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1. Bioactivity‐driven fungal metabologenomics identifies antiproliferative stemphone analogs and their biosynthetic gene cluster

   Ayon, Navid J; Earp, Cody E; Gupta, Raveena; Butun, Fatma A; Clements, Ashley E; Lee, Alexa G; Dainko; David; Robey, Matthew T; Khin, Manead; et al (August 2024, Metabolomics)

   Introduction Fungi biosynthesize chemically diverse secondary metabolites with a wide range of biological activities. Natu- ral product scientists have increasingly turned towards bioinformatics approaches, combining metabolomics and genomics to target secondary metabolites and their biosynthetic machinery. We recently applied an integrated metabologenomics workflow to 110 fungi and identified more than 230 high-confidence linkages between metabolites and their biosynthetic pathways. Objectives To prioritize the discovery of bioactive natural products and their biosynthetic pathways from these hundreds of high-confidence linkages, we developed a bioactivity-driven metabologenomics workflow combining quantitative chemical information, antiproliferative bioactivity data, and genome sequences. Methods The 110 fungi from our metabologenomics study were tested against multiple cancer cell lines to identify which strains produced antiproliferative natural products. Three strains were selected for further study, fractionated using flash chromatography, and subjected to an additional round of bioactivity testing and mass spectral analysis. Data were overlaid using biochemometrics analysis to predict active constituents early in the fractionation process following which their bio- synthetic pathways were identified using metabologenomics. Results We isolated three new-to-nature stemphone analogs, 19-acetylstemphones G (1), B (2) and E (3), that demonstrated antiproliferative activity ranging from 3 to 5 μM against human melanoma (MDA-MB-435) and ovarian cancer (OVACR3) cells. We proposed a rational biosynthetic pathway for these compounds, highlighting the potential of using bioactivity as a filter for the analysis of integrated—Omics datasets. Conclusions This work demonstrates how the incorporation of biochemometrics as a third dimension into the metabolog- enomics workflow can identify bioactive metabolites and link them to their biosynthetic machinery. 

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2. New approaches to secondary metabolite discovery from anaerobic gut microbes

   https://doi.org/10.1007/s00253-024-13393-y  

   Butkovich, Lazarina_V; Vining, Oliver_B; O’Malley, Michelle_A (January 2025, Applied Microbiology and Biotechnology)

   AbstractThe animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilizeinsilicotools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism. Key points• Gut microbial secondary metabolites have therapeutic and biotechnological potential• Culture- and (meta)genome-based workflows drive gut anaerobe metabolite discovery• Anaerobic biofoundries enable high-throughput workflows for metabolite discovery Graphical abstract 

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3. Genomic insights into the evolution of secondary metabolism of Escovopsis and its allies, specialized fungal symbionts of fungus-farming ants

   https://doi.org/10.1128/msystems.00576-24  

   Berasategui, Aileen; Salem, Hassan; Moller, Abraham G; Christopher, Yuliana; Vidaurre_Montoya, Quimi; Conn, Caitlin; Read, Timothy D; Rodrigues, Andre; Ziemert, Nadine; Gerardo, Nicole (July 2024, mSystems)

   The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.Microbial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts’ potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts. 

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4. Metabologenomics reveals strain-level genetic and chemical diversity of Microcystis secondary metabolism

   https://doi.org/10.1128/msystems.00334-24  

   Yancey, Colleen E; Hart, Lauren; Hefferan, Sierra; Mohamed, Osama G; Newmister, Sean A; Tripathi, Ashootosh; Sherman, David H; Dick, Gregory J (July 2024, mSystems)

   van_der_Hooft, Justin_J J (Ed.)

   ABSTRACT Microcystisspp. are renowned for producing the hepatotoxin microcystin in freshwater cyanobacterial harmful algal blooms around the world, threatening drinking water supplies and public and environmental health. However,Microcystisgenomes also harbor numerous biosynthetic gene clusters (BGCs) encoding the biosynthesis of other secondary metabolites, including many with toxic properties. Most of these BGCs are uncharacterized and currently lack links to biosynthesis products. However, recent field studies show that many of these BGCs are abundant and transcriptionally active in natural communities, suggesting potentially important yet unknown roles in bloom ecology and water quality. Here, we analyzed 21 xenicMicrocystiscultures isolated from western Lake Erie to investigate the diversity of the biosynthetic potential of this genus. Through metabologenomic andin silicoapproaches, we show that theseMicrocystisstrains contain variable BGCs, previously observed in natural populations, and encode distinct metabolomes across cultures. Additionally, we find that the majority of metabolites and gene clusters are uncharacterized, highlighting our limited understanding of the chemical repertoire ofMicrocystisspp. Due to the complex metabolomes observed in culture, which contain a wealth of diverse congeners as well as unknown metabolites, these results underscore the need to deeply explore and identify secondary metabolites produced byMicrocystisbeyond microcystins to assess their impacts on human and environmental health.IMPORTANCEThe genusMicrocystisforms dense cyanobacterial harmful algal blooms (cyanoHABs) and can produce the toxin microcystin, which has been responsible for drinking water crises around the world. While microcystins are of great concern,Microcystisalso produces an abundance of other secondary metabolites that may be of interest due to their potential for toxicity, ecological importance, or pharmaceutical applications. In this study, we combine genomic and metabolomic approaches to study the genes responsible for the biosynthesis of secondary metabolites as well as the chemical diversity of produced metabolites inMicrocystisstrains from the Western Lake Erie Culture Collection. This unique collection comprisesMicrocystisstrains that were directly isolated from western Lake Erie, which experiences substantial cyanoHAB events annually and has had negative impacts on drinking water, tourism, and industry. 

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5. Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity

   https://doi.org/10.1128/aem.00600-24  

   Harper, Christopher P; Day, Anna; Tsingos, Maya; Ding, Edward; Zeng, Elizabeth; Stumpf, Spencer D; Qi, Yunci; Robinson, Adam; Greif, Jennifer; Blodgett, Joshua AV (May 2024, Applied and Environmental Microbiology)

   Reguera, Gemma (Ed.)

   ABSTRACT Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.<bold>IMPORTANCE</bold>Polycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, includingStreptomycesandLysobacterspp.Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity. 

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