Toxin Biosynthesis Research Articles

Phylogenetic relationships and genetic divergence of paralytic shellfish toxin- and cylindrospermopsin- producing Cylindrospermopsis and raphidiopsis

Cylindrospermopsis and Raphidiopsis (C/R group) are closely related species responsible for cyanobacterial blooms worldwide. Paralytic shellfish toxins (PSTs) and cylindrospermopsins (CYNs) have been identified in different C/R group strains. However, the evolutionary relationship between PST- and CYN-producing strains has not been systematically evaluated. In this study, C/R group strains and their toxin biosynthesis genes were evaluated by phylogenetic analysis and sequence comparison. None of the tested strains are able to produce PSTs and CYNs simultaneously. The C/R group strains were clustered into five clades, including two non-toxic, two CYN-producing and one PST-producing clades. A high degree of similarity was observed for rpoC1 (> 96%) and ITS-L (> 97%) sequences within each clade with the exception of the ITS-L (87% to 100%) region in CYN-producing R. curvata, which has been shown to contain variable sequence insertions. Genomic analysis revealed that sxtY and sxtZ could be found in both toxic and non-toxic strains. The transposase gene IS4 was only observed in strains from the PST-producing clade. The sxt and cyr gene clusters share five gene families with similar functions. The amino acid sequences of the adenylyl-sulfate kinase genes, sxtO and cyrN, are more similar (45% to 81%) than other pairs of genes (8.0% to 40%). SxtO and CyrN proteins from C/R group strains forms an independent clade on the phylogenetic tree with a high degree of sequence similarity (78% to 100%). In conclusion, PST- and CYN- producing C/R group species can be classified into different clades based on their phylogenetic profile. The sxtO and cyrN genes have probably diverged from a single ancestral adenylyl-sulfate kinase gene, and may be specifically used for toxin biosynthesis in C/R group species.

Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression

BackgroundDolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology.ResultsIn D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5′ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions.ConclusionsBoth of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

Substrate Promiscuity of a Paralytic Shellfish Toxin Amidinotransferase.

Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins, and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.

Transcriptomic Profile and Sexual Reproduction-Relevant Genes of Alexandrium minutum in Response to Nutritional Deficiency.

Alexandrium minutum is a typical marine toxic dinoflagellate responsible for producing paralytic shellfish poisoning (PSP) toxins. Until now, we know little about the genomic information of A. minutum, so a transcriptome study was conducted to clarify the physiological adaptations related to nutritional deficiency. Here, we performed RNA-Seq analysis to assess the gene expression patterns of A. minutum under N and P deficient conditions for 0 (control), 6, and 72 h. Main differences between the control and experimental groups were observed in hydrolase activity and fatty acid, lipid, protein, and P metabolism. Activities of photosystem I (PSI) and PSII were significantly down-regulated, and the endocytosis pathway (clathrin-dependent endocytosis) was significantly enriched under N and P stress compared with the control, indicating that A. minutum shifts its trophy pattern under N and P stress. We also identified several unigenes related to the process of sexual reproduction, including sex determination, sperm-egg recognition, sex differentiation, mating, and fertilization. Approximately 50% of the successfully annotated unigenes were differentially expressed between the short-term stimulated sample (6 h) and control (R). However, the expression level of most unigenes returned to normal levels after 72 h, indicating that N and P stress plays a limited role in the induction of sexual reproduction. Furthermore, the quantitative real-time PCR (qRT-PCR) results of the five representative sex-related unigenes were consistent with sequencing data, which confirmed the authenticity of transcriptomic analysis. Also, qRT-PCR analysis showed that the long and short form transcripts of the saxitoxin biosynthesis gene (sxtA) were down-regulated under the nutrient deficient condition compared with the control, indicating that N and P stress regulates sxtA expression. Overall, transcriptome analysis of A. minutum revealed that N and P deficiency induced responses associated with stress response, photosynthetic efficiency, toxin biosynthesis, and sexual reproduction. Our data indicate that algae change their trophic modes (to facultative mixotrophy) and related physiological reactions under stress conditions; this possibly represents an ecological adaption strategy in the algal life cycle.

Antifungal and antiaflatoxigenic activity of rosemary essential oil (Rosmarinus officinalis L.) against Aspergillus flavus

ABSTRACTThe increased risk to health by diverse pathologies, such as cancer, liver diseases, and endocrine alterations, caused by chemical residues in food, has led to the search for sustainable agricultural management alternatives, such as the use of essential oils for the development of natural and eco-friendly fungicides. The aim of this study was to evaluate the antifungal and antiaflatoxigenic activity of Rosmarinus officinalis L. essential oil (REO) against Aspergillus flavus Link. REO was obtained by hydrodistillation and its major components were identified as 1,8-cineole (eucalyptol, 52.2%), camphor (15.2%) and α-pinene (12.4%) by GC/MS and NMR. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were both 500 µg/mL. REO reduced the mycelial growth of A. flavus at a concentration of 250 µg/mL (15.3%). The results obtained from scanning electron microscopy (SEM) demonstrated a reduction in the size of conidiophores and in the thickness of hyphae in A. flavus caused by treatment with REO (250 µg/mL). The production of ergosterol and the biomass of mycelium were both reduced as the REO treatment concentration increased. The production of aflatoxins B1 and B2 was inhibited after treatment with 250 µg/mL REO, a concentration below the MIC/MFC, indicating that the antiaflatoxigenic effect of REO is independent of its antifungal effect and is likely due to its direct action upon toxin biosynthesis. The data demonstrated that REO may be used as an alternative to synthetic fungicides.

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth, photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.

Use of the Secreted Proteome of Trametes versicolor for Controlling the Cereal Pathogen Fusarium langsethiae.

Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals. F. langsethiae is the main producer, in Europe, of T2 and HT-toxins in small grain cereals, albeit often asymptomatic; this makes its control challenging. The European Union (EU) is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides, which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives (e.g., 128/2009), here we report the use of protein fractions, purified from the culture filtrate of the basidiomycete Trametes versicolor, for controlling F. langsethiae. T. versicolor, a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers. In this study, the exo-proteome of T. versicolor proved highly efficient in inhibiting the growth of F. langsethiae and the biosynthesis of the T2 toxin. Results are promising for its future use as a sustainable product to control F. langsethiae infection in cereals under field conditions.

Omics Analysis for Dinoflagellates Biology Research.

Dinoflagellates are important primary producers for marine ecosystems and are also responsible for certain essential components in human foods. However, they are also notorious for their ability to form harmful algal blooms, and cause shellfish poisoning. Although much work has been devoted to dinoflagellates in recent decades, our understanding of them at a molecular level is still limited owing to some of their challenging biological properties, such as large genome size, permanently condensed liquid-crystalline chromosomes, and the 10-fold lower ratio of protein to DNA than other eukaryotic species. In recent years, omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, have been applied to the study of marine dinoflagellates and have uncovered many new physiological and metabolic characteristics of dinoflagellates. In this article, we review recent application of omics technologies in revealing some of the unusual features of dinoflagellate genomes and molecular mechanisms relevant to their biology, including the mechanism of harmful algal bloom formations, toxin biosynthesis, symbiosis, lipid biosynthesis, as well as species identification and evolution. We also discuss the challenges and provide prospective further study directions and applications of dinoflagellates.

Sabotage at the Powerhouse? Unraveling the Molecular Target of 2-Isopropylbenzaldehyde Thiosemicarbazone, a Specific Inhibitor of Aflatoxin Biosynthesis and Sclerotia Development in Aspergillus flavus, Using Yeast as a Model System.

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.

A Sequence Resource of Autosomes and Additional Chromosomes in the Peach Pathotype of Alternaria alternata.

Alternaria alternata is a generally saprophytic fungus. Its genome consists of 10 autosomes, while some strains have one or two additional chromosomes, called a conditionally dispensable chromosome (CDC). A CDC is not required for reproduction but confers host-specific pathogenicity. We sequenced the genome of the peach pathotype of A. alternata using Nanopore and assembled it into 20 sequences. The 10 largest sequences corresponded to 10 gapless sequences of A. solani autosomes, and 1 sequence was a mitochondrial genome. Nine other sequences may be derived from CDCs because of lack of similarity with autosomes of other Alternaria spp. The sequence information could provide novel insights into genomes of Alternaria spp. and on the biosynthesis of a novel host-specific toxin in the peach pathotype of A. alternata.

The Genetic Basis of Toxin Biosynthesis in Dinoflagellates.

In marine ecosystems, dinoflagellates can become highly abundant and even dominant at times, despite their comparatively slow growth rates. One factor that may play a role in their ecological success is the production of complex secondary metabolite compounds that can have anti-predator, allelopathic, or other toxic effects on marine organisms, and also cause seafood poisoning in humans. Our knowledge about the genes involved in toxin biosynthesis in dinoflagellates is currently limited due to the complex genomic features of these organisms. Most recently, the sequencing of dinoflagellate transcriptomes has provided us with valuable insights into the biosynthesis of polyketide and alkaloid-based toxin molecules in dinoflagellate species. This review synthesizes the recent progress that has been made in understanding the evolution, biosynthetic pathways, and gene regulation in dinoflagellates with the aid of transcriptomic and other molecular genetic tools, and provides a pathway for future studies of dinoflagellates in this exciting omics era.

Genetic diversity ofPseudomonas syringaepv.actinidiae, pathogen of kiwifruit bacterial canker

Bacterial canker disease of kiwifruit currently occurs in at least 15 countries, causing serious damage. The causative agent of the disease isPseudomonas syringaepv.actinidiae(Psa), which is genetically diverse and is currently classified into five biovars, namely, biovars 1, 2, 3, 5 and 6. In Japan, four biovars except biovar 2 have been found so far. These biovars have been confirmed to have differences in the virulence and composition of pathogenicity‐related genes, such as toxin biosynthesis and typeIIIeffector genes. Biovars 1 and 6 possess thetoxisland, a genomic island of approximately 38 kb, which contains phaseolotoxin biosynthesis genes (argK‐toxcluster) and is confirmed to have been acquired from other bacteria through horizontal transfers. Also, on the megaplasmid possessed by biovar 6, there exist coronatine biosynthesis genes, and biovar 6 has the ability to produce two phytotoxins, phaseolotoxin and coronatine. In 2014, biovar 3, considered to be of foreign origin, was confirmed for the first time in Japan. Biovar 5, whose virulence is relatively weak, is distributed only in a limited area. In addition to thetoxisland and various plasmids, a large number of mobile genetic elements are confirmed to be present on the Psa genomes, which might have played a major role in helping Psa to acquire new features. In order to understand how Psa acquired the ability to infect kiwifruit systemically, it is important to make polyphasic comparisons with related pathovars, such asP. syringaepv.theaeand pv.actinidifoliorum.

Enzymes that detoxify marine toxins

Potent microbial toxins found in shellfish are possible starting points for drug discovery, but analogues are needed for biological testing. Toxin-modification enzymes now suggest a new approach for producing these analogues. The biosynthesis of paralytic shellfish toxins.

CylA is a sequence-specific protease involved in toxin biosynthesis.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

Killer Yeasts And Their Application

Abstract A killer phenotype, associated with the production and secretion of killer toxins, is widespread among yeasts and in competitive conditions gives an advantage to killer yeast strains in relation to other, sensitive microorganisms colonizing the same ecological niche. Killer toxins are proteins, usually glycoproteins, that are able to kill strains of susceptible yeasts. Each killer toxin has unique properties that vary depending on the strain of yeast that produces it. These differences concern the location of genes that encode toxins, molecular weight, as well as mechanisms of action. Some strains of killer yeast are characterized by a wide range of antagonistic activity, inhibit the development of a number of yeast strains, as well as molds, and have been studied for many years in terms of their biotechnological potential. Killer yeast and its toxins can find potential application in many fields: in the production of food and beverages, especially during wine fermentation and maturation, in biological control of plant pathogens, in yeast biotyping and as new antifungal agents. 1. Introduction. 2. Biosynthesis and structure of killer toxins. 3. Properties of killer proteins. 4. The mechanism of action of killer toxins. 5. Use of killer yeasts and their toxins. 5.1. Application in viticulture. 5.2. Potential application in medicine. 5.3. Combating fungal diseases of plants. 5.4. Transgenic plants producing killer toxins. 5.5. Use of killer yeasts in the marine environment. 6. Summary

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

SummaryPerylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

ITRAQ-Based Quantitative Proteomic Analysis of a Toxigenic Dinoflagellate Alexandrium catenella at Different Stages of Toxin Biosynthesis during the Cell Cycle.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids that are produced mainly by marine dinoflagellates. PST biosynthesis in dinoflagellates is a discontinuous process that is coupled to the cell cycle. However, little is known about the molecular mechanism underlying this association. Here, we compared global protein expression profiles of a toxigenic dinoflagellate, Alexandrium catenella, collected at four different stages of toxin biosynthesis during the cell cycle, using an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach. The results showed that toxin biosynthesis occurred mainly in the G1 phase, especially the late G1 phase. In total, 7232 proteins were confidently identified, and 210 proteins exhibited differential expression among the four stages. Proteins involved in protein translation and photosynthetic pigment biosynthesis were significantly upregulated during toxin biosynthesis, indicating close associations among the three processes. Nine toxin-related proteins were detected, and two core toxin biosynthesis proteins, namely, sxtA and sxtI, were identified for the first time in dinoflagellates. Among these proteins, sxtI and ompR were significantly downregulated when toxin biosynthesis stopped, indicating that they played important roles in the regulation of PST biosynthesis. Our study provides new insights into toxin biosynthesis in marine dinoflagellates: nitrogen balance among different biological processes regulates toxin biosynthesis, and that glutamate might play a key modulatory role.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

Effect of Zingiber officinale Roscoe essential oil in fungus control and deoxynivalenol production of Fusarium graminearum Schwabe in vitro

ABSTRACTMembers of the Fusarium genus are capable of contaminating agricultural commodities, compromising the quality of maize and other grains, which leads to severe quality and yield losses. Contamination with mycotoxins is also a concern. Essential oils are possible alternatives to the use of synthetic pesticides for control of fungal contamination, as many have antifungal and anti-mycotoxigenic properties and are innocuous to human health. They also do not cause any sort of microbial resistance and do not promote environmental pollution. The aim of this study was to evaluate the antifungal and anti-mycotoxigenic effects of Zingiber officinale Roscoe essential oil (GEO) upon Fusarium graminearum Schwabe in vitro. The essential oil was extracted by hydrodistillation and analysed by GC/MS. Antifungal and anti-mycotoxigenic activities were assessed by HPLC/UV by quantifying ergosterol and deoxynivalenol (DON), respectively. Results indicated that GEO inhibited ergosterol production at a concentration of 1000 µg/mL and DON production at a concentration of 500 µg/mL, evidencing that the anti-mycotoxigenic effect is independent of the antifungal effect due to its probable direct action upon toxin biosynthesis.

C-H Hydroxylation in Paralytic Shellfish Toxin Biosynthesis.

The remarkable degree of synthetic selectivity found in Nature is exemplified by the biosynthesis of paralytic shellfish toxins such as saxitoxin. The polycyclic core shared by saxitoxin and its relatives is assembled and subsequently elaborated through the installation of hydroxyl groups with exquisite precision that is not possible to replicate with traditional synthetic methods. Here, we report the identification of the enzymes that carry out a subset of C-H functionalizations involved in paralytic shellfish toxin biosynthesis. We have shown that three Rieske oxygenases mediate hydroxylation reactions with perfect site- and stereoselectivity. Specifically, the Rieske oxygenase SxtT is responsible for selective hydroxylation of a tricyclic precursor to the famous natural product saxitoxin, and a second Rieske oxygenase, GxtA, selectively hydroxylates saxitoxin to access the oxidation pattern present in gonyautoxin natural products. Unexpectedly, a third Rieske oxygenase, SxtH, does not hydroxylate tricyclic intermediates, but rather a linear substrate prior to tricycle formation, rewriting the biosynthetic route to paralytic shellfish toxins. Characterization of SxtT, SxtH, and GxtA is the first demonstration of enzymes carrying out C-H hydroxylation reactions in paralytic shellfish toxin biosynthesis. Additionally, the reactions of these oxygenases with a suite of saxitoxin-related molecules are reported, highlighting the substrate promiscuity of these catalysts and the potential for their application in the synthesis of natural and unnatural saxitoxin congeners.