An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Gene Co-expression Network Analysis to Mine Genes Involved in Plant Secondary Metabolite Biosynthesis and Regulation.

Plants produce diverse specialized metabolites (SMs) that do not participate in plant growth and development but help them adapt to various environmental conditions. In addition to aiding in plant adaptation, different SMs serve as active ingredients for pharmaceutical and cosmetics products. However, despite their significant role in plant adaptation and industrial importance, the genes involved in the biosynthesis and regulation of many SMs remain largely unknown. This hinders deciphering the specific role of SMs in plant adaptation and limits their industrial utilization. Since many SMs pathway genes are expected to act in tight association with each other within a coexpression network, the network biology approach, such as weighted gene coexpression network analysis, could be used to identify the unknown genes. This chapter describes a workflow for constructing a gene coexpression network to identify genes that could be associated with the biosynthesis and regulation of SMs.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Transcriptional regulators of secondary metabolite biosynthesis in Streptomyces.

In the post-genome era, great progress has been made in metabolic engineering using recombinant DNA technology to enhance the production of high-value products by Streptomyces. With the development of microbial genome sequencing techniques and bioinformatic tools, a growing number of secondary metabolite (SM) biosynthetic gene clusters in Streptomyces and their biosynthetic logics have been uncovered and elucidated. In order to increase our knowledge about transcriptional regulators in SM of Streptomyces, this review firstly makes a comprehensive summary of the characterized factors involved in enhancing SM production and awakening SM biosynthesis. Future perspectives on transcriptional regulator engineering for new SM biosynthesis by Streptomyces are also provided.

Molecular mechanism of miRNA mediated biosynthesis of secondary metabolites in medicinal plants.

Plant secondary metabolites are important raw materials for the pharmaceutical industry, and their biosynthetic processes are subject to diverse and precise regulation by miRNA. The identification of miRNA molecules in medicinal plants and exploration of their mechanisms not only contribute to a deeper understanding of the molecular genetic mechanisms of plant growth, development and resistance to stress, but also provide a theoretical basis for elucidating the pharmacological effects of authentic medicinal materials and constructing bioreactors for the synthesis of medicinal secondary metabolite components. This paper summarizes the research reports on the discovery of miRNA in medicinal plants and their regulatory mechanisms on the synthesis of secondary metabolites by searching the relevant literature in public databases. It summarizes the currently discovered miRNA and their functions in medicinal plants, and summarizes the molecular mechanisms regulating the synthesis and degradation of secondary metabolites. Furthermore, it provides a prospect for the research and development of medicinal plant miRNA. The compiled information contributes to a comprehensive understanding of the research progress on miRNA in medicinal plants and provides a reference for the industrial development of related secondary metabolite biosynthesis.

FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

Streptomyces sungeiensis SD3 as a Microbial Chassis for the Heterologous Production of Secondary Metabolites.

We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1. The unique placement of S. sungeiensis SD3 in the phylogenetic tree designates it as a type strain, setting it apart from other frequently employed Streptomyces chassis. This distinction makes it the preferred chassis for expressing biosynthetic gene clusters (BGCs) derived from strains within the same phylogenetic or neighboring phylogenetic clade. The successful expression of secondary biosynthetic pathways from a closely related yet slow-growing strain underscores the utility of S. sungeiensis SD3 as a heterologous expression chassis. Validation of CRISPR/Cas9-assisted genetic tools for chromosomal deletion and insertion paved the way for further strain improvement and BGC refactoring through rational genome editing. The addition of S. sungeiensis SD3 to the heterologous chassis toolkit will facilitate the discovery and production of secondary metabolites.

OSMAC Strategy: A promising way to explore microbial cyclic peptides.

Microbial secondary metabolites are pivotal for the development of novel drugs. However, conventional culture techniques, have left a vast array of unexpressed biosynthetic gene clusters (BGCs) in microorganisms, hindering the discovery of metabolites with distinct structural features and diverse biological functions. To address this limitation, several innovative strategies have been emerged. The "One Strain Many Compounds" (OSMAC) strategy, which involves altering microbial culture conditions, has proven to be particularly effective in mining numerous novel secondary metabolites for the past few years. Among these, microbial cyclic peptides stand out. These peptides often comprise rare amino acids, unique chemical structures, and remarkable biological function. With the advancement of the OSMAC strategy, a plethora of new cyclic peptides have been identified from diverse microbial genera. This work reviews the progress in mining novel compounds using the OSMAC strategy and the applications of this strategy in discovering 284 microbial cyclic peptides from 63 endophytic strains, aiming to offer insights for the further explorations into novel active cyclic peptides.

AwSclB regulates a network for Aspergillus westerdijkiae asexual sporulation and secondary metabolism independent of the fungal light control.

As a prevalent pathogenic fungus, Aspergillus westerdijkiae poses a threat to both food safety and human health. The fungal growth, conidia production and ochratoxin A (OTA) in A. weterdijkiae are regulated by many factors especially transcription factors. In this study, a transcription factor AwSclB in A. westerdijkiae was identified and its function in asexual sporulation and OTA biosynthesis was investigated. In addition, the effect of light control on AwSclB regulation was also tested. The deletion of AwSclB gene could reduce conidia production by down-regulation of conidia genes and increase OTA biosynthesis by up-regulation of cluster genes, regardless under light or dark conditions. It is worth to note that the inhibitory effect of light on OTA biosynthesis was reversed by the knockout of AwSclB gene. The yeast one-hybrid assay indicated that AwSclB could interact with the promoters of BrlA, ConJ and OtaR1 genes. This result suggests that AwSclB in A. westerdijkiae can directly regulate asexual conidia formation by activating the central developmental pathway BrlA-AbaA-WetA through up-regulating the expression of AwBrlA, and promote the light response of the strain by activating ConJ. However, AwSclB itself is unable to respond to light regulation. This finding will deepen our understanding of the molecular regulation of A. westerdijkiae development and secondary metabolism, and provide potential targets for the development of new fungicides.

Discovering the secondary metabolic potential of Saccharothrix.

Rare actinomycetes are highly valued as potential sources of novel bioactive secondary metabolites. Among these rare actinomycetes, the genus Saccharothrix is particularly noteworthy due to its ability to produce a diverse range of bioactive secondary metabolites. With the continuous sequencing of bacterial genomes and the rapid development of bioinformatics technologies, our knowledge of the secondary metabolic potential of Saccharothrix can become more comprehensive, but this space has not been reviewed or explored. This review presents a detailed overview of the chemical structures and bioactivities of 138 Saccharothrix-derived secondary metabolites, which are classified into five distinct groups based on their biosynthetic pathways. Furthermore, we delve into experimentally characterized biosynthetic pathways of nine bioactive metabolites. By utilizing a combination of cheminformatic and bioinformatic approaches, we attempted to establish connections between the metabolite families and the biosynthetic gene cluster families encoded by Saccharothrix strains. Our analysis provides a comprehensive perspective on the secondary metabolites that can be linked to corresponding BGCs and highlights the underexplored biosynthetic potential of Saccharothrix. This review also provides guidance for the targeted discovery and biosynthesis of novel natural products from Saccharothrix.

Complexity of fungal polyketide biosynthesis and function.

Where does one draw the line between primary and secondary metabolism? The answer depends on the perspective. Microbial secondary metabolites (SMs) were at first believed not to be very important for the producers because they are dispensable for growth under laboratory conditions. However, such compounds become important in natural niches of the organisms, and some are of prime importance for humanity. Polyketides are an important group of SMs with aflatoxin as a well-known and well-characterized example. In Aspergillus spp., all 34 afl genes encoding the enzymes for aflatoxin biosynthesis are located in close vicinity on chromosome III in a so-called gene cluster. This led to the assumption that most genes required for polyketide biosynthesis are organized in gene clusters. Recent research, however, revealed an enormous complexity of the biosynthesis of different polyketides, ranging from individual polyketide synthases to a gene cluster producing several compounds, or to several clusters with additional genes scattered in the genome for the production of one compound. Research of the last decade furthermore revealed a huge potential for SM biosynthesis hidden in fungal genomes, and methods were developed to wake up such sleeping genes. The analysis of organismic interactions starts to reveal some of the ecological functions of polyketides for the producing fungi.

Fungal secondary metabolism is governed by an RNA-binding protein CsdA/RsdA complex.

Production of secondary metabolites is controlled by a complicated regulatory network in eukaryotic cells. Several layers of regulators are involved in this process, ranging from pathway-specific regulation, to epigenetic control, to global regulation. Here, we discover that interaction of an RNA-binding protein CsdA with a regulator RsdA coordinates fungal secondary metabolism. Employing a genetic deletion approach and transcriptome analysis as well as metabolomics analysis, we reveal that CsdA and RsdA synergistically regulate fungal secondary metabolism comprehensively. Mechanistically, comprehensive genetic and biochemical studies prove that RsdA and CsdA co-localize in the nucleus and physically interact to achieve their functions. In particular, we demonstrate that CsdA mediates rsdA expression by binding specific motif "GUCGGUAU" of its pre-mRNA at a post-transcriptional level. We thus uncover a mechanism in which RNA-binding protein physically interacts with, and controls the expression level of, the RsdA to coordinate fungal secondary metabolism.

The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

Induction of Fungal Secondary Metabolites by Co-Culture with Actinomycete Producing HDAC Inhibitor Trichostatins.

A recently bioinformatic analysis of genomic sequences of fungi indicated that fungi are able to produce more secondary metabolites than expected. Despite their potency, many biosynthetic pathways are silent in the absence of specific culture conditions or chemical cues. To access cryptic metabolism, 108 fungal strains isolated from various sites were cultured with or without Streptomyces sp. 13F051 which mainly produces trichostatin analogues, followed by comparison of metabolic profiles using LC-MS. Among the 108 fungal strains, 14 produced secondary metabolites that were not recognized or were scarcely produced in mono-cultivation. Of these two fungal strains, Myrmecridium schulzeri 15F098 and Scleroconidioma sphagnicola 15S058 produced four new compounds (1-4) along with a known compound (5), demonstrating that all four compounds were produced by physical interaction with Streptomyces sp. 13F051. Bioactivity evaluation indicated that compounds 3-5 impede migration of MDA-MB-231 breast cancer cells.

Fungal BGCs for Production of Secondary Metabolites: Main Types, Central Roles in Strain Improvement, and Regulation According to the Piano Principle.

Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, and immunosuppressants. A single fungus species in response to various signals can produce 100 or more secondary metabolites. Such signaling is possible due to the coordinated regulation of several dozen biosynthetic gene clusters (BGCs), which are mosaically localized in different regions of fungal chromosomes. Their regulation includes several levels, from pathway-specific regulators, whose genes are localized inside BGCs, to global regulators of the cell (taking into account changes in pH, carbon consumption, etc.) and global regulators of secondary metabolism (affecting epigenetic changes driven by velvet family proteins, LaeA, etc.). In addition, various low-molecular-weight substances can have a mediating effect on such regulatory processes. This review is devoted to a critical analysis of the available data on the "turning on" and "off" of the biosynthesis of secondary metabolites in response to signals in filamentous fungi. To describe the ongoing processes, the model of "piano regulation" is proposed, whereby pressing a certain key (signal) leads to the extraction of a certain sound from the "musical instrument of the fungus cell", which is expressed in the production of a specific secondary metabolite.

Integration of Transcriptomic and Metabolomic Profiles Provides Insights into the Influence of Nitrogen on Secondary Metabolism in Fusarium sacchari.

Nitrogen availability might play an essential role in plant diseases by enhancing fungal cell growth and influencing the expression of genes required for successful pathogenesis. Nitrogen availability could modulate secondary metabolic pathways as evidenced by the significant differential expression of several core genes involved in mycotoxin biosynthesis and genes encoding polyketide synthase/nonribosomal peptide synthetases, cytochrome P450 and carbohydrate-active enzymes in Fusarium sacchari, grown on different nitrogen sources. A combined analysis was carried out on the transcript and metabolite profiles of regulatory metabolic processes and the virulence of Fusarium sacchari grown on various nitrogen sources. The nitrogen regulation of the gibberellin gene cluster included the metabolic flux and multiple steps of gibberellin synthesis. UHPLC-MS/MS-based metabolome analysis revealed the coordination of these related transcripts and the accumulation of gibberellin metabolites. This integrated analysis allowed us to uncover additional information for a more comprehensive understanding of biological events relevant to fungal secondary metabolic regulation in response to nitrogen availability.

Global analyses of biosynthetic gene clusters in phytobiomes reveal strong phylogenetic conservation of terpenes and aryl polyenes.

There are gaps in our understandings on how did the evolutionary relationships among members of the phytobiomes shape their ability to produce tremendously complex specialized metabolites under the influence of plant host. To determine these relationships, we investigated the phylogenetic conservation of biosynthetic gene clusters (BGCs) on a global collection of 4,519 high-quality and nonredundant (out of 12,181) bacterial isolates and metagenome-assembled genomes from 47 different plant hosts and soil, by adopting three independent phylogenomic approaches (D-test, Pagel's λ, and consenTRAIT). We report that the BGCs are phylogenetically conserved to varying strengths and depths in their different classes. We show that the ability to produce specialized metabolites qualifies as a complex trait, and the depth of conservation is equivalent to ecologically relevant complex microbial traits. Interestingly, terpene and aryl polyene BGCs had the strongest phylogenetic conservation in the phytobiomes, but not in the soil microbiomes. Furthermore, we showed that terpenes are largely uncharacterized in phytobiomes and pinpointed specific clades that harbor potentially novel terpenes. Taken together, this study sheds light on the evolution of specialized metabolites' biosynthesis potential in phytobiomes under the influence of plant hosts and presents strategies to rationally guide the discovery of potentially novel classes of metabolites. IMPORTANCE This study expands our understandings of the biosynthetic potential of phytobiomes by using such worldwide and extensive collection of microbiomes from plants and soil. Apart from providing such vital resource for the plant microbiome researchers, this study provides fundamental insights into the evolution of biosynthetic gene clusters (BGCs) in phytobiomes under the influence of plant host. Specifically, we report that the strength of phylogenetic conservation in microbiomes varies for different classes of BGCs and is influenced as a result of plant host association. Furthermore, our results indicate that biosynthetic potential of specialized metabolites is deeply conserved equivalent to other complex and ecologically relevant microbial traits. Finally, for the most conserved class of specialized metabolites (terpenes), we identified clades harboring potentially novel class of molecules. Future studies could focus on plant-microbe coevolution and interactions through specialized metabolites building upon these findings.

Complete genomes and comparative analyses of Streptomyces phages that influence secondary metabolism and sporulation.

Bacteria in the genus Streptomyces are found ubiquitously in nature and are known for the number and diversity of specialized metabolites they produce, as well as their complex developmental lifecycle. Studies of the viruses that prey on Streptomyces, known as phages, have aided the development of tools for genetic manipulation of these bacteria, as well as contributing to a deeper understanding of Streptomyces and their behaviours in the environment. Here, we present the genomic and biological characterization of twelve Streptomyces phages. Genome analyses reveal that these phages are closely related genetically, while experimental approaches show that they have broad overlapping host ranges, infect early in the Streptomyces lifecycle, and induce secondary metabolite production and sporulation in some Streptomyces species. This work expands the group of characterized Streptomyces phages and improves our understanding of Streptomyces phage-host dynamics.

Global analysis of the biosynthetic chemical space of marine prokaryotes.

BACKGROUND: Marine prokaryotes are a rich source of novel bioactive secondary metabolites for drug discovery. Recent genome mining studies have revealed their great potential to bio-synthesize novel secondary metabolites. However, the exact biosynthetic chemical space encoded by the marine prokaryotes has yet to be systematically evaluated. RESULTS: We first investigated the secondary metabolic potential of marine prokaryotes by analyzing the diversity and novelty of the biosynthetic gene clusters (BGCs) in 7541 prokaryotic genomes from cultivated and single cells, along with 26,363 newly assembled medium-to-high-quality genomes from marine environmental samples. To quantitatively evaluate the unexplored biosynthetic chemical space of marine prokaryotes, the clustering thresholds for constructing the biosynthetic gene cluster and molecular networks were optimized to reach a similar level of the chemical similarity between the gene cluster family (GCF)-encoded metabolites and molecular family (MF) scaffolds using the MIBiG database. The global genome mining analysis demonstrated that the predicted 70,011 BGCs were organized into 24,536 mostly new (99.5%) GCFs, while the reported marine prokaryotic natural products were only classified into 778 MFs at the optimized clustering thresholds. The number of MF scaffolds is only 3.2% of the number of GCF-encoded scaffolds, suggesting that at least 96.8% of the secondary metabolic potential in marine prokaryotes is untapped. The unexplored biosynthetic chemical space of marine prokaryotes was illustrated by the 88 potential novel antimicrobial peptides encoded by ribosomally synthesized and post-translationally modified peptide BGCs. Furthermore, a sea-water-derived Aquimarina strain was selected to illustrate the diverse biosynthetic chemical space through untargeted metabolomics and genomics approaches, which identified the potential biosynthetic pathways of a group of novel polyketides and two known compounds (didemnilactone B and macrolactin A 15-ketone). CONCLUSIONS: The present bioinformatics and cheminformatics analyses highlight the promising potential to explore the biosynthetic chemical diversity of marine prokaryotes and provide valuable knowledge for the targeted discovery and biosynthesis of novel marine prokaryotic natural products. Video Abstract.

Global Map of Specialized Metabolites Encoded in Prokaryotic Plasmids.

Plasmids are the main mobile elements responsible for horizontal gene transfer (HGT) in microorganisms. These replicons extend the metabolic spectrum of their host cells by carrying functional genes. However, it is still unknown to what extent plasmids carry biosynthetic gene clusters (BGCs) related to the production of secondary or specialized metabolites (SMs). Here, we analyzed 9,183 microbial plasmids to unveil their potential to produce SMs, finding a large diversity of cryptic BGCs in a few varieties of prokaryotic host taxa. Some of these plasmids harbored 15 or more BGCs, and many others were exclusively dedicated to mobilizing BGCs. We found an occurrence pattern of BGCs within groups of homologous plasmids shared by a common taxon, mainly in host-associated microbes (e.g., Rhizobiales, Enterobacteriaceae members). Our results add to the knowledge of the ecological functions and potential industrial uses of plasmids and shed light on the dynamics and evolution of SMs in prokaryotes. IMPORTANCE Plasmids are mobile DNA elements that can be shared among microbial cells, and they are useful for bringing to fruition some microbial ecological traits. However, it is not known to what extent plasmids harbor genes related to the production of specialized/secondary metabolites (SMs). In microbes, these metabolites are frequently useful for defense purposes, signaling, etc. In addition, these molecules usually have biotechnological and clinical applications. Here, we analyzed the content, dynamics, and evolution of genes related to the production of SMs in >9,000 microbial plasmids. Our results confirm that some plasmids act as a reservoir of SMs. We also found that some families of biosynthetic gene clusters are exclusively present in some groups of plasmids shared among closely related microbes. Host-associated bacteria (e.g., plant and human microbes) harbor the majority of specialized metabolites encoded in plasmids. These results provide new knowledge about microbial ecological traits and might enable the discovery of novel metabolites.