Discovery and characterization of novel poriferan biosynthetic pathways via next-generation sequencing
Date
2023-01-10Author
Sandoval, Kenneth
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Abstract
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.