KR20150075637A - Method for mass production of quercetin-3-O-galactose using PeF3GalGT gene in Escherichia coli - Google Patents

Abstract

The present invention relates to a composition for producing a quercetin-3-O-galactose compound containing an expression vector including a gene for coding a Petunia hybrida UDP-dependent glycosyltransferase (PeF3GalGT) protein, and an expression vector including a UDP-galactose synthetic gene, and a Escherichia coli in which a UDP-galactose epimerase (galE) gene is deleted, and to a Escherichia coli transformed by an expression vector including gene for coding the PeF3GalGT protein, and an expression vector including the galE gene to produce a quercetin-3-O-galactose compound.

Description

A method for mass production of quercetin-3-O-galactose using the PeF3GalGT gene derived from petunia in Escherichia coli (method for mass production of quercetin-3-O-galactose using PeF3GalGT gene in Escherichia coli)

The present invention relates to a mass production method of quesetin- 3- O -galactose using the peanut- derived glycosyltransferase PeF3GalGT gene, and more particularly to a method for mass production of petunia-derived glycosyltransferase PeF3GalGT (Petunia hybrida UDP-dependent glycosyltransferase) An expression vector containing a coding gene, a UDP-galactose synthesis Expression vector containing a gene and galE (UDP-galactose epimerase) Gene Quebec paroxetine -3- O containing a deletion of E. coli-expressed, including a gene coding for a lactose go (quercetin-3- O -galactose) compounds for preparing compositions and Petunia transferase PeF3GalGT glycoprotein derived vector and UDP- O -galactose compound by transforming with an expression vector containing a galactose synthase gene to produce a quercetin-3- O -galactose compound.

In general, natural products can be enzymatically modified by introducing genes useful for microorganisms such as E. coli, which are well established in culture methods and genetic engineering techniques. This method is called biotransformation. Bioconversion has the advantage of saving costly cofactors in the middle of the reaction.

Flavonoids, which have attracted much attention as new drug candidates, are one of the many secondary metabolites produced by plants. Flavonoids have been reported to have various physiological activities such as antioxidant, anti-cancer, anti-inflammatory and anti-allergic effects. The glycosylation of these flavonoids is known to increase the solubility and at the same time increase the water absorption. Such natural structural changes cause changes in physiological activity. Typical structural modification methods involve the use of glycosyltransferases, which transfer a molecule of glucose to the hydroxyl group (-OH).

Quercetin (3,3 ', 4', 5,7-pentahydroxyflavone) has a strong antioxidant activity (Middlton et al., 2000, Pharmacol . Rev. 52: 673-751), it is known that it has various physiological functions such as inhibition of aggregation of platelets and adhesion inhibition, vasodilation, and anti-cancer action. In particular, quercetin glycosides (quercetin-4'-β-D-glucoside and quercetin-3,4'-β-D-glucoside (Quercetin-3,4'-β-D-glucoside) has been reported to be superior in absorbability (Hollman et al., 1998, Arch. Toxicol. Suppl. 20: 237-248). Similarly, quercetin-3-β-D-glucoside (quercetin-3-β-D-glucoside) in which glucose is β-linked to the 3-carbon position of quercetin is more absorbent than quercetin or (Morand et al., 2000, Free Raf. Res. 33: 667-676).

The transglycosylase utilizes a nucleotide sugar as a donor. The present invention analyzes the nucleic acid sugar metabolism pathway of E. coli to make a new structure of the nucleic acid sugar in E. coli, GalE derived from Escherichia coli that can be used as a substrate Gene and rice-derived OsUGE ( Oryza sativa UDP-glucose epimerase ) gene was introduced into E. coli. The OsUGE gene makes UDP-D-galactose using UDP-D-glucose, and the UDP-D-galactose thus produced is transformed into glucose It is used for glycation of cetin. The present invention was used as a substrate Quebec paroxetine, through a reaction as described above Quebec paroxetine -3- O - ethylamine galactose (Quercetin-3- O -galactose).

The bioconversion method (enzymatic synthesis method) for synthesizing these substances has various advantages compared with the chemical synthesis method because it has position selectivity and chiral selectivity which are difficult to be modified by a chemical synthesis method. Therefore, the bioconversion using E. coli with the gene encoding the Petunia flavonoid galactosyltransferase PeF3GalGT protein of the present invention can be a new method for producing quercetin - 3- O -galactose.

Korean Patent No. 1282329 discloses a 'chemical mass synthesis method of quercetin-3-glucoside', Korean Patent Publication No. 2010-0001907 discloses a process for preparing quercetin in a routine using Ramposydays derived from Aspergillus niger, -3-glucoside " has been disclosed. However, in the case of Escherichia coli, the sugar chain transferase PeF3GalGT There is no description about a method for mass production of quercetin-3- O -galactose using a gene.

The present invention has been made in view of the above needs, and the present inventors have found that quercetin-3- O -galactose is mass-produced in E. coli transformed with an expression vector containing the peanut- derived glycosyltransferase PeF3GalGT gene Respectively. In particular, in the present invention, when Escherichia coli BL21 (DE3) lacking the galE gene for converting UDP-glucoside to UDP-galactose is used, when E. coli is transformed into an expression vector containing a gene encoding PeF3GalGT protein , And at the same time, UDP-galactose synthesis Escherichia coli-derived galE Gene and rice-derived OsUGE ( Oryza sativa UDP-glucose epimerase) gene was expressed in E. coli, respectively. As a result, it was confirmed that quercetin-3- O- galactose was produced in E. coli expressing OsUGE gene derived from rice.

In order to solve the above problems, the present invention provides an expression vector comprising a gene encoding a Petunia hybrida UDP-dependent glycosyltransferase (PeF3GalGT) protein, a gene encoding UDP-galactose synthase Expression vector containing a gene and galE (UDP-galactose epimerase) Provides a galactose (quercetin-3- O -galactose) compounds for preparing compositions Quebec paroxetine -3- O containing a gene deletion of E. coli.

In addition,

a) Petunia-derived glycosyltransferase PeF3GalGT ( Petunia transforming E. coli with an expression vector comprising a gene encoding a hybrida UDP-dependent glycosyltransferase protein;

b) transforming the expression vector into E. coli with an expression vector containing a UDP-galactose synthase gene; And

It provides a galactose (Preparation method of quercetin-3- O -galactose) compounds - c) -3- O Quebec paroxetine comprising the step of culturing the transformed E. coli with addition of Quebec paroxetine as a substrate.

In addition, the present invention relates to the use of the petunia-derived glycosyltransferase PeF3GalGT ( Petunia hybrida U -galactosyltransferase (UDP-dependent glycosyltransferase) protein, and an expression vector containing a UDP-galactose synthase gene to produce quercetin-3- O- galactose ) ≪ / RTI > compound.

With the method of the galactose production, Quebec paroxetine -3- O - - Quebec paroxetine -3- O using the Petunia PeF3GalGT transferase gene derived from sugar of the present invention a large amount of galactose (quercetin-3- O- galactose) It can be used for various foods, medicines and cosmetics using it as a functional ingredient.

Figure 1 is a schematic illustration of the synthesis of quercetin-3- O -galactose from the substrate quercetin.
FIG. 2 is a result of analysis of a reaction product obtained by biotransformation of quercetin to a substrate using high-performance liquid chromatography (HPLC) using a metabolically-regulated Escherichia coli transformed with a gene encoding a peptidyl-derived glycosyltransferase PeF3GalGT protein . (A); pEGX vector, (B); A pEGX vector in which a gene encoding PeF3GalGT protein is cloned, S; Quercetin, P; Quercetin-3-O-galactose.
FIG. 3 is a result of mass spectrometry (MS) of a reaction product obtained by bioconversion of quercetin to a substrate using a metabolically-regulated E. coli transformed with a gene encoding a peptidase-derived glycosyltransferase PeF3GalGT protein. (A); A pEGX vector in which a gene encoding PeF3GalGT protein is cloned, (B); pEGX vector
4 Petunia galE gene or rice plant with a gene derived from the E. coli derived per transition encoding an enzyme protein PeF3GalGT OsUGE (Oryza sativa UDP-glucose epimerase) gene in E. coli. A; A compound produced by using Escherichia coli BL21 containing the gene (qGEX) encoding PeF3GalGT protein and galE (pCDF); B; The gene encoding the PeF3GalGT protein (qGEX) and Compounds produced using Escherichia coli BL21 containing OsUGE (pDCF).
FIG. 5 is a graph showing the effect of quercetin-1 in the culture-inducing culture conditions of E. coli BL21 (DE3) transformed with a pGEX vector containing a gene encoding a peptidyl-derived glycosyltransferase PeFGalGT protein and a pCDF vector containing a rice-derived OsUGE gene, 3-O-galactose. ≪ / RTI >

In order to accomplish the object of the present invention, the present invention relates to a method for producing a petunia-derived sugar transferase PeF3GalGT ( Petunia hybrida UDP-dependent glycosyltransferase (UDP-dependent glycosyltransferase) protein, a UDP-galactose synthase An expression vector containing the gene and galE (UDP-galactose epimerase) provides paroxetine Quebec -3-O- galactose (quercetin-3-O-galactose ) compound composition for preparing a gene containing the deletion of E. coli.

The quercetin-3-O-galactose compound of the present invention has the following general formula (1).

In the composition according to an embodiment of the present invention, the range of the PeF3GalGT protein includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from Petunia hybrida and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2.

In addition, the gene coding for the PeF3GalGT protein includes both genomic DNA and cDNA. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene homologue has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . ≪ / RTI > "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway and the left promoter of the phage lambda (pL 貫 promoter) can be used as a regulatory region.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4 · λB ,? -charon,?? z1, and M13), or a virus (e.g., SV40, etc.).

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

In the composition according to an embodiment of the present invention, the E. coli in which the galE gene has been deleted may be, but is not limited to, Escherichia coli BL21 (DE3).

In the present invention, UDP-galactose synthesis for converting UDP-glucoside to UDP-galactose Escherichia coli BL21 (DE3) lacking the gene was used. Therefore, in order to produce quercetin-3- O -galactose in E. coli, UDP-galactose synthesis And an expression vector containing the gene were simultaneously transformed.

In the composition according to an embodiment of the present invention, the UDP-galactose biosynthesis Gene may be a galE gene of E. coli origin having the base sequence of SEQ ID NO: 3 nucleotide sequence of OsUGE gene or sequence number of a rice plant origin having four, and preferably one OsUGE gene of rice origin having the base sequence of SEQ ID NO: 3 But is not limited thereto.

In addition,

a) Petunia-derived glycosyltransferase PeF3GalGT ( Petunia transforming E. coli with an expression vector comprising a gene encoding a hybrida UDP-dependent glycosyltransferase protein;

b) To the transformed Escherichia coli, UDP-galactose synthesis Transforming an expression vector comprising the gene; And

provides a galactose (Preparation method of quercetin-3- O -galactose) compounds - c) Quebec paroxetine -3- O in E. coli comprising the step of adding to the substrate Quebec paroxetine and culturing the transformed E. coli.

In the method according to one embodiment of the present invention, the peanut-derived glycosyltransferase PeF3GalGT protein may be composed of the amino acid sequence of SEQ ID NO: 2. Also, functional equivalents of the amino acid sequence are included within the scope of the present invention, and the details are as described above.

The method of delivering the vector of the present invention into a host cell can be carried out by a CaCl ₂ method, a Han method (Hanahan, 1983, J. Mol. Biol. 166: 557-580) and an electroporation method when the host cell is a prokaryotic cell . ≪ / RTI >

In the method according to one embodiment of the present invention, the E. coli may be E. coli used for mass production of recombinant protein, preferably E. coli with deletion of galE gene, more preferably E. coli BL21 (DE3) But is not limited thereto.

In the method according to another embodiment of the present invention, the protein expression inducing culture conditions of the transformed E. coli for the production of quesetin-3- O -galactose are at a temperature of 15 to 30 DEG C, a culturing time of 12 to 72 hours, The cell concentration OD ₆₀₀ = 2 to 10, preferably 17 to 19 ° C, the culture time 12 to 20 hours, the cell concentration is the OD ₆₀₀ = 2 to 5, most preferably 18 ° C, the 18 hour The culture and cell concentration OD ₆₀₀ = 3, but are not limited thereto.

The present invention also relates to a method for producing quetiene-3, which is transformed with an expression vector comprising a gene encoding a petunia-derived glycosyltransferase PeF3GalGT ( Petunia hybrida UDP-dependent glycosyltransferase) and an expression vector comprising a UDP- O -galactose < / RTI > compounds.

In Escherichia coli according to one embodiment of the present invention, the peanut-derived glycosyltransferase PeF3GalGT protein may be composed of the amino acid sequence of SEQ ID NO: 2. Also, functional equivalents of the amino acid sequence are included within the scope of the present invention, and the details are as described above.

In E. coli according to an embodiment of the present invention, the E. coli may be E. coli used for mass production of a recombinant protein, preferably E. coli having a galE gene deleted, more preferably E. coli BL21 (DE3) But is not limited thereto.

In E. coli according to one embodiment of the present invention, the UDP-galactose synthesis The gene is derived from rice having the nucleotide sequence of SEQ ID NO: 3 OsUGE gene or a galE derived from Escherichia coli having the nucleotide sequence of SEQ ID NO: 4 Derived from rice having the nucleotide sequence of SEQ ID NO: 3, But are not limited to, the OsUGE gene.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Petunia Transglycosylase  Gene Cloning  And Escherichia coli Expression Vector Production

Petunia Transglycosylase  Isolation of genes

Petunia whole plants were finely ground with liquid nitrogen and total DNA was isolated. Total isolated DNA EcoRI restriction enzymes and inserted into the seat created by the forward (5'-ATG GAATTC GATGTCCAATTACCATGTTGC-3 '; SEQ ID NO: 5, underlined; EcoRI position), and reverse (5'-TTCACGTCCAGCAATTCCTTGAGCCTCAACAT-3 '; SEQ ID NO: 6) in combination with primers forward (5'-ATGTTGAGGCTCAAGGAATTGCTGGACGTGAA-3'; SEQ ID NO: 7) and a NotI restriction enzyme digit reverse (5'-CAT made by inserting the GCGGCCGC TTAATTGCAAGAGGTGATA-3 '; SEQ ID NO: 8, underlined; NotI Restriction enzyme site) primers were used to perform PCR (Polymerase Chain Reaction). PCR was carried out using primers of SEQ. ID. NO. 5 and SEQ. ID NO. 8 as a template to isolate the transgenic enzyme gene for the pertussis gene.

PCR was performed by repeating the cycle of 94 ° C. for 60 seconds, 55 ° C. for 60 seconds, and 72 ° C. for 90 seconds by repeating the above 40 cycles of Taq DNA polymerase with Taq DNA polymerase and PCR was carried out by encoding PeF3GalGT protein And amplified the gene corresponding to about 1.3 kbp. The amplified DNA was transferred to the E. coli cloning vector pGEMT-easy to determine the base sequence.

PeF3GalGT  Creation of vectors to express genes encoding proteins

In order to express the gene coding for the cloned transgenic protein of the pertussis glycosyltransferase gene, the gene coding for the glycosyltransferase PeF3GalGT protein isolated by inserting the EcoRI and NotI restriction enzyme sites was digested with EcoRI and NotI , and pGEX 5X -3 to construct a vector for expressing the gene encoding PeF3GalGT protein.

Example  2. PeF3GalGT  Protein-encoding Generation of new substances using gene expression and bioconversion

Escherichia coli DH5a An expression vector containing a gene encoding PeF3GalGT protein was transformed. The transformed E. coli expressing the gene encoding the PeF3GalGT protein was seeded in LB medium supplemented with 50 mu g / ml ampicillin. E. coli strain cultured overnight was inoculated into fresh LB medium and cultured to an absorbance value of 0.8 at 600 nm. IPTG was added to the cultured Escherichia coli at a final concentration of 1 mM, and the protein was expressed at 18 DEG C for 18 hours. The cultured Escherichia coli was centrifuged to discard the supernatant, and the recovered Escherichia coli was adjusted to 3.0 at an absorbance of 600 nm on M9 medium supplemented with 50 μg / ml of ampicillin and 2% glucose. The substrate quercetin was added to a final concentration of 100 μM and incubated at 25 ° C for 24 hours with shaking. The supernatant was extracted with ethyl acetate. The extracted reaction product was dried using a vacuum condenser dryer, and the reaction product was again dissolved in DMSO and used for high performance liquid chromatography (HPLC) analysis.

As a result, the generation of a new compound from quercetin was confirmed (Fig. 2), and the molecular weight of the substance was confirmed by mass spectrometry. As a result, 464-Da Sized material. NMR analysis revealed that the structure of the product was quercetin-3- O- galactose.

Example  3. Metabolic regulation by E. coli Quercetin -3- O - Galactose  Comparison of production

The metabolism of the E. coli were adjusted to increase the production of galactose (quercetin-3- O -galactose) - Quebec paroxetine -3- O. UDP-galactose is not present in Escherichia coli BL21 (DE3) because the galE gene which converts UDP-glucose to UDP-galactose is deficient. Therefore, in the present invention, galactosidase activity of E. coli, which synthesizes UDP-galactose using UDP-glucose as a substrate, The OsUGE gene cloned from the gene or rice was introduced into the pCDF vector and co-expressed with the gene encoding PeF3GalGT protein in E. coli BL21 (DE3). As a result, Escherichia coli BL21 (DE3) transformants into which OsUGE was introduced showed galE It was found that quesetin-3- O -galactose increased about 1.85-fold more than E. coli to which the gene was introduced (FIG. 4).

Also, PeF3GalGT The gene encoding the protein and OsUGE The synthesis of quesetin-3- O- galactose from quercetin was examined using Escherichia coli BL21 (DE3) into which a gene was introduced. After induction at 18 ° C for 20 hours, E. coli was collected and quercetin was added using M9 containing 2% of glucose and 50 μg / ml of ampicillin and spectinomycin at an absorbance of 600 nm and 3 at 30 ° C. Lt; / RTI > Quercetin was added at 100 μM every 0, 3, 6, 9, 12, 24, and 36 hours, and finally 700 μM was added. As a result, it was confirmed that 277.9 mg / l of quercetin-3- O -galactose was produced after 72 hours (FIG. 5).

<110> Konkuk University Industrial Cooperation Corp <120> Method for mass production of quercetin-3-O-galactose using          PeF3GalGT gene in Escherichia coli <130> PN13383 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1356 <212> DNA <213> Petunia hybrida <400> 1 atgtccaatt accatgttgc tgttctagca tttccttttg caacacatgc tggcctttta 60 cttggacttg tacaaaggct agcaaatgca ttacctaacg tgacatttac attctttaac 120 acgtccaaat caaattcttc attattcact actcctcatg acaacaacat taaaccgttt 180 aatatctccg atggcgtccc ggagggttac gtggtcggaa aaggaggtat tgaagcgctt 240 atagggttgt tctttaagtc tgctaaagag aatattcaaa atgctatggc agctgctgtg 300 gaggaatcgg gaaagaagat tacttgtgtt atggcagatg catttatgtg gttttctggt 360 gagattgctg aggaattgag cgttggttgg atccctttat ggacttctgc tgctggatca 420 ctctctgttc atgtttacac tgatctcatt agagaaaatg ttgaggctca aggaattgct 480 ggacgtgaag atgaaattct aacattcatt ccaggatttg ccgaattacg gctaggtagt 540 ttgcctagtg gagttgtctc tggagacttg gaatcaccat tttccgtaat gcttcacaaa 600 atgggcaaaa caataggaaa agccactgcc ttgccagtaa actcctttga agaactagac 660 cctccaattg ttgaagatct caagtccaaa ttcaataact tcctcaatgt tggtcccttt 720 aacttaacta ccccaccacc ctcagcaaac attacagatg aatatgggtg cattgcatgg 780 ctagacaaac aagaaccagg ctcagtggct tacataggat ttggcacggt ggctacacca 840 ccacctaatg agctaaaagc tatggctgaa gcacttgaag aaagtaaaac tccttttctt 900 tggtcactta aagacctttt caaatcattt tttccagaag ggttcttgaa aagaactagt 960 gagtatggta aaattgtgtc atgggcacca caagtacaag ttcttagtca tggttcagtt 1020 ggtgttttta taaatcattg tggatggaac tctgttttgg agagtatagc tgctggtgtg 1080 cctgtgattt gcaggccatt ttttggagat catcaattga atgcttggat ggttgagaaa 1140 gtatggaaaa ttggagtgaa aattgaagga ggagttttta ctaaagatgg aacaatgctt 1200 gcacttgact tggttttatc aaaaaccaaa aggaatacaga aattgaaaca acaaattggg 1260 atgtataaag agcttgctct aaatgctgtt ggaccaagtg ggagctctac tgaaaatttc 1320 aagaaattgg ttgatattat cacctcttgc aattaa 1356 <210> 2 <211> 451 <212> PRT <213> Petunia hybrida <400> 2 Met Ser Asn Tyr His Val Ala Val Leu Ala Phe Pro Phe Ala Thr His   1 5 10 15 Ala Gly Leu Leu Leu Gly Leu Val Gln Arg Leu Ala Asn Ala Leu Pro              20 25 30 Asn Val Thr Phe Thr Phe Phe Asn Thr Ser Lys Ser Asn Ser Ser Leu          35 40 45 Phe Thr Thr Pro His Asp Asn Asn Ile Lys Pro Phe Asn Ile Ser Asp      50 55 60 Gly Val Pro Glu Gly Tyr Val Gly Lys Gly Gly Ile Glu Ala Leu  65 70 75 80 Ile Gly Leu Phe Phe Lys Ser Ala Lys Glu Asn Ile Gln Asn Ala Met                  85 90 95 Ala Ala Ala Val Glu Glu Ser Gly Lys Lys Ile Thr Cys Val Met Ala             100 105 110 Asp Ala Phe Met Trp Phe Ser Gly Glu Ile Ala Glu Glu Leu Ser Val         115 120 125 Gly Trp Ile Pro Leu Trp Thr Ser Ala Ala Gly Ser Leu Ser Val His     130 135 140 Val Tyr Thr Asp Leu Ile Arg Glu Asn Val Glu Ala Gln Gly Ile Ala 145 150 155 160 Gly Arg Glu Asp Glu Ile Leu Thr Phe Ile Pro Gly Phe Ala Glu Leu                 165 170 175 Arg Leu Gly Ser Leu Pro Ser Gly Val Val Ser Gly Asp Leu Glu Ser             180 185 190 Pro Phe Ser Val Met Leu His Lys Met Gly Lys Thr Ile Gly Lys Ala         195 200 205 Thr Ala Leu Pro Val Asn Ser Phe Glu Glu Leu Asp Pro Pro Ile Val     210 215 220 Glu Asp Leu Lys Ser Lys Phe Asn Asn Phe Leu Asn Val Gly Pro Phe 225 230 235 240 Asn Leu Thr Thr Pro Pro Ser Ala Asn Ile Thr Asp Glu Tyr Gly                 245 250 255 Cys Ile Ala Trp Leu Asp Lys Gln Glu Pro Gly Ser Val Ala Tyr Ile             260 265 270 Gly Phe Gly Thr Val Ala Thr Pro Pro Asn Glu Leu Lys Ala Met         275 280 285 Ala Glu Ala Leu Glu Glu Ser Lys Thr Pro Phe Leu Trp Ser Leu Lys     290 295 300 Asp Leu Phe Lys Ser Phe Phe Pro Glu Gly Phe Leu Lys Arg Thr Ser 305 310 315 320 Glu Tyr Gly Lys Ile Val Ser Trp Ala Pro Gln Val Gln Val Leu Ser                 325 330 335 His Gly Ser Val Gly Val Phe Ile Asn His Cys Gly Trp Asn Ser Val             340 345 350 Leu Glu Ser Ile Ala Gly Val Val Pro Ile Cys Arg Pro Phe Phe         355 360 365 Gly Asp His Gln Leu Asn Ala Trp Met Val Glu Lys Val Trp Lys Ile     370 375 380 Gly Val Lys Gly Gly Gly Gly Val Phe Thr Lys Asp Gly Thr Met Leu 385 390 395 400 Ala Leu Asp Leu Val Leu Ser Lys Thr Lys Arg Asn Thr Glu Leu Lys                 405 410 415 Gln Gln Ile Gly Met Tyr Lys Glu Leu Ala Leu Asn Ala Val Gly Pro             420 425 430 Ser Gly Ser Ser Thr Glu Asn Phe Lys Lys Leu Val Asp Ile Ile Thr         435 440 445 Ser Cys Asn     450 <210> 3 <211> 1065 <212> DNA <213> Oryza sativa <400> 3 atggtttcgg ccttgttgcg gacgatcctg gtgacgggcg gcgccggcta catcggcagc 60 cacaccgtcc tccagcttct ccaactcggc ttccgcgttg tcgtcctcga caacctcgac 120 aacgcctccg agctcgccat cctccgcgtc agggaactcg ccggacacaa cgccaacaac 180 ctcgacttcc gcaaggttga cctccgcgac aagcaagcgt tggaccaaat cttctcctct 240 caaaggtttg aggctgtcat ccattttgcc gggctgaaag ctgttggcga gagcgtgcag 300 aagcccctgc tttactacga caacaacctc atcggcacca tcactctcct gcaggtcatg 360 gccgcacatg gctgcaccaa gctggtgttc tcatcatccg caactgtcta cgggtggccc 420 aaggaggtgc cctgcactga agaatcccca ctttgtgcaa tgaaccccta cggcagaaca 480 aagctggtaa tcgaagacat gtgccgggat ctgcatgcct cagacccaaa ctggaagatc 540 atactgctcc gatacttcaa ccctgttgga gctcacccaa gcgggtacat tggtgaggac 600 ccctgcggca tcccaaacaa cctcatgccc ttcgtccagc aggtcgctgt tggcaggagg 660 ccggccctta ccgtctatgg aaccgactac aacaccaagg atggaactgg ggttcgtgac 720 tatatccatg ttgttgatct agcggatggt catatcgccg cgttaaggaa gctctatgaa 780 gattctgata gaataggatg tgaggtgtac aatctgggca ctggaaaggg gacatctgtg 840 ctggaaatgg ttgcagcatt cgagaaagct tctggaaaga aaatcccgct tgtatttgct 900 ggacgaaggc ctggagatgc cgagatcgtt tacgctcaaa ctgccaaagc tgagaaggaa 960 ctgaaatgga aggcaaaata cggggtagag gagatgtgca gggacctgtg gaattgggcg 1020 agcaagaacc cctacgggta tggatcgccg gacagtagca actga 1065 <210> 4 <211> 1017 <212> DNA <213> Escherichia coli <400> 4 atgagagttc tggttaccgg tggtagcggt tacattggaa gtcatacctg tgtgcaatta 60 ctgcaaaacg gtcatgatgt catcattctt gataacctct gtaacagtaa gcgcagcgta 120 ctgcctgtta tcgagcgttt aggcggcaaa catccaacgt ttgttgaagg cgatattcgt 180 aacgaagcgt tgatgaccga gatcctgcac gatcacgcta tcgacaccgt gatccacttc 240 gccgggctga aagccgtggg cgaatcggta caaaaacccc tggaatatta cgacaacaat 300 gtcaacggca ctctgcgcct gattagcgcc atgcgcgccg ctaacgtcaa aaactttatt 360 tttagctcct ccgccaccgt ttatggcgat cagcccaaaa ttccatacgt tgaaagcttc 420 ccgaccggca caccgcaaag cccttacggc aaaagcaagc tgatggtgga acagatcctc 480 accgatctgc aaaaagccca gccggactgg agcattgccc tgctgcgcta cttcaacccg 540 gttggcgcgc atccgtcggg cgatatgggc gaagatccgc aaggcattcc gaataacctg 600 atgccataca tcgcccaggt tgctgtaggc cgtcgcgact cgctggcgat ttttggtaac 660 gattatccga ccgaagatgg tactggcgta cgcgattaca tccacgtaat ggatctggcg 720 gacggtcacg tcgtggcgat ggaaaaactg gcgaacaagc caggcgtaca catctacaac 780 ctcggcgctg gcgtaggcaa cagcgtgctg gacgtggtta atgccttcag caaagcctgc 840 ggcaaaccgg ttaattatca ttttgcaccg cgtcgcgagg gcgaccttcc ggcctactgg 900 gcggacgcca gcaaagccga ccgtgaactg aactggcgcg taacgcgcac actcgatgaa 960 atggcgcagg acacctggca ctggcagtca cgccatccac agggatatcc cgattaa 1017 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atggaattcg atgtccaatt accatgttgc 30 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttcacgtcca gcaattcctt gagcctcaac at 32 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 catgcggccg cttaattgca agaggtgata 30 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atgttgaggc tcaaggaatt gctggacgtg aa 32

Claims (11)

An expression vector containing a gene encoding a Petunia hybrida UDP-dependent glycosyltransferase (PeF3GalGT) protein, a UDP-galactose synthase Quebec paroxetine -3- O which is an expression vector and galE (UDP-galactose epimerase) genes containing the gene containing the deletion of E. coli-galactose (quercetin-3- O -galactose) compounds for preparing the composition. The method of claim 1, wherein the petunia-derived sugar transferase PeF3GalGT proteins Quebec paroxetine, characterized in that it contains the amino acid sequence of SEQ ID NO: 2 -3- O - galactose (quercetin-3- O -galactose) compounds for preparing the composition. The method according to claim 1, wherein the UDP-galactose synthesis Gene Quebec paroxetine, characterized in that the galE gene of E. coli origin having the base sequence of SEQ ID NO: 4 or the gene OsUGE rice origin having a nucleotide sequence of SEQ ID NO: 3 -3- O - galactose (quercetin-3- O -galactose. The method of claim 1, wherein the galE Quebec paroxetine -3- O, characterized in that the gene is a deletion of E. coli is E. coli BL21 (DE3) - galactose (quercetin-3- O -galactose) compounds for preparing the composition. a) Petunia-derived glycosyltransferase PeF3GalGT ( Petunia transforming E. coli with an expression vector comprising a gene encoding a hybrida UDP-dependent glycosyltransferase protein;
b) To the transformed Escherichia coli, UDP-galactose synthesis Transforming an expression vector comprising the gene; And
The method of galactose (quercetin-3- O -galactose) compounds - c) the transformed E. coli by the Quebec paroxetine in E. coli comprising the step of adding to the substrate Quebec paroxetine -3- O were cultured. The method of claim 5, wherein the Petunia transferase PeF3GalGT glycoprotein derived from Quebec paroxetine -3- O, characterized in that it contains the amino acid sequence of SEQ ID NO: 2 - The method of galactose (quercetin-3- O -galactose) compound . In the E. coli galE (UDP-galactose epimerase) -3- O Quebec paroxetine, characterized in that the gene is a deletion of the E. coli of claim 5, wherein - galactose (quercetin-3- O -galactose) to obtain a compound. An expression vector containing a gene encoding a Petunia hybrida UDP-dependent glycosyltransferase (PeF3GalGT) protein and a UDP-galactose synthase It is transformed with the expression vector containing the gene Quebec paroxetine -3- O - galactose (quercetin-3- O -galactose) of E. coli to produce a compound. 9. The method of claim 8 wherein the sugar derived from Petunia transferase PeF3GalGT proteins Quebec paroxetine -3- O, characterized in that it contains the amino acid sequence of SEQ ID NO: 2 to produce galactose (quercetin-3- O -galactose) compound Escherichia coli. 9. The Escherichia coli producing a quercetin-3- O -galactose compound according to claim 8, wherein the Escherichia coli is Escherichia coli lacking the galE (UDP-galactose epimerase) gene. 11. The method of claim 10, wherein the UDP-galactose synthesis The gene is derived from rice having the nucleotide sequence of SEQ ID NO: 3 OsUGE gene or a galE derived from Escherichia coli having the nucleotide sequence of SEQ ID NO: 4 3- O -galactose compound, wherein the quercetin-3- O -galactose compound is quercetin-3- O -galactose.

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