Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.

Differentiation of secondary metabolite profiles in closely related plant species provide clues for unravelling biosynthetic pathways and regulatory circuits, an area that is still under-investigated. Cucurbitacins, a group of bitter and highly oxygenated tetracyclic triterpenes, are mainly produced by the plant family Cucurbitaceae. These compounds have similar structures, but differ in their anti-tumor activities and eco-physiological roles. By comparative analyses of the genomes of cucumber, melon, and watermelon, we uncovered conserved syntenic loci encoding metabolic genes for distinct cucurbitacins. Characterization of the cytochrome P450s (CYPs) identified from these loci enabled us to unveil a novel multi-oxidation CYP for the tailoring of the cucurbitacin core skeleton as well as two other CYPs responsible for the key structural variations among cucurbitacins C, B and E. We also discovered a syntenic gene cluster of transcription factors that regulate the tissue-specific biosynthesis of cucurbitacins and that may confer the loss of bitterness phenotypes associated with convergent domestication of wild cucurbits. This study illustrates the potential to exploit comparative genomics to identify enzymes and transcription factors that control the biosynthesis of structurally related yet unique natural products.

The overuse of antibiotics in animal agriculture and medicine has caused a series of potential threats to public health. Macleaya cordata is a medicinal plant species from the Papaveraceae family, providing a safe resource for the manufacture of antimicrobial feed additive for livestock. The active constituents from M. cordata are known to include benzylisoquinoline alkaloids (BIAs) such as sanguinarine (SAN) and chelerythrine (CHE), but their metabolic pathways have yet to be studied in this non-model plant. The active biosynthesis of SAN and CHE in M. cordata was first examined and confirmed by feeding C-labeled tyrosine. To gain further insights, we de novo sequenced the whole genome of M. cordata, the first to be sequenced from the Papaveraceae family. The M. cordata genome covering 378 Mb encodes 22,328 predicted protein-coding genes with 43.5% being transposable elements. As a member of basal eudicot, M. cordata genome lacks the paleohexaploidy event that occurred in almost all eudicots. From the genomics data, a complete set of 16 metabolic genes for SAN and CHE biosynthesis was retrieved, and 14 of their biochemical activities were validated. These genomics and metabolic data show the conserved BIA metabolic pathways in M. cordata and provide the knowledge foundation for future productions of SAN and CHE by crop improvement or microbial pathway reconstruction.