KR101434602B1 - Method for Preparing 3―Ｏ―Xylosyl Quercetin from Quercetin Using Microorganism Mutants - Google Patents

Abstract

The invention in quercetin 3- O (quercetin) using the mutant microorganism-xylene to room quercetin (3- O -xylosyl quercetin) the present invention relates to a method of making, and more particularly, to trehalose biosynthesis pathway UDP- xylene (UDP-xylose biosynthtic pathway) and a gene coding for glycosyltransferase were introduced into microorganism variants and microorganism variants having the ability to produce 3- O -xylosylcerecetin in quercetin, O -xylosyl quercetin. ≪ / RTI >
The 3- O -xylosyl quercetin produced using the gene encoding the enzyme related to the UDP-xylose biosynthetic pathway of the present invention and the microorganism variant into which the gene coding for glycosyltransferase is introduced has anti-cancer effect, It can be used as a food and cosmetic composition because it has effects such as anti-aging, whitening and anti-stress.

Description

Method for preparing 3-O-xylosyl quercetin from quercetin using microorganism variants [0001] The present invention relates to a method for preparing 3-O-xylosyl quercetin from quercetin using microorganism variants,

The invention in quercetin 3- O (quercetin) using the mutant microorganism-xylene to room quercetin (3- O -xylosyl quercetin) the present invention relates to a method of making, and more particularly, to trehalose biosynthesis pathway UDP- xylene (UDP-xylose a gene coding for an enzyme associated with the biosynthtic pathway and a gene coding for glycosyltransferase are introduced and microorganism variants and microorganism variants having the ability to produce 3- O -xylosylcerecetin in quercetin are used 0.0 > 3- O -xylosyl quercetin < / RTI > in quercetin.

Among the various phytochemicals derived from plants, flavonoids are yellow-based pigments widely distributed in foods. Two phenyl groups mediate the C ₃ chain to form a C ₆ -C ₃ -C ₆ carbon skeleton And it is often present in the form of a glycoside through several sugars and ether linkages. Flavonoids have antimicrobial, anticancer, antiviral, antiallergic and anti-inflammatory activities. In the field of medicine, new flavonoid substances are extracted and the diseases to be treated are diversified to be used for various diseases such as liver disease, diabetes, rheumatoid arthritis, various cancers, , An allergic disease, atherosclerosis, vascular diseases, myocardial diseases, stress and depression are known to have pharmacological activity.

Quercetin is a flavonoid known to be effective in antioxidant, anti-inflammatory and memory enhancement and is known to be found in vegetables such as apples, apricots, grapes and berries, fruits or broccoli, lettuce, tomatoes and onions. Quercetin, like other flavonoids, has antioxidant properties that remove active oxygen, maintain the proper amount of cells and glutathione, maintain anti-inflammatory and anti-allergic effects, regulate histamine release and inflammatory responses, and arteriosclerosis (Jeremy Appleton, ND et al . , Natural (2002)) . In addition , quercetin has been shown to inhibit intestinal glucose uptake Medicine Journal , 2 (1): 1, 2010).

Quercetin, like flavonoid compounds, has problems such as heat instability, instability of oxidizing conditions, and insolubility to water. However, quercetin glycosides are known to have such a problem to be reduced and bioavailability to be increased, But rather in the form of glycosides combined with several types of single or multiple sugars, such as glucose, rhamnose, galactose and arabinose, rather than form.

Methods for producing flavonoids, isoflavonoids, and anthocyanins and the like as microorganism fermentation products by transforming microorganisms such as E. coli , Streptomyces , and Saccharomyces have been studied, and quercetin Such as UDP-glucose, sucrose, TDP-rhamnose, TDP-allose, and UDP-xylose, which are used for glycosylation, A method of transforming a microorganism so that the sugar donor is overexpressed in cells has been studied (Malla, S., et al ., Appl . Environ . Microbiol . , 78: 684, 2011; Park, SR et al . , J. Microbiol . Biotechnol ., 21: 1143. 2011; Trantas, E et al . , Metab . Eng ., 11: 355, 2009; Yan, Y. meat al . , Appl . Environ . Microbiol. , ≪ / RTI > 71: 3617, 2005; Katsuyama, Y. meat al . , Chem . Biol . , 14: 613, 2007).

On the other hand, the inventors of the present invention using a microorganism mutant was transformed in the same manner as 3- O - Lam nosil quercetin (rhamnosyl quercetin), 3- O - Lam nosil campaign roll (rhamnosyl kaempferol), 3- O - allo Allosyl quercetin (Simkhada, D. et al . , Biotechnol Bioeng . , 107: 154, 2010), Naryn genin 7- O - Giles the side (naringenin 7- O -xyloside) (Simkhada , D. et al . , Mol Cells . , 28: 397, 2009), a stylized flower successfully allocated, the over-expression to the enzyme associated with the biosynthesis of UDP-Ara4FN the Arabidopsis thaliana derived dangjeon used as the enzyme donor and a sugar, such as E. coli quercetin in quercetin -3- O - Ara4FN (Kim, BG et < RTI ID = 0.0 > al . , & Lt ; / RTI > Chembiochem ., 11: 2389, 2010).

Therefore, the present inventors have made intensive efforts to develop a method for producing 3- O -xylosylcerecetin in quercetin. As a result, the inventors have found that a gene encoding an enzyme related to the UDP-xylose biosynthetic pathway and a gene encoding glycosyltransferase O -xylosyl quercetin was produced in quercetin at a high conversion rate when the introduced microorganism variant was used, thereby completing the present invention.

It is an object of the present invention to provide a microorganism variant and microorganism variant having an ability to produce 3- O -xylosylcerecetin in quercetin by introducing a gene encoding an enzyme associated with the UDP-xylose biosynthetic pathway and a gene encoding glycosyltransferase, O -xylosyl quercetin by using quercetin.

In order to accomplish the above object, the present invention provides a method for producing UDP-xylose from glucose-6-phosphate and a method for synthesizing a gene encoding a glycosyltransferase-encoding gene, -3 gene is introduced to provide a microorganism variant having the ability to produce 3- O -xylosylcercetin in quercetin.

The present invention also relates to a method for producing 3- O -xylosyl quercetin, comprising: (a) culturing said microorganism variant in a medium containing quercetin to produce 3- O -xylosyl quercetin; And (b) the resulting 3- O - recovering the quercetin in xylene at room quercetin 3- O - provides a method for producing yarn quercetin as characters.

The 3- O -xylosyl quercetin produced using the gene encoding the enzyme related to the UDP-xylose biosynthetic pathway of the present invention and the microorganism variant into which the gene coding for glycosyltransferase is introduced has anti-cancer effect, It can be used as a food and cosmetic composition because it has effects such as anti-aging, whitening and anti-stress.

FIG. 1 is a schematic diagram showing the step of generating 3- O -xylosyl quercetin by glycosylation of xylose by quercetin by biosynthesis pathway of UDP-xylose and glycosyl transferase.
FIG. 2 is a schematic diagram of a method for producing 3- O -xylosylcerquercetin in a transformed microorganism variant.
Figure 3 is a 3- O production - an analysis using a chromatographic method the yarn quercetin in xylene data (A; 3- O - xylene chamber TLC analysis data from the quercetin, a B: quercetin (●), and 3- O - HPLC analysis data of xylosyl quercetin (*)).
Figure 4 is a 3- O production - the data concerning the mass analysis chamber quercetin in xylene (A: 3- O - xylene room quercetin fragmentation pattern of a, B: 3- O - of the yarn to Giles quercetin ESI-MS / MS analysis data, C: ESI-MS / MS analysis data of quercetin).
FIG. 5 shows data on the degree of production of 3- O -xylosylcerecetin when quercetin was treated at various concentrations.
FIG. 6 shows data on the degree of production of 3- O -xylosylcercetin in LB medium, TB medium and M9 medium.
Figure 7 is data confirming the degree of production of 3- O -xylosylcherchetin in S1, S2, S3 and S4 variants.
8 is data confirming the degree of production of 3- O -xylosylcerecetin in a scale-up system.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect of the invention, the invention is glucose-6-phosphate arGt encoding a trehalose (UDP-xylose) path and the glycosyl transferase raised (glycosyltransferase) that the biosynthesis as xylene from UDP- (glucose-6-phosphate) - 3 gene into a microorganism variant having the ability to produce 3- O -xylosylcercetin in quercetin.

In the present invention, a pathway for biosynthesis of UDP-xylose is a gene encoding phosphoglucomutase, glucose-1-phosphate uridyl lyransferase, A gene encoding UDP-glucose dehydrogenase, and a gene encoding UDP-glucuronic acid decarboxylase. The gene encoding UDP-glucose dehydrogenase may be, for example, have.

In the present invention, a gene encoding phosphoglucomutase, a gene encoding glucose-1-phosphate uridylltransferase, a gene encoding UDP-glucose dihydrogenase (UDP- glucose dehydrogenase, and UDP-glucuronic acid decarboxylase are nfa44530 , galU, calS8 and calS9 , respectively.

The above UDP-xylose biosynthetic pathway converts glucose-6-phosphate into glucose-1-phosphate glucose-1-phosphate and finally converts UDP-xylose into glucose- And the like.

In the present invention, the microorganism variant may be a gene encoding glucose phosphate isomerase and a gene encoding glucose-6-phosphate dehydrogenase. Or more is further defective.

In the present invention, a gene encoding glucose phosphate isomerase and a gene encoding glucose-6-phosphate dehydrogenase are referred to as pgi And zwf .

In the present invention, a 'deletion' of a gene means a state in which the gene is deleted on a chromosome or a plasmid so that the gene can not produce a protein encoded by the gene.

The above glucose phosphate isomerase and glucose-6-phosphate dehydrogenase are prepared by reacting glucose-6-phosphate with fructose-6-phosphate phosphogluconolactone and used for glycolysis and pentose phosphate pathway. When two genes are defected, glycolysis and 5-phosphogluconolactone are converted into 6-phosphogluconolactone. The pentose phosphate pathway is blocked and most of the glucose-6-phosphate is used in the UDP-xylose biosynthetic pathway, resulting in increased production of UDP-xylose.

In the present invention, the microorganism is Escherichia coli ), but the present invention is not limited thereto.

In the present invention, the above-mentioned phosphoglucomutase is preferably a phosphogluconase selected from the group consisting of Nocardia farcinica IFM10152. < / RTI >

In the present invention, the above-mentioned glucose-1-phosphate glassy 1-phosphate uridyl lransferase is preferably Escherichia coli K-12. < / RTI >

In the present invention, the UDP-glucose dehydrogenase and the UDP-glucuronic acid decarboxylase may be used in combination with a micromonospora echinospora ssp.).

In the present invention, the glycosyltransferase may be Arabidopsis thaliana . < / RTI >

The glycosyltransferase may be characterized in that UDP-xylose produced through a UDP-xylose biosynthetic pathway is used to divide quercetin into 3- O -xylosyl quercetin.

In one embodiment of the present invention, in order to overexpress UDP-xylose in E. coli, phosphoglucomutase ( nfa44530 ), an enzyme associated with the UDP-xylose pathway, glucose-1-phosphate glassylytransferase (glucose-1-phosphate uridylyltransferase, g alU), UDP- glucose-dihydro-rise centipede (UDP-glucose dehydrogenase, calS8) and UDP- glucuronic dicarboxylate raised (UDP-glucuronic acid decarboxylase, calS9 ) and a glycosyl transferase Reyes and a gene of glycosyltransferase was cloned to prepare a plasmid. In addition, the pgi gene coding for glucose phosphate isomerase and the zwf gene encoding glucose-6-phosphate dehydrogenase Escherichia coli mutants in which one or more of the genes were further deleted were finally prepared to produce S1, S2, S3 and S4 variants (Table 2).

In another aspect of the present invention, the present invention provides a method for producing 3- O -xylosyl quercetin, comprising: (a) culturing the microorganism variant in a medium containing quercetin to produce 3- O -xylosyl quercetin; And (b) the resulting 3- O - recovering the quercetin in xylene at room quercetin 3- O - relates to a process for producing quercetin chamber with xylene.

In one embodiment of the present invention, the transformed E. coli mutant is produced by converting glucose to glucose-6-phosphate and then generating UDP-xylose by an enzyme associated with the over-expressed UDP-xylose biosynthetic pathway and adding quercetin , The glycosyltransferase overexpressed in E. coli mutant is converted to 3- O -xylosyl quercetin by using UDP-xylose produced as described above as a sugar donor, and finally, 3- O -xylosyl quercetin can be prepared (Fig. 2).

To confirm that the transformed Escherichia coli mutant of the present invention produced 3- O -xylosyl quercetin in quercetin, quercetin was added to the S1 mutant and cultured and analyzed by the TLC method. As a result, the R _f value was 0.5 3- O -xylosyl quercetin was produced in the S1 mutant (FIG. 3A). As a result, the retention time of the standard substance of quercetin and the standard substance of 3- O- xylosyl quercetin ) Showed 14.2 min and 5.8 min, indicating an increase in the conversion rate from quercetin to 3- O -xylosyl quercetin according to the incubation time of the S1 variants (Fig. 3B)

In addition, the production of 3- O - for quantitative check of the actual quercetin as xylene, ESI-MS / MS (electrospray ionization tandem mass spectrometry) was analyzed by the method, a total of m / z values of the isolated compound is 433 to S1 quercetin by the mutant ^{([M-xyl] - m} / z = 301) it was found that an acylated in xylene of, 3- O - were compared with the standard of the actual quercetin as xylene, produced by the production process of the invention O -xylosyl quercetin was confirmed to be consistent with the reference material (FIG. 4 and Table 3).

In order to increase the conversion yield of 3- O -xylosyl quercetin in quercetin using the transformed E. coli mutant of the present invention, 100 μM, 200 μM, 300 μM, 500 μM and 1000 μM of quercetin were added to obtain 3- O -xylosyl quercetin The conversion yield of 3- O -xylosylcerecetin was decreased due to cytotoxicity when high concentration of quercetin was added. When cultured for 48 hours with 100 μM of quercetin, the conversion yield of 3- O -xylosyl quercetin was produced (Fig. 5). When S1 mutants were cultured using LB medium, TB medium, and M9 medium, 11.46 μM 3- O -xyloin was obtained when mutants were cultured in TB medium, which were 1.43 times and 3.44 times higher than LB medium and M9 medium, respectively (Fig. 6). ≪ tb >< TABLE >

In order to increase the production of UDP-xylose in the transformed E. coli mutant of the present invention, Escherichia coli mutants (S2, S3 and S4) lacking the pgi gene and the zwf gene were prepared and the conversion of 3- O -xylosylcerecetin As a result, it was confirmed that the S2 mutant had a conversion rate of 28.6%, 3.6 times higher than the S1 mutant, and 1.7 times and 1.32 times higher than the S3 mutant and S4 mutant, respectively 7). In addition, it was confirmed that the mutant in which the pgi gene was deleted showed a higher conversion yield of 3- O -xylosyl quercetin than the mutant in which the zwf gene was deleted.

When using a S2 variant showed a high conversion yield of the room as quercetin in xylene of culture with quercetin in 3ℓ fermenter, 3- O in quercetin-3- O in the present invention in confirming the quercetin in the chamber quercetin conversion yield in Giles As a result, it was confirmed that 3- O -xylosyl quercetin was converted at a yield of about 98% (127.6 mg) when cultured for 6 hours (FIG. 8).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 . UDP - Xylose  Pathway-related enzymes and Glycosyltransferase  Production of transformed E. coli mutants

1-1: UDP - Xylose  Enzymes involved in the biosynthesis process and Glycosyltransferase end Cloned  Plasmid production

In order to overexpress UDP-xylose in E. coli, phosphoglucomutase ( nfa44530 ), glucose-1-phosphate uridyllyl transferase (g alU), UDP- glucose-dihydro-rise centipede (UDP-glucose dehydrogenase, calS8) and UDP- glucuronic dicarboxylate raised (UDP-glucuronic acid decarboxylase, calS9 ) and glycosyl by cloning the gene transfer raise (glycosyltransferase) plasmid Respectively.

First, the nfa44530 (GenBank accession No. BAD59304, SEQ ID NO: 1) gene and galU (GenBank accession No. BAA36104, SEQ ID NO: 2) gene are nfa44530 F / nfa44530 R (SEQ ID NOS: 3 and 4) and galU F / galU R Nos. 5 and 6) was amplified by PCR using a pair of primers, restriction sites Eco (MCS1 of each pETDuet-1 vector RI / Hin dIII) and was cloned into the MCS2 (restriction sites Eco RV / Kpn I) pET- nfa44530 -galU plasmid.

calS8 (GenBank accession No.AAM70332.1 SEQ ID NO: 7) gene and c alS9 (Genbank accession No.AAM70333.1, SEQ ID NO: 8) gene calS8 F / calS8 R (SEQ ID NO: 9 and 10) and calS9 F / R calS9 ( Bam HI / Hin dIII) and MCS2 ( NdeI / XhoI ) of the pCDFDuet-1 vector, respectively, and then cloned into pCDF- calS89 ( SEQ ID NO: The plasmid Respectively.

In order to produce a galU integrated vector (integration vector), the gene containing the galU -IF / galU -IR after using the primer pair (SEQ ID NO: 13 and 14) were amplified by PCR, pLOI2223 integration vector Eco RI / Bam HI restriction enzyme site to construct pLLO2223- galU plasmid.

Arabidosis thaliana) a transferase per derived arGt -3 (GenBank accession No. AF360160, SEQ ID NO: 15) gene was amplified by PCR using the arGt -3 F / arGt -3 R (SEQ ID NO: 16 and 17) primers , and cloned into the Eco RI / Xho I site of pET28a (+) vector to construct pET28- arGt- 3 plasmid.

1-2: Pgi  Gene and zwf The gene Deficient Mutant  making

In order to increase the production of the 3- O -xylosyl quercetin of the present invention, the pgi gene encoding glucose phosphate isomerase and glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase) zwf coding for the dehydrogenase) Escherichia coli mutants were produced in which one or more of the genes were further deleted. pg (gene number, SEQ ID NO: 18) and zwf (gene number, SEQ ID NO: 19) genes were cloned into the FLP / FRT system and Del pgi F / Del pgi R (SEQ ID NOs: 20 and 21) Using the zwf F Del / Del pzwf R (SEQ ID NO: 22 and 23) primers finally pgi Gene and zwf And a microorganism mutant in which the gene was deleted was obtained.

The oligonucleotide sequence used in the preparation of microorganism variants having the ability to produce 3- O -xylosylcercetin in quercetin primer Oligonucleotide Sequences (5 ' -3 ') nfa -44530 F (SEQ ID NO: 3) GAATTC GATGATCCTGATGACGAGCCGGG nfa -44530 R (SEQ ID NO: 4) AAGCTT TCACGGCTGGAGCGCCTTGCC galU F (SEQ ID NO: 5) GATATC GATGGCTGCCATTAATACGAAAGT galU R (SEQ ID NO: 6) GGTACC TTACTTCTTAATGCCCATCTCTTC calS8 F (SEQ ID NO: 9) AGC GGATCC CATCATGCCGTTCCTTCC calS8 R (SEQ ID NO: 10) AGC AAGCTT TCACCTTCCAATGCCGC calS9 F (SEQ ID NO: 11) AAC CATATG CCCAGATCCCTGGTCACC calS9 R (SEQ ID NO: 12) AGT CTCGAG CTACCTGACGACCAGTCC galU - IF (SEQ ID NO: 13) AGCGAATTCATGGCTGCCATTAATACG galU - IR (SEQ ID NO: 14) GCA GGATCC TTACTTCTTAATGCCCAT argt -3 F (SEQ ID NO: 16) AGC GAATTC ATGACCAAATTCTCCGAG argt - 3R (SEQ ID NO: 17) AGC CTCGAG CTAAACTTTCACAATTTC Del pgi F (SEQ ID NO: 20) TTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCTA GTGTAGGCTGGAGCTGCTTC Del pgi R (SEQ ID NO: 21) GCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATG ACATATGAATATCCTCCTTAG Del zwf F (SEQ ID NO: 22) CTGGCTTAAGTACCGGGTTAGTTAACTTAAGGAGAATGAC GTGTAGGCTGGAGCTGCTTC Del zwf R (SEQ ID NO: 23) ATGTTACCGGTAAAATAACCATAAAGGATAAGCGCAGAT ACATATGAATATCCTCCTTAG

1-3. Construction of Transformants Using Recombinant Plasmids

The recombinant plasmids prepared above were introduced into Escherichia coli ( BL21) and Escherichia coli mutants lacking the pgi gene and the zwf gene under various conditions to construct a transformant (Table 2). S1 mutant wild-type (wild type) was introduced into the gene nfa44530, galU, calS8 and calS9 in E. coli, S2 variants were introduced nfa44530, galU, calS8 and calS9 gene in E. coli (BL21 / Δ pgi) with a pgi gene is deficient . Also, S3 variant was introduced into nfa44530, galU, calS8 and calS9 gene in E. coli (BL21 / Δ zwf) with the zwf gene is deficient, S4 variant is the galU gene transfer using the integrated vector (integration vector) and pgi It was introduced and calS8 calS9 gene in E. coli (BL21 / Δ pgi), which gene is deficient.

Construction of Transformants Using Recombinant Plasmids strains  Description S1 BL21 (DE3) / nfa44530 - galU / calS89 / arGt -3 S2 BL21 (DE3) / Δ pgi / nfa44530 - galU / calS89 / arGt -3 S3 BL21 (DE3) / Δ zwf / nfa44530 - galU / calS89 / arGt -3 S4 galU integrated E. coli BL21 (DE3) / Δ pgi / calS89 / arGt -3

Experimental Example  1: Transformed microorganism Mutant  In the quercetin used, 3- O - Xylosyl  Manufacture of quercetin

Experiments were performed to confirm the production of 3- O -xylosylcerecetin in quercetin using the transformed microorganism variants in Example 1-3.

S1 mutants were inoculated in 3 ml of LB liquid medium containing antibiotics, and then cultured overnight at 7 DEG C and 220 rpm. 500 쨉 l of the cultured S1 mutant was inoculated into 5 ml of LB medium and cultured at 37 째 C until OD (600 nm) reached 0.6. Then, 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and cultured at 20 ° C. for 3 hours. Then, 100 μM of quercetin was further added and incubated at 20 ° C. for 60 hours with shaking at 220 rpm. During the incubation, 300 μl was taken every 12 hours, treated with twice the volume of ethyl acetate (JUNSEI, Japan), and then the solvent was evaporated to dryness and concentrated. The concentrated material was dissolved in 500 μl of methanol and used for analysis such as TLC and HPLC.

Experimental Example  2. Microorganisms Mutant  3- O - Xylosyl  Analysis of Quercetin

2-1. TLC  And HPLC Lt; RTI ID = O - Xylosyl  Analysis of Quercetin

O -xylosyl quercetin prepared using the S1 mutant of Experimental Example 1 was analyzed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).

The specimens loaded on the TLC plate were run on a normal system in a closed TLC chamber under the conditions of ethyl acetate: methanol: water: toluene (10: 1.5: 1.3: 0.2) and the spots were visualized by UV illumination. Reversed phase HPLC analysis was carried out using 70% water (0.1% trifluroacetic acid buffer) and 30% acetonitrile using a UV detector (330 nm) connected to a C18 column (Mightysil, ODS Hypersil; 4.6 × 250 mm; Lt; RTI ID = 0.0 > ml / min. ≪ / RTI >

As a result of TLC analysis, it was confirmed that 3- O -xylosyl quercetin was produced in the S1 mutant ( _Rf value: 0.5) (FIG. 3A). As a result of HPLC analysis, quercetin standards and 3- O- xylosyl residence time (retention time) of the reference material of quercetin is quercetin 3- O in accordance with the incubation time of the S1 mutant with 14.2 minutes and 5.8 minutes was found to increase the conversion rate as quercetin chamber in xylene (FIG. 3B)

For the quantitative determination of the results, an accurate quercetin calibration curve was identified at various concentration conditions and analyzed by ESI-MS / MS (electrospray ionization tandem mass spectrometry) using a Thermo Finnigan TSQ 7000 mass spectrometer.

As a result, the total of m / z values of the isolated compounds by the S1 mutant with quercetin 433 ^{([M-xyl] - m} / z = 301) it was found that an acylated in xylene of, 3- O - room in Giles As a result of comparing with quercetin standard materials, it was confirmed that 3- O -xylosyl quercetin produced by the production method of the present invention agrees with the standard material (FIG. 4).

2-2: 3-O- xylosyl quercetin  Purification and structure analysis

In order to purify and analyze the structure of 3- O -xylosyl quercetin prepared using the S1 mutant, the sample of Experimental Example 1 was purified using silica gel column chromatography using an isocratic solvent system. The partially purified result is ^{Amberlite ® XAD7HP (20-60 mesh; Sigma} -Aldrich) using a non-ionic and was freeze-dried to yield the resin of the absorbent, NMR spectra (aVarian Unity Inova 300 MHz, FT-NMR) structure (Table 3).

NMR analysis of 3- O -xylosyl quercetin prepared using microbial mutants Position of Carbon 13C NMR chemical Shifts  ( Parts  per Million ) C-2 156.7 C-3 134.5 C-4 177.9 C-5 161.48 C-6 101.8 C-7 164.14 C-8 93 C-9 157.5 C-10 98.1 C-1 ' 121.2 C-2 ' 115.2 C-3 ' 144.7 C-4 ' 148 C-5 ' 114.6 C-6 ' 121.1 C-1 " 104.1 C-2 " 70.4 C-3 " 71.5 C-4 " 70.3 C-5 " 70.2

Experimental Example  3. Microorganisms Mutant  Quercetin-3- O - Xyloside Determination of optimal reaction conditions to produce

In order to establish optimal reaction conditions for producing quercetin-3- O -xyloside in quercetin using the transformed E. coli mutant of the present invention, the S1 mutant was cultured in the same manner as in Experimental Example 1, The concentrations were 100 μM, 200 μM, 300 μM, 500 μM and 1000 μM, and cultured at 20 ° C. for 60 hours.

As a result, it was confirmed that 3- O -xylosyl quercetin was abundantly produced at 8.01 μM when quercetin was added at 100 μM for 48 hours compared with other conditions (FIG. 5). This can be attributed to the lack of cell growth due to cytotoxicity when high concentrations of quercetin are added.

In order to determine the optimal reaction conditions, S1 mutants were cultured in the same manner as in Experimental Example 1 using LB medium, TB medium, and M9 medium. As a result, when the mutants were cultured in TB medium, the mutants were 1.43 O -xylosyl quercetin (FIG. 6).

Experimental Example  4. Microorganisms of various conditions Mutant  Using quercetin 3- O - Preparation of xylosyl quercetin

In order to increase the conversion yield of 3- O -xylosyl quercetin in quercetin by using the transformed E. coli mutant of the present invention, as in Example 1, the pgi gene encoding glucose phosphate isomerase (S2, S3 and S4) lacking the zwf gene coding for glucose-6-phosphate dehydrogenase were prepared and subjected to the same procedure as in Experimental Example 1 using TB medium After cultivation, the conversion yield to 3- O -xylosyl quercetin was confirmed.

As a result, it was confirmed that the S2 mutant had a conversion rate of 28.6%, which was 3.6 times higher than the S1 mutant, and 1.7 and 1.32 times higher conversion efficiency than the S3 and S4 mutants, respectively (FIG. 7) . Also, pgi It was confirmed that the mutant lacking the gene had a higher conversion yield of 3- O -xylosyl quercetin than the mutant lacking the zwf gene.

Experimental Example  5. Scale up using fermenter ( scale - up ) From quercetin in the system 3- O -character Work Manufacture of losyl quercetin

When the S2 mutant showing a high conversion yield was cultured in a 3 L fermenter, an experiment was conducted to confirm the conversion yield of 3- O -xylosyl quercetin in quercetin.

S2 mutants were inoculated in 300 ml of TB liquid medium containing antibiotics, and then cultured overnight at 7 DEG C and 220 rpm. The cultured S2 mutant was added to the fermenter in sterile condition with 3 L sterilized TB medium. The pH was kept at 7.0 using ammonium hydroxide (28%; DAEJUNG, Korea) and maintained at 95% or more of dissolved oxygen (DO) and kept below 20%. When the absorbance at 600 nm was at least 5, 0.2 mM IPTG was added and cultured for 5 hours. Then, quercetin was added to a final concentration of 100 μM and cultured at 25 ° C for 48 hours. Glucose solution was fed to the medium during incubation and sampled at 4 hour intervals for HPLC analysis.

As a result, it was confirmed that 3- O -xylosyl quercetin was produced at a yield of about 98% (127.6 mg) when cultured for 36 hours (FIG. 8).

Culture conditions and conversion yield of 3- O -xylosylcerecetin Substrate Quercetin 100 M Fermentation Conditions Media TB Volume 3ℓ Temperature 25 ^o C pH 7.0 Dissolved Oxygen 90% (> 20% throughout the experiment) Duration 36h Product calculated 98% Yield 127 mg

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Sunmoon University Industry-University Cooperation Foundation <120> Method for Preparing 3-O-Xylosyl Quercetin from Quercetin Using          Microorganism Mutants <130> P12-B113 <160> 23 <170> Kopatentin 2.0 <210> 1 <211> 1689 <212> DNA <213> Escherichia coli K-12 nfa44530 <400> 1 tcacggctgg agcgccttgc ccaccatctc ctcggcggcg gcctggacct gggcgaggtg 60 ctcggggccc tggaacgact cggcgtagat cttgtacttg tcctcggtgc cggagggccg 120 ggccgcgaac caggcgttct cggtggtcac cttcaacccg cccaggggcg cgccgttgcc 180 gggggcgcgg gtgagcacgg cggtgatcgg ctcgccggcg atctcttcgc tggtgatcat 240 gtccggtgtc aactgcgaaa gcagcttctt ctgttccgcg ccggccggtg cgtcgatgcg 300 ggcgtaggcg gggctgccgt accggcgctc gagttcgccg tagcgggccg agggggtctg 360 gccggtgacg gcggcgatct cggcggccag cagggccagc aggatgccgt ccttgtcggt 420 ggtccacacg gtgccgtcca tgcgcaggaa cgacgcgccc gcgctctcct cgccgccgaa 480 agcgagactg ccgctgaaca atccgggcac gaaccatttg aagccgaccg gcacctcgtg 540 cacctggcgg ccgagcacgc tcaccacccg gtccaccatc gacgaggtga cgacggtctt 600 gccgatcttg gtcagcgcgt cccagcccat ccggttggcg accaggtatt cgatggcgac 660 ggccaggaag tggttggggt tcatcaggcc accgtcgggg gtgacgatgc cgtgccggtc 720 ggcgtcggcg tcgttgccgg tggagatgtc gtagtcgtcc ttgatcgcga cgagcccggc 780 catggcgtag cgggaggag gatccatgcg gatcttgccg tcgctgtcga gggtcatgaa 840 acgccaggtg gggtcgacga acgggttgac gacctcgagc tcgaggtcgt agcgctggcc 900 gatctcctcc cagtagtcga cgctggcgcc gcccatcggg tcggcgccga gccggatgcc 960 cgcgccgcga atcgcgtcca ggttcagcac attcggcagg tcggcgatgt agtggtcgag 1020 gtagtcgtag cgttcgacgc cggtggccag cgcctgctgg taggtggcgc gacgcacccc 1080 tgtcagcccg tcggccagca gttcgttggc gcgggcggcg atggcgtcgg tgacgccggt 1140 gtcggccgga ccaccgtgcg gggggttgta tttgaagccg ccgtcgcggg gcgggttgtg 1200 tgagggggtc accacgatgc cgtcggcctg gtgcttggtg ccgccgcggt tgtgccgcag 1260 ccggcgtgg ctcagcgccg gggtgggggt gtagcggtcg cgggcgtcga tcatggcggt 1320 gacgccgttg ccggccagca cctcgagggc ggtggtccag gcgggctcgg acagcgcgtg 1380 ggtgtcgcgg gccaggtaga ccgggcccgt gatgccgcgc gtggcacggt attccacgat 1440 cgcctgggtg atggcgagga tgtgggcttc gttgaacgcg cagtccaggc tggacccgcg 1500 gtgtcccgag gtgccgaaca ccactcgctg tgccggatcc cgcggatcgg gcacgcggct 1560 gaagtaggcg gtcaccaggt gcgcgatgtc gtccaggtcg ctcgcgcgcg cgggacgccc 1620 ggctcgatcg tgggccatgc tcgggtctcc cttccaggtc ggctgcgccc ggctcgtcat 1680 caggatcat 1689 <210> 2 <211> 909 <212> DNA <213> Escherichia coli K-12 galU <400> 2 atggctgcca ttaatacgaa agtcaaaaaa gccgttatcc ccgttgcggg attaggaacc 60 aggatgttgc cggcgacgaa agccatcccg aaagagatgc tgccacttgt cgataagcca 120 ttaattcaat acgtcgtgaa tgaatgtatt gcggctggca ttactgaaat tgtgctggtt 180 acacactcat ctaaaaactc tattgaaaac cactttgata ccagttttga actggaagca 240 atgctggaaa aacgtgtaaa acgtcaactg cttgatgaag tgcagtctat ttgtccaccg 300 ccgtgacta ttatgcaagt tcgtcagggt ctggcgaaag gcctgggaca cgcggtattg 360 tgtgctcacc cggtagtggg tgatgaaccg gtagctgtta ttttgcctga tgttattctg 420 gatgaatatg aatccgattt gtcacaggat aacctggcag agatgatccg ccgctttgat 480 gaaacgggtc atagccagat catggttgaa ccggttgctg atgtgaccgc atatggcgtt 540 gtggattgca aaggcgttga attagcgccg ggtgaaagcg taccgatggt tggtgtggta 600 gaaaaaccga aagcggatgt tgcgccgtct aatctcgcta ttgtgggtcg ttacgtactt 660 agcgcggata tttggccgtt gctggcaaaa acccctccgg gagctggtga tgaaattcag 720 ctcaccgacg caattgatat gctgatcgaa aaagaaacgg tggaagccta tcatatgaaa 780 gggaagagcc atgactgcgg taataaatta ggttacatgc aggccttcgt tgaatacggt 840 attcgtcata acacccttgg cacggaattt aaagcctggc ttgaagaaga gatgggcatt 900 aagaagtaa 909 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> nfa44530F <400> 3 gaattcgatg atcctgatga cgagccggg 29 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> nfa44530R <400> 4 aagctttcac ggctggagcg ccttgcc 27 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> galUF <400> 5 gatatcgatg gctgccatta atacgaaagt 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> gall <400> 6 ggtaccttac ttcttaatgc ccatctcttc 30 <210> 7 <211> 1362 <212> DNA <213> Nocardia farcinica IFM10152 calS8 <400> 7 atgccgttcc ttcccgaccc gggcgaaccg tccccgctga aggtggtcat cgccggcgcc 60 ggctacgtcg gcacctgtct cgccgtcacc ctcgccggcc gcggcgccga ggtggtcgcg 120 gtcgacagcg acccgggcac cgtcgcggac ctgcgggccg gccggtgccg gctgcccgag 180 cccggcctgg ccggcgccgt ccgggacctc gccgcgaccg gacggctgac ggcgagcacg 240 tcgtacgacc cggtcggcgc ggcggacgtg gtgatcgtga cggtcggcac cccgaccgac 300 gccggccacg agatggtcac cgaccagctc gtcgcggcgt gcgagcagat cgccccgcgg 360 ctgcgcgccg ggcaactggt gatcctcaag tcgacggtct ccccgggcac cacccggacc 420 ctcgtcgcgc ccctgctgga gagcggcggg ctggtgcacg agcgcgactt cgggctggcc 480 ttctgcccgg agcggctcgc cgagggggtg gcgctggcgc aggtgcggac gctgccggtg 540 gtggtgggtg ggtgcggccc gcgcagcgcc gccgcggccg aacggttctg gcggtccgcg 600 ctcggcgtcg acgtccggca ggtgccgtcg gccgagtccg ccgaggtggt caagctcgcg 660 accaactggt ggatcgacgc gaacgtggcg atcgccaacg aactcgcccg gtactgcgcg 720 gtgctggggg tggacgtcct cgacgtgatc ggcgcggcga acaccctgcc caagggcagc 780 agcatggtga acctgctgct gccgggggtg ggtgtcggcg gctcctgcct gacgaaggac 840 ccgtggatgg cgtggcggga cggccgggac cggggcgtgc ccctgcgcac ggtcgagacg 900 gcccgcgcgg tcaacgacga catgccccgc cacaccgccg ccgtcatcgc cgacgagctg 960 gtcaagctgg gacgggatcg gaacgacacg acgatcgccg tgctcggcgc ggcgttcaag 1020 aacgacaccg gcgacgtccg caacaccccg gtgcgcgggg tcgtggcggc gctgcgcgac 1080 agcggcttcc gggtccggat cttcgacccg ctggccgatc ccgccgagat cgtcgcccgg 1140 ttcggcaccg cgccggcggc gagcctggac gaggcggtga gcggggcggg ctgcctggcc 1200 ttcctcgccg ggcaccgcca gttccacgag ctcgacttcg gcgccctggc cgagcgggtg 1260 gcgagccct gcctggtctt cgacggccgc atgcacctcc cgccggcgcg catccgcgag 1320 ctgcaccggt tcggcttcgc ctaccgcggc attggaaggt ga 1362 <210> 8 <211> 990 <212> DNA <213> Nocardia farcinica IFM10152 calS9 <400> 8 gtgcccagat ccctggtcac cggcggcttc ggcttcgtcg gcagtcacgt cgtcgaacgg 60 ctggtccgcc ggggtgacga ggtcgtcgtc tacgacctcg ccgacccgcc gcccgacctg 120 gagcacccgc cgggcgcgat ccggcacgtc cgcggcgacg tccgggacgc cgacgggctg 180 gcggccgccg ccaccggcgt ggacgaggtc taccacctcg cggcggtcgt cggcgtcgac 240 cggtacctca gccggccgct ggacgtggtc gagatcaacg tggacggcac ccggaacgcg 300 ttgcgcgccg cactgcgcgc cggtgcccgg gtcgtggtgt ccagcaccag cgaggtgtac 360 gggcgcaatc cgcgggtgcc gtggcgggag gacgacgacc gggtgctcgg cagcacggcg 420 acggaccggt ggtcgtactc gacgagcaag gcggcggccg agcacctggc cttcgccttc 480 caccggcagg agggcctgcc ggtgacggtg ctgcggtact tcaacgtcta cggcccacgc 540 cagcgcccgg cgtacgtcct cagccgcacc gtcgcccgcc tgctgcgggg cgttccgccc 600 gtggtgtacg acgacggccg ccagacgcgg tgcttcacct ggatcgacga ggcggccgag 660 gcgaccctgc tggccgccgc ccacccgcgg gccgtcggcg agtgtttcaa catcggcagc 720 agcgtggaga ccaccgtcgc cgaggcggtc cggctggccg gcacggtggc cggggtgccg 780 gtggcggccc agaccgcgga caccggagcc gggctcggcg cccgctacca ggacattccc 840 cgccgcgtac cggactgcgg caaggccgcc gcgctgctgg actggcgggc ccgggtgccg 900 ctggtgaccg gcctgcgccg gaccgtcgag tgggcccgcc gcaacccgtg gtggaccgcc 960 caggccgacg acggactggt cgtcaggtag 990 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> calS8F <400> 9 agcggatccc atcatgccgt tccttcc 27 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> calS8R <400> 10 agcaagcttt caccttccaa tgccgc 26 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> calS9F <400> 11 aaccatatgc ccagatccct ggtcacc 27 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> calS9R <400> 12 agtctcgagc tacctgacga ccagtcc 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> galU-IF <400> 13 agcgaattca tggctgccat taatacg 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> galU-IR <400> 14 gcaggatcct tacttcttaa tgcccat 27 <210> 15 <211> 1533 <212> DNA <213> Arabidopsis thaliana putative UDP glucose: flavonoid 3-o-glucosyltransferase <400> 15 atcattcact cacgacacta accatgacca aattctccga gccaatcaga gactcccacg 60 tggcagttct cgcgtttttc cccgttggcg ctcatgccgg tcctctctta gccgtcactc 120 gccgtctcgc cgccgcttct ccctccacca tcttttcttt cttcaacacc gcaagatcaa 180 acgcgtcgtt gttctcctct gatcatcccg agaacatcaa ggtccacgac gtctctgacg 240 gtgttccgga gggaaccatg ctcgggaatc cactggagat ggtcgagctg tttctcgaag 300 cggctccacg tattttccgg agcgaaatcg cggcggcaga gatagaagtt ggaaagaaag 360 tgacatgcat gctaacagat gccttcttct ggttcgcagc ggacatagcg gctgagctga 420 acgcgacttg ggttgccttc tgggccggcg gagcaaactc actctgtgct catctctaca 480 ctgatctcat cagagaaacc atcggtctca aagatgtgag tatggaagag acattagggt 540 ttataccagg aatggagaat tacagagtta aagatatacc agaggaagtt gtatttgaag 600 atttggactc tgttttccca aaggctttat accaaatgag tcttgcttta cctcgtgcct 660 ctgctgtttt catcagttcc tttgaagagt tagaacctac attgaactat aacctaagat 720 ccaaacttaa acgtttcttg aacatcgccc ctctcacgtt attatcttct acatcggaga 780 aagagatgcg tgatcctcat ggctgctttg cttggatggg gaagagatca gctgcttctg 840 tagcgtacat tagcttcggc accgtcatgg aacctcctcc tgaagagctt gtggcgatag 900 cacaagggtt ggaatcaagc aaagtgccgt ttgtttggtc gctgaaggag aagaacatgg 960 ttcatctacc aaaagggttt ttggatcgga caagagagca agggatagtg gttccttggg 1020 ctccacaagt ggaactgctg aaacacgagg caatgggtgt gaatgtgaca cattgtggat 1080 gt; cggataatag gctcaacgga agagcagtgg aggttgtgtg gaaggttgga gtgatgatgg 1200 ataatggagt cttcacgaaa gaaggatttg agaagtgttt gaatgatgtt tttgttcatg 1260 atgatggtaa gacgatgaag gctaatgcca agaagcttaa agaaaaactc caagaagatt 1320 tctccatgaa aggaagctct ttagagaatt tcaaaatatt gttggacgaa attgtgaaag 1380 tttaggttgt ttacaacata attgaagact ataaatgtga tttcaaaatt aacaaaatca 1440 tagtcaaata agtgtgtgcc tattctattt tgtcaaagta agagtgtatg gatctattct 1500 atttcaaagg gttgttgaaa aaaaaaaaaa aaa 1533 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> argt-3F <400> 16 agcgaattca tgaccaaatt ctccgag 27 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> argt-3R <400> 17 agcctcgagc taaactttca caatttc 27 <210> 18 <211> 1650 <212> DNA <213> Escherichia coli phosphoglucoseisomerase <400> 18 atgaaaaaca tcaatccaac gcagaccgct gcctggcagg cactacagaa acacttcgat 60 gaaatgaaag acgttacgat cgccgatctt tttgctaaag acggcgatcg tttttctaag 120 ttctccgcaa ccttcgacga tcagatgctg gtggattact ccaaaaaccg catcactgaa 180 gagacgctgg cgaaattaca ggatctggcg aaagagtgcg atctggcggg cgcgattaag 240 tcgatgttct ctggcgagaa gatcaaccgc actgaaaacc gcgccgtgct gcacgtagcg 300 ctgcgtaacc gtagcaatac cccgattttg gttgatggca aagacgtaat gccggaagtc 360 aacgcggtgc tggagaagat gaaaaccttc tcagaagcga ttatttccgg tgagtggaaa 420 ggttataccg gcaaagcaat cactgacgta gtgaacatcg ggatcggcgg ttctgacctc 480 ggcccataca tggtgaccga agctctgcgt ccgtacaaaa accacctgaa catgcacttt 540 gtttctaacg tcgatgggac tcacatcgcg gaagtgctga aaaaagtaaa cccggaaacc 600 acgctgttcc tggtagcatc taaaaccttc accactcagg aaactatgac caacgcccat 660 agcgcgcgtg actggttcct gaaagcggca ggtgatgaaa aacacgttgc aaaacacttt 720 gcggcgcttt ccaccaatgc caaagccgtt ggcgagtttg gtattgatac tgccaacatg 780 ttcgagttct gggactgggt tggcggccgt tactctttgt ggtcagcgat tggcctgtcg 840 attgttctct ccatcggctt tgataacttc gttgaactgc tttccggcgc acacgcgatg 900 gacaagcatt tctccaccac gcctgccgag aaaaacctgc ctgtactgct ggcgctgatt 960 ggcatctggt acaacaattt ctttggtgcg gaaactgaag cgattctgcc gtatgaccag 1020 tatatgcacc gtttcgcggc gtacttccag cagggcaata tggagtccaa cggtaagtat 1080 gttgaccgta acggtaacgt tgtggattac cagactggcc cgattatctg gggtgaacca 1140 ggcactaacg gtcagcacgc gttctaccag ctgatccacc agggaaccaa aatggtaccg 1200 tgcgatttca tcgctccggc tatcacccat aacccgctct ctgatcatca ccagaaactg 1260 ctgtctaact tcttcgccca gaccgaagcg ctggcgtttg gtaaatcccg cgaagtggtt 1320 ggcaggaat atcgtgatca gggtaaagat ccggcaacgc ttgactacgt ggtgccgttc 1380 aaagtattcg aaggtaaccg cccgaccaac tccatcctgc tgcgtgaaat cactccgttc 1440 agcctgggtg cgttgattgc gctgtatgag cacaaaatct ttactcaggg cgtgatcctg 1500 aacatcttca ccttcgacca gtggggcgtg gaactgggta aacagctggc gaaccgtatt 1560 ctgccagagc tgaaagatga taaagaaatc agcagccacg atagctcgac caatggtctg 1620 attaaccgct ataaagcgtg gcgcggttaa 1650 <210> 19 <211> 1477 <212> DNA <213> Escherichia coli glucose 6-phosphate-1-dehydrogenase <400> 19 atggcggtaa cgcaaacagc ccaggcctgt gacctggtca ttttcggcgc gaaaggcgac 60 cttgcgcgtc gtaaattgct gccttccctg tatcaactgg aaaaagccgg tcagctcaac 120 ccggacaccc ggattatcgg cgtagggcgt gctgactggg ataaagcggc gtataccaaa 180 gttgtccgcg aggcgctcga aactttcatg aaagaaacca ttgatgaagg tttatgggac 240 accctgagtg cacgtctgga tttttgtaat ctcgatgtca atgacactgc tgcattcaac 300 cgtctcggcg cgatgctgga tcaaaaaaat cgtatcacca ttaactactt tgccatgccg 360 cccagcactt ttggcgcaat ttgcaaaggg cttggcgagg caaaactgaa tgctaaaccg 420 gcacgcgtag tcatggagaa accgctgggg acgtcgctgg cgacctcgca ggaaatcaat 480 gatcaggttg gcgaatactt cgaggagtgc caggtttacc gtatcgacca ctatcttggt 540 aaagaaacgg tgctgaacct gttggcgctg cgttttgcta actccctgtt tgtgaataac 600 tgggacaatc gcaccattga tcatgttgag attaccgtgg cagaagaagt ggggatcgaa 660 gggcgctggg gctattttga taaagccggt cagatgcgcg atatgatcca aaaccacctg 720 ctgcaaattc tctgcatgat tgcgatgtct ccgccatctg acctgagcgc agacagcatc 780 cgcgatgaaa aagtgaaagt actgaagtcc ctgcgccgca tcgaccgctc caacgtacgc 840 gaaaaaaccg tacgtgggca atatactgcg ggcttcgccc agggcaaaaa agtgccggga 900 tatctggaag aagagggcgc gaacaagagc agcaatacag aaaccttcgt ggcgatccgc 960 gtcgacattg ataactggcg ctgggccggt gtgccattct acctgcgtac tggtaaacgt 1020 ctgccgacca aatgttctga agtcgtggtc tatttcaaaa cacctgaact gaatctgttt 1080 aaagagtcgt ggcaggatct gccgcagaat aaactgacta tccgtctgca acctgatgaa 1140 ggcgtggata tccaggtact gaataaagtt cctggccttg accacaaaca taacctgcaa 1200 atcaccaagc tggatctgag ctattcagaa acctttaatc agacgcatct ggcggatgcc 1260 tatgaacgtc tgctgctgga aaccatgcgt ggtattcagg cactgtttgt tcgtcgcgac 1320 gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 1380 gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 1440 acccgtgatg gtcgttcctg gaatgagttt gagtaat 1477 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> DelpgiF <400> 20 ttccaaagtc acaattctca aaatcagaag agtattgcta gtgtaggctg gagctgcttc 60                                                                           60 <210> 21 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> DelpgiR <400> 21 gcggcgtgaa cgccttatcc ggcctacata tcgacgatga catatgaata tcctccttag 60                                                                           60 <210> 22 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> DelzwfF <400> 22 ctggcttaag taccgggtta gttaacttaa ggagaatgac gtgtaggctg gagctgcttc 60                                                                           60 <210> 23 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> DelzwfR <400> 23 atgttaccgg taaaataacc ataaaggata agcgcagata catatgaata tcctccttag 60                                                                           60

Claims (11)

The present invention relates to a microorganism having the ability to produce glucose-6-phosphate from glucose, which comprises (i) a gene encoding phosphoglucomutase, (ii) glucose-1-phosphate free DNA ligase (UDP-glucose dehydrogenase), and (iv) a UDP-glucuronic acid decarboxylase (UDP-glucuronic acid decarboxylase) gene encoding a UDP-glucose dehydrogenase. (UDP-xylose) from glucose-6-phosphate containing a gene coding for a glycosyltransferase and an arGt-3 gene encoding glycosyltransferase Wherein the microorganism has the ability to produce 3- O -xylosylcercetin in quercetin.
delete The method according to claim 1, wherein the gene coding for phosphoglucomutase, the gene encoding glucose-1-phosphate uridylltransferase, the gene encoding UDP-glucose dihydrogenase (UDP- -glucose dehydrogenase and UDP-glucuronic acid decarboxylase are nfa44530, galU, calS8, and calS9 , respectively. The microorganism variant according to claim 1, wherein the gene coding for UDP-glucuronic acid decarboxylase is nfa44530, galU, calS8 and calS9 .
2. The method according to claim 1, wherein at least one of a gene encoding glucose phosphate isomerase and a gene encoding glucose-6-phosphate dehydrogenase is further added Wherein the microorganism is defective.
5. The method according to claim 4, wherein the gene encoding glucose phosphate isomerase and the gene encoding glucose-6-phosphate dehydrogenase are pgi And zwf .
The microorganism according to claim 1, wherein the microorganism is Escherichia coli ). &lt; / RTI &gt;
The microorganism variant according to claim 1, wherein the phosphoglucomutase is derived from Nocardia farcinica IFM10152.
The method according to claim 1, wherein the glucose-1-phosphate uridyl lyransferase is Escherichia coli K-12. &lt; / RTI &gt;
The method according to claim 1, wherein the UDP-glucose dehydrogenase and the UDP-glucuronic acid decarboxylase are derived from Micromonospora echinospora ssp. Wherein the microorganism is a microorganism.
The method of claim 1, wherein the glycosyltransferase is Arabidopsis thaliana. &lt; / RTI &gt;
O -xylosyl quercetin in quercetin comprising the steps of:
(a) culturing the microorganism mutant microorganism variant of any one of claims 1 to 3 in a culture medium containing quercetin to produce 3-O-xylosyl quercetin; And
(b) recovering the resulting 3- O -xylosyl quercetin.

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