CN108866020A

CN108866020A - Glycosyl transferase, mutant and its application

Info

Publication number: CN108866020A
Application number: CN201710344730.7A
Authority: CN
Inventors: 周志华; 严兴; 王平平; 魏维; 李晓东
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2017-05-16
Filing date: 2017-05-16
Publication date: 2018-11-23
Also published as: CN110462033A; WO2018210208A1; JP2020520244A; KR102418138B1; KR20200016268A; JP7086107B2

Abstract

The present invention relates to glycosyl transferase, mutant and its applications.Specifically, a kind of external glycosylation process, including step are provided：In the presence of glycosyl transferase, the glycosyl of glycosyl donor is transferred on the position the C-3 hydroxyl of tetracyclic triterpenoid；To form glycosylated tetracyclic triterpenoid；Wherein, the glycosyl transferase is the glycosyl transferase as shown in SEQ ID NO.4 or its derived peptides；Or the glycosyl transferase as shown in SEQ ID NO.21 or its derived peptides.

Description

Glycosyl transferase, mutant and its application

Technical field

The present invention relates to biotechnologys and phytobiology field, in particular it relates to close for ginseng saponin Rh 2 At glycosyl transferase, glycosyl transferase mutant and its application.

Background technique

Ginsenoside is the main active substances in araliaceae ginseng plant (such as ginseng, Radix Notoginseng, American Ginseng), in recent years Come in cucurbitaceous plant Radix Notoginseng it has also been found that some ginsenosides.Currently, scientist is from plants such as ginseng, Radix Notoginseng both at home and abroad In isolated at least 100 kinds of saponin(es, ginsenoside and belong to triterpenoid saponin.Some of ginseng saponin(es are proven to have widely Physiological function and medical value：Including the functions such as antitumor, immunological regulation, antifatigue, shield heart, protect liver.Many of saponin(e is Through can such as improve the qi deficiency of tumor patient with ginseng sapoglycoside Rg 3 monomer drug Shenyi capsule as main component for clinic Shape improves body's immunity.It is a kind of health medicine for mentioning with ginseng saponin Rh 2 monomer modern good fortune capsule as main component High immunity of organisms enhances disease resistance.

From structure, ginsenoside is the bioactive small molecule that sapogenin is formed after glycosylation.Ginseng soap The sapogenin of glycosides only has limited several, the mainly protopanoxadiol of dammarane type and protopanaxatriol and oleanane The botany bar gum of type.In addition to the difference of sapogenin, the difference between ginsenoside in structure is mainly reflected in the different sugar of sapogenin In baseization modification.The sugar chain of ginsenoside is typically incorporated on the hydroxyl of C3, C6 or C20 of sapogenin, and glycosyl can be grape Sugar, rhamnose, xylose and arabinose.

Different glycosyl binding sites, sugar chain composition and length generate ginsenoside in physiological function and medical value Great difference.For example, ginsenoside Rb1, Rd and Rc are the saponin(e using protopanoxadiol as sapogenin, the difference between them Not Zhi Shi it is glycosyl modified on difference, but the physiological function between them just has many difference.Rb1 has stable central nervous member The function of system, and the function of Rc is to inhibit the function of central nervous metasystem, the physiological function of Rb1 is very extensive, and Rd But there was only very limited several functions.

Rare ginsenoside refers to the saponin(e that content is extremely low in ginseng.Ginseng saponin Rh 2 (3-O- β-(D- Glucopyranosyl) -20 (S)-protopanaxadiol) saponin(e that belongs to protopanoxadiol class, in the position C-3 of sapogenin A glucosyl group is connected on hydroxyl.The a ten thousandth or so of the content of ginseng saponin Rh 2 only about ginseng dry weight, still, Ginseng saponin Rh 2 has good anti-tumor activity, is one of most important anti-tumor active ingredient in ginseng, is able to suppress swollen Tumor cell growth, inducing apoptosis of tumour cell, anti-tumor metastasis.Research shows that ginseng saponin Rh 2 is able to suppress lung cancer Cells 3LL (mice), Morris liver cancer cells (rats), B-16melanoma cells (mice), and The increment of HeLa cells (human).Clinically, ginseng saponin Rh 2 and radiotherapy or chemotherapy combined treatment, can be enhanced radiotherapy With the effect of chemotherapy.In addition, ginseng saponin Rh 2 also has antiallergy, the function of immunity of organisms is improved, NO and PGE is inhibited to produce The effects of raw inflammation.

The function of glycosyl transferase is by the glycosyl transfer on glycosyl donor (nucleoside diphosphate sugar, such as UDP-glucose) Onto different glycosyl acceptors.According to the difference of amino acid sequence, glycosyl transferase has 94 families at present.It has been sequenced at present Plant Genome in, it was found that hundreds of above different glycosyl transferase.The glycosyl acceptor of these glycosyl transferases includes Sugar, rouge, albumen, nucleic acid, antibiotic and other small molecules.The glycosyl transferase that saponin glycosyl is participated in ginseng, makees With being the glycosyl on glycosyl donor to be transferred on the hydroxyl of C3, C6 or C20 of sapogenin or aglycon, so that being formed has not With the saponin(e of medical value.

This field still lacks a kind of effective method for producing rare ginsenoside Rh2, Ginsenoside F2 at present, therefore compels A variety of specifical and efficient glycosyl transferases will be developed by being essential.

Summary of the invention

It is an object of the invention to provide a kind of glycosyl transferase and its applications, for synthesizing rare ginsenoside Rh2, people Join saponin(e F2.

The first aspect of the present invention provides a kind of isolated polypeptide, and the amino acid sequence of the isolated polypeptide is right It should be in 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:The amino acid residue that amino acid sequence shown in 19 is the 322nd is non-Ala.

In another preferred example, the isolated polypeptide：

I) has such as SEQ ID NO:Amino acid sequence shown in 19 and the 222nd amino acid residue be non-Gln and/or 322nd amino acid residue is non-Ala, or

Ii) have i) defined by sequence pass through one or several amino acid residues, preferably 1-20, more preferable 1-15 A, more preferable 1-10,1-3 more preferable, most preferably 1 replacing, missing or adding for amino acid residue and the sequence formed, And have substantially i) defined by separation polypeptide function by the polypeptide that separates derived from i).

In another preferred example, the isolated polypeptide have i) defined by sequence by one or several amino acid it is residual Base, preferably 1-20,1-15 more preferable, 1-10 more preferable, 1-3 more preferable, most preferably 1 amino acid residue addition And the sequence formed, and have substantially i) defined by separation polypeptide function by the polypeptide that separates derived from i).

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

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

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

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

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

In another preferred example, the isolated polypeptide：

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

Iv) passes through one or several amino acid residues, preferably 1-20, more preferable 1- with sequence defined by iii) 15, more preferable 1-10,1-3 more preferable, most preferably 1 replacing, missing or adding for amino acid residue and the sequence formed Column, and substantially with iii) defined by separation polypeptide function by iii) derived from the polypeptide that separates.

In another preferred example, the isolated polypeptide passes through one or several amino acid with sequence defined by iii) Residue, preferably 1-20 1-15 more preferable, 1-10 more preferable, 1-3 more preferable, most preferably 1 amino acid residue add The sequence for adding and being formed, and substantially with iii) defined by separation polypeptide function by iii) derived from the polypeptide that separates.

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

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

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

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

In another preferred example, the amino acid sequence of the isolated polypeptide is in SEQ ID NO:Amino acid sequence shown in 19 The 222nd amino acid residue be His, in SEQ ID NO:The amino acid residue that amino acid sequence shown in 19 is the 322nd is Val。

In another preferred example, the isolated polypeptide is glycosyl transferase.

In another preferred example, the glycosyl transferase derives from panax species.

In another preferred example, the glycosyl transferase derives from ginseng, American Ginseng and/or Radix Notoginseng.

In another preferred example, 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 ammonia Base acid residue, preferably 1-20,1-15 more preferable, 1-10 more preferable, 1-3 more preferable, most preferably 1 amino acid residue Replace, miss or add and form or add formed after signal peptide sequence and spreading out with glycosyl transferase activity Raw polypeptide；

(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.:The homology of amino acid sequence shown in 21 >= 85% (preferably >=90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), and turn with glycosyl Move the derived peptides of enzymatic activity.

In another preferred example, the sequence (c) is served as reasons (a) or (b) is added to sequence label, signal sequence or secretion Fusion protein is formed by after signal sequence.

In another preferred example, the glycosyl transferase activity, which refers to, to be transferred to tetracyclic triterpenes for the glycosyl of glycosyl donor Activity on the position the C-3 hydroxyl of compound.

In another preferred example, the glycosyl transferase can improve the yield of ginseng saponin Rh 2 and/or Ginsenoside F2.

In another preferred example, in artificial constructed bacterial strain, the glycosyl transferase can improve the production of ginseng saponin Rh 2 Amount；Preferably, 5-150% is improved；It is highly preferred that improving 10-100%；It is highly preferred that improving 20-80%；Most preferably, it improves 28-70%.

In another preferred example, the artificial constructed bacterial strain is selected from the group：Wine brewing yeast strain, coli strain, Pichi strain, fission yeast bacterial strain, kluyveromyces yeast strains.

The second aspect of the present invention, provides a kind of isolated polynucleotides, and the polynucleotides are selected from the group below Sequence：

(A) nucleotide sequence of polypeptide described in the first aspect of the present invention 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 add 1-60 (preferably 1-30, more preferably 1-10) nucleotide are added to be formed by nucleotide sequence；

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

The third aspect of the present invention, provides a kind of carrier, and the carrier contains described in the second aspect of the present invention Polynucleotides.

In another preferred example, the carrier includes expression vector, shuttle vector, integration vector.

The fourth aspect of the present invention, provides the purposes of the polypeptide of separation described in the first aspect of the present invention, it is used for The following reaction of catalysis, or be used to prepare the following catalyst formulations reacted of catalysis：

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

In another preferred example, the glycosyl donor includes nucleoside diphosphate sugar selected from the group below：UDP-glucose, ADP- glucose, TDP- glucose, CDP- glucose, GDP- glucose, UDP- acetyl group glucose, ADP- acetyl group grape Sugar, TDP- acetyl group glucose, CDP- acetyl group glucose, GDP- acetyl group 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- galactolipin, ADP- galactolipin, TDP- galactolipin, CDP- galactolipin, GDP- galactolipin, UDP- arabinose, ADP- arabinose, TDP- arabinose, CDP- arabinose, GDP- arabinose, UDP- rhamnose, ADP- rhamnose, TDP- rhamnose, CDP- rhamnose, GDP- rhamnose or other nucleoside diphosphate hexoses or Nucleoside diphosphate pentose, or combinations thereof.

In another preferred example, the glycosyl donor includes uridine 5'-diphosphate selected from the group below (UDP) sugar：UDP- grape Sugar, UDP- xylose, UDP- galacturonic acid, UDP- galactolipin, UDP- arabinose, UDP- rhamnose or other two phosphorus of uridine Sour hexose or uridine 5'-diphosphate pentose, or combinations thereof.

In another preferred example, the isolated polypeptide is for being catalyzed following reactions or being used to prepare the following reactions of catalysis Catalyst formulations：

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

The polypeptide is selected from SEQ ID NO.:4 or SEQ ID NO.:Polypeptide shown in 21 or its derived peptides.

In another preferred example, the glycosyl is selected from：Glucosyl group, galacturonic acidic group, xylose glycosyl, galactolipin Base, aralino, rhamnopyranosyl and other hexose-baseds or pentose base.

In another preferred example, the reaction product of the reaction (A) and/or (B) includes but is not limited to：S configuration or R structure Dammarane type four-ring triterpenoid class compound, the lanolin alkane type tetracyclic triterpenoid, apotirucallane type Fourth Ring of type Triterpene, root of gansui alkane type tetracyclic triterpenoid, cycloartane (cyclic-ahltin alkane) type tetracyclic triterpenoid, cucurbitane four Ring triterpene compound or chinaberry alkane type tetracyclic triterpenoid.

The fifth aspect of the present invention provides a kind of external glycosylation process, including step：

In the presence of glycosyl transferase, the glycosyl of glycosyl donor is transferred to the position the C-3 hydroxyl of tetracyclic triterpenoid On；To form glycosylated tetracyclic triterpenoid；

Wherein, the glycosyl transferase is polypeptide or its derived peptides described in the first aspect of the present invention.

In another preferred example, 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 Replacing, missing or adding for residue and formed or added being formed after signal peptide sequence and there is glycosyl transferase activity Derived peptides；Or

Amino acid sequence and SEQ ID NO.:4 or SEQ ID NO.:Homology >=85% of 21 amino acid sequences is (preferably Ground >=90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and with glycosyl transferase activity Derived peptides；

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

The sixth aspect of the present invention provides a kind of method of progress glycosyl catalysis reaction, including step：Of the invention Under the conditions of polypeptide described in first aspect or its derived peptides are existing, glycosyl catalysis reaction is carried out.

In another preferred example, the method further includes step：

In the presence of the polypeptide described in glycosyl donor and the first aspect of the present invention or its derived peptides, formula (I) is changed It closes object and is converted into the formula (II) compound.

In another preferred example, formula (I) compound is protopanoxadiol PPD, and formula (II) compound is ginseng Saponin(e Rh2；Or

Formula (I) compound is Compound K, and formula (II) compound is Ginsenoside F2.

In another preferred example, the method further includes that the polypeptide and its derived peptides are separately added into catalysis instead It answers；And/or

The polypeptide and its derived peptides is added to catalysis reaction simultaneously.

In another preferred example, the method further includes by the nucleotide sequence of encoding glycosyltransferases and reaching Ma dilute two Alcohol and/or key gene in protopanoxadiol metabolic pathway of synthesizing and/or other glycosyltransferase genes are in host cell Coexpression, to obtain described formula (II) compound.

In another preferred example, formula (II) compound is ginseng saponin Rh 2 or Ginsenoside F2.

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

In another preferred example, the method also includes：Additive for adjusting enzymatic activity is provided into reaction system.

In another preferred example, the additive for adjusting enzymatic activity is：Improve enzymatic activity or inhibitory enzyme activity Additive.

In another preferred example, the additive for adjusting enzymatic activity is selected from the group：Ca²⁺、Co²⁺、Mn²⁺、Ba²⁺、 Al³⁺、Ni²⁺、Zn²⁺Or Fe²⁺。

In another preferred example, the additive for adjusting enzymatic activity is：Ca can be generated²⁺、Co²⁺、Mn²⁺、 Ba²⁺、Al³⁺、Ni²⁺、Zn²⁺Or Fe²⁺Substance.

In another preferred example, the glycosyl donor is nucleoside diphosphate sugar, is selected from the group：UDP-glucose, ADP- Glucose, TDP- glucose, CDP- glucose, GDP- glucose, UDP- acetyl group glucose, ADP- acetyl group glucose, TDP- acetyl group glucose, CDP- acetyl group glucose, GDP- acetyl group glucose, UDP- xylose, ADP- xylose, TDP- wood Sugar, CDP- xylose, GDP- xylose, UDP- xylose, UDP- galacturonic acid, ADP- galacturonic acid, TDP- galacturonic acid, CDP- galacturonic acid, GDP- galacturonic acid, UDP- galactolipin, ADP- galactolipin, TDP- galactolipin, CDP- galactolipin, GDP- galactolipin, UDP- arabinose, ADP- arabinose, TDP- arabinose, CDP- arabinose, GDP- arabinose, UDP- rhamnose, ADP- rhamnose, TDP- rhamnose, CDP- rhamnose, GDP- rhamnose or other nucleoside diphosphate hexoses or Nucleoside diphosphate pentose, or combinations thereof.

In another preferred example, the glycosyl donor is uridine 5'-diphosphate sugar, is selected from the group：UDP-glucose, UDP- Xylose, UDP- galacturonic acid, UDP- galactolipin, UDP- arabinose, UDP- rhamnose or other uridine 5'-diphosphate hexoses or Uridine 5'-diphosphate pentose, or combinations thereof.

In another preferred example, the pH of reaction system is：PH4.0-10.0, preferably pH are 5.5-9.0.

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

In another preferred example, the described key gene up in the dilute glycol metabolic pathway of synthesizing of Ma includes (but unlimited In)：Dammarendiol synthase gene.

In another preferred example, the key gene in the protopanoxadiol metabolic pathway of synthesizing includes (but unlimited In)：Dammarendiol synthase gene, the cytochrome P450 gene CYP716A47 of protopanoxadiol synthesis and its reduction Enzyme gene, or combinations thereof.

In another preferred example, the substrate of the glycosyl catalysis reaction is formula (I) compound, and the product is formula (II) compound.

The seventh aspect of the present invention, provides a kind of genetically engineered host cell, and the host cell contains this Polynucleotides described in the second aspect of the present invention are integrated in carrier described in the third aspect of invention or its genome.

In another preferred example, the glycosyl transferase is that polypeptide described in the first aspect of the present invention or its derivative are more Peptide.

In another preferred example, the nucleotide sequence of the glycosyl transferase is encoded as according to the second aspect of the invention.

In another preferred example, the cell is prokaryotic cell or eukaryocyte.

In another preferred example, the host cell is eukaryocyte, such as yeast cells or plant cell.

In another preferred example, the host cell is brewing yeast cell.

In another preferred example, the host cell prokaryotic cell, such as Escherichia coli.

In another preferred example, the host cell is ginseng-cell.

In another preferred example, the host cell is not the cell of naturally-produced formula (II) compound.

In another preferred example, the host cell is not the thin of naturally-produced ginseng saponin Rh 2 or Ginsenoside F2 Born of the same parents.

In another preferred example, the host cell contains the key gene packet in protopanoxadiol metabolic pathway of synthesizing Include (but being not limited to)：Dammarendiol synthase gene, protopanoxadiol synthesis cytochrome P450 gene CYP716A47 and Its reductase gene, or combinations thereof.

The eighth aspect of the present invention provides the purposes of host cell described in the seventh aspect of the present invention, is used to prepare Enzymatic reagent, or production glycosyl transferase or as activated cell or generate glycosylated tetracyclic triterpenoid.

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

In another preferred example, the host cell is used to produce formula by the glycosylation to formula (I) compound (II) compound.

In another preferred example, the host cell is used for by protopanoxadiol PPD and/or Compound K's Glycosylation produces ginseng saponin Rh 2 and/or Ginsenoside F2.

The ninth aspect of the present invention provides a kind of method for generating genetically modified plants, including step：By of the invention Genetically engineered host cell described in seven aspects is regenerated as plant, and the genetically engineered host cell is to plant Object cell.

In another preferred example, the genetically engineered host cell is selected from：Ginseng-cell, American ginseng cell, three Seven cells, tobacco cell.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 glycosyltransferase gene Pn50 agarose gel electrophoresis figure.

Fig. 2 glycosyltransferase gene Pn50 is catalyzed ginsenoside TLC analysis chart.

Fig. 3 is to construct the HPLC analysis chart that recombinant Saccharomyces cerevisiae bacterial strain produces rare ginsenoside Rh2 using Pn50.

Specific embodiment

The present inventor after extensive and in-depth study, provides Radix Notoginseng glycosyl transferase Pn50 (SEQ ID NO. for the first time:4) With mutant 8E7 (the SEQ ID NO. of the glycosyl transferase UGTPg45 in ginseng source:21) it glycosylates and is catalyzed in terpenoid And the application in new saponin formation.

Specifically, glycosyl transferase of the invention can specifically and efficiently be catalyzed tetracyclic triterpenoids compound substrate) and/or Glycosyl from glycosyl donor is transferred on the position the C-3 hydroxyl of tetracyclic triterpenoid.It especially can be efficiently by protoplast Ginseng glycol PPD is converted into rare ginsenoside Rh2 with anti-tumor activity, by rare ginsenoside Compound K (in PPD The position C20 have one it is glycosyl modified) be converted into product Ginsenoside F2.Unexpectedly, to the saccharomyces cerevisiae for producing protopanoxadiol Recombinant Saccharomyces cerevisiae bacterial strain ZWBY04RS-Pn50 energy constructed by the glycosyltransferase gene Pn50 in Radix Notoginseng source is imported in bacterial strain Synthesize rare ginsenoside Rh2, and the bacterial strain with the glycosyltransferase gene UGTPg45 for importing wild type ginseng source ZWBY04RS-UGTPg45 is compared, the 28% (45.55/35.66-1 of output increased of bacterial strain ZWBY04RS-Pn50 ginseng saponin Rh 2 =28%)；UGTPg45 is transformed using random mutation artificial synthesized rare constructed by the mutant gene 8E7 of acquisition Ginseng saponin Rh 2 bacterial strain ZWBY04RS-8E7, Rh2 yield is compared to bacterial strain ZWBY04RS- constructed by utilization UGTPg45 UGTPg45 yield improves 70% (60.48/35.66-1=70%).

The present invention also provides conversion and catalysis process.Glycosyl transferase of the invention can also with dammarendiol and/or In protopanoxadiol metabolic pathway of synthesizing key enzyme (such as dammarendiol synthase gene PgDDS, protopanoxadiol synthesis Cytochrome P450 gene CYP716A47 and its reductase gene PgCPR1) co-expressed in host cell, or be applied to It prepares in the genetically engineered cell of ginseng saponin Rh 2, applied to the bacterial strain for constructing artificial synthesized rare ginsenoside Rh2.

In addition, glycosyl transferase of the invention can also in dammarendiol and/or protopanoxadiol metabolic pathway of synthesizing Key enzyme co-expressed in host cell, applied to the bacterial strain for constructing artificial synthesized rare ginsenoside Rh2.On this basis Complete the present invention.

Definition

As used herein, term " active peptides ", " polypeptide and its derived peptides of the invention ", " enzyme of the invention ", " sugar Based transferase " or " glycosyl transferase of the invention " are used interchangeably, and have those of ordinary skill in the art normally understood Meaning.Glycosyl transferase of the present invention has the position the C-3 hydroxyl that the glycosyl of glycosyl donor will be transferred to tetracyclic triterpenoid On activity.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention 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.

In a preference of the invention, glycosyl transferase of the present invention：

In a preference of the invention, glycosyl transferase of the present invention have i) defined by sequence pass through one or several A amino acid residue, preferably 1-20 are a, 1-15 more preferable, 1-10 more preferable, more preferable 1-3 is a, most preferably 1 amino acid The addition of residue and the sequence formed, and have substantially i) defined by separation polypeptide function separated derived from i) it is more Peptide.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention is corresponding to SEQ ID NO: 222nd amino acid residue of amino acid sequence shown in 19 is selected from at least one of following amino acid：His,Asn,Gln,Lys And Arg.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention is corresponding to SEQ ID NO: 222nd amino acid residue of amino acid sequence shown in 19 is His.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention is corresponding to SEQ ID NO: 322nd amino acid residue of amino acid sequence shown in 19 is selected from at least one of following amino acid：Val,Ile,Leu,Met And Phe.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention is corresponding to SEQ ID NO: 322nd amino acid residue of amino acid sequence shown in 19 is Val.

In a preference of the invention, the amino acid sequence of glycosyl transferase of the present invention is corresponding to SEQ ID NO: 222nd amino acid residue of amino acid sequence shown in 19 is His, is corresponding to SEQ ID NO:Amino acid sequence shown in 19 322nd amino acid residue is Val.

In a preference of the invention, glycosyl transferase of the present invention is selected from the group：

In a particular embodiment, glycosyl transferase activity of the present invention, which refers to, to be transferred to Fourth Ring for the glycosyl of glycosyl donor Activity on the position the C-3 hydroxyl of triterpene compound.

In a preference of the invention, the new glycosyltransferase gene Pn50 of one cloned from Radix Notoginseng is utilized This new glycosyl transferase can be catalyzed a variety of dammarane type ginsenosides C3 glycosylations.

By analyzing Radix Notoginseng transcript profile data, it is therefrom spliced to the glycosyltransferase gene sequence an of overall length, And it is named as Pn50.It is cloned on cloning vector PMDT-18T, then designs expression primer and be building up to Bacillus coli expression On carrier pET28a, make its inducing expression in Escherichia coli.Albumen obtained can be catalyzed the pure and mild Compound K of protoplast two C3 position hydroxyl glycosylation.

The UGTPg45 in Radix Notoginseng glycosyltransferase gene Pn50 replacement ginseng source can be substantially improved Rh2 yield and (mention It is high 28%).

In another preference of the invention, a kind of glycosyl transferase mutant protein 8E7 is provided.The glycosyl turns Move the mutant protein for the wild type gene UGTPg45 that enzyme mutant albumen is ginseng source, the mutation glycosyl transferase base Because Rh2 yield (improving 70%) can be substantially improved in the wild type gene UGTPg45 in 8E7 replacement ginseng source.

Those of ordinary skill in the art are not difficult to know, in some regions of polypeptide, such as insignificant area change minority ammonia Base acid residue will not substantially change bioactivity, for example, suitably replacing the sequence that certain amino acid obtain will not influence it Activity is (reference can be made to Watson etc., Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/ Cummings Pub.Co.P224).Therefore, those of ordinary skill in the art can implement this replacement and ensure gained molecule Still there is required bioactivity.

Therefore, polypeptide of the invention can correspond to SEQ ID NO:222nd ammonia of amino acid sequence shown in 19 Base acid residue is non-Gln and/or is corresponding to SEQ ID NO:The amino acid residue that amino acid sequence shown in 19 is the 322nd is non- Make the function and activity that are further mutated and still have glycosyl transferase of the present invention on the basis of Ala.Such as glycosyl of the invention Its amino acid sequence of transferase (a) such as SEQ ID NO:Shown in 21；Or (b) pass through one or more comprising sequence defined by (a) A amino acid residue, preferably 1-20 are a, 1-15 more preferable, 1-10 more preferable, more preferable 1-3 is a, most preferably 1 amino acid Replacing, missing or adding for residue and the sequence formed, and it is basic with polypeptide function defined by (a) derived from (a) Polypeptide.

In the present invention, glycosyl transferase of the invention includes and amino acid sequence such as SEQ ID NO:Glycosyl shown in 21 Transferase is compared, and has at most 20, preferably at most 10, then good at most 3, more preferably at most 2, most preferably at most 1 A amino acid is by the mutant that amino acid with similar or analogous properties is replaced and is formed.The mutant of these conservative variations can According to, such as carry out amino acid substitution shown in following table and generate.

Original residue	Representative substitution residue	It is preferred to replace residue
			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	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；Ala	Leu

The present invention also provides the polynucleotides for encoding polypeptide of the present invention.Term " polynucleotides of coding polypeptide " can be Polynucleotides including encoding this polypeptide, be also possible to further include additional code and/or non-coding sequence polynucleotides.

Therefore, " containing " used herein, " having " or " comprising " include "comprising", " mainly by ... constitute ", " base On this by ... constitute " and " by ... constitute "；" mainly by ... constitute ", " substantially by ... constitute " and " by ... structure At " belong to the subordinate concept of " containing ", " having " or " comprising ".

Corresponding to SEQ ID NO:222nd/322 amino acid residues of amino acid sequence shown in 19

Those of ordinary skill in the art know, can make in the amino acid sequence of some albumen to some amino acid residues Various mutation out, such as the mutant for replacing, adding or deleting, but obtaining remain to the function or activity that have former albumen.Therefore, Those of ordinary skill in the art can make certain change to the specifically disclosed amino acid sequence of the present invention and obtain still with required Active mutant, then in this mutant with SEQ ID NO:222nd/322 ammonia of amino acid sequence shown in 19 The corresponding amino acid residue of base acid residue may not be just the 222nd/322, but the obtained mutant should be fallen in In protection scope of the present invention.

The term as used herein " corresponding to " has the normally understood meaning of those of ordinary skill in the art.Specifically, " corresponding to " indicates specific bit of the two sequences after homology or sequence identity compare, in a sequence and another sequence Set corresponding position.Therefore, just " correspond to SEQ ID NO:222nd/322 amino of amino acid sequence shown in 19 For sour residue ", if in SEQ ID NO:One end of amino acid sequence shown in 19 adds 6-His label, then gained mutation Correspond to SEQ ID NO in body:The 222nd/322 of amino acid sequence shown in 19 may be the 228th/328；And such as Fruit deletes SEQ ID NO:A few amino acids residue in amino acid sequence shown in 19, then corresponding to SEQ in gained mutant ID NO:The 222nd/322 of amino acid sequence shown in 19 may be the 220th/320, etc..For another example if one Item has the sequence and SEQ ID NO of 400 amino acid residues:20-420 of amino acid sequence shown in 19 are with higher Homology or sequence identity, then corresponding to SEQ ID NO in gained mutant:The 222nd of amino acid sequence shown in 19/ 322 may be the 202nd/302.

In a particular embodiment, the homology or sequence identity can be 80% or more, preferably 90% or more, More preferable 95%-98%, most preferably 99% or more.

Measurement sequence homology known to a person of ordinary skill in the art or the method for the phase same sex include but is not limited to：It calculates Machine molecular biology (Computational Molecular Biology), Lesk, A.M. are compiled, Oxford University Press, knob About, 1988；Biological computation：Informatics and Genome Project (Biocomputing:Informatics and Genome Projects), Smith, D.W. are compiled, academic press, New York, and 1993；The computer of sequence data analyzes (Computer Analysis of Sequence Data), first part, Griffin, A.M. and Griffin, H.G. are compiled, Humana Press, New Jersey, 1994；Sequence in molecular biology analyzes (Sequence Analysis in Molecular Biology), von Heinje, G., academic press, 1987 and sequence analysis primer (Sequence Analysis Primer), Gribskov, M. and Devereux, J. compile M Stockton Press, New York, 1991 and Carillo, H. with Lipman, D., SIAM J.Applied Math., 48:1073(1988).The preferred method for measuring the phase same sex will be in the sequence of test Maximum matching is obtained between column.The method of the measurement phase same sex is compiled in publicly available computer program.It is preferred to survey The computer program means for determining the phase same sex between two sequences include but is not limited to：GCG program bag (Devereux, J. etc., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. etc., 1990).The public can obtain from NCBI and other sources BLASTX program (BLAST handbook, Altschul, S. etc., NCBI NLM NIH Bethesda, Md.20894；Altschul, S. Deng 1990).Well known Smith Waterman algorithm can also be used for the measurement phase same sex.

If not otherwise indicated, ginsenoside and sapogenin mentioned herein, the ginseng soap of the C20 position S and/or R configuration that are Glycosides and sapogenin.

As used herein, " isolated polypeptide " refer to the polypeptide substantially free of natural relative other albumen, Lipid, carbohydrate or other materials.Those skilled in the art can purify the polypeptide with the purified technology of protein of standard.Substantially Upper pure polypeptide can generate single master tape in non-reducing polyacrylamide gel.The purity of the polypeptide can also use amino Acid sequence is further analyzed.

Active peptides of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention can be day The product or chemically synthesized product so purified, or use recombinant technique from protokaryon or eucaryon host (for example, bacterium, ferment Female, plant) in generate.According to host used in recombinant production scheme, polypeptide of the invention can be glycosylated, or can be It is nonglycosylated.Polypeptide of the invention may also include or not include the methionine residues of starting.

The invention also includes the segments of the polypeptide, derivative and analogue.As used herein, term " segment ", " derivative Object " and " analog ", which refer to, is kept substantially the identical biological function of the polypeptide or active polypeptide.

Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more conservative or non-conservation ammonia Base acid residue (preferably conservative amino acid) substituted polypeptide, and can be can also for such substituted amino acid residue Be not by genetic code encoding, or (ii) in one or more amino acid residues with substituent group polypeptide, or (iii) mature polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion institute's shape At polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide (such as leader sequence or secretion that are formed Sequence or for purifying the sequence of this polypeptide or proprotein sequence, or the fusion protein with the formation of antigen I gG segment).According to Teaching herein, these segments, derivative and analogue belong to scope known to those skilled in the art.

There is glycosyl transferase activity in active peptides of the invention, and one or more of reaction can be catalyzed：

The polypeptide sequence is SEQ ID NO.:4,SEQ ID NO.:21 or its derived peptides, which further includes tool Have and shown polypeptide SEQ ID NO. with the same function:4,SEQ ID NO.:The variant form of 21 sequences.These variation shapes Formula includes (but being not limited to)：One or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1- 10) missing, insertion and/or the substitution of amino acid, and in C-terminal and/or N-terminal addition one or several (usually 20 More preferably it is within 5 within preferably 10 within a) amino acid.For example, in the art, with similar performance or phase As amino acid when being replaced, do not usually change the function of protein.For another example, in C-terminal and/or N-terminal addition one A or several amino acid will not generally also change the function of protein.The term further includes active fragment and the work of albumen of the present invention Property derivative.The present invention also provides the analogs of the polypeptide.The difference of these analogs and natural polypeptides can be amino acid Difference in sequence is also possible to not influence the difference on the modified forms of sequence, or haves both at the same time.These polypeptides include day Right or induction genetic variant.Induction variant can be obtained by various technologies, such as by radiating or being exposed to mutagens And random mutagenesis is generated, it can also pass through the technology of site-directed mutagenesis or other known molecular biology.Analog further includes having Different from the analog of the residue (such as D- amino acid) of natural L-amino acids, and with non-naturally occurring or synthesis amino The analog of sour (such as β, gamma-amino acid).It should be understood that polypeptide of the invention is not limited to enumerated representative polypeptide.

Modification (not changing primary structure usually) form include：The chemical derivative form of internal or external polypeptide such as acetyl Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.It further include being modified to improve its antiprotease hydrolysis property or excellent The polypeptide of solubility property is changed.

The aminoterminal or c-terminus of Pn50,8E7 polypeptide or its derived peptides of the invention can also be containing one or more more Peptide fragment, as protein tag.Any suitable label may be used to the present invention.For example, the label can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These Label can be used for purifying albumen.Table 1 lists some of which label and its sequence.

Table 1

Label	Residue number	Sequence
			Poly-Arg	5-6 (usual 5)	RRRRR
Poly-His	2-10 (usual 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-TagII	8	WSHPQFEK
			C-myc	10	WQKLISEEDL
GST	220	6 nextLVPRGS

In order to make the protein secretion expression (be such as secreted into extracellular) of translation, can also Pn50,8E7 polypeptide or its spread out Add upper signal peptide sequence, such as pelB signal peptide in the amino amino end of raw polypeptide.Signal peptide is in polypeptide from intracellular point It can be cut out during secreting out.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide Coding region sequence can be with SEQ ID NO.:3 or SEQ ID NO.:Coding region sequence shown in 22 is identical or degeneracy Variant.As used herein, " variant of degeneracy " refers to that coding has SEQ ID NO. in the present invention:4 or SEQ ID NO.:The polypeptide of amino acid sequence shown in 21 or its derived peptides, but with SEQ ID NO.:4 or SEQ ID NO.:21 or its spread out The coded sequence of raw polypeptide, preferably SEQ ID NO.:3 or SEQ ID NO.:The differentiated nucleic acid sequence of sequence shown in 22.

Encode SEQ ID NO.:4 or SEQ ID NO.:The multicore glycosides of the mature polypeptide of polypeptide or its derived peptides shown in 21 Acid includes：The coded sequence of encoding mature polypeptide；The coded sequence of mature polypeptide and various additional coding sequences；Mature polypeptide Coded sequence (and optional additional coding sequence) and non-coding sequence.

The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, Missing or insertion, but not from substantially change its encode polypeptide function.

The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under stringent condition (or stringent condition) with The interfertile polynucleotides of polynucleotides of the present invention.In the present invention, " stringent condition " refers to：(1) strong in lower ion Hybridization and elution under degree and higher temperature, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant when (2) hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) are only identical between two sequences Property at least just hybridizes at 90% or more, more preferably 95% or more.Also, the polypeptide of interfertile polynucleotide encoding with SEQ ID NO.:4 or SEQ ID NO.:Mature polypeptide shown in 21 has identical biological function and activity.

The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence.As used herein, the length of " nucleic acid fragment " is extremely Contain 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably at least 100 nucleosides less It is more than acid.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid with determine and/or separation coding Pn50,8E7 polypeptide or its The polynucleotide of derived peptides.

Polypeptide and polynucleotides in the present invention preferably provide in a separate form, are more preferably purified to homogeneous.

Pn50,8E7 polypeptide or its derived peptides nucleotide full length sequence of the invention or its segment can usually be expanded with PCR Increasing method, recombination method or artificial synthesized method obtain.It, can disclosed related nucleotide according to the present invention for PCR amplification method Sequence, especially open reading frame sequence carry out design primer, and with the commercially available library cDNA or by well known by persons skilled in the art The library cDNA prepared by conventional method expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice Or multiple PCR amplification, then the segment that each time amplifies is stitched together by proper order again.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.

At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

It is optimized for obtaining gene of the invention using round pcr DNA amplification/RNA method.Especially it is difficult from text When obtaining the cDNA of overall length in library, RACE method (end RACE-cDNA rapid amplification) preferably is used, the primer for PCR It can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA segment of amplification is such as separated and purified by gel electrophoresis.

The present invention also relates to the carriers comprising polynucleotides of the invention, and more with carrier or Pn50,8E7 of the invention Peptide or the genetically engineered host cell of the coded sequence of its derived peptides, and generate through recombinant technique of the present invention The method of polypeptide.

By the recombinant dna technology of routine, it can be used to express or produce weight using polynucleotide sequence of the invention Pn50,8E7 polypeptide or its derived peptides of group.In general there are following steps：

(1) with coding Pn50,8E7 polypeptide of the invention or the polynucleotides (or variant) of its derived peptides, or with containing There are the recombinant expression carrier conversion of the polynucleotides or suitable host cell of transduceing；

(2) host cell cultivated in suitable culture medium；

(3) it is separated from culture medium or cell, protein purification.

In the present invention, the polynucleotide sequence for encoding Pn50,8E7 polypeptide or its derived peptides can be plugged into recombinant expression load In body.Term " recombinant expression carrier " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, feeds Newborn zooblast virus such as adenovirus, retrovirus or other carriers.As long as can replicate and stablize, Ren Hezhi in host Grain and carrier can be used.One important feature of expression vector is to usually contain replication orgin, promoter, marker gene and turn over Translate control element.

Method well-known to those having ordinary skill in the art can be used to construct the coding containing Pn50,8E7 polypeptide or its derived peptides The expression vector of DNA sequence dna and suitable transcription/translation control signal.These methods include recombinant DNA technology in vi, DNA conjunction At technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to refer to Lead mRNA synthesis.The representative example of these promoters has：Lac the or trp promoter of Escherichia coli；λ bacteriophage PL promoter； Eukaryotic promoter includes CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse transcription The promoter that the LTRs and some other known controllable gene of virus are expressed in protokaryon or eukaryotic or its virus.Table It further include the ribosome bind site and transcription terminator of translation initiation up to carrier.

In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg White (GFP), or tetracycline or amicillin resistance for Escherichia coli.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable When host cell, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell；Fungal cell's such as yeast；Plant cell；The insect cell of drosophila S2 or Sf9；CHO, COS, 293 cells or Bowes are black The zooblast etc. of plain oncocyte.

When polynucleotides of the invention are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier Transcription can be made to be enhanced.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 base-pairs, acts on and open Mover is to enhance the transcription of gene.Can for example include 100 to 270 base-pairs in replication origin advanced stage side SV40 enhancer, in the polyoma enhancer of replication origin advanced stage side and adenovirus cancers etc..

Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Using

The purposes of active peptides or glycosyl transferase Pn50,8E7 polypeptide of the present invention or its derived peptides include (but It is not limited to)：Specifically and efficiently the glycosyl from glycosyl donor is transferred on the position the C-3 hydroxyl of tetracyclic triterpenoid. Can especially formula (I) compound be converted to the formula (II) compound, such as converted protopanoxadiol PPD to antitumor The more excellent rare ginsenoside Rh2 of activity；Ginsenoside F2 is converted by Compound K.

The tetracyclic triterpenoids compound includes but is not limited to：The dammarane type of S configuration or R configuration, lanolin alkane type, The tetracyclic triterpenes such as root of gansui alkane type, cycloartane (cyclic-ahltin alkane) type, apotirucallane type, cucurbitane, chinaberry alkane type Close object.

The present invention provides a kind of commercial catalytic processes, including：Under conditions of providing glycosyl donor, with of the invention Pn50,8E7 polypeptide or its derived peptides obtain ginseng saponin Rh 2 and Ginsenoside F2.Specifically, institute in (A) reaction Polypeptide is selected from SEQ ID NO.:4 or SEQ ID NO.:Polypeptide shown in 21 or its derived peptides；It is used in (B) reaction Polypeptide be SEQ ID NO.:Polypeptide shown in 4 or its derived peptides.

In a preference of the invention, provide glycosyl transferase Pn50 and glycosyl using aforementioned Radix Notoginseng turn a kind of Move the method that enzyme mutant albumen 8E7 synthesizes ginseng saponin Rh 2 in saccharomyces cerevisiae.

The glycosyl donor is nucleoside diphosphate sugar, is selected from the group：UDP-glucose, ADP- glucose, TDP- grape Sugar, CDP- glucose, GDP- glucose, UDP- acetyl group glucose, ADP- acetyl group glucose, TDP- acetyl group glucose, CDP- acetyl group glucose, GDP- acetyl group glucose, UDP- xylose, ADP- xylose, TDP- xylose, CDP- xylose, UDP- wood Sugar, GDP- xylose, UDP- galacturonic acid, ADP- galacturonic acid, TDP- galacturonic acid, CDP- galacturonic acid, GDP- Galacturonic acid, UDP- galactolipin, ADP- galactolipin, TDP- galactolipin, CDP- galactolipin, GDP- galactolipin, UDP- are Arabic Sugar, ADP- arabinose, TDP- arabinose, CDP- arabinose, GDP- arabinose, UDP- rhamnose, ADP- rhamnose, TDP- rhamnose, CDP- rhamnose, GDP- rhamnose or other nucleoside diphosphate hexoses or nucleoside diphosphate pentose or its group It closes.

The glycosyl donor is preferably uridine 5'-diphosphate sugar, is selected from the group：UDP-glucose, UDP- xylose, UDP- mouse Lee's sugar, UDP- galacturonic acid, UDP- galactolipin, UDP- arabinose or other uridine 5'-diphosphate hexoses or uridine 5'-diphosphate Pentose, or combinations thereof.

In the method, enzymatic activity additive (additive for improving enzymatic activity or inhibitory enzyme activity) can also be added. The additive of the enzymatic activity can be selected from the group：Ca²⁺、Co²⁺、Mn²⁺、Ba²⁺、Al³⁺、Ni²⁺、Zn²⁺Or Fe²⁺；Or for can be with Generate Ca²⁺、Co²⁺、Mn²⁺、Ba²⁺、Al³⁺、Ni²⁺、Zn²⁺Or Fe²⁺Substance.

The pH condition of the method is：PH4.0-10.0, preferably pH6.0-pH8.5, more preferable 8.5.

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

The present invention also provides a kind of compositions, it contains a effective amount of active peptides or glycosyl transferase of the invention On Pn50,8E7 polypeptide or its derived peptides and bromatology or industrial acceptable carrier or excipient.This kind of carrier packet Include (but being not limited to)：Water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.

The substance for adjusting glycosyl transferase activity of the present invention can be also added in the composition.It is any that there is raising enzyme activity The substance of sexual function is available.Preferably, the substance for improving glycosyl transferase activity of the invention is selected from sulfydryl Ethyl alcohol.In addition, many substances can reduce enzymatic activity, including but not limited to：Ca²⁺、Co²⁺、Mn²⁺、Ba²⁺、Al³⁺、Ni²⁺、Zn²⁺With Fe²⁺；Or hydrolyzable forms Ca after being added to substrate²⁺、Co²⁺、Mn²⁺、Ba²⁺、Al³⁺、Ni²⁺、Zn²⁺And Fe²⁺Substance.

After obtaining Pn50,8E7 polypeptide or its derived peptides of the invention, those skilled in the art apply in which can be convenient should Enzyme turns the effect of glycosyl, the especially transglycosylation to Ma dilute glycol, protopanoxadiol is reached to play.As of the invention excellent Mode is selected, the method for two kinds of formation rare ginsenosides is additionally provided, one of this method includes：With Pn50 of the present invention, 8E7 polypeptide or its derived peptides handle the substrate of glycosyl to be turned, the substrate include up to the dilute glycol of Ma, protopanoxadiol and its The tetracyclic triterpenoids such as derivative.Preferably, under the conditions of pH3.5-10, with Pn50,8E7 polypeptide or its derivative The substrate of polypeptide enzymatic treatment glycosyl to be turned.Preferably, under the conditions of 30-105 DEG C of temperature, with Pn50,8E7 polypeptide or its The substrate of derived peptides enzymatic treatment glycosyl to be turned.

The two of this method include：Pn50,8E7 polypeptide of the present invention or its derived peptides gene are transferred to and can be synthesized In the engineering bacteria (for example, yeast or colibacillus engineering) of protopanoxadiol PPD, alternatively, by Pn50,8E7 polypeptide or its spread out Raw polypeptide gene with up to the dilute glycol of Ma, the key gene in protopanoxadiol PPD metabolic pathway of synthesizing and optionally other glycosyls Transferase gene coexpression in host cell (such as yeast cells or Escherichia coli), obtains and directly produces rare ginsenoside The recombinant bacterium of Rh2 and/or Ginsenoside F2.Alternatively, by Pn50,8E7 polypeptide or the coding nucleotide sequence of its derived peptides with Dammarendiol and/or key enzyme in protopanoxadiol PPD metabolic pathway of synthesizing and optionally other glycosyl transferases and The key enzyme of synthesis UDP- rhamnose co-expresses in host cell, be applied to construct artificial synthesized rare ginsenoside Rh2 and The recombinant bacterial strain of Ginsenoside F2.

The described key gene up in the dilute glycol metabolic pathway of synthesizing of Ma includes but is not limited to：Dammarendiol synthesis Enzyme gene.

In another preferred example, the key gene in the protopanoxadiol metabolic pathway of synthesizing includes (but unlimited In)：The cytochrome P450 gene CYP716A47 and its go back that dammarendiol synthase gene PgDDS, protopanoxadiol synthesize Nitroreductase gene, or combinations thereof.Or the isoenzymes and combinations thereof of the above various enzymes.Wherein, dammarendiol synzyme is by epoxy Squalene (saccharomyces cerevisiae itself synthesis) is converted into dammarendiol, and Cytochrome P450 CYP716A47 and its reductase again will Dammarendiol is converted into protopanoxadiol PPD.(Han et.al, plant&cell physiology, 2011,52.2062- 73)

Main advantages of the present invention：

(1) present invention is planted compared to traditional dependent on Panax using the method for saccharomyces cerevisiae production ginseng saponin Rh 2 Object extracts and the method for glycosyl hydrolase has many advantages, such as that at low cost, the period is short, quality is stable；

(2) the glycosyl transferase Pn50 that the present invention is obtained from Radix Notoginseng for the first time can be catalyzed PPD and Rh2, catalysis CK synthesis F2, and the synthesis that being conducted into can be more efficient compared to wild type glycosyl transferase UGTPg45 in ginseng in production PPD bacterial strain Rare ginsenoside Rh2；

(3) present invention is by obtaining mutant gene to wild type glycosyl transferase UGTPg45 random mutation in ginseng 8E7, rare people can be significantly improved compared to wild type glycosyl transferase UGTPg45 in ginseng by being conducted into produce in PPD bacterial strain Join saponin(e Rh2 combined coefficient.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are calculated by weight.

By implementation method in detail below, it will be further appreciated that specific implementation process of the invention.

The clone of 1. Radix Notoginseng glycosyltransferase gene Pn50 of embodiment

Synthesis such as sequence Pn50 cloning primer F, SEQ ID NO:1 (ATGGAGAGAGAAATGTTGAGCA) and Pn50 clone Primer R, SEQ ID NO:Two primers of 2 (TCAGGAGGAAACAAGCTTTGAA).With the RNA reverse transcription extracted from Radix Notoginseng The cDNA of acquisition is template, carries out PCR using primer as above.Archaeal dna polymerase selects the high-fidelity of precious bioengineering Co., Ltd KOD archaeal dna polymerase.PCR amplification program is：94℃2min；94 DEG C of 15s, 58 DEG C of 30s, 68 DEG C of 2min, totally 35 recycle；68 DEG C 10min is down to 10 DEG C.PCR product is detected through agarose gel electrophoresis, the result is shown in Figure 1.

In ultraviolet lower irradiation, target dna band is cut.Then Axygen Gel Extraction Kit (AEYGEN is used Company) DNA fragmentation that DNA is the glycosyltransferase gene amplified is recycled from Ago-Gel.Utilize precious bioengineering The PCR product of recycling is cloned into PMDT carrier, institute's structure by the PMD18-T Cloning Kit of (Dalian) Co., Ltd (Takara) The carrier built is named as PMDT-Pn50.The gene order of Pn50 is obtained through sequencing.

Pn50 gene has SEQ ID NO:3 nucleotide sequence.From SEQ ID NO:35 ' the 1-1368 nucleosides in end Acid is the open reading frame (Open Reading Frame, ORF) of Pn50, from SEQ ID NO:The 1-3 nucleosides at 35 ' ends Acid is the initiation codon ATG of Pn50 gene, from SEQ ID NO:The 1366-1368 nucleotide at 35 ' ends are Pn50 base The terminator codon TGA of cause.Glycosyl transferase Pn50 gene encodes a protein Pn50 containing 455 amino acid, has SEQ ID NO:4 amino acid residue sequence is 51.1kDa with the theoretical molecular weight size of software prediction to the protein, etc. Electricity point pI is 5.10.From SEQ ID NO:The 332-375 amino acids of 4 aminoterminal are that glycosyl transferase PSPG guards function Domain.

Pn50 nucleotide sequence SEQ ID NO:3

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGC CCAATGATGCAATTCGTCAAGCGTTTAGCCTGGAAAGGCGTGCGAATCACGATAGTTCTTCCGGCTGAGATTCGAGA TTCTATGCAAATAAACAACTCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGATGATGAGATGCCAT ACAGCATGCGGGCTTATATGGGAGTTGTAAAGCTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAAA ACAAATGGCTACCCTGTTAATTTGCTAGTGGTCGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGG GGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGA AGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAAATGATTTGCCAATT ATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGAT CTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCAATGGCCAATTACATCCATCG GACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGACATTAATTTCTACAAGGCA GACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAA GCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACACAGCAAATATCACTTCATATGGGTTG TTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGCAGAGGAAAAAGGCCTAATAGTGAGTTGG TGCCCTCAACTCGAAGTTTTGTCACATAAATCTATAGGTAGTTTTATGACTCATTGTGGTTGGAACTCGACGGTTGA GGCATTGAGTTTGGGCGTGCCAATGGTGGCAGTGCCACAACAGTTTGATCAGCCTGTTAATGCCAAGTATATCGTGG ATGTATGGCGAATTGGGGTTCAGGTTCCGATTGGTGAAAATGGGGTTCTTTTGAGGGGAGAAGTTGCTAACTGTATA AAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGTGGAGGC AATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

Pn50 amino acid sequence SEQ ID NO:4

MEREMLSKTHIMFIPFPAQGHMSPMMQFVKRLAWKGVRITIVLPAEIRDSMQINNSLINTECISFDFDK DDEMPYSMRAYMGVVKLKVTNKLSDLLEKQKTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNA RLGKLKIPPEEGLTSVSLPSIPLLGRNDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSQW PITSIGPLIPSMYLDKQLPNDKDNDINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKY HFIWVVIESERTKLSSDFLAEAEEKGLIVSWCPQLEVLSHKSIGSFMTHCGWNSTVEALSLGVPMVAVPQQFDQPVN AKYIVDVWRIGVQVPIGENGVLLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKLVS S

2. Radix Notoginseng glycosyltransferase gene Pn50 of embodiment in expression in escherichia coli

Synthesis such as sequence Pn50 expression primer F SEQ ID NO:5 (GGATCCATGGAGAGAGAAATGTTGAGCA) and Pn50 expression primer R SEQ ID NO:Two primers of 6 (CTCGAGTCAGGAGGAAACAAGCTTTGAA).In drawing for synthesis Add two restriction enzyme sites of BamH I and Xho I on object F/R respectively, PCR is carried out as template using the cDNA being extracted from plants.DNA Polymerase selects the KOD archaeal dna polymerase of the high-fidelity of precious bioengineering Co., Ltd.PCR amplification program is：94℃2min；94 DEG C 15s, 58 DEG C of 30s, 68 DEG C of 2min, totally 35 circulations；68 DEG C of 10min are down to 10 DEG C.PCR product is examined through agarose gel electrophoresis It surveys.In ultraviolet lower irradiation, target dna band is cut.Then Axygen Gel Extraction Kit (AEYGEN company) is used The DNA fragmentation that DNA is the glycosyltransferase gene amplified is recycled from Ago-Gel.By two PCR products of recycling It is connect with after BamH I and Xho I digestion with respectively with same with the pET28a after BamH I and Xho I digestion, connection product turns Change Escherichia coli EPI300 competent cell, the Escherichia coli bacteria liquid after conversion is coated on addition 50ug/mL kanamycins On LB plate, and further pass through PCR and digestion verification recombinant clone.It is laggard that each one of clone of selection extracts recombinant plasmid Row sequence verification, by inducing expression, inducing expression side in recombinant plasmid transformed e. coli bl21 (DE3) after sequence verification is correct Method is：It is seeded in the LB test tube containing 50ug/mL kanamycins overnight from plate picking monoclonal, takes 1% to be seeded to 50ml The IPTG induction of final concentration of 0.1mM is added in 37 DEG C of shake cultures to OD600 in triangular flask for 0.6-0.7, induces at 18 DEG C 16h.12000g, 3min are collected and are cracked thallus after every gram of weight in wet base thallus addition 10ml PBS buffer of thallus is resuspended, in centrifuging and taking Clearly as crude enzyme liquid.

3. Radix Notoginseng glycosyltransferase gene Pn50 of embodiment is catalyzed different substrate reactions and its product detection

It is as follows to configure glycosyl transferase Pn50 catalystic converter system (100 μ L)：

2h is reacted under 37 DEG C of water-baths.Isometric n-butanol extracting is added after reaction, n-butanol phase is taken, through vacuum After concentration, reaction product is dissolved in 10 μ L methanol, is as a result detected with TLC or HPLC.Result can be seen that the present invention from Fig. 2 Used in glycosyl transferase Pn50 can be catalyzed protopanoxadiol PPD and form a kind of new product (formula A is shown in reaction), Migration position on TLC plate is consistent with the migration position of Rh2, it was demonstrated that such new product is ginseng saponin Rh 2.Furthermore Pn50 is also It can be catalyzed compound K, the product of generation speculates to be people according to the regiospecificity of migration position and Pn50 on TLC plate Join saponin(e F2 (Fig. 2).

Embodiment 4. synthesizes rare ginsenoside using the glycosyltransferase gene Pn50 in Radix Notoginseng source in saccharomyces cerevisiae Rh2

(1) the glycosyltransferase gene Pn50 in Radix Notoginseng source can be catalyzed C3 hydroxyl glycosyls of protopanoxadiol be combined to it is dilute There is ginseng saponin Rh 2, synthesizes rare ginsenoside Rh2 in saccharomyces cerevisiae to realize, the present invention constructs one plant first and can give birth to Produce the saccharomyces cerevisiae chassis cell of protopanoxadiol.Dammarendiol synthase gene is imported into wild type Saccharomyces cerevisiae PgDDS, the cytochrome P450 gene CYP716A47 and its reductase gene PgCPR1 of protopanoxadiol synthesis, utilizes wine brewing The 2,3- epoxy squalene of the mevalonate pathway synthesis of yeast itself can synthesize protopanoxadiol.By to artificial constructed Protopanoxadiol route of synthesis is optimized including to synthesis rate-limiting step optimization, precursor supply optimization etc. obtains plant height production The Wine brewing yeast strain ZWBY04RS of protopanoxadiol.

(2) synthesis such as sequence SEQ ID NO:12 primers of 7-18 obtain glycosyltransferases expression in the method for PCR Promoter, terminator, ORF, selection markers, upstream and downstream homology arm segment, PCR method is the same as embodiment 1.By above-mentioned PCR fragment with And after each 100ng of ORF of UGTPg45 is mixed, the LiAc/ssDNA method for transformation of saccharomyces cerevisiae routine, conversion recombination wine brewing are utilized Yeast strain ZWBY04RS obtains recombinant Saccharomyces cerevisiae bacterial strain ZWBY04RS-UGTPg45.It is similar by above-mentioned PCR fragment and After each 100ng of the ORF of Pn50 is mixed, using the LiAc/ssDNA method for transformation of saccharomyces cerevisiae routine, recombinant Saccharomyces cerevisiae is converted Bacterial strain ZWBY04RS obtains recombinant Saccharomyces cerevisiae bacterial strain ZWBY04RS-Pn50.

Promoter primer F SEQ_ID_NO.7

TAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGAGATT

Promoter primer R SEQ_ID_NO.8

ACTGTCAAGGAGGGTATTCTGGGCCTCCATGTCGCTGCTATATAACAGTTGAAATTTGGATAAGAACAT

ORF primers F SEQ_ID_NO.9

GCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAGAATTACAATAGT

ORF primer R SEQ_ID_NO.10

TCTGGTGAGGATTTACGGTATGATCATGCTGGTAAGCTTCGTTA

Terminator primers F SEQ_ID_NO.11

GAAAAGAAGATAATATTTTTATATAATTATATTAATCTCAGGAGGAAACAAGCTTTGAA

Terminator primer R SEQ_ID_NO.12

GCATAGCAATCTAATCTAAGTTTTAATTACAAAATGGAGAGAGAAATGTTGAGCAAAAC

Selection markers primers F SEQ_ID_NO.13

CGCGTTGAGAAGATGTTCTTATCCAAATTTCAACTGTTATATAGCAGCGA

Selection markers primer R SEQ_ID_NO.14

TTACTTCTTGCAGACATCAGACATACTATTGTAATTCTCGACACTGGATGGCGGCGTTA

Upstream homology arm primers F SEQ_ID_NO.15

GGCTGTCGCCATTCAAGAGCAGATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC

Upstream homology arm primer R SEQ_ID_NO.16

CATAATGTGAGTTTTGCTCAACATTTCTCTCTCCATTTTGTAATTAAAACTTAGATTAG

Downstream homology arm primers F SEQ_ID_NO.17

CCCAAAGCTAAGAGTCCCATTTTATTC

Downstream homology arm primer R SEQ_ID_NO.18

GAGTAGAAACATTTTGAAGCTATCTGCTCTTGAATGGCGACAGCCTATTGCCCCAGTGT

(3) solid medium is configured：Configure culture medium：1%Yeast Extract (yeast extract), 2%Peptone (albumen Peptone), 2%Dextrose (glucose) (glucose), 2% agar powder.

Configure fluid nutrient medium：Configure culture medium：1%Yeast Extract (yeast extract), 2%Peptone (albumen Peptone), 2%Dextrose (glucose) (glucose).

Recombinant Saccharomyces cerevisiae the bacterium ZWBY04RS-UGTPg45 and ZWBY04RS- that picking is crossed in solid medium tablets Pn50, overnight (30 DEG C, 250rpm, 16h) respectively at the test tube shake culture containing 5mL fluid nutrient medium；Thalline were collected by centrifugation, It is transferred in the 50mL triangular flask of 10mL fluid nutrient medium, adjusts OD600 to 0.05,30 DEG C, sent out within 250rpm shake culture 4 days Ferment product.A parallel laboratory test is arranged to each plant of recombination yeast in this method simultaneously.

The extraction and detection of protopanoxadiol and rare ginsenoside Rh2：100 μ L fermentation is drawn from 10mL fermentation liquid Liquid shakes cracking yeast with Fastprep, and isometric n-butanol extracting is added, then steams n-butanol under vacuum conditions It is dry.With the yield for passing through HPLC testing goal product after the dissolution of 100 μ L methanol.HPLC result is shown in Fig. 3.

The glycosyltransferase gene UGTPg45 institute structure in ginseng source is imported into the Wine brewing yeast strain for producing protopanoxadiol The recombinant Saccharomyces cerevisiae bacterial strain ZWBY04RS-UGTPg45 built can synthesize rare ginsenoside Rh2, yield 35.66mg/L. Recombination constructed by the glycosyltransferase gene Pn50 in Radix Notoginseng source is imported into the Wine brewing yeast strain for producing protopanoxadiol to make Brewer yeast bacterial strain ZWBY04RS-Pn50 can synthesize rare ginsenoside Rh2, yield 45.55mg/L.

The building in the glycosyl transferase UGTPg45 random mutation library in 5. ginseng source of embodiment

Using plasmid UGTPg45-pMD18T as template, GeneMorph II Random Mutagenesis Kit is used (Agilent Technology), UGTPg45 random mutation primers F SEQ ID NO:23 (Gcatagcaatctaatctaagttttaattacaaaatggagagagaaatgttgagcaa aac) and UGTPg45 dash forward at random Become primer R SEQ ID NO:24(Gaaaagaagataatatttttatataattatattaatctcaggaggaaacaagcttt Gaa fallibility PCR) is carried out for primer, program is as follows：95 DEG C of 2min initial denaturations；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 3min 15s, 30 circulations；72 DEG C extend 10min eventually.1ug is added according to kit specification, 1.5ug, 2ug template is to prominent Variability is groped.It is tapped and recovered PCR product, with Taq enzyme plus A, is connected on carrier pMD18T, Escherichia coli TOP10 is converted. Random 10 positive colony sequencings of picking determine that mutation rate in 1-2 base/corresponding template consumption of gene is 1.5ug.Subsequent reality It tests and carries out the random mutation library that fallibility PCR builds UGTPg45 using the condition.

Use primer SEQ ID NO:25 (7 primers F of segment) (Acactggggcaataggctgtcgccattcaagagcag ) and SEQ ID NO atagcttcaaaatgtttctactc:26 (7 primer R of segment) (Cataatgtgagttttgctcaacatttctctctccattttgtaattaaaacttagat tag), with genes of brewing yeast Group DNA is that template PCR obtains segment 7, uses primer SEQ ID NO:27 (8 primers Fs of segment) (Ttgatgagttcatttcaaagcttgtttcctcctgagattaatataattatataaaa ata) and SEQ ID NO:28 (8 primer R of segment) (Actgtcaaggagggtattctgggcctccatgtcgctgctatataacagttgaaatt tgg) is to make Brewer yeast genomic DNA is that template PCR obtains segment 8, uses primer SEQ ID NO:29 (9 primers Fs of segment) (Cccaaagctaagagtcccattttattc) and SEQ ID NO:30 (9 primer R of segment) (Gaagagtaaaaaaggagtagaaacattttgaagctatctgctcttgaatggcgaca gcc), SEQ ID NO:31 (pieces 10 primers Fs of section) (Tgtcgattcgatactaacgccgccatccagtgtcgagaattacaatagtatgtctg atg) and SEQ ID NO:32 (10 primer R of segment) (Tctggtgaggatttacggtatg) are obtained by template PCR of saccharomyces cerevisiae genome DNA Segment 9 and 10.Use primer SEQ ID NO:33 (11 primers Fs of segment) (Aagatgttcttatccaaatttcaactgttatatagcagcgacatggaggcccagaa tac) and SEQ ID NO:34 (11 primer R of segment) (tacttcttgcagacatcagacatactattgtaattctcgacactggatggcggcgt tag) with Plasmid PLKAN is that template PCR obtains segment 11.By above-mentioned segment 7-11 equimolar than mixing, PPD superior strain is converted (100ug/ml G418 is added) in ZWBY04RS, coating YPD plate, and 30 DEG C are cultivated 2 days, the ferment of UGTPg45 mutated gene to be expressed Female transformant is grown.Similar, conversion wild type UGTPg45 building yeast transformant is as control.

Every hole 600ul YPD culture medium (100ug/ml G418 is added) is added in 96 orifice plates, picking yeast monoclonal arrives In culture medium, 30 DEG C 280rpm shake culture 1 day.6ul culture is shifted to one piece of new 96 containing 600ul YPD culture medium In orifice plate, 30 DEG C 280rpm shake culture 3 days.600ul n-butanol is added in every hole, covers serum cap blend compounds band and seals, and rotates Extract 3h.4000rpm is centrifuged 10min, draws 150ul n-butanol mutually into one piece of 96 new orifice plate, HPLC carries out product measurement.

It is compared using its Rh2 yield of rare ginsenoside Rh2 bacterial strain ZWBY04RS-8E7 constructed by mutant gene 8E7 In improving 70% using bacterial strain ZWBY04RS-UGTPg45 yield constructed by UGTPg45, reach 60.48mg/L.

The wild type gene UGTPg45 has SEQ ID NO:20 nucleotide sequence.From SEQ ID NO:The 5 ' of 20 Holding 1-1374 nucleotide is the open reading frame of UGTPg45, from SEQ ID NO:20 5 ' end 1-3 nucleotide be The initiation codon ATG of UGTPg45 gene, from SEQ ID NO:The 1371-1374 nucleotide at 20 5 ' ends are UGTPg45 The terminator codon TGA of gene.Glycosyl transferase UGTPg45 gene encodes the protein containing 457 amino acid UGTPg45 has SEQ ID NO:19 amino acid residue sequence, with the theoretical molecular weight size of software prediction to the protein It is 5.10 for 51.1kDa, isoelectric point pI.From SEQ ID NO:The 332-375 amino acids of 19 aminoterminal are glycosyl transfer Enzyme PSPG conserved functional domains.

UGTPg45 amino acid sequence SEQ_ID_NO.19

MEREMLSKTHIMFIPFPAQGHMSPMMQFAKRLAWKGLRITIVLPAQIRDFMQITNPLINTECISFDFDK DDGMPYSMQAYMGVVKLKVTNKLSDLLEKQRTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNA RLGKLKIPPEEGLTSVSLPSIPLLGRDDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSQW PITSIGPLIPSMYLDKQLPNDKDNGINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKY HFIWVVIESERTKLSSDFLAEAEAEEKGLIVSWCPQLQVLSHKSIGSFMTHCGWNSTVEALSLGVPMVALPQQFDQP ANAKYIVDVWQIGVRVPIGEEGVVLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKL VSS

UGTPg45 nucleotide sequence SEQ_ID_NO.20

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGC CCAATGATGCAATTCGCCAAGCGTTTAGCCTGGAAAGGCCTGCGAATCACGATAGTTCTTCCGGCTCAAATTCGAGA TTTCATGCAAATAACCAACCCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGACGATGGGATGCCAT ACAGCATGCAGGCTTATATGGGAGTTGTAAAACTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAGA ACAAATGGCTACCCTGTTAATTTGCTAGTGGTTGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGG GGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGA AGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAGATGATTTGCCAATT ATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGAT CTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCAATGGCCAATTACATCCATCG GACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGGCATTAATTTCTACAAGGCA GACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAA GCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACATAGCAAATATCACTTCATATGGGTTG TTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGCAGAGGCAGAGGAAAAAGGCCTAATAGTG AGTTGGTGCCCTCAACTCCAAGTTTTGTCACATAAATCTATAGGGAGTTTTATGACTCATTGTGGTTGGAACTCGAC GGTTGAGGCATTGAGTTTGGGCGTGCCAATGGTGGCACTGCCACAACAGTTTGATCAGCCTGCTAATGCCAAGTATA TCGTGGATGTATGGCAAATTGGGGTTCGGGTTCCGATTGGTGAAGAGGGGGTTGTTTTGAGGGGAGAAGTTGCTAAC TGTATAAAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGT GGAGGCAATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

The mutant gene 8E7 has SEQ ID NO:22 nucleotide sequence.From SEQ ID NO:22 5 ' ends the The open reading frame that 1-1374 nucleotide are 8E7, from SEQ ID NO:The 1-3 nucleotide at 22 5 ' ends are 8E7 gene Initiation codon ATG, from SEQ ID NO:The 1371-1374 nucleotide at 22 5 ' ends are the termination codon of 8E7 gene Sub- TGA.Glycosyl transferase 8E7 gene encodes a protein 8E7 containing 457 amino acid, has SEQ ID NO:21 Amino acid residue sequence.

8E7 amino acid sequence SEQ_ID_NO.21

MEREMLSKTHIMFIPFPAQGHMSPMMQFAKRLAWKGLRITIVLPAQIRDFMQITNPLINTECISFDFDK DDGMPYSMQAYMGVVKLKVTNKLSDLLEKQRTNGYPVNLLVVDSLYPSRVEMCHQLGVKGAPFFTHSCAVGAIYYNA RLGKLKIPPEEGLTSVSLPSIPLLGRDDLPIIRTGTFPDLFEHLGNQFSDLDKADWIFFNTFDKLENEEAKWLSSHW PITSIGPLIPSMYLDKQLPNDKDNGINFYKADVGSCIKWLDAKDPGSVVYASFGSVKHNLGDDYMDEVAWGLLHSKY HFIWVVIESERTKLSSDFLAEVEAEEKGLIVSWCPQLQVLSHKSIGSFMTHCGWNSTVEALSLGVPMVALPQQFDQP ANAKYIVDVWQIGVRVPIGEEGVVLRGEVANCIKDVMEGEIGDELRGNALKWKGLAVEAMEKGGSSDKNIDEFISKL VSS

8E7 nucleotide sequence SEQ_ID_NO.22

ATGGAGAGAGAAATGTTGAGCAAAACTCACATTATGTTCATCCCATTCCCAGCTCAAGGCCACATGAGC CCAATGATGCAATTCGCCAAGCGTTTAGCCTGGAAAGGCCTGCGAATCACGATAGTTCTTCCGGCTCAAATTCGAGA TTTCATGCAAATAACCAACCCATTGATCAACACTGAGTGCATCTCCTTTGATTTTGATAAAGACGATGGGATGCCAT ACAGCATGCAGGCTTATATGGGAGTTGTAAAACTCAAGGTCACAAATAAACTGAGTGACCTACTCGAGAAGCAAAGA ACAAATGGCTACCCTGTTAATTTGCTAGTGGTTGATTCATTATATCCATCTCGGGTAGAAATGTGCCACCAACTTGG GGTAAAAGGAGCTCCATTTTTCACTCACTCTTGTGCTGTTGGTGCCATTTATTATAATGCTCGCTTAGGGAAATTGA AGATACCTCCTGAGGAAGGGTTGACTTCTGTTTCATTGCCTTCAATTCCATTGTTGGGGAGAGATGATTTGCCAATT ATTAGGACTGGCACCTTTCCTGATCTCTTTGAGCATTTGGGGAATCAGTTTTCAGATCTTGATAAAGCGGATTGGAT CTTTTTCAATACTTTTGATAAGCTTGAAAATGAGGAAGCAAAATGGCTATCTAGCCATTGGCCAATTACATCCATCG GACCATTAATCCCTTCAATGTACTTAGACAAACAATTACCAAATGACAAAGACAATGGCATTAATTTCTACAAGGCA GACGTCGGATCGTGCATCAAGTGGCTAGACGCCAAAGACCCTGGCTCGGTAGTCTACGCCTCATTCGGGAGCGTGAA GCACAACCTCGGCGATGACTACATGGACGAAGTAGCATGGGGCTTGTTACATAGCAAATATCACTTCATATGGGTTG TTATAGAATCCGAACGTACAAAGCTCTCTAGCGATTTCTTGGCAGAGGTAGAGGCAGAGGAAAAAGGCCTAATAGTG AGTTGGTGCCCTCAACTCCAAGTTTTGTCACATAAATCTATAGGGAGTTTTATGACTCATTGTGGTTGGAACTCGAC GGTTGAGGCATTGAGTTTGGGCGTGCCAATGGTGGCACTGCCACAACAGTTTGATCAGCCTGCTAATGCCAAGTATA TCGTGGATGTATGGCAAATTGGGGTTCGGGTTCCGATTGGTGAAGAGGGGGTTGTTTTGAGGGGAGAAGTTGCTAAC TGTATAAAGGATGTTATGGAGGGGGAAATAGGGGATGAGCTTAGAGGGAATGCTTTGAAATGGAAGGGGTTGGCTGT GGAGGCAATGGAGAAAGGGGGTAGCTCTGATAAGAATATTGATGAGTTCATTTCAAAGCTTGTTTCCTCCTGA

The above results show using the glycosyltransferase gene Pn50 in Radix Notoginseng source in the present invention or to wild type glycosyl The glycosyltransferase gene UGTPg45 in the mutant gene 8E7 replacement ginseng source that transferase gene transformation obtains can be substantially The combined coefficient and yield of rare ginsenoside Rh2 are promoted, there is significant beneficial effect.

It discusses

Currently, the transcriptome analysis of American ginseng and Radix Notoginseng, researcher has discovered that a large amount of glycosyl by ginseng Transferase candidate gene, but the glycosyl transferase of only only a few is verified the synthesis for taking part in ginsenoside.To in Radix Notoginseng The glycosyl transferase for participating in ginseng saponin formation does not have been reported that so far.Since Radix Notoginseng also synthesizes identical ginsenoside, three are excavated On the one hand seven source glycosyl transferases can make us be better understood by these two types of induction biosynthesis ginsenoside route of synthesis, another Aspect can be studied for the synthetic biology of ginsenoside and provide richer element, be of great significance.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Shanghai Inst. of Life Science, CAS

<120>Glycosyl transferase, mutant and its application

<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 agggggtagc 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 acatttctct ctccattttg 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 ccccagtgt 59

<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 ctatagggag 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 ctatagggag 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 acatttctct ctccattttg 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

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：

(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；

(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；

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

(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：

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.