Terpenoids are the largest and most diverse group of plant specialized metabolites. Within the plant, these metabolites often function as tools for biological interactions, such as pollinator attraction, herbivore repellants, mediation of the microbial community, and plant-plant communication. Additionally, recent work has uncovered evidence that terpenoids may also be important in how plants handle abiotic stresses such as drought. Humans have recognized the extensive bioactivities of plant-derived terpenoids for thousands of years and have put them to use as fragrances, flavorings, medicines, insect repellants, and psychoactives. With modern genetic sequencing tools, the past two decades have ushered in a large-scale effort to unravel the biosynthetic pathways towards terpenoids, especially those with industrially relevant bioactivities. Since specialized terpenoid biosynthesis is often lineage-specific, this typically involves non-model plant species. In addition to contributing to our basic understanding of plant specialized metabolism, biosynthetic pathway studies can also identify enzymes which are prime candidates for biotechnological production of useful terpenoids. The numerous chiral centers and oxidations of these compounds often prevent facile access via traditional organic synthesis methods. In this dissertation, I investigate diterpenoid biosynthesis in the mint (Lamiaceae) family plant Callicarpa americana (American Beautyberry), which has several reported bioactive diterpenoids. Many of the findings in this plant become a starting point for further elucidation of diterpenoid biosynthesis in related species across the mint family. First, I elucidate the gateway enzymes in diterpenoid biosynthesis based on the generation of a high-quality genome for C. americana. This laid the foundation for further study of diterpenoid biosynthesis as well as leading to the discovery of a large diterpenoid biosynthetic gene cluster (BGC) in the genome. Next, I℗ investigate this BGC along with my coauthor Abby Bryson and we find that a version of this BGC is present in at least six additional Lamiaceae species genomes. Based on the evolutionary distance of these species, we conclude that this BGC has been conserved from an early Lamiaceae ancestor and has played an important role in the evolution of diterpene biosynthesis in this plant family. Functional characterization of the BGC genes in C. americana reveals the pathway to a previously inaccessible terpene backbone, (+)-kaurene, in addition to diterpene synthases and cytochrome P450s which catalyze formation of abietane diterpenoids. In the last two chapters, I search for enzymes involved in the clerodane-type diterpene pathways in C. americana. I discover along with coauthor Nick Schlecht that a set of orthologous P450s classified as CYP76BK1s are key to formation of furanoclerodanes in all but one Lamiaceae subfamily, including C. americana. Second, I generate additional transcriptomic resources for two additional Callicarpa species as well as a trichome-specific dataset for C. americana. This enables discovery of three short-chain dehydrogenases which also play a key role in furanoclerodane biosynthesis in C. americana. Together, this work represents an important advance in understanding diterpenoid biosynthesis in C. americana as well as the wider Lamiaceae family.℗
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