Doctor of Philosophy

dissertationDrug discovery and development from marine invertebrates has been fraught with two key problems, namely, the variability of occurrence and limited supply. Bacteria in symbiosis with marine invertebrates have been shown to produce most bioactive natural products isolated from these organisms, and thus are central to addressing questions of occurrence and issues of supply. Specifically, the factors that influence symbiosis influence the distribution and supply of natural products. This dissertation sought to address these two problems through studies in symbiosis and supply of symbiotic natural products. First, the global patterns of chemical symbiosis in marine ascidians, a group of highly prolific producers of natural products, were examined. Symbiosis in ascidians is shown to be host-specific (meaning that similar species of invertebrates contain similar bacterial symbionts); further, microbiomes are shown to be equally diverse regardless of location. Secondary metabolism was also found to be host-specific, but is more sensitive to biogeographical factors as evidenced by the increase in the potency of the secondary metabolites in tropical regions. To address the supply of rare natural products, heterologous expression was used to produce useful quantities of a group of symbiotic natural products, cyanobactins. Using metabolic engineering, a platform was developed to supply cyanobactins in high-titer, and its usefulness showcased in the discovery of ! novel activities of these natural products. Another facet of the supply problem is the substantial difficulty involved in synthesizing derivatives of natural products, which generally requires total chemical synthesis. On this aspect of the supply problem, the capacity of the cyanobactin pathway to generate unprecendented structural diversity by the incorporation of non-proteinogenic amino acids into this multistep, substrate-tolerant biosynthetic pathway was demonstrated

A New Polyunsaturated Brominated Fatty Acid from a Haliclona Sponge

A new polyunsaturated brominated fatty acid possessing acetylenic bonds 1 was isolated from the Indonesian sponge Haliclona sp. The structure of compound 1 was elucidated by analyzing its spectral data. It showed moderate cytotoxicity against cultured cells

An Efficient Method for the In Vitro Production of Azol(in)e-Based Cyclic Peptides

Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6–9 residues representing 11 out of the 20 canonical amino acids

Quorum sensing signalling alters virulence potential and population dynamics in complex microbiome-host interactomes

Despite the discovery of the first N-acyl homoserine lactone (AHL) based quorum sensing (QS) in the marine environment, relatively little is known about the abundance, nature and diversity of AHL QS systems in this diverse ecosystem. Establishing the prevalence and diversity of AHL QS systems and how they may influence population dynamics within the marine ecosystem, may give a greater insight into the evolution of AHLs as signaling molecules in this important and largely unexplored niche. Microbiome profiling of Stelletta normani and BD1268 sponge samples identified several potential QS active genera. Subsequent biosensor-based screening of a library of 650 marine sponge bacterial isolates identified 10 isolates that could activate at least one of three AHL biosensor strains. Each was further validated and profiled by Ultra-High Performance Liquid Chromatography Mass Spectrometry, with AHLs being detected in 8 out of 10 isolate extracts. Co-culture of QS active isolates with S. normani marine sponge samples led to the isolation of genera such as Pseudomonas and Paenibacillus, both of which were low abundance in the S. normani microbiome. Surprisingly however, addition of AHLs to isolates harvested following co-culture did not measurably affect either growth or biofilm of these strains. Addition of supernatants from QS active strains did however impact significantly on biofilm formation of the marine Bacillus sp. CH8a sporeforming strain suggesting a role for QS systems in moderating the microbemicrobe interaction in marine sponges. Genome sequencing and phylogenetic analysis of a QS positive Psychrobacter isolate identified several QS associated systems, although no classical QS synthase gene was identified. The stark contrast between the biodiverse sponge microbiome and the relatively limited diversity that was observed on standard culture media, even in the presence of QS active compounds, serves to underscore the extent of diversity that remains to be brought into culture

A new antimicrobial fatty acid from the calcareous sponge Paragrantia cf. waguensis

A new acetylenic fatty acid, 1, has been isolated from the title sponge. The structure of the molecule was elucidated to contain an enyne and a thiophene by spectroscopic methods. Compound 1 showed a weak cytotoxic effect against NBT-72 rat bladder epithelial cells (IC50 > 20 mu g/ml), and antimicrobial activity with minimal-inhibitory concentrations (MIC) of 64 and 128 mu g/ml against Staphylococcus aureus and Escherichia coli, respectively

Aestuaramides, a Natural Library of Cyanobactin Cyclic Peptides Resulting from Isoprene-Derived Claisen Rearrangements

We report 12 cyanobactin cyclic peptides, the aestuaramides, from the cultivated cyanobacterium <i>Lyngbya aestuarii</i>. We show that aestuaramides are synthesized enzymatically as reverse <i>O</i>-prenylated tyrosine ethers that subsequently undergo a Claisen rearrangement to produce forward <i>C</i>-prenylated tyrosine. These results reveal that a nonenzymatic Claisen rearrangement dictates isoprene regiochemistry in a natural system. They also reveal one of the mechanisms that organisms use to generate structurally diverse compound libraries starting from simple ribosomal peptide pathways (RiPPs)