Phenylphenalenones and Linear Diarylheptanoid Derivatives are Biosynthesized via Parallel Routes in Musella lasiocarpa, the Chinese Dwarf Banana

Phenylphenalenones (PPs) are complex polycyclic natural products that play an important role in the chemical defense system of banana and plantain (Musaceae). Although suggestions for how plants synthesize the PP scaffold were first proposed more than 50 years ago, no biosynthetic information is yet available at the enzyme level. Here, we use transcriptomic data from seeds of Musella lasiocarpa, the Chinese dwarf banana, to identify five biosynthetic genes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to the two types of major aromatic seed metabolites, the PPs and the linear diarylheptanoid derivatives. Furthermore, through multiple rounds of feeding potential intermediates to M. lasiocarpa root protein extract, followed by high-resolution mass spectrometry profiling, product isolation, and NMR-based elucidation, we demonstrate the stepwise conversion of a dihydrocurcumin-type precursor to the PP 4-hydroxylachnanthocarpone. In contrast to the commonly hypothesized Diels-Alder cyclization mechanism, we propose an unexpected two-step cyclization route to the PP scaffold.