We hypothesize that the secondary metabolite pathways in cyanobacteria can be manipulated by applying transcriptional promoters, either of a genetic or chemical nature (elicitors), to produce more secondary metabolites with potential pharmaceutical utility.

Tyrosinase, an important oxidase involved in the primary immune response in humans, can sometimes become problematic as it can catalyze undesirable oxidation reactions. Therefore, for decades there has been a strong pharmaceutical interest in the discovery of novel inhibitors of this enzyme. Recent studies have also indicated that tyrosinase inhibitors can potentially be used in the treatment of melanoma cancer. Over the years, a number of new tyrosinase inhibitors have been isolated from various natural sources, although marine natural products (MNPs) have contributed only a small number of promising candidates. In this regard, the exploration of tyrosinase inhibitors from marine cyanobacteria has been especially understudied. This research therefore mainly focused on the discovery of new tyrosinase inhibitors of marine cyanobacterial and algal origins. A colorimetric fungal tyrosinase inhibitory assay was used to screen extracts, fractions, and pure compounds for anti-tyrosinase activity. Scytonemin monomer (ScyM), the monomeric structure of the well-known cyanobacterial sunscreen pigment scytonemin (Scy), was found to have the highest tyrosinase inhibitory score and was more potent than the commercial standard inhibitor kojic acid (KA). Consequently, ScyM has become our top compound and we explored a series of follow-up studies on its structure, inhibitory power, and mode of inhibition.

In 1969 Weinheimer and Spraggins reported the isolation of prostaglandins from the gorgonian coral Plexaura homomalla. This was a remarkable discovery for being one of the first reports of a marine natural product and for its significance to human health, where prostaglandins were being studied for their obstetric effects. It foreshadowed a new pipeline of drug discovery from the oceans; as well as highlighting many difficulties of drug discovery in general, and specific problems of working with marine samples. The isolation of a compound of interest from P. homomalla demanded that questions be asked about sustainability and ecology; were there other sources of the prostaglandins to be discovered, could this species be cultured or harvested sustainably? Further work on P. homomalla revealed a minor analog, 5-trans-15(S)-PGA2, demonstrating one of the most persistent problems in natural products chemistry, the presence of compounds that are difficult or impossible to detect at the low concentration which they are found in nature. Decades later, with thousands of marine natural products reported from sources across the tree of life, many of these problems still exist. New analytical tools have been developed, and old ones have become far more sensitive, but this has not erased these difficulties. In fact they have often simply shown that the unexpected diversity of biologically active compounds produced below the previous limit of detection. Every tool and technique developed, or improved, to survey the metabolome offers new data, but none provides a complete picture. To be successful a natural products workflow must incorporate the concept of `orthogonality', application of multiple overlapping and complementing techniques to provide a more comprehensive survey of the target metabolome. The work in this dissertation exemplifies this concept, where the study of various cyanobacterial strains began with one technique, such as NMR structure guided isolation, which led to another, such as mass spectrometry fragmentation analysis, when new tools became necessary to describe an organism's metabolic capacity. This work has revealed novel compounds, shown unprecedented diversity in known families of compounds, and improved our understanding of the mechanism of action of an active compound.

The natural world is an extraordinary source of diverse organisms and natural product compounds. The use of natural products and their derivatives has been a recurring theme in the discovery and development of new therapeutics. The marine world is vast and contains incredible biological and chemical diversity. Therefore, the oceans have the potential for new discoveries of compounds with medicinal applications. One group of marine organism that has been shown to be a prolific producer of bioactive compounds is the cyanobacteria. This work focuses on the study of new compounds from marine cyanobacteria for their chemical diversity and biological activity. The work in this dissertation focuses on the discovery and characterization of compounds from two different species of cyanobacteria, Leptolyngbya sp. and Moorena producens. These were collected from American Samoa and Puerto Rico, respectively. The characterization of these compounds was achieved through the integration of a variety of techniques including mass spectrometry, advanced NMR technology and genomic information. This analysis led to the characterization of a new ionophore from the Leptolygbya sp. The collection of Moorena producens afforded new analogues of the bioactive compound, curacin A. 2D NMR was an essential aspect in these studies. Therefore, the optimization of different NMR techniques for faster data acquisition was studied in this dissertation for its applications on lower concentrations of natural products. This work highlights the novelty of compounds that can be discovered by the application of the multiple techniques. Additionally, it demonstrates the reduction in time that is possible with new techniques for the experimental acquisition of NMR data. Finally, this dissertation adds to the body of knowledge of the chemistry that originates from the world’s oceans.

Organisms throughout the natural world have evolved different methods for biosynthesis of natural products, utilized in their native environs for defense, structure and signaling. One such method is the employment of modular polyketide synthase and non-ribosomal peptide synthetase enzymes to produce molecules from acetate and amino acid building blocks. The combinatorial capacity of these modules, combined with associated tailoring enzymes and unique protein-protein interaction strategies, has enabled a wide diversity of chemical structures and scaffolds. Many secondary metabolites or semi-synthetic derivatives have strong affinity for enzymatic targets and are used therapeutically to treat certain diseases. Photosynthetic marine filamentous cyanobacteria are prominent sources of bioactive natural products and present unique enzymatic methods for generating new chemical structures. This thesis describes the use of chemical biology techniques, including biochemistry, genomics, stable-isotope labeled feeding studies, NMR and mass spectrometry-based isolation and structure elucidation to explore and understand these novel methods for generating chemical structure diversity in cyanobacteria. Following an introduction and background in chapter one, chapter two describes new strategies for biosynthesis in the production of type A malyngamides in the species Okeania hirsuta sp. PAB10Feb10-1 and sp. PAP21Jun06-1, including neofunctionalization of a lipoic acid synthesis enzyme and ketoreductase domain inactivation. Chapter three of this thesis describes an unprecedented combinatorial approach to natural products biosynthesis in Moorea producens sp. ASI16Jul14-2: identical protein-protein interaction motifs guide a polyketide synthase-generated fatty acid tail to disparate downstream NRPS modules in the same biosynthetic gene cluster, representing a new mechanism for generating combinatorial chemical diversity in the productions of vatiamides A-F. Chapter four of this thesis highlights investigations toward understanding the biosynthesis of the t-butyl group in apratoxin A, a cyclic lipopeptide derived from cyanobacterium Moorea bouillonii PNG5-198. Lastly, chapter five presents a comprehensive overview of the findings described here, and discusses future avenues for research in microbial biosynthesis and natural products drug discovery as a whole.

Filamentous marine cyanobacteria are known to produce a variety of structurally diverse and biologically active secondary metabolites, many of which are being studied for a wide spectrum of applications, ranging from biofuels to pharmaceuticals. The development of a stable heterologous expression host for cyanobacterial biosynthetic pathways would alleviate current bottlenecks in natural products research, such as the low yield of many of these potentially valuable compounds from field-collected extracts. Additionally, it would allow for a deeper investigation of the expression and genetic regulation of these complex pathways while providing a platform in which to investigate pathways from strains for which no genetic tools may yet be developed. To date, there are no known model filamentous cyanobacterial strains of marine origin. In this study we demonstrate the first known transformation and expression of a self-replicating broad-host-range vector and a knockout vector, carrying two reporter genes, in a filamentous marine cyanobacterium.

The tropical marine benthic filamentous cyanobacterium Moorena bouillonii and the snapping shrimp Alpheus frontalis engage in a charismatic symbiosis, in which the shrimp weaves the cyanobacterial filaments into tubes and hollow chambers in and around coral reefs. Pairs of A. frontalis live in and feed upon these M. bouillonii structures. M. bouillonii produces a vast arsenal of biologically active and structurally diverse natural products, suggesting that A. frontalis may benefit from an associational defense. It is unclear, however, how M. bouillonii may benefit from this partnership, and what impact the symbiosis may have on the surrounding reef. Also unknown is what role chemistry plays in directing the interactions between the shrimp and cyanobacterium. Furthermore, the breadth of chemodiversity currently known from M. bouillonii that originates from a small collection of discrete locations, paired with the broad distribution of the M. bouillonii – A. frontalis symbiosis across the Indian Ocean and the western tropical Pacific raises the question of what more chemical diversity there is to be discovered in relation to this association. Field surveys and photographic documentation were employed to better understand how M. bouillonii – A. frontalis colonies are distributed across scleractinian coral reefs in the Mariana Islands, and revealed that colonies tend to inhabit interstices in the reef. Nutrient analysis of paired water samples and culturing experiments suggested that A. frontalis may benefit M. bouillonii via an enriched nutrient environment. Competing hypotheses on why shrimp are found in association with certain chemotypes of the cyanobacterium propelled a series of behavioral assays and comparative gas chromatography – mass spectrometry experiments to ultimately yield a lead compound for a shrimp attractant waterborne chemical cue produced by M. bouillonii. Chemogeographic investigations utilizing liquid chromatography – tandem mass spectrometry facilitated the discovery of a regionally specific family of natural products, while also improving understanding of how M. bouillonii chemodiversity is distributed across geographic space and generating paths forward for future discovery. Development of a new bioinformatic tool provided substantial improvements to M. bouillonii genome contiguity, allowing for new insights into the concentrations of potential biosynthetic diversity across compound classes.

Numerous advantages of systematic genomics have been leveraged in other areas, such as transcriptomics, proteomics and human microbiome studies, but it has yet to be effective in stimulating the increased discovery of natural products (secondary metabolites).1 Since the discovery of the first microbial genome (2002), the surprisingly high percentage of cryptic biosynthetic pathways per genome (genes not connected to any known secondary metabolite, aka natural product) has continued as a consistent theme.2 Cyanobacteria from benthic environments are prolific producers of natural products but genomes from these organisms remain scarce in public repositories. Hence, this dissertation focused on sequencing and performing genomic analysis in prolific cyanobacteria collected from several tropical benthic ecosystems around the planet. First, we analyzed the relationship between a cyanobacterium host and its associated heterotrophic microbiome. By evaluation the non-axenic culture of Moorea producens JHB, we characterized a novel uncultured acidobacteria heterotroph living with the cyanobacterial host.3 This heterotroph was only 85% similar to the closest cultured representative, it presented a large number of genes encoding for transcriptional regulators and it was auxotrophic for several proteogenic amino acids. Next, we expanded our genome comparison by deeper analyzing 4 Moorea genomes. Our genome comparison revealed that Moorea, already a prolific producer of secondary metabolites, harbored an even richer metabolic potential, four times above the cyanobacterial average. We observed that Moorea conserved its primary metabolism while evolved an intricate secondary metabolism machinery, accounting for 20% of their genomic content. These findings were very promising for future genome mining efforts in those four strains. Thereby, we further expanded our sequencing efforts by sampling 165 metagenomes of filamentous marine cyanobacteria collected from around the globe. Our new metagenomic pipeline was able to generated 85 high-quality genomes, including 32 Moorea and 29 Okeania strains. Our genome comparison highlighted that these two genera are among the most diverse and prolific producers of natural products in our dataset (comparing pairwise 506 cyanobacterial genomes, 425 from the NCBI database). Gene networking revealed the abundance of unique (only encountered in a single strain) and “extended families” (found in several strains). Using genomes with paired mass spectrometry data, we correlated the distribution of gene families with the distribution of metabolic families and automatically annotated a new dehydromicrosclerodermin B homologue.