CN114410665A

CN114410665A - Gene for efficiently catalyzing biosynthesis of gallic acid methyl ester and application thereof

Info

Publication number: CN114410665A
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: 2022-04-29
Anticipated expiration: 2041-12-27
Also published as: CN114410665B

Abstract

The invention discloses a gene for efficiently catalyzing biosynthesis of gallic acid methyl ester 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) CsTA gene with a nucleotide sequence shown as SEQ ID NO.1 or MrTA gene with a nucleotide sequence shown as SEQ ID NO. 2; (2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and/or added in the nucleotide sequence in (1) and the same functional protein is expressed; (3) a nucleotide sequence which has more than 90% of homology 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 gallic acid methyl ester, clones and verifies the genes CsTA and MrTA for catalyzing biosynthesis of gallic acid methyl ester from waxberries of tea trees, and also provides recombinant plasmids, transgenic engineering bacteria and recombinant proteins containing the genes CsTA and MrTA respectively.

Description

Gene for efficiently catalyzing biosynthesis of gallic acid methyl ester 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 gallic acid methyl ester and application thereof.

Background

Methyl gallate is a gallated derivative in many plants, and has strong antifungal activity on two main tea plant diseases, namely anthracnose pathogenic bacteria and wheel spot pathogenic bacteria; medically, some phenolic compounds, including methyl gallate, have been found to be most effective against proteus mirabilis and staphylococcus saprophyticus pathogens in the urinary tract, which provides scientific basis for treating gastric, skin, respiratory and urinary tract diseases in traditional medicine; in addition, methyl gallate is also a main anti-HIV-1 compound in the bark of the pith and has the potential of developing novel HIV-1 inhibitors; methyl gallate as an important flavonoid substance can induce plant defense reaction in tobacco, and the compound methyl gallate can improve the immunity of plants, and can provide a new idea for green agriculture, crop disease management and crop disease control by intentionally stimulating the plant immunity.

However, methyl gallate has been synthesized only by chemical methods so far, and has a complicated process, a long time consumption, a large variety of raw materials and the generation of environmental-polluting chemical substances; the problems of low content of gallic acid methyl ester in plant bodies, high purification difficulty, low efficiency and the like also limit the obtaining of the compound; gallic acid-related methyltransferases have also 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, and lays 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 gallic acid methyl ester and application thereof.

The invention is realized by adopting the following technical scheme:

the invention provides a gene for efficiently catalyzing biosynthesis of gallic acid methyl ester, and the gene is a plant TA family gene.

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

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

(2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and/or added in the nucleotide sequence in (1) and the same functional protein is expressed;

(3) a nucleotide sequence which has more than 90% of homology 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 obtained by separating and cloning fresh tea leaves and waxberry.

The further improvement is that the amino acid sequences of the coding 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 containing the gene for efficiently catalyzing the biosynthesis of the gallic acid methyl ester.

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

The invention also provides an engineering bacterium, which is characterized in that the engineering bacterium is an escherichia coli BL21(DE3) strain containing the recombinant vector pRSFDuet-1-CsTA or pRSFDuet-1-MrTA.

The invention also provides an application of the gene in catalyzing biosynthesis of gallic acid methyl ester.

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

The invention has the beneficial effects that:

the invention provides two genes CsTA and MrTA for efficiently catalyzing biosynthesis of gallic acid methyl ester, coding proteins and application thereof, and clones and verifies the genes CsTA and MrTA for catalyzing biosynthesis of gallic acid methyl ester from waxberries of tea trees. The invention provides two high-efficiency and safe gallic acid methyl ester biosynthesis technologies, optimizes the optimal addition amount of a substrate for enzyme reaction and the optimal reaction conditions of enzyme by using a bioengineering method, and provides a foundation for realizing the commercial production of gallic acid methyl ester. The gallic acid methyl ester is used as an important secondary metabolite, has the characteristics of resisting fungi, HIV-1, inflammation and oxidation, enhancing plant defense and the like, has been widely accepted and utilized in various scientific research fields, and lays a solid foundation for the biosynthesis of medicinal compounds.

Drawings

FIG. 1 is an SDS-PAGE protein electrophoretic analysis diagram of CsTA and MrTA recombinant proteins in an example 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 result diagram of HPLC analysis of the enzymatic activity products catalyzed by CsTA and MrTA recombinant proteins using EGCG as a substrate in the embodiment of the present invention.

FIG. 3 is a result diagram of HPLC analysis of the enzymatic activity products catalyzed by CsTA and MrTA recombinant proteins using PGG as a substrate in the embodiment of the present invention.

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

FIG. 5 is a graph showing the optimal reaction temperature gradient of each of the recombinant proteins CsTA and MrTA under the optimal pH condition in the example of the present invention.

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

FIG. 7 is a graph showing the reaction time gradient when the recombinant proteins CsTA and MrTA of the present invention each use PGG as a substrate.

FIG. 8 is a flow chart of the method for producing gallic acid methyl ester by catalyzing EGCG or PGG and methanol reaction with recombinant proteins CsTA and MrTA in vitro through enzyme reaction in the embodiment of the present invention.

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

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.

1. Material

(1) Plant material: fresh tea leaves and fresh waxberry leaves are collected from Shucha (Camellia sinensis (L.) O.Kuntze.var. sinensiscultivar Shucha) and waxberry (Myricarba (Lour.) S.etZucc.), immediately frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for later use.

(2) Competent E.coli cell DH 5. alpha. was cloned and the host strain BL21 was expressed (DE 3).

(3) LB culture medium: weighing 5g of yeast extract, 10g of tryptone and 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/L NaOH solution, adding water to a constant volume of 1L, and performing autoclaving to obtain a liquid culture medium; adding 3g of agar powder into each 200mLLB liquid culture medium, mixing, and sterilizing with high pressure steam at 121 deg.C for 15 min.

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

(5) Isopropyl thio-beta-D-galactoside (IPTG,1mol/L) 2.383g of the powder was weighed out and dissolved in 10mL of sterile water, filtered and sterilized, and then subpackaged into small tubes and stored at-20 ℃.

(6) Protein purification buffer (0.1mol/L phosphate buffer): weighing 17.907gNa₂HPO₄·12H₂O and 2.925g NaCl, made to a volume of 500ml with pure water, which was solution I, 3.1202g NaH was weighed₂PO₄·2H₂O and 1.17g of NaCl, using pure water to fix the volume to 200ml, wherein the volume is solution II, using II to adjust the pH value to 7.4, using the solution II to finally obtain a loading buffer solution, and adding 1.02g of imidazole into each 100ml of the loading buffer solution to obtain the solution, namely the elution buffer solution.

Unless otherwise indicated, the examples were carried out according to conventional experimental conditions or according to the manufacturer's instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

2. Method of producing a composite material

2.1 cloning and expression of Gene combinations for concerted catalysis of methyl gallate biosynthesis

2.1.1 cloning of CsTA and MrTA genes

(1) According to the open reading frame sequences of the two genes CsTA and MrTA, specific primers of multiple cloning sites BamH I and Pst I with an expression vector pRSFDuet-1 are designed.

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 Shucha early fresh leaves and the waxberry fresh leaves, and reverse transcription is carried out on the total RNA by utilizing reverse transcriptase to obtain a tea tree cDNA template and a waxberry cDNA template.

(3) Respectively taking tea tree cDNA and waxberry cDNA as templates, and respectively amplifying by using a CsTA primer and an MrTA primer, 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 extension at 72 ℃ for 6min to obtain a PCR product.

(4) The PCR product was purified using the DNA purification recovery kit, and the product was finally ligated to expression plasmid pRSFDuet-1 by one-step cloning to obtain pRSFDuet-1-CsTA and pRSFDuet-1-MrTA recombinant plasmids, and the structural maps thereof are shown in FIG. 9.

2.1.2 prokaryotic expression and protein purification of genes CsTA and MrTA

The prokaryotic expression and functional verification techniques used in this embodiment are those commonly used or fully understood by those skilled in the art.

(1) Respectively transforming pRSFDuet-1-CsTA and pRSFDuet-1-MrTA recombinant plasmids into an escherichia coli BL21(DE3) expression host bacterium, adding 200 mu L LB liquid medium, culturing at 37 ℃ and 180r/min for 60min, then coating the bacterium liquid on an LB plate containing 50 mu g/mL Kan +, and performing inverted culture at 37 ℃;

(2) selecting positive colonies after PCR verification, and inoculating the positive colonies into 100mL of sterilized LB liquid culture medium for amplification culture until OD600 is approximately equal to 0.6;

(3) adding 90uL IPTG into the successfully amplified bacterial liquid, culturing at 16 ℃ for 24h, collecting thalli, adding 10-15mL of sample loading buffer solution, fully suspending the thalli, carrying out ultrasonic disruption for 30min in an ultrasonic disruptor at 25% power, and centrifuging at 12000rpm to collect supernatant; recombinant proteins were purified using an amylose resin affinity column with a His tag (affinity chromatography on an amylase resin, New England Biolabs, MA, USA), and protein expression and purification effects were examined using 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 proteins were 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) 50 mul of enzyme activity reaction system: comprises 50mM phosphate buffer solution (pH 7.0), 1-3 μ g CsTA or 1-3 μ g MrTA purified protein, 0.8mM EGCG or 0.4mM PGG and 2M methanol, water bath reaction at 40-45 deg.C for 5min, adding equal volume of methanol, shaking and mixing uniformly to denature enzyme, 13000g centrifuging for 20min, and detecting with ultra high performance liquid chromatograph and mass spectrum. The synthesis process of EGCG is shown in the upper graph of FIG. 8, and the synthesis process of PGG is shown in the lower graph of FIG. 8.

The method for detecting the enzyme reaction product comprises the following steps: agilent HPLC system, mobile phase: phase A of 1% acetic acid water, phase B of pure acetonitrile, flow rate of 0.4mL/min, column model PoroshellHPH-C18column (2.7 μm, 4.6X 100mm), detector wavelength of 280nm, sample size of 5 μ l, gradient elution method: from 0min to 5min, phase B increased from 1% to 10%; from 5min to 20min, phase B rises from 10% to 35%; from 20min to 21min, phase B decreased from 35% to 10%, from 21min to 23min, phase B decreased from 10% to 1%, from 23min to 25min, maintaining phase B at 1% for equilibration of the column. And determining an enzyme reaction product according to the peak time of the standard product and the maximum ultraviolet absorption peak.

Mass spectrometry method is the same as above, mass spectrometry conditions: electrospray, negative ion mode, collection of compounds with mass to charge ratio of 100-1700. According to the identification and analysis of the product peak by the standard substance and the characteristic ion fragment, the results are shown in fig. 2 and fig. 3, when EGCG and methanol or PGG and methanol are respectively used as substrates, no product is generated after boiling protein is added in the reaction system, and when the CsTA protein or MrTA protein is added, the substrate EGCG is almost completely consumed to generate the product methyl gallate, gallic acid GA and epigallocatechin EGC or tetragalloyl glucose TeGG, and the peak time of the obtained product methyl gallate and the standard substance is completely consistent with the mass spectrum information, so that the substance is the methyl gallate.

(2) Determining the optimal reaction pH range: to exclude the influence of the buffer difference on the enzyme activity, 4 buffers having different buffer intervals were selected to search for the optimal buffer and pH range for each of the two proteins. Respectively, citrate sodium salt buffer solution (pH4.0-6.0), phosphate buffer solution (pH6.0-8.0), Tris-hydrochloric acid buffer solution (pH7.0-10.5) and glycine-NaOH buffer solution (pH 9.0-11.0), 1-3 μ g CsTA or 1-3 μ g MrTA purified protein, 0.8mM EGCG or 0.4mM PGG and 2M methanol, reacting in water bath at 40-45 deg.C for 3-5min, adding equal volume of methanol, shaking, mixing to denature enzyme, centrifuging at 13000g for 20min, and detecting peak area with ultra high performance liquid chromatograph, the result is shown in FIG. 4, and the optimum pH range of the two proteins is 8.0-9.0.

(3) Determining the optimal reaction temperature range: in order to maximize the efficiency of the enzyme reaction, 10, 20, 30, 35, 40, 45, 50, 60, and 70 ℃ were selected for the enzyme reaction, and the results are shown in FIG. 5, where the optimal temperature ranges for both proteins were 45-50 ℃.

2.2.2 substrate conversion assay for CsTA and MrTA recombinant proteins

Under the optimal condition, different reaction time gradients are set for in vitro enzyme activity experiments to judge the conversion rates of the two proteins to the substrate. As can be seen from the change curves of the substrate and the product in the enzyme reaction system of FIG. 6: when EGCG is used as a substrate, in a reaction system of CsTA, 1 mu g of protein can reach the substrate conversion rate of more than 90 percent when the reaction is carried out for 8 min; in the reaction system of MrTA, the conversion rate can reach more than 90 percent within 5min of reaction of 1 mu g of protein.

As can be seen from the change curves of the substrate and the product in the enzyme reaction system of FIG. 7: when PGG is used as a substrate, in a CsTA reaction system, 3 mu g of protein can reach a substrate conversion rate of more than 200% after reacting for 15 min; in the reaction system of MrTA, 3 mu g of protein can reach the conversion rate of more than 300 percent when reacting for 15 min. This provides a basic condition for the subsequent production of gallic acid methyl ester by enzyme engineering.

The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.

Sequence listing

<110> agriculture university of Anhui

<120> gene for efficiently catalyzing biosynthesis of gallic acid methyl ester and application thereof

<141> 2021-12-27

<160> 4

<170> SIPOSequenceListing 1.0

<210> 3

<211> 724

<212> DNA

<213> tea (tea)

<400> 3

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> waxberry (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 (tea)

<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> waxberry (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. The gene for efficiently catalyzing biosynthesis of gallic acid methyl ester is characterized in that the gene is a plant TA family gene.

2. The gene for efficiently catalyzing biosynthesis of gallic acid methyl ester according to claim 1, wherein said TA family gene is any one of the following sequences:

3. The gene for efficiently catalyzing biosynthesis of gallic acid methyl ester according to claim 1, wherein said gene CsTA and gene MrTA are isolated and cloned from fresh leaves of Camellia sinensis and fresh leaves of Myrica rubra respectively.

4. The gene for efficiently catalyzing biosynthesis of gallic acid methyl ester according to claim 1, wherein the amino acid sequences of the encoded proteins of said gene CsTA and said gene MrTA are shown in SEQ ID No.3 and SEQ ID No.4, respectively.

5. An expression cassette comprising the gene highly catalyzing biosynthesis of gallic acid methyl ester according to any one of claims 1 to 4.

6. A recombinant plant expression vector, wherein the vector combination comprises a recombinant vector pRSFDuet-1-CsTA or pRSFDuet-1-MrTA or a combination thereof obtained by recombining the gene capable of efficiently catalyzing the biosynthesis of gallic acid methyl ester according to any one of claims 1 to 4 onto a pRSFDuet-1 vector.

7. An engineered bacterium, which is Escherichia coli BL21(DE3) strain comprising the recombinant vector pRSFDuet-1-CsTA or pRSFDuet-1-MrTA of claim 6.

8. Use of the gene of any one of claims 1-4 in catalyzing the biosynthesis of gallic acid methyl ester.

9. A method for biosynthesis of methyl gallate, characterized in that a recombinant protein CsTA or MrTA of any one of 1 to 4 of the genes is added to a reaction system containing EGCG or PGG and methanol as a substrate, and methyl gallate is biosynthesized by an enzymatic reaction.