Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Highlights of biosynthetic enzymes and natural products from symbiotic cyanobacteria.

Covering: up to 2023Cyanobacteria have long been known for their intriguing repertoire of natural product scaffolds, which are often distinct from other phyla. Cyanobacteria are ecologically significant organisms that form a myriad of different symbioses including with sponges and ascidians in the marine environment or with plants and fungi, in the form of lichens, in terrestrial environments. Whilst there have been several high-profile discoveries of symbiotic cyanobacterial natural products, genomic data is scarce and discovery efforts have remained limited. However, the rise of (meta-)genomic sequencing has improved these efforts, emphasized by a steep increase in publications in recent years. This highlight focuses on selected examples of symbiotic cyanobacterial-derived natural products and their biosyntheses to link chemistry with corresponding biosynthetic logic. Further highlighted are remaining gaps in knowledge for the formation of characteristic structural motifs. It is anticipated that the continued rise of (meta-)genomic next-generation sequencing of symbiontic cyanobacterial systems will lead to many exciting discoveries in the future.

Streptomyces sp. BV410: Interspecies cross-talk for staurosporine production.

AIMS: Sequencing and genome analysis of two co-isolated streptomycetes, named BV410-1 and BV410-10, and the effect of their co-cultivation on the staurosporine production. METHODS AND RESULTS: Identification of two strains through genome sequencing and their separation using different growth media was conducted. Sequence analysis revealed that the genome of BV410-1 was 9.5 Mb, whilst that of BV410-10 was 7.1 Mb. AntiSMASH analysis identified 28 biosynthetic gene clusters (BGCs) from BV410-1, including that responsible for staurosporine biosynthesis, whilst 20 BGCs were identified from BV410-10. The addition of cell-free supernatant from BV410-10 monoculture to BV410-1 fermentations improved the staurosporine yield from 8.35 mg L-1 up to 15.85 mg L-1 , whilst BV410-10 monoculture ethyl acetate extract did not have the same effect. Also, there was no improvement in staurosporine production when artificial mixed cultures were created using three different BV410-1 and BV410-10 spore ratios. CONCLUSIONS: The growth of BV410-10 was inhibited when the two strains were grown together on agar plates. Culture supernatants of BV410-10 showed potential to stimulate staurosporine production in BV410-1, but overall co-cultivation attempts did not restore the previously reported yield of staurosporine produced by the original mixed isolate. SIGNIFICANCE AND IMPACT OF STUDY: This work confirmed complex relations between streptomycetes in soil that are difficult to recreate under the laboratory conditions. Also, mining of streptomycetes genomes that mainly produce known bioactive compounds could still be the fruitful approach in search for novel bioactive molecules.

Biosynthesis of cyanobacterin, a paradigm for furanolide core structure assembly.

The γ-butyrolactone motif is found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin, which has a highly functionalized γ-butyrolactone core structure. The enzymatic machinery that assembles cyanobacterin and structurally related natural products (herein termed furanolides) has remained elusive for decades. Here, we elucidate the biosynthetic process of furanolide assembly. The cyanobacterin biosynthetic gene cluster was identified by targeted bioinformatic screening and validated by heterologous expression in Escherichia coli. Full functional evaluation of the recombinant key enzymes in vivo and in vitro, individually and in concert, provided in-depth mechanistic insights into a streamlined C-C bond-forming cascade that involves installation of compatible reactivity at seemingly unreactive Cα positions of amino acid precursors. Our work extends the biosynthetic and biocatalytic toolbox for γ-butyrolactone formation, provides a general paradigm for furanolide biosynthesis and sets the stage for their targeted discovery, biosynthetic engineering and enzymatic synthesis.

Recent developments in quantitative PCR for monitoring harmful marine microalgae.

Marine microalgae produce a variety of specialised metabolites that have toxic effects on humans, farmed fish, and marine wildlife. Alarmingly, many of these compounds bioaccumulate in the tissues of shellfish and higher trophic organisms, including species consumed by humans. Molecular methods are emerging as a potential alternative and complement to the conventional microscopic diagnosis of toxic or otherwise harmful microalgal species. Quantitative PCR (qPCR) in particular, has gained popularity over the past decade as a sensitive, rapid, and cost-effective method for monitoring harmful microalgae. Assays targeting taxonomic marker genes provide the opportunity to identify and quantify (or semi-quantify) microalgal species and importantly to pre-empt bloom events. Moreover, the discovery of paralytic shellfish toxin biosynthesis genes in dinoflagellates has enabled researchers to directly monitor toxigenic species in coastal waters and fisheries. This review summarises the recent developments in qPCR detection methods for harmful microalgae, with emphasis on emerging toxin gene monitoring technologies.

Final Destination? Pinpointing Hyella disjuncta sp. nov. PCC 6712 (Cyanobacteria) Based on Taxonomic Aspects, Multicellularity, Nitrogen Fixation and Biosynthetic Gene Clusters.

Unicellular cyanobacteria inhabit a wide range of ecosytems and can be found throughout the phylum offering space for taxonomic confusion. One example is strain PCC 6712 that was described as Chlorogloea sp. (Nostocales) and later assigned to the genus Chroococcidiopsis (Chroococcidiopsidales). We now show that this strain belongs to the order Pleurocapsales and term it Hyella disjuncta based on morphology, genome analyses and 16S-23S ITS rRNA phylogeny. Genomic analysis indicated that H. disjuncta PCC 6712 shared about 44.7% orthologue genes with its closest relative H. patelloides. Furthermore, 12 cryptic biosynthetic gene clusters (BGCs) with potential bioactivity, such as a mycosporine-like amino acid BGC, were detected. Interestingly, the full set of nitrogen fixation genes was found in H. disjuncta PCC 6712 despite its inability to grow on nitrogen-free medium. A comparison of genes responsible for multicellularity was performed, indicating that most of these genes were present and related to those found in other cyanobacterial orders. This is in contrast to the formation of pseudofilaments-a main feature of the genus Hyella-which is weakly expressed in H. disjuncta PCC 6712 but prominent in Hyella patelloides LEGE 07179. Thus, our study pinpoints crucial but hidden aspects of polyphasic cyanobacterial taxonomy.

Symphyonema bifilamentata sp. nov., the Right Fischerella ambigua 108b: Half a Decade of Research on Taxonomy and Bioactive Compounds in New Light.

Since 1965 a cyanobacterial strain termed 'Fischerella ambigua 108b' was the object of several studies investigating its potential as a resource for new bioactive compounds in several European institutes. Over decades these investigations uncovered several unique small molecules and their respective biosynthetic pathways, including the polychlorinated triphenyls of the ambigol family and the tjipanazoles. However, the true taxonomic character of the producing strain remained concealed until now. Applying a polyphasic approach considering the phylogenetic position based on the 16S rRNA and the protein coding gene rbcLX, secondary structures and morphological features, we present the strain 'Fischerella ambigua 108b' as Symphyonema bifilamentata sp. nov. 97.28. Although there is the type species (holotype) S. sinense C.-C. Jao 1944 there is no authentic living strain or material for genetic analyses for the genus Symphyonema available. Thus we suggest and provide an epitypification of S. bifilamentata sp. nov. 97.28 as a valid reference for the genus Symphyonema. Its affiliation to the family Symphyonemataceae sheds not only new light on this rare taxon but also on the classes of bioactive metabolites of these heterocytous and true-branching cyanobacteria which we report here. We show conclusively that the literature on the isolation of bioactive products from this organism provides further support for a clear distinction between the secondary metabolism of Symphyonema bifilamentata sp. nov. 97.28 compared to related and other taxa, pointing to the assignment of this organism into a separate genus.

Comparative proteomics of the toxigenic diazotroph Raphidiopsis raciborskii (cyanobacteria) in response to iron.

Raphidiopsis raciborskii is an invasive bloom-forming cyanobacteria with the flexibility to utilize atmospheric and fixed nitrogen. Since nitrogen-fixation has a high requirement for iron as an ezyme cofactor, we hypothesize that iron availability would determine the success of the species under nitrogen-fixing conditions. This study compares the proteomic response of cylindrospermopsin-producing and non-toxic strains of R. racibroskii to reduced iron concentrations, under nitrogen-fixing conditions, to examine any strain-specific adaptations that might increase fitness under these conditions. We also compared their proteomic responses at exponential and stationary growth phases to capture the changes throughout the growth cycle. Overall, the toxic strain was more competitive under Fe-starved conditions during exponential phase, with upregulated growth and transport-related proteins. The non-toxic strain showed reduced protein expression across multiple primary metabolism pathways. We propose that the increased expression of porin proteins during the exponential growth phase enables toxic strains to persist under Fe-starved conditions with this ability providing a potential explanation for the increased fitness of cylindrospermoipsin-producing strains during unfavourable environmental conditions.

Antarctic desert soil bacteria exhibit high novel natural product potential, evaluated through long-read genome sequencing and comparative genomics.

Actinobacteria and Proteobacteria are important producers of bioactive natural products (NP), and these phyla dominate in the arid soils of Antarctica, where metabolic adaptations influence survival under harsh conditions. Biosynthetic gene clusters (BGCs) which encode NPs, are typically long and repetitious high G + C regions difficult to sequence with short-read technologies. We sequenced 17 Antarctic soil bacteria from multi-genome libraries, employing the long-read PacBio platform, to optimize capture of BGCs and to facilitate a comprehensive analysis of their NP capacity. We report 13 complete bacterial genomes of high quality and contiguity, representing 10 different cold-adapted genera including novel species. Antarctic BGCs exhibited low similarity to known compound BGCs (av. 31%), with an abundance of terpene, non-ribosomal peptide and polyketide-encoding clusters. Comparative genome analysis was used to map BGC variation between closely related strains from geographically distant environments. Results showed the greatest biosynthetic differences to be in a psychrotolerant Streptomyces strain, as well as a rare Actinobacteria genus, Kribbella, while two other Streptomyces spp. were surprisingly similar to known genomes. Streptomyces and Kribbella BGCs were predicted to encode antitumour, antifungal, antibacterial and biosurfactant-like compounds, and the synthesis of NPs with antibacterial, antifungal and surfactant properties was confirmed through bioactivity assays.

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

BACKGROUND: Dolichospermum 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. RESULTS: In 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. CONCLUSIONS: Both 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.

Streptomyces sp. BV410 isolate from chamomile rhizosphere soil efficiently produces staurosporine with antifungal and antiangiogenic properties.

Applying a bioactivity-guided isolation approach, staurosporine was separated and identified as the active principle in the culture extract of the new isolate Streptomyces sp. BV410 collected from the chamomile rhizosphere. The biotechnological production of staurosporine by strain BV410 was optimized to yield 56 mg/L after 14 days of incubation in soy flour-glucose-starch-mannitol-based fermentation medium (JS). The addition of FeSO4 significantly improved the staurosporine yield by 30%, while the addition of ZnSO4 significantly reduced staurosporine yield by 62% in comparison with the starting conditions. Although staurosporine was first isolated in 1977 from Lentzea albida (now Streptomyces staurosporeus) and its potent kinase inhibitory effect has been established, here, the biological activity of this natural product was assessed in depth in vivo using a selection of transgenic zebrafish (Danio rerio) models, including Tg(fli1:EGFP) with green fluorescent protein-labeled endothelial cells allowing visualization and monitoring of blood vessels. This confirmed a remarkable antiangiogenic activity of the compound at doses of 1 ng/ml (2.14 nmol/L) which is below doses inducing toxic effects (45 ng/ml; 75 nmol/L). A new, efficient producing strain of commercially significant staurosporine has been described along with optimized fermentation conditions, which may lead to optimization of the staurosporine scaffold and its wider applicability.

Discovery of the Streptoketides by Direct Cloning and Rapid Heterologous Expression of a Cryptic PKS II Gene Cluster from Streptomyces sp. Tü 6314.

Genome sequencing and bioinformatic analysis have identified numerous cryptic gene clusters that have the potential to produce novel natural products. Within this work, we identified a cryptic type II PKS gene cluster (skt) from Streptomyces sp. Tü 6314. Facilitated by linear plus linear homologous recombination-mediated recombineering (LLHR), we directly cloned the skt gene cluster using the Streptomyces site-specific integration vector pSET152. Direct cloning allowed for rapid heterologous expression in Streptomyces coelicolor, leading to the identification and structural characterization of six polyketides (three known compounds and new streptoketides), four of which exhibit anti-HIV activities. Our study shows that the pSET152 vector can be directly used for LLHR, expanding the Rec/ET direct cloning toolbox and providing the possibility for rapid heterologous expression of gene clusters from Streptomyces.

Bypassing Biocatalytic Substrate Limitations in Oxidative Dearomatization Reactions by Transient Substrate Mimicking.

Enzymatic oxidative dearomatization is an efficient way to generate chiral molecules from simple arenes. One example is the flavin-dependent monooxygenase SorbC involved in sorbicillinoid biosynthesis. However, SorbC requires a long-chain keto substituent at its phenolic substrate, thus preventing its application beyond the synthesis of natural sorbicillinoids or close structural analogues. This work describes an approach to broaden the accessible product spectrum of SorbC by employing an ester functionality mimicking the natural substrate structure during enzymatic oxidation.

Antifungal potential of bacterial rhizosphere isolates associated with three ethno-medicinal plants (poppy, chamomile, and nettle).

The objective of the present study was to isolate Actinobacteria, preferably Streptomyces spp. from the rhizosphere soils of three ethno-medicinal plants collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and Urtica dioica) and to screen their antifungal activity against Candida spp. Overall, 103 sporulating isolates were collected from rhizosphere soil samples and determined as Streptomyces spp. Two different media and two extraction procedures were used to facilitate identification of antifungals. Overall, 412 crude cell extracts were tested against Candida albicans using disk diffusion assays, with 42% (43/103) of the strains showing the ability to produce antifungal agents. Also, extracts inhibited growth of important human pathogens: Candida krusei, Candida parapsilosis, and Candida glabrata. Based on the established degree and range of antifungal activity, nine isolates, confirmed as streptomycetes by 16S rRNA sequencing, were selected for further testing. Their ability to inhibit Candida growth in liquid culture, to inhibit biofilm formation, and to disperse pre-formed biofilms was assessed with active concentrations from 8 to 250 µg/mL. High-performance liquid chromatographic profiles of extracts derived from selected strains were recorded, revealing moderate metabolic diversity. Our results proved that rhizosphere soil of ethno-medicinal plants is a prolific source of streptomycetes, producers of potentially new antifungal compounds.

Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.

BACKGROUND: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. RESULTS: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. CONCLUSIONS: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

Re-evaluation of paralytic shellfish toxin profiles in cyanobacteria using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

To date Paralytic shellfish toxin (PST) variants in cyanobacteria have primarily been characterized using high performance liquid chromatography coupled with fluorescence detection. In this study we re-evaluated the PST profiles of five cyanobacterial cultures (Dolichospermum circinale AWQC131C, Aphanizomenon sp. NH-5, Raphidiopsis raciborskii T3, Scytonema cf. crispum CAWBG524 and CAWBG72) and one environmental sample (Microseria wollei) using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 35 different PST variants were detected. D. circinale contained the highest number of variants (23), followed by S. cf. crispum CAWBG72 (21). Many of the variants detected in the cultures/environmental sample had not been reported from these strains previously: D. circinale (14 variants), S. cf. crispum CAWBG72 (16), S. cf. crispum CAWBG524 (9), Aphanizomenon sp. (9), R. raciborskii (7), and M. wollei (7). Of particular interest was the detection of M-toxins (Aphanizomenon sp., R. raciborskii, D. circinale). These have previously only been identified from shellfish where they were thought to be metabolites. Well-characterized PST variant profiles are essential for research investigating the genetic basis of PST production, and given that the toxicity of each variants differs, it will assist in refining risk assessments.

Bioinformatic, phylogenetic and chemical analysis of the UV-absorbing compounds scytonemin and mycosporine-like amino acids from the microbial mat communities of Shark Bay, Australia.

Shark Bay, Western Australia is a World Heritage area with extensive microbial mats and stromatolites. Microbial communities that comprise these mats have developed a range of mitigation strategies against changing levels of photosynthetically active and ultraviolet radiation, including the ability to biosynthesise the UV-absorbing natural products scytonemin and mycosporine-like amino acids (MAAs). To this end, the distribution of photoprotective pigments within Shark Bay microbial mats was delineated in the present study. This involved amplicon sequencing of bacterial 16S rDNA from communities at the surface and subsurface in three distinct mat types (smooth, pustular and tufted), and correlating this data with the chemical and molecular distribution of scytonemin and MAAs. Employing UV spectroscopy and MS/MS fragmentation, mycosporine-glycine, asterina and an unknown MAA were identified based on typical fragmentation patterns. Marker genes for scytonemin and MAA production (scyC and mysC) were amplified from microbial mat DNA and placed into phylogenetic context against a broad screen throughout 363 cyanobacterial genomes. Results indicate that occurrence of UV screening compounds is associated with the upper layer of Shark Bay microbial mats, and the occurrence of scytonemin is closely dependent on the abundance of cyanobacteria.

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.

Direct Pathway Cloning Combined with Sequence- and Ligation-Independent Cloning for Fast Biosynthetic Gene Cluster Refactoring and Heterologous Expression.

The need  for new pharmacological lead structures, especially against drug resistances, has led to a surge in natural product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural product pathways have thus been developed. Direct pathway cloning (DiPaC) is an emerging synthetic biology strategy that utilizes long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture. As a proof of principle, the majority of the cyanobacterial hapalosin gene cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression. The complementation of DiPaC with SLIC depicts a time and cost-efficient method for simple capture and expression of new natural product pathways.