Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway.
Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivated taxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrations of sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recently attracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costunolide is considered the common precursor of the STLs and three enzymes are involved in its biosynthetic pathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolide synthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), in a set of ∼19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta. The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 and CYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates in Nicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes. The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast with the corresponding terpene synthases.
Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Torino, Via L. da Vinci 44, 10095 Grugliasco, Italy.
Kaouthar
K
Eljounaidi
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands; Laboratory of Plant Physiology, Wageningen University, P.O. Box 658, 6700AR Wageningen, The Netherlands.
Katarina
K
Cankar
Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Torino, Via L. da Vinci 44, 10095 Grugliasco, Italy.
Cinzia
C
Comino
Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Torino, Via L. da Vinci 44, 10095 Grugliasco, Italy.
Andrea
A
Moglia
Université de Lorraine, UMR 1121 Agronomie et Environnement, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy, France; INRA, UMR 1121 Agronomie et Environnement, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy, France.
Alain
A
Hehn
Université de Lorraine, UMR 1121 Agronomie et Environnement, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy, France; INRA, UMR 1121 Agronomie et Environnement, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre-lès-Nancy, France.
Frédéric
F
Bourgaud
Laboratory of Plant Physiology, Wageningen University, P.O. Box 658, 6700AR Wageningen, The Netherlands.
Harro
H
Bouwmeester
PTP/Rice Genomics Unit, Via Einstein, 26900 Lodi, Italy.
Barbara
B
Menin
Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Torino, Via L. da Vinci 44, 10095 Grugliasco, Italy.
Sergio
S
Lanteri
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands. Electronic address: jules.beekwilder@wur.nl.
Jules
J
Beekwilder
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D031942:Cynara / Q000201:enzymology* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017343:Genes, Plant; D006900:Hydroxylation; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
D007783:Lactones; D010940:Plant Proteins; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; C002602:costunolide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase
Costunolide; Cynara cardunculus; Germacrene A; N. benthamiana transient expression; P450 enzymes; Sesquiterpene lactones