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Bioactivity-guided fractionation (BGF) has historically been a fruitful natural product discovery workflow. However, it is plagued by increasing rediscovery rates in recent years and new methods capable of exploring the natural product chemical space more broadly and more efficiently is in urgent need. Chemical structure metagenomics as one such method is the theme of this Perspective. It emphasizes a chemical-structure-centered viewpoint toward natural product research. Key to chemical structure metagenomics is the ability to predict the structure of a natural product based on its biosynthetic gene sequences, which facilitated the discovery of numerous new bioactive molecules and helped uncover oversampled/underexplored niches of decades of BGF based discovery. While microbial nonribosomal peptides have been the focus of chemical structure metagenomics efforts thus far, it is in principle applicable to other natural product families. The future outlook of this new approach will also be discussed.

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes. [ABSTRACT FROM AUTHOR]

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Marine cyanobacterial bioactives, due to their diverse chemistry and promising pharmacological properties, hold significant potential as therapeutic agents. Here, we comprehensively review the scientific state‐of‐the‐art relating to marine cyanobacterial bioactives, including bioprospecting, preclinical and clinical studies, mechanisms of action, safety and toxicity considerations, sustainability, and conservation. We discuss challenges in and opportunities for developing marine cyanobacterial bioactives as therapeutics; we underscore their potential in treating life‐threatening health ailments, such as cancer and neurodegenerative, inflammatory and viral diseases. We further articulate the significant potential of marine cyanobacterial bioactives for improving human health, and identify future research directions underpinning their development as therapeutics. [ABSTRACT FROM AUTHOR]

Background: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth's surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. Results: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. Conclusions: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity. [ABSTRACT FROM AUTHOR]

Over the last several years, there has been increased interest from academia and the pharmaceutical/biotech industry in the development of vaccine adjuvants for new and emerging vaccine modalities. Despite this, vaccine adjuvant development still has some of the longest timelines in the pharmaceutical space, from discovery to clinical approval. The reasons for this are manyfold and range from complexities in translation from animal to human models, concerns about safety or reactogenicity, to challenges in sourcing the necessary raw materials at scale. In this review, we will describe the current state of the art for many adjuvant technologies and how they should be approached or applied in the development of new vaccine products. We postulate that there are many factors to be considered and tools to be applied earlier on in the vaccine development pipeline to improve the likelihood of clinical success. These recommendations may require a modified approach to some of the common practices in new product development but would result in more accessible and practical adjuvant-containing products. [ABSTRACT FROM AUTHOR]

Abstract Marine cyanobacterial bioactives, due to their diverse chemistry and promising pharmacological properties, hold significant potential as therapeutic agents. Here, we comprehensively review the scientific state‐of‐the‐art relating to marine cyanobacterial bioactives, including bioprospecting, preclinical and clinical studies, mechanisms of action, safety and toxicity considerations, sustainability, and conservation. We discuss challenges in and opportunities for developing marine cyanobacterial bioactives as therapeutics; we underscore their potential in treating life‐threatening health ailments, such as cancer and neurodegenerative, inflammatory and viral diseases. We further articulate the significant potential of marine cyanobacterial bioactives for improving human health, and identify future research directions underpinning their development as therapeutics.

Bioactivity-guided fractionation (BGF) has historically been a fruitful natural product discovery workflow. However, it is plagued by increasing rediscovery rates in recent years and new methods capable of exploring the natural product chemical space more broadly and more efficiently is in urgent need. Chemical structure metagenomics as one such method is the theme of this Perspective. It emphasizes a chemical-structure-centered viewpoint toward natural product research. Key to chemical structure metagenomics is the ability to predict the structure of a natural product based on its biosynthetic gene sequences, which facilitated the discovery of numerous new bioactive molecules and helped uncover oversampled/underexplored niches of decades of BGF based discovery. While microbial nonribosomal peptides have been the focus of chemical structure metagenomics efforts thus far, it is in principle applicable to other natural product families. The future outlook of this new approach will also be discussed., Competing Interests: None to declare., (Copyright © 2024, Ma and Chu.)

Background: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth’s surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. Results: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. Conclusions: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity.

Over the last several years, there has been increased interest from academia and the pharmaceutical/biotech industry in the development of vaccine adjuvants for new and emerging vaccine modalities. Despite this, vaccine adjuvant development still has some of the longest timelines in the pharmaceutical space, from discovery to clinical approval. The reasons for this are manyfold and range from complexities in translation from animal to human models, concerns about safety or reactogenicity, to challenges in sourcing the necessary raw materials at scale. In this review, we will describe the current state of the art for many adjuvant technologies and how they should be approached or applied in the development of new vaccine products. We postulate that there are many factors to be considered and tools to be applied earlier on in the vaccine development pipeline to improve the likelihood of clinical success. These recommendations may require a modified approach to some of the common practices in new product development but would result in more accessible and practical adjuvant-containing products.

To enhance the discovery of novel natural products, various innovations have been developed to aid in the cultivation of previously unculturable microbial species. One approach involving the microencapsulation of bacteria has been gaining popularity as a new cultivation technique, with promising applications. Previous studies demonstrated the success of bacterial encapsulation; however, they highlighted that a key limitation of encapsulating bacteria within agarose is the high temperature required for encapsulation. Encapsulation of bacteria within agarose typically requires a temperature high enough to maintain the flow of agarose through microfluidic devices without premature gelation. Given the sensitivity of many bacterial taxa to temperature, the effect of various agarose-based encapsulating matrices on marine bacterial viability was assessed to further develop this approach to bacterial culture. It was determined that lowering the temperature of encapsulation via the use of low-gelling-temperature agarose, as well as the addition of nutrients to the matrix, significantly improved the viability of representative marine sediment bacteria in terms of abundance and metabolic activity. Based on these findings, the use of low-gelling-temperature agarose with supplemental nutrients is recommended for the encapsulation of marine bacteria obtained from temperate habitats. [ABSTRACT FROM AUTHOR]

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.

Fungi are well known for production of antibiotics and other bioactive secondary metabolites, that can be served as pharmaceuticals, therapeutic agents and industrially useful compounds. However, compared with the characterization of prokaryotic biosynthetic gene clusters (BGCs), less attention has been paid to evaluate fungal BGCs. This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators, as well as removal of intron sequences, and this substantially slow down the workflow in natural product discovery. It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories, such as the yeast Saccharomyces cerevisiae. Here, we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring, followed by outlooking prominent opportunities and challenges for future advancement.

Cycads, which primarily consist of the families Cycadaceae and Zamiaceae, possess intrinsic therapeutic attributes that are prominently expressed across their morphological spectrum, including roots, leaves, flowers, and seeds. In Chinese traditional medicine, the leaves of cycads are particularly revered for their profound healing capabilities. This meticulous review engages with existing literature on cycads and presents insightful avenues for future research. Over 210 phytoconstituents have been isolated and identified from various cycad tissues, including flavonoids, azoxy metabolites, sterols, lignans, non-proteogenic amino acids, terpenoids, and other organic constituents. The contemporary pharmacological discourse highlights the antineoplastic, antimicrobial, and antidiabetic activities inherent in these ancient plants, which are of particular importance to the field of oncology. Despite the prevalent focus on crude extracts and total flavonoid content, our understanding of the nuanced pharmacodynamics of cycads lags considerably behind. The notoriety of cycads derived toxicity, notably within the context of Guam's neurological disease cluster, has precipitated an established emphasis on toxicological research within this field. As such, this critical review emphasizes nascent domains deserving of academic and clinical pursuit, whilst nested within the broader matrix of current scientific understanding. The systematic taxonomy, traditional applications, phytochemical composition, therapeutic potential, and safety profile of cycads are holistically interrogated, assimilating an indispensable repository for future scholarly inquiries. In conclusion, cycads stand as a veritable treasure trove of pharmacological virtue, displaying remarkable therapeutic prowess and holding vast promise for ongoing scientific discovery and clinical utilization. Cycads represent a rich, therapeutic resource, exhibiting exceptional medicinal efficacy and enormous potential for continued exploration and application. [Display omitted] • Up to now, more than 210 phytochemicals have been identified from cycads. • Cycads is a double-edged sword, and its toxicity should not be overlooked. • Cycads has great medical development and application value. [ABSTRACT FROM AUTHOR]

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

To enhance the discovery of novel natural products, various innovations have been developed to aid in the cultivation of previously unculturable microbial species. One approach involving the microencapsulation of bacteria has been gaining popularity as a new cultivation technique, with promising applications. Previous studies demonstrated the success of bacterial encapsulation; however, they highlighted that a key limitation of encapsulating bacteria within agarose is the high temperature required for encapsulation. Encapsulation of bacteria within agarose typically requires a temperature high enough to maintain the flow of agarose through microfluidic devices without premature gelation. Given the sensitivity of many bacterial taxa to temperature, the effect of various agarose-based encapsulating matrices on marine bacterial viability was assessed to further develop this approach to bacterial culture. It was determined that lowering the temperature of encapsulation via the use of low-gelling-temperature agarose, as well as the addition of nutrients to the matrix, significantly improved the viability of representative marine sediment bacteria in terms of abundance and metabolic activity. Based on these findings, the use of low-gelling-temperature agarose with supplemental nutrients is recommended for the encapsulation of marine bacteria obtained from temperate habitats.

Microbial marine natural products hold significant potential for the discovery of new bioactive therapeutics such as antibiotics. Unfortunately, this discovery is hindered by the inability to culture the majority of microbes using traditional laboratory approaches. While many new methods have been developed to increase cultivability, a high‐throughput in situ incubation chamber capable of simultaneously isolating individual microbes while allowing cellular communication has not previously been reported. Development of such a device would expedite the discovery of new microbial taxa and, thus, facilitate access to their associated natural products. In this study, this concept is achieved by the development of a new device termed by the authors as the microbe domestication (MD) Pod. The MD Pod enables single‐cell cultivation by isolating marine bacterial cells in agarose microbeads produced using microfluidics, while allowing potential transmission of chemical signals between cells during in situ incubation in a chamber, or "Pod," that is deployed in the environment. The design of the MD Pod was optimized to ensure the use of biocompatible materials, allow for simple assembly in a field setting, and maintain sterility throughout incubation. The encapsulation process was designed to ensure that the viability of marine sediment bacteria was not adversely impacted by the encapsulation process. The process was validated using representative bacteria isolated from temperate marine sediment samples: Marinomonas polaris, Psychrobacter aquimaris, and Bacillus licheniformis. The overall process appeared to promote metabolic activity of most representative species. Thus, microfluidic encapsulation of marine bacteria and subsequent in situ incubation in the MD Pod is expected to accelerate marine natural products discovery by increasing the cultivability of marine bacteria. [ABSTRACT FROM AUTHOR]

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)‐Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha‐(E)‐Dhb. It displays narrow‐spectrum activity against vancomycin‐resistant Enterococcus faecium. In‐vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria. [ABSTRACT FROM AUTHOR]

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium , a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi. [ABSTRACT FROM AUTHOR]

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.

ABSTRACT Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world’s population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis. Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis. This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis. This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets. IMPORTANCE Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae , carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications. [ABSTRACT FROM AUTHOR]

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces. Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs)were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1–N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of “inducer” microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.

Microorganisms, plants, and animals alike have specialized acquisition pathways for obtaining metals, with microorganisms and plants biosynthesizing and secreting small molecule natural products called siderophores and metallophores with high affinities and specificities for iron or other non-iron metals, respectively. This chapter details a novel approach to discovering metal-binding molecules, including siderophores and metallophores, from complex samples ranging from microbial supernatants to biological tissue to environmental samples. This approach, called Native Metabolomics, is a mass spectrometry method in which pH adjustment and metal infusion post-liquid chromatography are interfaced with ion identity molecular networking (IIMN). This rule-based data analysis workflow that enables the identification of metal-binding species based on defined mass (m/z) offsets with the same chromatographic profiles and retention times. Ion identity molecular networking connects compounds that are structurally similar by their fragmentation pattern and species that are ion adducts of the same compound by chromatographic shape correlations. This approach has previously revealed new insights into metal binding metabolites, including that yersiniabactin can act as a biological zincophore (in addition to its known role as a siderophore), that the recently elucidated lepotchelin natural products are cyanobacterial metallophores, and that antioxidants in traditional medicine bind iron. Native metabolomics can be conducted on any liquid chromatography-mass spectrometry system to explore the binding of any metal or multiple metals simultaneously, underscoring the potential for this method to become an essential strategy for elucidating biological metal-binding molecules., (Copyright © 2024. Published by Elsevier Inc.)

The quest for novel natural products has recently focused on the marine environment as a source for novel microorganisms. Although isolation of marine-derived actinomycete strains is now common, understanding their distribution in the oceans and their adaptation to this environment can be helpful in the selection of isolates for further novel secondary metabolite discovery. This study explores the taxonomic diversity of marine-derived actinomycetes from distinct environments in the coastal areas of the Yucatan Peninsula and their adaptation to the marine environment as a first step towards novel natural product discovery. The use of simple ecological principles, for example, phylogenetic relatedness to previously characterized actinomycetes or seawater requirements for growth, to recognize isolates with adaptations to the ocean in an effort to select for marine-derived actinomycete to be used for further chemical studies. Marine microbial environments are an important source of novel bioactive natural products and, together with methods such as genome mining for detection of strains with biotechnological potential, ecological strategies can bring useful insights in the selection and identification of marine-derived actinomycetes for novel natural product discovery. [ABSTRACT FROM AUTHOR]

2,2′-Bipyridine (2,2′-BP) is the unique molecular scaffold of the bioactive natural products represented by caerulomycins (CAEs) and collismycins (COLs). CAEs and COLs are highly similar in the chemical structures in which their 2,2′-BP cores typically contain a di- or tri-substituted ring A and an unmodified ring B. Here, we summarize the CAE and COL-type 2,2′-BP natural products known or hypothesized to date: (1) isolated using methods traditional for natural product characterization, (2) created by engineering the biosynthetic pathways of CAEs or COLs, and (3) predicted upon bioinformatics-guided genome mining. The identification of these CAE and COL-type 2,2′-BP natural products not only demonstrates the development of research techniques and methods in the field of natural product chemistry but also reflects the general interest in the discovery of CAE and COL-type 2,2′-BP natural products. [ABSTRACT FROM AUTHOR]

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of "inducer" microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS. [ABSTRACT FROM AUTHOR]

Since the onset of microbiology in the late 19th century, scientists have been growing microorganisms almost exclusively as pure cultures, resulting in a limited and biased view of the microbial world. Only a paradigm shift in cultivation techniques – from axenic to mixed cultures – can allow a full comprehension of the (chemical) communication of microorganisms, with profound consequences for natural product discovery, microbial ecology, symbiosis, and pathogenesis, to name a few areas. Three main technical advances during the last decade are fueling the realization of this revolution in microbiology: microfluidics, next-generation 3D-bioprinting, and single-cell metabolomics. These technological advances can be implemented for large-scale, systematic cocultivation studies involving three or more microorganisms. In this review, we present recent trends in microbiology tools and discuss how these can be employed to decode the chemical language that microorganisms use to communicate. [ABSTRACT FROM AUTHOR]

Coinciding with the increase in sequenced bacteria, mining of bacterial genomes for biosynthetic gene clusters (BGCs) has become a critical component of natural product discovery. The order Myxococcales, a reputable source of biologically active secondary metabolites, spans three suborders which all include natural product producing representatives. Utilizing the BiG-SCAPE-CORASON platform to generate a sequence similarity network that contains 994 BGCs from 36 sequenced myxobacteria deposited in the antiSMASH database, a total of 843 BGCs with lower than 75% similarity scores to characterized clusters within the MIBiG database are presented. This survey provides the biosynthetic diversity of these BGCs and an assessment of the predicted chemical space yet to be discovered. Considering the mere snapshot of myxobacteria included in this analysis, these untapped BGCs exemplify the potential for natural product discovery from myxobacteria.

In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents.

Marine-derived actinomycetes have demonstrated an ability to produce novel compounds with medically relevant biological activity. Studying the diversity and biogeographical patterns of marine actinomycetes offers an opportunity to identify genera that are under environmental pressures, which may drive adaptations that yield specific biosynthetic capabilities. The present study describes research efforts to explore regions of the Atlantic Ocean, specifically around the Madeira Archipelago, where knowledge of the indigenous actinomycete diversity is scarce. A total of 400 actinomycetes were isolated, sequenced and screened for antimicrobial and anticancer activities. The three most abundant genera identified were Streptomyces, Actinomadura and Micromonospora. Phylogenetic analyses of the marine OTUs isolated indicated that the Madeira Archipelago is a new source of actinomycetes adapted to life in the ocean. Phylogenetic differences between offshore (>100m from shore) and nearshore (

The biosynthetically well-studied landomycin A cluster has been used to demonstrate the unbalancing of gene transcription as an efficient method for the generation of new compounds. Landomycin A structural genes were decoupled from the native regulators LanI and LanK and placed under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. In contrast to their native quantitative and temporal regulation, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article. [ABSTRACT FROM AUTHOR]

The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products. [ABSTRACT FROM AUTHOR]

Microbial natural products are an evolved resource of bioactive small molecules, which form the foundation of many modern therapeutic regimes. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a class of natural products which have attracted extensive interest for their diverse chemical structures and potent biological activities. Genome sequencing has revealed that the vast majority of genetically encoded natural products remain unknown. Many bioinformatic resources have therefore been developed to predict the chemical structures of natural products, particularly nonribosomal peptides and polyketides, from sequence data. However, the diversity and complexity of RiPPs have challenged systematic investigation of RiPP diversity, and consequently the vast majority of genetically encoded RiPPs remain chemical "dark matter." Here, we introduce an algorithm to catalog RiPP biosynthetic gene clusters and chart genetically encoded RiPP chemical space. A global analysis of 65,421 prokaryotic genomes revealed 30,261 RiPP clusters, encoding 2,231 unique products. We further leverage the structure predictions generated by our algorithm to facilitate the genome-guided discovery of a molecule from a rare family of RiPPs. Our results provide the systematic investigation of RiPP genetic and chemical space, revealing the widespread distribution of RiPP biosynthesis throughout the prokaryotic tree of life, and provide a platform for the targeted discovery of RiPPs based on genome sequencing. [ABSTRACT FROM AUTHOR]

Marine-derived actinomycetes have demonstrated an ability to produce novel compounds with medically relevant biological activity. Studying the diversity and biogeographical patterns of marine actinomycetes offers an opportunity to identify genera that are under environmental pressures, which may drive adaptations that yield specific biosynthetic capabilities. The present study describes research efforts to explore regions of the Atlantic Ocean, specifically around the Madeira Archipelago, where knowledge of the indigenous actinomycete diversity is scarce. A total of 400 actinomycetes were isolated, sequenced, and screened for antimicrobial and anticancer activities. The three most abundant genera identified were Streptomyces, Actinomadura, and Micromonospora. Phylogenetic analyses of the marine OTUs isolated indicated that the Madeira Archipelago is a new source of actinomycetes adapted to life in the ocean. Phylogenetic differences between offshore (>100m from shore) and nearshore (<100m from shore) populations illustrates the importance of sampling offshore in order to isolate new and diverse bacterial strains. Novel phylotypes from chemically rich marine actinomycete groups like MAR4 and the genus Salinispora were isolated. Anticancer and antimicrobial assays identified Streptomyces, Micromonospora, and Salinispora as the most biologically active genera. This study illustrates the importance of bioprospecting efforts at unexplored regions of the ocean to recover bacterial strains with the potential to produce novel and interesting chemistry. [ABSTRACT FROM AUTHOR]

Background: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Results: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. Conclusion: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery. [ABSTRACT FROM AUTHOR]