CN114606212A - Conus clematis-derived coumarin synthase, gene, vector and application thereof - Google Patents
Conus clematis-derived coumarin synthase, gene, vector and application thereof Download PDFInfo
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- CN114606212A CN114606212A CN202210312834.0A CN202210312834A CN114606212A CN 114606212 A CN114606212 A CN 114606212A CN 202210312834 A CN202210312834 A CN 202210312834A CN 114606212 A CN114606212 A CN 114606212A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a clematis terniflora-derived coumarin synthase, a gene, a vector and application thereof, and belongs to the field of genetic engineering. The amino acid sequence of the clematis terniflora coumarin synthase CtCOSY is shown in SEQ ID No. 1. The invention obtains the CtCOSY gene from clematis terniflora by screening, the base sequence is shown in SEQ ID NO.2, and the recombinant escherichia coli expressing the enzyme is constructed, has the capability of catalyzing 2, 4-dihydroxycinnamic acid to synthesize umbelliferone together with 4-coumaric acid coenzyme A, and has important significance for promoting the application of high-yield coumarin in industry.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to clematis terniflora coumarin synthase and a gene, and also relates to a recombinant expression vector containing the gene and application of the recombinant expression vector.
Background
Clematis terniflora DC is a traditional Chinese medicinal material distributed in Asian countries such as China, Japan, Korea and the like, and coumarin is one of main medicinal components, is used for resisting diseases such as inflammation and hypertension and has high medicinal value. An important cyclozyme is needed in the synthesis process of coumarin: coumarin synthase (COSY), belonging to BAHD acyltransferase family, participates in phenylpropane synthesis pathway, and can catalyze cis-trans isomerization and lactonization of 2-hydroxy-p-coumaroyl-CoA to generate simple coumarin umbelliferone. The umbelliferone has anti-inflammatory effect and certain function of preventing liver fibrosis, and is used as the basic structure of coumarin compound to form the core structure of complex coumarin compound.
According to previous reports, researchers only represent coumarin synthase in model plant Arabidopsis thaliana, and research key enzyme on a synthesis path, namely coumarin synthase, which is novel and efficient in catalyzing spontaneous isomerization and lactonization of coumarin in other medicinal plants has important theoretical and application significance.
The clematis terniflora is used as a traditional medicinal material and has high medicinal value, and the coumarin synthase participates in a coumarin synthesis way and is one of important enzymes for improving the yield of coumarin. Therefore, the deep research on the coumarin synthase can deepen the understanding on the synthetic pathway of the medicinal component coumarin, improve the yield of the coumarin and solve the problem of low effective components in medicinal plants.
Disclosure of Invention
The invention aims to provide a potential coumarin synthase (CtCOSY) from clematis terniflora, which is (a) or (b):
(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) and (b) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a) and having coumarin synthase activity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides clematis terniflora-derived coumarin synthase, which is specifically (a) or (b), wherein:
(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) and (b) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a) and having coumarin synthase activity.
In a second aspect, the present invention provides a gene encoding the coumarin synthase described in the first aspect, wherein the base sequence of the gene is shown in SEQ ID No. 2.
In a third aspect, the present invention provides a recombinant expression vector obtained by inserting the gene of the second aspect into an expression vector.
Preferably, in the third aspect, the expression vector is a pET28a (+) plasmid.
In a fourth aspect, the present invention provides an E.coli comprising the recombinant expression vector of any one of the above third aspects.
As the fourth aspect, Escherichia coli using BL21(DE3) as the host bacteria.
In a fifth aspect, the present invention provides a method for producing coumarin synthase, specifically: inoculating the escherichia coli in the fourth aspect into a culture medium for overnight culture, expressing a gene encoding the coumarin synthase, collecting an expressed protein, and purifying to obtain the clematis terniflora-derived coumarin synthase.
Preferably, IPTG is added to the medium after inoculation of the Escherichia coli, and expression is induced overnight at 18-20 ℃.
Preferably, the collected expressed protein is purified by a nickel column to obtain a purified protein.
In a sixth aspect, the present invention provides a use of coumarin synthase produced by the method according to claim 7 for the catalytic production of umbelliferone.
The invention has the beneficial effects that: the invention provides a coumarin synthase gene CtCOSY cloned by taking clematis terniflora leaves as a biological source, and constructs recombinant escherichia coli expressing the enzyme. The gene can code and generate coumarin synthase, and the protein of the coumarin synthase is one of key enzymes in a coumarin metabolic pathway. The recombinant escherichia coli has the capability of catalyzing 2, 4-dihydroxycinnamic acid to synthesize umbelliferone together with 4-coumaric acid coenzyme A, has important significance for promoting the application of high-yield coumarin in industry, and can solve the problem of low effective components in medicinal plants.
Drawings
FIG. 1 is the PCR electrophoresis of clematis terniflora coumarin synthase CtCOSY gene.
FIG. 2 is an SDS-PAGE picture of coumarin synthase CtCOSY expressed and purified in Escherichia coli.
FIG. 3 is the HPLC chart of the enzyme activity reaction of coumarin synthase CtCOSY and negative control, and the mass spectrogram of the final product.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The technical characteristics in the embodiments of the present invention can be combined correspondingly without mutual conflict.
Example 1: obtaining of Ctccosy gene cDNA full-length sequence of clematis terniflora
Taking leaves of clematis terniflora, extracting total RNA, and performing reverse transcription to obtain cDNA. The extraction of total RNA was performed according to the instructions of the RNA extraction kit of the Huayuyang. Reverse transcription was performed according to the 5X All-In-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) instructions.
BLAST analysis is carried out according to the existing transcriptome data and the AtCOSY amino acid sequence in NCBI, a sequence with the highest similarity is selected, and an open reading frame of the sequence is used as a template to design a primer:
CtCOSY-F：5’-CACACACTCTAAAGTATCAGC-3’,
CtCOSY-R：5’-ACCGAGAACCTCACTTCAGTA-3’。
then using the Clematis terniflora leaf cDNA as a template
PCR amplification was performed with the Hot Start High-Fidelity 2X Master Mix in 50. mu.l:
after the amplification product is subjected to 1% agarose gel electrophoresis, the CtCOSY gene sequence is successfully cloned out, and the result is shown in figure 1.
The amplified product was subjected to gel cutting by an OMEGA-bio-tek gel recovery kit to recover a PCR product, which was then ligated to pClone007 vector and then transferred to E.coli DH 5. alpha. competent cells plated on LB solid medium containing 50mg/L ampicillin, and cultured overnight at 37 ℃. And (3) selecting a monoclonal colony, shaking the colony in an LB liquid culture medium containing the same antibiotics at 37 ℃ for 8h, and then sequencing to obtain a nucleotide sequence of the CtCOSY gene in the clematis terniflora leaf, wherein the nucleotide sequence is shown as SEQ ID NO. 2.
The CtCOSY gene can code coumarin synthase CtCOSY in clematis terniflora, wherein the coumarin synthase CtCOSY is a protein consisting of an amino acid sequence shown in SEQ ID No. 1. In the subsequent embodiment, the CtCOSY gene is used for coding coumarin synthase (CtCOSY) from clematis terniflora and catalytically synthesizing the coumarin compound umbelliferone.
Example 2: construction of recombinant plasmid
On the basis of the embodiment 1, according to the nucleotide sequence of the CtCOSY gene in the clematis terniflora leaves, a primer is designed, the CtCOSY is cloned into an escherichia coli expression vector pET28a (+), EcoRI and HindIII are selected as enzyme cutting sites, and the primer is designed, wherein the nucleotide sequence of the EcoRI I-F: 5'-CCGGAA TTCATGGAATTAGAAATCAA-3', Hind III-R: 5'-CCCAAGCTTTCAGTATTTGCTGAGAA-3' are provided. According to
A50. mu.l system of Hot Start High-Fidelity 2X Master Mix was used for PCR amplification. Then, gel recovery is carried out according to the instruction of an OMEGA-bio-tek gel recovery kit, the vector pET28a (+) and gel recovery products are subjected to double enzyme digestion by EcoR I and Hind III respectively, and the reaction system is as follows:
after reaction at 37 ℃ for 2 hours, gel recovery was performed after agarose gel electrophoresis, and then recombinant vector construction was performed using T4 DNA Lignase from Takara, in which the purified CtCOSY gene (shown in SEQ ID NO. 2) and pET28a (+) were cleaved according to 3: 1 (molar ratio) ligation was carried out overnight at 16 ℃ in the following ligation system:
conventionally transforming Escherichia coli competence DH5 alpha, coating on a plate containing kanamycin, culturing overnight at 37 ℃, inoculating a single colony to an LB culture medium containing kanamycin for culturing, and obtaining a recombinant plasmid pET28a-CtCOSY after sequencing identification.
The coding gene sequence of tobacco 4-coumaric acid coenzyme A ligase provided by NCBI database was synthesized and ligated to pET28a (+) vector to obtain recombinant plasmid pET28a-Nt4 CL.
Example 3: inducible expression and purification of recombinant proteins
On the basis of example 2, synthesized recombinant plasmids pET28a-CtCOSY, pET28a-Nt4CL and pET28a are transformed into competent Escherichia coli BL21(DE3) in a conventional way, and cultured overnight at 37 ℃, and after colony sequencing is correctly selected, BL-pET28a-CtCOSY, BL-pET28a-Nt4CL and BL-pET28a are obtained. The three recombinant plasmids contained in Escherichia coli were inoculated into LB medium containing 50mg/L of mycin at a ratio of 1:100, cultured at 37 ℃ until OD600 of the bacterial solution became 0.4-1.0, IPTG was added to the bacterial solution to a final concentration of 0.1-0.8mM, and cultured overnight at 18-20 ℃. The subsequent steps were all performed at 4 ℃ and the cells were collected by centrifugation, then dissolved in a resuspension solution (20mM HEPES,500mM NaCl,20mM imidazole, 1mM PMSF, pH 7.5), sonicated and centrifuged at 14,000g for 20 min. The supernatant was purified using a HisTrap column, washed with an eluent containing 20mM HEPES pH 7.5, 500mM NaCl, 400mM imidazole, and the eluted protein was solubilized with 100mM Tris pH 7.4. Protein concentration was determined by the Bradford method, and the purified protein was subjected to SDS-PAGE, and the results are shown in FIG. 2. The protein is coumarin synthase CtCOSY from clematis terniflora, and the amino acid sequence of the protein is shown in SEQ ID No. 1.
Example 4: establishment of in vitro enzyme activity reaction for generating umbelliferone based on coumarin synthase
On the basis of example 3, an in vitro enzyme-activated reaction system of coumarin synthase containing Tris-HCl pH 7.4100mM, MgCl2 pH 5mM, ATP 5mM, CoA 400. mu.M, 2-Hydroxy-p-coumaric acid 800. mu.M was designed, and different proteins were added as different reaction systems: 1) 30. mu.g Nt4CL1 and 30. mu.g CtCOSY protein were added as a mixed system, 2) 30. mu.g Nt4CL as control 1, 3) 30. mu.g pET protein as control 2. The reaction was carried out at room temperature for 2h in the absence of light, then stopped with an equal volume of methanol, centrifuged at 14000g for 10min and the supernatant removed.
Example 5: detection of umbelliferone
The results were analyzed by HPLC on the basis of example 4, on a Waters 2695 Alliance HPLC system using a C18 column (250 mm. times.4.6 mm inner diameter, 5mm) at a column temperature of 35 ℃ and a flow rate of 1.0ml min-1. Mobile phase: buffer a (containing 1% acetonitrile and 0.5% ammonium acetate, pH 7), buffer B (containing 99% acetonitrile and 0.5% ammonium acetate, pH 7). Gradient elution: after 5 minutes equilibration with 90% buffer a, buffer a decreased from a 90% gradient to 55% in 15 minutes, then the system held buffer a 55% for 5 minutes, then buffer a increased from a 55% gradient to 90% in 5 minutes and held for 5 minutes. The spectra were measured at a wavelength of 324 nm. The flow rate of LC-MS/MS was 1ml min-1 and the temperature was 35 ℃. The solvent system was the same as the HPLC method, buffer A was changed to 0.1% ammonium acetate and buffer B was acetonitrile. MS/MS spectra were measured using PeakView. V1.6 software. The results are shown in fig. 3, and the comparison with the mass spectrum of umbelliferone proves that the synthesized coumarin synthase CtCOSY can catalyze the production of umbelliferone, which is consistent with the expected results.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Sequence listing
<110> institute of basic medicine and tumor (research) of Chinese academy of sciences
<120> clematis terniflora-derived coumarin synthase, gene, vector and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 444
<212> PRT
<213> Clematis terniflora DC)
<400> 1
Met Glu Leu Glu Ile Lys Gln Thr Ile Leu Ile Ser Pro Ala Ser Pro
1 5 10 15
Pro Phe Asp His Asp His Ile Leu Ser Leu Ser His Leu Asp Asn Asp
20 25 30
Arg Asn Val Gln Val Asn Phe Arg Tyr Val Arg Ala Tyr Ala Asn Thr
35 40 45
Thr Thr Glu Thr Ala Ser Ser Ser Asp Pro Val His Val Ile Ser Glu
50 55 60
Ala Leu Gly Lys Ala Leu Val His Tyr Tyr Pro Phe Ala Gly Thr Leu
65 70 75 80
Arg Arg Arg Leu His Asp His Arg Leu Glu Leu Phe Cys Ala Ala Gly
85 90 95
Gln Thr Ala Pro Leu Ile Ser Ala Gly Val Asn Arg Thr Leu Asp Ser
100 105 110
Val Asn Tyr Leu Asp Asn Pro Ala Asp Pro Phe Leu Glu Gln Leu Val
115 120 125
Pro Asn Pro Asn Pro Asp Asp Ser Leu Leu His Pro Phe Val Leu Gln
130 135 140
Val Thr Val Phe Gln Cys Gly Gly Phe Thr Leu Gly Ala Ser Ile His
145 150 155 160
His Ser Met Cys Asp Gly Leu Gly Ser Thr Gln Phe Phe Asn Val Met
165 170 175
Ala Glu Phe Ala Arg Gly Gly Thr Gln Pro Ser Val Gln Ala Val Trp
180 185 190
Asn Arg Ser Ser Leu Leu Gly Pro Arg Asp Pro Pro Arg Val Glu Val
195 200 205
Pro Phe His Glu Phe Leu Gly Leu Asp Lys Glu Phe Ser Pro Tyr Ser
210 215 220
Arg Ser Ser Glu Asp Val Val Arg Glu Cys Phe Asp Val Lys Asp Gly
225 230 235 240
Trp Val Glu Arg Phe Lys Ala Ala Leu Lys Glu Glu Ser Gly Met Ser
245 250 255
Phe Thr Thr Phe Glu Ala Leu Gly Ala Phe Ile Trp Arg Ala Arg Val
260 265 270
Lys Ala Cys Lys Leu Pro Glu Asp Glu Lys Val Lys Phe Ala Tyr Ser
275 280 285
Ile Asn Ile Arg Arg Ile Leu Lys Pro Ala Leu Pro Phe Gly Tyr Trp
290 295 300
Gly Asn Gly Cys Val Thr Met Tyr Ala Gln Ala Ser Ala Lys Glu Leu
305 310 315 320
Val Glu Gln Pro Leu Trp Lys Thr Ala Glu Leu Ile Asn Lys Ser Lys
325 330 335
Arg Asn Ala Thr Asp Glu Tyr Val Arg Ser Phe Ile Asp Phe Gln Glu
340 345 350
Leu His Tyr Ala Glu Gly Ile Thr Ala Gly Lys Glu Val Ser Gly Phe
355 360 365
Thr Asp Trp Arg His Leu Gly His Ser Thr Val Asp Phe Gly Trp Gly
370 375 380
Gly Pro Val Thr Val Leu Pro Leu Ser Arg His Leu Leu Gly Ser Ser
385 390 395 400
Glu Ile Cys Phe Phe Leu Pro Tyr Ser Ser Val Ser Gln Gly Lys Lys
405 410 415
Asp Gly Phe Lys Val Leu Val Ser Leu Pro Gln Asn Ala Leu Pro Ala
420 425 430
Phe Lys Val Asp Met Glu Asn Phe Leu Ser Lys Tyr
435 440
<210> 2
<211> 1335
<212> DNA
<213> Clematis terniflora DC)
<400> 2
atggaattag aaatcaaaca aacaatcctc atctcccctg cttctccgcc gttcgatcac 60
gaccacatcc tatctctctc tcacctagac aacgaccgta atgttcaagt caatttccgt 120
tacgtccggg cgtatgctaa cactaccact gaaaccgcca gctcatcgga tcccgtccac 180
gtcatctccg aagccctcgg caaagctctc gttcattact accctttcgc cgggactctc 240
cgtcgccgct tacatgatca tcggctcgag ctcttctgcg ctgctggtca aactgcgccg 300
ttaatatccg ctggagtgaa tcgtacactt gactcagtga attatcttga taacccggcc 360
gatcctttcc tcgaacagtt ggtgcctaat cctaacccgg acgattctct actccacccc 420
ttcgttctcc aagtgacagt atttcaatgc ggagggttca cactcggtgc atcgattcac 480
cactcgatgt gcgatggact cgggtcgact cagttcttta atgtgatggc tgagttcgca 540
cgtggaggaa ctcagccgtc tgttcaagct gtgtggaatc gttcgagtct gctcggaccg 600
agagatccgc cacgtgttga ggtgccgttt catgagtttc ttggcttaga taaagagttt 660
tcgccgtatt caaggtcgag tgaggatgtg gttcgtgaat gctttgatgt gaaggatggg 720
tgggtggagc ggttcaaggc ggctttgaag gaggaatctg ggatgagctt tactaccttt 780
gaagcgttgg gtgctttcat atggcgggcc agagtgaagg cttgtaagtt acccgaagat 840
gagaaagtga agtttgcata ctcaatcaat attcgaagga tacttaagcc agcactcccc 900
tttggttact ggggcaatgg ttgtgtgacg atgtatgctc aagccagtgc gaaagagttg 960
gtggagcaac ctctatggaa aacagccgaa cttataaaca agagcaagcg taacgctacc 1020
gatgaatatg tgagatcatt tattgacttt caagaattac attatgctga ggggatcaca 1080
gcggggaaag aagtaagtgg gttcacagat tggagacatc ttggtcactc gactgtggac 1140
tttggatggg gaggtcctgt gactgtgttg cccttatcgc gccacctact tggcagtagt 1200
gaaatttgtt tcttcttacc ttattcttct gtgagccagg gaaagaaaga tggatttaag 1260
gttttggtgt cattgcctca gaatgcatta cctgctttca aggttgatat ggagaacttt 1320
ctcagcaaat actga 1335
Claims (10)
1. A clematis terniflora-derived coumarin synthase characterized by being (a) or (b), wherein:
(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) and (b) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a) and having coumarin synthase activity.
2. A gene encoding the coumarin synthase according to claim 1, characterized in that the base sequence is as shown in SEQ ID No. 2.
3. A recombinant expression vector obtained by inserting the gene according to claim 2 into an expression vector.
4. The recombinant expression vector of claim 3, wherein the expression vector is the pET28a (+) plasmid.
5. An Escherichia coli comprising the recombinant expression vector of claim 3 or 4.
6. Escherichia coli according to claim 5, wherein BL21(DE3) is used as host bacterium.
7. A method for producing coumarin synthase, characterized in that Escherichia coli as claimed in claim 6 is inoculated into a culture medium and cultured overnight, the gene encoding the coumarin synthase is expressed, and the coumarin synthase derived from Clematis terniflora is obtained after collection and purification of the expressed protein.
8. The method according to claim 7, wherein IPTG is added to the medium after inoculation of the E.coli, and expression is induced overnight at 18-20 ℃.
9. The method of claim 7, wherein the collected expressed protein is purified by a nickel column to obtain a purified protein.
10. Use of coumarin synthase produced according to the method of claim 7 for the catalytic production of umbelliferone.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732434A (en) * | 2009-11-05 | 2010-06-16 | 浙江大学 | Method for inducing increase in varierty and content of secondary metabolite in clematis terniflora (medicinal plant) |
| CN112725256A (en) * | 2021-02-22 | 2021-04-30 | 湖南省农产品加工研究所 | Recombinant escherichia coli and method for biosynthesizing diosmetin by using recombinant escherichia coli |
| CN112877349A (en) * | 2021-01-25 | 2021-06-01 | 南京林业大学 | Recombinant expression vector, genetic engineering bacterium containing recombinant expression vector and application of recombinant expression vector |
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2022
- 2022-03-28 CN CN202210312834.0A patent/CN114606212B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732434A (en) * | 2009-11-05 | 2010-06-16 | 浙江大学 | Method for inducing increase in varierty and content of secondary metabolite in clematis terniflora (medicinal plant) |
| CN112877349A (en) * | 2021-01-25 | 2021-06-01 | 南京林业大学 | Recombinant expression vector, genetic engineering bacterium containing recombinant expression vector and application of recombinant expression vector |
| CN112725256A (en) * | 2021-02-22 | 2021-04-30 | 湖南省农产品加工研究所 | Recombinant escherichia coli and method for biosynthesizing diosmetin by using recombinant escherichia coli |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "ACCESSION NO. VERSION KAF9609964.1", GENBANK * |
| GENBANK: "ACCESSION NO. VERSION PIA30537.1", GENBANK * |
| 张娟;刘林德;王蕾;魏丽娟;: "天然香豆素类物质的利用和研究进展", 鲁东大学学报(自然科学版), no. 03 * |
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