Discovery and characterization of novel poriferan biosynthetic pathways via next-generation sequencing

Sponges of the phylum Porifera are benthic, filter feeding animals which can be found in waters throughout the world. Because of these qualities, consistent challenges they face include exposure to pathogenic microorganisms, spatial competition with other benthics, and predation from more mobile animals. To protect themselves, many sponges have developed complex chemical defences which display antimicrobial, antifouling, and antifeeding purposes. In turn, isolated chemical compounds, or natural products, from these organisms have been shown to exhibit activity towards clinically relevant targets such as pathogenic microorganisms, parasites, and tumoral cells. While derived from an animal, it has been shown that many sponge natural products are actually produced by associated microorganisms living on or within the host. Traditionally, such natural products would be isolated and characterized via chemical extraction, purification, structure elucidation, and bioassays. However, advances in next-generation sequencing and heterologous expression have produced an alternative process to drug discovery. First, the genome of an organism is sequenced and genes responsible for known or unknown natural products are identified in silico via a process known as genome mining. Second, these genes are cloned and expressed in a heterologous expression system in vivo to determine their connection to a natural product. This second approach to drug discovery has been used to identify the biosynthetic origin of natural products from many organisms including sponges. The overall aim of this project was to employ this genomics-driven approach to drug discovery to identify genes responsible for new and known natural products with possible therapeutic and biotechnological applications from Irish sponges. A specific focus was to identify the biosynthetic origin of a family of compounds known as 3-alkylpyridine alkaloids (3-APs) which are highly limited to sponges of the Order Haplosclerida. These compounds are hypothesized to be produced via a polyketide synthase (PKS) which accepts nicotinic acid (NTA) as a starter unit. Two species found in Ireland, Haliclona indistincta and Haliclona viscosa, are known sources of 3-APs and thus were chosen to test this hypothesis. Furthermore, chemical extracts from these species have exhibited selective cytotoxicity towards tumoral cells which may be attributed to their 3-APs. However, it was a secondary goal of this project to also identify alternative genes responsible for new natural products such as sterols and larger, bioactive proteins. Based on these goals, I first sequenced the transcriptomes of H. indistincta and H. viscosa, mapped protein-coding genes to metabolic pathways, and identified a pathway to produce NTA from tryptophan in both species. However, a source of the alkyl chain was not identified as no complete pathway for polyketide or fatty caid biosynthesis could be identified. To overcome the limitations of transcriptomics, I then sequenced and mined the metagenome of H. indistincta. This allowed for the identification of a single megasynthase gene, a nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS), which fit the PKS hypothesis on 3-AP biosynthesis. This NRPS-PKS gene appears to originate from a chromosome of H. indistincta rather than its associated microbiota which is unheard of in the field of marine natural products. By analysing publicly available sponge genomes and transcriptomes, I was able to identify similar enzymes in other sponges of the classes Demospongiae and Homoscleromorpha. This indicates that sponges themselves should not be discounted in comparison to their associated microbiota as sources of nitrogenous natural products. Because in silico predictive analysis could not determine whether the H. indistincta NRPS-PKS could create 3-APs, I attempted heterologous expression of the entire gene in Saccharomyces cerevisiae with the intention of in vitro functional characterization. No heterologous expression was observed which may indicate the presence of undetected introns within the synthesized gene. Finally, an alternative source of bioactive molecules from H. indistincta and H. viscosa was identified: an array of genes encoding for actinoporin like proteins (ALPs). By characterizing the ALPs with in silico methods, I predicted that one likely has membrane binding and cytolytic capabilities similar to actinoporins from cnidarians. These ALP genes are widespread in the phylum Porifera and thus represent an untapped source of bioactive proteins with potential applications.

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NUI Galway

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Attribution-NonCommercial-NoDerivs 3.0 Ireland
CC BY-NC-ND 3.0 IE

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University of Galway Theses (PhD Theses)