20 research outputs found
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
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)
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Host control of symbiont natural product chemistry in cryptic populations of the tunicate Lissoclinum patella.
Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions
Bioactive peptides from Gemmula speciosa venom: Isolation, purification, and sequencing
Turrids comprise the largest group of venomous gastropods in superfamily Conacea, the others being augers (Terebridae) and cones (Conidae). They are a hallmark of diversity with some turrid shells looking like cones and others resembling mitrids, fasciolarids or buddinids; although one feature common among them is the presence of a notch or sinus in the body whorl.
Turrids are carnivorous marine gastropods preying largely on polychaete worms. Their well-developed venom apparatus endows them the ability to effectively capture their prey through a specialized delivery system of their venom via a radula, thereby eliciting effects like paralysis prior to swallowing their victim. This feeding mechanism, also prevalent in Conus species, where they paralyze their prey by blocking voltage-gated ion channels on nerve membranes with venom toxins, had been well characterized more than two decades ago. The biologically active venom toxins were found to be highly structured cysteine-rich peptides with physiological targets on prey or predators/competitors (Olivera, 2002). Unlike the coniids, however, the turrid venom toxins, as well as their physiological targets, functions and applications, are largely unknown and are only beginning to be explored.
As part of a continuing effort to gain understanding of the turrid toxinology, this study aims to isolate, purify, and characterize bioactive components of the venom duct of Philippine turrid Gemmula speciosa, which could be a promising source of neuractive peptides for therapeutic applications. Preliminary results on the isolation, purification and biochemical characterization of venom components are reported
Ribosomal Route to Small-Molecule Diversity
The cyanobactin ribosomal peptide (RP) natural product
pathway
was manipulated to incorporate multiple tandem mutations and non-proteinogenic
amino acids, using eight heterologous components simultaneously expressed
in Escherichia coli. These studies
reveal the potential of RPs for the rational synthesis of complex,
new small molecules over multiple-step biosynthetic pathways using
simple genetic engineering
Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from Didemnidae animals not included in Figure S3.
<p>The COXI sequences for <i>L. patella</i> amimals L2, L5 and L6 used to make this tree were obtained from the respective mitochondrial genome assembly from Illumina sequencing data. The inset identity matrix shows the pairwise nucleotide identities of sequences in this clade, indicating that <i>L. patella</i> sequence AB602781.1 likely is a group B animal.</p
Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona savignyi</i> acting as the outgroup.
<p>Note: the <i>Didemnum vexillum</i> clade is collapsed for space. The Didemnidae COXI genes found in the NCBI database cover two non-overlapping regions of the gene (see Main Text), and therefore two separate trees were constructed (for the other tree, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g004" target="_blank">Figure 4</a>).</p
Collection locations, phylogeny and divergence of <i>Lissoclinum patella</i> individuals collected across areas of the Southeastern Pacific between 2002 and 2011.
<p>(a) Collection sites, with a portion of the phylogenetic tree based on mitochondrial cytochrome c oxidase 1 (COXI) genes overlaid, with individuals colored by clades that diverge by 5% or more in their nucleotide sequence, as shown in (b).</p