Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

Göpfert, Jens; MacNevin, Gillian; Ro, Dae‐Kyun; Spring, Otmar

doi:10.1186/1471-2229-9-86

Cited by 82 publications

(87 citation statements)

References 73 publications

(84 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Sunflower has GAS and GAO (20,21), and its capitate glandular trichomes produce abundant STLs of costunolide-type C6-C7 trans-lactone (16); however, its trichome ESTs and the ESTs from several related Helianthus spp. do not contain LsCOS.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism

Ikezawa

Göpfert

Nguyen

et al. 2011

Journal of Biological Chemistry

Self Cite

101

148

View full text Add to dashboard Cite

Sesquiterpene lactones (STLs) are terpenoid natural products possessing the ␥-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 ␥-lactone group in many reported bioactivities of STLs, the biosynthetic origins of the ␥-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 (␣ or ␤) hydroxylation on a carbon of GAA adjacent to its carboxylic acid at C12 is responsible for the ␥-lactone formation. Here, we report two cytochrome P450 monooxygenases (P450s) capable of catalyzing 6␣-and 8␤-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 8␤-hydroxylation of GAA, but the STL was not formed by spontaneous lactonization. Subsequently, we identified the closest homolog of the GAA 8␤-hydroxylase from lettuce and discovered 6␣-hydroxylation of GAA by the recombinant enzyme. The resulting 6␣-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.

show abstract

Section: Discussionmentioning

confidence: 99%

“…This result suggested that the latex may not be the correct cellular site where the transcription of STL biosynthetic genes occurs. On the other hand, almost exclusive expression of GAS in sunflower trichomes has been observed (20). Accordingly, H. annuus cv.…”

Section: Construction Of Cdna Library From Sunflower Capitatementioning

confidence: 99%

Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism

Ikezawa

Göpfert

Nguyen

et al. 2011

Journal of Biological Chemistry

Self Cite

101

148

View full text Add to dashboard Cite

show abstract

“…The production of the hydrocarbon germacrene A in the EPY300 strain expressing previously characterized lettuce GAS has been demonstrated (21,24). For in vivo catalytic coupling of GAS and GAO, open reading frames of GAO with a FLAG epitope tag and A. annua CPR with a cMyc epitope tag under Gal10 and Gal1 promoters, respectively, were placed in the pESC-URA dual expression vector.…”

Section: Isolation Of Gao and Pathway Reconstitution In Engineeredmentioning

confidence: 99%

“…Although the involvement of germacrene A acid is apparent in sesquiterpenoid metabolisms of sunflower, costus, chicory, and lettuce on the basis of chemical structures and/or in vitro enzyme assays (19,20,24,27), the biochemical relevance of germacrene A acid in B. spinosa has not been addressed. Thus, the presence of germacrene A acid was examined by (Ϫ)LC-SIM (selective ion mode) from the ethyl acetate extract of B. spinosa (leaf and flower).…”

Section: Isolation Of Gao and Pathway Reconstitution In Engineeredmentioning

confidence: 99%

“…A number of GAS genes have been isolated and characterized in Asteraceae (21)(22)(23)(24); however, the biochemical mechanism of germacrene A C12 oxidation and other downstream modifications have not been fully understood. Based on cell-free assays in chicory, it has been proposed that three distinct enzymes, germacrene A C12 hydroxylase (P450), germacrene alcohol dehydrogenase, and germacrene aldehyde dehydrogenase, are involved in the biosynthesis of germacrene A acid in chicory (19).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Nguyen

Göpfert

Ikezawa

et al. 2010

Journal of Biological Chemistry

Self Cite

112

152

View full text Add to dashboard Cite

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ϳ8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

show abstract

Broad diversity in monoterpene–sesquiterpene balance across wild sunflowers: Implications of leaf and floral volatiles for biotic interactions

Bahmani

Robinson

Majumder

et al. 2022

American J of Botany

View full text Add to dashboard Cite

Premise As plant lineages diversify across environmental gradients, species are predicted to encounter divergent biotic pressures. This study investigated the evolution of volatile secondary metabolism across species of Helianthus. Methods Leaves and petals of 40 species of wild Helianthus were analyzed via gas chromatography–mass spectrometry to determine volatile secondary metabolite profiles. Results Across all species, 500 compounds were identified; 40% were sesquiterpenes, 18% monoterpenes, 3% diterpenes, 4% fatty acid derivatives, and 35% other compounds such as phenolics and small organic molecules. Qualitatively, annuals and species from more arid western climates had leaf compositions with a higher proportion of total monoterpenes, while erect perennials and species from more mesic eastern habitats contained a higher proportion of total sesquiterpenes. Among species, mass‐based leaf monoterpene and sesquiterpene abundance were identified as largely orthogonal axes of variation by principal component analysis. Profiles for leaves were not strongly correlated with those of petals. Conclusions Volatile metabolites were highly diverse among wild Helianthus, indicating the value of this genus as a model system and rich genetic resource. The independence of leaf and petal volatile profiles indicates a low level of phenotypic integration between vegetative and reproductive structures, implying vegetative defense and reproductive defense or pollinator attraction functions mediated by terpene profiles in these two organs can evolve without major trade‐offs. The major biosynthetic pathways for the major terpenes in wild Helianthus are already well described, providing a road map to deeper inquiry into the drivers of this diversity.

show abstract

Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

Cited by 82 publications

References 73 publications

Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism

Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Broad diversity in monoterpene–sesquiterpene balance across wild sunflowers: Implications of leaf and floral volatiles for biotic interactions

Contact Info

Product

Resources

About