Project Summary Soft corals (Alcyonaria) are a remarkable source of natural products (NPs) with pharmaceutically relevant biological activity, and complex scaffolds that are unique to marine eukaryotes with no terrestrial sources of structurally related compounds. The composition of secondary metabolites isolated from soft corals is dominated by a diverse suite of bioactive diterpenes with over 50 unique scaffolds and >1500 unique diterpenoids isolated to date. Many coral-derived diterpenes have potent and selective biological activity, including the xenicane, acalycixeniolide F, which displays cytotoxicity against human leukemia cell line K562 (LC50 = 200 ng/mL) via an undetermined mode of action. However, despite the promise these compounds exhibit as lead structures for the development of novel medicines, the pharmaceutical potential of coral diterpenes remains untapped as neither total synthesis nor isolation from aquaculture have provided sufficient material to enable effective in vivo clinical testing. Through bioinformatic analysis of published soft coral genomes and transcriptomes, our group has recently identified a series of terpene synthases that synthesize the hydrocarbon backbones of multiple coral- specific diterpenes, including xeniaphyllene, the precursor to the archetypal coral diterpenes, the xenicanes. Here, I propose to use the biosynthesis of the xenicanes as model for the development of a sustainable platform for the microbial production of bioactive coral diterpenes. This proposal aims to optimize the production of xenicane diterpenes through characterization and directed evolution of enzymes involved in the synthesis and oxidation of the xenicane scaffold, culminating in the reconstitution of xenicane biosynthesis through pathway engineering in methylotrophic yeast, Pichia pastoris. Terpene synthases are often the rate-limiting step in terpenoid biosynthesis due to their low catalytic efficiency and instability. In Aim 1, I will use a high-throughput, colorimetric substrate competition assay to screen error- prone PCR-generated xeniaphyllene terpene synthase variants for improved activity and stability. To access the core scaffold common to all xenicanes, the fused cyclobutane ring in xeniaphyllene must undergo an oxidative carbon-carbon bond cleavage. In the recently published chromosomal level genome assembly of a Xenia sp., we found a suite of cytochromes P450 co-localized with the xeniaphyllene synthase. In Aim 2, I will explore the oxidative chemistry of these cytochromes P450 in search of the unprecedented carbon–carbon lyase. Finally, the reconstitution of xenicane biosynthesis will be explored in Aim 3, wherein I will use Universal Loop Assembly (uLoop) to efficiently design and construct a refactored biosynthetic pathway for fermentative production of xenicanes in P. pastoris. This research will provide invaluable insights into the enzymology behind the construction of the biologically active xenicane coral dite...