Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria

Abstract

Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.Paralytic shellfish poisoning (PSP) toxins are among the world's most potent and pervasive toxins and are considered a serious toxicological health risk that may affect humans, animals, and ecosystems worldwide (18,36). These toxins block voltage-gated sodium and calcium channels and prolong the gating of potassium channels (21, 53, 59), preventing the transduction of neuronal signals. It has been estimated that more than 2,000 human cases of PSP occur globally every year at a mortality rate of 15% (16). Moreover, coastal blooms of productive microorganisms result in millions of dollars of economic damage due to PSP toxin contamination of seafood and the continuous requirement for costly biotoxin monitoring programs. Early warning systems to anticipate the occurrence of paralytic shellfish toxin (PST)-producing algal blooms, such as PCR and enzyme-linked immunosorbent assay-based screening, are as yet unavailable due to the lack of data on the genetic basis of PST production.Saxitoxin (STX) is a tricyclic perhydropurine alkaloid that can be substituted at various positions, leading to more than 30 naturally occurring STX analogues (4,5,28,32,33,63). Although STX biosynthesis seems complex and unique, organisms from two kingdoms, including certain species of marine dinoflagellates and freshwater cyanobacteria, are capable of producing these toxins, apparently by the same biosynthetic route (47). In spit...

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

“…In detail, we have confirmed the presence of sxtA, domain A4, in 40 strains of 8 species of STX-producing dinoflagellates, and did not identify it in 67 strains of 54 species of non STX-producing species representing different dinoflagellate families (Table 1). These results confirmed the hypothesis that sxtA4 is an essential domain for saxitoxin synthesis (Kellmann et al, 2008;Stüken et al, 2011;Murray et al, 2011a,b). Conversely, only those species which could produce this toxin possessed this domain, apart from one interesting anomaly: all four strains of Alexandrium australiense (formerly A. tamarense, Group V), only two of which have been found to produce STXs, possess the domain sxtA4 Murray et al, 2012a,b).…”

“…STXs are produced by freshwater prokaryotic cyanobacteria and a group of marine eukaryotic protists. The genes putatively involved in STX biosynthesis (sxt), involving $30 biosynthetic steps, are now known from $6 cyanobacterial genera (Kellmann et al, 2008;Mihali et al, 2009Mihali et al, , 2011Moustafa et al, 2009;Stucken et al, 2010).…”

“…They have molecular genetic characteristics typical of dinoflagellates: a conserved 22 bp spliced leader sequence at the 5' end of the transcribed mRNA; eukaryotic poly A tails; a GC content of 62-69%, similar to that reported for Alexandrium genomes, compared to 43% for cyanobacterial sxtA genes; dinoflagellate-related signal peptides; and a high genomic copy number of $10 2 copies cell À1 , a feature typical of dinoflagellates Murray et al, 2011a;Orr et al, 2013). A complement of 14 'core' genes (sxtA-sxtI, sxtP-sxtR, sxtS, and sxtU) is common between the sxt clusters of cyanobacterial STX-producing strains (Murray et al, 2011b), of which eight (sxtA, sxtB, sxtD, sxtG, sxtH or sxtT, sxtI, sxtS, and sxtU) may be directly implicated in STX synthesis (Kellmann et al, 2008). In cyanobacteria, the gene cluster appears to have an ancient origin, and both gene duplication and positive selection have played a role in its evolution (Murray et al, 2011b).…”

“…sxtA has four catalytic domains, with predicted activities similar to a SAM-dependent methyltransferase (sxtA1), related N-acetyltransferase (sxtA2), acyl carrier protein (sxtA3) and an amidinotransferase (sxtA4) (Kellmann et al, 2008). sxtA in dinoflagellates shows two isoforms: one of which comprises four domains, sxtA1-sxtA4, while the other one encompasses only the domains sxtA1-sxtA3 , and may represent a duplicated, paralogs copy of sxtA1.…”

First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

“…The effectivity of ion channel blockage depends on the structural characteristics of each given PST variant, 57 of which have been identified (Wiese et al, 2010). The biosynthesis of all PSTs is directed by a suite of biosynthetic genes (sxt), first described in C. raciborskii strain T3 (Kellmann et al, 2008). Since then, toxinspecific molecular genetic analysis has been employed in the study of PST-producing cyanobacteria in culture and in the field.…”