John A. Kalaitzis

On the origins and biosynthesis of tetrodotoxin

The potent neurotoxin tetrodotoxin (TTX) has been identified from taxonomically diverse marine organisms. TTX possesses a unique cage-like structure, however, its biosynthesis has yet to be elucidated. Biosynthetic studies in the TTX-producing newt Taricha torosa, and in bacterial genera, including Vibrio, have proven inconclusive. Indeed, very few studies have been performed that address the cellular production of TTX. Here we review the sources of TTX described to date and provide evidence for the biosynthesis of TTX by symbiotic microorganisms in higher taxa. Chemical and genetic based biosynthesis studies of TTX undertaken thus far are discussed and we outline approaches which may be useful for expanding upon the current body of knowledge. The complex biosynthesis of structurally similar toxins, that reveal clues into the biosynthetic pathway of TTX, is also presented.

Biosynthesis of toxic naturally-occurring seafood contaminants

Outbreaks of human illness caused by the consumption of contaminated seafood, continues to be a major problem particularly for the shellfish industry. Toxins from marine, brackish and freshwater environments, which are often produced as a result of harmful algal blooms, have been implicated as the causative agents of these poisonings. Commonly, poisoning events have been grouped into one of six classes, Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Ciguatera Fish Poisoning (CFP), Azaspiracid Shellfish Poisoning (AZP), and Amnesiac Shellfish Poisoning (ASP). The causative agents of these specific poisonings along with their biosyntheses are discussed in this review. The highly unusual and complex structures of most common seafood toxins have made them interesting targets for biosynthetic studies. Many of the toxins presented are biosynthesized via complex pathways that have been elucidated either through isotope labelled precursor feeding studies and/or characterization of the genes encoding the producing organisms biosynthetic machinery. Feeding studies key to our understanding of a particular toxins biosynthesis, such as the incorporation of unusual precursors, as well as unique biosynthetic pathways and rare chemical mechanisms involved in the assembly process are highlighted. More recently, however, modern genomics-based techniques have been used for the elucidation of biosynthetic pathways and these are presented in the context of polyketide, non-ribosomal peptide, and hybrid pathway derived, toxin assembly.

Differential accumulation of paralytic shellfish toxins from Alexandrium minutum in the pearl oyster, Pinctada imbricata

To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml(-1), the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml(-1). When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9+/-4.47 microg STX-equivalent 100 g(-1) biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0+/-2.20 microg STX-equivalent 100 g(-1) biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.

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.

Bioactive Natural Products from Papua New Guinea Marine Sponges

The discovery of novel natural products for drug development relies heavily upon a rich biodiversity, of which the marine environment is an obvious example. Marine natural product research has spawned several drugs and many other candidates, some of which are the focus of current clinical trials. The sponge megadiversity of Papua New Guinea is a rich but underexplored source of bioactive natural products. Here, we review some of the many natural products derived from PNG sponges with an emphasis on those with interesting biological activity and, therefore, drug potential. Many bioactive natural products discussed here appear to be derived from non‐ribosomal peptide and polyketide biosynthesis pathways, strongly suggesting a microbial origin of these compounds. With this in mind, we also explore the notion of sponge‐symbiont biosynthesis of these bioactive compounds and present examples to support the working hypothesis.

Discovery, Biosynthesis, and Rational Engineering of Novel Enterocin and Wailupemycin Polyketide Analogues

The marine actinomycete Streptomyces maritimus produces a structurally diverse set of unusual polyketide natural products including the major metabolite enterocin. Investigations of enterocin biosynthesis revealed that the unique carbon skeleton is derived from an aromatic polyketide pathway which is genetically coded by the 21.3 kb enc gene cluster in S. maritimus. Characterization of the enc biosynthesis gene cluster and subsequent manipulation of it via heterologous expression and/or mutagenesis enabled the discovery of other enc-based metabolites that were produced in only very minor amounts in the wild type. Also described are techniques used to harness the enterocin biosynthetic machinery in order to generate unnatural enc-derived polyketide analogues. This review focuses upon the molecular methods used in combination with classical natural products detection and isolation techniques to access minor metabolites of the S. maritimus secondary metabolome.

Unequivocal 13C NMR assignment of cyclohexadienyl rings in bromotyrosine-derived metabolites from marine sponges

Bromotyrosine‐derived compounds are commonly isolated from Verongida sponges and are a major class of marine natural products. Here we report on the unequivocal 13C NMR assignment of the brominated carbons at positions C‐2 and C‐4 of the cyclohexadiene ring, two carbons whose resonances are often incorrectly assigned. Interpretation of HMBC data acquired for a series of known bromotyrosine analogues, which included ianthesine E (1), aerothionin (2), 11‐hydroxyaerothionin (3), and 11,19‐dideoxyfistularin‐3 (4), allowed us to unequivocally assign the carbons in question, C‐2 and C‐4, through the observance of unique HMBC correlations from the C‐1 hydroxyl proton. Here we present the complete 2D NMR data sets recorded in DMSO‐d6 for 2–4 that were used to confirm the assignment and establish the working model. Using this model, a survey of the literature revealed that many members of this structure class had been wrongly assigned. This paper serves to reassign those compounds whose 13C NMR assignment at positions C‐2 and C‐4 of the cyclohexadiene ring should be reversed. Copyright

Combined genetic and bioactivity-based prioritization leads to the isolation of an endophyte-derived antimycobacterial compound

To initiate a genetic and bioactivity‐based screening programme of culturable endophytes to identify micro‐organisms capable of producing bioactive polyketides and peptides.

Genome-Guided Discovery of Natural Products and Biosynthetic Pathways from Australia’s Untapped Microbial Megadiversity

Historically microbial natural product biosynthesis pathways were elucidated mainly by isotope labelled precursor directed feeding studies. Now the genetics underpinning the assembly of microbial natural products biosynthesis is so well understood that some pathways and their products can be predicted from DNA sequences alone. The association between microbial natural products and their biosynthesis gene clusters is now driving the field of ‘genetics guided natural product discovery’. This account overviews our research into cyanotoxin biosynthesis before the genome sequencing era through to some recent discoveries resulting from the mining of Australian biota for natural product biosynthesis pathways.

Alternariol 9-O-methyl ether dimethyl sulfoxide monosolvate.

The title compound (systematic name: 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one dimethyl sulfoxide monosolvate), C15H12O5·C2H6OS, was isolated from an unidentified endophytic fungus (belonging to class Ascomycetes) of Taxus sp. In the crystal, both the alternariol 9-O-methyl ether (AME) and the dimethyl sulfoxide (DMSO) molecules exhibit crystallographic mirror symmetry. One of the hydroxy groups makes bifurcated hydrogen bonds, viz. an intramolecular bond with the carbonyl group and an intermolecular bond with the carbonyl group in an inversion-related AME molecule. In the crystal, the AME molecules are organized into stacks parallel with the b axis by π–π interactions between centrosymmetrically related molecules [the distance between the centroid of the central ring and the centroid of the methoxy-substituted benzene ring in the next molecule of the stack is 3.6184 (5) Å]. Pairs of DMSO molecules, linked via centrosymmetric C—H⋯O contacts, are inserted into the voids created by the AME molecules, making O—H⋯O and C—H⋯O contacts with the hosts.