CN106544310A - A kind of engineering bacteria of gynostemma pentaphylla glycosyl transferase and its construction method and application - Google Patents
A kind of engineering bacteria of gynostemma pentaphylla glycosyl transferase and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of engineering bacteria of gynostemma pentaphylla glycosyl transferase and its construction method and application.Gynostemma pentaphylla glycosyl transferase disclosed by the invention, can be catalyzed protopanoxadiol synthesis rare ginsenoside CK.Its entitled UGTGp3.The present invention also discloses that the method for synthesis rare ginsenoside CK and application simultaneously.The present invention has carried out Function Identification by will come from the glycosyltransferase gene of the gynostemma pentaphylla heterogenous expression in Escherichia coli, external enzymatic reaction.ESI mass spectrums are carried out to its product and HPLC detections all show higher signal.Ginsenoside prepared by the present invention has yield high, and accessory substance is few, the advantages of easy industrialized production, is that the synthesis of gypenoside heterologous organisms is laid a good foundation.
Description
Technical field
The invention belongs to biological pharmacy technical field, is related to a kind of using the transfer of Escherichia coli heterogenous expression gynostemma pentaphylla glycosyl
Enzyme synthesizes the method for rare ginsenoside in vitro, also relates to the gynostemma pentaphylla glycosyl transferase in synthesis rare ginsenoside
Application in CK.
Background technology
Gynostemma pentaphylla is Curcurbitaceae gynostemma pentaphyllum genus herbaceous perennial vine plant, contains various medicinal ingredients in gynostemma pentaphylla, its
In topmost effective component be gypenoside.Gypenoside structure is tetracyclic triterpene dammarane type, with ginsenoside
Structure is identical.Gypenoside is made up of two parts, is by glycosides base and the large biological molecule that formed of combining with glycosyl.So far
It was found that there is eight kinds of gypenosides to be identical, and this eight kinds of saponin(es in structure with protopanaxadiol-type's ginsenoside
Total amount reaches 25% of total saposins or so, and wherein has six kinds of its contents of composition higher than identical component corresponding in ginseng, because
This has extensive pharmacological action.Gypenoside has various special pharmacological actions, with reduction blood fat, antitumor, drop
Blood sugar, anti-ageing pharmacological action of waiting for a long time, and without the resistance to the action of a drug, have no side effect.Gynostemma pentaphylla is researched and developed all in health food and novel medicine
There is huge application prospect, be a kind of new medicine food dual purpose plant resource.The metabolic pathway of research gypenoside, carries out saponin(e
Heterologous organisms synthesis, so as to the yield for improving gypenoside is significant.At present with regard to the research of gypenoside
Total saposins level is concentrated mainly on, the research to single saponin(e is relatively fewer, by glycosylation modified obtaining is carried out to substrate
Saponin(e has monistic feature, and rare gypenoside is also obtained.Therefore, a large amount of highly purified gypenosides are obtained
It is the focus of current research.
At present, the method for producing ginsenoside is mainly the extraction from cultivated ginseng.And the growth cycle of ginseng is long, and carry
Take the technological process complexity of ginsenoside, low yield, easily cause environmental pollution.And glycosidase rule possess stereoselectivity,
Yield is high, accessory substance is few and the advantages of can obtaining rare ginsenoside.It is considered as that production rare ginsenoside is most potential
Method.
With the continuous development of the technologies such as sequencing, gene base resource enriches constantly, the present inventor make great efforts to have found out with from
Gynostemma pentaphylla shifts the gynostemma pentaphylla glycosyl transferase of sugar activity to ginsenoside.And confirm the gynostemma pentaphylla glycosyl transferase UGTGp3
To there is transglycosylation activity with the glucose of the 20th of PPD by glycosidic bond, so as to complete the present invention.
The content of the invention
It is an object of the present invention to develop a kind of glycosyl transferase, the glycosyl transferase is by glycosidic bond and protoplast
The glucose of the 20th of ginseng glycol PPD has transglycosylation activity, so as to generate rare ginsenoside CK.
By means of Characteristic Sequences Method, PSPG boxes are a marks of Secondary metabolites glycosyl transferase to the present inventor
The key property of property, can screen glycosyltransferase gene by PSPG sequences.And with reference to database resources such as NCBI, filter out
May catalysis protopanoxadiol 20 hydroxyl glycosylations glycosyltransferase gene.The gene that preliminary screening is arrived is big
Expressed in enterobacteria expression system, purifying obtains crude enzyme liquid.The experiment proved that the gene can be catalyzed protopanoxadiol 20
Position hydroxyl glycosylation, generates a large amount of, single rare ginsenoside CK.
On the other hand, the invention provides a kind of glycosyl is combined to the glycosyl transferase and its coding of rare ginsenoside CK
Gene.The gene order of the glycosyl transferase is SEQ ID NO.4.
The third aspect, the invention provides a kind of gynostemma pentaphylla glycosyltransferase proteins matter, which is to being connected by glycosidic bond
There is sugared transferase active in the glucose of the 20th of the PPD classes ginsenoside.
Fourth aspect, invention further provides one kind synthesizes Ginsenoside compound K in vitro using gynostemma pentaphylla glycosyl transferase
Method.The method with protopanoxadiol and glycosyl donor UDPG as raw material, in the catalysis of gynostemma pentaphylla glycosyl transferase
Under, there is glycosylation in the 20th hydroxyl of protopanoxadiol, generate rare ginsenoside CK.Experimental result shows：Using this
The Ginsenoside compound K generated by bright synthetic method, Jing after processing, purity can reach 98%.
Finally, present invention also offers carrying comprising the recombinant vector for encoding the gynostemma pentaphylla glycosyl transferase, and the restructuring
The transformant of body.The recombinant vector is the recombinant vector of the arbitrary gene in modern technologies, such as use in this experiment
PET-28a plasmids.The carrier fusion has hexahistine label, readily returns using Ni-NTA His- binding resins posts
Target protein needed for receiving.
The transformant of the recombinant vector, that is, refer to the host cell of recombinant vector.That what is for example used in this experiment is big
EnterobacteriaE.coliBL21（DE3）（NEB companies）.But it is not limited only to this.
For achieving the above object the invention discloses following technology contents：
A kind of Recombinant organism of expression gynostemma pentaphylla glycosyl transferase UGTGp3, it is characterised in that can be with heterogenous expression
Gynostemma pentaphylla glycosyl transferase, the entitled H2493 of genetic engineering bacterium.
The construction method of the Recombinant organism of expression gynostemma pentaphylla glycosyl transferase UGTGp3 of the present invention, its
It is characterised by carrying out in accordance with the following steps：
1）With bioinformatics method, BLAST pair is carried out by glycosyl transferase sequence related to known ginsenoside function
Than predicting the glycosyltransferase gene that can form gypenoside, being named ascomp61。
2）Gynostemma pentaphylla total serum IgE is extracted, gynostemma pentaphylla cDNA library is obtained by reverse transcription；
3）Design special primer, then from gynostemma pentaphylla cDNA libraries, amplification obtains gypenoside glycosyltransferase genecomp61；
4）Prepare containing gypenoside glycosyltransferase genecomp61Recombinant plasmid, obtain build metabolic pathway matter
Grain；
5）By Host Strains Escherichia coliE.coliBL21（DE3）（NEB companies）Lysogenization process is carried out, by step 4）Obtained
Recombinant plasmid transformed in lysogen, acquisition can express the bacillus coli gene engineering of gynostemma pentaphylla glycosyl transferase UGTGp3
Bacterium.The nucleotide sequence such as SEQ ID NO.1-SEQ of the cloning primer of wherein described structure gynostemma pentaphylla glycosyl transferase UGTGp3
Shown in ID NO.2.
The present invention further discloses a kind of gynostemma pentaphylla glycosyl transferase UGTGp3 protein, it is characterised in that with by sugar
Glycosidic bond is connected to the gynostemma pentaphylla glycosyltransferase proteins matter with sugared transferase active of the PPD of the glucose on the 20th carbon,
Amino acid sequence shown in SEQ ID NO. 3.
The method that the present invention further discloses the external synthesis CK of gynostemma pentaphylla glycosyl transferase glycosylation, its feature exist
In, with protopanoxadiol PPD and glycosyl donor UDPG as substrate, under the catalysis of gynostemma pentaphylla glycosyl transferase, protoplast
There is glycosylation in the C20 positions of ginseng glycol, generate rare ginsenoside CK.
The sequence of SEQ ID NO1-4 disclosed by the invention is as follows：
CCGCTCGAGTCAAGATACATTATTATCAAGTACA （SEQ ID NO.1）
CGGGATCCATGAAGAAGATGGATCTTGTTTTC（SEQ ID NO.2）
MKKMDLVFIPGPGIGHLSSTVQIANLLIDRNHHLFITILIINLPSSTNLNTQKHESLSIDRIQFITLPQQPLG
DASKMRLIYKYVMESNKQSVRDTVAKLPHQLAGFVLDMFCSTMIDVANEFGVPSYTFFTSGAGYLSFTLHLQKLYDF
HDNQTFKQFLHSDIEIPGFTNPIPSKAIPIVYLNEDTADWLHEITRRFRETKGILVNTFSELESQVLNSFFQVTTSD
SGFPTVYAVGPILDLNKKNSQTSSSTDGILKWLDEQPPSSVVFLCFGSRGSFDKNQVKEIAQALERSGYRFLWSLRK
PPPAGEYQFPSEYENVEEVLPEGFLDRTADIGRVIGWAPQTEILAHPATGGFVSHCGWNSTLESIWFGVPIGTWPLY
AEQHFNAFEMVVELGLAVEISWDCELFGNNGVIVSAEKIENGIRKLMEGDSDVRKMVKVKSEESRKSMMEGGSSFSS
LNRFIDDVLDNNVS（SEQ ID NO.3）
（SEQ ID NO.4）
ATGAAGAAGATGGATCTTGTTTTCATCCCCGGCCCAGGAATCGGCCATCTCTCATCCACC
GTCCAAATCGCCAACCTCCTCATCGATCGGAATCATCATCTCTTCATCACAATCCTCATC
ATCAATCTTCCTTCATCAACTAACTTAAACACCCAAAAACACGAATCTCTCTCTATCGAC
AGAATCCAATTCATCACTCTCCCTCAACAACCTCTCGGCGACGCCTCTAAAATGCGCCTC
ATCTATAAATATGTTATGGAAAGCAATAAACAGAGTGTCAGAGACACTGTCGCCAAACTT
CCCCATCAGCTCGCCGGGTTCGTCCTTGATATGTTCTGCTCAACCATGATCGATGTTGCC
AATGAATTTGGAGTTCCTTCTTATACATTCTTCACTTCTGGTGCTGGGTATCTTTCTTTC
ACTCTTCATCTTCAAAAGCTTTACGATTTTCATGATAACCAAACATTCAAACAGTTTCTG
CATTCGGATATTGAGATTCCGGGTTTTACTAATCCGATTCCGAGTAAAGCGATTCCTATC
GTCTATCTCAACGAAGATACGGCTGATTGGCTGCATGAGATCACTAGAAGGTTTAGAGAAACCAAGGGTATTC
TAGTAAACACGTTTTCCGAACTTGAATCACAAGTCTTGAACTCGTTTTTTCAAGTCACGACATCCGATTCCGGGTTT
CCGACTGTTTATGCAGTAGGACCTATTTTG
GATCTGAATAAGAAAAACTCTCAAACATCATCATCGACTGACGGTATACTCAAATGGCTC
GATGAACAACCTCCGTCATCGGTCGTATTCCTCTGTTTCGGGAGCAGAGGAAGCTTTGAT
AAAAACCAAGTGAAGGAAATTGCTCAAGCTCTAGAGCGAAGTGGATACCGATTCTTGTGGTCGTTAAGAAAGC
CCCCACCAGCTGGTGAATATCAATTCCCGAGTGAATATGAAAATGTAGAAGAGGTTCTTCCAGAGGGATTTTTGGAT
CGAACGGCGGATATCGGAAGAGTCATCGGGTGGGCACCGCAAACGGAGATCTTAGCGCACCCGGCAACGGGTGGGTT
TGTATCTCATTGCGGTTGGAATTCGACGTTAGAGAGTATATGGTTTGGGGTACCGATTGGGACATGGCCATTGTATG
CAGAACAACATTTTAATGCTTTTGAAATGGTTGTGGAATTGGGTTTAGCTGTGGAGATTTCTTGGGATTGTCAGACG
GAATTGTTTGGTAATAACGGAGTAATTGTGAGTGCGGAGAAGATTGAAAATGGGATTCGAAAATTAATGGAGGGAGA
TAGTGATGTGAGGAAGATGGTGAAAGTTAAAAGTGAAGAGAGTAGAAAGAGTATGATGGAAGGTGGATCTTCGTTTA
GTTCATTGAATCGCTTTATTGATGATGTACTTGATAATAATGTATCTTGA。
By the enforcement of above technical scheme, the engineering bacteria of gynostemma pentaphylla glycosyl transferase disclosed by the invention and its structure side
Method with application had good effect be：
1）Glycosyl transferase of the present invention successfully can synthesize rare ginsenoside CK in vitro.It is a kind of new producer
Method, flow process are simple, and yield is higher, consume low.
2）Gynostemma pentaphylla glycosyltransferase gene of the present invention enriches current glycosyl transferase database.
Description of the drawings
Fig. 1 is plasmid pET-28a（+）The collection of illustrative plates of-comp61, for expressing gynostemma pentaphylla glycosyl transferase UGTGp3；
Fig. 2 is the mass spectrum of protopanoxadiol glycation product（MS）Spectrogram；
Fig. 3 is the HPLC spectrograms of protopanoxadiol and its glycation product.
Specific embodiment
Below by the specific embodiment narration present invention.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment be interpreted as it is illustrative, and it is unrestricted the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, various changes that the material component and consumption in these embodiments is carried out or change
Belong to protection scope of the present invention.
In following examples of the present invention, material used, reagent, instrument and method are in this area without specified otherwise
Conventional material, reagent, instrument and method, can be obtained by commercial channel；Such as Tryptone (tryptone), Yeast
Extract（Yeast extract）, Agar（Agar）；Kanamycins,E.coliDH5 α etc..
In the present invention, plasmid extraction is using raw work bioengineering（Shanghai）The SanPrep pillar DNAs of Co., Ltd are little
Amount extraction agent box（Catalog NO.：B518191）, it is using raw work bioengineering to cut glue reclaim（Shanghai）Co., Ltd
SanPrep gel extraction kits（Catalog NO.：B518131）, the connection of DNA fragmentation is using fermentas companies
T4 DNA Ligase（Catalog NO.：EL0014）, the pfu DNA expanded using fermentas companies of DNA fragmentation
polymerase（Catalog NO.：EP0571）, the Fast digested using fermentas companies of PCR plasmid templates
DigestXhoI（Catalog NO.：FD0694）, BamHI（Catalog NO.：FD0054）The electroporated experiments of E.coli make
With the electroporation of Bio-Rad（Catalog NO.：165-2100）.
Embodiment 1
From the clone of the glycosyl transferase UGTGp3 of gynostemma pentaphylla
By means of the gopher based on compound, chemical reaction, with reference to the resource of the databases such as NCBI, and the PSPG of plant
The principles such as box, having screened may the glycosylated glycosyltransferase gene of catalysis protopanoxadiol.Using primer SEQ ID NO.1-
SEQ ID NO.2, and pass through PCR from gynostemma pentaphylla cDNA library amplification gene using polymerase.Fragment after amplification carries out cutting glue
Purifying, and carries out double digestion with XhoI and BamHI, the fragment after digestion and the plasmid for also passing through XhoI and BamHI double digestions
pET-28a（+）Plasmid is attached, by carrier：Purpose fragment is 1 in molar ratio:3 ratio mixing, adds T4 DNA
After Ligase, at 22 DEG C, enzyme connects 5h, connection product conversionE.coliDH5 α, and screening positive clone is carried out on card receives plate,
Sequence verification.Obtain recombinant plasmid pET-28a（+）-comp61.
Embodiment 2
The foundation of E. coli expression strains and the purifying of gynostemma pentaphylla glycosyl transferase UGTGp3
Recombinant expression plasmid is transformed into into Host StrainsE.coliBL21(DE3)（NEB companies）Competent cell, obtains gynostemma pentaphylla
The recombinant strains of glycosyl transferase.Culture recombinant bacterium to OD be 0.6～0.8 when, add 0.1mM IPTG, in 16 DEG C of low temperature
Abduction delivering 20h.4 DEG C, 5500rpm is collected by centrifugation cell, and cell is carried out ultrasonication.Ni-NTA His- are utilized to tie to which
Resin is purified.
Embodiment 3
In vitro enzymes are tested
External reaction system is prepared according to described, the experiment of glycosyl transferase is carried out.
At 30 DEG C, after the culture reaction of 3h is carried out to reactant mixture, same volume n-butanol is added to make its terminating reaction.
Embodiment 4
The detection of product
Reaction system is extracted with n-butanol, after the organic phase vacuum drying of extraction, adds methyl alcohol back dissolving.Carry out electron spray ionisation matter
Spectrum（ESI）Detection, as a result as shown in Fig. 2 result shows the molecular ion peak that (m/z) 622.68 is glc-O- protopanoxadiols,
As a result with expected product glc-O- protopanoxadiols（623）Molecular weight be consistent.
Instrument：Finnigan
LCQ Advantage MAX ion trap mass spectrometer
(Thermo Electron,CA)
Ionization mode：Negative electrospray ionization pattern；
Electron spray scope：400-500 m/z；
Dryer temperature：250 ℃；
Atomisation pressure：45 psi；
Capillary voltage：4500 V；
Sample size：0.2 mL/min；
The mass spectrogram of product is as shown in Fig. 2 judge according to molecular weight, mono-glycosylated product of the compound for protopanoxadiol
Glc-O- protopanoxadiols (m/z) 623.
Reaction system is extracted with n-butanol, after the organic phase vacuum drying of extraction, adds methyl alcohol back dissolving.Carry out HPLC detections
Analysis is as shown in Figure 3 with quantitative result.
Using Shimadzu LC-20A prominence system (Shimadzu, Kyoto, Japan), use
Shodex C18-120-5 4E column (5 μm of 4.6mm × 250mm), according to the flow velocity of 800 μ l per minute, 0 min,
35% acetonitrile, 50 min, 90% acetonitrile, 55 min, 90% acetonitrile, 55-65 min, 35% acetonitrile.203 nm of Detection wavelength.
Fig. 3 results show：
（1）Standard items CK is in 35 min or so appearance；
（2）Negative control is not added with enzyme liquid does not have product to occur in 35 min；
（3）There is product peak to occur in 35 min after addition enzyme liquid reaction, and peak value is higher.
Embodiment 5
With protopanoxadiol PPD and glycosyl donor UDPG as substrate, under the catalysis of gynostemma pentaphylla glycosyl transferase, protoplast
There is glycosylation in the C20 positions of ginseng glycol, generate rare ginsenoside CK.By the proportions 100mL's in embodiment 3
Reactant liquor.Under the conditions of 30 DEG C, react 48 hours.Add same volume n-butanol terminating reaction.Take upper organic phase rotation
Evaporation drying, with silica column purification, eluant, eluent is chloroform:Methyl alcohol（85:15）, every 5 mL equal portions collection, the sample of collection are carried out
ESI and HPLC analyses (condition is the same as those described above), obtains protopanoxadiol glycation product part, purity<90%.
Above-mentioned collection part is purified further with Sep-Pak tC18 posts (Waters), water (A) and acetonitrile
(B) as eluant, eluent, using gradient elution (20% B, 40% B, 50% B, 60% B, 65% B, 70% B, 75% B,
80%B, 85%B, 90%B, 100%B), when 70% and 75% acetonitrile is eluted, protopanoxadiol glycation product part is obtained,
Purity reaches 98%.White powder is obtained in 40 DEG C of rotary evaporations or freeze-drying.
Above example is only illustrating technical scheme, rather than is limited, although with reference to above-mentioned reality
Apply example to be described in detail the present invention, for the person of ordinary skill of the art, still can be to aforementioned enforcement
Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic.And these are changed or replace
Change, do not make the essence of appropriate technical solution depart from the scope and spirit of claimed technical solution of the invention.
SEQUENCE LISTING
<110>Nankai University
<120>A kind of engineering bacteria of gynostemma pentaphylla glycosyl transferase and its construction method and application
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 34
<212> DNA
<213>Artificial sequence
<400> 1
ccgctcgagt caagatacat tattatcaag taca 34
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence
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cgggatccat gaagaagatg gatcttgttt tc 32
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<212> PRT
<213>Gynostemma pentaphylla glycosyltransferase proteins matter
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Met Lys Lys Met Asp Leu Val Phe Ile Pro Gly Pro Gly Ile Gly His
1 5 10 15
Leu Ser Ser Thr Val Gln Ile Ala Asn Leu Leu Ile Asp Arg Asn His
20 25 30
His Leu Phe Ile Thr Ile Leu Ile Ile Asn Leu Pro Ser Ser Thr Asn
35 40 45
Leu Asn Thr Gln Lys His Glu Ser Leu Ser Ile Asp Arg Ile Gln Phe
50 55 60
Ile Thr Leu Pro Gln Gln Pro Leu Gly Asp Ala Ser Lys Met Arg Leu
65 70 75 80
Ile Tyr Lys Tyr Val Met Glu Ser Asn Lys Gln Ser Val Arg Asp Thr
85 90 95
Val Ala Lys Leu Pro His Gln Leu Ala Gly Phe Val Leu Asp Met Phe
100 105 110
Cys Ser Thr Met Ile Asp Val Ala Asn Glu Phe Gly Val Pro Ser Tyr
115 120 125
Thr Phe Phe Thr Ser Gly Ala Gly Tyr Leu Ser Phe Thr Leu His Leu
130 135 140
Gln Lys Leu Tyr Asp Phe His Asp Asn Gln Thr Phe Lys Gln Phe Leu
145 150 155 160
His Ser Asp Ile Glu Ile Pro Gly Phe Thr Asn Pro Ile Pro Ser Lys
165 170 175
Ala Ile Pro Ile Val Tyr Leu Asn Glu Asp Thr Ala Asp Trp Leu His
180 185 190
Glu Ile Thr Arg Arg Phe Arg Glu Thr Lys Gly Ile Leu Val Asn Thr
195 200 205
Phe Ser Glu Leu Glu Ser Gln Val Leu Asn Ser Phe Phe Gln Val Thr
210 215 220
Thr Ser Asp Ser Gly Phe Pro Thr Val Tyr Ala Val Gly Pro Ile Leu
225 230 235 240
Asp Leu Asn Lys Lys Asn Ser Gln Thr Ser Ser Ser Thr Asp Gly Ile
245 250 255
Leu Lys Trp Leu Asp Glu Gln Pro Pro Ser Ser Val Val Phe Leu Cys
260 265 270
Phe Gly Ser Arg Gly Ser Phe Asp Lys Asn Gln Val Lys Glu Ile Ala
275 280 285
Gln Ala Leu Glu Arg Ser Gly Tyr Arg Phe Leu Trp Ser Leu Arg Lys
290 295 300
Pro Pro Pro Ala Gly Glu Tyr Gln Phe Pro Ser Glu Tyr Glu Asn Val
305 310 315 320
Glu Glu Val Leu Pro Glu Gly Phe Leu Asp Arg Thr Ala Asp Ile Gly
325 330 335
Arg Val Ile Gly Trp Ala Pro Gln Thr Glu Ile Leu Ala His Pro Ala
340 345 350
Thr Gly Gly Phe Val Ser His Cys Gly Trp Asn Ser Thr Leu Glu Ser
355 360 365
Ile Trp Phe Gly Val Pro Ile Gly Thr Trp Pro Leu Tyr Ala Glu Gln
370 375 380
His Phe Asn Ala Phe Glu Met Val Val Glu Leu Gly Leu Ala Val Glu
385 390 395 400
Ile Ser Trp Asp Cys Glu Leu Phe Gly Asn Asn Gly Val Ile Val Ser
405 410 415
Ala Glu Lys Ile Glu Asn Gly Ile Arg Lys Leu Met Glu Gly Asp Ser
420 425 430
Asp Val Arg Lys Met Val Lys Val Lys Ser Glu Glu Ser Arg Lys Ser
435 440 445
Met Met Glu Gly Gly Ser Ser Phe Ser Ser Leu Asn Arg Phe Ile Asp
450 455 460
Asp Val Leu Asp Asn Asn Val Ser
465 470
<210> 4
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<212> DNA
<213>Artificial sequence
<400> 4
atgaagaaga tggatcttgt tttcatcccc ggcccaggaa tcggccatct ctcatccacc 60
gtccaaatcg ccaacctcct catcgatcgg aatcatcatc tcttcatcac aatcctcatc 120
atcaatcttc cttcatcaac taacttaaac acccaaaaac acgaatctct ctctatcgac 180
agaatccaat tcatcactct ccctcaacaa cctctcggcg acgcctctaa aatgcgcctc 240
atctataaat atgttatgga aagcaataaa cagagtgtca gagacactgt cgccaaactt 300
ccccatcagc tcgccgggtt cgtccttgat atgttctgct caaccatgat cgatgttgcc 360
aatgaatttg gagttccttc ttatacattc ttcacttctg gtgctgggta tctttctttc 420
actcttcatc ttcaaaagct ttacgatttt catgataacc aaacattcaa acagtttctg 480
cattcggata ttgagattcc gggttttact aatccgattc cgagtaaagc gattcctatc 540
gtctatctca acgaagatac ggctgattgg ctgcatgaga tcactagaag gtttagagaa 600
accaagggta ttctagtaaa cacgttttcc gaacttgaat cacaagtctt gaactcgttt 660
tttcaagtca cgacatccga ttccgggttt ccgactgttt atgcagtagg acctattttg 720
gatctgaata agaaaaactc tcaaacatca tcatcgactg acggtatact caaatggctc 780
gatgaacaac ctccgtcatc ggtcgtattc ctctgtttcg ggagcagagg aagctttgat 840
aaaaaccaag tgaaggaaat tgctcaagct ctagagcgaa gtggataccg attcttgtgg 900
tcgttaagaa agcccccacc agctggtgaa tatcaattcc cgagtgaata tgaaaatgta 960
gaagaggttc ttccagaggg atttttggat cgaacggcgg atatcggaag agtcatcggg 1020
tgggcaccgc aaacggagat cttagcgcac ccggcaacgg gtgggtttgt atctcattgc 1080
ggttggaatt cgacgttaga gagtatatgg tttggggtac cgattgggac atggccattg 1140
tatgcagaac aacattttaa tgcttttgaa atggttgtgg aattgggttt agctgtggag 1200
atttcttggg attgtcagac ggaattgttt ggtaataacg gagtaattgt gagtgcggag 1260
aagattgaaa atgggattcg aaaattaatg gagggagata gtgatgtgag gaagatggtg 1320
aaagttaaaa gtgaagagag tagaaagagt atgatggaag gtggatcttc gtttagttca 1380
ttgaatcgct ttattgatga tgtacttgat aataatgtat cttga 1425
Claims (7)
1. a kind of Recombinant organism of expression gynostemma pentaphylla glycosyl transferase UGTGp3, it is characterised in that can be with heterologous table
Up to gynostemma pentaphylla glycosyl transferase, the entitled H2493 of genetic engineering bacterium.
2. the construction method of the Recombinant organism of gynostemma pentaphylla glycosyl transferase UGTGp3 is expressed described in claim 1,
It is characterized in that carrying out in accordance with the following steps：
With bioinformatics method, BLAST contrasts are carried out by glycosyl transferase sequence related to known ginsenoside function,
The glycosyltransferase gene that can form gypenoside is predicted, is named ascomp61；Gynostemma pentaphylla total serum IgE is extracted, by anti-
Transcription obtains gynostemma pentaphylla cDNA library；Design special primer, then from gynostemma pentaphylla cDNA libraries, amplification obtains gypenoside
Glycosyltransferase genecomp61；Prepare containing gypenoside glycosyltransferase genecomp61Recombinant plasmid, obtain structure
Build the plasmid of metabolic pathway；By Host Strains Escherichia coliE.coliBL21（DE3）Lysogenization process is carried out, by step 4）Obtained
To in lysogen, acquisition can express the bacillus coli gene work of gynostemma pentaphylla glycosyl transferase UGTGp3 to the recombinant plasmid transformed for obtaining
Journey bacterium.
3. the construction method described in claim 2, it is characterised in that the structure gynostemma pentaphylla glycosyl transferase UGTGp3 gram
The nucleotide sequence of grand primer is as shown in SEQ ID NO.1-SEQ ID NO.2.
4. a kind of gynostemma pentaphylla glycosyl transferase UGTGp3 protein, it is characterised in that with glycosidic bond is connected on the 20th carbon
Glucose PPD the gynostemma pentaphylla glycosyltransferase proteins matter with sugared transferase active, be shown in SEQ ID NO. 3
Amino acid sequence.
5. the method for the external synthesis CK of a kind of gynostemma pentaphylla glycosyl transferase glycosylation, it is characterised in that with protopanoxadiol PPD and
Glycosyl donor UDPG is substrate, under the catalysis of gynostemma pentaphylla glycosyl transferase, the C of protopanoxadiol20There is glycosyl in position
Change reaction, generate rare ginsenoside CK.
6. a kind of glycosyl is combined to the gene of the glycosyl transferase and its coding of rare ginsenoside CK, the glycosyl transferase
Gene order is SEQ ID NO.4.
7. the Recombinant organism that gynostemma pentaphylla glycosyl transferase UGTGp3 is expressed described in claim 1 is preparing external conjunction
Application into rare ginsenoside CK in terms of.
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