CN114410665B

CN114410665B - Gene for efficiently catalyzing biosynthesis of methyl gallate and application thereof

Info

Publication number: CN114410665B
Application number: CN202111610349.3A
Authority: CN
Inventors: 高丽萍; 陈一凡; 代新龙; 夏涛
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2024-01-16
Anticipated expiration: 2041-12-27
Also published as: CN114410665A

Abstract

The invention discloses a gene for efficiently catalyzing biosynthesis of methyl gallate and application thereof, belonging to the technical field of molecular biology and metabolic engineering, wherein the gene is a plant TA family gene, and the TA family gene is any one of the following sequences: (1) A CsTA gene with a nucleotide sequence shown as SEQ ID NO.1 or an MrTA gene with a nucleotide sequence shown as SEQ ID NO. 2; (2) A nucleotide sequence having the nucleotide sequence of (1) substituted, deleted and/or added with one or more nucleotides and expressing the same functional protein; (3) A nucleotide sequence which has a homology of 90% or more with the nucleotide sequence of any one of (1) and (2) and expresses the same functional protein. The invention provides genes CsTA and MrTA for catalyzing biosynthesis of methyl gallate, which are cloned and verified from waxberry of tea trees, and recombinant plasmids, transgenic engineering bacteria and recombinant proteins respectively containing the CsTA and MrTA genes.

Description

Gene for efficiently catalyzing biosynthesis of methyl gallate and application thereof

Technical Field

The invention relates to the fields of molecular biology and metabolic engineering, in particular to a gene for efficiently catalyzing biosynthesis of methyl gallate and application thereof.

Background

Gallic acid methyl ester is a gallized derivative in many plants, and has stronger antifungal activity on two main tea plant diseases, namely anthracnose pathogenic bacteria and round spot pathogenic bacteria; some phenolic compounds, including methyl gallate, have been found to be most effective against Proteus mirabilis and Staphylococcus saprophyticus pathogens in medicine, providing a scientific basis for the treatment of gastric, skin, respiratory and urinary tract diseases in traditional medicine; in addition, the gallic acid methyl ester is also a main anti-HIV-1 compound in the stem bark of the Indian hemp stem, and has the potential of developing novel HIV-1 inhibitors; the gallic acid methyl ester is used as an important flavonoid substance which can induce plant defense reaction in tobacco, and the compound can improve the immunity of plants, so that the deliberate stimulation of plant immunity can provide a new idea for green agriculture, crop disease management and crop disease control.

However, until now, the methyl gallate can only be synthesized by a chemical method, the procedures are complicated, the time is long, and the raw materials are used in various types and are accompanied with chemical substances which pollute the environment; the compound is limited to be obtained due to the low content of the methyl gallate in the plant body, high purification difficulty, low efficiency and other problems; gallic acid-related methyltransferases have not been reported in plants. Therefore, the invention adopts a biosynthesis mode to obtain two proteins, which can efficiently catalyze the synthesis of the methyl gallate, thereby laying a foundation for realizing the commercial production of the methyl gallate.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a gene for efficiently catalyzing biosynthesis of methyl gallate and application thereof.

The invention is realized by adopting the following technical scheme:

the invention provides a gene for catalyzing biosynthesis of methyl gallate with high efficiency, wherein the gene is a plant TA family gene.

The further improvement is that the gene is CsTA, or MrTA, or the combination of the gene and the gene, and the TA family gene is any one of the following sequences:

(1) A CsTA gene with a nucleotide sequence shown as SEQ ID NO.1 or an MrTA gene with a nucleotide sequence shown as SEQ ID NO. 2;

(2) A nucleotide sequence having the nucleotide sequence of (1) substituted, deleted and/or added with one or more nucleotides and expressing the same functional protein;

(3) A nucleotide sequence which has a homology of 90% or more with the nucleotide sequence of any one of (1) and (2) and expresses the same functional protein.

The further improvement is that the gene CsTA and the gene MrTA are isolated and cloned from fresh leaves of tea trees and waxberries.

The further improvement is that the amino acid sequences of the encoding proteins of the gene CsTA and the gene MrTA are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also provides an expression cassette comprising the gene for efficiently catalyzing the biosynthesis of the methyl gallate.

The invention also provides a recombinant plant expression vector, which comprises a recombinant vector pRSFDuet-1-CsTA obtained by recombining the gene for efficiently catalyzing the biosynthesis of the methyl gallate onto the pRSFDuet-1 vector, or pRSFDuet-1-MrTA, or a combination of the two.

The invention also provides an engineering bacterium, which is characterized by comprising the recombinant vector pRSFDuet-1-CsTA or pRSFDuet-1-MrTA and being an escherichia coli BL21 (DE 3) strain.

The invention also provides application of the gene in catalyzing biosynthesis of methyl gallate.

The invention also provides a biosynthesis method of the methyl gallate, which is characterized in that the co-expression recombinant protein of the gene combination or any recombinant protein CsTA or MrTA separated and purified in the engineering bacteria combination is added into a reaction system containing EGCG or PGG and methanol as a substrate, and the methyl gallate is biosynthesized through an enzyme catalytic reaction.

The beneficial effects of the invention are as follows:

the invention provides two genes CsTA and MrTA which catalyze the biosynthesis of methyl gallate at high efficiency, encoding proteins and application thereof, and the CsTA and MrTA genes which catalyze the biosynthesis of methyl gallate are cloned and verified from waxberry of tea trees. The invention provides two high-efficiency and safe methyl gallate biosynthesis technologies, optimizes the optimal addition amount of a substrate for enzyme reaction and the optimal reaction condition of the enzyme by utilizing a bioengineering method, and provides a basis for realizing commercial production of methyl gallate. The gallic acid methyl ester is taken as an important secondary metabolic substance, has the characteristics of antifungal, anti-HIV-1, anti-inflammatory, antioxidant, plant defense enhancement and the like, has been widely accepted and utilized in various scientific research fields, and lays a solid foundation for biosynthesis of medicinal compounds.

Drawings

FIG. 1 is a diagram showing SDS-PAGE analysis of CsTA and MrTA recombinant proteins according to an embodiment of the present invention; wherein M is a protein Marker; the two bands are the purified CsTA and MrTA recombinant proteins, respectively.

FIG. 2 is a graph showing the results of HPLC analysis of the enzymatic activity product catalyzed by CsTA and MrTA recombinant proteins using EGCG as a substrate in the examples of the present invention.

FIG. 3 is a graph showing the results of HPLC analysis of the enzymatic activity product catalyzed by CsTA and MrTA recombinant proteins using PGG as a substrate in the examples of the present invention.

FIG. 4 is a graph showing the pH gradient of the optimal reaction of recombinant proteins CsTA and MrTA under different buffer conditions, respectively, in an example of the present invention.

FIG. 5 is a graph showing the optimal reaction temperature gradient of recombinant proteins CsTA and MrTA under optimal pH conditions, respectively, in an embodiment of the present invention.

FIG. 6 is a graph showing the time gradient of the reaction of recombinant proteins CsTA and MrTA each using EGCG as a substrate in the examples of the present invention.

FIG. 7 is a graph showing the time gradient of the reaction of recombinant proteins CsTA and MrTA using PGG as a substrate, respectively, according to the embodiment of the present invention.

FIG. 8 is a flow chart of the recombinant proteins CsTA and MrTA in vitro for catalyzing EGCG or PGG to react with methanol to generate methyl gallate by enzyme reaction in an embodiment of the invention.

FIG. 9 is a structural diagram of recombinant expression vector pRSFDuet-1 in the examples of the present invention.

Detailed Description

The invention is further described with reference to the following detailed drawings in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the implementation of the invention easy to understand.

1. Material

(1) Plant material: early tea leaf comfort (l.) o.kuntze.var.sinenscultiva shochazao), fresh tea leaves and Yang Meixian leaves of bayberry (myricaubra) s.etzucc were collected, immediately frozen with liquid nitrogen and stored in a-80 ℃ refrigerator for later use.

(2) Competent E.coli cells DH 5. Alpha. Were cloned and host strain BL21 (DE 3) was expressed.

(3) LB medium: weighing 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, adding 950mL of pure water, performing ultrasonic treatment until the yeast extract is fully dissolved, adjusting the pH to 7.0 by using 1mol/LNaOH solution, adding water to a constant volume of 1L, and performing high-pressure sterilization to obtain a liquid culture medium; LB solid medium, 3g agar powder is added into each 200mLLB liquid medium, and the mixture is uniformly mixed, and the mixture is sterilized by high-pressure steam at 121 ℃ for 15min.

(4) Kanamycin mother liquor (50 mg/mL): 0.5g of kanamycin is weighed, dissolved in 10mL of sterilized water, filtered, sterilized, packaged in small tubes and stored at-20 ℃.

(5) Isopropyl thio-beta-D-galactoside (IPTG, 1 mol/L) is prepared by weighing 2.383g of powder, dissolving in 10mL of sterilized water, filtering, sterilizing, sub-packaging into small tubes, and preserving at-20deg.C.

(6) Protein purification buffer (0.1 mol/L phosphate buffer): weighing 17.907g of Na ₂ HPO ₄ ·12H ₂ O and 2.925g NaCl, and the volume was set to 500ml with pure water, which is solution I, 3.1202g NaH was weighed ₂ PO ₄ ·2H ₂ O and 1.17g NaCl, the volume is fixed to 200ml by pure water, the solution is solution II, the pH value is adjusted to 7.4 by II, the finally obtained solution is a loading buffer solution, 1.02g imidazole is added into every 100ml loading buffer solution, and the obtained solution is an elution buffer solution.

Unless otherwise indicated, the examples were conducted under conventional experimental conditions or under conditions recommended by the manufacturer's instructions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

2. Method of

2.1 cloning and expression of Gene combinations that synergistically catalyze the biosynthesis of methyl gallate

2.1.1 Cloning of CsTA and MrTA genes

(1) Based on the open reading frame sequences of the two genes CsTA and MrTA, the specific primers of BamHI and PstI were designed for the multiple cloning site with the expression vector pRSFDuet-1.

The primer sequences are shown below:

CsTA forward primer:

5’-CATCACCATCATCACCACAGCCAGGATCCGATGGATTCAATAGCCC-3’；

CsTA reverse primer:

5’-GCCGCAAGCTTGTCGACCTGCAGTCAATTTTCATTTACG-3’；

MrTA forward primer:

5’-CATCACCATCATCACCACAGCCAGGATCCGATGGCGTCAAGCACTGG-3’；

MrTA reverse primer:

5’-GCCGCAAGCTTGTCGACCTGCAGTCAAGTTATG-3’；

(2) According to the specification of the polysaccharide polyphenol total RNA extraction kit, total RNA is respectively extracted from the early fresh leaves of Shucha and the fresh leaves of Myrica rubra, and reverse transcription is carried out on the total RNA by reverse transcriptase to obtain a tea tree cDNA template and a Myrica rubra cDNA template.

(3) Respectively using tea tree cDNA and waxberry cDNA as templates, and respectively amplifying with a primer of CsTA and a primer of MrTA, wherein the amplification procedure is as follows: denaturation at 98℃for 10s, annealing at 62℃for 15s, extension at 72℃for 30s,30 cycles, and further extension at 72℃for 6min to obtain PCR products.

(4) The PCR product was purified using a DNA purification recovery kit, and the product was finally ligated to the expression plasmid pRSFDuet-1 by a one-step cloning method to obtain pRSFDuet-1-CsTA and pRSFDuet-1-MrTA recombinant plasmids, the structure of which is shown in FIG. 9.

2.1.2 prokaryotic expression of the genes CsTA and MrTA and protein purification

The prokaryotic expression and functional verification techniques used in this example are those commonly used or well understood by those of ordinary skill in the art.

(1) Respectively transferring pRSFDuet-1-CsTA and pRSFDuet-1-MrTA recombinant plasmids into escherichia coli BL21 (DE 3) expression host bacteria, adding 200 mu L of LB liquid culture medium, culturing at 37 ℃ for 60min at 180r/min, coating the bacterial liquid on an LB plate containing 50 mu g/mL Kan+, and culturing at 37 ℃ in an inverted manner;

(2) After PCR verification, positive colonies are selected, inoculated into 100mL of sterilized LB liquid medium for expansion culture until OD600 is approximately equal to 0.6;

(3) Adding 90uL of IPTG into the successfully expanded bacterial liquid, culturing for 24 hours at 16 ℃, collecting bacterial cells, adding 10-15mL of loading buffer solution, fully suspending bacterial cells, placing in an ultrasonic crusher, ultrasonically crushing for 30 minutes at 25% power, and centrifugally collecting supernatant at 12000 rpm; recombinant proteins were purified using His-tagged amylose resin affinity columns (affinity chromatography on an amylase resin, new England Biolabs, MA, USA) and protein expression and purification were tested by SDS-PAGE methods commonly used in the art, and the results are shown in FIG. 1. The two purer protein bands were MrTA and CsTA, respectively, and the purified protein was used for further enzymatic analysis.

2.2 Enzyme activity function verification of CsTA and MrTA coexpression recombinant protein

2.2.1 Detection of enzymatic Activity of CsTA and MrTA proteins and determination of optimal condition Range

(1) Enzyme activity reaction system 50 μl: comprises 50mM phosphate buffer (pH 7.0), 1-3 μg CsTA or 1-3 μg MrTA purified protein, 0.8mM EGCG or 0.4mM PGG and 2M methanol, reacting in water bath at 40-45deg.C for 5min, adding equal volume of methanol, shaking, mixing, thoroughly denaturing the enzyme, centrifuging at 13000g for 20min, and detecting with ultra high performance liquid chromatograph and mass spectrum. The EGCG synthesis process is shown in the upper diagram in FIG. 8, and the PGG synthesis process is shown in the lower diagram in FIG. 8.

The method for detecting the enzyme reaction product comprises the following steps: agilent HPLC system, mobile phase: phase A1% acetic acid water, phase B pure acetonitrile, flow rate 0.4mL/min, column model PooshellHPH-C18 column (2.7 μm, 4.6X100 mm), detector wavelength 280nm, sample injection amount 5 μl, gradient elution method: from 0min to 5min, phase b rises from 1% to 10%; from 5min to 20min, phase b rises from 10% to 35%; from 20min to 21min, phase B was reduced from 35% to 10%, from 21min to 23min, phase B was reduced from 10% to 1%, from 23min to 25min, and phase B was maintained at 1% for equilibration of the column. And determining the enzyme reaction product according to the peak emergence time and the maximum ultraviolet absorption peak of the standard substance.

Mass spectrometry method is the same as above, mass spectrometry conditions: electrospray, negative ion mode, collecting compounds with mass-to-charge ratio of 100-1700. According to the results of identifying and analyzing the product peaks by the standard substance and the characteristic ion fragments, when EGCG and methanol or PGG and methanol are respectively used as substrates, no product is produced after boiling protein is added into a reaction system, and when CsTA protein or MrTA protein is added, the substrate EGCG is almost completely consumed to produce the product of methyl gallate, gallic acid GA and epigallocatechin EGC or tetragalloylglucose TeGG, and the peak-out time and mass spectrum information of the obtained product of methyl gallate and the standard substance completely agree with each other to prove that the product is the gallic acid methyl ester.

(2) Determination of the optimal reaction pH range: in order to eliminate the influence on the enzyme activity caused by different buffers, 4 buffers with different buffer intervals were selected to find the optimal buffers and pH ranges of the two proteins. Sodium citrate buffer (pH 4.0-6.0), phosphate buffer (pH 6.0-8.0), tris-hydrochloric acid buffer (pH 7.0-10.5) and glycine-NaOH buffer (pH 9.0-11.0), wherein 1-3 μg CsTA or 1-3 μg MrTA purified protein, 0.8mM EGCG or 0.4mM PGG and 2M methanol are reacted in a water bath at 40-45 ℃ for 3-5min, added with equal volume of methanol, and mixed uniformly by shaking to fully denature enzyme, 13000g is centrifuged for 20min, and peak area detection is carried out by an ultra-high performance liquid chromatograph, and the result is shown in figure 4, wherein the optimal pH range of the two proteins is 8.0-9.0.

(3) Determination of the optimal reaction temperature range: in order to maximize the efficiency of the enzyme reaction, the enzyme reaction was detected at 10, 20, 30, 35, 40, 45, 50, 60, and 70℃respectively, and the optimal temperature ranges of both proteins were 45-50℃as shown in FIG. 5.

2.2.2 Substrate conversion detection of CsTA and MrTA recombinant proteins

Under the optimal condition, the conversion rate of the two proteins to the substrate is judged by setting different reaction time gradients for in vitro enzyme activity experiments. From the curves of substrate and product changes in the enzyme reaction system of FIG. 6, it can be seen that: when EGCG is used as a substrate, 1 mug protein is in a reaction system of CsTA, and the substrate conversion rate can reach more than 90% when the reaction is carried out for 8 min; in the reaction system of MrTA, 1 mug protein can reach more than 90% in 5min.

From the curves of substrate and product changes in the enzyme reaction system of FIG. 7, it can be seen that: when PGG is used as a substrate, 3 mug protein is in a reaction system of CsTA, and the substrate conversion rate can reach more than 200% after 15min of reaction; in the reaction system of MrTA, the conversion rate can reach more than 300% when the reaction is carried out for 15min by 3 mug protein. The method provides a basic condition for the subsequent production of the methyl gallate by utilizing enzyme engineering.

While the invention has been described in detail with respect to the general description and specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Sequence listing

<110> Anhui university of agriculture

<120> gene for high-efficiency catalysis of methyl gallate biosynthesis and application thereof

<141> 2021-12-27

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 724

<212> DNA

<213> tea (teas)

<400> 1

caagatcaac ggaccagatc acaagctccc cctcctcgtt cactaccacg gtggaggctt 60

ctgcatggga tcctccctcg acaccgtcac tctcagattc ctcacctccc tcgcctccca 120

agcccacttg atcgctatct ccgttgacta caggctcgcc ccggagcacc cattacccat 180

cgcgtatgaa gactcctggt ccgcgttgca gtggatcgct acccactcta acggccaagg 240

acccgatccc tggctaaacc agtacgcgga tttcggtcgg gttttcctgg cgggagagag 300

tgccggggca aatatagccc accaagtggc ggttcgggtt ggcactgtcg gattggaggg 360

tttcatgcca cgtggagtga ttataattca cccctatttt gcgggtagtg aaccggacaa 420

gatgatccag tatttgtatc cggggagtag tgggtcggag gatgacccga atttgagccc 480

caaggaggat ccgaatttga cgaagatggg ttgctccaag gtgattgtgt tcgtggcaga 540

gaaggatcgg ttgaaaccga gaggcgtgga ttactatgag acattgaaaa atagtggttg 600

ggaaggtagg gtggaatttg tggaggataa aggggaggac cactgtttcc atatgtttag 660

tcccaatagt gataaggttg tgggtttgat gcaaaagttg ggtactttcg taaatgaaaa 720

ttga 724

<210> 2

<211> 906

<212> DNA

<213> Myrica rubra (Myrica rubra)

<400> 2

atggcgtcaa gcactggtga gataacccat gatttcccac ctttcttcaa agtatacaaa 60

gatggccgca tagagaggta catgacccat gacccagtcc ccgcgaggct agacccaagc 120

acgggaatac aatccaaaga cgtggtgatc tcgccggaag ccggtgtgtc ggcccgaatc 180

ttcatcccca aaatcaacgg cccggatcaa aagctgccgc taatcgtcca ctaccacggg 240

ggaggcttct gcgttggatc tccgttcgat acgatttccg agagatttct cacatctttg 300

gtctcccaag cgaacgtaat agttgtctct gtcgactaca ggttagcccc agagcaccca 360

ctaccgattg catatgaaga ctcctgggcc gcgctgcagt ggattgcgtc tcattctaac 420

gggcaaggac ccgaaccgag tctcaacgag tacgtggatt tcggtcgggt tttcttgttg 480

ggcgagagcg ctggcgccaa catagccaac tacgtggcag tccaagcagg tgcgattgga 540

ttgcctggcc tgaagatcgt tggggcactt atagtgcacc ccttctttgg gagcaaggag 600

cgggatgaaa tgtacacgtt tctgtgtccc acgagctcag ggtgtgagga tgacccgaaa 660

ctgaacccgg cggttgatcc gaatttgtcg accatggcgt gcgcgaaggt cctggtatgt 720

gtggcggaga aagacgggct gaaagataga ggggtggcct actgcgagac tttacgtaag 780

agcggttggg gcggttctgt ggaattattc gaaaccaaag gagaagacca ctgctttcat 840

atgttcggcg aaggggaaaa cgtcgtgtcg ttgacgaaaa agtttgtcga ctttatcata 900

acttga 906

<210> 3

<211> 299

<212> PRT

<213> tea (teas)

<400> 3

Met Asp Ser Ile Ala His Asp Phe Pro Pro Phe Phe Arg Val His Lys

1 5 10 15

Asp Gly Arg Val Glu Arg Phe Met Val Ser Asp Tyr Val Pro Pro Ala

20 25 30

Val Asp Pro Lys Thr Gly Val Glu Phe Lys Asp Thr Leu Ile Ser Pro

35 40 45

Glu Thr Gly Val Lys Ala Arg Ile Phe Leu Pro Lys Ile Asn Gly Pro

50 55 60

Asp His Lys Leu Pro Leu Leu Val His Tyr His Gly Gly Gly Phe Cys

65 70 75 80

Met Gly Ser Ser Leu Asp Thr Val Thr Leu Arg Phe Leu Thr Ser Leu

85 90 95

Ala Ser Gln Ala His Leu Ile Ala Ile Ser Val Asp Tyr Arg Leu Ala

100 105 110

Pro Glu His Pro Leu Pro Ile Ala Tyr Glu Asp Ser Trp Ser Ala Leu

115 120 125

Gln Trp Ile Ala Thr His Ser Asn Gly Gln Gly Pro Asp Pro Trp Leu

130 135 140

Asn Gln Tyr Ala Asp Phe Gly Arg Val Phe Leu Ala Gly Glu Ser Ala

145 150 155 160

Gly Ala Asn Ile Ala His Gln Val Ala Val Arg Val Gly Thr Val Gly

165 170 175

Leu Glu Gly Phe Met Pro Arg Gly Val Ile Ile Ile His Pro Tyr Phe

180 185 190

Ala Gly Ser Glu Pro Asp Lys Met Ile Gln Tyr Leu Tyr Pro Gly Ser

195 200 205

Ser Gly Ser Glu Asp Asp Pro Asn Leu Ser Pro Lys Glu Asp Pro Asn

210 215 220

Leu Thr Lys Met Gly Cys Ser Lys Val Ile Val Phe Val Ala Glu Lys

225 230 235 240

Asp Arg Leu Lys Pro Arg Gly Val Asp Tyr Tyr Glu Thr Leu Lys Asn

245 250 255

Ser Gly Trp Glu Gly Arg Val Glu Phe Val Glu Asp Lys Gly Glu Asp

260 265 270

His Cys Phe His Met Phe Ser Pro Asn Ser Asp Lys Val Val Gly Leu

275 280 285

Met Gln Lys Leu Gly Thr Phe Val Asn Glu Asn

290 295

<210> 4

<211> 301

<212> PRT

<213> Myrica rubra (Myrica rubra)

<400> 4

Met Ala Ser Ser Thr Gly Glu Ile Thr His Asp Phe Pro Pro Phe Phe

1 5 10 15

Lys Val Tyr Lys Asp Gly Arg Ile Glu Arg Tyr Met Thr His Asp Pro

20 25 30

Val Pro Ala Arg Leu Asp Pro Ser Thr Gly Ile Gln Ser Lys Asp Val

35 40 45

Val Ile Ser Pro Glu Ala Gly Val Ser Ala Arg Ile Phe Ile Pro Lys

50 55 60

Ile Asn Gly Pro Asp Gln Lys Leu Pro Leu Ile Val His Tyr His Gly

65 70 75 80

Gly Gly Phe Cys Val Gly Ser Pro Phe Asp Thr Ile Ser Glu Arg Phe

85 90 95

Leu Thr Ser Leu Val Ser Gln Ala Asn Val Ile Val Val Ser Val Asp

100 105 110

Tyr Arg Leu Ala Pro Glu His Pro Leu Pro Ile Ala Tyr Glu Asp Ser

115 120 125

Trp Ala Ala Leu Gln Trp Ile Ala Ser His Ser Asn Gly Gln Gly Pro

130 135 140

Glu Pro Ser Leu Asn Glu Tyr Val Asp Phe Gly Arg Val Phe Leu Leu

145 150 155 160

Gly Glu Ser Ala Gly Ala Asn Ile Ala Asn Tyr Val Ala Val Gln Ala

165 170 175

Gly Ala Ile Gly Leu Pro Gly Leu Lys Ile Val Gly Ala Leu Ile Val

180 185 190

His Pro Phe Phe Gly Ser Lys Glu Arg Asp Glu Met Tyr Thr Phe Leu

195 200 205

Cys Pro Thr Ser Ser Gly Cys Glu Asp Asp Pro Lys Leu Asn Pro Ala

210 215 220

Val Asp Pro Asn Leu Ser Thr Met Ala Cys Ala Lys Val Leu Val Cys

225 230 235 240

Val Ala Glu Lys Asp Gly Leu Lys Asp Arg Gly Val Ala Tyr Cys Glu

245 250 255

Thr Leu Arg Lys Ser Gly Trp Gly Gly Ser Val Glu Leu Phe Glu Thr

260 265 270

Lys Gly Glu Asp His Cys Phe His Met Phe Gly Glu Gly Glu Asn Val

275 280 285

Val Ser Leu Thr Lys Lys Phe Val Asp Phe Ile Ile Thr

290 295 300

Claims

1.MrTAThe application of the gene in catalyzing the biosynthesis of the methyl gallate is characterized in thatMrTAThe nucleotide sequence of the gene is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein,MrTAthe gene catalysis methyl gallate biosynthesis steps are as follows: adding into a reaction system containing EGCG or PGG and methanol as substrateMrTAThe recombinant protein MrTA of the gene is used for biologically synthesizing the methyl gallate through an enzyme catalytic reaction.