Quassinoids are highly oxidized triterpenoids with modified skeletons found only in the plant family Simaroubaceae. Many quassinoids show bioactivities with high values in pharmaceutical, agricultural, and industrial applications. Due to their limited natural sources and the challenges in chemical synthesis, the applications of quassinoids have been hindered. Understanding quassinoid biosynthesis is crucial for their production using synthetic biology. However, the molecular and biochemical basis of quassinoid biosynthesis is yet to be explored. The aim of this thesis is to use the invasive plant tree of heaven (Ailanthus altissima) as an accessible model plant to elucidate the quassinoid biosynthetic pathway. A combination of metabolic, co-expression, and homology analysis was applied to identify gene candidates. The candidates were screened using agrobacteria-mediated transient expression in Nicotiana benthamiana coupled with chromatography and mass spectrometry analysis. Large-scale agroinfiltration was performed to collect heterologously produced metabolites for NMR analysis. Enzyme catalytic mechanisms were characterized by in vitro assays using yeast (Saccharomyces cerevisiae) microsomes. By employing these methods, the first five steps of quassinoid biosynthesis in tree of heaven were elucidated. A 2,3-oxidosqualene cyclase (AaOSC2) catalyzes the first committed step, producing tirucalla-7,24-dien-3β-ol (1). Subsequently, three cytochrome P450s (AaCYP71CD4, AaCYP71BQ17, and AaCYP88A154) oxidize tirucalla-7,24-dien-3β-ol (1) to dihydroniloticin (2), melianol (3), and 7,8-epoxymelianol (4). Two isomerases (AaISM1 and AaISM2) rearrange the carbon skeleton of 7,8-epoxymelianol (4) to generate two different compounds, isomeliandiol (5), which is the key intermediate of quassinoids and the related triterpenoids limonoids, and protoglabretal (6), which belongs to a different class of triterpenoids, glabretanes. The occurrence of skeletal rearrangements of cyclized triterpenoids differs from canonical triterpenoid biosynthesis, in which triterpenoid skeletons remain unaltered post-cyclization. Knowledge of Δ8,7-sterol isomerases (8,7SI), from which AaISM1 and AaISM2 are derived, were leveraged to study skeletal rearranging mechanisms. Yeast complementation assays revealed that AaISM1 and AaISM2 no longer complement ergosterol biosynthesis like plant 8,7SIs. Site-directed mutagenesis showed that residues essential to 8,7SI are crucial in AaISM1 and AaISM2 activities. In conclusion, the elucidation of these biosynthetic steps confirmed the protolimonoid origin hypothesis of quassinoids and unveiled a process for triterpenoid skeletal rearrangements in nature. Moreover, the enzymes described here are crucial for sustainable biotechnological production of quassinoids. Last, the exploration of AaISM1 and AaISM2 catalytic mechanisms laid the foundation for tailoring cyclized triterpenoid skeletons to expand the chemical space of accessible triterpenoids via enzyme engineering.

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