PROJECT SUMMARY Biotic processes in the oceans introduce various halogenated molecules in the environment and in the human exposome. Some of these halogenated molecules possess favorable pharmaceutical activities making them attractive drug candidates while many of these naturally produced marine halogenated molecules are potent toxins and pollutants. Understanding, at the organismal, molecular, and atomistic levels how these halogenated molecules are naturally constructed in the oceans is the principal motivation of the research program described herein. Seaweeds and filter feeding marine benthic invertebrates such as sponges are well validated to be exceptionally prolific producers of halogenated natural products. Contrary to prokaryotic natural product biochemistry, our understanding of how these eukaryotes biosynthesize natural products is far less developed due to challenges in culturing and genetically interrogating these organisms. This is the key scientific challenge that this proposal seeks to address in order to deliver seaweed- and sponge-derived halogenated natural products using biogenetic means. Progress envisaged here is predicated upon two key intellectual drivers. The first of these is the sequencing of eukaryotic transcriptomes, rather than genomes, to circumvent the eukaryotic genome complexity. The second driver is to design natural product biosynthetic schemes based on intermediates that are mined from untargeted metabolomic datasets and then use these rationalized schemes to guide the mining of eukaryotic transcriptomes for biosynthetic enzyme discovery. Specifically, halogenated intermediates and halogenating enzymes are used as diagnostic signatures in this workflow. Interdisciplinary competence in genomics, biochemistry, synthetic biology, and metabolomics allows the program participants to not only interrogate biogenetic pathways for the production of marine eukaryote- derived halogenated pharmacophores and pollutants, but to also use the genetic dark matter locked away in marine holobiont metagenomes to produce new-to-nature halogenated molecules with favorable pharmaceutical bioactivities. The program design also embraces opportunities to discover and characterize new halogenation enzymology and adapt halogenases as general purpose biocatalysts. Research described here is both molecule focused, in that, it will lead to the understanding of how key halogenated molecules of interest are constructed in marine sponge biomes, while concomitantly embracing method-development and engineering opportunities.