Terpenoids are the largest class of specialized metabolites in plants, with widespread uses ranging from fragrances and cosmetics to biofuels, antifeedants, and pharmaceuticals. Terpenoids are derived from a small set of prenyl diphosphate substrates which are cyclized into different terpene scaffolds by terpene synthases. These scaffolds are then modified by various tailoring enzymes-typically starting with cytochrome P450s-into functionalized terpenoids. Given the structural complexity of many of these metabolites, total chemical synthesis is often challenging to achieve at a relevant scale and cost, and as such, biosynthetic methods are increasingly being employed as an alternative for their production. The work presented in this dissertation describes the elucidation of two terpenoid biosynthetic pathways and the repurposing of known pathways to convert synthetic substrates not found in nature. First, three steps constituting the full biosynthetic pathway to leubethanol, an antimicrobial diterpenoid active against multidrug-resistant TB, was elucidated in the Texas Sage (Leucophyllum frutescens). Second, seven steps in the biosynthetic pathway towards structurally complex diterpenoid alkaloids were elucidated in the Siberian Larkspur (Delphinium grandiflorum). Third, twenty-four terpene synthases were screened for activity against twenty synthetic substrate analogs not found in nature, resulting in fifty-six new products and demonstrating the ability to derivatize terpene scaffolds through the derivatization of a starting substrate. In all, this work expands access to different classes of terpenoids through the elucidation of biosynthetic pathways and semi-biosynthesis of terpene scaffolds not found in nature, allowing for more feasible and sustainable production of these structurally complex compounds.
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