

gaggtagagg cagaggaaaa aggcctaata gtgagttggt gccctcaact ccaagttttg 1020

tcacataaat ctataggggag ttttatgact cattgtggtt ggaactcgac ggttgaggca 1080

ttgagtttgg gcgtgccaat ggtggcactg ccacaacagt ttgatcagcc tgctaatgcc 1140

aagtatatcg tggatgtatg gcaaattggg gttcgggttc cgattggtga agagggggtt 1200

gttttgaggg gagaagttgc taactgtata aaggatgtta tggaggggga aataggggat 1260

gagcttagag ggaatgcttt gaaatggaag gggttggctg tggaggcaat ggagaaaggg 1320

ggtagctctg ataagaatat tgatgagttc atttcaaagc ttgtttcctc ctga 1374

<210> 23

<211> 59

<212>DNA

<213> Artificial sequence

<400> 23

gcatagcaat ctaatctaag ttttaattac aaaatggaga gagaaatgtt gagcaaaac 59

<210> 24

<211> 59

<212>DNA

<213> Artificial sequence

<400> 24

gaaaagaaga taatattttt atataattat attaatctca ggaggaaaca agctttgaa 59

<210> 25

<211> 59

<212>DNA

<213> Artificial sequence

<400> 25

acactggggc aataggctgt cgccattcaa gagcagatag cttcaaaatg tttctactc 59

<210> 26

<211> 59

<212>DNA

<213> Artificial sequence

<400> 26

cataatgtga gttttgctca aatttctct ctccatttg taattaaaac ttagattag 59

<210> 27

<211> 59

<212>DNA

<213> Artificial sequence

<400> 27

ttgatgagtt catttcaaag cttgtttcct cctgagatta atataattat ataaaaata 59

<210> 28

<211> 59

<212>DNA

<213> Artificial sequence

<400> 28

actgtcaagg agggtattct gggcctccat gtcgctgcta tataacagtt gaaatttgg 59

<210> 29

<211> 27

<212>DNA

<213> Artificial sequence

<400> 29

cccaaagcta agagtcccat tttattc 27

<210> 30

<211> 59

<212>DNA

<213> Artificial sequence

<400> 30

gaagagtaaa aaaggagtag aaacattttg aagctatctg ctcttgaatg gcgacagcc 59

<210> 31

<211> 59

<212>DNA

<213> Artificial sequence

<400> 31

tgtcgattcg atactaacgc cgccatccag tgtcgagaat tacaatagta tgtctgatg 59

<210> 32

<211> 22

<212>DNA

<213> Artificial sequence

<400> 32

tctggtgagg atttacggta tg 22

<210> 33

<211> 59

<212>DNA

<213> Artificial sequence

<400> 33

aagatgttct tatccaaatt tcaactgtta tatagcagcg acatggaggc ccagaatac 59

<210> 34

<211> 59

<212>DNA

<213> Artificial sequence

<400> 34

tacttcttgc agacatcaga catactattg taattctcga cactggatgg cggcgttag 59

Claims (13)

1. a kind of isolated polypeptide, which is characterized in that the amino acid sequence of the isolated polypeptide is corresponding to SEQ ID NO: 222nd amino acid residue of amino acid sequence shown in 19 is non-Gln and/or is corresponding to SEQ ID NO:Amino shown in 19 The amino acid residue that acid sequence is the 322nd is non-Ala.

2. a kind of isolated polypeptide, which is characterized in that the polypeptide is selected from the group：

(a) there is SEQ ID NO.:4 or SEQ ID NO.:The polypeptide of amino acid sequence shown in 21；

(b) by SEQ ID NO.:4 or SEQ ID NO.:The polypeptide of amino acid sequence shown in 21 passes through one or several amino acid Residue, preferably 1-20 1-15 more preferable, 1-10 more preferable, 1-3 more preferable, most preferably 1 amino acid residue take Generation, missing or addition and formed or addition signal peptide sequence after formed and with glycosyl transferase activity derivative it is more Peptide；

(c) in sequence containing (a) or (b) described in polypeptide sequence derived peptides；

(d) amino acid sequence and SEQ ID NO.:4 or SEQ ID NO.:Amino acid sequence shown in 21 homology >=85% (compared with Goodly >=90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), and have glycosyl transferase living The derived peptides of property.

3. a kind of isolated polynucleotides, which is characterized in that the polynucleotides are sequence selected from the group below：

(A) nucleotide sequence of polypeptide as claimed in claim 1 or 2 is encoded；

(B) coding such as SEQ ID NO.:4 or SEQ ID NO.:The nucleotide sequence of polypeptide or its derived peptides shown in 21；

(C) such as SEQ ID NO.:3 or SEQ ID NO.:Nucleotide sequence shown in 22；

(D) with SEQ ID NO.:3 or SEQ ID NO.:Homology >=90% of sequence shown in 22 is (preferably >=91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or nucleotide sequence 99%)；

(E) in SEQ ID NO.:3 or SEQ ID NO.:5 ' the ends and/or 3 ' ends of nucleotide sequence shown in 22 truncate or addition 1- 60 (preferably 1-30, more preferably 1-10) nucleotide are formed by nucleotide sequence；

(F) nucleotide sequence complementary with (A)-(E) any nucleotide sequence.

4. a kind of carrier, which is characterized in that the carrier contains polynucleotides as claimed in claim 3.

5. the purposes of isolated polypeptide as claimed in claim 1 or 2, which is characterized in that it be used to be catalyzed following reaction, or by with The following catalyst formulations reacted are catalyzed in preparation：

(i) glycosyl from glycosyl donor is transferred on the position the C-3 hydroxyl of tetracyclic triterpenoid.

6. purposes as claimed in claim 5, which is characterized in that the isolated polypeptide is for being catalyzed following reactions or being used for Preparation is catalyzed the catalyst formulations of following reactions：

Wherein, R1 is H or OH；R2 is H or OH；R3 is H or glycosyl；R4 is glycosyl.

7. a kind of external glycosylation process, which is characterized in that including step：

In the presence of glycosyl transferase, the glycosyl of glycosyl donor is transferred on the position the C-3 hydroxyl of tetracyclic triterpenoid；From And form glycosylated tetracyclic triterpenoid；

Wherein, the glycosyl transferase is polypeptide of any of claims 1 or 2 or its derived peptides.

8. the method for claim 7, which is characterized in that the derived peptides are selected from：

By SEQ ID NO.:4 or SEQ ID NO.:The polypeptide of amino acid sequence shown in 21 passes through one or several amino acid residues Replace, miss or add and form or add formed after signal peptide sequence and spreading out with glycosyl transferase activity Raw polypeptide；Or

Amino acid sequence and SEQ ID NO.:4 or SEQ ID NO.:21 amino acid sequences homology >=85% (preferably >= 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and the derivative with glycosyl transferase activity 90%, Polypeptide；

Wherein, the glycosyl transferase activity refers to the position C-3 that the glycosyl of glycosyl donor can be transferred to tetracyclic triterpenoid Activity on hydroxyl.

9. a kind of method for carrying out glycosyl catalysis reaction, which is characterized in that including step：In polypeptide of any of claims 1 or 2 Or under the conditions of its derived peptides is existing, glycosyl catalysis reaction is carried out.

10. method as claimed in claim 9, which is characterized in that the substrate of the glycosyl catalysis reaction is formula (I) compound, And the product is formula (II) compound.

11. a kind of genetically engineered host cell, which is characterized in that the host cell contains as claimed in claim 4 Polynucleotides as claimed in claim 3 are integrated in carrier or its genome.

12. the purposes of host cell described in claim 11, which is characterized in that be used to prepare enzymatic reagent, or production sugar Based transferase as activated cell or generates glycosylated tetracyclic triterpenoid.

13. a kind of method for generating genetically modified plants, which is characterized in that including step：By hereditary work described in claim 11 The host cell of journey is regenerated as plant, and the genetically engineered host cell is plant cell.