TY - JOUR
T1 - Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway
AU - Eljounaidi, Kaouthar
AU - Cankar, Katarina
AU - Comino, Cinzia
AU - Moglia, Andrea
AU - Hehn, Alain
AU - Bourgaud, Frederic
AU - Bouwmeester, Harro
AU - menin, Barbara
AU - Lanteri, Sergio
AU - Beekwilder, Jules
N1 - © 2014 Elsevier Ireland Ltd. All rights reserved
PY - 2014/3/13
Y1 - 2014/3/13
N2 - 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.
AB - 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.
KW - Cynara cardunculus
KW - Sesquiterpene lactones
KW - P450 enzymes
KW - Germacrene A
KW - N. benthamiana transient expression
KW - Costunolide
U2 - 10.1016/j.plantsci.2014.03.007
DO - 10.1016/j.plantsci.2014.03.007
M3 - Article
SP - 59
EP - 68
JO - Plant science
JF - Plant science
SN - 0168-9452
ER -