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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 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.

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.

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.

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.

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.

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.

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.

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 (