Vinblastine, a potent anticancer drug, is produced by (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

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.

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.

Background: Natural rubber biosynthesis in lettuce (Lactuca sativa) and other plants remains elusive. Results: An unusual cis-prenyltransferase-like protein interacts with and tethers a cis-prenyltransferase on endoplasmic reticulum, and its RNAi-silencing eliminates natural rubber. Conclusion: cis-Prenyltransferase-like protein is a necessary component in natural rubber biosynthesis in lettuce. Significance: The results presented here suggest hetero-protein complexes are involved in natural rubber biosynthesis.