Doctor of Philosophy (Ph.D.)
Degree Granting Department
Bill J. Baker, Ph.D.
Laura J. Blair, Ph.D.
Theresa Evans-Nguyen, Ph.D.
Wayne C. Guida, Ph.D.
Alison E. Murray, Ph.D.
Antarctica, Ascidian, Cnidaria, Deep-Sea Ireland, Host-Associated Bacteria, Marine Natural Products
Microorganisms and invertebrate animals from cold water marine environments, such as in Antarctica and in Ireland’s deep sea, are a rich source of secondary metabolites. In this dissertation, research was centered around secondary metabolism and natural product biosynthesis. The projects include the delineation of biosynthetic gene clusters hypothesized to be responsible for the biosynthesis of the palmerolides, stratification of the microbiome of the Antarctic ascidian Synoicum adareanum, genomic and peptidomic analysis of a host-associated Antarctic Pseudovibrio sp., and isolation and characterization of secondary metabolites from the Irish deep-sea coral Drifa sp.
Palmerolide A, the principle secondary metabolite associated with S. adareanum, is a potent V-ATPase inhibitor with selectivity for malignant melanoma cell lines; however, drug development efforts have not been feasible due to a lack of supply. Identification of the producing organism and of the biosynthetic gene cluster could solve the supply issue through biotechnological methods of heterologous gene expression and large scale microbial cultivation efforts. The putative biosynthetic gene cluster responsible for palmerolide A production has now been identified and delineated in a host-associated bacterium. The biosynthetic gene cluster is a hybrid Type-I PKS/NRPS with several interesting non-canonical features, including a truncated condensation termination domain, a luciferase-like monooxygenase that is hypothesized to play a biosynthetic role as a hydroxylase, and a HMG-CoA synthetase cassette that installs a β-branch. Additionally, five variants of the pal biosynthetic gene cluster were identified, some of which explain the architectural diversity seen in the family of palmerolides.
The microbiome of S. adareanum was stratified through statistical analysis in efforts to identify the microbial producer of palmerolide A. A suite of ecological statistics was used for analysis of the ASV occurrence data. Twenty-one amplicon sequence variants (ASVs) were persistent in at least 80% of the 63 samples collected. These core members demonstrated coherence across samples. Genomic associations with members of the core microbiome, rather than correlations with concentrations of palmerolide A, has led to the positive identification of the producing organism.
Another member of the Synoicum adareanum core microbiome is Pseudovibrio sp. str. TunPSC04-5.I4, which possesses a genome replete with biosynthetic potential. In this research, several non-ribosomal peptide synthetase biosynthetic gene clusters were identified. Bacterial cultures were analyzed using peptidomic methods. The research focused on the triangulation of the biosynthetic gene clusters, compound structure predictions, and bioinformatic peptide sequencing tools to develop a new method of drug discovery.
Additionally, the secondary metabolome of four samples of a deep-sea Drifa sp. was evaluated using NMR-guided fractionation methods. This resulted in the discovery of a new natural product, NEA-1. The diterpene represents new carbon scaffold containing a chromene core with a highly substituted benzopyran ring system with an aldehyde, methyl, and two phenol features on the aromatic ring, along with a monoterpene tail. Molecular networking was also performed, which suggests the presence of natural analogues. This is a potential source for further discovery. Additionally, steroid natural products were isolated from the Drifa sp. samples.
Scholar Commons Citation
Avalon, Nicole Elizabeth, "Biosynthetic Gene Clusters, Microbiomes, and Secondary Metabolites in Cold Water Marine Organisms" (2020). USF Tampa Graduate Theses and Dissertations.