US 201003234O2A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0323402 A1 Ono et al. (43) Pub. Date: Dec. 23, 2010

(54) UDP-GLUCURONYL TRANSFERASE AND Publication Classification POLYNUCLEOTDE ENCODING THE SAME (51) Int. Cl. CI2P 9/60 (2006.01) (75) Inventors: Eiichiro Ono, Osaka (JP); Akio C7H 2L/04 (2006.01) Noguchi, Osaka (JP); Yuko Fukui, C7H 2L/00 (2006.01) Osaka (JP); Masako Mizutani, CI2N 9/10 (2006.01) Osaka (JP) CI2N 15/63 (2006.01) CI2N I/2 (2006.01) Correspondence Address: (52) U.S. Cl...... 435/75:536/23.2:536/23.1; 435/193; GREENBLUM & BERNSTEIN, P.L.C. 1950 ROLAND CLARKE PLACE 435/320.1; 435/252.33 RESTON, VA 20191 (US) (57) ABSTRACT The present invention provides a novel UDP-glucuronosyl (73) Assignee: SUNTORY HOLDINGS transferase and a polynucleotide encoding the same (for LIMITED, Osaka (JP) example, a polynucleotide comprising a polynucleotide con sisting of one nucleotide sequence selected from the group (21) Appl. No.: 12/678,161 consisting of the nucleotide sequence at positions 1 to 1359 in the nucleotide sequence represented by SEQ ID NO: 4, the (22) PCT Fled: Sep. 29, 2008 nucleotide sequence at positions 1 to 1365 in the nucleotide PCT NO.: PCT/UP2008/O67613 sequence represented by SEQ ID NO: 10, the nucleotide (86) sequence at positions 1 to 1371 in the nucleotide sequence S371 (c)(1), represented by SEQID NO: 12, and the nucleotide sequence (2), (4) Date: May 12, 2010 at positions 1 to 1371 in the nucleotide sequence represented by SEQ ID NO: 22; or a polynucleotide comprising a poly (30) Foreign Application Priority Data nucleotide encoding a protein having one amino acid sequence selected from the group consisting of SEQID NOS: Oct. 12, 2007 (JP) ...... 2007-267050 5, 11, 13 and 23), etc. This provides a novel UDP-glucurono Mar 17, 2008 (JP) ...... 2008-067.185 syltransferase with a broad substrate specificity. Patent Application Publication Dec. 23, 2010 Sheet 1 of 8 US 2010/0323402 A1

Formatted Alignments

AmUGTogi Oaa 450 SEUGTaa 448 445 434 Patent Application Publication Dec. 23, 2010 Sheet 2 of 8 US 2010/0323402 A1

Fig. 2

BaiCalein a a a 3.33. is Myricetins Naringenin AureuSidin Genistein DaidZein Formononetin Catechin Epigallocatechin gallate ESCuletin Coniferyl alcohol ReSVeratrol SC1 SC2

Relative activity (%) Patent Application Publication Dec. 23, 2010 Sheet 3 of 8 US 2010/0323402 A1

Fig. 3

BaiCalein SCutellarein Apigenin Luteolin TriCetin DiOSmetin Chrysoeriol Vitexin SOVitexin Orientin Kaempferol QuerCetin Naringenin AureuSidin Genistein Daidzein Formononetin Catechin Epigallocatechin gallate ESCulletin Coniferyl alcohol ReSVeratrol SC 1 SC2 EC2 O 20 40 60 80 100 120 Relative activity (%) Patent Application Publication Dec. 23, 2010 Sheet 4 of 8 US 2010/0323402 A1

Baicalein SCutellarein Apigenin Luteolin TriCetin DiOSmetin Chrysoeriol Vitexin ISOVitexin Orientin Kaempferol QuerCetin Naringenin Aureusidin Genistein Daidzein Formononetin Catechin Epigallocatechin gallate ESCulletin Coniferyl alcohol ReSVeratrol SC1 SC2 EC2 O 20 40 60 80 100 120

Relative activity (%) Patent Application Publication Dec. 23, 2010 Sheet 5 of 8 US 2010/0323402 A1

Fig. 5

BaiCalein SCutellarein Apigenin Luteolin TriCetin DioSmetin Chrysoeriol Vitexin SOVitexin Orientin Kaempferol QuerCetin Naringenin AureuSidin Genistein DaidZein Formononetin Catechin Epigallocatechin gallate ESCulletin Caffeic acid Resveratrol SC1 SC2 EC2 O 20 40 60 80 100 120 Relative activity (%) Patent Application Publication Dec. 23, 2010 Sheet 6 of 8 US 2010/0323402 A1

Fig. 6

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UDP-GLUCURONYLTRANSFERASE AND is registered in GenBank (Accession No. AB042277) but its POLYNUCLEOTIDE ENCODING THE SAME function remains unconfirmed. 0008. On the other hand, it is known that flavone 7-glucu FIELD OF THE INVENTION ronides which are more diverse than skullcap or “wogon' are accumulated in Perilla frutescens a red-leaf variety with a 0001. The present invention relates to a UDP-glucurono dietary experience (Literature 7: Yamazaki, M. et al. Phy Syltransferase, a polynucleotide encoding the same, a vector tochemistry 62,987-998. 2003). containing the same, a transformant, and so on. Literatures: BACKGROUND OF THE INVENTION 0009. 1. Day, A. J. et al., Free Radic. Res., 35,941-952, 0002 Polyphenolic plant secondary metabolites including 2001 flavonoids and lignans with a rich dietary experience have (0010 2. Moon, J. H. et al., Free Radical Biology & Medi attracted attention as functional materials over the years due cine, 30, 1274-1285, 2001 to their functional properties represented by their antioxida 0011 3. O'Leary, K. A. et al., Biochemical Pharmacology, tive activities, and are already commercially available as 65, 479–491, 2003 health foods. For example, quercetin (flavonoid), OTPP (fla 0012 4. Vander Woude, H. et al., Chem. Res. Toxicol., 17, vonoid), Sesamin (lignan), etc. are representative materials 1520-1530, 2004 for health foods. 0013 5. Gao, Z. et al., Biochemica et Biophysica Acta, 0003. The biosynthetic pathway of flavonoids in plant 1472, 643-650, 1999 cells has been studied since old times. Biosynthetic enzymes (0014) 6. Nagashima S. et al., Phytochemistry, 53,533-538, that catalyze the metabolic pathway and genes encoding the 2OOO. enzymes are isolated, leading to a better understanding of (0015 7. Yamazaki, M. et al., Phytochemistry, 62.987-998, their molecular mechanisms. 2003 0004. On the other hand, knowledge is insufficient on how the plant secondary metabolites would be metabolized to DISCLOSURE OF THE INVENTION exhibit their functions, after their in vivo uptake. 0005. It is known that glycosylation of plant secondary Problems To Be Solved by the Invention metabolites is generally catalyzed by an enzyme belonging to 0016 Under these circumstances, it has been desired to the superfamily called UDP-glycosyltransferase (UGT), irre identify a novel UDP-glucuronosyltransferase having a spective of types of Sugars (glucose, rhamnose, glucuronic broader Substrate specificity and a gene encoding the same. acid, galactose, etc.). Further in the studies of Sesamin, the secondary metabolites are shown to be present in vivo as Means of Solving the Problem glucuronides via catechol metabolites. It is thus considered that the glucuronides would play a part in developing the in 0017. The present invention has been made in view of the Vivo functions of plant secondary metabolites. foregoing circumstances and provides the following UDP 0006. It is confirmed that four monoglucuronides are glucuronosyltransferases and polynucleotides encoding the present as the metabolites of quercetin in mammals (Q-3- same, as well as vectors bearing the same, transformants, and GlcA, Q-7-GlcA, Q-3'-GlcA and Q-4'-GlcA) (Literature 1: SO. O. Day, AJ et al. Free Radic. Res. 35,941-952, 2001, Literature 0018 (1) A polynucleotide of any one of (a) through (f) 2: Moon, J. H. et al. Free Radical Biology & Medicine 30, below: 1274-1285, 2001, Literature 3: O'Leary, K. A. et al. Bio 0019 (a) a polynucleotide comprising a polynucleotide chemical Pharmacology 65, 479–491, 2003, and Literature 4: consisting of one nucleotide sequence selected from the van der Woude, H. et al. Chem. Res. Toxicol. 17, 1520-1530, group consisting of the nucleotide sequence at positions 1 to 2004); in order to understand these functions in vivo, it is 1359 in the nucleotide sequence represented by SEQID NO: necessary to obtain a sufficient amount of compounds to 4, the nucleotide sequence at positions 1 to 1365 in the nucle examine their activities. However, any appropriate UDP-glu otide sequence represented by SEQID NO: 10, the nucleotide curonosyltransferase showing a broad Substrate specificity is sequence at positions 1 to 1371 in the nucleotide sequence unknown so far, and it was actually impossible to chemically represented by SEQID NO: 12, and the nucleotide sequence synthesize a binding site-specific reaction product. at positions 1 to 1371 in the nucleotide sequence represented 0007. The radix of Labiatae Scutellaria baicalensis is by SEQID NO: 22: called skullcap or “wogon' in Japanese, and it is known that 0020 (b) a polynucleotide comprising a polynucleotide 7-glucuronides of highly antioxidative are accumu encoding a protein having one amino acid sequence selected lated therein. According to the borderline of pharmaceuticals from the group consisting of SEQID NOs: 5, 11, 13 and 23; to non-pharmaceuticals, the radix of Scutellaria baicalensis 0021 (c) a polynucleotide comprising a polynucleotide is classified into the pharmaceuticals (Literature 5: Gao, Z. et encoding a protein consisting of an amino acid sequence with al. Biochimica et Biophysica Acta 1472, 643-650. 1999). To deletion, substitution, insertion and/or addition of 1 to 15 date, Sb7GAT is purified from Labiatae Scutellaria baicallen amino acids in one amino acid sequence selected from the sis as flavone 7-glucuronosyltransferase; this enzyme acts group consisting of SEQID NOs: 5, 11, 13 and 23, and having only on flavones with Substituents such as hydroxyl group at a UDP-glucuronosyltransferase activity; the ortho position of the 7-OH flavones (baicalein, scutella 0022 (d) a polynucleotide comprising a polynucleotide rein, etc.) but does not act on apigenin and luteolin which are encoding a protein having an amino acid sequence having a the major flavones and further not on quercetin which is one homology of at least 80% to one amino acid sequence of flavonols (Literature 6: Nagashima S. et al., Phytochemis selected from the group consisting of SEQID NOs: 5, 11, 13 try 53,533-538, 2000). A gene corresponding to this Sb2OAT and 23 and having a UDP-glucuronosyltransferase activity; US 2010/0323402 A1 Dec. 23, 2010

0023 (e) a polynucleotide comprising a polynucleotide 0032 (5) The polynucleotide according to (1) above, encoding a protein that hybridizes under Stringent conditions which comprises a polynucleotide consisting of the nucle with a polynucleotide consisting of a nucleotide sequence otide sequence at positions 1 to 1371 in the nucleotide complementary to one nucleotide sequence selected from the sequence represented by SEQID NO: 12. group consisting of the nucleotide sequence at positions 1 to 0033 (6) The polynucleotide according to (1) above, 1359 in the nucleotide sequence represented by SEQID NO: which comprises a polynucleotide consisting of the nucle 4, the nucleotide sequence at positions 1 to 1365 in the nucle otide sequence at positions 1 to 1371 in the nucleotide otide sequence represented by SEQID NO: 10, the nucleotide sequence represented by SEQID NO: 22. sequence at positions 1 to 1371 in the nucleotide sequence 0034 (7) The polynucleotide according to (1) above, represented by SEQID NO: 12, and the nucleotide sequence which comprises a polynucleotide encoding the protein con at positions 1 to 1371 in the nucleotide sequence represented sisting of the amino acid sequence represented by SEQ ID by SEQID NO: 22, and has a UDP-glucuronosyltransferase NO: 5. activity; and, 0035 (8) The polynucleotide according to (1) above, 0024 (f) a polynucleotide comprising a polynucleotide which comprises a polynucleotide encoding the protein con encoding a protein that hybridizes under Stringent conditions sisting of the amino acid sequence represented by SEQ ID with a polynucleotide consisting of a nucleotide sequence NO: 11. complementary to the nucleotide sequence of a polynucle 0036 (9) The polynucleotide according to (1) above, otide encoding a protein consisting of one amino acid which comprises a polynucleotide encoding the protein con sequence selected from the group consisting of SEQID NOs: sisting of the amino acid sequence represented by SEQ ID 5, 11, 13 and 23, and has a UDP-glucuronosyltransferase NO: 13. activity. 0037 (10) The polynucleotide according to (1) above, 0025 (2) The polynucleotide according to (1) above, which comprises a polynucleotide encoding the protein con which is any one of (g) through () below: sisting of the amino acid sequence represented by SEQ ID 0026 (g) a polynucleotide comprising a polynucleotide NO: 23. encoding a protein consisting of an amino acid sequence with 0038 (11) The polynucleotide according to any one of (1) deletion, Substitution, insertion and/or addition of not greater to (10) above, which is a DNA. than 10 amino acids (i.e. 0-10amino acids) in one amino acid 0039 (12) A protein encoded by the polynucleotide sequence selected from the group consisting of SEQID NOs: according to any one of (1) to (11) above. 5, 11, 13 and 23, and having a UDP-glucuronosyltransferase 0040 (13) A vector comprising the polynucleotide activity; according to any one of (1) to (11) above. 0027 (h) a polynucleotide comprising a polynucleotide 0041 (14) A transformant, wherein the polynucleotide encoding a protein having an amino acid sequence having a according to any one of (1) to (11) above is introduced. homology of at least 90% to one amino acid sequence 0042 (15) A transformant, wherein the vector according selected from the group consisting of SEQID NOs: 5, 11, 13 to (13) above is introduced. and 23, and having a UDP-glucuronosyltransferase activity; 0043 (16) A method for producing the protein of claim 12, 0028 (i) a polynucleotide comprising a polynucleotide which comprises using the transformant according to (14) or encoding a protein that hybridizes under high Stringent con (15) above. ditions with a polynucleotide consisting of a nucleotide 0044) (17) A method for producing a glucuronide, which sequence complementary to one nucleotide sequence comprises forming the glucuronide from UDP-glucuronic selected from the group consisting of the nucleotide sequence acid and a flavonoid using the protein according to (12) above at positions 1 to 1359 in the nucleotide sequence represented as a catalyst. by SEQID NO: 4, the nucleotide sequence at positions 1 to 1365 in the nucleotide sequence represented by SEQID NO: Advantageous Effect of the Invention 10, the nucleotide sequence at positions 1 to 1371 in the 0045. The polynucleotide of the present invention is useful nucleotide sequence represented by SEQID NO: 12, and the for the production of a novel UDP-glucuronosyltransferase nucleotide sequence at positions 1 to 1371 in the nucleotide by introducing the polynucleotide into, e.g., a transformant. sequence represented by SEQ ID NO: 22, and has a UDP In a preferred embodiment of the invention, the UDP-glucu glucuronosyltransferase activity; and, ronosyltransferase has a broad Substrate specificity and an 0029 () a polynucleotide comprising a polynucleotide activity of glucuronidation of diverse glycosyl acceptor Sub encoding a protein that hybridizes under high Stringent con ditions with a polynucleotide consisting of a nucleotide Strates. sequence complementary to the nucleotide sequence of a polynucleotide encoding a protein consisting of one amino BRIEF DESCRIPTION OF DRAWINGS acid sequence selected from the group consisting of SEQID 0046 FIG. 1 shows alignments of the amino acid NOs: 5, 11, 13 and 23, and has a UDP-glucuronosyltrans sequences of AmUGTcg 10 derived from Antirrhinum majus, ferase activity. S1 UGT derived from Scutellaria laete violacea V. yakusinen 0030 (3) The polynucleotide according to (1) above, sis, PflugT50 derived from Perilla frutescens a red-leaf vari which comprises a polynucleotide consisting of the nucle ety and Sb7GAT derived from Scutellaria baicalensis. otide sequence at positions 1 to 1359 in the nucleotide 0047 FIG. 2 shows the results of analysis on the specific sequence represented by SEQID NO: 4. ity of glycosyl acceptor substrates for PflugT50. 0031 (4) The polynucleotide according to (1) above, 0048 FIG. 3 shows the results of analysis on the specific which comprises a polynucleotide consisting of the nucle ity of glycosyl acceptor substrates for S1 UGT. otide sequence at positions 1 to 1365 in the nucleotide 0049 FIG. 4 shows the results of analysis on the specific sequence represented by SEQID NO: 10. ity of glycosyl acceptor substrates for AmuGTcg10. US 2010/0323402 A1 Dec. 23, 2010

0050 FIG. 5 shows the results of analysis on the specific 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino ity of glycosyl acceptor substrates for SiOGT23. acid(s) is/are deleted, substituted, inserted and/or added and 0051 FIG. 6 shows the results of LC-MS analysis on the having the UDP-glucuronosyltransferase activity. In general, products in the reaction solution of S1 UGT and SC1, wherein the number of deletions, Substitutions, insertions, and/or the arrows denote the products. additions is preferably smaller. Such proteins include a pro 0052 FIG. 7 shows the results of MS analysis on the tein having an amino acid sequence having a homology of products in the reaction of SC1 and S1 UGT1. approximately 80% or higher, 81% or higher, 82% or higher, 0053 FIG. 8 shows the results of LC analysis on the prod 83% or higher, 84% or higher, 85% or higher, 86% or higher, ucts in the reaction of quercetin and S1 UGT 1. 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, SEQUENCE LISTING FREETEXT 95% or higher, 96% or higher, 97% or higher, 98% or higher, 0054 SEQID NO: 1 synthetic DNA 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or 0.055 SEQID NO: 2 synthetic DNA higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 0056 SEQID NO: 6 synthetic DNA 99.7% or higher, 99.8% or higher, or 99.9% or higher, to one 0057 SEQID NO: 7 synthetic DNA amino acid sequence selected from the group consisting of 0.058 SEQID NO: 8 synthetic DNA SEQ ID NOs: 5, 11, 13 and 23, and having the UDP-glucu 0059 SEQID NO: 9 synthetic DNA ronosyltransferase activity. As the homology percentage 0060 SEQID NO: 14 synthetic DNA described above is higher, the protein is preferred in general. 0061 SEQID NO: 15 synthetic DNA 0072. As used herein, the term “UDP-glucuronosyltrans 0062 SEQID NO: 16 synthetic DNA ferase activity” refers to an activity of catalyzing the reaction 0063 SEQID NO: 17 synthetic DNA that involves the glucuronidation of hydroxyl groups in fla 0064 SEQID NO: 18 synthetic DNA vonoids, Stilbenes, lignans, etc. (glucuronidation of, e.g., a 0065 SEQID NO: 19 synthetic DNA flavone at the position 7-OH) to form glucuronides. 0.066 SEQID NO: 20 synthetic DNA 0073. The UDP-glucuronosyltransferase activity can be 0067 SEQID NO: 21 synthetic DNA assayed by reacting, for example, UDP-glucuronic acid with 0068 SEQID NO: 24 synthetic DNA a glycosyl acceptor Substrate (e.g., a flavone) in the presence 0069 SEQID NO: 25 synthetic DNA of an enzyme to be assayed and analyzing the reaction prod uct by HPLC (cf., EXAMPLES described below for more BEST MODE FOR CARRYING OUT THE details). INVENTION 0074 The present invention further includes (e) a poly nucleotide comprising a polynucleotide encoding a protein 0070 Hereinafter, the UDP-glucuronosyltransferases of that hybridizes under stringent conditions with a polynucle the invention, the polynucleotide encoding the same, the vec otide consisting of a nucleotide sequence complementary to tor bearing the same, the transformant and so on are described one nucleotide sequence selected from the group consisting in detail. of the nucleotide sequence at positions 1 to 1359 in the nucle otide sequence represented by SEQID NO: 4, the nucleotide 1. Polynucleotide of the Invention sequence at positions 1 to 1365 in the nucleotide sequence 0071 First, the present invention provides (a) a polynucle represented by SEQID NO: 10, the nucleotide sequence at otide (specifically a DNA, hereinafter sometimes merely positions 1 to 1371 in the nucleotide sequence represented by referred to as “DNA) comprising a polynucleotide consist SEQID NO: 12, and the nucleotide sequence at positions 1 to ing of one nucleotide sequence selected from the group con 1371 in the nucleotide sequence represented by SEQID NO: sisting of the nucleotide sequence at positions 1 to 1359 in the 22, and has the UDP-glucuronosyltransferase activity; and (f) nucleotide sequence represented by SEQID NO: 4, the nucle a polynucleotide comprising a polynucleotide encoding a otide sequence at positions 1 to 1365 in the nucleotide protein that hybridizes under Stringent conditions with a poly sequence represented by SEQ ID NO: 10, the nucleotide nucleotide consisting of a nucleotide sequence complemen sequence at positions 1 to 1371 in the nucleotide sequence tary to the nucleotide sequence of a polynucleotide encoding represented by SEQID NO: 12, and the nucleotide sequence a protein consisting of one amino acid sequence selected from at positions 1 to 1371 in the nucleotide sequence represented the group consisting of SEQID NOs: 5, 11, 13 and 23, and has by SEQ ID NO: 22; and (b) a polynucleotide comprising a the UDP-glucuronosyltransferase activity. polynucleotide encoding a protein having one amino acid (0075. As used herein, the “polynucleotide” refers to a sequence selected from the group consisting of SEQID NOS: DNA or RNA 5, 11, 13 and 23. The DNA targeted in the present invention is 0076. As used herein, the term “polynucleotide that not limited only to the DNA encoding UDP-glucuronosyl hybridizes under stringent conditions” refers to, for example, transferase described above but also includes other DNAS a polynucleotide obtained by colony hybridization, plaque encoding a protein functionally equivalent to this protein. The hybridization, Southern hybridization or the like, using as a functionally equivalent protein is, for example, (c) a protein probe all or part of a polynucleotide consisting of a nucleotide consisting of an amino acid sequence with deletion, Substitu sequence complementary to one nucleotide sequence tion, insertion and/or addition of 1 to 15 amino acids in one selected from the group consisting of the nucleotide sequence amino acid sequence selected from the group consisting of at positions 1 to 1359 in the nucleotide sequence represented SEQID NOs: 5, 11, 13 and 23, and having a UDP-glucurono by SEQID NO: 4, the nucleotide sequence at positions 1 to Syltransferase activity. Such a protein includes a protein con 1365 in the nucleotide sequence represented by SEQID NO: sisting of one amino acid sequence selected from the group 10, the nucleotide sequence at positions 1 to 1371 in the consisting of SEQID NOs: 5, 11, 13 and 23 wherein 1 to 15, nucleotide sequence represented by SEQID NO: 12, and the 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, nucleotide sequence at positions 1 to 1371 in the nucleotide US 2010/0323402 A1 Dec. 23, 2010

sequence represented by SEQID NO: 22 or a polynucleotide J. Mol. Biol., 215:403, 1990). When a nucleotide sequence is consisting of a nucleotide sequence complementary to the sequenced using BLASTN, the parameters are, for example, nucleotide sequence of a polynucleotide encoding one amino score=100 and word length=12. When an amino acid acid sequence selected from the group consisting of SEQID sequence is sequenced using BLASTX, the parameters are, NOs: 5, 11, 13 and 23. The hybridization method may be a for example, score=50 and word length=3. When BLAST and method described in, for example, Sambrook & Russell, Gapped BLAST programs are used, default parameters for Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring each of the programs are employed. Harbor, Laboratory Press 2001, Ausubel, Current Protocols I0081. The polynucleotide of the invention described in Molecular Biology, John Wiley & Sons 1987-1997, etc. above can be acquired by known genetic engineering means 0077. As used herein, the “stringent conditions' may be or known synthetic means. any of low-stringent conditions, medium-stringent condi tions or high-stringent conditions. The “low-stringent condi 2. Protein of the Invention tions” are, for example, 5xSSC, 5x Denhardt’s solution, 0.5% I0082 In a further embodiment, the present invention also SDS, 50% formamide and 32° C. The “medium-stringent provides the protein encoded by any one of the polynucle conditions” are, for example, 5xSSC, 5x Denhardt’s solution, otides (a) through (f) described above. The protein of the 0.5% SDS, 50% formamide and 42°C. The “high-stringent invention which is preferred is a protein consisting of an conditions” are, for example, 5xSSC, 5x Denhardt’s solution, amino acid sequence with deletion, Substitution, insertion 0.5% SDS, 50% formamide and 50° C. Under these condi and/or addition of 1 to 15 amino acids in one amino acid tions, as the temperature is higher, a DNA with higher homol sequence selected from the group consisting of SEQID NOs: ogy is expected to be obtained efficiently at higher tempera 5, 11, 13 and 23, and having a UDP-glucuronosyltransferase ture, although multiple factors are involved in the activity. Such a protein includes a protein consisting of an hybridization stringency including temperature, probe con amino acid sequence wherein amino acid residues with the centration, probe length, ionic strength, time, salt concentra number described above are deleted, substituted, inserted tion and the like. Those skilled in the art may realize similar and/or added in one amino acid sequence selected from the stringency by appropriately selecting these factors. group consisting of SEQID NOs: 5, 11, 13 and 23 and having 0078. When a commercially available kit is used for the UDP-glucuronosyltransferase activity. The protein also hybridization, for example, Alkphos Direct Labeling includes a protein having the amino acid sequence having the Reagents (manufactured by Amersham Pharmacia) can be homology described above to one amino acid sequence used. In this case, according to the attached protocol, a mem selected from the group consisting of SEQID NOs: 5, 11, 13 brane is incubated with a labeled probe overnight, the mem and 23 and having the UDP-glucuronosyltransferase activity. brane is washed with a primary wash buffer containing 0.1% These proteins may be obtained by using site-directed (w/v) SDS at 55° C. and the hybridized DNA can then be mutagenesis described in Sambrook & Russell, Molecular detected. Cloning: A Laboratory Manual, Vol. 3, Cold Spring Harbor, 0079. Other polynucleotides that can be hybridized Laboratory Press 2001, Ausubel, Current Protocols in include DNAs having a homology of approximately 60% or Molecular Biology, John Wiley & Sons 1987-1997, Nuc. higher, approximately 70% or higher, 71% or higher, 72% or Acids Res., 10,6487 (1982), Proc. Natl. Acad. Sci. USA, 79, higher, 73% or higher, 74% or higher, 75% or higher, 76% or 6409 (1982), Gene, 34,315 (1985), Nuc. Acids Res., 13,4431 higher, 77% or higher, 78% or higher, 79% or higher, 80% or (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc. higher, 81% or higher, 82% or higher, 83% or higher, 84% or 0083. The deletion, substitution, insertion and/or addition higher, 85% or higher, 86% or higher, 87% or higher, 88% or of one or more amino acid residues in an amino acid sequence higher, 89% or higher, 90% or higher, 91% or higher, 92% or of the protein of the invention means that one or a plurality of higher, 93% or higher, 94% or higher, 95% or higher, 96% or amino acid residues are deleted, Substituted, inserted and/or higher, 97% or higher. 98% or higher, 99% or higher, 99.1% added at one or a plurality of positions in the same amino acid or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, sequence. Two or more types of deletion, Substitution, inser 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or tion and addition may occur concurrently. higher or 99.9% or higher, to a DNA encoding one nucleotide I0084 Examples of amino acid residues which are mutu sequence selected from the group consisting of the nucleotide ally substitutable are given below. Amino acid residues in the sequence at positions 1 to 1359 in the nucleotide sequence same group are mutually substitutable. represented by SEQ ID NO: 4, the nucleotide sequence at I0085 Group A: leucine, isoleucine, norleucine, valine, positions 1 to 1365 in the nucleotide sequence represented by norvaline, alanine, 2-aminobutanoic acid, methionine, o-me SEQ ID NO: 10, the nucleotide sequence at positions 1 to thylserine, t-butylglycine, t-butylalanine and cyclohexylala 1371 in the nucleotide sequence represented by SEQID NO: nine; Group B: aspartic acid, glutamic acid, isoaspartic acid, 12 and the nucleotide sequence at positions 1 to 1371 in the isoglutamic acid, 2-aminoadipic acid and 2-aminosuberic nucleotide sequence represented by SEQID NO: 22, or one acid; Group C: asparagine and glutamine; Group D: lysine, amino acid sequence selected from the group consisting of arginine, ornithine, 2,4-diaminobutanoic acid and 2,3-diami SEQ ID NOs: 5, 11, 13 and 23, as calculated by homology nopropionic acid; Group E: proline, 3-hydroxyproline and search software, such as FASTA and BLAST using default 4-hydroxyproline; Group F: serine, threonine and parameters. homoserine; and Group G: phenylalanine and tyrosine. 0080 Homology between amino acid sequences or nucle I0086. The protein of the present invention may also be otide sequences can be determined by using algorithm produced by chemical synthesis methods such as the Fmoc BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, method (fluorenylmethyloxycarbonyl method) and the tRoc 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90:5873, method (t-butyloxycarbonyl method). In addition, peptide 1993). Programs called BLASTN and BLASTX based on synthesizers available from Advanced ChemTech, Perkin BLAST algorithm have been developed (Altschul, S. F. et al., Elmer, Pharmacia, Protein Technology Instrument, Synth US 2010/0323402 A1 Dec. 23, 2010 ecell-Vega, PerSeptive, Shimadzu Corp., etc. may also be kaempferol, naringenin, aureusidin, formononetin, etc., a used for the chemical synthesis. coumarine Such as esculetin, etc., a stilbene Such as resvera 0087 Herein, the protein of the invention is a UDP-glu trol, etc., and shows a potent activity as compared to other curonosyltransferase. The term “UDP-glucuronosyltrans glycosyl acceptor Substrates, especially when the glycosyl ferase' catalyzes the reaction of transferring the glucuronyl acceptor Substrate is a flavonoid Such as baicalein, Scutella group from UDP-glucuronic acid as a glycosyl donor onto a rein, luteolin, tricetin, kaempferol, aureusidin, etc. glycosyl acceptor substrate to form the glucuronide and UDP. In the present invention, the glycosyl acceptor Substrate is, for 3. Vector and Transformant Bearing the Same example, a flavonoid, a stilbene and alignan. 0088. The flavonoid includes flavones, flavonols, fla 0095. In another embodiment, the present invention pro Vanones, isoflavones, flavone C-glycosides, aurones, cat vides the expression vector comprising the polynucleotide of echins, and the like. Among them, examples of the flavones the present invention. The vector of the invention comprises include baicalein, Scutellarein, apigenin, luteolin, tricetin, any one of (a) through (f) described above. Preferably, the diosmetin, chrysoeriol, etc. Examples of the flavonols include expression vector of the invention comprises any one of the quercetin, myricetin, kaempferol, etc. An example of the polynucleotides (g) through (). More preferably, the expres flavanones is naringenin. Examples of the isoflavones are sion vector of the invention comprises a polynucleotide com genistein, daidzein and formononetin. Examples of the fla prising one polynucleotide selected from the group consisting Vone C-glycosides include Vitexin, isoVitexin and orientin. of a polynucleotide consisting of the nucleotide sequence at An example of the aurones is aureusidin. Examples of the positions 1 to 1359 in the nucleotide sequence represented by catechins are catechin and epigallocatechin gallate. SEQID NO: 4, a polynucleotide consisting of the nucleotide 0089. The stilbene includes resveratrol and its glycoside sequence at positions 1 to 1365 in the nucleotide sequence piceid, etc. represented by SEQID NO: 10, a polynucleotide consisting 0090 The lignan includes (+)-pinoresinol, (+)-piperitol, of the nucleotide sequence at positions 1 to 1371 in the nucle (+)-sesaminol, (+)-secoisolariciresinol, (+)-sesamin catechol otide sequence represented by SEQ ID NO: 12 and a poly 1) (SC1), (+)-sesamin catechol 2 (SC2), (+)-episesamin cat nucleotide consisting of the nucleotide sequence at positions echol 2 (EC2), matairesinol, etc. 1 to 1371 in the nucleotide sequence represented by SEQID 0091 For example, the UDP-glucuronosyltransferase NO: 22, or one polynucleotide selected from the group con having the amino acid sequence of SEQID NO: 5 (PflugT50) sisting of a polynucleotide encoding a protein consisting of shows the activity when the glycosyl acceptor substrate is a the amino acid sequence of SEQID NO: 5, a polynucleotide flavonoid Such as baicalein, apigenin, scutellarein, luteolin, encoding a protein consisting of the amino acid sequence of tricetin, dioSmetin, chrysoeriol, quercetin, myricetin, SEQID NO: 11, a polynucleotide encoding a protein consist kaempferol, naringenin, aureusidin, etc., a stilbene Such as ing of the amino acid sequence of SEQ ID NO: 13 and a resveratrol, etc., alignan Such as SC1, etc., and shows a potent polynucleotide encoding a protein consisting of the amino activity as compared to other glycosyl acceptor Substrates, acid sequence of SEQID NO. 23. especially when the glycosyl acceptor Substrate is baicalein, 0096. The vector of the invention is generally constructed apigenin, Scutellarein, luteolin, tricetin, dioSmetin, chryso to contain an expression cassette comprising (i) a promoter eriol, quercetin and aureusidin. that can be transcribed in a host cell, (ii) any of the polynucle 0092. The UDP-glucuronosyltransferase having the otides described in (a) to () above that is linked to the pro amino acid sequence of SEQID NO: 11 (S1UGT) shows the moter, and (iii) a signal that functions in the host cell with activity when the glycosyl acceptor Substrate is a flavonoid respect to the transcription termination and polyadenylation Such as baicalein, apigenin, Scutellarein, luteolin, tricetin, of RNA molecule. The vector thus constructed is introduced diosmetin, chrysoeriol, quercetin, kaempferol, naringenin, into a host cell. To construct the expression vector, methods genistein, daidzein, formononetin, myricetin, etc., a couma using a plasmid, phage or cosmid are used but are not par rine such as esculetin, etc., a stilbene Such as resveratrol, etc., ticularly limited. a lignan Such as SC1, SC2, EC2, etc., and shows a potent 0097 Specific types of the vector are not particularly lim activity as compared to other glycosyl acceptor Substrates, ited, and vectors capable of expressing in a host cell can be especially when the glycosyl acceptor Substrate is baicalein Suitably selected. That is, a Suitable promoter sequence may and apigenin. be chosen depending upon the type of the host cell to reliably 0093. The UDP-glucuronosyltransferase having the express the polynucleotide of the invention, and a vector amino acid sequence of SEQ ID NO: 13 (AmUGTcg10) obtained by incorporating this sequence and the polynucle shows the activity when the glycosyl acceptor Substrate is a otide of the present invention into various plasmids or the like flavonoid Such as baicalein, apigenin, scutellarein, luteolin, may be used as an expression vector. tricetin, dioSmetin, chrysoeriol, quercetin, kaempferol, nar 0098. The expression vector of the present invention con ingenin, aureusidin, etc., a stilbene Such as resveratrol, etc., tains an expression control region (for example, a promoter, a and shows a potent activity as compared to other glycosyl terminator, and/or a replication origin, etc.) depending on the acceptor Substrates, especially when the glycosyl acceptor type of a host to be introduced. A conventional promoter (for Substrate is a flavonoid Such as baicalein, apigenin, Scutella example, trc promoter, tac promoter, lac promoter, etc.) is rein, luteolin, tricetin, dioSmetin, chrysoeriol, kaempferol, used as a promoter for a bacterial expression vector. As a naringenin, etc. promoter for yeast, there are used, for example, a glyceralde 0094. The UDP-glucuronosyltransferase having the hyde 3-phosphate dehydrogenase promoter, PH05 promoter, amino acid sequence of SEQ ID NO. 23 (SiOGT23) shows etc. As a promoter for fungi there are used, for example, the activity when the glycosyl acceptor Substrate is a fla amylase, trpC, etc. Additionally, a viral promoter (e.g., SV40 vonoid such as baicalein, apigenin, Scutellarein, luteolin, early promoter, SV40 late promoter, etc.) is used as a pro tricetin, dioSmetin, chrysoeriol, isoVitexin, quercetin, moter for animal-derived host cell. US 2010/0323402 A1 Dec. 23, 2010

0099. The expression vector preferably contains at least above can be performed by a conventional method of sepa one selective marker. The marker available includes an aux ration and purification. The separation and purification meth otrophic marker (ura5, niaD), a drug-resistant marker (hygro ods including ammonium Sulfate precipitation, gel filtration mycin, Zeocin), a geneticin-resistant marker (G418r), a cop chromatography, ion exchange chromatography, affinity per-resistant gene (CUP1) (Marinet al., Proc. Natl. Acad. Sci. chromatography, reversed phase high performance liquid USA, 81, 337, 1984), a cerulenin resistant gene (fas2m, chromatography, dialysis, and ultrafiltration, etc. may be used PDR4) (Junji Inokoshi et al., Biochemistry, 64, 660, 1992: singly or in a Suitable combination. and Hussain et al., Gene, 101: 149, 1991), and the like. 0100. The present invention provides the transformant in 5. Method of Producing the Glucuronide which the polynucleotide described in any of (a) to () above 0107 The present invention further provides a method of is introduced. producing the glucuronide using the protein of the present 0101. A method of preparing (method of producing) the invention. The protein of the invention catalyzes the reaction transformant is not particularly limited and includes, for of transferring the glucuronic acid from UDP-glucuronic acid example, a method which comprises introducing the recom to a glycosyl acceptor Substrate (e.g., a flavonoid, a stilbene or binant vector into a host followed by transformation. The host alignan) and therefore, the glucuronide can be produced from cell used herein is not particularly limited and various known the glycosyl acceptor Substrate and UDP-glucuronic acid by cells may be preferably used. Specific examples are bacteria using the protein of the invention. The glycosyl acceptor Such as Escherichia coli, etc., yeast (budding yeast Saccha substrate is preferably a flavonoid. romyces cerevisiae, fission yeast Schizosaccharomyces 0108. The glucuronide can be produced, for example, by pombe), nematode (Caenorhabditis elegans), oocyte of Afri preparing a solution containing 1 mM glycosyl acceptor Sub can clawed frog (Xenopus laevis), etc. Culture media and strate, 2 mMUDP-glucuronic acid, 50 mM calcium phos conditions suitable for the host cells above are well known in phate buffer (pH 7.5) and 20 uM of the protein of the inven the art. The organism to be transformed is not particularly tion is prepared and reacting them at 30° C. for 30 minutes. limited, and includes various microorganisms, plants and ani The glucuronide can be isolated/purified from this solution by mals given as examples of the host cells above. known methods. Specifically, e.g., ammonium Sulfate pre 0102 For transformation of the host cell, there may be cipitation, gel filtration chromatography, ion exchange chro used generally known methods. For example, methods that matography, affinity chromatography, reversed phase high can be used include but not limited to the electroporation performance liquid chromatography, dialysis, ultrafiltration, method (Mackenzie D. A. et al., Appl. Environ. Microbiol. etc. can be used alone or in an appropriate combination. 66, 4655-4661, 2000), the particle delivery method (method 0109 The glucuronide thus obtained is useful as a reagent described in JPA 2005-287.403 “Method of Breeding Lipid for inspecting the in vivo functions, an antioxidant, etc. (Gao, Producing Fungus'), the spheroplast method (Proc. Natl. Z., Huang, K., Yang. X., and Xu, H. (1999) Biochimica et Acad. Sci. USA, 75: 1929 (1978)), the lithium acetate method Biophysica Acta, 1472, 643-650). (J. Bacteriology, 153: 163 (1983)), and methods described in 0110. The present invention is described in more details Proc. Natl. Acad. Sci. USA, 75: 1929 (1978), Methods in with reference to EXAMPLES below but is not deemed to be yeast genetics, 2000 Edition: A Cold Spring Harbor Labora limited thereto. tory Course Manual, etc. Example 1 4. Method of Producing the Protein of the Invention 0103) In yet another embodiment, the present invention 1. Outline of Example 1 provides a method of producing the protein of the present 0111. In this EXAMPLE, Sb7GAT homologue gene invention using the transformants described above. (S1UGT) was cloned from Scutellaria laeteviolacea V. 0104 Specifically, the protein of the invention may be yakusimensis by PCR and using this gene as a probe, it was obtained by isolating and purifying the protein of the inven attempted to isolate 7-glucuronosyltransferase of the fla tion from the culture of the transformant described above. As vonoid from the cDNA library of Perilla frutescens a red-leaf used herein, the culture refers to any one of a culture broth, variety, which accumulates the 7-glucuronide of flavonoid. cultured bacteria or cultured cells, and the homogenate of 0112. As a result of screening, PflugT50 having an activ cultured bacteria or cultured cells. Conventional methods ity to transfer the glucuronic acid to the 7-hydroxy group of a may be used to isolate and purify the protein of the invention. flavone was identified in glycosyltransferase PflugT derived 0105 Specifically, when the protein of the invention accu from Perilla frutescens a red-leaf variety. This PflugT50 had mulates within cultured bacteria or within cultured cells, a the glucuronosyltransfer activity not only to flavones such as crude extract of the protein of the invention may be obtained baicalein, Scutellarein, apigenin, luteolin, etc. but also to by culturing the bacteria or cells, then disrupting the bacterial quercetin which is a flavonol. Accordingly, the use of or cells using a conventional technique (e.g., ultrasonication, PflugT50 enables to glucuronidate the 7-position of various lysozymes, freezing and thawing, etc.) and applying a con flavonoids including flavones in vivo. ventional method such as centrifugation or filtration. When 0113 Also, S1 UGT derived from Scutellaria laeteviola the protein of the invention is accumulated in the culture cea V. yakusinensis, which was isolated as a screening probe, broth, the culture Supernatant containing the protein of the showed the glucuronosyl transfer activity to various fla invention can be obtained, after completion of the incubation, vonoids. Scrophulariaceae Antirrhinum majus was also by separating the bacteria or cells from the culture Superna searched for UGT (UDP-glucuronosyltransferase) highly tant in a conventional manner (e.g., centrifugation, filtration, homologous to PflugT50 and S1 UGT to identify etc.). AmUGTcg10 as UGT from Antirrhinum majus. The 0106 Purification of the protein of the invention contained AmUGTcg10 protein expressed in Escherichia coli, which is in the extract or culture Supernatant obtained as described UGT derived from Antirrhinum majus, showed the glucu US 2010/0323402 A1 Dec. 23, 2010 ronosyltransfer activity to apigenin, quercetin and naringe PCR was performed by reacting at 94° C. for 5 minutes and nin. It was therefore shown that PflugT50 from Perilla frute then amplified for 30 cycles with each cycle consisting of scens a red-leaf variety, S1 UGT from Scutellaria reacting at 94° C. for a minute, 53°C. for a minute and 72°C. laeteviolacea V. yakusinensis and AmUGTcg10 from Anti for 2 minutes. After the S1 UGT fragment was confirmed to be rrhinum maius described above had the activity to transfer the labeled by agarose electrophoresis, this DIG-labeled frag 7-glucuronic acid of flavonoids. ment was used for the following experiment as a probe for hybridization. 2. Isolation of Glucosyltransferase Gene from the Perilla Frutescens cDNA Library (2) Hybridization (1) Preparation of Probe 0119 Using the S1 UGT probe described above, the cDNA 0114. The molecular biological method used in this library derived from Perilla frutescens (species: aka-chiri EXAMPLE was in accordance with the method described in men-jiso or red-leaved perilla) (Yonekura-Sakakibara, K. et Molecular Cloning (Sambrook et al. Cold Spring Harbour al., Plant Cell Physiol. 41, 495-502. 2000) was screened by Laboratory Press, 2001), unless otherwise indicated in detail. using the Non-Radioisotope DIG-Nucleic Acid Detection 0115. After total RNA was extracted from the radix of System (Roche Diagnostics) in accordance with the condi Scutellaria laeteviolacea V. yakusimensis using RNeasy Plant tions recommended by the manufacturer. Mini Kit (QIAGEN), SuperScriptTM First-Strand Synthesis I0120) A hybridization buffer (5 xSSC, 30% formamide, System for RT-PCR (Invitrogen Corp.) was used under the 50 mM sodium phosphate buffer (pH 7.0), 1% SDS, 2% conditions recommended by the manufacturer to synthesize blocking reagent (Roche), 0.1% lauroylsarcosine and 80 g/ml cDNA from 1 g of total RNA. Based on the sequence of salmon sperm DNA) was used for prehybridization at 40°C. Sb7GAT from Scutellaria baicalensis (GenBank accession for an hour, and the denatured probe was then added, followed No. AB042277), primer-Fw (SEQID NO: 1) and primer-RV by incubation overnight. The membrane was rinsed in 4xSSC (SEQID NO: 2) were designed. Using the primers, PCR was wash buffer containing 1% SDS at 58° C. for 30 minutes. performed using as a template cDNA from Scutellaria laete Approximately 1x10 pful plaques were screened to obtain violacea V. yakusinensis. 300 positive clones.

SEO ID NO: 1: (3) Gene Identification S1UGT-FW : 5'-AAACATATGGCGGTGCTGGCGAAGTTC-3' 0121 The positive clones were purified to a single plaque (the underlined is the NdeI site) by secondary screening. Using a primer pair of M13RV and M13M4 (-20), the inserted fragment was amplified to deter SEO ID NO: 2: mine the DNA sequence of the inserted part. Using the puta S1UGT-Rw : 5'-TTTTGATCATTAATCCCGAGTGGCGTGAAG-3 tive amino acid sequence deduced based on the determined (the underlined is the Bcl site) DNA sequence, database search was performed by Blast X to obtain perilla UGT (PflugT50) having high homology to 0116 Specifically, the PCR solution (50 ul) was composed S1 UGT and Sb7GAT (SEQID NOs: 4 and 5). of 1 Jul of cDNA (Scutellaria laeteviolacea V. yakusimensis), 0.122 The results of analysis on the obtained full-length 1x Taq buffer (TakaRa), 0.2 mM dNTPs, 0.4 pmol each/ul of PflugT50, S1UGT and Sb7GAT on the CLUSTAL-W pro the primers (SEQID NOs: 1 and 2) and 2.5U of rTaq poly gram (MACVECTOR 7.2.2 software. Accerly Corporation) merase. PCR was performed by reacting at 94° C. for 3 strongly suggested that both S1UGT and Sb7GAT were minutes and then amplified for 30 cycles with each cycle incomplete ORF (open reading frame) missing the 3'- and consisting of reacting at 94° C. for a minute, 53° C. for a minute and 72° C. for 2 minutes. 5'-regions. So, rapid amplification of cDNA end (hereinafter 0117 The amplified fragment was inserted into the mul RACE) was conducted using a Gene Racer Kit (Invitrogen), ticloning site of pCR-TOPOII vector (Invitrogen), and the according to the protocol recommended by the manufacturer nucleotide sequence of the inserted fragment was determined to amplify the 5’- and 3'-regions of S1 UGT. For RACE, the by the primer walking method using synthetic oligonucle following primer sets specific to each S1 UGT gene were used otide primer with DNA Sequencer Model 3100 (Applied (SEQID NOs: 6 to 9). Biosystems). As a result of the analysis on the nucleotide sequence obtained by CLUSTAL-W Program (MACVEC SEO ID NO: 6: GR-S1UGT-Rv: TOR 7.2.2 software. Accerly Corporation), it was confirmed 5'-TGG GAG GCA AAC CAG GGA, TCT CCA CAA that the partial sequence of UGT from Scutellaria laetevio lacea V. yakusinensis highly homologous to Scutellaria SEO ID NO: 7 : S1UGT-nest-Riv: baicalensis was obtained (SEQ ID NO:3). This UGT from 5 - AAT CAT CCA. AAT. CTT TAA GGT Scutellaria laeteviolacea V. yakusinensis was used as a tem SEO ID NO: 8: GR-S1UGT-Fw : plate for the screening probe in the form of S1 UGT partial 5 - AGA AGG GGT GTG TTC TCC GCT GAG CAA Sequence. SEO ID NO: 9: S1UGT-nest-Fw : 0118. The fragment obtained by RT-PCR was labeled by 5 - GAA CAG CGG TCA CAG ATT TCT using the Non-Radioisotope DIG-Nucleic Acid Detection System (Roche Diagnostics) in accordance with the condi I0123. The nucleotide sequences of the respective ampli tions recommended by the manufacturer. Specifically, the fied products were determined by the primer walking method PCR solution (50 ul) was composed of 1 Jul of each cDNA, 1 x using synthetic oligonucleotide primers to obtain the S1UGT Taq buffer (TakaRa), 0.2 mM dNTPs, 0.4 pmol eachful of the gene including the full-length ORF and the amino acid primers (SEQID NOs: 1 and 2) and 2.5U of rTaq polymerase. sequence (SEQID NOs: 10 and 11). US 2010/0323402 A1 Dec. 23, 2010

0.124. In addition to Perilla frutescens and Scutellaria The eluate was concentrated and freeze dried. This fraction laeteviolacea V. yakusinensis, 7-glucuronides of apigenin, was purified by preparatory HPLC below to give 0.4 mg of which is one of flavones, are reported also on Scrophulari Scutellarein (aglycone). aceae, Antirrhinum majus (Harborne, J. B. Phytochemistry, 2. 327-334. 1963). Since the functionally unknown Antirrhinum Conditions for Preparatory HPLC majus glucosyltransferase AmUGTcg 10 represented by SEQ ID NOs: 12 and 13, which were previously isolated, shows a 0.133 Column: Develosil C-30-UG5, 20 mmx250 mm high homology to PflugT50 and S1 UGT isolated in this I0134) Moving phase: A 0.1% TFA, B 0.05% EXAMPLE, AmCGTcg10 was also a candidate gene for TFA/90% CHCN enzyme analysis (Ono, E. et al., Proc. Natl. Acad. Sci. USA 0.135 Flow rate: 6 ml/min. 103, 11075-11080. 2006). 0.136 Gradient: B15->B70% (60 min), B70% iso (10 0.125. The alignments of AmuGTcg10 from Antirrhinum min) majus, S1 UGT from Scutellaria laeteviolacea V. yakusinen 0.137 Detected: A330 mm sis and PflugT50 from Perilla frutescens obtained above are shown in FIG. 1. In FIG. 1, Sb7GAT from Scutellaria 4. Expression of Recombinant Perilla Frutescens baicalensis is also included. Glucosyltransferase and Its Activity Assay (1) Construction of Expression Vector 3. Extraction and Purification of Flavonoids from the 0.138 cDNAs bearing the full-length ORF of 3 glucosyl Leaves of Perilla Frutescens transferases of PflugT50 from Perilla frutescens, S1UGT from Scutellaria laeteviolacea V. yakusimensis and (1) Purification of Scutellarein 7-Glucuronide AmUGTcg10 from Antirrhinum majus, having a high homol 0126. After 144.4 g of green-leaved perilla (Toyohashi ogy to each other, were amplified using the primer sets spe Greenhouse Association in Aichi) corresponding to 200 cific to the respective genes (PflugT50, SEQID NOs: 14-15; leaves were ground into powders in liquid nitrogen, the pow S1UGT, SEQ ID NOs: 16-17; AmuGTcg10, SEQID NOs: ders were immersed in 1.5 L of 50% CHCN and 0.1% 18-19). cDNAs synthesized using total RNAs extracted from HCOOH overnight and then filtered through a celite pad. The the leaves of Perilla frutescens, the radix of Scutellaria laete filtrate was concentrated under reduced pressure. The con violacea V. yakusimensis and the petals of Antirrhinum maius centrate was loaded on 600 ml of CHP-20P and stepwise were used as templates, respectively. eluted twice with 300 ml each of water, 10, 20, 30, 40 and 50% CHCN/HO. Each fraction from 10%-2 to 50%-1 in which elution of polyphenols was observed was concen SEO ID NO : 14: Pf UGT5 O-fw: trated, frozen and dried, which was subjected to HPLC analy 5'-AAACATATGGAAGGCGTCATACTTC-3 sis. The yield was 10%-2 (12.1 mg), 20%-1 (52.5 mg), 20%-2 (the underlined is the NdeI site) (122.4 mg), 30%-1 (227.5 mg), 30%-2 (262.8 mg), 40%-1 (632.4 mg), 40%-2 (192.0 mg) and 50%-1 (113.2 mg). In the SEO ID NO : 15: Pf UGT5 O-rv: 40%-1 fraction, roSmarinic acid was contained in a high s' - TTTTGATCATTAATCACGAGTTACGGAATC-3' purity and the other fractions all contained flavones. The (the underlined is the Bcl site) 30%-2 fraction was purified by preparatory HPLC below to SEO ID NO: 16: S1UGT-fw: give 16 mg of Scutellarein 7-glucuronide. 5'-AAACATATGGAGGACACGATTGTTATC-3'

Conditions for Preparatory HPLC (the underlined is the NdeI site) 0127 Column: Develosil C-30-UG5, 20 mmx250 mm SEO ID NO : 17: S1UGT-rv: 0128 Moving phase: A 0.1% TFA, B 0.05% s' - TTCATATGTCAATCCCTCGTGGCCAGAAG-3' TFA/90% CHCN (the underlined is the NdeI site) 0129. Flow rate: 6 ml/min. 0130 Gradient: B20->B60% (100 min), B60% iso (20 SEQ ID NO: 18: AmOGTcg10-fw: min) 5'-AAACATATGGAGGACACTATCGTTCTC-3' 0131 Detected: A280 nm. (the underlined is the NdeI site) (2) Hydrolysis of Scutellarein 7-Glucuronide and Purifica SEQ ID NO: 19: AmOGTcg10-rv: tion of Aglycone 5'-TTGGATCCTTAAGAAACCACCATATCAAC-3' (the underlined is the BamHI site) 0.132. From scutellarein 7-glucuronide obtained in (1) above, 3 mg was dissolved in 100 uL of DMSO and the I0139 PCR (KOD -Plus-, TOYOBO) was carried out, after solution was divided into 15 ml falcon tubes in half. To each reacting at 94°C. for 2 minutes, by repeating 35 cycles with tube 10 mL of HO, 2.5 mL of 0.2MNaacetate buffer (pH 5.0) each cycle consisting of reacting at 94°C. for 15 seconds, 50° and 0.8 ml of B-glucronidase/arylsulfatase (EC3.2.1.31/EC3. C. for 30 seconds and 68°C. for 1.5 minutes. The amplified 1.6.1, Roche Diagnostics GmbH) solution were added, fol DNA fragments were subcloned into pCR-Blunt II-TOPO lowed by incubation at 37°C. for 2 hours. After completion of vector (Zero Blunt TOPO PCR Cloning Kit, Invitrogen) and the reaction, the solution mixture was loaded onto Sep-Pak-C the nucleotide sequence was confirmed by an ABI 3100 Avant 18 (20cc). After washing with 20 ml of water and then with 20 (Applied Biosystems). With respect to PflugT50, unmethy ml of 10% EtOH to remove salts and proteins, the aglycone lated plasmid was obtained by transformation of the positive formed was eluted with 40 ml of 80% CHCN+0.05%TFA. strains obtained. The plasmids obtained were digested with US 2010/0323402 A1 Dec. 23, 2010

Ndel and BclI in the case of Pf JGT50, with Nde in S1UGT detection wavelength: 280 and 350 nm. Respective charac and with NdeI and BamHI in AmUGTcg10, respectively. The terizations and results are shown below. resulting DNA fragments of about 1.5 kb were ligated into pET-15 digested with Ndel and BamHI in the case of Analysis of Glycosyl Acceptor Substrate Specificity PflugT50 and AmUGTcg10 and with Ndel in the case of 0142. In accordance with the method described above, the SJUGT. specificity of various glycosyl acceptor Substrates was ana lyzed (FIGS. 2 to 4). (2) Culture of Recombinant Escherichia Coli and Purification 0.143 FIG. 2 shows the results of analysis on the specific of Protein ity of glycosyl acceptor substrates for PflugT50. In FIG. 2, the relative activity to each Substrate is shown, taking the 0140 Escherichia coli BL21 (DE3) was transformed with activity to scutellarein as 100%. As a result of the analysis, each plasmid obtained in 4-(1) above. The transformant PflugT50 showed the highest activity to scutellarein and obtained was shake cultured overnight at 37°C. in 4 ml of LB reacted well with flavones other than glycosides (baicalein medium (10 g/l tryptone, 5 g/l yeast extract, 1 g/l NaCl) (relative activity to scutellarein, 73%), apigenin (44%), luteo supplemented with 50 ug/ml of ampicillin. The culture broth, lin (41%), tricetin (87.9%), diosmetin (62%) and chrysoeriol 4 ml, which reached the stationary phase, was inoculated into (18%)). Furthermore, PflugT50 showed the activity also to 80 ml of the medium with the same composition, followed by flavonols (quercetin (26%), myricetin (9%) and kaempferol shake culture at 37° C. When the cell turbidity (OD) (3%)) and flavanones (naringenin (3%)) and (aureusidin reached approximately 0.5, 0.4 mM IPTG (isopropyl-3- (40%)). However, PflugT50 showed no activity to flavone thiogalactopyranoside) in a final concentration was added to C-glycosides (vitexin, isoVitexin and orientin), isoflavones the medium, followed by shake culture at 22°C. for 20 hours. (genistein, daidzein and formononetin), catechins (catechin All of the subsequent procedures were performed at 4°C. The and epigallocatechin gallate), coumarines (esculetin) and transformants cultured were centrifuged (7,000xg, 15 mins.) phenylpropanoids (coniferyl alcohol). In HPLC, the reaction to collect the cells, and 2 ml/g cell of Buffer S(20 mM sodium products of Scutellarein, baicalein, apigenin and quercetin phosphate buffer (pH 7.4), 20 mMimidazole, 0.5 MNaCl, 14 coincided in terms of retention time with the respective 7-glu mM f-mercaptoethanol) was added to the cells for suspen curonosylated products. 014.4 FIG. 3 shows the results of analysis on the specific Sion. Subsequently, ultrasonication was performed (15 secs.x ity of glycosyl acceptor substrates for S1 UGT. In FIG. 3, the 8) followed by centrifugation (15,000xg, 10 mins.). To the relative activity to each substrate is shown, taking the activity supernatant obtained, 0.12% (w/v) polyethyleneimine in a to scutellarein as 100%. As a result of the analysis, S1UGT final concentration was added to Suspend the cells, which was showed the highest activity to scutellarein but lower activities then allowed to stand for 30 minutes. The mixture was cen to flavones (apigenin (3%), luteolin (0.6%), tricetin (1.1%), trifuged (15,000xg, 10 mins.) and the supernatant was recov diosmetin (0.3%), chrysoeriol (0.02%)) other than baicalein ered as a crude enzyme solution. The crude enzyme solution (relative activity to scutellarein, 63%). The activities to fla was applied to His SpinTrap (GE Healthcare), which had Vonols (quercetin (1%), kaempferol (14%), flavanones (nar been equilibrated with Buffer S, and centrifuged (70xg, 30 ingenin (6%)), isoflavones (genistein (1%), daidzein (0.3%) secs.). After washing with 600 ul of Buffer S, the protein and formononetin (0.08%)) and coumarines (esculetin bound to the column was stepwise eluted with 600 ul each of (0.3%)) were low. S1 UGT showed no activity on flavone Buffer S containing 100, 200 and 500 mM imidazole. The C-glycosides (Vitexin, isoVitexin and orientin), aurones (au buffer in each fraction eluted was replaced by 20 mM potas reusidin), catechins (catechin and epigallocatechingallate) or sium phosphate buffer (pH 7.5) containing 14 mM f-mercap phenylpropanoids (coniferyl alcohol). In other words, it was toethanol, using Microcon YM-30 (Amicon). As a result of Suggested that modification at the ortho-position of the 7 SDS-PAGE analysis, the protein of expected size was con position of flavonoids would be important for the activity of firmed in the fractions eluted with 100 mM and 200 mM S1 UGT. In HPLC, the reaction products of scutellarein, imidazole. These fractions were mixed and used for analysis. baicalein and apigenin coincided in terms of retention time The objective proteins were not single in these fractions. with the respective 7-glucuronosylated products. On the other hand, pluralities of the products were detected with quercetin and one of them coincided in terms of retention time with the (3) Enzyme Reaction and Conditions for HPLC Analysis 7-glucuronosylated product. 0141 Standard reaction conditions are as follows. A reac 0145 FIG. 4 shows the results of analysis on the specific tion solution (2 mMUDP-glucuronic acid, 100 uM glycosyl ity of glycosyl acceptor substrates for AmUGTcg10. In FIG. acceptor substrate, 50 mM potassium phosphate buffer (pH 4, the relative activity to each Substrate is shown, taking the 7.5), enzyme solution), 50 ul, was prepared and the enzyme activity to scutellarein as 100%. As a result of the analysis, solution was added to initiate the reaction at 30° C. for 30 AmUGTcg10 showed the highest activity to scutellarein and minutes. The reaction was stopped by adding 50 ul of 0.5% reacted also with flavones other than glycosides (baicalein TFA in CHCN and provided for HPLC analysis. The condi (relative activity to scutellarein, 22%), apigenin (40%), luteo tions for HPLC areas follows: column, Develosil C30-UG-5 lin (34%), tricetin (23.8%), diosmetin (26%) and chrysoeriol (4.6x150 mm); eluant A, 0.1% TFA/HO; eluant B, 0.08% (18%)). AmUGTcg10 also showed the activities to flavonols TFA/90% CHCN; conditions A for elution (except for (quercetin (4%) and kaempferol (28%)), flavanones (narin aurone, catechin, coumarine and phenylpropanoid), 0 min/ genin (15%)) and aurones (aureusidin (2%)). However, 5% B-s 18 min/100% eluant B18.1 min/5% B-s25 min/5% AmUGTcg10 showed no activity to flavone C-glycosides B; conditions B for elution (aurone, catechin, coumarine and (Vitexin, isoVitexin and orientin), isoflavones (genistein, phenylpropanoid), 0 min/5% B->20 min/50% eluant daidzein and formononetin), catechins (catechin and epigal B->20.5 min/5% B->25 min/5% B; flow rate, 1 ml/min: locatechin gallate), coumarines (esculetin) and phenylpro US 2010/0323402 A1 Dec. 23, 2010 panoids (coniferyl alcohol). In HPLC, the reaction products majus, which are structurally similar as described above, of scutellarein, baicalein, apigenin and quercetin coincided in were identified. Glucosyltransferases from PflugT50 and terms of retention time with the respective 7-glucuronosy AmUGTcg10 had a broad glycosyl acceptor Substrate speci lated products. ficity as compared to Sb7GAT from Scutellaria baicalensis 0146 The foregoing results reveal that PflugT50 and and had the 7-glucuronosyl transfer activity of various fla AmUGTcg10 show a low specificity, whereas S1UGT vonoids such as flavones, flavonols, aurones, etc. On the other showed a high specificity, to flavonoids such as flavones, etc. hand, S1 UGT showed a substrate specificity similar to The results further reveal that PflyGT50 derived from Perilla Sb7GAT. By using these glucosyltransferases, the 7-position frutescens shows a particularly low specificity, i.e., is an of flavonoids can be glucuronidated at a low cost. enzyme with a broader range of Substrate specificity. 0151. Therefore, glucuronides including quercetin 7-glu cronide, which are present in Vivo, can be mass-produced in Analysis of Glycosyl Donor Substrate Specificity vitro, which enables to assess their physiological activities. 0147 Using apigenin as a glycosyl acceptor Substrate, Example 2 specificity to various UDP-activated glycosyl donors was analyzed. As a result, PflugT50, S1UGT and AmuGTcg10 1. Outline of Example 2 showed the activity only to UDP-glucuronic acid but showed 0152. In this EXAMPLE, SiOGT23 highly homologous to no activity on UDP-glucose and UDP-galactose. S1 UGT from Scutellaria laeteviolacea V. yakusimensis and 0148. The foregoing results reveal that these enzymes are AmUGTcg12 from Antirrhinum majus was identified from 7-glucuronosyltransferase highly specific for flavones (fla Lamiales Pedaliaceae Sesamum indicum and it was con Vone 7-glucuronosyltransferase). firmed that its Escherichia coli expression protein had the glucuronosyltransfer activity to scutellarein and luteolin. Analysis of pH and Temperature Characteristics 0149 Temperature stability was analyzed as follows. An 2. Identification of SiOGT23 Gene enzyme solution was treated at 15 to 55° C. for an hour and 0153. Paying attention to the sequence conservation of then cooled to 4°C. Using the sample cooled, the residual 7-glucuronosyltransferase genes (UGT genes) of flavonoids activity was determined under standard reaction conditions. from Scutellaria laeteviolacea V. yakusinensis and Antirrhi After 167 mM final concentration of a buffer solution (pH 4. num majus, genes having a high homology to these genes 4.5, 5, Acetate-NaOH: pH 5.5, 6, 6.5, 7, 7.5, NaH2PO were searched, and sesame UGT gene SiOGT23 showing a NaOH: pH 8, 8.5, 9, Tris-HCl) was added to the enzyme high homology to these UGT genes was found also in Lami solution, the mixture was treated at 4°C. for an hour and then ales Pedaliaceae Sesamum indicum. Since SiOGT23 isolated 500 mM final concentration of potassium phosphate buffer from the sesame cDNA library had no full-length sequence, (pH 7.5) was added thereto. Using the sample treated, the the full-length sequence of Si UGT23 (SEQID NOs: 22 and residual activity was determined understandard reaction con 23) was determined by the RACE method described in ditions. The reaction was performed understandard reaction EXAMPLE 1, 2(3), using the primers of SEQIDNOs: 20 and conditions where the reaction temperature was set at 10 to 55° 21 described below. C. to analyze the reaction temperature dependency. Reaction pH dependency was analyzed under standard reaction condi SEO ID NO: 2O: GR-SiOGT23-Rv tions using a buffer solution (pH 4, 4.5, 5, Acetate-NaOH: pH 5 " - GGCCAAACGCGCCGGAGCTGATGTAGA-3' 5.5, 6, 6.5, 7, 7.5, NaHPO-NaOH: pH 8, 8.5, 9, Tris-HCl) in place of potassium phosphate buffer (pH 7.5). As a result of SEO ID NO: 21: SiOGT23-nest-Riv the foregoing analyses, PflugT50 was stable at pH ranging 5'-AGTGGGTATATTCAAGCCTGT-3' from 7 to 9 and showed the maximum catalytic activity at pH between 7 and 8 but showed little activity in an acidic region. 3. Expression of SiOGT23 Derived from Sesamum PflugT50 was stable at 30° C. or lower (an hour, pH 7.5) and Indicum and Its Activity Assay the reaction optimum temperature was 30°C. in the activity assay for 30 minutes. S1 UGT was stable at pH ranging from 0154 cDNA containing full-length ORF of Si UGT23 4.5 to 8 and showed the maximum catalytic activity at pH derived from Sesamum indicum was amplified by a gene between 7 and 8 but showed little activity in an acidic region. specific primer set (SEQID NOs: 24 and 25). cDNA synthe This enzyme was stable at 40°C. or lower (an hour, pH 7.5) sized using total RNA extracted from Sesame seeds was used and the reaction optimum temperature was 35° C. in the as a template. SiOGT23 was incorporated into Escherichia coli expression vector as in UGT of Perilla frutescens, Scutel activity assay for 30 minutes. AmUGTcg10 was stable at pH laria laeteviolacea V. yakusinensis and Antirrhinum majus in ranging from 7.5 to 9.5 and showed the maximum catalytic EXAMPLE 1 and recombinant Si UGT23 protein was activity at pH between 8.5 and 9.5 but showed little activity in expressed in the same way to assay the enzyme activity. an acidic region. This enzyme was stable at 30° C. or lower (an hour, pH 7.5) and the reaction optimum temperature was 45° C. in the activity assay for 30 minutes. These properties SEO ID NO: 24: SiOGT23-fw: were similar to known GT involved in plant secondary 5 - CACCATATGGAAGACACCGTTGTCCTCTA-3' metabolism. (the underlined is the NdeI site) 5. Results SEO ID NO: 25: SiUGT23-rv: 0150. PflugT50 derived from Perilla frutescens, S1UGT 5 - GGATCCTAACATCACTCAAACCCGAGTCA-3' derived from Scutellaria laeteviolacea V. yakusinensis and (the underlined is the BamHI site) AmUGTcg10 derived from Scrophulariaceae, Antirrhinum US 2010/0323402 A1 Dec. 23, 2010

0155 FIG. 5 shows the results of analysis on the specific 0.165 Based on the results above, it was confirmed that the ity of glycosyl acceptor substrates for SiOGT23. In FIG. 5, glucuronides of Stilbenes and lignans could be produced by the relative activity to each Substrate is shown, taking the using PflugT50 from Perilla frutescens a red-leaf variety, activity to scutellarein as 100%. As a result of the analysis, S1 UGT from Scutellaria laeteviolacea V. yakusimensis, SiOGT23 showed the highest activity to scutellarein and AmUGTcg10 from Antirrhinum majus and SiOGT23 from reacted also with the following flavones (baicalein (relative Sesamum indicum, in addition to diverse flavonoids. activity to scutellarein (hereinafter the same), 24%), apigenin (7%), luteolin (29%), tricetin (14.0%), diosmetin (4%), chry Soeriol (6%) and isovitexin (0.3%)). In addition, SiOGT23 Example 4 showed the activities to flavonols (quercetin (4%) and kaempferol (18%)), flavanones (naringenin (2%)), aurones (0166 S1 UGT derived from Scutellaria laeteviolacea V. (aureusidin (13%)), coumarines (esculetin (0.03%) and the yakusimensis, when reacted with quercetin (QU), gave a plu following isoflavones (formononetin (0.03%)). However, rality of products in addition to the 7-glucuronic acid (FIG. 8). SiOGT23 showed no activity to flavone C-glycosides (vitexin 0167. They are characteristic products that are not found and orientin) other than isoVitexin, isoflavones (genistein, with SiOGT23 derived from Sesamum indicum, Daidzein) other than formononetin, catechins (catechin and AmUGTcg10 derived from Antirrhinum majus and epigallocatechin gallate) and phenylpropanoids (caffeic PflugT50 derived from Perilla frutescens. As a result of the acid). In HPLC, the reaction products of scutellarein, baica LC-MS analysis, quercetin diglucuronide showing m/z =653. lein, apigenin and quercetin coincided in terms of retention 1009 M-H (CHOs, calcd. 653.0990, err. 2.9ppm), in time with the respective 7-glucuronosylated products. which two glucuronic acids would be transferred to quercetin 0156 The foregoing results revealed that SiOGT23 from Sesamum indicum was found to be a 7-glucuronosyltrans during the retention time of 7 to 9 minutes, was detected. ferase of flavonoids showing a low specificity for flavonoids During the retention time of 9 to 11 minutes, 3 types of such as flavones, etc., as in PfiT50 from Perilla frutescens quercetin monoglucuronides showing m/z. 477.06 M-H, and AmCGTcg10 from Antirrhinum majus. in which one glucuronic acid would be transferred to querce tin, were detected. The component showing R.T. =10.12 min Example 3 utes coincided with the reaction product with GAT of Perilla frutescens, which was purified and structurally analyzed, and (O157. Using recombinant proteins of PflugT50 derived was found to be quercetin 7-O-glucuronide. Furthermore, the from Perilla frutescens, S1UGT derived from Scutellaria laeteviolacea V.yakusinensis and AmUGTcg10 derived from component showing R.T. = 10.34 minutes coincided with the Antirrhinum majus as well as recombinant protein of major flavonol from grape leaves, which was purified and SiOGT23 derived from Sesamum indicum, the glucuronosyl structurally analyzed, and was found to be quercetin 3-O- transfer activities to Stilbenes and lignans, which are plant glucronide (Hmamouch, M. et al. (1996) Am. J. Enol Vitic. polyphenols, were examined. The enzyme reaction was per 47, 186-192). The component showing R.T. = 11.3 minutes, formed under the same conditions as described above, using which is the main reaction product, was found to be quercetin resveratrol as a substrate for stilbenes and as substrates for 3'-O-glucronide, after purification on reversed phase HPLC lignans, (+)-pinoresinol, (+)-piperitol, (+)-sesaminol, (+)- and structural analysis by NMR. It was confirmed that the 3 secoisolariciresinol, (+)-sesamin catechol 1 (SC1), (+)-sesa major peaks represented 7-, 3- and 3'-monoglucronides of min catechol 2 (SC2) and (+)-episesamin catechol 2 (EC2) quercetin, respectively. (Nakai M, et al. (2003).J. Agric Food Chem. 51, 1666-1670). (0168 The conditions for LC-MS areas follows. 0158. As a result of the HPLC analysis, the products were obtained in all of the reaction solutions of PflugT50 from (0169 Column: YMC-pack polymer C18, 2 mmx 150 mm, Perilla frutescens a red-leaf variety, S1UGT from Scutellaria 6 um laete violacea V. yakusimensis, AmCGTcg10 from Antirrhi (0170 Moving phase: A: H.O. B: CHCN, C; 2.5% num majus and SiOGT23 from Sesamum indicum (cf., FIGS. HCOOH, 0.2 ml/min. 2 to 5). As for lignans, new products were obtained when SC1 (0171 Gradient: B10%B50% (10 minutes), B50% (5min and SC2 were used as substrates. As a result of the LC-MS utes), C4% analysis on the reaction of S1 UGT with SC 1 in which the 0172 Detected: A350 nm. largest numbers of the products were obtained, SC1 was (0173 MS conditions: Q-TOF-Premier (Micromass, eluted at R.T. = 12.8 minutes and the reaction product was confirmed to be the SC1 monoglucuronide showing R.T. Manchester, UK) =10.8 minutes (FIG. 6) and m/z 517.1328 M-H 0.174 V mode, negative, capillary voltage: 3.0 KV, cone (CHO, calcd. 517.1346, err. -2.5 ppm) (FIG. 7). LC voltage: 30V MS was performed under the following conditions. 0.175. The foregoing results reveal that a variety of quer 0159 Column: Develosil C30-UG3, 3 mmx 150 mm cetin glucuronides can be obtained from quercetin by using (0160 Moving phase: A: HO, B: CHCN, C; 2.5% S1 UGT from Scutellaria laeteviolacea V. yakusimensis. HCOOH, 0.2 ml/min. (0161 Gradient: B20%->B70% (10 mins.), B70% (5 INDUSTRIAL APPLICABILITY mins.), C4% (0162. Detected: A280 nm. 0176 The UDP-glucuronosyltransferase of the present (0163 MS conditions: Q-TOF-Premier (Micromass, invention has a broad substrate specificity and is useful for the Manchester, UK) production of various glucuronides. The glucuronides pro 0164 V mode, negative, capillary voltage: 2.3 KV, cone duced are useful as reagents for inspecting the in vivo func voltage: 45V tions, antioxidants, etc.

US 2010/0323402 A1 Dec. 23, 2010 15

- Continued

Ile Thir Ile Luell Ser Ser Ala Asp Ser Ala Ala Ala Ser Wall Ser 35 4 O 45

Thir Luell Pro Ser Ile Thir Tyr Arg Arg Luell Pro Pro Wall Ala Ile Pro SO 55 6 O

Pro Asp Ser Ile Asn Pro Wall Glu Ala Phe Phe Glu Ile Pro Arg 65 70

Lell Glin Asn Pro Asn Lell Arg Wall Ala Luell Glu Glu Ile Ser Glin 85 90 95

Thir Arg Ile Arg Ala Phe Wall Ile Asp Phe Phe Asn Ser Ala Phe 105 11 O

Glu Wall Ser Thir Ser Lell Ser Ile Pro Thir Phe Tyr Wall Ser Thir 115 12 O 125

Gly Ser Ala Gly Wall Ile Phe Luell Tyr Phe Pro Thir Thir Asp Glu 13 O 135 14 O

Thir Wall Ala Thir Asp Ile Gly Asp Luell Arg Asp Phe Lell Glu Phe Pro 145 150 155 160

Gly Ser Pro Ile Ile His Ser Ser Asp Luell Pro Glin Lell Thir Phe Phe 1.65 17O 17s

Arg Arg Ser Asn Wall Phe His Met Luell Asp Thir Ser Lys Asn Met 18O 185 19 O

Glin Ser Ser Gly Ile Lell Thir Asn Gly Phe Asp Ala Met Glu Phe 195

Arg Ala Glu Ala Lell Thir Asn Gly Luell Cys Wall Pro Asn Gly Pro 21 O 215 22O

Thir Pro Pro Wall Tyr Lell Wall Gly Pro Luell Wall Ala Gly Ser Asn Ala 225 23 O 235 24 O

Asp His Glu Lell Luell Trp Luell Asp Arg Glin Pro Ser 245 250 255

Ser Wall Wall Phe Lell Phe Gly Arg Arg Gly Lell Phe Ser Gly 26 O 265 27 O

Glin Luell Arg Glu Met Ala Wall Ala Luell Glu Arg Ser Gly Tyr Arg Phe 285

Lell Trp Ser Wall Arg Asn Pro Pro Glu Asn Arg Ser Pro Ala Glu Asp 29 O 295 3 OO

Pro Asp Luell Asp Glu Lell Lell Pro Glu Gly Phe Lell Glu Arg Thir Lys 3. OS 310 315

Asp Ile Gly Phe Wall Wall Ser Trp Ala Pro Glin Glu Wall Luell 3.25 330 335

Ser His Asp Ala Wall Ala Gly Phe Wall Thir His Gly Arg Ser Ser 34 O 345 35. O

Ile Luell Glu Ala Lell Wall Asn Gly Pro Met Ile Gly Trp Pro Met 355 360 365

Ala Glu Glin Arg Met Asn Wall Phe Met Wall Asp Glu Met 37 O 375

Wall Ala Luell Pro Lell Glu Glu Glu Glu Asp Gly Phe Wall Thir Ala Wall 385 390 395 4 OO

Glu Luell Glu Arg Lell Arg Glin Luell Met Glu Ser Thir Gly Arg 4 OS 41O 415

Asp Wall Arg His Arg Wall Ala Glu Met Ala Ala Ala Thir Ala Ala 42O 425 43 O US 2010/0323402 A1 Dec. 23, 2010 16

- Continued Met Gly Glu Asin Gly Ser Ala Val Val Ala Lieu. Arg Llys Phe Ile Asp 435 44 O 445 Ser Val Thr Arg Asp 450

<210s, SEQ ID NO 6 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 6 tgggaggcaa accagggat.c ticgacaa 27

<210s, SEQ ID NO 7 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OO > SEQUENCE: 7 aatcatccaa atctittaagg t 21

<210s, SEQ ID NO 8 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 8 agaaggggtg tdttct cogc tigagcaa. 27

<210s, SEQ ID NO 9 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 9 gaac agcggt cacagatttic t 21

<210s, SEQ ID NO 10 &211s LENGTH: 1446 &212s. TYPE: DNA <213> ORGANISM: Scutellaria sp. 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1365)

<4 OOs, SEQUENCE: 10 atg gag gac acg att gtt at C tac acc acg ccg gag cac ctgaac acc 48 Met Glu Asp Thir Ile Val Ile Tyr Thr Thr Pro Glu. His Lieu. Asn Thr 1. 5 1O 15 atg gog gtg ct c goc aag titc atc agc aaa cac cac ccc toc gtc. ccc 96 Met Ala Val Lieu Ala Lys Phe Ile Ser Lys His His Pro Ser Val Pro 2O 25 3O

US 2010/0323402 A1 Dec. 23, 2010 18

- Continued gag gtg Ctg agt cac gac tog gtggggggg ttc gtc act cac tdt ggg O56 Glu Val Lieu Ser His Asp Ser Val Gly Gly Phe Val Thr His Cys Gly 34 O 345 35. O cgg agc ticc att tog gaa ggg gtg togg ttt ggg gtg ccg atg at C ggg 104 Arg Ser Ser Ile Ser Glu Gly Val Trp Phe Gly Val Pro Met Ile Gly 355 360 365 tgg ccg gtg gaC gcg gag cag aag ttgaat cqa aca gtg ttg gtg gag 152 Trp Pro Val Asp Ala Glu Gln Lys Lieu. Asn Arg Thr Val Lieu Val Glu 37 O 375 38O gala atg cag gtg gC9 Ctg cc.g. atg gag gag gC9 gag get gig titC gtg 2OO Glu Met Glin Val Ala Leu Pro Met Glu Glu Ala Glu Gly Gly Phe Val 385 390 395 4 OO acg gcg gct gag Ctg gag aaa C9a gtt aga gag titg atg gag tog aag 248 Thir Ala Ala Glu Lieu. Glu Lys Arg Val Arg Glu Lieu Met Glu Ser Lys 4 OS 41O 415 gtgggg aag gC9 gtg agg caa cqa gtC ggit gaa titg aaa to tcg gCC 296 Val Gly Lys Ala Val Arg Glin Arg Val Gly Glu Lieu Lys Cys Ser Ala 42O 425 43 O agg gca gcg gtg acg ggg aat gga to C tog Cta agt gat titt aaa aag 344 Arg Ala Ala Val Thr Gly Asn Gly Ser Ser Lieu. Ser Asp Phe Llys Llys 435 44 O 445 titt citt ctd gcc acg agg gat tdat catata citct coat ct c cqt cattga 395 Phe Lieu. Lieu Ala Thr Arg Asp 450 45.5 att cataaaa gtatttittaa gacaagttitt tdtgagagaa taagttaatc a 446

<210s, SEQ ID NO 11 &211s LENGTH: 45.5 212. TYPE: PRT <213> ORGANISM: Scutellaria sp. <4 OOs, SEQUENCE: 11 Met Glu Asp Thir Ile Val Ile Tyr Thr Thr Pro Glu. His Lieu. Asn Thr 1. 5 1O 15 Met Ala Val Lieu Ala Lys Phe Ile Ser Lys His His Pro Ser Val Pro 2O 25 3O

Ile Ile Lieu. Ile Ser Thir Ala Ala Glu Ser Ala Ala Ala Ser Ile Ala 35 4 O 45 Ala Val Pro Ser Ile Thr Tyr His Arg Lieu Pro Leu Pro Glu Ile Pro SO 55 6 O Pro Ser Lieu. Thir Lys Asp Arg Val Glu Lieu. Phe Phe Glu Lieu Pro Arg 65 70 7s 8O Lieu. Ser Asn Pro Asn Lieu. Arg Lieu Ala Lieu. Glin Glu Ile Ser Glin Lys 85 90 95 Ala Arg Ile Arg Ala Phe Val Ile Asp Phe Phe Cys Asn Ala Ala Phe 1OO 105 11 O Glu Val Ser Thr Ser Leu Ser Ile Pro Thr Phe Tyr Tyr Phe Ser Ser 115 12 O 125 Gly Ser Pro Thr Ala Thr Lieu Val Lieu. His Phe Glin Thr Lieu. Asp Glu 13 O 135 14 O Thir Ile Pro Gly Asp Lieu Lys Asp Lieu. Asp Asp Phe Val Glu Ile Pro 145 150 155 160 Gly Leu Pro Pro Ile Tyr Ser Lieu. Asp Ile Pro Val Ala Leu Lieu. Thr 1.65 17O 17s US 2010/0323402 A1 Dec. 23, 2010 19

- Continued Arg Glin Ser Lieu Val Tyr Glin Ser Ser Val Asp Ile Ser Lys Asn Lieu 18O 185 19 O Arg Llys Ser Ala Gly Phe Lieu Val Asn Gly Phe Asp Ala Lieu. Glu Phe 195 2OO 2O5 Arg Ala Lys Glu Ala Ile Val Asn Gly Lieu. CyS Val Pro Asn Gly Pro 21 O 215 22O Thr Pro Pro Val Tyr Phe Ile Gly Pro Leu Val Gly Asp Val Asp Ala 225 23 O 235 24 O Lys Ala Gly Gly Glu Glu. His Glu. Cys Lieu. Arg Trp Lieu. Asp Thr Glin 245 250 255 Pro Ser Lys Ser Val Ile Phe Lieu. Cys Phe Gly Arg Arg Gly Val Phe 26 O 265 27 O Ser Ala Glu Gln Lieu Lys Glu Thir Ala Val Ala Lieu. Glu Asn. Ser Gly 27s 28O 285 His Arg Phe Leu Trp Ser Val Arg Asn Pro Pro Glu Ile Met Lys Asn 29 O 295 3 OO Ser Asp Glu Pro Asp Lieu. Asp Glu Lieu. Lieu Pro Glu Gly Phe Lieu. Glu 3. OS 310 315 32O Arg Thr Lys Asp Arg Gly Phe Val Ile Llys Ser Trp Ala Pro Gln Lys 3.25 330 335 Glu Val Lieu Ser His Asp Ser Val Gly Gly Phe Val Thr His Cys Gly 34 O 345 35. O Arg Ser Ser Ile Ser Glu Gly Val Trp Phe Gly Val Pro Met Ile Gly 355 360 365 Trp Pro Val Asp Ala Glu Gln Lys Lieu. Asn Arg Thr Val Lieu Val Glu 37 O 375 38O Glu Met Glin Val Ala Leu Pro Met Glu Glu Ala Glu Gly Gly Phe Val 385 390 395 4 OO Thir Ala Ala Glu Lieu. Glu Lys Arg Val Arg Glu Lieu Met Glu Ser Lys 4 OS 41O 415 Val Gly Lys Ala Val Arg Glin Arg Val Gly Glu Lieu Lys Cys Ser Ala 42O 425 43 O Arg Ala Ala Val Thr Gly Asn Gly Ser Ser Lieu. Ser Asp Phe Llys Llys 435 44 O 445 Phe Lieu. Lieu Ala Thr Arg Asp 450 45.5

<210s, SEQ ID NO 12 &211s LENGTH: 1374 &212s. TYPE: DNA <213> ORGANISM: Antirrhinum majus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1371)

<4 OOs, SEQUENCE: 12 atg gag gac act atc gtt ct c tac got to a goa gag cac citt aac to c 48 Met Glu Asp Thir Ile Val Lieu. Tyr Ala Ser Ala Glu. His Lieu. Asn. Ser 1. 5 1O 15 atg ct a ct a ct c gagc aaa citc atc aac aaa cac cac ccc aca atc. tcc 96 Met Lieu. Lieu. Lieu. Gly Llys Lieu. Ile Asn Llys His His Pro Thir Ile Ser 2O 25 3O gtc gcc att atc agc acc gcc cca aac goc goc got agt to c gtc gcc 144 Wall Ala Ile Ile Ser Thr Ala Pro Asn Ala Ala Ala Ser Ser Wall Ala 35 4 O 45

US 2010/0323402 A1 Dec. 23, 2010 21

- Continued tgt ggg agg agt t c at a ttg gala gcg gtg tcg titt ggg gtg ccg atg 104 Cys Gly Arg Ser Ser Ile Lieu. Glu Ala Val Ser Phe Gly Val Pro Met 355 360 365 atc ggg togg ccg at a tac gcg gag cag agg atgaat agg gtg tt C atg 152 Ile Gly Trp Pro Ile Tyr Ala Glu Glin Arg Met Asn Arg Val Phe Met 37 O 375 38O gtg gag gag atg aag gttg gC9 ttg cag ttg gat gag gttg gag gala gig 2OO Val Glu Glu Met Llys Val Ala Lieu. Glin Lieu. Asp Glu Val Glu Glu Gly 385 390 395 4 OO ttic gtg gcg gC9 gtg gaa ttg gag aag aga gtg aag gag titg atg gat 248 Phe Val Ala Ala Val Glu Lieu. Glu Lys Arg Val Lys Glu Lieu Met Asp 4 OS 41O 415 tcg aag aat ggg aga gcg gtt agg cag aga gtg aag gag atg aaa gtg 296 Ser Lys Asn Gly Arg Ala Val Arg Glin Arg Val Lys Glu Met Llys Val 42O 425 43 O gC9 gct gag gtg gC9 gtt gala aag gt get tog tica gtt gtg gC9 ttg 344 Ala Ala Glu Val Ala Val Glu Lys Gly Gly Ser Ser Val Val Ala Lieu 435 44 O 445 caa cqc titt gtt gat atg gtg gtt tot taa 374 Glin Arg Phe Val Asp Met Val Val Ser 450 45.5

<210s, SEQ ID NO 13 &211s LENGTH: 457 212. TYPE: PRT <213> ORGANISM: Antirrhinum majus <4 OOs, SEQUENCE: 13 Met Glu Asp Thir Ile Val Lieu. Tyr Ala Ser Ala Glu. His Lieu. Asn. Ser 1. 5 1O 15 Met Lieu. Lieu. Lieu. Gly Llys Lieu. Ile Asn Llys His His Pro Thir Ile Ser 2O 25 3O

Wall Ala Ile Ile Ser Thr Ala Pro Asn Ala Ala Ala Ser Ser Wall Ala 35 4 O 45 Asp Wall Ala Ala Ile Ser Tyr Glin Glin Lieu Lys Pro Ala Thr Lieu Pro SO 55 6 O Ser Asp Lieu. Thir Lys Asn Pro Ile Glu Lieu Phe Phe Glu Ile Pro Arg 65 70 7s 8O Lieu. His Asn Pro Asn Lieu. Lieu. Glu Ala Lieu. Glu Glu Lieu. Ser Lieu Lys 85 90 95 Ser Llys Val Arg Ala Phe Val Ile Asp Phe Phe Cys Asn Pro Ala Phe 1OO 105 11 O Glu Val Ser Thr Ser Lieu. Asn Ile Pro Thr Tyr Phe Tyr Val Ser Ser 115 12 O 125 Gly Ala Phe Gly Lieu. Cys Gly Phe Lieu. His Phe Pro Thr Ile Asp Glu 13 O 135 14 O Thr Val Glu Lys Asp Ile Gly Glu Lieu. Asn Asp Ile Lieu. Glu Ile Pro 145 150 155 160 Gly Cys Pro Pro Val Lieu Ser Ser Asp Phe Pro Lys Gly Met Phe Phe 1.65 17O 17s Arg Llys Ser Asn. Thir Tyr Llys His Phe Lieu. Asp Thr Ala Lys Asn Met 18O 185 19 O Arg Arg Ala Lys Gly Ile Val Val Asn Ala Phe Asp Ala Met Glu Phe 195 2OO 2O5 US 2010/0323402 A1 Dec. 23, 2010 22

- Continued Arg Ala Lys Glu Ala Lieu Val Asn. Asn Lieu. CyS Val Pro Asn. Ser Pro 21 O 215 22O Thr Pro Pro Val Phe Leu Val Gly Pro Leu Val Gly Ala Ser Thir Thr 225 23 O 235 24 O Thir Lys Thir Thir Asn. Glu Gln His Glu. Cys Lieu Lys Trp Lieu. Asp Wall 245 250 255 Glin Pro Asp Arg Ser Val Ile Phe Lieu. Cys Phe Gly Arg Arg Gly Lieu. 26 O 265 27 O Phe Ser Ala Asp Glin Lieu Lys Glu Ile Ala Ile Gly Lieu. Glu Asn. Ser 27s 28O 285 Gly His Arg Phe Leu Trp Ser Val Arg Cys Pro Pro Ser Llys Pro Asn 29 O 295 3 OO Ser Tyr Asn. Thir Asp Pro Asp Lieu. Asp Glu Lieu Lleu Pro Glu Gly Phe 3. OS 310 315 32O Lieu. Ser Arg Thr Glu Thir Arg Gly Phe Val Ile Llys Ser Trp Ala Pro 3.25 330 335 Gln Lys Glu Val Lieu Ser His Gly Ala Val Gly Gly Phe Val Thr His 34 O 345 35. O Cys Gly Arg Ser Ser Ile Lieu. Glu Ala Val Ser Phe Gly Val Pro Met 355 360 365 Ile Gly Trp Pro Ile Tyr Ala Glu Glin Arg Met Asn Arg Val Phe Met 37 O 375 38O Val Glu Glu Met Llys Val Ala Lieu. Glin Lieu. Asp Glu Val Glu Glu Gly 385 390 395 4 OO Phe Val Ala Ala Val Glu Lieu. Glu Lys Arg Val Lys Glu Lieu Met Asp 4 OS 41O 415 Ser Lys Asn Gly Arg Ala Val Arg Glin Arg Val Lys Glu Met Llys Val 42O 425 43 O Ala Ala Glu Val Ala Val Glu Lys Gly Gly Ser Ser Val Val Ala Lieu 435 44 O 445 Glin Arg Phe Val Asp Met Val Val Ser 450 45.5

<210s, SEQ ID NO 14 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 14 aaacatatgg aaggcgt.cat acttic 25

<210s, SEQ ID NO 15 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 15 ttittgat cat taatcacgag titacggaatc 3 O

<210s, SEQ ID NO 16 US 2010/0323402 A1 Dec. 23, 2010 23

- Continued

&211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 16 aalacatatgg aggacacgat tittatc 27

<210s, SEQ ID NO 17 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 17 tt catatgtc. aatccct cqt ggc.ca.gaag 29

<210s, SEQ ID NO 18 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 18 aaacatatgg aggacactat cqttct c 27

<210s, SEQ ID NO 19 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 19 ttggat.cctt aagaaaccac catat caac 29

<210s, SEQ ID NO 2 O &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 2O ggccaaacgc gcc.ggagctg atgtaga 27

<210s, SEQ ID NO 21 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 21 agtgggtata ttcaa.gc.ctg. it 21

US 2010/0323402 A1 Dec. 23, 2010 26

- Continued Lieu. Asn. Asn Pro Asn Val Ser Lys Ala Lieu. Glin Glu Ile Ser Glin Lys 85 90 95 Ser Arg Ile Lys Ala Phe Val Ile Asp Phe Phe Cys Asn Pro Val Phe 1OO 105 11 O Glu Val Ser Thr Gly Lieu. Asn Ile Pro Thr Tyr Phe Tyr Ile Ser Ser 115 12 O 125 Gly Ala Phe Gly Lieu. Cys Pro Phe Lieu. Asn Phe Pro Thr Ile Glu Glu 13 O 135 14 O Thr Val Pro Gly Asp Lieu Ala Asp Lieu. Asn Asp Phe Val Glu Ile Pro 145 150 155 160 Gly Cys Pro Pro Val His Ser Ser Asp Phe Pro Glu Ala Met Ile His 1.65 17O 17s Arg Llys Ser Asn. Ile Tyr Llys His Phe Met Asp Ala Ala Arg Asn Met 18O 185 19 O Ala Lys Ser Thr Gly Asn Lieu Val Asn Ala Phe Asp Ala Lieu. Glu Phe 195 2OO 2O5 Arg Ala Lys Glu Ala Lieu. Ile Asin Gly Lieu. Cys Ile Pro Asn Ala Pro 21 O 215 22O Thr Pro Pro Val Tyr Lieu Val Gly Pro Leu Val Gly Asp Ser Asn Arg 225 23 O 235 24 O Asn Asn Gly Cys Ile Gln His Glu. Cys Lieu Lys Trp Lieu. Asp Ser Glin 245 250 255 Pro Ser Lys Ser Val Ile Phe Lieu. Cys Phe Gly Arg Arg Gly Lieu Phe 26 O 265 27 O Ser Val Glu Gln Lieu Lys Glu Met Ala Lieu. Gly Lieu. Glu Asn. Ser Gly 27s 28O 285 Tyr Arg Phe Leu Trp Ser Val Arg Ser Pro Pro Gly Lys Glin Asn Ser 29 O 295 3 OO Ala Ala Ala Glu Pro Asp Lieu. Asp Glu Lieu. Lieu Pro Llys Gly Phe Lieu 3. OS 310 315 32O Glu Arg Thir Lys Asp Arg Gly Phe Ile Ile Llys Ser Trp Ala Pro Glin 3.25 330 335 Thr Glu Val Lieu Ser His Asp Ser Val Gly Gly Phe Val Thr His Cys 34 O 345 35. O Gly Arg Ser Ser Ile Lieu. Glu Ala Val Ser Lieu. Gly Val Pro Met Ile 355 360 365 Gly Trp Pro Leu Tyr Ala Glu Glin Arg Met Asn Arg Val Phe Met Val 37 O 375 38O Glu Glu Met Llys Val Ala Lieu Pro Lieu. Glu Glu Thir Ala Asp Gly Lieu. 385 390 395 4 OO Val Thir Ala Val Glu Lieu. Glu Lys Arg Val Arg Glin Lieu Met Asp Ser 4 OS 41O 415 Glin Thr Gly Arg Ala Val Arg His Arg Val Thr Glu Lieu Lys Ser Ser 42O 425 43 O Ala Ala Ala Ala Val Arg Lys Asn Gly Ser Ser Lieu Val Ala Lieu. Glin 435 44 O 445 Asn Phe Ile Ala Ser Val Thr Arg Val 450 45.5

<210s, SEQ ID NO 24 &211s LENGTH: 29 &212s. TYPE: DNA US 2010/0323402 A1 Dec. 23, 2010 27

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 24 caccatatgg aagacaccgt tdtcct cta 29

<210s, SEQ ID NO 25 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 25 ggat.cctaac at cact caaa ccc.gagtica 29

<210s, SEQ ID NO 26 &211s LENGTH: 441 212. TYPE: PRT <213> ORGANISM: Scutellaria baicallensis

<4 OOs, SEQUENCE: 26 Met Ala Val Lieu Ala Lys Phe Ile Ser Lys Asn His Pro Ser Val Pro 1. 5 1O 15

Ile Ile Ile Ile Ser Asn Ala Pro Glu Ser Ala Ala Ala Ser Wall Ala 2O 25 3O Ala Ile Pro Ser Ile Ser Tyr His Arg Lieu Pro Leu Pro Glu Ile Pro 35 4 O 45 Pro Asp Met Thr Thr Asp Arg Val Glu Lieu Phe Phe Glu Lieu. Pro Arg SO 55 6 O Lieu. Ser Asn Pro Asn Lieu. Lieu. Thir Ala Lieu. Glin Glin Ile Ser Glin Lys 65 70 7s 8O Thir Arg Ile Arg Ala Val Ile Lieu. Asp Phe Phe Cys Asn Ala Ala Phe 85 90 95 Glu Val Pro Thr Ser Lieu. Asn Ile Pro Thr Tyr Tyr Tyr Phe Ser Ala 1OO 105 11 O Gly Thr Pro Thr Ala Ile Lieu. Thir Lieu. Tyr Phe Glu Thir Ile Asp Glu 115 12 O 125 Thir Ile Pro Val Asp Lieu. Glin Asp Lieu. Asn Asp Tyr Val Asp Ile Pro 13 O 135 14 O Gly Leu Pro Pro Ile His Cys Lieu. Asp Ile Pro Val Ala Leu Ser Pro 145 150 155 160 Arg Llys Ser Lieu Val Tyr Lys Ser Ser Val Asp Ile Ser Lys Asn Lieu 1.65 17O 17s Arg Arg Ser Ala Gly Ile Lieu Val Asn Gly Phe Asp Ala Lieu. Glu Phe 18O 185 19 O Arg Ala Ile Gly Ser His Ser Glin Arg Pro Met His Phe Lys Gly Pro 195 2OO 2O5 Thr Pro Pro Val Tyr Phe Ile Gly Pro Leu Val Gly Asp Val Asp Thr 21 O 215 22O Lys Ala Gly Ser Glu Glu. His Glu. Cys Lieu. Arg Trp Lieu. Asp Thr Glin 225 23 O 235 24 O Pro Ser Lys Ser Val Val Phe Lieu. Cys Phe Gly Arg Arg Gly Val Phe US 2010/0323402 A1 Dec. 23, 2010 28

- Continued

245 250 255

Ser Ala Glin Lell Glu Thir Ala Ala Ala Lell Glu Asn. Ser Gly 26 O 265 27 O

His Arg Phe Lieu. Trp Ser Wall Arg Asn Pro Pro Glu Lieu. Lys Ala 27s 285

Thr Gly Ser Asp Glu Pro Asp Lieu. Asp Glu Luell Leul Pro Glu Gly Phe 29 O 295 3 OO

Lell Glu Arg Thir Asp Arg Gly Phe Wall Ile Lys Ser Trp Ala Pro 3. OS 310 315

Glin Glu Wall Lell Ala His Asp Ser Wall Gly Gly Phe Wall Thr His 3.25 330 335

Gly Arg Ser Ser Wall Ser Glu Gly Wall Trp Phe Gly Wall Pro Met 34 O 345 35. O

Ile Gly Trp Pro Wall Asp Ala Glu Luell Arg Luell Asn Arg Ala Wal Met 355 360 365

Wall Asp Asp Luell Glin Wall Ala Luell Pro Luell Glu Glu Glu Ala Gly Gly 37 O 375

Phe Wall Thir Ala Ala Glu Lell Glu Arg Wall Arg Glu Luell Met Glu 385 390 395 4 OO

Thir Ala Gly Lys Ala Val Arg Glin Arg Wall. Thir Glu Lieu. Lys Luell 4 OS 415

Ser Ala Arg Ala Ala Wall Ala Glu Asn Gly Ser Ser Leu Asn Asp Luell 425 43 O Llys Phe Lieu. His Ala Thr Arg Asp 435 44 O

1. A polynucleotide of any one of (a) through (f) below: a polynucleotide consisting of a nucleotide sequence (a) a polynucleotide comprising a polynucleotide consist complementary to one nucleotide sequence selected ing of one nucleotide sequence selected from the group from the group consisting of the nucleotide sequence at consisting of the nucleotide sequence at positions 1 to positions 1 to 1359 in the nucleotide sequence repre 1359 in the nucleotide sequence represented by SEQID sented by SEQ ID NO: 4, the nucleotide sequence at NO: 4, the nucleotide sequence at positions 1 to 1365 in positions 1 to 1365 in the nucleotide sequence repre the nucleotide sequence represented by SEQID NO: 10, sented by SEQ ID NO: 10, the nucleotide sequence at the nucleotide sequence at positions 1 to 1371 in the positions 1 to 1371 in the nucleotide sequence repre nucleotide sequence represented by SEQID NO: 12, and sented by SEQID NO: 12, and the nucleotide sequence the nucleotide sequence at positions 1 to 1371 in the at positions 1 to 1371 in the nucleotide sequence repre nucleotide sequence represented by SEQID NO: 22: sented by SEQID NO: 22, and has a UDP-glucurono (b) a polynucleotide comprising a polynucleotide encod Syltransferase activity; and, ing a protein having one amino acid sequence selected (f) a polynucleotide comprising a polynucleotide encoding from the group consisting of SEQID NOs: 5, 11, 13 and a protein that hybridizes under Stringent conditions with 23; a polynucleotide consisting of a nucleotide sequence (c) a polynucleotide comprising a polynucleotide encoding complementary to the nucleotide sequence of a poly a protein consisting of an amino acid sequence with nucleotide encoding a protein consisting of one amino deletion, substitution, insertion and/or addition of 1 to acid sequence selected from the group consisting of 15 amino acids in one amino acid sequence selected SEQ ID NOs: 5, 11, 13 and 23, and has a UDP-glucu from the group consisting of SEQID NOs: 5, 11, 13 and ronosyltransferase activity. 23, and having a UDP-glucuronosyltransferase activity; 2. The polynucleotide according to claim 1, which is any (d) a polynucleotide comprising a polynucleotide encod one of (g) through () below: ing a protein having an amino acid sequence having a (g) a polynucleotide comprising a polynucleotide encod homology of at least 80% to one amino acid sequence ing a protein consisting of an amino acid sequence with selected from the group consisting of SEQID NOs: 5, deletion, substitution, insertion and/or addition of not 11, 13 and 23 and having a UDP-glucuronosyltrans greater than 10 amino acids in one amino acid sequence ferase activity; selected from the group consisting of SEQID NOs: 5, (e)a polynucleotide comprising a polynucleotide encoding 11, 13 and 23, and having a UDP-glucuronosyltrans a protein that hybridizes under Stringent conditions with ferase activity; US 2010/0323402 A1 Dec. 23, 2010 29

(h) a polynucleotide comprising a polynucleotide encod 5. The polynucleotide according to claim 1, which com ing a protein having an amino acid sequence having a prises a polynucleotide consisting of the nucleotide sequence homology of at least 90% to one amino acid sequence at positions 1 to 1371 in the nucleotide sequence represented by SEQID NO: 12. selected from the group consisting of SEQID NOs: 5, 6. The polynucleotide according to claim 1, which com 11, 13 and 23, and having a UDP-glucuronosyltrans prises a polynucleotide consisting of the nucleotide sequence ferase activity; at positions 1 to 1371 in the nucleotide sequence represented (i) a polynucleotide comprising a polynucleotide encoding by SEQID NO: 22. a protein that hybridizes under high Stringent conditions 7. The polynucleotide according to claim 1, which com with a polynucleotide consisting of a nucleotide prises a polynucleotide encoding the protein consisting of the sequence complementary to one nucleotide sequence amino acid sequence represented by SEQID NO: 5. 8. The polynucleotide according to claim 1, which com Selected from the group consisting of the nucleotide prises a polynucleotide encoding the protein consisting of the sequence at positions 1 to 1359 in the nucleotide amino acid sequence represented by SEQID NO: 11. sequence represented by SEQID NO: 4, the nucleotide 9. The polynucleotide according to claim 1, which com sequence at positions 1 to 1365 in the nucleotide prises a polynucleotide encoding the protein consisting of the sequence represented by SEQID NO: 10, the nucleotide amino acid sequence represented by SEQID NO: 13. sequence at positions 1 to 1371 in the nucleotide 10. The polynucleotide according to claim 1, which com sequence represented by SEQID NO: 12, and the nucle prises a polynucleotide encoding the protein consisting of the otide sequence at positions 1 to 1371 in the nucleotide amino acid sequence represented by SEQID NO. 23. sequence represented by SEQ ID NO: 22, and has a 11. The polynucleotide according to claim 1, which is a UDP-glucuronosyltransferase activity; and, DNA. 12. A protein encoded by the polynucleotide according to () a polynucleotide comprising a polynucleotide encoding claim 1. a protein that hybridizes under high Stringent conditions 13. A vector comprising the polynucleotide according to with a polynucleotide consisting of a nucleotide claim 1. sequence complementary to the nucleotide sequence of 14. A transformant, wherein the polynucleotide according a polynucleotide encoding a protein consisting of one to claim 1 is introduced. amino acid sequence selected from the group consisting 15. A transformant, wherein the vector according to claim of SEQ ID NOs: 5, 11, 13 and 23, and has a UDP 13 is introduced. glucuronosyltransferase activity. 16. A method for producing a protein encoded by the polynucleotide according to claim 1, which comprises using 3. The polynucleotide according to claim 1, which com a transformant, wherein the polynucleotide according to prises a polynucleotide consisting of the nucleotide sequence claim 1 is introduced. at positions 1 to 1359 in the nucleotide sequence represented 17. A method for producing a glucuronide, which com by SEQID NO: 4. prises forming the glucuronide from UDP-glucuronic acid 4. The polynucleotide according to claim 1, which com and a flavonoid using the protein according to claim 12 as a prises a polynucleotide consisting of the nucleotide sequence catalyst. at positions 1 to 1365 in the nucleotide sequence represented by SEQID NO: 10.