CN114317480B - Glycosyl transferase for catalytically synthesizing salidroside, and coding gene and application thereof - Google Patents

Glycosyl transferase for catalytically synthesizing salidroside, and coding gene and application thereof Download PDF

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CN114317480B
CN114317480B CN202210023456.4A CN202210023456A CN114317480B CN 114317480 B CN114317480 B CN 114317480B CN 202210023456 A CN202210023456 A CN 202210023456A CN 114317480 B CN114317480 B CN 114317480B
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hydroxytyrosol
glu
salidroside
ser
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CN114317480A (en
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虞沂
曹应龙
刘亚婷
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Hubei Carbon Yuan Materia Medica Biotechnology Co ltd
Wuhan University WHU
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Abstract

The invention provides a glycosyl transferase for catalytically synthesizing salidroside, and a coding gene and application thereof. The glycosyl transferase can catalyze the reaction of glycosyl donor UDP-glucose and acceptor hydroxytyrosol to generate hydroxyl salidroside, has the characteristics of high regioselectivity and single product, can be used as a synthesis element to be applied to the in vitro enzyme catalysis or in vivo reconstruction synthesis of the hydroxyl salidroside or the phenylethanoid glycoside compound taking the hydroxyl salidroside as a framework, and also lays a foundation for deeply analyzing the complete biosynthesis way of the phenylethanoid glycoside compound.

Description

Glycosyl transferase for catalytically synthesizing salidroside, and coding gene and application thereof
Technical Field
The invention relates to the technical field of synthesis of hydroxy salidroside, in particular to glycosyltransferase for in vitro catalytic synthesis of hydroxy salidroside, and a coding gene and application thereof.
Background
The molecular formula of the hydroxyl salidroside is C 14 H 20 O 8 Molecular weight is 316.11582, CAS number is 76873-99-9, and research reports that the compound has good neuroprotective effect (Ying-Guo Liu et al, 2015), and a high-efficiency method for synthesizing salidroside in vitro is urgently needed to be developed.
Disclosure of Invention
Based on the above, there is a need for a glycosyltransferase for in vitro catalytic synthesis of hydroxysalidroside, and a coding gene and application thereof.
The invention adopts the following technical scheme:
the invention provides a glycosyl transferase for catalyzing hydroxytyrosol to generate salidroside, which has the sequence shown in a) and b):
a) a protein consisting of an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;
b) a derivative protein of which the amino acid sequence in a) is substituted, deleted and/or added with one or more amino acid residues and which has the enzymatic activity of catalyzing hydroxytyrosol to generate hydroxytyrosol.
The invention also provides a coding gene of the glycosyltransferase for catalyzing hydroxytyrosol to generate salidroside, and the gene sequence is shown as the following c) or d):
c) a DNA molecule as shown in SEQ ID NO.3 or SEQ ID NO. 4;
d) hybridizing under stringent conditions with the DNA sequence defined in c) and encoding a DNA molecule having the enzymatic activity of catalyzing hydroxytyrosol to produce hydroxytryptaside.
The invention also provides an expression cassette, a recombinant vector or a recombinant bacterium containing the coding gene of the glycosyltransferase for catalyzing hydroxytyrosol to generate hydroxytyrosol glycoside.
The invention also provides recombinant escherichia coli, which comprises the coding gene of the glycosyl transferase for catalyzing hydroxytyrosol to generate hydroxytyrosol.
The invention also provides application of the glycosyltransferase for catalyzing hydroxytyrosol to generate hydroxytyrosol to a process for catalyzing hydroxytyrosol to generate hydroxytyrosol in vitro.
The invention also provides a synthetic method of the hydroxyl salidroside, which comprises the following steps: adding substrates hydroxytyrosol, UDPG and the glycosyltransferase into a buffer solution respectively to obtain a mixed solution; and (3) reacting the mixed solution at 25-35 ℃, and adding ice methanol to quench the reaction.
In some of these embodiments, the buffer is Tris-HCl buffer, pH 7.4.
In some embodiments, the mixture further comprises ascorbic acid.
In some embodiments, the mixture comprises hydroxytyrosol, UDPG, and glycosyltransferase at a concentration of 1 mM: 2 mM: 5 μ M.
The invention has the beneficial effects that:
compared with the prior art, the invention discovers and provides glycosyltransferase for the first time, and the glycosyltransferase can efficiently catalyze the reaction of glycosyl donor UDP-glucose and acceptor hydroxytyrosol to generate single glycosylation product, namely the hydroxysalidroside.
Drawings
FIG. 1 shows the gel diagram of the purification detection of OfT8GT1 and OfT8GT3 protein expression.
FIG. 2 shows that glycoside glycosyltransferases OfT8GT1 and OfT8GT3 catalyze the production of hydroxytyrosol to salidroside.
FIG. 3 is the primary and secondary mass spectrum analysis of the product hydroxy salidroside.
FIG. 4 is the nuclear magnetic hydrogen spectrum of the product hydroxy salidroside.
FIG. 5 is the nuclear magnetic carbon spectrum of the product hydroxy salidroside.
FIG. 6 is the nuclear magnetic two-dimensional HSQC spectrum of the product hydroxy salidroside.
FIG. 7 is a nuclear magnetic two-dimensional HMBC spectrogram of the product hydroxy salidroside.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
Description of key material sources:
plasmid pET-28a (+), purchased from Novagen. Plasmid extraction and gel recovery kits were purchased from OMEGA. Coli TOP10 and BL21(DE3) are competent, purchased from organisms of the Peking Okame family.
Restriction enzymes were purchased from New England Biolabs.
The reagents used in the usual media were purchased from Oxford, Amresco and national pharmaceutical group Chemicals, Inc.
All antibiotics used were purchased from Sigma-Aldrich.
The LB medium formulation is 10g/L Tryptone, 5g/L Yeast extract and 10g/L NaCl.
Chemical preparation of solutions for E.coli competence:
TFB I (100mL) solution: KAC (5M)0.6mL, CaCl 2 (1M)1mL，KCl(1M)10mL， MnCl 2 (1M)5mL, glycerol (100%) 15mL, sterile water 68.4 mL.
TFB II (100mL) solution: MOPS (1M)1mL, CaCl 2 7.5mL (1M), 1mL KCl (1M), 15mL glycerol (100%), and 75.5mL sterile water.
The experimental reagents which are not particularly described in the invention are all conventional reagents in the field, and can be prepared according to a conventional method in the field or obtained commercially.
Example 1 in vitro catalytic Synthesis of Hydroxysalidroside enzymes and genes encoding same.
The inventor team finds out through a large amount of research that: the osmanthus fragrans contains glycosyltransferase OfT8GT1 or glycosyltransferase OfT8GT3, and can catalyze the reaction of glycosyl donor UDP-glucose and acceptor Hydroxytyrosol (Hydroxytyrosol) to generate a single glycosylation product: salidroside Hydroxysalidroside (Hydroxysalidroside).
Wherein the amino acid sequence of glycosyltransferase OfT8GT1 is as follows:
MGSIAGNDKPHAVCIPYPAQGHINPMFNLAKLLHHQGFYITFVNTEFN HKRLLKSRGPAALDGLPDFRFETIPDGLPSSDGDATQDIPSLCESTTKTCLA PFCSLLTKLNNAAPVMPPVTCIVSDGAMSFTLKAAEQFGLPEVLLWTTSA CGFLGYTQYANLIERGYTPLKEMSQVTNGYLETTIDWIPGMKDIRLRDLP TFLRTTDSNDIMLNFVLREIEAVPKAKAIILNTFDALEHDVLDALSSMNPVI YSVGPLQLMMNHIQNEKHTLTSSSLWKEELECIKWLDKKEPNSVVYVNF GSIAVVTAQQLTEFAWGLANSKKSFLWIIRPDLVAGDLAMLPPEFVTETED RSMLISWCPQEEVLKHPAIGGFLTHSGWNSTIESIVAGVPLICWPFFAEQQT NCRYSCVEWGMGMEIDNNVKRDEVEELVKELMDGEKGMKMKEKAME WKKKAEEATAPGGSSFMNFEELVKLLQ(SEQ ID NO.1)。
the amino acid sequence of glycosyltransferase OfT8GT3 is as follows:
MGSISLLEKPHVVCIPFPAQGHINPMLKLAKLLHQKGFHVTFVNTEFN HGRLLKSRGPDSLKGLASFRFETIPDGLPISDADATQDIASLCESTTNTCLV PFKDLLARLNDTISSNVPPVSCIISDGVMSFTLEAAEELRIPEVLLWTTSAC GFLAYTQYAKLIEEGYVPLKDASYLTNGYLDTVIADEIPGMEGMRLKDIP SFIRTTSLDDYMVKFVLQETERARKASAIILNTFEDLEHDCVKTLSSILPPV YAIGPLHFLQKDVKDKSLEILGSNLWKEDQHCLEWLDSKEPKSVVYINFG SITVMTPKQLIEFAWGLANSGQTFLWIIRPDLVTGDDAIFPPEFLEATKERS LLANWCPQEKVLSHPSIGGFLTHSGWNSTLESIGSGVPMICWPFFAEQQT NCWFSCTKWGIGMEIDSDVKRDEVEILVKELMIGEKGKEMKKRALEWM KLAKDSAKNSEGSSHKNLEKLINQVLLCTKL(SEQ ID NO.2)。
wherein, the nucleotide sequence of the gene coded by the glycosyltransferase OfT8GT1 is as follows:
ATGGGTAGCATTGCGGGTAACGACAAACCGCATGCGGTTTGCATCC CGTACCCGGCGCAGGGTCACATTAACCCGATGTTCAACCTGGCGAAAC TGCTGCACCACCAAGGCTTCTATATCACCTTTGTGAACACCGAGTTTAA CCACAAGCGTCTGCTGAAAAGCCGTGGTCCGGCGGCGCTGGATGGCC TGCCGGATTTCCGTTTTGAAACCATTCCGGATGGTCTGCCGAGCAGCGA TGGTGATGCGACCCAGGATATTCCGAGCCTGTGCGAAAGCACCACCAA GACCTGCCTGGCGCCGTTCTGCAGCCTGCTGACCAAACTGAACAACGC GGCGCCGGTGATGCCGCCAGTTACCTGCATCGTGAGCGATGGTGCGAT GAGCTTCACCCTGAAGGCGGCGGAGCAATTTGGCCTGCCGGAAGTTCT GCTGTGGACCACCAGCGCGTGCGGTTTTCTGGGCTACACCCAGTATGC GAACCTGATCGAGCGTGGTTACACCCCGCTGAAGGAAATGAGCCAAG TGACCAACGGTTATCTGGAAACCACCATCGACTGGATTCCGGGCATGA AAGACATTCGTCTGCGTGATCTGCCGACCTTCCTGCGTACCACCGACA GCAACGATATCATGCTGAACTTTGTTCTGCGTGAGATTGAAGCGGTGCC GAAAGCGAAGGCGATCATTCTGAACACCTTCGATGCGCTGGAGCACGA CGTTCTGGATGCGCTGAGCAGCATGAACCCGGTTATCTACAGCGTGGG TCCGCTGCAGCTGATGATGAACCACATTCAAAACGAAAAGCACACCCT GACCAGCAGCAGCCTGTGGAAAGAGGAGCTGGAGTGCATCAAATGGC TGGACAAGAAAGAACCGAACAGCGTGGTTTATGTGAACTTCGGTAGCA TTGCGGTGGTTACCGCGCAGCAACTGACCGAGTTCGCGTGGGGCCTGG CGAACAGCAAGAAAAGCTTTCTGTGGATCATTCGTCCGGACCTGGTTG CGGGCGATCTGGCGATGCTGCCGCCGGAATTCGTGACCGAAACCGAAG ATCGTAGCATGCTGATCAGCTGGTGCCCGCAGGAAGAGGTGCTGAAAC ACCCGGCGATTGGTGGCTTTCTGACCCACAGCGGTTGGAACAGCACCA TCGAGAGCATTGTTGCGGGCGTTCCGCTGATCTGCTGGCCGTTCTTTGC GGAACAGCAAACCAACTGCCGTTACAGCTGCGTTGAGTGGGGTATGG GCATGGAAATTGACAACAACGTGAAGCGTGATGAGGTTGAGGAACTG GTGAAGGAGCTGATGGACGGTGAAAAAGGCATGAAAATGAAGGAGAA AGCGATGGAATGGAAGAAAAAGGCGGAGGAAGCGACCGCGCCGGGT GGCAGCAGCTTCATGAACTTTGAGGAACTGGTGAAACTGCTGCAATGA （SEQ ID NO.3）。
the nucleotide sequence of the gene encoding glycosyltransferase OfT8GT3 is as follows:
ATGGGTAGCATCAGCCTGCTGGAGAAGCCGCACGTGGTTTGCATCC CGTTCCCGGCGCAGGGCCACATTAACCCGATGCTGAAGCTGGCGAAAC TGCTGCACCAAAAAGGTTTCCACGTGACCTTTGTTAACACCGAATTTA ACCACGGCCGTCTGCTGAAGAGCCGTGGTCCGGACAGCCTGAAAGGC CTGGCGAGCTTCCGTTTTGAAACCATCCCGGACGGTCTGCCGATTAGC GACGCGGATGCGACCCAGGATATCGCGAGCCTGTGCGAAAGCACCAC CAACACCTGCCTGGTGCCGTTCAAGGACCTGCTGGCGCGTCTGAACGA TACCATTAGCAGCAACGTGCCGCCGGTTAGCTGCATCATTAGCGATGGT GTGATGAGCTTCACCCTGGAAGCGGCGGAGGAACTGCGTATCCCGGA AGTGCTGCTGTGGACCACCAGCGCGTGCGGCTTTCTGGCGTACACCCA GTATGCGAAGCTGATTGAGGAAGGTTACGTGCCGCTGAAAGACGCGA GCTACCTGACCAACGGCTATCTGGACACCGTTATCGCGGATGAGATTCC GGGTATGGAAGGCATGCGTCTGAAGGATATCCCGAGCTTCATTCGTACC ACCAGCCTGGACGATTACATGGTGAAGTTTGTTCTGCAAGAAACCGAA CGTGCGCGTAAAGCGAGCGCGATCATTCTGAACACCTTCGAGGACCTG GAACACGATTGCGTGAAAACCCTGAGCAGCATCCTGCCGCCGGTTTAT GCGATTGGTCCGCTGCACTTTCTGCAGAAGGACGTTAAGGATAAAAGC CTGGAGATCCTGGGCAGCAACCTGTGGAAAGAGGACCAACACTGCCT GGAATGGCTGGATAGCAAGGAGCCGAAAAGCGTGGTTTATATCAACTT CGGTAGCATTACCGTGATGACCCCGAAGCAGCTGATCGAATTCGCGTG GGGTCTGGCGAACAGCGGCCAAACCTTTCTGTGGATCATTCGTCCGGA CCTGGTTACCGGCGACGATGCGATTTTCCCGCCGGAGTTTCTGGAAGC GACCAAGGAACGTAGCCTGCTGGCGAACTGGTGCCCGCAGGAGAAAG TGCTGAGCCACCCGAGCATTGGTGGCTTCCTGACCCACAGCGGTTGGA ACAGCACCCTGGAAAGCATCGGTAGCGGCGTTCCGATGATTTGCTGGC CGTTCTTTGCGGAGCAGCAAACCAACTGCTGGTTTAGCTGCACCAAAT GGGGTATCGGCATGGAAATTGACAGCGATGTGAAGCGTGACGAGGTG GAAATCCTGGTTAAAGAGCTGATGATTGGCGAGAAGGGCAAAGAAAT GAAGAAACGTGCGCTGGAATGGATGAAGCTGGCGAAAGATAGCGCGA AGAACAGCGAAGGCAGCAGCCACAAGAACCTGGAGAAACTGATCAA CCAAGTTCTGCTGTGCACCAAACTGTGA（SEQ ID NO.4）。
example 2 construction method of E.coli expression vector
The embodiment provides a construction method of an escherichia coli expression vector, which comprises the following steps:
s1, synthesizing genes aiming at the gene sequences of glycosyltransferase OfT8GT1 and glycosyltransferase OfT8GT3, and designing amplification primers required by constructing an Escherichia coli expression vector. The primer sequences are shown in the following table:
Figure BDA0003463535700000061
Figure BDA0003463535700000071
s2, using the plasmids of the synthesized genes as templates, amplifying the target fragment by using primers according to the following PCR reaction system and reaction program:
PCR reaction system
Components Dosage (mu L)
10×PCR Buffer for KOD-Plus-Neo 5
2mMdNTPs 5
25mM MgSO 4 3
Primer (F end) 1.5
Primer (R end) 1.5
Form panel 1
distilled water 29
KOD-Plus-Neo 1
Totol 50
PCR reaction procedure: pre-denaturation at 94 ℃ for 2min, denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 30s, extension at 62 ℃ for 1min, amplification cycle for 30 times, and final extension at 68 ℃ for 10 min.
After the PCR reaction, 2. mu.L of the reaction product was electrophoresed through 0.8% agarose gel to determine whether the size of the target band was correct. After the detection is correct, agarose gel electrophoresis is carried out to recover a PCR product.
S3, the pS1300T-Flag vector was linearized with restriction enzymes EcoRI and SacI, and the digested linearized vector was recovered by agarose gel electrophoresis.
S4, In-fusion clone recombination and transformation screening to identify positive clones: adding a 15-20 nt homologous sequence of a vector to the end of a primer according to the In-fusion instruction, purifying the amplified DNA fragment, mixing the purified DNA fragment with the linearized vector according to the molar ratio of 3:1, adding 5 multiplied reaction mixed enzyme solution, and reacting at 50 ℃ for 30-50 min. The resulting product was ready for transformation of e.coli TOP10 competence.
Mixing the DNA to be transformed with competent cell (DNA: competent cell <1/10), ice-bath for 25 min; carrying out heat shock at 37 ℃ for 2min (or 42 ℃ for 90s), quickly transferring the tube into an ice bath, and cooling the cells for 3-5 min; adding 600 mu L of LB culture medium, and pre-culturing for 45-60 min at 37 ℃; an appropriate volume (200 μ Ι _) was spread evenly on LA solid plates containing the corresponding antibiotic; and (3) inverting the plate, and culturing in an incubator at 37 ℃ for about 12-16 h to form bacterial colonies.
And (3) inoculating a single colony on the culture plate into a kanamycin-resistant LB culture medium for overnight culture, extracting a plasmid for enzyme digestion identification, and sending the plasmid which is verified to be correct to a sequencing company for sequencing as shown in figure 1.
EXAMPLE 3 protein expression and purification
Selecting single colony of Escherichia coli BL21(DE3) containing protein expression recombinant vector, culturing in 5mL LB (containing kanamycin) at 37 deg.C and 200rpm for 12 h; transferring the strain into 0.5L LB culture medium, culturing at 37 deg.C and 200rpm until OD600 value is 0.6; adding 0.1-0.5 mL of 1M IPTG (final concentration is 0.5mM) to perform induction expression at 18 ℃ for 18-20 h, collecting thalli at 5000rpm for 10min, washing twice by combining with a Buffer, and then placing the thalli at-80 ℃ for storage or directly crushing for purifying protein.
The collected bacteria are dissolved again by Binding buffer (50mM Tris-HCl, pH 7.4, 200mM NaCl, 1mM EDTA, 25mM imidazole), cells are crushed by a high-pressure homogenizer, the cells are crushed by 15000rpm high-speed low-temperature centrifugation for 60min, supernatant is taken and transferred to a 50mL centrifuge tube, 1mL nickel beads (Ni-reset) are taken from an elution tube, 10mL MiliQ is firstly used for eluting the nickel beads, 5mL Binding buffer is then used for eluting, the centrifuged supernatant is added, protein with His affinity label is combined on a Ni column by natural flow rate elution, after the elution is repeated twice, Washing buffer (20 mM-HCl, pH 7.4, 200mM NaCl) containing 50-300mM imidazole is used for elution, the dosage of imidazole with each concentration is 2 times of column volume, and eluent is collected.
After elution, SDS-PAGE gel is run to detect whether target protein exists in each collection tube, the solution containing the target protein is dialyzed by using stripe buffer (20mM Tris-HCl, pH 7.4,100mM NaCl and 5% glycerol), concentration is carried out, the concentrated protein solution is divided into 20-50 mu L after concentration is measured, and the concentrated protein solution is frozen and stored at minus 80 ℃.
As can be seen from the SDS-PAGE result shown in FIG. 1, the purified OfT8GT1 and OfT8GT3 proteins have higher purity.
Example 4 in vitro enzymatic reaction
OfT8GT1 or OfT8GT3 (100 μ L): 50mM Tris-HCl (pH 7.4), 2mM UDPG, 1mM Hydroxytyrosol (Hydroxypyrosol), 2mM ascorbic acid and 5. mu.M purified enzyme OfT8GT1 or OfT8GT 3. The enzymatic reaction is carried out in a water bath kettle at 30 ℃, and after the reaction is carried out for 1h, 100 mu L of ice methanol is added to quench the reaction.
The quenched reaction product was centrifuged at 13,000rpm for 30min, and the supernatant was filtered through a 0.22 μ L filter and placed in a sample vial with an lined tube for subsequent HPLC or LC-MS detection.
Detection conditions are as follows: the column was a reversed phase C18 column (150X 4.6mm, Agilent), mobile phase A was water (0.1% formic acid), mobile phase B was acetonitrile (0.1% formic acid), flow rate was 0.8mL/min, 10. mu.L of sample was injected, detection wavelength was set at 280nm, elution conditions were:
0-5min:B 5％-10％；
5-15min:B 10-20％；
15-17min:B 20-80％；
17-25min:B 5％。
from the results of HPLC, it can be seen that the reaction products of the OfT8GT1 and OfT8GT3 reaction systems all produced product peaks with the same retention time (fig. 2).
And (3) structural identification: the reaction products shown after HPLC detection were analyzed by LC-HR-MS using a Thermo Scientific LTQ XL Orbitrap as a platform, ESI ion source, using a chromatographic column and mobile phase conditions identical to those of HPLC detection.
MS detection conditions were used of Heated ESI Temperature 40 ℃, Sneath Gas Flow Rate 30arb, Aux Gas Flow Rate5 arb, I spread valve 3.5kV, Capillary Temperature 270 ℃, Capillary valve 35V, Tube Lens 110V.
From the LC-HR-MS results, it can be seen that the reaction product of the OfT8GT1, OfT8GT3 reaction system produced characteristic ion 317.12309, and the secondary mass spectrum fragment of the molecular ion was consistent with the hydroxy salidroside (fig. 3).
To further confirm the glycosylation and hydroxylation sites of the products of the OfT8GT1, OfT8GT3 reaction systems, the products were isolated in large quantities and subjected to one-dimensional and two-dimensional nuclear magnetic characterization. From the results of one-dimensional and two-dimensional NMR detection, the structures of the products of the OfT8GT1 and OfT8GT3 reaction systems are determined to be consistent with those of salidroside (fig. 4-7).
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Hubei carbon element and herbal Biotech Co., Ltd
<120> glycosyltransferase for catalytic synthesis of hydroxysalidroside, and coding gene and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 477
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Gly Ser Ile Ala Gly Asn Asp Lys Pro His Ala Val Cys Ile Pro
1 5 10 15
Tyr Pro Ala Gln Gly His Ile Asn Pro Met Phe Asn Leu Ala Lys Leu
20 25 30
Leu His His Gln Gly Phe Tyr Ile Thr Phe Val Asn Thr Glu Phe Asn
35 40 45
His Lys Arg Leu Leu Lys Ser Arg Gly Pro Ala Ala Leu Asp Gly Leu
50 55 60
Pro Asp Phe Arg Phe Glu Thr Ile Pro Asp Gly Leu Pro Ser Ser Asp
65 70 75 80
Gly Asp Ala Thr Gln Asp Ile Pro Ser Leu Cys Glu Ser Thr Thr Lys
85 90 95
Thr Cys Leu Ala Pro Phe Cys Ser Leu Leu Thr Lys Leu Asn Asn Ala
100 105 110
Ala Pro Val Met Pro Pro Val Thr Cys Ile Val Ser Asp Gly Ala Met
115 120 125
Ser Phe Thr Leu Lys Ala Ala Glu Gln Phe Gly Leu Pro Glu Val Leu
130 135 140
Leu Trp Thr Thr Ser Ala Cys Gly Phe Leu Gly Tyr Thr Gln Tyr Ala
145 150 155 160
Asn Leu Ile Glu Arg Gly Tyr Thr Pro Leu Lys Glu Met Ser Gln Val
165 170 175
Thr Asn Gly Tyr Leu Glu Thr Thr Ile Asp Trp Ile Pro Gly Met Lys
180 185 190
Asp Ile Arg Leu Arg Asp Leu Pro Thr Phe Leu Arg Thr Thr Asp Ser
195 200 205
Asn Asp Ile Met Leu Asn Phe Val Leu Arg Glu Ile Glu Ala Val Pro
210 215 220
Lys Ala Lys Ala Ile Ile Leu Asn Thr Phe Asp Ala Leu Glu His Asp
225 230 235 240
Val Leu Asp Ala Leu Ser Ser Met Asn Pro Val Ile Tyr Ser Val Gly
245 250 255
Pro Leu Gln Leu Met Met Asn His Ile Gln Asn Glu Lys His Thr Leu
260 265 270
Thr Ser Ser Ser Leu Trp Lys Glu Glu Leu Glu Cys Ile Lys Trp Leu
275 280 285
Asp Lys Lys Glu Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser Ile
290 295 300
Ala Val Val Thr Ala Gln Gln Leu Thr Glu Phe Ala Trp Gly Leu Ala
305 310 315 320
Asn Ser Lys Lys Ser Phe Leu Trp Ile Ile Arg Pro Asp Leu Val Ala
325 330 335
Gly Asp Leu Ala Met Leu Pro Pro Glu Phe Val Thr Glu Thr Glu Asp
340 345 350
Arg Ser Met Leu Ile Ser Trp Cys Pro Gln Glu Glu Val Leu Lys His
355 360 365
Pro Ala Ile Gly Gly Phe Leu Thr His Ser Gly Trp Asn Ser Thr Ile
370 375 380
Glu Ser Ile Val Ala Gly Val Pro Leu Ile Cys Trp Pro Phe Phe Ala
385 390 395 400
Glu Gln Gln Thr Asn Cys Arg Tyr Ser Cys Val Glu Trp Gly Met Gly
405 410 415
Met Glu Ile Asp Asn Asn Val Lys Arg Asp Glu Val Glu Glu Leu Val
420 425 430
Lys Glu Leu Met Asp Gly Glu Lys Gly Met Lys Met Lys Glu Lys Ala
435 440 445
Met Glu Trp Lys Lys Lys Ala Glu Glu Ala Thr Ala Pro Gly Gly Ser
450 455 460
Ser Phe Met Asn Phe Glu Glu Leu Val Lys Leu Leu Gln
465 470 475
<210> 2
<211> 485
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Gly Ser Ile Ser Leu Leu Glu Lys Pro His Val Val Cys Ile Pro
1 5 10 15
Phe Pro Ala Gln Gly His Ile Asn Pro Met Leu Lys Leu Ala Lys Leu
20 25 30
Leu His Gln Lys Gly Phe His Val Thr Phe Val Asn Thr Glu Phe Asn
35 40 45
His Gly Arg Leu Leu Lys Ser Arg Gly Pro Asp Ser Leu Lys Gly Leu
50 55 60
Ala Ser Phe Arg Phe Glu Thr Ile Pro Asp Gly Leu Pro Ile Ser Asp
65 70 75 80
Ala Asp Ala Thr Gln Asp Ile Ala Ser Leu Cys Glu Ser Thr Thr Asn
85 90 95
Thr Cys Leu Val Pro Phe Lys Asp Leu Leu Ala Arg Leu Asn Asp Thr
100 105 110
Ile Ser Ser Asn Val Pro Pro Val Ser Cys Ile Ile Ser Asp Gly Val
115 120 125
Met Ser Phe Thr Leu Glu Ala Ala Glu Glu Leu Arg Ile Pro Glu Val
130 135 140
Leu Leu Trp Thr Thr Ser Ala Cys Gly Phe Leu Ala Tyr Thr Gln Tyr
145 150 155 160
Ala Lys Leu Ile Glu Glu Gly Tyr Val Pro Leu Lys Asp Ala Ser Tyr
165 170 175
Leu Thr Asn Gly Tyr Leu Asp Thr Val Ile Ala Asp Glu Ile Pro Gly
180 185 190
Met Glu Gly Met Arg Leu Lys Asp Ile Pro Ser Phe Ile Arg Thr Thr
195 200 205
Ser Leu Asp Asp Tyr Met Val Lys Phe Val Leu Gln Glu Thr Glu Arg
210 215 220
Ala Arg Lys Ala Ser Ala Ile Ile Leu Asn Thr Phe Glu Asp Leu Glu
225 230 235 240
His Asp Cys Val Lys Thr Leu Ser Ser Ile Leu Pro Pro Val Tyr Ala
245 250 255
Ile Gly Pro Leu His Phe Leu Gln Lys Asp Val Lys Asp Lys Ser Leu
260 265 270
Glu Ile Leu Gly Ser Asn Leu Trp Lys Glu Asp Gln His Cys Leu Glu
275 280 285
Trp Leu Asp Ser Lys Glu Pro Lys Ser Val Val Tyr Ile Asn Phe Gly
290 295 300
Ser Ile Thr Val Met Thr Pro Lys Gln Leu Ile Glu Phe Ala Trp Gly
305 310 315 320
Leu Ala Asn Ser Gly Gln Thr Phe Leu Trp Ile Ile Arg Pro Asp Leu
325 330 335
Val Thr Gly Asp Asp Ala Ile Phe Pro Pro Glu Phe Leu Glu Ala Thr
340 345 350
Lys Glu Arg Ser Leu Leu Ala Asn Trp Cys Pro Gln Glu Lys Val Leu
355 360 365
Ser His Pro Ser Ile Gly Gly Phe Leu Thr His Ser Gly Trp Asn Ser
370 375 380
Thr Leu Glu Ser Ile Gly Ser Gly Val Pro Met Ile Cys Trp Pro Phe
385 390 395 400
Phe Ala Glu Gln Gln Thr Asn Cys Trp Phe Ser Cys Thr Lys Trp Gly
405 410 415
Ile Gly Met Glu Ile Asp Ser Asp Val Lys Arg Asp Glu Val Glu Ile
420 425 430
Leu Val Lys Glu Leu Met Ile Gly Glu Lys Gly Lys Glu Met Lys Lys
435 440 445
Arg Ala Leu Glu Trp Met Lys Leu Ala Lys Asp Ser Ala Lys Asn Ser
450 455 460
Glu Gly Ser Ser His Lys Asn Leu Glu Lys Leu Ile Asn Gln Val Leu
465 470 475 480
Leu Cys Thr Lys Leu
485
<210> 3
<211> 1434
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgggtagca ttgcgggtaa cgacaaaccg catgcggttt gcatcccgta cccggcgcag 60
ggtcacatta acccgatgtt caacctggcg aaactgctgc accaccaagg cttctatatc 120
acctttgtga acaccgagtt taaccacaag cgtctgctga aaagccgtgg tccggcggcg 180
ctggatggcc tgccggattt ccgttttgaa accattccgg atggtctgcc gagcagcgat 240
ggtgatgcga cccaggatat tccgagcctg tgcgaaagca ccaccaagac ctgcctggcg 300
ccgttctgca gcctgctgac caaactgaac aacgcggcgc cggtgatgcc gccagttacc 360
tgcatcgtga gcgatggtgc gatgagcttc accctgaagg cggcggagca atttggcctg 420
ccggaagttc tgctgtggac caccagcgcg tgcggttttc tgggctacac ccagtatgcg 480
aacctgatcg agcgtggtta caccccgctg aaggaaatga gccaagtgac caacggttat 540
ctggaaacca ccatcgactg gattccgggc atgaaagaca ttcgtctgcg tgatctgccg 600
accttcctgc gtaccaccga cagcaacgat atcatgctga actttgttct gcgtgagatt 660
gaagcggtgc cgaaagcgaa ggcgatcatt ctgaacacct tcgatgcgct ggagcacgac 720
gttctggatg cgctgagcag catgaacccg gttatctaca gcgtgggtcc gctgcagctg 780
atgatgaacc acattcaaaa cgaaaagcac accctgacca gcagcagcct gtggaaagag 840
gagctggagt gcatcaaatg gctggacaag aaagaaccga acagcgtggt ttatgtgaac 900
ttcggtagca ttgcggtggt taccgcgcag caactgaccg agttcgcgtg gggcctggcg 960
aacagcaaga aaagctttct gtggatcatt cgtccggacc tggttgcggg cgatctggcg 1020
atgctgccgc cggaattcgt gaccgaaacc gaagatcgta gcatgctgat cagctggtgc 1080
ccgcaggaag aggtgctgaa acacccggcg attggtggct ttctgaccca cagcggttgg 1140
aacagcacca tcgagagcat tgttgcgggc gttccgctga tctgctggcc gttctttgcg 1200
gaacagcaaa ccaactgccg ttacagctgc gttgagtggg gtatgggcat ggaaattgac 1260
aacaacgtga agcgtgatga ggttgaggaa ctggtgaagg agctgatgga cggtgaaaaa 1320
ggcatgaaaa tgaaggagaa agcgatggaa tggaagaaaa aggcggagga agcgaccgcg 1380
ccgggtggca gcagcttcat gaactttgag gaactggtga aactgctgca atga 1434
<210> 4
<211> 1458
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgggtagca tcagcctgct ggagaagccg cacgtggttt gcatcccgtt cccggcgcag 60
ggccacatta acccgatgct gaagctggcg aaactgctgc accaaaaagg tttccacgtg 120
acctttgtta acaccgaatt taaccacggc cgtctgctga agagccgtgg tccggacagc 180
ctgaaaggcc tggcgagctt ccgttttgaa accatcccgg acggtctgcc gattagcgac 240
gcggatgcga cccaggatat cgcgagcctg tgcgaaagca ccaccaacac ctgcctggtg 300
ccgttcaagg acctgctggc gcgtctgaac gataccatta gcagcaacgt gccgccggtt 360
agctgcatca ttagcgatgg tgtgatgagc ttcaccctgg aagcggcgga ggaactgcgt 420
atcccggaag tgctgctgtg gaccaccagc gcgtgcggct ttctggcgta cacccagtat 480
gcgaagctga ttgaggaagg ttacgtgccg ctgaaagacg cgagctacct gaccaacggc 540
tatctggaca ccgttatcgc ggatgagatt ccgggtatgg aaggcatgcg tctgaaggat 600
atcccgagct tcattcgtac caccagcctg gacgattaca tggtgaagtt tgttctgcaa 660
gaaaccgaac gtgcgcgtaa agcgagcgcg atcattctga acaccttcga ggacctggaa 720
cacgattgcg tgaaaaccct gagcagcatc ctgccgccgg tttatgcgat tggtccgctg 780
cactttctgc agaaggacgt taaggataaa agcctggaga tcctgggcag caacctgtgg 840
aaagaggacc aacactgcct ggaatggctg gatagcaagg agccgaaaag cgtggtttat 900
atcaacttcg gtagcattac cgtgatgacc ccgaagcagc tgatcgaatt cgcgtggggt 960
ctggcgaaca gcggccaaac ctttctgtgg atcattcgtc cggacctggt taccggcgac 1020
gatgcgattt tcccgccgga gtttctggaa gcgaccaagg aacgtagcct gctggcgaac 1080
tggtgcccgc aggagaaagt gctgagccac ccgagcattg gtggcttcct gacccacagc 1140
ggttggaaca gcaccctgga aagcatcggt agcggcgttc cgatgatttg ctggccgttc 1200
tttgcggagc agcaaaccaa ctgctggttt agctgcacca aatggggtat cggcatggaa 1260
attgacagcg atgtgaagcg tgacgaggtg gaaatcctgg ttaaagagct gatgattggc 1320
gagaagggca aagaaatgaa gaaacgtgcg ctggaatgga tgaagctggc gaaagatagc 1380
gcgaagaaca gcgaaggcag cagccacaag aacctggaga aactgatcaa ccaagttctg 1440
ctgtgcacca aactgtga 1458
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctttaagaag gagatatacc atgggtagca ttgcgggta 39
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tggtgctcga gtgcggccgc ttgcagcagt ttcaccagt 39
<210> 7
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctttaagaag gagatatacc atgggtagca tcagcctgct 40
<210> 8
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggtgctcga gtgcggccgc cagtttggtg cacagcaga 39

Claims (9)

1. A glycosyl transferase for catalyzing hydroxytyrosol to generate hydroxytryptaside is a protein composed of amino acid sequences shown in SEQ ID NO.1 or SEQ ID NO. 2.
2. The coding gene of the glycosyl transferase of claim 1 catalyzing hydroxytyrosol to produce salidroside, wherein the gene is a DNA molecule with the sequence as shown in SEQ ID NO.3 or SEQ ID NO. 4.
3. An expression cassette, a recombinant vector or a recombinant bacterium comprising a gene encoding the glycosyltransferase of claim 2 catalyzing hydroxytyrosol to produce hydroxytyrosol.
4. A recombinant Escherichia coli comprising the gene encoding the glycosyltransferase of claim 2 which catalyzes the production of hydroxytyrosol to form hydroxytryptoroside.
5. The use of the glycosyltransferase of claim 1 that catalyzes the production of hydroxytyrosol to produce hydroxytyroside in an in vitro process for the production of hydroxytyrosol to produce hydroxytyroside.
6. A synthetic method of hydroxyl salidroside is characterized by comprising the following steps: adding substrates hydroxytyrosol, UDPG and the glycosyltransferase of claim 1 into the buffer solution respectively to obtain mixed solution; and (3) reacting the mixed solution at 25-35 ℃, and adding ice methanol to quench the reaction.
7. The method for synthesizing salidroside according to claim 6, wherein said buffer is Tris-HCl buffer at pH 7.4.
8. The method for synthesizing hydroxysalidroside according to claim 7, wherein said mixture further comprises ascorbic acid.
9. The method for synthesizing hydroxysalidroside according to any one of claims 6 to 8, wherein the concentration of hydroxytyrosol, UDPG and glycosyltransferase in the mixture is 1 mM: 2 mM: 5 μ M.
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