title: Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.
abstract: Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark.
authors.forename: Nethaji J
authors.initials: NJ
authors.lastname: Gallage
authors.affiliation: Evolva A/S, Lersø Parkallé 42-44, 5th floor, DK-2100 Copenhagen, Denmark.
authors.forename: Esben H
authors.initials: EH
authors.lastname: Hansen
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark.
authors.forename: Rubini
authors.initials: R
authors.lastname: Kannangara
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark.
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark.
authors.forename: Mohammed Saddik
authors.initials: MS
authors.lastname: Motawia
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jørgensen
authors.affiliation: AU Flakkebjerg, Danish Centre for Food and Agriculture, University of Aarhus, Forsøgsvej, DK-4200 Slagelse, Denmark.
authors.forename: Inger
authors.initials: I
authors.lastname: Holme
authors.affiliation: AU Flakkebjerg, Danish Centre for Food and Agriculture, University of Aarhus, Forsøgsvej, DK-4200 Slagelse, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Hebelstrup
authors.affiliation: Centre de Coopération Internationale en Recherche Agronomique pour le Dévelopement, UMR PVBMT, 97410 Saint Pierre, La Réunion, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Grisoni
authors.affiliation: 1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [4] Carlsberg Laboratory, Gamle Carlsberg Vej 10, Valby DK-2500, Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
mesh_terms: D001547:Benzaldehydes / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031669:Vanilla / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001547:Benzaldehydes; D003373:Coumaric Acids; D010940:Plant Proteins; C004999:ferulic acid; C100058:vanillin