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WO2006016395A1 - Udp-glucuronyle transferase et gene codant pour celle-ci - Google Patents

Udp-glucuronyle transferase et gene codant pour celle-ci Download PDF

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Publication number
WO2006016395A1
WO2006016395A1 PCT/JP2004/011437 JP2004011437W WO2006016395A1 WO 2006016395 A1 WO2006016395 A1 WO 2006016395A1 JP 2004011437 W JP2004011437 W JP 2004011437W WO 2006016395 A1 WO2006016395 A1 WO 2006016395A1
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Prior art keywords
udp
enzyme
gene
bpugt
seq
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Japanese (ja)
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Toru Nakayama
Shinya Sawada
Hirokazu Suzuki
Tokuzo Nishino
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Tohoku University NUC
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Tohoku University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention relates to a novel UDP-dalcronyltransferase and a gene thereof.
  • UDP-Daluronyltransferase is responsible for sugar chain synthesis and detoxification of various compounds in the body of mammals, and its detoxification action has been extensively studied due to its pharmaceutical importance. I'm going. Dalcronyl transferase is an enzyme that plays a central role in glucuronidation and detoxification mechanisms, and by dalucuronylating various toxic compounds such as steroids, bilirubin and other hydrophobic endogenous substances, phenol and drugs. Increase the water solubility of these compounds and promote their excretion into urine and bile.
  • soybean-derived dalcuronyltransferase is It is thought to have properties similar to those of vertebrate dalcronyltransferase, such as requiring metal ions for the expression of activity in proteins.
  • rye and Scarab dalcronyltransferases differ from known animal dalcronyltransferases in that they are water-soluble proteins and do not require metal cofactors for their expression of activity.
  • the results are available (see Non-Patent Documents 7 and 8). From this, it is possible that these enzymes have different functions from the known mammalian gnolecronyltransferases.
  • dalcronyltransferase derived from Pisum sativum
  • glucoronyltransferase NpGUTl
  • Nicotiana plumbaginifolia NpGUTl
  • UDP-glucuronic acid can be used as a substrate, and except for the fact that it is expressed in the mitotic phase of the root growth point, including the identity of its sugar residue receptor, NpGUTl is only speculated to be a sequence homology dalcronyltransferase, and no enzymatic knowledge of these expression products has been obtained.
  • anthocyanins are a type of flavonoid compound, and are a general term for anthocyanidins and compounds that are modified with sugars, methyl groups, acyl groups, sulfuric acid, gnoretathion, etc., such as red, blue, purple, and black purple. It is a pigment found in purple shiso, red cabbage and colored leaves.
  • anthocyanins are extremely abundant, and hundreds of types of anthocyanins have been isolated from plants and their structures have been determined in previous studies. These anthocyanins exist in the living body in a glycosylated form. Most of them have been modified with glucose, and there are a few that have been modified with galactose-rhamnose. No one having a lucuronyl group has been found. However, cyanidin 3-0- (6 "-0-malonyl-2" -0-glucuronyl darcoside), isolated from the red daisy (Bellis perennis) petal in 1994, is the only anthocyanin and has a darcronyl group. It is an unusual dye in the structure (see Non-Patent Documents 11 and 12).
  • Plant flavonoid compounds such as anthocyanins have antioxidant, antibacterial, carcinogenic, arteriosclerotic, pile caries, halitosis, liver function, visual acuity, etc. In general, these actions are said to be expressed in the form of aglycones, but flavonoid aglycones are generally poorly water-soluble and in particular anthocyanin aglycones (antocyanidins). ) Is unstable in aqueous solution, etc. Intestinal absorption of these compounds is also carried out in the form of glycosides and their modified forms (eg, acylated forms) and has a half-life in vivo.
  • glycosides and their modified products have been shown to be longer. Therefore, glucosides of flavonoid compounds and their modifications are stabilized and absorbed by these compounds. These compounds can play an effective role in improving the functions of these compounds, such as improving their properties, controlling their physiological activities, and slowing the release of active substances.Gnoreclonylation is an effective flavonoid that can be used for such purposes. Although it is a decoration means, the enzyme catalyst and its gene that can achieve this have not been obtained yet.
  • Non-Patent Document 1 C. D. King, G. R. Rios, M. D. Green, and T. R. Tephly.
  • Non-Patent Document 3 Akira Iyanagi Biochemistry Vol. 75, No. 3, 234-241, (2003)
  • Non-Patent Document 4 Christopher D. King, Mitchell D. Green, Gladys R. Rios, Birgit L. CofFman, Ida S. Owens, Warren P. Bishop, and Thomas R. Tephly. The
  • Non-Patent Document 5 Sahidan B. Senafi, Douglas J. Clarke, and Brian Burchell.
  • Non-Special Reference 6 Yasunori Kurosawa, Hidenari Takahara, and Masakazu Shiraiwa.UDP-glucuronicacid: soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds.Planta, 215, 620-629 (2002)
  • UDP-glucuronate flavone-glcuronosyltransferases from primary leaves of Secale cereale.Phytochemistry, 27, 1261-1267 (1988)
  • Non-Special Terms 8 lgeyuki Nagashima, Masao Hiratani, and Takafumi Yoshikawa. Purification and characterization of UDP-glucuronate: baicalein
  • Non-Patent Document 9 Ho-Hyung Woo, Marc J. Orbach, Ann M. Hirsch, and Martha C. Hawes. Meristem— localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa.Plant Cell , 11, 2303-2315 (1999)
  • Non-Patent Literature 0 Hiroaki Iwai, Nobutaka. Masaoka, Tadashi Ishii, and Shinobu Satoh. A pectin glucuronyltransferase gene is essentinal for intercellular attachment in the plantmeristem. PNAS, 99, 16319—16324 (2002)
  • Non-Patent Document 11 Kenjiro Toki, Norio Saito, and Toshio Honda.Three cyanidin 3-glucuronylglucosides from red flower of Bellis perennis.Phytochemistry, 30, 3769-3771 (1991)
  • Patent Document 12 Norio Saito, Kenjiro Toki, Toshio Honda, and Koshiro Kawase. And yanidin 3-malonylglucuronylglucoside in Bellis and Cyani in 3-malonylglucoside in
  • the present invention aims to obtain an enzyme capable of darcronylation of anthocyanin and its gene, and cyanidin which is a darcronylated anthocyanin accumulated in a red daisy petal.
  • 3-0- (6 "_0-malonyl-2" -0-dalcronyl dalcoside), a gene that is expected to cause darcronylation of dalcronyl transferase (BpUGT) Cloning was also performed.
  • the present invention has at least one amino acid sequence selected from SEQ ID NO: 1 (FIG. 1), SEQ ID NO: 2 (FIG. 2) and SEQ ID NO: 3 (FIG. 3) according to claim 1. Itfe child is.
  • a gene derived from red daisy which is the base sequence represented by SEQ ID NO: 4 (FIG. 4) according to claim 2 and having the 1313th base sequence as an amino acid translation region.
  • genes are preferably genes encoding UDP-dalcronyltransferase.
  • the present invention also includes at least one amino acid sequence selected from SEQ ID NO: 1 (FIG. 1), SEQ ID NO: 2 (FIG. 2) and SEQ ID NO: 3 (FIG. 3) according to claim 1.
  • UDP Gnoreclonyl transferase.
  • the UD p -dalcronyl group transfer derived from red daisy comprising the base sequence represented by SEQ ID NO: 4 (FIG. 4) according to claim 2 and having the base sequence as an amino acid translation region
  • An enzyme (BpUGT) is provided.
  • reaction catalyzed by BpUGT is specifically represented by the following formula (1).
  • a UDP-dalcronyltransferase (BpUGT) derived from red daisy and its gene.
  • BpUGT UDP-dalcronyltransferase
  • UDP-glucuronic acid, trifluoroacetic acid (TFA), and acetonitrile were purchased from Nacalai Testa.
  • Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Daishin Chemical.
  • the anthocyanin substrate used was the one isolated and purified at the Faculty of Horticulture, Minamikyushu University.
  • Reaction solution (0.5 mg / mL cyanidin 3-0-6 "-0-malonylglucoside) in an Epenpendorf tube I 0.01% trifluoroacetic acid 'methanol 5 / i L, 0.5 mg / mL UDP-glucuronic acid 3 ⁇ L of aqueous solution, 20 mM potassium phosphate buffer (pH 7.0, final volume of 100 ⁇ L) was prepared.
  • the reaction solution was pre-incubated with a dry thermo unit DTU-2B (TAITEC) at 30 ° C for 5 min, and the reaction was started by adding the enzyme preparation.
  • TITEC dry thermo unit
  • reaction was stopped by adding 200 / i L of ice-cooled 0.5% trifluoroacetic acid aqueous solution.
  • the produced reaction product was analyzed using reverse phase high performance liquid chromatography (HPLC). The amount of the enzyme preparation used for the enzyme reaction was adjusted so that the amount of the reaction product was 10% or less of the initial amount of the substrate.
  • the reaction product is reverse phase HPLC (DYNAMAX HPLC system; UV-VIS detector,
  • Quantification was performed using a SHIMADZU SPD-10A VP; column, flow rate 0.7 mL / min; detection wavelength, 520 nm.
  • the separation conditions are as shown in Table 1.
  • Solvents in 3G and 3G5G methods are A solution, 0.5% trifluoroacetic acid aqueous solution; B solution, 50% acetonitrile aqueous solution containing 0.5% trifluoroacetic acid, and Qu and IsoF method solvents are A solution, 0.1% trifluoroacetic acid aqueous solution; Liquid B, a 90% acetonitrile solution containing 0.1% trifluoroacetic acid.
  • PVPP Polybulupolypyrrolidone
  • PMSF phenylmethylsulfonyl fluoride
  • 2-ME 2-mercaptoethanol
  • EDTA ethylenediamine tetraacetic acid
  • ethylene glycol glycerol
  • CHAPS 2- [ (3-Cholamidopropyl) -dimethylammonio] -1-propanesulfonic acid
  • Nacalai Tester The distributor of the column for enzyme purification was described each time. The starting material, Digi Ichibana, was collected (stored immediately after flowering) and stored (-80 ° C) during the market period (late winter winter).
  • BpUGT activity Evaluation was made based on dalcronyl group transfer activity using benzidine 3-0-6 "-0-malonyldarcoside and UDP-glucuronic acid as substrates.
  • the total amount of the enzyme preparation obtained in Amersham Bioscience was loaded (1 mL / min), and the column was washed with buffer MQ.A (50 mL). Next, 0 to 100% linear concentration gradient of buffer MQ.B (300 mM potassium phosphate buffer H 7.5, 10% glycerol, 0.1% (w / v) CHAPS, 0.2% 2-ME) (for 350 min) The protein bound to the column was eluted.
  • buffer MQ.B 300 mM potassium phosphate buffer H 7.5, 10% glycerol, 0.1% (w / v) CHAPS, 0.2% 2-ME
  • RESOURCE PHE (Amasham Amersham) equilibrated with buffer RP.A (100 mM potassium phosphate buffer pH 7.5, 20% saturated ammonium sulfate, 0.2% 2-ME, 0.1% (w / v) CHAPS)
  • SDS-PAGE was performed according to Laemmli's method, and a separation gel having an acrylamide concentration of 10% was used. Electrophoresis was performed at a constant current of 20 mA.
  • Protein was quantified using Protein Assay Kit (BioRad). Usi serum albumin (Sigma) was used as a standard protein. [0031] SDS-PAGE of the obtained purified enzyme preparation showed a uniform protein band corresponding to 54 kDa, and this protein band appeared and disappeared in SDS-PAGE in the fractions after each chromatography. Was consistent with that of BpUGT activity, so it was considered to be the main body of BpUGT activity responsible for the alklonylation of anthocyanins in red digi.
  • mammalian-derived dalcronyltransferase is a membrane protein localized in the endoplasmic reticulum, and it has been clarified in previous studies that plant-derived darcronyltransferase is also derived from Koganebana. Baicalein 7-0-Dalcronyltransferase and the like form dimers.
  • this enzyme was considered to be a water-soluble protein because it did not require a protein solubilizing reagent such as a surfactant during extraction and could be easily extracted with an aqueous solvent.
  • BpUGT activity was observed at a position corresponding to 49 kDa, suggesting that this enzyme is a monomeric protein with a molecular mass of 54 kDa.
  • Poly (A) +-RNA was obtained from the daisy petals disrupted in liquid nitrogen using the Straight A's mRNA Isolation System (Novagen). The resulting poly (A) + RNA is in a bowl shape,
  • a double-stranded cDNA library was prepared using the ZAP-cDNA Synthesis Kit (STRATAGENE).
  • the resulting 7-stranded cDNA was purified using SizeSep 400 Spin Columns (Amersham Pharmacia Biotech) and then cloned into the Uni-ZAP XR insertion vector according to the prescription of the ZAP-cDNA Synthesis Kit. This was packaged in phage using the Gigapack III Gold Cloning Kit (STRATAGENE) to construct a daisy petal cDNA fuzzy library.
  • I Inosine
  • R A or G
  • Y C or T
  • H A or C or T
  • D A or G or T
  • RT-PCR using primers F1 and Rl was performed using poly (A) + RNA obtained from a daisy petal as a saddle type.
  • the QIAGEN OneSt RT-PCR Kit was used, and the composition of the reaction solution was in accordance with the kit prescription. Reaction conditions are 50 ° C, 30 minutes; 94 ° C, 15 minutes; [94 ° C, 15 seconds; 50 ° C, 15 seconds; 72 ° C, 30 seconds] X 35 cycles, 72 ° C, 10 minutes went.
  • reaction mixture 1 st PCR product, 100 ng; ExTaq buffer, 1 X; dNTPs, 200 mM; primer Fl, 1 ⁇ 1; primer R2, 1 ⁇ M; ExTaq polymerase (TaKaRa ), 2.5 U; reaction conditions 94 ° C, 2 minutes; [94 ° C, 15 seconds; 55 ° C, 15 seconds; 72 ° C, 30 seconds] X 30 cycles, 72 ° C, 7 minutes) .
  • the amplified DNA fragment was TA cloned into the pCR2.1-TOPO vector (TOPO TA cloning Kit, Invitrogen), and the base sequence was analyzed by a DNA sequencer (BECKMAN COULTER CEQ 2000XL DNA Analysis System).
  • a DNA sequencer BECKMAN COULTER CEQ 2000XL DNA Analysis System.
  • the obtained 965 bp PCR product showed the highest homology with the known flavonoid glycosyltransferase gene. Based on this, it was judged that there is a high possibility that this partial cDNA fragment is a part of the BpUGT gene.
  • a DIG (digoxigenin) -labeled probe was prepared by PCR DIG Probe Synthesis Kit (Roche Biochemicals).
  • Hybridization buffer containing the above DIG-labeled probe (100 ng / mL) is used as a hybridization buffer (30% formamide, 5xSSC, 0.02% SDS, 2% blocking reagent (Roche Diagnostics), 0.1% N_ Lauroyl sarcosine) at 37 ° C for 16 hours. Wash the membrane with washing solution (0.1 X SSC, 0.1% SDS) (65.C, 15 minutes x 2), then use DIG Labeling and Detection KIT (Roche Diagnostics) to test positive plaques as recommended by the manufacturer. As a result, 16 positive clones were finally obtained.
  • the conserved sequence common to the glycosyltransferase superfamily was present in the deduced amino acid sequence of the BpUGT gene (SEQ ID NO: 2: Fig. 2).
  • the black line is a conserved sequence of glycosyltransferase and is assumed to be a sugar nucleotide recognition site. This conserved sequence is thought to be the binding site for sugar nucleotides.
  • Enzymes belonging to the GT family are broadly divided into GT-A and GT-B due to their structure. The biggest difference between GT-A and GT-B is the position of the conserved sequence that seems to be the sugar nucleotide binding site.
  • glycosyltransferase superfamily has been classified into 73 families (James A. ampbell, Giaeon J. Davies, Vincent Bulone and Bernard Henrissat. A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid Sequence similarities. See Biochem J. 326,929—942 (1997)).
  • this enzyme is located away from mammalian-derived dalcuronyltransferases, and is a plant-derived GT population. Belonged ( Figure 3). Based on this, it was considered that gnorechronyl transferase activity was obtained by evolution different from that of mammal-derived gnoleclonyl transferase known to have this enzyme power.
  • Clusters have been found and each has the following characteristics.
  • Cluster I contains many enzymes that transfer the darcosyl group to the 3-position of flavonols or anthocyanins.
  • Cluster II consists of an enzyme that transfers the dalcosyl group to the 5-position of anthocyanin and other enzymes with unknown functions.
  • cluster III has not been clarified, it is considered to be an enzyme having a broad substrate specificity and transferring a gnorecosinole group in a site-specific manner.
  • the homology is about 40-50%.
  • the homology is about 20%.
  • the BpUGT gene did not belong to any of these three clusters and was closely related to the flavonoid-rhamnosyltransferase. These transglycosylation reactions catalyzed by BpUGT and rhamnosyltransferase both have the property of adding a new sugar residue to the sugar residue already bound to the flavonoid aglycone. If more types of enzymes are discovered, there is a possibility of forming new clusters.
  • Agar, Galactose, Glucose, Polyethyleneimine (PEI), Glycerol, Adenine Sanoleate, Uracil, Lithium Acetate, EDTA, Polyethylene Glycol 3000, and various amino acids were purchased from Nakarai Tester.
  • Yeast Nitrogen Base without amino acids, Bacto peptone, and yeast extract were purchased from DIFCO. Restriction enzymes were purchased from TaKaRa or New England Biolabs.
  • the obtained transformant was cultured with shaking (30 ° C, 160 rpm) for 3 days in 50 mL of SD Trp dropout medium prepared by the method recommended by pESC Yeast Epitope Tagging Vectors (STRATAGENE). When cell turbidity (OD) reaches 0.5, centrifuge (6,000 X g, 15 minutes)
  • the cells were collected and washed twice with 0.1 mL of SG dropout medium prepared by the method recommended by the kit.
  • the cells were resuspended in SG Trp dropout medium 50 mL, was inoculated suspension 4 mL to SG Trp dropout medium 4 L, the main culture was carried out (30 ° C, 160 rpm) 0 7 days in this condition After culturing for 10 days, when the OD force became S1.4-1.5, the cells were collected using centrifugation (6,000 X g, 15 minutes).
  • the cells were stored frozen at -30 ° C.
  • the beads and the extract were separated by centrifugation (> 1000 X g, 1 minute), and the extract was centrifuged again (10,000 X g, 20 minutes).
  • the supernatant was supplemented with PEI to a final concentration of 0.3%, stirred for about 10 minutes, and then centrifuged (10,000 ⁇ g, 20 minutes).
  • the obtained supernatant was recovered as a crude enzyme solution. By the method already described, it was confirmed that BpUGT activity was present in the crude enzyme solution.
  • the enzyme reaction was carried out by the method described in Example 1, and the product was purified and recovered by HPLC. Then, acetonitrile was removed using an aspirator, loaded onto HPLC again and eluted with methanol. The obtained sample was concentrated to 1 mL with an evaporator, and a part of the obtained sample was subjected to HPLC analysis and FAB-MS analysis to identify BpUGT recombinant reaction products. In HPLC analysis, the retention time of the peak considered to be a reaction product was consistent with the main pigment in Daisy 1 petal.
  • PCR reaction conditions were (50 ° C, 20 min; 95 ° C, 15 min, then [94 ° C, 15 sec; 55 ° C, 20 sec; 72 ° C, 10 sec] X 35 cycles; then 95 ° C 0 seconds; 65 ° C, 15 seconds), calibration curves were prepared each time.
  • a calibration curve was prepared with the horizontal axis representing the number of cycles and the vertical axis representing fluorescence intensity, and the number of copies of the BpUGT gene transcript in each daisy tissue was determined using this standard curve.
  • Fig. 4 shows the transcription amount (A) and specific activity (B) of the BpUGT gene in each daisy tissue.
  • Acetic acid, glycine, hydrochloric acid (HC1), tris (hydroxymethyl) aminomethane (Tris), hydroxylated rhodium (KOH), N-ethylmaleimide (NEM), jetyl pyrocarbonate (DEPC), 30% Monia water and metal salts were purchased from Nacalai Testa.
  • UDP-glucose, UDP-galactose, uridine, UMP, UDP, UTP, / 3-estradiol, 17-estradiol, 1_naphthol, 2-naphthol, 4-methylumbelliferone, p-nitrophenol, and human UDP-Dalcronyltransferase 1A1 (UGT1A1) was purchased from Sigma.
  • Silica gel 60F (MERCK) was used for the TLC plate.
  • the purified enzyme preparation was characterized for dalcronyl group transfer activity using 3-0-6 "-0-malonyl darcoside and UDP-glucuronic acid as substrates.
  • the buffer solution in the reaction solution described above was changed, and the pH dependence of the BpUGT recombinant was evaluated.
  • a 20 mM acetic acid-NaOH buffer solution was used at pH 4.0 ⁇ 6.0, and a 20 mM phosphoric acid-KOH buffer solution was used at H 6.0-7.5.
  • H 7.5-9.0 20 mM Tris-HC1 buffer was used, and for pH 9.0-11.0, 20 mM glycine-KOH buffer was used.
  • the enzyme preparation was diluted (10 times) with the above-mentioned buffer solution at each pH and incubated at 20 ° C for 17 h.
  • the residual activity of the enzyme preparation after the treatment was measured by the method described in Example 1, and the pH stability of the BpUGT recombinant was evaluated.
  • the optimal reaction pH of this enzyme was 8.0 (FIG. 5A) and was stable at pH 7 (FIG. 5B).
  • the K was 455 ⁇ .
  • the V of this enzyme is 1,300 nmol / mg / s, which is already m max.
  • This enzyme showed the highest activity against cyanidin 3-0-6 "-0_malonyl darcoside (relative activity 100%), and also showed an effective activity against the following anthocyanins: pelargonidin 3- ⁇ -6 "-0_malonyl darcoside (86% relative activity), cyanidin 3-0-darcoside (31%), delphidinidine 3 _0 -darcoside (14%).
  • flavonoid compounds accept dalcronyl groups could not be a body (relative activity, less than 0.1%): cyanidin 3_0-3 ", 6" - ⁇ dimalonino legnorecoside, peranolegonidin 3, 5- ⁇ _dignolecoside, peranolegonidin 3- ⁇ -6,, - ⁇ -malonyl darcoside-5-0 -darcoside, quercetin 3-0 -darcoside, quercetin 3-0-6, -O-malonyldarcoside, daidzin, genistin, daidzein 7-0-6, 0-malonyldarcoside, cyanidin, quercetin, daidzein, genistein.
  • P-nitrophenyl J3 -D -Darcoside could not be a substrate.
  • UDP- [U- 14 C] glucuronic acid (0.02 ⁇ Ci / ⁇ L, 225 mCi / mmol) 5 ⁇ L and final concentration of 3 mM of each sugar residue receptor in 20 mM Tris-maleate buffer pH 7.4 Added (total volume 20 ⁇ L).
  • UGT1AK 5 mg protein / mL) 10 ⁇ L, (0.034 U equivalent) was added to each tube to initiate the reaction. The reaction was stopped at 30 ° C for 30 minutes and 100% ethanol was added to stop the reaction.
  • the enzyme preparation was diluted (20 times) with a buffer containing final concentrations of 5 mM NEM and 1 mM DEPC and incubated at 20 ° C. for 20 minutes.
  • the enzyme preparation was similarly treated with a buffer solution containing no NEM.
  • NEM an alkyl group reagent specific for the SH group.
  • the enzyme activity was measured with an Atsy system containing 0.1 mM of various divalent metal ions, and compared with the enzyme activity under the same conditions without the metal ion, each metal ion relative to the malonyl group transfer activity was measured. The effect was investigated.
  • this enzyme contains malonic acid, which is a part of cyanidin 3_0-6 "- ⁇ -malonyldarcoside, It was not disturbed by gnole course.
  • this enzyme does not require these cofactors for its catalytic activity, and although inhibition of activity by metal ions was observed, activation was not observed.
  • the enzyme was treated with 5 mM EDTA for 20 minutes, the enzyme activity was not affected, suggesting that the enzyme does not require metal ions for its catalytic activity. .
  • the activity was not detected in the presence of Hg 2+ and Cu 2+ , but in recent reports, anthocyanin force g 2+ or Cu 2+ forms a metal complex and precipitates, which makes it colorless. Therefore, these metal ions may not act on the enzyme but may act on the anthocyanin substrate.
  • FIG. 1 shows the base sequence (SEQ ID NO: 1) of the enzyme gene according to the present invention.
  • the first underline in the sequence indicates the translation start codon, and the last underline indicates the translation stop codon.
  • FIG. 2 is an amino acid sequence (SEQ ID NO: 2) of an enzyme according to the present invention. Underlined sequences represent the conserved sequences in glycosyltransferase series 1.
  • FIG. 3 is a phylogenetic tree of glycosyltransferase family 1.
  • Figures I, II, and III show the cluster (s) of the plant glycosyltransferase family 1.
  • the GeneBank accession number of each enzyme in FIG. 3 is as follows. Tobacco IslOa, U32643; Tobacco Is5a, U32644; tomato Twil, X85138; Dorotheanthus 5GT,
  • FIG. 5 is a graph showing the pH dependence and pH stability of BpUGT.
  • Figure 5 (A) shows the pH dependence of BpUGT (30 ° C, 20 minutes), and
  • Fig. 5 (B) shows the pH stability (20 ° C, 12 hours treatment).
  • each plot shows age: 20 mM acetic acid-NaOH; ⁇ : 20 mM phosphoric acid-KOH; A: 20 mM
  • FIG. 6 shows the specificity for the sugar residue donor.

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Abstract

La présente invention a trait à l'UDP-glucuronyle transférase (BpUGT) dérivée de la pâquerette vivace (Bellis perennis); à un gène codant pour la BpUGT; et à un transformant comportant le gène. Ainsi la glucuronylation d'un composé flavonoïde peut être effectuée pour réaliser des améliorations fonctionnelles pour ce composé, telle que la stabilisation de composé flavonoïde, l'accroissement d'absorption, le contrôle de l'activité physiologique et la libération prolongée de substance active. Outre l'UDP-glucuronyle transférase (BpUGT) dérivée de la pâquerette vivace (Bellis perennis); à un gène codant pour la BpUGT, l'invention a trait à des moyens de modification efficaces applicables au contrôle de génie génétique métabolique, la régulation de couleur ou de ton et la stabilisation de développement de couleur de fleur selon la technique de génie génétique. En particulier, l'invention a trait à une enzyme comportant une séquence d'acides aminés de SEQ ID NO: 1 et une enzyme codée par le gène spécifié dans SEQ ID NO: 2.
PCT/JP2004/011437 2004-08-09 2004-08-09 Udp-glucuronyle transferase et gene codant pour celle-ci Ceased WO2006016395A1 (fr)

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PCT/JP2004/011437 WO2006016395A1 (fr) 2004-08-09 2004-08-09 Udp-glucuronyle transferase et gene codant pour celle-ci
JP2006531072A JPWO2006016395A1 (ja) 2004-08-09 2004-08-09 Ｕｄｐ−グルクロニル基転移酵素およびその遺伝子

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JP2008048612A (ja) * 2006-08-22 2008-03-06 Bio Taxol:Kk 糖転移酵素遺伝子
US9562251B2 (en) 2010-06-02 2017-02-07 Evolva Sa Production of steviol glycosides in microorganisms
US9631215B2 (en) 2011-08-08 2017-04-25 Evolva Sa Recombinant production of steviol glycosides
US9957540B2 (en) 2013-02-06 2018-05-01 Evolva Sa Methods for improved production of Rebaudioside D and Rebaudioside M
US10017804B2 (en) 2013-02-11 2018-07-10 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
US10364450B2 (en) 2015-01-30 2019-07-30 Evolva Sa Production of steviol glycoside in recombinant hosts
US10421983B2 (en) 2014-08-11 2019-09-24 Evolva Sa Production of steviol glycosides in recombinant hosts
US10612064B2 (en) 2014-09-09 2020-04-07 Evolva Sa Production of steviol glycosides in recombinant hosts
KR20200061140A (ko) * 2018-11-23 2020-06-02 경북대학교 산학협력단 리코리시딘을 포함하는 ｕｇｔ 활성 평가용 조성물 및 그의 용도
US10815514B2 (en) 2016-05-16 2020-10-27 Evolva Sa Production of steviol glycosides in recombinant hosts
US10837041B2 (en) 2015-08-07 2020-11-17 Evolva Sa Production of steviol glycosides in recombinant hosts
US10947515B2 (en) 2015-03-16 2021-03-16 Dsm Ip Assets B.V. UDP-glycosyltransferases
US10982249B2 (en) 2016-04-13 2021-04-20 Evolva Sa Production of steviol glycosides in recombinant hosts
US11396669B2 (en) 2016-11-07 2022-07-26 Evolva Sa Production of steviol glycosides in recombinant hosts

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
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WOO H H ET AL: "Meristem-Localized Inducible Expression of a UPD-Glycosyltransferase Gene Is Essential for growth and Development in Pea and Alfalfa", THE PLANT CELL, vol. 11, December 1999 (1999-12-01), pages 2303 - 2315, XP002904271 *

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008048612A (ja) * 2006-08-22 2008-03-06 Bio Taxol:Kk 糖転移酵素遺伝子
EP2575432B1 (fr) * 2010-06-02 2019-08-14 Evolva, Inc. Production de glycosides de stéviol par recombinaison
US9562251B2 (en) 2010-06-02 2017-02-07 Evolva Sa Production of steviol glycosides in microorganisms
EP3593633A1 (fr) * 2010-06-02 2020-01-15 Evolva, Inc. Production recombinante de glycosides de stéviol
US10392644B2 (en) 2010-06-02 2019-08-27 Evolva Sa Production of steviol glycosides in microorganisms
US12428628B2 (en) 2010-06-02 2025-09-30 Danstar Ferment Ag Recombinant production of steviol glycosides
US10435730B2 (en) 2011-08-08 2019-10-08 Evolva Sa Recombinant production of steviol glycosides
US12416034B2 (en) 2011-08-08 2025-09-16 Danstar Ferment Ag Recombinant production of steviol glycosides
US9631215B2 (en) 2011-08-08 2017-04-25 Evolva Sa Recombinant production of steviol glycosides
US9957540B2 (en) 2013-02-06 2018-05-01 Evolva Sa Methods for improved production of Rebaudioside D and Rebaudioside M
US10612066B2 (en) 2013-02-06 2020-04-07 Evolva Sa Methods for improved production of rebaudioside D and rebaudioside M
US11530431B2 (en) 2013-02-06 2022-12-20 Evolva Sa Methods for improved production of Rebaudioside D and Rebaudioside M
US10017804B2 (en) 2013-02-11 2018-07-10 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
US11021727B2 (en) 2013-02-11 2021-06-01 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
US11168343B2 (en) 2014-08-11 2021-11-09 Evolva Sa Production of steviol glycosides in recombinant hosts
US10421983B2 (en) 2014-08-11 2019-09-24 Evolva Sa Production of steviol glycosides in recombinant hosts
US10612064B2 (en) 2014-09-09 2020-04-07 Evolva Sa Production of steviol glycosides in recombinant hosts
US12123042B2 (en) 2014-09-09 2024-10-22 Danstar Ferment Ag Production of steviol glycosides in recombinant hosts
US11466302B2 (en) 2014-09-09 2022-10-11 Evolva Sa Production of steviol glycosides in recombinant hosts
US10364450B2 (en) 2015-01-30 2019-07-30 Evolva Sa Production of steviol glycoside in recombinant hosts
US11041183B2 (en) 2015-01-30 2021-06-22 Evolva Sa Production of steviol glycoside in recombinant hosts
US11807888B2 (en) 2015-01-30 2023-11-07 Evolva Sa Production of steviol glycoside in recombinant hosts
US10947515B2 (en) 2015-03-16 2021-03-16 Dsm Ip Assets B.V. UDP-glycosyltransferases
US11459548B2 (en) 2015-03-16 2022-10-04 Dsm Ip Assets B.V. UDP-glycosyltransferases
US10837041B2 (en) 2015-08-07 2020-11-17 Evolva Sa Production of steviol glycosides in recombinant hosts
US11821015B2 (en) 2016-04-13 2023-11-21 Evolva Sa Production of steviol glycosides in recombinant hosts
US10982249B2 (en) 2016-04-13 2021-04-20 Evolva Sa Production of steviol glycosides in recombinant hosts
US10815514B2 (en) 2016-05-16 2020-10-27 Evolva Sa Production of steviol glycosides in recombinant hosts
US11396669B2 (en) 2016-11-07 2022-07-26 Evolva Sa Production of steviol glycosides in recombinant hosts
KR20200061140A (ko) * 2018-11-23 2020-06-02 경북대학교 산학협력단 리코리시딘을 포함하는 ｕｇｔ 활성 평가용 조성물 및 그의 용도
KR102138548B1 (ko) 2018-11-23 2020-07-28 경북대학교 산학협력단 리코리시딘을 포함하는 ｕｇｔ 활성 평가용 조성물 및 그의 용도

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