title: Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.
abstract: Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.
authors.affiliation: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Weinberg 3, 06120 Halle-Saale, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Brückner
authors.affiliation: Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
authors.forename: Dragana
authors.initials: D
authors.lastname: Božić
authors.affiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain; Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Manzano
authors.affiliation: Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Papaefthimiou
authors.affiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain; Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: Irini
authors.initials: I
authors.lastname: Pateraki
authors.affiliation: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Weinberg 3, 06120 Halle-Saale, Germany.
authors.forename: Ulschan
authors.initials: U
authors.lastname: Scheler
authors.affiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain; Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
authors.forename: Albert
authors.initials: A
authors.lastname: Ferrer
authors.affiliation: Plant Research International, Wageningen University and Research Centre, The Netherlands; Netherlands Metabolomics Centre, The Netherlands.
authors.forename: Ric C H
authors.initials: RC
authors.lastname: de Vos
authors.affiliation: Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
authors.forename: Angelos K
authors.initials: AK
authors.lastname: Kanellis
authors.affiliation: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Weinberg 3, 06120 Halle-Saale, Germany. Electronic address: Alain.Tissier@ipb-halle.de.
authors.forename: Alain
authors.initials: A
authors.lastname: Tissier
mesh_terms: D045784:Abietanes / Q000096:biosynthesis*; D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D055162:Biocatalysis; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D027542:Rosmarinus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D064201:Trichomes / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D045784:Abietanes; D019883:Alkyl and Aryl Transferases; C021459:kaurene synthetase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)
keywords: Carnosic acid; Diterpene synthases; Lamiaceae; Miltiradiene; Phenolic diterpenes; Rosmarinus officinalis