Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereoselective hydroxylations in sesquiterpene lactone metabolism

Abstract

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.

FIGURE 1.

Costunolide biosynthetic pathway in Asteraceae. COS, costunolide synthase.

FIGURE 2.

Structures of STLs found in various Asteraceae plants. A, four representative structures of STLs with distinct regio- and stereo-characteristics of γ-lactone rings are shown, and their sesquiterpene backbones are labeled in red. B, structures of STLs found in sunflower cv. HA300 and lettuce. Santonin is from Artemisia maritima (Anthemideae, Asteroideae); parthenin is from Parthenium hysterophorus (Heliantheae, Asteroideae); xanthatin is from Xanthium strumarium (Heliantheae, Asteroideae); helenalin is from Arnica montana (Heliantheae, Asteroideae); niveusin B and haageanolide are from H. annuus (Heliantheae, Asteroideae); and lactucopicrin is from L. sativa (Cichorieae, Cichorioideae). Tribe and subfamily are given in parentheses.

FIGURE 3.

Biochemical and chemical characterizations of germacrene A acid 8β-hydroxylase. A and B, (±)LC-MS analyses of C12 (HaG8H) enzymatic products are shown. Microsomes from the yeast expressing C12 and CPR catalyzes the synthesis of a new compound (labeled as 3) with [M-H₂O+H]⁺ ion at m/z 233 in A and with [M-H]⁻ ion at m/z 249 in B. The 6-hydroxy-GAA (compound 2) was prepared by alkaline hydrolysis of authentic costunolide standard (1). The identity of 2 was confirmed by reverting it to costunolide. The chemical identity of the peak marked by asterisk is unknown. The compound marked by the star in A (inset) is a minor compound displaying m/z 233, but it showed different retention time from the costunolide (8.03 min versus 7.66 min). C, structures of the new compound (3) purified from the in vivo feeding assay (8β-hydroxygermacrene A acid) and its rearranged product in an acidic condition (8β-hydroxyilicic acid). In the 8β-hydroxyilicic acid, the stereochemistry of a C15 methyl and a hydroxyl group attached to C4 could not be determined due to NMR signal overlapping.

FIGURE 4.

Biochemical and chemical characterization of costunolide synthase. A, (+)LC-MS scan at m/z 233 demonstrated that compounds 1 and 2 showed identical retention times with costunolide and 6-hydroxygermacrene A acid, respectively. Asterisks indicate unknown compounds, which are likely due to the unspecific activities of LsCOS. B, the structures of the standards are depicted. C, metabolite profile of the culture extraction from the EPY300 strain expressing GAS, LsGAO, CPR, and with or without LsCOS by (+)LC-MS scan at m/z 233. D, product ion scans of the costunolide standard and compound 1 by (+)LC-MS-MS showed identical fragmenting patterns. Diamonds indicate the parental ion at m/z 233.

FIGURE 5.

Identifications of the homologous genes for HaG8H (CYP71BL1) and LsCOS (CYP71BL2) in Asteraceae. The Asteraceae phylogeny was adapted from Panero and Funk (15). Estimated percentages of species constituting Asteraceae plants in three major subfamilies are given in parentheses. Shown are: Matricaria recutita (German Chamomile); Chrysanthemum coronarium (Garland chrysanthemum); two Arnica species are A. montana and A. chamissonis; three Helianthus species are H. annuus, H. argophyllus, and H. ciliaris; Xanthium strumarium; two Cichorium species are Cichorium intybus and C. endivia; T. officinale is Taraxacum officinale; and five Lactuca species are L. sativa, L. serriola, L. saligna, L. virosa, and L. perennis; Barnadesia spinosa.