Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast.
Identifieur interne : 000044 ( Main/Exploration ); précédent : 000043; suivant : 000045Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast.
Auteurs : Takao Ohashi [Japon] ; Yuka Hasegawa [Japon] ; Ryo Misaki [Japon] ; Kazuhito Fujiyama [Japon]Source :
- Applied microbiology and biotechnology [ 1432-0614 ] ; 2016.
English descriptors
- KwdEn :
- Citrus (enzymology), Citrus sinensis (enzymology), Cloning, Molecular, Disaccharides (metabolism), Enzymes, Flavanones (biosynthesis), Flavanones (metabolism), Flavonoids (biosynthesis), Flavonoids (metabolism), Glucosides (metabolism), Glycosides (biosynthesis), Glycosylation, Glycosyltransferases (isolation & purification), Glycosyltransferases (metabolism), Humans, Plant Proteins (genetics), Plant Proteins (metabolism), Quercetin (analogs & derivatives), Quercetin (metabolism), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Rhamnose (metabolism), Schizosaccharomyces (enzymology), Schizosaccharomyces (genetics), Schizosaccharomyces (metabolism), Substrate Specificity.
- MESH :
- chemical , analogs & derivatives : Quercetin.
- chemical , biosynthesis : Flavanones, Flavonoids, Glycosides.
- chemical , genetics : Plant Proteins.
- chemical , isolation & purification : Glycosyltransferases, Recombinant Proteins.
- chemical , metabolism : Disaccharides, Flavanones, Flavonoids, Glucosides, Glycosyltransferases, Plant Proteins, Quercetin, Recombinant Proteins, Rhamnose.
- enzymology : Citrus, Citrus sinensis, Schizosaccharomyces.
- genetics : Schizosaccharomyces.
- metabolism : Schizosaccharomyces.
- Cloning, Molecular, Enzymes, Glycosylation, Humans, Substrate Specificity.
Abstract
Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.
DOI: 10.1007/s00253-015-6982-6
PubMed: 26433966
Affiliations:
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Le document en format XML
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last="Hasegawa">Yuka Hasegawa</name><affiliation wicri:level="1"><nlm:affiliation>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871</wicri:regionArea><wicri:noRegion>565-0871</wicri:noRegion></affiliation></author><author><name sortKey="Misaki, Ryo" sort="Misaki, Ryo" uniqKey="Misaki R" first="Ryo" last="Misaki">Ryo Misaki</name><affiliation wicri:level="1"><nlm:affiliation>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871</wicri:regionArea><wicri:noRegion>565-0871</wicri:noRegion></affiliation></author><author><name sortKey="Fujiyama, Kazuhito" sort="Fujiyama, Kazuhito" uniqKey="Fujiyama K" first="Kazuhito" last="Fujiyama">Kazuhito Fujiyama</name><affiliation wicri:level="1"><nlm:affiliation>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan. fujiyama@icb.osaka-u.ac.jp.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871</wicri:regionArea><wicri:noRegion>565-0871</wicri:noRegion></affiliation></author></analytic><series><title level="j">Applied microbiology and biotechnology</title><idno type="eISSN">1432-0614</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Citrus (enzymology)</term><term>Citrus sinensis (enzymology)</term><term>Cloning, Molecular</term><term>Disaccharides (metabolism)</term><term>Enzymes</term><term>Flavanones (biosynthesis)</term><term>Flavanones (metabolism)</term><term>Flavonoids (biosynthesis)</term><term>Flavonoids (metabolism)</term><term>Glucosides (metabolism)</term><term>Glycosides (biosynthesis)</term><term>Glycosylation</term><term>Glycosyltransferases (isolation & purification)</term><term>Glycosyltransferases (metabolism)</term><term>Humans</term><term>Plant Proteins (genetics)</term><term>Plant Proteins (metabolism)</term><term>Quercetin (analogs & derivatives)</term><term>Quercetin (metabolism)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Rhamnose (metabolism)</term><term>Schizosaccharomyces (enzymology)</term><term>Schizosaccharomyces (genetics)</term><term>Schizosaccharomyces (metabolism)</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analogs & derivatives" xml:lang="en"><term>Quercetin</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Flavanones</term><term>Flavonoids</term><term>Glycosides</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Plant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Glycosyltransferases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Disaccharides</term><term>Flavanones</term><term>Flavonoids</term><term>Glucosides</term><term>Glycosyltransferases</term><term>Plant Proteins</term><term>Quercetin</term><term>Recombinant Proteins</term><term>Rhamnose</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Citrus</term><term>Citrus sinensis</term><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cloning, Molecular</term><term>Enzymes</term><term>Glycosylation</term><term>Humans</term><term>Substrate Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.</div></front></TEI><affiliations><list><country><li>Japon</li></country></list><tree><country name="Japon"><noRegion><name sortKey="Ohashi, Takao" sort="Ohashi, Takao" uniqKey="Ohashi T" first="Takao" last="Ohashi">Takao Ohashi</name></noRegion><name sortKey="Fujiyama, Kazuhito" sort="Fujiyama, Kazuhito" uniqKey="Fujiyama K" first="Kazuhito" last="Fujiyama">Kazuhito Fujiyama</name><name sortKey="Hasegawa, Yuka" sort="Hasegawa, Yuka" uniqKey="Hasegawa Y" first="Yuka" last="Hasegawa">Yuka Hasegawa</name><name sortKey="Misaki, Ryo" sort="Misaki, Ryo" uniqKey="Misaki R" first="Ryo" last="Misaki">Ryo Misaki</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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