Biosynthesis of Marine Toxins

Keywords : Biosynthesis, Brevetoxin, Domoic acid, Harmful algal bloom, Marine toxin, Saxitoxin, Tetrodotoxin

Introduction

Natural toxins from the marine environment have long fascinated scientists due to their extraordinary chemical structures and potent biological properties. Marine neurotoxins, in particular, have revealed the function and modulated the activity of numerous essential cellular proteins, such as ion channels and receptor proteins. However, toxin-producing oceanic harmful algal blooms continue to dramatically harm the environment, human health, and livelihoods, as seen during the devastating Karenia brevis bloom off Southwest Florida in 2019.

In contrast to freshwater systems, where cyanobacteria are the primary large-scale toxin producers, the major producers of marine neurotoxins are eukaryotic organisms, such as dinoflagellates and diatoms, which possess much larger genomes. This has slowed our understanding of marine toxin biosynthesis at the molecular level due to the lack of genomic data and tools. In freshwater cyanobacteria, biosynthetic pathways to most major cyanotoxins have been established, aided by smaller genomes and recognizable gene clusters. This allows for environmental monitoring of cyanobacterial toxin transcription since the biosynthetic genes have been identified.

Despite these challenges, substantial progress has been made in connecting marine toxins to their biosynthetic genes. This review focuses on advances in understanding the biosynthetic pathways of domoic acid, kainic acid, saxitoxin, tetrodotoxin, and large polyether compounds such as brevetoxin. Studies on these toxins have revealed unusual and interesting enzymology, which may facilitate biocatalytic production methods and improved environmental monitoring in the future.

Domoic Acid

Domoic acid is a potent neurotoxin produced primarily by diatoms of the Pseudo-nitzschia genus and some red algae. It acts as an agonist of ionotropic glutamate receptors, promoting calcium influx into neurons and leading to excitotoxicity. Domoic acid was first discovered in the 1950s from the red algae Chondria armata but gained notoriety after a major Pseudo-nitzschia multiseries bloom in 1987 in Prince Edward Island, Canada, which caused amnesic shellfish poisoning in humans.

Research has focused on elucidating the biosynthetic route of domoic acid due to its human health implications. Early studies suggested it was derived from geranyl diphosphate and L-glutamic acid, but the responsible enzymes were unknown. A transcriptomic-based approach identified a gene cluster (dabA–D) upregulated under domoic acid-producing conditions. These genes encode a terpene cyclase (dabA), a hypothetical protein (dabB), an α-ketoglutarate-dependent dioxygenase (dabC), and a cytochrome P450 oxidase (dabD).

The first committed step is catalyzed by DabA, which performs N-geranylation of L-glutamic acid to produce N-geranyl-L-glutamic acid (NGG). DabD then catalyzes three successive oxidations of the 7′ carbon of the prenyl chain, followed by DabC, which cyclizes the intermediate to yield isodomoic acid A. A final isomerization step, by an as-yet-unidentified enzyme, converts isodomoic acid A to domoic acid. Isolation and feeding studies support this biosynthetic proposal, with NGG identified as a key intermediate.

Kainic Acid

Kainic acid, another prominent kainoid natural product, was originally isolated from the marine red algae Digenea simplex. It shares a pyrrolidine core with domoic acid but has a shorter C4 moiety. Like domoic acid, kainic acid is an ionotropic glutamate receptor agonist, though less potent, and has been used clinically to treat parasitic worm infections.

Genome sequencing of kainic acid-producing red algae revealed gene clusters homologous to dabA and dabC, named kabA and kabC. Heterologous expression and in vitro assays showed that KabA catalyzes N-prenylation of L-glutamic acid using dimethylallyl diphosphate to yield prekainic acid. KabC then cyclizes this intermediate to generate kainic acid, also forming kainic acid lactone as a byproduct. The discovery of the biosynthetic gene cluster enabled a scalable, efficient biotransformation route for kainic acid production.

Saxitoxin

Paralytic shellfish toxins (PSTs), including saxitoxin, are a family of over 50 related alkaloid compounds with two guanidine moieties. They are produced by marine dinoflagellates, freshwater, and brackish water cyanobacteria. PSTs block voltage-gated sodium channels, leading to paralytic shellfish poisoning in humans and wildlife.

The saxitoxin biosynthetic gene cluster was first discovered in cyanobacteria, with similar genes found in dinoflagellates, suggesting horizontal gene transfer. The biosynthesis begins with SxtA, a polyketide synthase-like enzyme with four domains, which produces arginine ethyl ketone, the first committed intermediate. SxtG, an amidinotransferase, transfers an amidino group from arginine, and the product undergoes spontaneous cyclodehydration and further modifications, including cyclization, carbamylation, and hydroxylation, to yield β-saxitoxinol. Rieske oxygenases (SxtH, SxtT, and GxtA) catalyze specific hydroxylations, and sulfotransferases (SxtN and SxtSUL) add sulfate groups, generating the diversity of PSTs. While most biochemical work has used cyanobacterial enzymes, homologous genes and intermediates have been characterized in dinoflagellates.

Tetrodotoxin

Tetrodotoxin (TTX) is a potent sodium channel blocker with a unique dioxaadamantane and cyclic guanidine structure, found in pufferfish, newts, and other animals. TTX poisoning typically results from ingestion of contaminated animals and can cause muscle paralysis and death.

Despite decades of research, the biosynthetic origin of TTX remains unclear, and no biosynthetic gene has been identified. Its distribution among diverse animals and unusual structure suggest a bacterial origin, potentially acquired through diet or symbiosis. Several bacteria have been isolated from TTX-containing animals and shown to produce low levels of toxin, but reliable laboratory production has not been achieved. Feeding studies and the identification of guanidino-containing monoterpenes in newts and bicyclic guanidino compounds in pufferfish suggest different biosynthetic pathways in terrestrial and marine organisms. The biosynthesis of TTX remains a major unsolved question in natural product chemistry.

Brevetoxin and Other Polyether Toxins

Marine polyether toxins, such as brevetoxins, ciguatoxins, palytoxins, and maitotoxins, are characterized by their massive, complex structures. These neurotoxins are primarily produced by dinoflagellate microalgae, but some haptophytes also produce polyether toxins.

Isotopic tracer experiments support a polyketide origin for these compounds, with dinoflagellates confirmed as authentic producers. The regularity of trans-fused ring systems led to the hypothesis that polyepoxide precursors undergo epoxide-opening cascade reactions to form the polyether ladder structures. Although biomimetic chemistry supports this, the biosynthetic scheme remains unproven.

Dinoflagellate polyether biosynthesis differs from bacterial systems, with limited starter and extender units and unusual mechanisms such as Favorskii-like rearrangements, β-alkylation, and the ‘odd-even’ methylation rule. The genetic basis of polyether biosynthesis is difficult to resolve due to the large, complex genomes of dinoflagellates, which lack transcriptional regulation and contain repetitive gene arrays. Transcriptomic studies have identified numerous polyketide synthase (PKS) genes, including multimodular assembly line proteins, but definitive links to toxin biosynthesis are still lacking.

Conclusions and Outlook

Significant progress has been made in elucidating the biosynthetic pathways for several prominent marine toxins, especially for domoic acid, kainic acid, and saxitoxins. The discovery of gene clusters and new enzymatic reactions has advanced our understanding. However, the biosynthesis of TTX and polyether toxins remains enigmatic, primarily due to challenges in genetics and biochemistry of the producing organisms. Continued advances in genomics, transcriptomics, and biochemical approaches are expected to further unravel the biosynthetic stories of these fascinating marine natural products in the coming years.