Cyanobacterial Natural Products Research Articles

Advancing cyanobacterial biotechnology: new frontiers in natural product discovery and production

Cyanobacteria, as oxygenic phototrophs, offer significant potential for sustainable biotechnology applications. Cyanobacterial natural products, with antimicrobial, anticancer, and plant growth-promoting properties, hold promise in pharmaceuticals, agriculture, and environmental remediation. By leveraging advanced technologies, cyanobacteria can significantly impact various industries, supporting the green biotechnology agenda. Recent advancements in integrated omics, orphan gene cluster activation, genetic manipulation, and chemo-enzymatic methods are expanding their biotechnological relevance. Omics technologies revolutionize cyanobacterial natural product research by facilitating biosynthetic gene cluster identification. Heterologous expression and pathway reconstitution enable complex natural product production, while high-titer strategies like metabolic engineering enhance yields. Interdisciplinary research and technological progress position cyanobacteria as valuable sources of bioactive compounds, driving sustainable biotechnological practices forward.

Impacts of cyanobacterial metabolites on fish: Socioeconomic and environmental considerations

AbstractThe increasing global population has led to rising demand for food, particularly protein. As an excellent source of protein, fish play a crucial role in meeting this demand, making aquaculture a highly impactful industry. Floating cages have been developed as a method of fish management and production to achieve high productivity and volume. However, these intensive fish farming practices can contribute to eutrophication, leading to changes in primary producers and promoting the excessive proliferation of cyanobacteria. Cyanobacteria blooms pose significant consequences to aquatic ecosystems, including potential risks to fish exposed to harmful cyanobacterial metabolites. Cyanobacterial metabolites encompass various chemical classes, such as terpenoids, carotenoids, alkaloids, cyanopeptides, amino acids, organophosphates, macrolides, and lipopolysaccharides. Some of these compounds' toxicity and impact on fish farms still need to be better understood. Cyanotoxins and off‐flavour compounds in fish farms can pose risks to water and fish quality and potential health hazards for humans throughout the food chain. Therefore, cyanobacteria in fish farms can have significant economic, environmental, and public health implications. This review examines the concerns associated with cyanobacterial natural products in fish farming, including off‐flavours, known cyanotoxins, and other potentially toxic compounds, while exploring their socioeconomic and environmental risks.

Harnessing the potential: advances in cyanobacterial natural product research and biotechnology.

Covering: 2000 to 2023Cyanobacteria produce a variety of bioactive natural products that can pose a threat to humans and animals as environmental toxins, but also have potential for or inspire pharmaceutical use. As oxygenic phototrophs, cyanobacteria furthermore hold great promise for sustainable biotechnology. Yet, the necessary tools for exploiting their biotechnological potential have so far been established only for a few model strains of cyanobacteria, while large untapped biosynthetic resources are hidden in slow-growing cyanobacterial genera that are difficult to access by genetic techniques. In recent years, several approaches have been developed to circumvent the bottlenecks in cyanobacterial natural product research. Here, we summarize current progress that has been made in unlocking or characterizing cryptic metabolic pathways using integrated omics techniques, orphan gene cluster activation, use of genetic approaches in original producers, heterologous expression and chemo-enzymatic techniques. We are mainly highlighting genomic mining concepts and strategies towards high-titer production of cyanobacterial natural products from the last 10 years and discuss the need for further research developments in this field.

Up to Date of Cyanobacterial Natural Products

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.

A Review of the Antimicrobial Properties of Cyanobacterial Natural Products.

The development of multiple-drug-resistant pathogens has prompted medical research toward the development of new and effective antimicrobial therapies. Much research into novel antibiotics has focused on bacterial and fungal compounds, and on chemical modification of existing compounds to increase their efficacy or reactivate their antimicrobial properties. In contrast, cyanobacteria have been relatively overlooked for antibiotic discovery, and much more work is required. This may be because some cyanobacterial species produce environmental toxins, leading to concerns about the safety of cyanobacterial compounds in therapy. Despite this, several cyanobacterial-derived compounds have been identified with noteworthy inhibitory activity against bacterial, fungal and protozoal growth, as well as viral replication. Additionally, many of these compounds have relatively low toxicity and are therefore relevant targets for drug development. Of particular note, several linear and heterocyclic peptides and depsipeptides with potent activity and good safety indexes have been identified and are undergoing development as antimicrobial chemotherapies. However, substantial further studies are required to identify and screen the myriad other cyanobacterial-derived compounds to evaluate their therapeutic potential. This study reviews the known phytochemistry of cyanobacteria, and where relevant, the effects of those compounds against bacterial, fungal, protozoal and viral pathogens, with the aim of highlighting gaps in the literature and focusing future studies in this field.

Antibiotic Potential of the Ambigol Cyanobacterial Natural Product Class and Simplified Synthetic Analogs.

The ambigols are cyanobacterial natural products characterized by three polychlorinated aromatic building blocks connected by biaryl and biaryl ether bridges. All ambigols known to date possess promising biological activities. Most significantly, ambigol A was reported to have antibacterial activity against Gram-positive bacteria, such as Bacillus megaterium and B. subtilis. We established a diverse compound library for in-depth biological evaluation building on our previous bio- and total synthetic research on this natural product family. To explore the antimicrobial potential in detail and to determine initial structure-activity relationships of this product class, a large set of dimeric and trimeric compounds were screened against selected bacterial and Candida target strains. Our results reveal exceptional antibiotic activity of the ambigols, especially against challenging clinical isolates.

Utility of Spirulina as Immunity Booster Agent

Spirulina is a single-cell protein that is high in important minerals and vitamins and can be used to make functional foods. Spirulina is a single-cell protein that contains all of the essential amino acids.It contains important minerals and vitamins and can be used to make functional cuisine. Indeed, one of the most serious issues in the food business is the use of manufactured food additives, which raise the chance of cancer.As a result, attempts are currently being made around the globe to isolate new and safe antioxidants from natural sources.Among these, cyanobacterial natural products are a significant source of novel drug molecules. Natural bioactive products have therapeutic value in and of themselves, but they are also used as building blocks to make synthetic analogues. Spirulina contains protein (70-55%), carbs (25-25%), important fatty acids (18%), vitamins, minerals, and colours such as carotene, chlorophyll A, and phycocyanin. Clearly, the advent of important cyanobacterial properties Spirulina can serve as an appropriate replacement for many antimicrobial substances and synthetic antioxidants that not only pose no danger to the consumer but can also enhance consumer health. Spirulina's nutritional value, bioactive characteristics, and immunological uses have all been addressed in this study. According to current study, spirulina supplements have been recognised by worldwide certification as a safe nutritious and dietary addition.

Incorporation and modification of fatty acids in cyanobacterial natural products biosynthesis.

Fatty acid-derived alkyl chains are often found in natural products, where they can exert a number of different functions, most notably biological membrane interactions. Such alkyl chains are difficult to modify regio- and stereoselectively, since most positions are distant from any directing functional group. Chemical and biochemical diversification of these moieties is therefore a challenge, and most organisms do not modify alkyl moieties to a great extent. Still, one particular group of microorgansims - cyanobacteria - display not only a large number of fatty acid-incorporating natural products, but also modify these to a great extent. Here, we provide an overview of the unique fatty acid metabolism of cyanobacteria in the context of natural products biosynthesis. We cover the diverse range of fatty acid incorporation mechanisms that these organisms use to recruit and commit fatty acids to natural products biosynthetic pathways. A variety of alkyl chain decorations and modifications that are found in cyanobacterial natural products are highlighted, illustrating the rich enzymatic arsenal that these organisms have evolved to diversify fatty acid-derived alkyl chains.

Metabolomics Applied to Cyanobacterial Toxins and Natural Products.

The biological and chemical diversity of Cyanobacteria is remarkable. These ancient prokaryotes are widespread in nature and can be found in virtually every habitat on Earth where there is light and water. They are producers of an array of secondary metabolites with important ecological roles, toxic effects, and biotechnological applications. The investigation of cyanobacterial metabolites has benefited from advances in analytical tools and bioinformatics that are employed in metabolomic analyses. In this chapter, we review selected articles highlighting the use of targeted and untargeted metabolomics in the analyses of secondary metabolites produced by cyanobacteria. Here, cyanobacterial secondary metabolites have been didactically divided into toxins and natural products according to their relevance to toxicological studies and drug discovery, respectively. This review illustrates how metabolomics has improved the chemical analysis of cyanobacteria in terms of speed, sensitivity, selectivity, and/or coverage, allowing for broader and more complex scientific questions.

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.

Description of Aliinostoc alkaliphilum sp. nov. (Nostocales, Cyanobacteria), a New Bioactive Metabolite-Producing Strain from Salina Verde (Pantanal, Brazil) and Taxonomic Distribution of Bioactive Metabolites in Nostoc and Nostoc-like Genera

Cyanobacteria are a group of oxygenic photosynthetic prokaryotes found in almost all habitats on earth including those characterized as extreme environments. It has been observed that the number of studies dealing with the biodiversity of extremophilic cyanobacteria is limited while studies exploring their bioactive potential are even scarcer. The taxonomy of three Nostoc-like cyanobacterial strains isolated from a shallow lake in Brazil was studied by applying a polyphasic approach. The bioactive potential of the strains was also evaluated using antimicrobial susceptibility testing. The metabolites present in the bioactive HPLC fractions were identified by UPLC/ESI/Q-TOF. Based on our phylogenetic inferences in combination with morphological and ecological information, we describe Aliinostoc alkaliphilum sp. nov., exhibiting antibacterial and antifungal activities. The main bioactive metabolite in all three strains was nocuolin A, which represents the first report of this metabolite in Aliinostoc. Our phylogenetic studies also revealed that many bioactive metabolite-producting strains that are currently assigned to Nostoc belong to other distinct evolutionary lineages. These findings highlight the importance of polyphasic approach studies in both cyanobacterial taxonomy and natural product discovery programs.

Discovery of Novel Tyrosinase Inhibitors From Marine Cyanobacteria.

Tyrosinase, an important oxidase involved in the primary immune response in humans, can sometimes become problematic as it can catalyze undesirable oxidation reactions. Therefore, for decades there has been a strong pharmaceutical interest in the discovery of novel inhibitors of this enzyme. Recent studies have also indicated that tyrosinase inhibitors can potentially be used in the treatment of melanoma cancer. Over the years, many new tyrosinase inhibitors have been discovered from various natural sources; however, marine natural products (MNPs) have contributed only a small number of promising candidates. Therefore, in this study we focused on the discovery of new MNP tyrosinase inhibitors of marine cyanobacterial and algal origins. A colorimetric tyrosinase inhibitory assay was used to screen over 4,500 marine extracts against mushroom tyrosinase (A. bisporus). Our results revealed that scytonemin monomer (ScyM), a pure compound from our compound library and also the monomeric last-step precursor in the biosynthesis of the well-known cyanobacterial sunscreen pigment “scytonemin,” consistently showed the highest tyrosinase inhibitory score. Determination of the half maximal inhibitory concentration (IC50) further indicated that ScyM is more potent than the commonly used commercial inhibitor standard “kojic acid” (KA; IC50 of ScyM: 4.90 μM vs. IC50 of KA: 11.31 μM). After a scaled-up chemical synthesis of ScyM as well as its O-methyl analog (ScyM-OMe), we conducted a series of follow-up studies on their structures, inhibitory properties, and mode of inhibition. Our results supported ScyM as the second case ever of a novel tyrosinase inhibitory compound based on a marine cyanobacterial natural product. The excellent in vitro performance of ScyM makes it a promising candidate for applications such as a skin-whitening agent or an adjuvant therapy for melanoma cancer treatment.

Abstract 3916: Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome

Abstract Coibamide A is a cyclopeptide natural product with potent antiproliferative activity against cultured human cancer cells and subcutaneous U87-MG glioblastoma tumors in mice. We have recently determined that coibamide A binds to the Sec61 translocon channel at a site that is close, but not identical, to the binding site of the peptide/polyketide natural product apratoxin A. As the majority of secreted proteins in mammalian cells are trafficked through the conventional secretory pathway via the Sec61 translocon, this raised the possibility that coibamide A and apratoxin A block import and secretion of different Sec61 client proteins. In the present study, we analyzed and compared the secretome of human U87-MG glioblastoma cells in the presence and absence of both marine natural products. U87-MG glioblastoma cells were treated with coibamide A (5 nM), apratoxin A (5 nM), or vehicle for 24 h. The growth media was then replaced with serum-free media for a further 24 h. Tryptic proteomic samples were generated from cell-free conditioned media and analyzed on a LC-coupled Orbitrap Fusion mass spectrometer. The high-resolution Orbitrap mass analyzer was used to both identify precursor ions for tandem mass (MS2) analysis in the ion trap mass analyzer and to quantitate those ions (MS1). Secreted proteins were identified from peptide–spectrum matches to the human protein database using a decoy database to assign an FDR and quantified from their MS1 ion intensities. Comparison of mass spectrometric data against the human secretome database revealed 175 proteins in the U87-MG cell secretome that were identified with high confidence. Differences between coibamide A and apratoxin A treatment were not significant, however both Sec61 inhibitors induced significant suppression of the U87-MG secretome relative to vehicle-treated cells. Treatment with coibamide A or apratoxin A resulted in the reduction of 75% of quantifiable secretome (131 proteins). Of the remaining 44 proteins, 34 are directed to intracellular organelles or contain a motif associated with retention in the endoplasmic reticulum and 10 were not suppressed with treatment but were hypothesized to be Sec61 clients. Malignant glioblastoma cells secrete proteins into the extracellular space and use complex intercellular signaling mechanisms for a survival advantage. Our results indicate that broad-acting inhibitors of Sec61-dependent co-translational translocation block the progression of a significant percentage of the U87-MG glioblastoma cell secretome including several proteins that have previously been detected as biomarkers in the cerebrospinal fluid, blood, or urine of brain tumor patients. Taken together, our work illustrates the feasibility of inducing a reversible suppression of numerous (undrugged) extracellular signals by targeting Sec61-dependent protein biogenesis in the secretory pathway with drug-like molecules. Citation Format: Daphne R. Mattos, Jeffrey D. Serrill, Philip R. Gafken, Kerry L. McPhail, Walter K. Vogel, Jane E. Ishmael. Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3916.

Targeted Discovery of Amantamide B, an Ion Channel Modulating Nonapeptide from a South China Sea Oscillatoria Cyanobacterium.

Amantamide B (1) is a new linear nonapeptide analogue of the cyanobacterial natural product amantamide A (2), and both have methyl ester and butanamide termini. These compounds were discovered in this study from the organic extract of a tropical marine filamentous cyanobacterium, Oscillatoria sp., collected around the Paracel Islands in the South China Sea. The use of LC-MS/MS molecular networking for sample prioritization and as an analytical dereplication tool facilitated the targeted isolation of 1 and 2. These molecules were characterized by spectroscopy and spectrometry, and configurational assignments were determined using chemical degradation and chiral-phase HPLC analysis. Compounds 1 and 2 modulated spontaneous calcium oscillations without notable cytotoxicity at 10 μM in short duration in vitro testing on primary cultured neocortical neurons, a model system that evaluates neuronal excitability and/or the potential activity on Ca2+ signaling. Both molecules were also found to be moderately cytotoxic in longer duration bioassays, with in vitro IC50 values of 1-10 μM against CCRF-CEM human T lymphoblastoid cells and U937 human histiocytic lymphoma cells. These formerly undiscovered bioactivities of known compound 2 expand upon its previously reported function as a selective CXCR7 agonist among 168 GPCR targets.

Uncovering the Bioactive Potential of a Cyanobacterial Natural Products Library Aided by Untargeted Metabolomics.

The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) holds a vast number of cyanobacteria whose chemical richness is still largely unknown. To expedite its bioactivity screening we developed a natural products library. Sixty strains and four environmental samples were chromatographed, using a semiautomatic HPLC system, yielding 512 fractions that were tested for their cytotoxic activity against 2D and 3D models of human colon carcinoma (HCT 116), and non-cancerous cell line hCMEC/D3. Six fractions showed high cytotoxicity against 2D and 3D cell models (group A), and six other fractions were selected by their effects on 3D cells (group B). The metabolome of each group was organized and characterized using the MolNetEnhancer workflow, and its processing with MetaboAnalyst allowed discrimination of the mass features with the highest fold change, and thus the ones that might be bioactive. Of those, mass features without precedented identification were mostly found in group A, indicating seven possible novel bioactive molecules, alongside in silico putative annotation of five cytotoxic compounds. Manual dereplication of group B tentatively identified nine pheophytin and pheophorbide derivatives. Our approach enabled the selection of 7 out of 60 cyanobacterial strains for anticancer drug discovery, providing new data concerning the chemical composition of these cyanobacteria.

Heterologous production of cyanobacterial compounds.

Cyanobacteria produce a plethora of compounds with unique chemical structures and diverse biological activities. Importantly, the increasing availability of cyanobacterial genome sequences and the rapid development of bioinformatics tools have unraveled the tremendous potential of cyanobacteria in producing new natural products. However, the discovery of these compounds based on cyanobacterial genomes has progressed slowly as the majority of their corresponding biosynthetic gene clusters (BGCs) are silent. In addition, cyanobacterial strains are often slow-growing, difficult for genetic engineering, or cannot be cultivated yet, limiting the use of host genetic engineering approaches for discovery. On the other hand, genetically tractable hosts such as Escherichia coli, Actinobacteria, and yeast have been developed for the heterologous expression of cyanobacterial BGCs. More recently, there have been increased interests in developing model cyanobacterial strains as heterologous production platforms. Herein, we present recent advances in the heterologous production of cyanobacterial compounds in both cyanobacterial and noncyanobacterial hosts. Emerging strategies for BGC assembly, host engineering, and optimization of BGC expression are included for fostering the broader applications of synthetic biology tools in the discovery of new cyanobacterial natural products.

Total Synthesis of the Ambigols: A Cyanobacterial Class of Polyhalogenated Natural Products.

The first total synthesis of all members of the cyanobacterial natural product class of the ambigols is described. Key steps of the synthetic strategy are the formation of sterically demanding mono- and bis-iodonium salts to install the required biaryl ether structural elements and Suzuki cross-coupling giving straightforward access to the biaryl bonds. The synthetic methods are also utilized to construct unnatural or hypothetical ambigols that are still awaiting discovery from Nature.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S.elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S.elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

Discovery, Total Synthesis, and SAR of Anaenamides A and B: Anticancer Cyanobacterial Depsipeptides with a Chlorinated Pharmacophore.

New modified depsipeptides and geometric isomers, termed anaenamides A (1a) and B (1b), along with the presumptive biosynthetic intermediate, anaenoic acid (2), were discovered from a marine cyanobacterium from Guam. Structures were confirmed by total synthesis. The alkylsalicylic acid fragment and the C-terminal α-chlorinated α,β-unsaturated ester are novelties in cyanobacterial natural products. Cancer cell viability assays indicated that the C-terminal unit serves as the pharmacophore and that the double-bond geometry impacts the cytotoxicity.

Marine Cyanobacteria: A Source of Lead Compounds and their Clinically-Relevant Molecular Targets.

The prokaryotic filamentous marine cyanobacteria are photosynthetic microbes that are found in diverse marine habitats, ranging from epiphytic to endolithic communities. Their successful colonization in nature is largely attributed to genetic diversity as well as the production of ecologically important natural products. These cyanobacterial natural products are also a source of potential drug leads for the development of therapeutic agents used in the treatment of diseases, such as cancer, parasitic infections and inflammation. Major sources of these biomedically important natural compounds are found predominately from marine cyanobacterial orders Oscillatoriales, Nostocales, Chroococcales and Synechococcales. Moreover, technological advances in genomic and metabolomics approaches, such as mass spectrometry and NMR spectroscopy, revealed that marine cyanobacteria are a treasure trove of structurally unique natural products. The high potency of a number of natural products are due to their specific interference with validated drug targets, such as proteasomes, proteases, histone deacetylases, microtubules, actin filaments and membrane receptors/channels. In this review, the chemistry and biology of selected potent cyanobacterial compounds as well as their synthetic analogues are presented based on their molecular targets. These molecules are discussed to reflect current research trends in drug discovery from marine cyanobacterial natural products.