germacrene-a and costunolide

Adaptive evolution of enzymes benefits from catalytic promiscuity. Sesquiterpene lactones (STLs) have diverged extensively in the Asteraceae, and studies of the enzymes for two representative STLs, costunolide and artemisinin, could provide an insight into the adaptive evolution of enzymes. Costunolide appeared early in Asteraceae evolution and is widespread, whereas artemisinin is a unique STL appearing in a single Asteraceae species,

Topics: Artemisinins; Asteraceae; Catalysis; Evolution, Molecular; Lactones; Mutation; Oxidoreductases; Plant Proteins; Sesquiterpenes; Sesquiterpenes, Germacrane; Substrate Specificity

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.

Topics: Alkyl and Aryl Transferases; Biocatalysis; Biosynthetic Pathways; Cloning, Molecular; Cynara; Cytochrome P-450 Enzyme System; Genes, Plant; Hydroxylation; Lactones; Mass Spectrometry; Nicotiana; Oxidoreductases; Phylogeny; Plant Proteins; Plants, Genetically Modified; Saccharomyces cerevisiae; Sequence Alignment; Sesquiterpenes; Sesquiterpenes, Germacrane; Substrate Specificity

Sesquiterpene lactones (STLs) are terpenoid natural products possessing the γ-lactone, well known for their diverse biological and medicinal activities. The occurrence of STLs is sporadic in nature, but most STLs have been isolated from plants in the Asteraceae family. Despite the implication of the γ-lactone group in many reported bioactivities of STLs, the biosynthetic origins of the γ-lactone ring remains elusive. Germacrene A acid (GAA) has been suggested as a central precursor of diverse STLs. The regioselective (C6 or C8) and stereoselective (α or β) hydroxylation on a carbon of GAA adjacent to its carboxylic acid at C12 is responsible for the γ-lactone formation. Here, we report two cytochrome P450 monooxygenases (P450s) capable of catalyzing 6α- and 8β-hydroxylation of GAA from lettuce and sunflower, respectively. To identify these P450s, sunflower trichomes were isolated to generate a trichome-specific transcript library, from which 10 P450 clones were retrieved. Expression of these clones in a yeast strain metabolically engineered to synthesize substrate GAA identified a P450 catalyzing 8β-hydroxylation of GAA, but the STL was not formed by spontaneous lactonization. Subsequently, we identified the closest homolog of the GAA 8β-hydroxylase from lettuce and discovered 6α-hydroxylation of GAA by the recombinant enzyme. The resulting 6α-hydroxy-GAA spontaneously undergoes a lactonization to yield the simplest form of STL, costunolide. Furthermore, we demonstrate the milligram per liter scale de novo synthesis of costunolide using the lettuce P450 in an engineered yeast strain, an important advance that will enable exploitation of STLs. Evolution and homology models of these two P450s are discussed.

Topics: Catalysis; Cytochrome P-450 Enzyme System; Gene Library; Helianthus; Lactones; Models, Chemical; Molecular Sequence Data; Phylogeny; Plant Proteins; Protein Conformation; Recombinant Proteins; Sesquiterpenes; Sesquiterpenes, Germacrane; Solvents; Stereoisomerism

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

Topics: Alkyl and Aryl Transferases; Biosynthetic Pathways; Chromatography, Liquid; Cichorium intybus; Cysteine; Cytochrome P-450 Enzyme System; Glutathione; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Nicotiana; Oxidation-Reduction; Oxidoreductases; Phylogeny; Plant Proteins; Sesquiterpenes; Sesquiterpenes, Germacrane; Tanacetum parthenium; Transformation, Genetic; Yeasts