Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus

Microbial and chemical diversity of tetrodotoxin producing marine animals

Marine molluscs are known to employ a variety of defence systems to improve their survivability, such as the production of bioactive molecules. Recently, microbes have been identified as the true producers of these compounds. Similarly, tetrodotoxin (TTX) is hypothesised to have a bacterial origin, however, much controversy still exists. This thesis attempts to shed further light on the biosynthetic origins of TTX with a focus on Hapalochalaena sp. and Pleurobranchaea maculata. This thesis also investigates the potential for biosynthesis of other natural products by bacteria living in association with these marine molluscs. Culture-based studies attempting to isolate TTX-producing bacterium within Hapalochalaena sp. and P. maculata were unable to isolate any such strains. Furthermore, we attempted to replicate the production of TTX by published TTX-producing bacteria, however, TTX was unable to be detected in these strains via spectrometric methods. Nevertheless, culture-independent methods were able to identify four taxa that were strongly correlated to TTX-concentration in P. maculata. Further experiments, however, are required to isolate or characterise these strains. Molecular screening for natural product biosynthesis genes, putatively involved in the biosynthesis of TTX and other natural products, revealed many candidate bacteria. These experiments also identified a bacterium with significant biosynthetic potential, Pseudoalteromonas sp. HM-SA03. Members of Pseudoalteromonas are known to produce many bioactive compounds. Mining of the HM-SA03 genome identified a total of seven novel NRPS and PKS biosynthesis gene clusters. Bioinformatic analysis of these gene clusters revealed putative novel pathways for the assembly of bromoalterochromide, alteramide and pseudoalterobactin-like compounds. However, production of these compounds in laboratory-cultures of Pseudoalteromonas sp. HM-SA03 could not be confirmed by chemical studies. Nonetheless, chemical studies were able to identify the production of eight diketopiperazines. These bioactive molecules have been observed to function as cell-signalling molecules, and are proposed to function as such in the complex microbial community identified within the Hapalochalaena octopus. Taken together, these results indicate that marine molluscs are a rich source of biosynthetically potent microbes that deserve further attention for the elucidation of new natural products

Directing the Heterologous Production of Specific Cyanobacterial Toxin Variants

Microcystins are globally the most commonly occurring freshwater cyanotoxins, causing acute poisoning and chronically inducing hepatocellular carcinoma. However, the detection and toxicological study of microcystins is hampered by the limited availability and high cost of pure toxin standards. Biosynthesis of microcystin variants in a fast-growing heterologous host offers a promising method of achieving reliable and economically viable alternative to isolating toxin from slow-growing cyanobacterial cultures. Here, we report the heterologous expression of recombinant microcystin synthetases in <i>Escherichia coli</i> to produce [d-Asp³]­microcystin-LR and microcystin-LR. We assembled a 55 kb hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster from <i>Microcystis aeruginosa</i> PCC 7806 using Red/ET recombineering and replaced the native promoters with an inducible P<i>tet</i>_<i>O</i> promoter to yield microcystin titers superior to <i>M. aeruginosa</i>. The expression platform described herein can be tailored to heterologously produce a wide variety of microcystin variants, and potentially other cyanobacterial natural products of commercial relevance

High-Titer Heterologous Production in <i>E. coli</i> of Lyngbyatoxin, a Protein Kinase C Activator from an Uncultured Marine Cyanobacterium

Many chemically complex cyanobacterial polyketides and nonribosomal peptides are of great pharmaceutical interest, but the levels required for exploitation are difficult to achieve from native sources. Here we develop a framework for the expression of these multifunctional cyanobacterial assembly lines in <i>Escherichia coli</i> using the lyngbyatoxin biosynthetic pathway, derived from a marine microbial assemblage dominated by the cyanobacterium <i>Moorea producens</i>. Heterologous expression of this pathway afforded high titers of both lyngbyatoxin A (25.6 mg L^–1) and its precursor indolactam-V (150 mg L^–1). Production, isolation, and identification of all expected chemical intermediates of lyngbyatoxin biosynthesis in <i>E. coli</i> also confirmed the previously proposed biosynthetic route, setting a solid chemical foundation for future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in <i>E. coli</i> also opens the possibility for future heterologous expression, characterization, and exploitation of other cyanobacterial natural product pathways