Spatial and temporal dynamics of marine natural products biosynthesis

The marine environment represents one of the most promising sources of novel, bioactive natural products. The process used in their discovery has been simplified and accelerated greatly in the 20th century with the advent of new technology, including spectroscopy, mass spectrometry, chromatography, in conjunction with bioassay -guided fractionation. However, more recent developments in genomics and metabolomics suggest that the biosynthetic capacity and interrelationships between organisms has been underappreciated and could likely yield many more important discoveries, both in medical value and in basic biological understanding. Contained in this dissertation are a series of unique experiments that harness the capacity of Matrix Assisted Laser Desorption Ionization (MALDI) to detect multiple metabolites concurrently from a single, small sample. This ability is used to capture the temporal dynamics of biosynthesis and turnover of secondary metabolites from cultured marine filamentous cyanobacteria, both in relation to each other and to primary metabolites. These temporal relationships are useful for improving compound yields from cultured organisms, tracking nitrogen in nutrient cycles, as well as providing an experimental tool to explore secondary metabolism in general. Also contained are the first images of natural products captured by MALDI imaging mass spectrometry, revealing complex chemical microenvironments in marine sponges and the distribution of known bioactive metabolites in marine cyanobacteria. Initial results also suggest that there is differential distribution of metabolites in cultured cyanobacteria. A third series of experiments exploiting the sensitivity of MALDI was conducted on single cells of filamentous marine cyanobacteria that are freed from the sheath and associated heterotrophs. The results reveal that many known natural products are found in single cells, further confirming their origin of biosynthesis; however, it appears not all cells from a filament contain the same metabolites. Nitrogen labeling experiments with these same preparations suggest the biosynthesis of the metabolites that are present occurs at similar rates. The last set of experiments employ Ion Mobility mass spectrometry on various preparations of marine filamentous cyanobacteria and show that this new technology can effectively separate out halogenated metabolites from complex mixtures, a powerful tool for the identification and discovery of bioactive metabolite