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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. 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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4. 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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5. Small molecule inducers of actinobacteria natural product biosynthesis

   https://doi.org/10.1093/jimb/kuad019  

   Alwali, Amir Y.; Parkinson, Elizabeth I. (August 2023, Journal of Industrial Microbiology and Biotechnology)

   Abstract  Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated with many natural products (NPs) being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. Specifically, they can increase titers of known NPs as well as enabling discovery of novel NPs typically produced at undetectable levels. These elicitors can be NPs, including antibiotics or hormones, or synthetic compounds. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of NPs from “silent” biosynthetic gene clusters. This review aims to highlight classes of molecules that induce secondary metabolite production in actinobacteria and to describe the potential mechanisms of induction. One-Sentence SummaryThis review describes chemical elicitors of actinobacteria natural products described to date and the proposed mechanisms of induction. 

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