CN114606212B

CN114606212B - Coumarin synthase from clematis terniflora, gene, vector and application thereof

Info

Publication number: CN114606212B
Application number: CN202210312834.0A
Authority: CN
Inventors: 朱玮; 陶明磊; 韩昊特; 田景奎; 李瑶函; 李守信; 刘胜志
Original assignee: Institute Of Basic Medicine And Oncology Chinese Academy Of Sciences Preparatory
Current assignee: Institute Of Basic Medicine And Oncology Chinese Academy Of Sciences Preparatory
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2023-06-30
Anticipated expiration: 2042-03-28
Also published as: CN114606212A

Abstract

The invention discloses coumarin synthase from clematis terniflora, genes, vectors and application thereof, and belongs to the field of genetic engineering. The amino acid sequence of the clematis terniflora coumarin synthase CtCOSY is shown as SEQ ID NO. 1. The CtCOSY gene derived from clematis terniflora is obtained through screening, the base sequence is shown as SEQ ID NO.2, recombinant escherichia coli expressing the enzyme is constructed, the recombinant escherichia coli has the capacity of catalyzing 2, 4-dihydroxycinnamic acid to synthesize umbelliferone together with 4-coumaric acid coenzyme A, and the recombinant escherichia coli has important significance in promoting the application of high-yield coumarin in industry.

Description

Coumarin synthase from clematis terniflora, gene, vector and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a clematis ternatus coumarin synthase and a gene, and a recombinant expression vector containing the gene and application thereof.

Background

The clematis terniflora (Clematis terniflora DC.) is a traditional Chinese medicinal material distributed in some Asian countries such as China, japan, korea and the like, coumarin is one of main medicinal components of the clematis ternate, is used for resisting diseases such as inflammation, hypertension and the like, and has higher medicinal value. An important ring synthase is required in the coumarin synthesis process: coumarin synthase (coumarin synthase, COSY), which belongs to the BAHD acyltransferase family, participates in the phenylpropane synthesis pathway, and is capable of catalyzing cis-trans isomerism and lactonization of 2-hydroxy-p-coumaroyl-CoA to produce simple coumarin umbelliferone. The umbelliferone has anti-inflammatory effect, has a certain function for preventing liver fibrosis, and is a core structure for forming a complex coumarin compound as a basic structure of the coumarin compound.

According to the previous report, researchers only characterize coumarin synthase in the Arabidopsis thaliana which is a model plant, but research on novel and high-efficiency coumarin synthase which is a key enzyme on a synthesis path for 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, has extremely high medicinal value, and coumarin synthase participates in a coumarin synthesis path, so that the clematis ternate is one of important enzymes for improving the coumarin yield. Therefore, the deep research on coumarin synthase can deepen the understanding of the synthetic pathway of the coumarin as a medicinal ingredient, improve the yield of the coumarin and solve the problem of lower effective ingredients in medicinal plants.

Disclosure of Invention

The invention aims to provide a potential coumarin synthase (coumarin synthase, ctCOSY) from clematis conical, which is (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;

(b) A protein derived from (a) having coumarin synthase activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a).

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the invention provides a coumarin synthase derived from clematis terniflora, in particular (a) or (b), wherein:

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;

In a second aspect, the invention provides a gene encoding the coumarin synthase of the first aspect, wherein the gene has a base sequence shown in SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant expression vector obtained by inserting the gene according to the second aspect into an expression vector.

As a preferred aspect of the above third aspect, the expression vector is pET28a (+) plasmid.

In a fourth aspect, the present invention provides E.coli comprising the recombinant expression vector according to any one of the above third aspects.

As a preferred embodiment of the fourth aspect, BL21 (DE 3) is used as the host bacterium.

In a fifth aspect, the invention provides a method for producing coumarin synthase, the method comprising: inoculating the escherichia coli in the fourth aspect into a culture medium for culturing overnight, expressing a gene encoding the coumarin synthase, collecting expressed proteins and purifying to obtain the coumarin synthase from the clematis terniflora.

As a preferable mode of the fifth aspect, IPTG is added to the medium after inoculating the E.coli, and the expression is induced at 18-20℃overnight.

Preferably, the collected expressed protein is purified by a nickel column to obtain a purified protein.

In a sixth aspect, the invention provides the use of coumarin synthase produced according to the method of claim 7 for the catalytic production of umbelliferone.

The invention has the beneficial effects that: the invention provides a method for cloning a biological source of clematis coni leaves to obtain a coumarin synthase gene CtCOSY, and constructing recombinant escherichia coli for expressing the enzyme. The gene can code and generate coumarin synthase, and the enzyme protein is one of key enzymes in 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 lower active ingredients in medicinal plants.

Drawings

FIG. 1 is a PCR electrophoresis diagram of the coumarin synthase CtCOSY gene of Clematis ternanthera.

FIG. 2 is a SDS-PAGE map of coumarin synthase CtCOSY after expression and purification in E.coli.

FIG. 3 is an HPLC chart of the enzyme activity of coumarin synthase CtCOSY and negative control, and mass spectrum of the final product.

Detailed Description

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.

Example 1: obtaining of full-length cDNA sequence of CtcCOSY gene of clematis terniflora

Taking the leaves of clematis terniflora, extracting total RNA, and reversely transcribing into cDNA. The extraction of total RNA was performed according to the instructions of the Wasabia RNA extraction kit. 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 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 cDNA of the clematis conica leaves as a template, using

PCR amplification was performed with Hot Start High-Fidelity 2X Master Mix, and the reaction system was 50. Mu.l:

after 1% agarose gel electrophoresis of the amplified product, the CtCOSY gene sequence was successfully cloned, and the result is shown in FIG. 1.

The amplified product was cut through an OMEGA-bio-tek gel recovery kit to recover the PCR product, followed by ligation of the recovered product onto pClone007 vector, followed by transfer into E.coli DH 5. Alpha. Competent cells, spread on LB solid medium containing 50mg/L ampicillin, and cultured overnight at 37 ℃. And (3) picking a monoclonal colony, vibrating in an LB liquid culture medium containing the same antibiotics at 37 ℃ for 8 hours, and sequencing to obtain the nucleotide sequence of the CtCOSY gene in the clematis ternatus leaves, wherein the nucleotide sequence is shown as SEQ ID No. 2.

The CtCOSY gene can code coumarin synthase CtCOSY in clematis ternatus, wherein the coumarin synthase CtCOSY is protein composed of an amino acid sequence shown in SEQ ID NO. 1. The subsequent examples will be directed to the catalytic synthesis of coumarin compounds umbelliferone using the CtCOSY gene to encode a coumarin synthase derived from clematis terniflora (coumarin synthase, ctCOSY).

Example 2: construction of recombinant plasmids

Based on example 1, primers were designed based on the nucleotide sequence of the CtCOSY gene in the leaf of Clematis Cone for use in preparing CtCOSY gramsE.coli expression vector pET28a (+) was cloned, ecoRI and HindIII were selected as cleavage sites, and primers EcoRI-F were designed: 5'-CCGGAA TTCATGGAATTAGAAATCAA-3' HindIII-R: 5'-CCCAAGCTTTCAGTATTTGCTGAGAA-3'. According to

PCR amplification was performed using a 50. Mu.l system from Hot Start High-Fidelity 2X Master Mix. Then, according to the specification of an OMEGA-bio-tek gel recovery kit, carrying out gel recovery, and respectively carrying out double digestion on a vector pET28a (+) and a gel recovery product by EcoRI and HindIII, wherein the reaction system is as follows:

after reaction at 37℃for 2 hours, gel recovery was performed after agarose gel electrophoresis, after which construction of recombinant vector was performed using T4 DNA Lignse of Takara, and the digested and purified CtCOSY gene (shown as SEQ ID NO. 2) and pET28a (+) were subjected to a procedure of 3:1 (molar ratio) at 16℃overnight connection, the connection system is as follows:

e.coli competent DH5 alpha is transformed conventionally, smeared on a plate containing kanamycin, cultured overnight at 37 ℃, and single-picked colonies are inoculated on LB culture medium containing kanamycin for culture, and the recombinant plasmid pET28a-CtCOSY is obtained after sequencing and identification.

The coding gene sequence of tobacco 4-coumarate-CoA ligase provided by NCBI database is synthesized and is connected to pET28a (+) vector to obtain recombinant plasmid pET28a-Nt4CL.

Example 3: inducible expression and purification of recombinant proteins

Based on example 2, the synthesized recombinant plasmids pET28a-CtCOSY and pET28a-Nt4CL and pET28a plasmid are cultured overnight at 37 ℃ to obtain BL-pET28a-CtCOSY and BL-pET28a-Nt4CL and BL-pET28a after the colony is properly sequenced by culturing the competent E.coli BL21 (DE 3) in a conventional manner. Three recombinant plasmids contained in escherichia coli are inoculated into LB culture medium containing 50mg/L of mycin according to a ratio of 1:100, the culture is carried out at 37 ℃ until the OD600 of bacterial liquid is 0.4-1.0, IPTG is added into the bacterial liquid to a final concentration of 0.1-0.8mM, and the culture is carried out at 18-20 ℃ overnight. The subsequent steps were all operated at 4℃and the cells were collected by centrifugation, then dissolved in a heavy suspension (20mM HEPES,500mM NaCl,20mM imidazole, 1mM PMSF, pH 7.5) and after ultrasonication centrifuged at 14,000g for 20min. The supernatant was purified by a HisTrap column, washed with an eluent containing 20mM HEPES pH 7.5, 500mM NaCl, 400mM imidazole, and eluted proteins were solubilized with 100mM Tris pH 7.4. Protein concentration was determined by the Bradford method and the purified proteins were subjected to SDS-PAGE electrophoresis, as shown in fig. 2. The protein is coumarin synthase CtCOSY derived from clematis terniflora, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

Example 4: establishment of in-vitro enzyme activity reaction for producing umbelliferone based on coumarin synthase

Based on example 3, an in vitro enzyme activity reaction system of coumarin synthase was designed, which contained pH 7.4100mM Tris-HCl,5mM MgCl2,5mM ATP,400. Mu.M CoA, 800. Mu.M 2-Hydroxy-p-coumatic acid, and different proteins were added as different reaction systems, respectively: 1) 30. Mu.g of Nt4CL1 and 30. Mu.g of CtCOSY protein were added as a mixed system, 2) 30. Mu.g of Nt4CL was added as a control 1, 3) 30. Mu.g of pET protein was added as a control 2. The reaction was kept at room temperature for 2h in the dark, 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, which was performed on a Waters 2695 Alliance HPLC system using a C18 column (250 mm. Times.4.6 mm inner diameter, 5 mm) with 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) and buffer B (containing 99% acetonitrile and 0.5% ammonium acetate, pH 7). Gradient elution: after equilibration of 90% buffer a for 5 minutes, buffer a was dropped from a 90% gradient to 55% in 15 minutes, then the system maintained buffer a for 55% for 5 minutes, then buffer a was raised from a55% gradient to 90% in 5 minutes and maintained for 5 minutes. The spectrum is 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 is the same as that of HPLC, buffer A is changed to 0.1% ammonium acetate, and buffer B is acetonitrile. MS/MS spectra were measured using peakview.v1.6 software. As shown in FIG. 3, mass spectrum comparison with umbelliferone proves that the coumarin synthase CtCOSY synthesized by the invention can catalyze the generation of umbelliferone, which is consistent with the expected result.

The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present 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, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.

Sequence listing

<110> basic medicine of national academy of sciences and tumor institute (Ji)

<120> coumarin synthase derived from clematis terniflora, gene, vector and use thereof

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<170> SIPOSequenceListing 1.0

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<211> 444

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<213> Cone clematis (Clematis terniflora DC.)

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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

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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

1. The coumarin synthase derived from clematis terniflora is characterized by being a protein consisting of an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the coumarin synthase of claim 1, wherein 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 a pET28a (+) plasmid.

5. An E.coli comprising the recombinant expression vector of claim 3 or 4.

6. The Escherichia coli according to claim 5, wherein BL21 (DE 3) is used as a host.

7. A method for producing coumarin synthase, characterized in that the coumarin synthase derived from clematis ternifolia is obtained by inoculating the escherichia coli of claim 6 into a culture medium and culturing overnight, expressing a gene encoding the coumarin synthase, collecting expressed proteins and purifying.

8. The method according to claim 7, wherein IPTG is added to the medium after inoculation of the E.coli and expression is induced at 18-20℃overnight.

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.