KR101780766B1 - Multi-monocistronic Vector For Producing Flavonol Glucoside and Recombinant Microorganism - Google Patents

Multi-monocistronic Vector For Producing Flavonol Glucoside and Recombinant Microorganism Download PDF

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KR101780766B1
KR101780766B1 KR1020160012889A KR20160012889A KR101780766B1 KR 101780766 B1 KR101780766 B1 KR 101780766B1 KR 1020160012889 A KR1020160012889 A KR 1020160012889A KR 20160012889 A KR20160012889 A KR 20160012889A KR 101780766 B1 KR101780766 B1 KR 101780766B1
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송재경
파라줄리 프라카스
프라사드 판데이 라메스
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선문대학교 산학협력단
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Abstract

The present invention relates to a multi-monocystronic vector for producing flavonol glucoside and a recombinant microorganism, and more particularly, to a recombinant microorganism which contains a gene encoding flavonol 3- O -glycosyl group transfer enzyme (UGT78K1) comprising a gene encoding (glk), phosphoglucoisomerase gene (pgm2) encoding the Muta kinase, a gene encoding the glucose facilitated diffusion protein (glf) and glucose 1-phosphate gene (galU) we code the group-transfer enzyme To a recombinant microorganism into which a multi-monocystronic expression vector has been introduced and a method for producing flavonol glucoside using the same.
When a recombinant microorganism into which a multi-monocystronic vector for producing flavonol glucoside according to the present invention is used is used, a large amount of flavonol glucoside is produced and used for various diseases including cardiovascular diseases, antioxidant activity, anti-obesity activity and anticancer activity And can be usefully used as a pharmaceutical composition.

Description

[0001] The present invention relates to a multi-monocistronic vector for producing Flavonol Glucoside and Recombinant Microorganism,

The present invention relates to a multi-monocystronic vector for producing flavonol glucoside and a recombinant microorganism, and more particularly, to a recombinant microorganism which contains a gene encoding flavonol 3- O -glycosyl group transfer enzyme (UGT78K1) comprising a gene encoding (glk), phosphoglucoisomerase gene (pgm2) encoding the Muta kinase, a gene encoding the glucose facilitated diffusion protein (glf) and glucose 1-phosphate gene (galU) we code the group-transfer enzyme To a recombinant microorganism into which a multi-monocystronic expression vector has been introduced and a method for producing flavonol glucoside using the same.

Flavonoids are secondary metabolites present in various plants, and these secondary metabolites accumulate in the tissues or organs of plants depending on the surrounding environmental conditions and stage of development. More than 8,000 naturally occurring flavonoids are found mostly in higher plants (Harborne JB et al ., Phytochemistry , 55: 481-504, 2000; Ververidis F, et al ., Biotechnol J , 2: 1214-1234, (Gattuso G et al ., Molecules, 12: 1641-1673, 2007), and is widely distributed in fruits, vegetables, peanuts, seeds, stems, flowers, roots, tea, wine and coffee. Flavonol, a kind of flavonoid, is divided into quercetin, chemerol, myrcetin, maurine and picetine according to phenolic -OH located in different skeletons from the 3-hydroxyflavone skeleton. As they are known to have various pharmacological activities ranging from prevention of cardiovascular diseases, antioxidant activity, anti-obesity activity and anticancer activity, there is a growing interest in the development and utilization of substances.

Most flavonoid drugs exist in the form of glycosides (Xiao J, Muzashvili TS, Georgiev MI et al., Biotechnol Adv. , 32: 1145-56, 2014). Glycocide is a molecule formed by combining sugars with non-carbohydrate components, and is an important factor in determining the solubility, bioactivity and bioavailability of natural products (Wang X et al., FEBS Lett , 583: 3303-3309, 2009). Depending on the kind of the sugar, glycosides can be distinguished from glucosides, lanosides, mannosides and fructosides, and can be distinguished from pyranoside and furanoside according to the ring structure.

On the other hand, microbial biotransformation (microbial biotransformation) is asbestos peogil Russ (Aspergillus), Bacillus (Bacillus), saccharose in my process (Saccharomyces), Streptomyces (Streptomyces) and E. coli life by using a microorganism, such as (E. coli) (Bartmanska A, Tronina T, Poplonski J, Huszcza E et al., Curr Drug Metab , 14: 1083-97, 2013), which can mass produce flavonoids and flavonol glycosides. In particular, Escherichia coli is the most widely used microorganism in industrial use and contributes to the production of therapeutic proteins, biochemicals, biofuels, cosmetics and functional food compositions.

Therefore, in order to highly express Flavonol glucoside conjugated with glucose and Flavonol in E. coli, the glucose promoting diffusion protein gene ( glf , glk ) derived from Zymomonas mobilis , which is necessary for the biosynthesis process of flavonol glucoside , O -glycosyltransferase (UGT78K1) gene derived from Glycine max ( Glycine max ) into a strain essential for gene expression. In order to stably express such a protein, a gene It is possible to simultaneously express a large number of genes in a single vector without causing an abnormal expression problem such as stop codon generation and frame shift in the cloning process at the same time as transforming multiple monocistronic vectors containing Multi-monocistronic gene expression that can be It is known that it is preferable for stable gene expression (Korean Patent Publication No. 10-2014-0042398).

Accordingly, the present inventors have made intensive efforts to more stably produce flavonol glucoside. As a result, they have produced a multi-monocistronic vector in which five genes necessary for the production of flavonol glucoside have been cloned , And stable gene expression was confirmed by an individual promoter (T7). Further, the multi-monocysteone vector was introduced into a microbial host to confirm stable production of flavonol glucoside from the microorganism host, and the present invention was completed.

It is an object of the present invention to provide a multi-monocistronic vector for the production of flavonol glucoside.

It is another object of the present invention to provide a recombinant microorganism into which a multi-monocystronic expression vector for producing flavonol glucoside is introduced and a method for producing flavonol glucoside using the recombinant microorganism.

In order to accomplish the above object, the present invention provides a gene encoding a flavonol 3- O -glycosyltransferase (UGT78K1), which comprises a gene encoding glucokinase ( glk ), a gene encoding a phosphoglucormutase monosystronic expression vector ( pgm2 ) for the production of flavonol glucoside, in which a gene ( glf ) encoding a glucose promoting diffusion protein and a gene ( galU ) encoding a glucose 1-phosphate uridylase- monocistronic vector).

The present invention also provides a recombinant microorganism into which the multi-monocystronic expression vector is introduced.

The present invention also provides a method for producing flavonoid glucoside, comprising culturing the recombinant microorganism in a flavonol-containing medium to produce flavonol glucoside; And recovering the resulting flavonol glucoside. The present invention also provides a method for preparing flavonol glucoside.

When a recombinant microorganism into which a multi-monocystronic vector for producing flavonol glucoside according to the present invention is used, it is possible to produce a large amount of flavonol glucoside and to produce various flavonol glucosides by various methods including prevention of cardiovascular diseases, antioxidant activity, Can be usefully utilized as a pharmaceutical composition.

Figure 1 is a schematic diagram of a recombinant glucose expression cassette and a piBR181 multi-monocystronic vector comprising it.
FIG. 2 shows the results of analysis of biotransformed picetin glucoside by HPLC-PDA and LC-QTOF-ESI / MS.
FIG. 3 shows the result of confirming the concentration of the strain and the flavonol bioconversion rate over time in order to confirm the optimum flavonol concentration through substrate inhibition.
FIG. 4 shows the results of comparing the bioconversion rates by glucose concentration in order to determine optimal glucose concentrations for flavonol bioconversion.
FIG. 5 shows the result of bioconversion of strains to confirm the bioconversion of flavonol according to the presence of the glf of the glucose promoting diffusion protein.
6 is a result of confirming the bioconversion pattern of the phytetin in the 3 L fermenter over time.
Figure 7 shows the conversion of pisetin (Fis), quercetin (Que), myristine (Myr), chemerol (Kmf) and morin to glucoside.

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 the present invention, in order to more stably produce flavonol glucoside, a multi-monocistronic vector in which five genes necessary for the production of flavonol glucoside have been cloned has been produced. As a result, T7), and it was also confirmed that the multi-monocysteone vector could be introduced into the microbial host to produce stable flavonol glucoside.

Accordingly, the present invention provides, in one aspect, a gene encoding a flavonol 3- O -glycosyltransferase (UGT78K1), a gene encoding glucokinase ( glk ), a gene encoding phosphoglucormutase pgm2), the gene encoding the glucose facilitated diffusion protein (glf) and glucose 1-phosphate our preparative flavonol glucosides which gene (galU) encoding the enzyme is inserted into a multi-group-transfer-tronic monomethyl cis expression vector (multi-monocistronic vector.

In the present invention, glucoside is a kind of glycoside, and is a generic name of a compound in which glucose and hemiacetal hydroxyl groups are bonded to each other in an ether phase.

In the present invention, the expression cassette is characterized in that it includes a minimal structure (Biobrick) necessary for expression and cloning of a gene such as a promoter, a ribosome binding site, a multicloning site and the like. More particularly, the expression cassette of the present invention comprises a T7 promoter, (b) a Lac operator, (c) a ribosome binding site (RBS), (d) a multiple cloning site ), (e) a T7 transcription terminator (TT), (f) a Bam HI cleavage site, and (g) an Xho I cleavage site.

In the present invention, a promoter is a site to which transcription regulatory elements bind, and may be composed of a core promoter, a proximal promoter, and a distal promoter.

The core promoter is the smallest part of the promoter required for transcription to occur. Refers to a portion within about 45 bases forward from the transcription start site, where RNA polymerase and general transcriptional regulatory factors are combined; The Proximymel promoter refers to a portion within about 250 bases forward from the transcription start site and is known to be a major part directly affecting transcription regulation; Distal Promoter Located far from the beginning of the transcription start site, it is generally less influential and secondary than the Proximymel promoter in regulating transcription.

The T7 promoter is a bacteriophage-derived promoter, and since E. coli RNA polymerase has a very low expression rate, it is a high-expression promoter commonly used when E. coli is used as a host for protein production. In addition, the T7 promoter is located at the lac operator, and the lac repressor binds to this region. In normal times, gene expression by T7 polymerase is inhibited. When the expression inducer such as IPTG is added, , And expression is terminated by recognizing the T7 expression ending site,

In the present invention, the Bgl II cleavage site is present at the front of the promoter of the expression system, the Spe I and Hind III cleavage sites are present at the multicloning site, and the Bam HI and Xho I cleavage sites are sequentially introduced after the transcription termination site But can be constructed on other restriction enzyme cuts if such a site is present in the sequence of the foreign gene to be inserted and is not suitable for cloning, such as cleaving the foreign gene in the cloning process, and such selection is obvious to those skilled in the art .

More specifically, the expression cassette according to the present invention is characterized in that at the multicloning site of the expression cassette, glf , glk, pgm2 , galU And UGT78K1 Followed by cloning to prepare a monocystronic vector. In this case, it is necessary to insert Xba I and Hind III recognition sites at both ends of each gene. Normally, by inserting a cleavage site artificially recognized by a specific restriction enzyme at the end of the gene amplification primer, Is obvious to those skilled in the art. In the present invention, as shown in Table 2, primers containing Xba I and Hind III restriction site sequences were used to amplify the gene.

The expression cassette according to the present invention may be supplemented with glf , glk , pgm2 , galU And UGT78K1 In order to insert, the blunt end or cohesive end cleavage planes obtained by treating the 5 'and 3' ends of the expression cassette with the same restriction enzymes of the 3 'and 5' ends of the respective gene fragments, respectively, are ligated to each other Can be manufactured.

In the present invention, the amplified enzyme gene fragments are inserted into the respective cognate sites of Xba I and Hind III artificially in the PCR process, treated with the restriction enzymes, and purified by the method described in Korean Patent Publication No. 10-2014-0042398 Treated with Spe I and Hind III so that the multicloning site of the vector piBR181 was cleaved and then ligated to each other to prepare a monocystic vector containing one gene. In order to prepare them again as a single vector, firstly, a vector into which glf is introduced is cleaved with Bgl II and Xho I, and only the gene fragment containing the expression cassette and glf is isolated therefrom. Then, the fragment is digested with Bam HI and Xho I glk was introduced into the vector, and glf And < RTI ID = 0.0 > glk . ≪ / RTI > Then, the vector was digested again with Bam HI and Xho I, and Bgl II and < RTI ID = 0.0 > The gene fragment containing the expression cassette and pgm2 isolated from the vector into which Xho I-treated pgm2 was introduced was ligated to prepare a vector into which expression cassettes each containing glf , glk and pgm2 were sequentially inserted. Similarly, the galU gene and the UGT78K1 gene were sequentially inserted into the above vector in the same manner to finally produce a multi- monocystronic vector containing all five genes (FIG. 1).

In the process of preparing the multi-monocystronic expression vector, when Xba I and Spe I, or the ends treated with Bam HI and Bgl II are ligated to each other, the sequence of the original restriction enzyme cleavage site is scar sequence ).

In the present invention, a restriction enzyme can also be used as a restriction endonuclease. An enzyme which recognizes a specific nucleotide sequence of a double-stranded DNA molecule and catalyzes cleavage of the part or its periphery Quot; Most restriction enzymes are characterized in that they cleave DNA at a position with a specific nucleotide sequence called a recognition site or restriction site, respectively.

Ligation usually occurs when the DNA end sequences cleaved by restriction enzymes are identical to each other, and can be cleaved with the same restriction enzyme even after ligation. However, in the case of Xba I and Spe I, there is a special case where the sequence is different from each other, but the ligation is possible but leaves a scar sequence which can not be cleaved by the enzyme after ligation , And restriction enzyme pairs capable of ligation-compatible even if Xba I- Spe I is different in the sequence of the restriction enzyme cleavage site, will be apparent to those skilled in the art.

Since the expression cassette of the present invention is shorter in distance from the promoter and the transcription termination site than the conventional expression cassette, it is possible to recycle the T7 polymerase in expressing the foreign gene, thus enabling multi-round transcription High expression can be realized.

The multi-monocystronic vector of the present invention may include two or more expression cassettes in which a foreign gene is inserted, preferably two or more and ten or fewer.

In the present invention, a vector is a carrier that stably transports a desired foreign gene fragment into a host cell, has autonomous replication function, can contain an antibiotic resistance gene, and has a multiple cloning site (MCS ), Which makes it easy to insert a foreign gene.

In the present invention, a multi-monocistronic expression vector may be mixed with a multi-monocystronine vector or a multi-monocystronic vector.

A cistron is a unit of microstructure that can perform a genetic function in a structural gene, and can be classified as a monocistron or a polycistron. Since monocistron determines the primary structure of one kind of polypeptide chain in a structural gene, the multi-monocystronic vector of the present invention is characterized in that it contains a large number of single cistron have.

The genes required for the biosynthesis of flavonol glucosides according to the present invention include Zymomonas mobilis mobilis) glucose-diffusion promoting protein gene derived from (glf, glk), phosphoglucoisomerase Muta dehydratase gene (pgm2), glucose 1-phosphate transferase we group- (galU) and Glycine max (Glycine max) Plastic derived bonol 3 O -glycosyl group transferase (UGT78K1) gene.

In another aspect, the present invention relates to a recombinant microorganism into which the multi-monocystronic expression vector is introduced.

In the present invention, the use of Escherichia coli (E. coli) BL21 (DE3) with the recombinant microorganism, but, if the microorganism used for microbial biotransformation is possible to use anything.

In the present invention, biotransformation, also referred to as biotransformation or biotransformation, refers to a biotransformation reaction that converts a substance introduced into a living body into another substance or binds with metabolites in the body.

In another aspect, the present invention provides a method for producing flavonol glucoside, comprising culturing the recombinant microorganism in a flavonol-containing medium to produce flavonol glucoside; And recovering the resulting flavonol glucoside. The present invention also relates to a method for producing flavonol glucoside.

In the present invention, the flavonol is picetin, chemerol, myrcetin, morin, and quercetin, but is not limited thereto.

In the present invention, the recombinant microorganism can be cultured at 20 캜 or 37 캜, but is not limited thereto.

When culturing the recombinant microorganism in the present invention, the medium to be used can be easily selected by those skilled in the art, and preferably LB medium can be used.

In the present invention, IPTG (Isopropyl beta-D-1-thiogalactopyranoside) may be used in the culturing process to induce gene expression. The substance is a substance that initiates foreign gene expression by effecting elimination of the lac repressor bound to the lac operator. The treatment method and treatment concentration of IPTG can be easily selected by a person skilled in the art.

In the present invention, a method for mass-culturing microorganisms for producing flavonol glucoside may be a shaking culture, a continuous culture, a batch culture, a fed-batch culture, and the like. It is not.

[Example]

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 embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example  One : Multi-monocystronic  Production of expression vector

1-1: Flavonol  Isolation of genes for glucoside production

The vectors, plasmids and strains used in the present invention are shown in Table 1.

Flavonols glucosidase gene (glucose facillitate diffusion protein) glf required for seed biosynthesis, glk (glucosekinase), pgm2 ( phosphoglucomutase), galU (glucose 1-phosphate uridyltransferase) and UGT78K1 (flavonol 3-O-glycosyltransferase ), to separate the table 2 primer and an ordinary PCR technique were used to obtain an enzyme gene fragment.

Vector and plasmid Explanation source pIBR181 Monosystronic vector modified from pET28a + and containing f1 pBR322 ori, Km r (kanamycin resistance gene) Invention piBR181-UGT78K1 PiBR181 vector containing UGT78K1 Invention piBR181-pgm2.galU.UGT78K1 A piBR181 vector containing pgm2.galU.UGT78K1 Invention piBR181-glf.glk.pgm2.galU.UGT78K1 A vector piBR181 containing glf.glk.pgm2.galU.UGT78K1 Invention piBR181-pGM2.galU. A piBR181 vector containing pgm2.galU Invention pET-Duet-pgm2.galu A pET-Duet vector containing pgm2.galU Invention Strain Explanation Strain number source Escherichia coli XL-1 Blue (MRF ') Cloning host Stratagene, USA E. coli BL21 (DE3) ompT hsdT hsdS (r B -m B -) gal (DE3) Novagen E. coli BL21 (DE3) / piBR181-UGT78K1 BL21 (DE3) strain containing piBR181-UGT78K1 S 1 Invention E. coli BL21 (DE3) pET-Duet-pgm2.galU piBR181.UGT78K1 BL21 (DE3) strain containing pET-Duet-pgm2.galU and piBR181-UGT78K1 S 2 Invention E. coli BL21 piBR181-pgm2.galU.UGT78K1 BL21 (DE3) strain containing piBR181-pgm2.galU.UGT78K1 S 3 Invention E. coli BL21 piBR181-glf.glk.pgm2.galU.UGT78K1 BL21 (DE3) strain containing piBR181-glf.glk.pgm2.galU.UGT78K1 S 4 Invention

primer Oligonucleotide  The sequence (5'-3 ') Restriction enzyme site glf-F (SEQ ID NO: 1) TCTAGA ATGAGTTCTGAAAGTAGTCAGGGTCTA Xba I glf-R (SEQ ID NO: 2) AAGCTT CTACTTCTGGGAGCGCCACATCTCCTC Hind III glk-F (SEQ ID NO: 3) TCTAGA ATGGAAATTGTTGCGATTGACATCGGT Xba I glk-R (SEQ ID NO: 4) AAGCTT TTATTCAACTTCAGAATATTTGTTGGC Hind III pgm2-F (SEQ ID NO: 5) TCTAGA ATGAGCTGGAGAACGAGCTATGAACGC Xba I pgm2-R (SEQ ID NO: 6) AAGCTT TTACGAATTTGAGGTCGCTTTTACAAT Hind III galU-F (SEQ ID NO: 7) TCTAGA ATGGCTGCCATTAATACGAAAGTCAAA Xba I galU-R (SEQ ID NO: 8) AAGCTT TTACTTCTTAATGCCCATCTCTTCTTC Hind III UGT78K1-F (SEQ ID NO: 9) TCTAGA ATGGATCATCAAAACAAACAC Xba I UGT78K1-R (SEQ ID NO: 10) AAGCTT TTAAGACCTAGAAATTACTTC Hind III

The five gene amplification products obtained by PCR were respectively digested with Xba I- Hind III and the piBR181 vector prepared by the method of Korean Patent Publication No. 10-2014-0042398 with Spe I- Hind III, respectively, and then inserted into the piBR181 vector Five individual expression cassettes (individual recombinant plasmids) containing each of the five gene fragments were prepared (Fig. 1).

1-2: Flavonol  For the production of glucosides Multi-monocystronic  Production of expression vector

In order to insert the above five genes into a single vector, the following fabrication process was performed.

The glf is cut in the introduced vector to the Bgl II and Xho I isolate a gene fragment comprising from which the expression cassette and glf, which was ligated to the introduction of a glk cutting the fragment with Bam HI and Xho I vector, glf And < RTI ID = 0.0 > glk . ≪ / RTI > Then, the vector was digested again with Bam HI and Xho I, and the gene fragment containing the expression cassette and pgm2 isolated from the vector into which pgm2 treated with Bgl II and Xho I had been introduced was ligated to this region to obtain glf , glk, and pgm2 were sequentially inserted into the expression cassette. Similarly, the galU gene and the UGT78K1 gene were sequentially inserted into the vector in the same manner to finally produce a multi- monocystronic vector containing all five genes (FIG. 1).

Example  2: Multi- Monosystronic  Using an expression vector Flavonol  Production of glucosides

2-1: Confirmation of Substrate Inhibition by Bioconversion of Pisatin Glucoside

Only single-pIBR181 UGT78K1 E. coli (strain S 1) the vector is introduced, including a UGT78K1 through the biotransformation of flavonols one kinds of bast paroxetine (fisetin) of was undertaken to determine the substrate inhibition. The different concentrations of phytetin (0.2 mM, 0.3 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM) dissolved in DMSO were biotransformed in 50 mL of the oil culture medium at 37 ° C. The mice were inoculated at 6-hour intervals until 24 hours, and then inoculated at 12-hour intervals until 60 hours. One mL of sample was taken in each flask and centrifuged at 12,000 rpm for 10 minutes. The supernatant corresponding to twice the volume of ethyl acetate was extracted, and the remaining cell pellet was mixed with water and the cell density was measured with a spectrophotometer having a UV absorbance of 600 nm. The ethyl acetate fraction was dried and then added again to 100 μL of methanol and analyzed by HPLC-PDA. The peak area in the HPLC-PDA analysis procedure was used to determine the biotransformed picetin glucoside.

As a result of HPLC-PDA analysis, the peak of biotransformed picetin glucoside was observed at 14.4 minutes. Further, through LC-QTOF-ESI / MS analysis, values ranging from [M + H] + m / z + to 449.1086 were confirmed (FIG. 2).

As a result of confirming the substrate inhibition effect on the flavonol biotransformation, the biotransformation products of picetin increased exponentially up to 48 hours and became static for 60 hours (FIG. 3). As a result, it was confirmed that phycetin was maximally bioconverted for 48 hours. In addition, the maximum conversion of picetine reached 58.9% when the cell concentration reached an OD 600 value of 3.5 at a picetin concentration of 0.3 mM. At a concentration of more than 0.3 mM of picetine, cell growth rate and substrate conversion gradually decreased. It can be seen that the higher the concentration of picetin, the more toxic to the culture environment of E. coli (FIG. 3).

2-2: Flavonol  Determine optimal glucose concentration for biotransformation

To determine the optimal glucose concentration for the strain at the time of bioconversion of flavonol, strain S 1 was cultured at 37 ° C until the cell concentration reached OD 600 0.5-0.7, and further cultured at 20 ° C for 20 hours , And the strains were inoculated into media containing three different concentrations of glucose (5%, 10%, 15%) and 0.3 mM of picetin, and the picetin bioconversion activity of E. coli was analyzed by HPLC-PDA.

As a result, it was confirmed that the conversion rate to picetin glucoside was the highest at 77.1% in the medium containing 10% glucose for 48 hours of culture time (FIG. 4).

2-3: Genes of Glucose Promoting Diffusion Protein glf ) Depending on whether Bioconversion ability  Confirm

There is no gene (glf) of glucose-diffusion promoting protein, a recombinant strain that contains a multi-vector (pET-Duet-pgm2.galU, piBR181 -UGT78K1) overexpressing UDP- glucose-free (S 2), glf multi-sheath mono tronic vector recombinant strains, including (piBR181-pgm2.galU.UGT78K1) (S 3 ) and including a multi-glf - mono cis tronic vector (piBR181-glf.glk.pgm2.galU.UGT78K1) recombinant strain (S 4), including To confirm the bioconversion ability of picetine. The recombinant strains were cultured under the same conditions as in Example 2-2. Samples were extracted at different time intervals and analyzed by HPLC-PDA.

As a result, the S 3 strain showed a conversion rate of 85%, which is higher than that of the S 2 strain showing 80% conversion. In addition, the strain S 4 exhibited a conversion rate of up to 100% (FIG. 5). This suggests that the promoter of glucose diffusion protein improves the conversion of flavonol to glucoside.

2-4: Scale-up Flavonol  Biotransformation

To determine the bioconversion of pysetin in a 3 L fermentor using S 4 strain, which is optimal for flavonol glucoside production. Glucose was added at an interval of 1 hour in the presence of 300 mg (0.35 mM) of picetin in a fermenter at 25 ° C, pH 7, and 10% glucose, and cultured for 36 hours. After 12 hours and 24 hours, the same amount of picetin was added. Samples were taken at intervals of 12 hours up to 60 hours and analyzed by HPLC-PDA.

As a result, it was confirmed that picetine was converted to picetin glucoside 100% when it passed 12 hours after culturing. The conversion rate was 100% even after 24 hours. However, thereafter, the rate of biotransformation of picetin decreased. The picetine injected after 24 hours did not completely convert to picetin glucoside until 60 hours. The picetine conversion rate between 48 hours and 60 hours remained constant. A total of 1.178 g (393 mg / L, 0.30 mM) of picetin glucoside was produced until 48 hours (FIG. 6).

2-5: Other Flavonol  Biological conversion confirmation

S 4 strains were used to determine the bioconversion patterns of flavonols other than picetin. 0.3 mM of chemperol, myristate, maurine, and quercetin were injected into a shaking flask containing S 4 strain cultured under the same conditions as in Example 2-2, and cultured for 48 hours.

As a result, Kemperol, quercetin and myristate showed biological conversion rates ranging from 95 to 100%, similar to picetine. On the other hand, the morin showed a biological conversion rate of 40% or less (Fig. 7).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-University Cooperation Foundation Sunmoon University <120> Multi-monocistronic Vector For Producing Flavonol Glucoside and          Recombinant Microorganism <130> P15-B133 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> glF-F <400> 1 tctagaatga gttctgaaag tagtcagggt cta 33 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> GlF-R <400> 2 aagcttctac ttctgggagc gccacatctc ctc 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> glK-F <400> 3 tctagaatgg aaattgttgc gattgacatc ggt 33 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Glk-R <400> 4 aagcttttat tcaacttcag aatatttgtt ggc 33 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pGM2-F <400> 5 tctagaatga gctggagaac gagctatgaa cgc 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pGM2-R <400> 6 aagcttttac gaatttgagg tcgcttttac aat 33 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> galU-F <400> 7 tctagaatgg ctgccattaa tacgaaagtc aaa 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> galU-R <400> 8 aagcttttac ttcttaatgc ccatctcttc ttc 33 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> UGT78K1-F <400> 9 tctagaatgg atcatcaaaa caaacac 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> UGT78K1-R <400> 10 aagcttttaa gacctagaaa ttacttc 27

Claims (3)

Flavonols 3- O - includes a gene encoding a glycosyl group transfer enzymes and genes (glf) to which the gene coding for glucokinase (glk), encoding the phosphoglucoisomerase Muta encoding the kinase gene, glucose-diffusion promoting protein and A multi-monocistronic vector for the production of flavonol glucoside in which a gene ( galU ) coding for the glucose 1-phosphate uridyl transferase is inserted.
A recombinant Escherichia coli having introduced the multi-monocystronic expression vector of claim 1.
A method for producing flavonol glucoside comprising the steps of:
(a) culturing the recombinant E. coli of claim 2 in a flavonol-containing medium to produce flavonol glucoside; And
(b) recovering the resulting flavonol glucoside.
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