title: Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.
abstract: Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
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: 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: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Halle (Saale), Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Brückner
authors.affiliation: Plant Research International, Wageningen University and Research Centre, The Netherlands; Netherlands Metabolomics Centre, Leiden, The Netherlands.
authors.forename: Ric C H
authors.initials: RC
authors.lastname: de Vos
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
authors.forename: Constantinos A
authors.initials: CA
authors.lastname: Tsoleridis
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: Katsarou
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: Antigoni
authors.initials: A
authors.lastname: Papanikolaou
authors.affiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain.
authors.forename: Irini
authors.initials: I
authors.lastname: Pateraki
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: Fani M
authors.initials: FM
authors.lastname: Chatzopoulou
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: Eleni
authors.initials: E
authors.lastname: Dimitriadou
authors.affiliation: Laboratory of Floriculture, School of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
authors.forename: Stefanos
authors.initials: S
authors.lastname: Kostas
authors.affiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Manzano
authors.affiliation: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, 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: Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Halle (Saale), Germany.
authors.forename: Alain
authors.initials: A
authors.lastname: Tissier
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi Thessaloniki, Greece.
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, 710 03 Heraklion, Greece.
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
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
mesh_terms: D045784:Abietanes / Q000096:biosynthesis*; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D027542:Rosmarinus / Q000201:enzymology* / Q000235:genetics; D027544:Salvia / Q000201:enzymology* / Q000235:genetics; D017423:Sequence Analysis, RNA
chemical_list: D045784:Abietanes; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; C018381:salvin