Harnessing regulatory networks in Actinobacteria for natural product discovery.

Microbes typically live in complex habitats where they need to rapidly adapt to continuously changing growth conditions. To do so, they produce an astonishing array of natural products with diverse structures and functions. Actinobacteria stand out for their prolific production of bioactive molecules, including antibiotics, anticancer agents, antifungals, and immunosuppressants. Attention has been directed especially towards the identification of the compounds they produce and the mining of the large diversity of biosynthetic gene clusters (BGCs) in their genomes. However, the current return on investment in random screening for bioactive compounds is low, while it is hard to predict which of the millions of BGCs should be prioritized. Moreover, many of the BGCs for yet undiscovered natural products are silent or cryptic under laboratory growth conditions. To identify ways to prioritize and activate these BGCs, knowledge regarding the way their expression is controlled is crucial. Intricate regulatory networks control global gene expression in Actinobacteria, governed by a staggering number of up to 1000 transcription factors per strain. This review highlights recent advances in experimental and computational methods for characterizing and predicting transcription factor binding sites and their applications to guide natural product discovery. We propose that regulation-guided genome mining approaches will open new avenues toward eliciting the expression of BGCs, as well as prioritizing subsets of BGCs for expression using synthetic biology approaches. ONE-SENTENCE SUMMARY: This review provides insights into advances in experimental and computational methods aimed at predicting transcription factor binding sites and their applications to guide natural product discovery.

Malignance-restriction activity exhibited by bioactive compounds of selected actinobacteria as silver nanoparticles against A549 lung cancer cell lines.

This article deals with the antibacterial and anticancer potential of secondary metabolites produced by actinomycetes also reported as actinobacteria, Microbacterium proteolyticum (MN560041), and Streptomycetes rochei, where preliminary studies were done with the well diffusion method. These actinobacteria's silver nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and UV-Visible spectroscopy. Anticancer was measured using the MTT test, reactive oxygen species (ROS) generation measured with DCFDA, mitochondrial membrane potential (MMP) measurement, and DAPI fluorescence intensity activity was measured in treated and non-treated cancerous cells. The IC50 value for 5-FU (a), LA2(O) (b), LA2(R) (c), LA2(ON) (d), and LA2(RN) (e) was obtained at 3.91 µg/mL (52.73% cell viability), 56.12 µg/mL (52.35% cell viability), 44.90 µg/mL (52.3% cell viability), 3.45 µg/mL (50.25% cell viability), and 8.05 µg/mL (48.72% cell viability), respectively. TEM micrographs revealed discrete, well-separated AgNPs particles of size 7.88 ± 2 to 12.86 ± 0.24 nm. Gas chromatography-mass spectrometry was also performed to detect the compounds in bioactive metabolites where n-hexadecanoic acid was obtained as the most significant one. MTT test showed a substantial decline in A549 cell viability (up to 48.72%), 2.75-fold increase in ROS generation was noticed in comparison to untreated A549 lung cancer cells when measured with DCFDA. A total of 0.31-fold decrease in MMP and 1.74-fold increase in DAPI fluorescence intensity compared to untreated A549 lung cancer cells suggests that the synthesized nanoparticles promote apoptosis in cancerous cells. Our findings suggests that the secondary metabolites of M. proteolyticum and S. rochei in nanoparticle form can be used as a significant compound against lung cancers.

Discovery of sesquiterpenoids from an actinomycete Crossiella cryophila through genome mining and heterologous expression.

Genome mining of the Actinomycete Crossiella cryophila facilitated the discovery of a minimal terpenoid biosynthetic gene cluster of cry consisting of a class I terpene cyclase CryA and a CYP450 monooxygenase CryB. Heterologous expression of cry allowed the isolation and characterization of two new sesquiterpenoids, ent-viridiflorol (1) and cryophilain (2). Notably, cryophilain (2) possesses a 5/7/3-fused tricyclic skeleton bearing a distinctive bridgehead hydroxy group. The combined in vivo and in vitro experiments revealed that CryA, the first ent-viridiflorol terpene cyclase, catalyzes farnesyl diphosphate to form the 5/7/3 sesquiterpene core scaffold and P450 CryB serves as a tailoring enzyme responsible for installing a hydroxy group at the bridgehead carbon.

The actinomycete Kitasatospora sp. SeTe27, subjected to adaptive laboratory evolution (ALE) in the presence of selenite, varies its cellular morphology, redox stability, and tolerance to the toxic oxyanion.

The effects of oxyanions selenite (SeO32-) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized.

Genomic insights and anti-phytopathogenic potential of siderophore metabolome of endolithic Nocardia mangyaensis NH1.

Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among actinobacteria, Nocardia is one of the most important agricultural and industrial bacteria. Screening and isolation of Nocardia related bacteria from extreme habitats such as endolithic environments are beneficial for practical applications in agricultural and environmental biotechnology. In this work, bioinformatics analysis revealed that a novel strain Nocardia mangyaensis NH1 has the capacity to produce structurally varied bioactive compounds, which encoded by non-ribosomal peptide synthases (NRPS), polyketide synthase (PKS), and post-translationally modified peptides (RiPPs). Among NRPS, five gene clusters have a sequence homology with clusters encoding for siderophore synthesis. We also show that N. mangyaensis NH1 accumulates both catechol- and hydroxamate-type siderophores simultaneously under iron-deficient conditions. Untargeted LC-MS/MS analysis revealed a variety of metabolites, including siderophores, lipopeptides, cyclic peptides, and indole-3-acetic acid (IAA) in the culture medium of N. mangyaensis NH1 grown under iron deficiency. We demonstrate that four CAS (chrome azurol S)-positive fractions display variable affinity to metals, with a high Fe3+ chelating capability. Additionally, three of these fractions exhibit antioxidant activity. A combination of iron scavenging metabolites produced by N. mangyaensis NH1 showed antifungal activity against several plant pathogenic fungi. We have shown that the pure culture of N. mangyaensis NH1 and its metabolites have no adverse impact on Arabidopsis seedlings. The ability of N. mangyaensis NH1 to produce siderophores with antifungal, metal-chelating, and antioxidant properties, when supplemented with phytohormones, has the potential to improve the release of macro- and micronutrients, increase soil fertility, promote plant growth and development, and enable the production of biofertilizers across diverse soil systems.

Effect of Precursors and Their Regulators on the Biosynthesis of Antibiotics in Actinomycetes.

During the life activities of microorganisms, a variety of secondary metabolites are produced, including antimicrobials and antitumor drugs, which are widely used in clinical practice. In addition to exploring new antibiotics, this makes it one of the research priorities of Actinomycetes to effectively increase the yield of antibiotics in production strains by various means. Most antibiotic-producing strains have a variety of functional regulatory factors that regulate their growth, development, and secondary metabolite biosynthesis processes. Through the study of precursor substances in antibiotic biosynthesis, researchers have revealed the precursor biosynthesis process and the mechanism by which precursor synthesis regulators affect the biosynthesis of secondary metabolites, which can be used to obtain engineered strains with high antibiotic production. This paper summarizes the supply of antibiotic biosynthesis precursors and the progress of research on the role of regulators in the process of precursors in biosynthesis. This lays the foundation for the establishment of effective breeding methods to improve antibiotic yields through the manipulation of precursor synthesis genes and related regulators.

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Screening biosurfactant-producing actinomycetes: Identification of Streptomyces sp. RP1 as a potent species for bioremediation.

This study aimed to isolate biosurfactant-producing and hydrocarbon-degrading actinomycetes from different soils using glycerol-asparagine and starch-casein media with an antifungal agent. The glycerol-asparagine agar exhibited the highest number of actinomycetes, with a white, low-opacity medium supporting pigment production and high growth. Biosurfactant analyses, such as drop collapse, oil displacement, emulsification, tributyrin agar test, and surface tension measurement, were conducted. Out of 25 positive isolates, seven could utilize both olive oil and black oil for biosurfactant production, and only isolate RP1 could produce biosurfactant when grown in constrained conditions with black oil as the sole carbon source and inducer, demonstrating in situ bioremediation potential. Isolate RP1 from oil-spilled garden soil is Gram-staining-positive with a distinct earthy odor, melanin formation, and white filamentous colonies. It has a molecular size of ~621 bp and 100% sequence similarity to many Streptomyces spp. Morphological, biochemical, and 16 S rRNA analysis confirmed it as Streptomyces sp. RP1, showing positive results in all screenings, including high emulsification activity against kerosene (27.2%) and engine oil (95.8%), oil displacement efficiency against crude oil (7.45 cm), and a significant reduction in surface tension (56.7 dynes/cm). Streptomyces sp. RP1 can utilize citrate as a carbon source, tolerate sodium chloride, resist lysozyme, degrade petroleum hydrocarbons, and produce biosurfactant at 37°C in a 15 mL medium culture, indicating great potential for bioremediation and various downstream industrial applications with optimization.

Actinobacterial chalkophores: the biosynthesis of hazimycins.

Copper is a transition metal element with significant effects on the morphological development and secondary metabolism of actinobacteria. In some microorganisms, copper-binding natural products are employed to modulate copper homeostasis, although their significance in actinobacteria remains largely unknown. Here, we identified the biosynthetic genes of the diisocyanide natural product hazimycin in Kitasatospora purpeofusca HV058, through gene knock-out and heterologous expression. Biochemical analyses revealed that hazimycin A specifically binds to copper, which diminishes its antimicrobial activity. The presence of a set of putative importer/exporter genes surrounding the biosynthetic genes suggested that hazimycin is a chalkophore that modulates the intracellular copper level. A bioinformatic survey of homologous gene cassettes, as well as the identification of two previously unknown hazimycin-producing Streptomyces strains, indicated that the isocyanide-based mechanism of copper homeostasis is prevalent in actinobacteria.

Effects of effective microorganisms on the physiological status, intestinal microbiome, and serum metabolites of Eriocheir sinensis / Efectos de microorganismos eficaces sobre el estado fisiológico, el microbioma intestinal y los metabolitos séricos de Eriocheir sinensis

The compound known as effective microorganisms (EMs) is widely used in aquaculture to improve water quality, but how they affect the health of Chinese mitten crab (Eriocheir sinensis) is unclear, especially in terms of intestinal microbiota and serum metabolites. In this study, we fed juvenile crabs with an EM-containing diet to explore the effects of EM on the physiological status, intestinal microbiome, and metabolites of E. sinensis. The activities of alanine aminotransferase and alkaline phosphatase were significantly enhanced by EM, indicating that EM supplementation effectively enhanced the antioxidant capacity of E. sinensis. Proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, and Actinobacteria were the main intestinal microbes in both the control and EM groups. Linear discriminant effect size analysis showed that Fusobacteriaceae, Desulfovibrio, and Morganella were biomarkers in the control group, and Exiguobacterium and Rhodobacteraceae were biomarkers in the EM group. Metabolomics analysis revealed that EM supplementation increased cellular energy sources and decreased protein consumption, and oxidative stress. Together, these results indicate that EM can optimize the intestinal microbiome and serum metabolites, thereby benefiting the health of E. sinensis.(AU)

Promoter engineering of natural product biosynthetic gene clusters in actinomycetes: concepts and applications.

Covering 2011 to 2022Low titers of natural products in laboratory culture or fermentation conditions have been one of the challenging issues in natural products research. Many natural product biosynthetic gene clusters (BGCs) are also transcriptionally silent in laboratory culture conditions, making it challenging to characterize the structures and activities of their metabolites. Promoter engineering offers a potential solution to this problem by providing tools for transcriptional activation or optimization of biosynthetic genes. In this review, we summarize the 10 years of progress in promoter engineering approaches in natural products research focusing on the most metabolically talented group of bacteria actinomycetes.

Marine Streptomyces-Derived Novel Alkaloids Discovered in the Past Decade.

Natural alkaloids originating from actinomycetes and synthetic derivatives have always been among the important suppliers of small-molecule drugs. Among their biological sources, Streptomyces is the highest and most extensively researched genus. Marine-derived Streptomyces strains harbor unconventional metabolic pathways and have been demonstrated to be efficient producers of biologically active alkaloids; more than 60% of these compounds exhibit valuable activity such as antibacterial, antitumor, anti-inflammatory activities. This review comprehensively summarizes novel alkaloids produced by marine Streptomyces discovered in the past decade, focusing on their structural features, biological activity, and pharmacological mechanisms. Future perspectives on the discovery and development of novel alkaloids from marine Streptomyces are also provided.

Marine actinomycete Streptomyces variabilis S26 as a biocontrol agent for vibriosis in shrimp larval rearing systems.

Indiscriminate use of antibiotics has led to the emergence of antibiotic-resistant microbes and the loss of natural flora in aquaculture systems necessitating the ban of many of these chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine-derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16 S ribosomal RNA (rRNA) gene sequencing was then checked for the production of antibiotic in five different fermentation media and the one which showed maximum production was chosen for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was highly significant for the response concerned that is, antimicrobial activity as evident from the Fisher's F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38%; soy powder, 0.88%; ammonium sulfate, 0.16% and salinity, 27.76‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in vibrios and conferred better protection to P. monodon in culture system compared with control.

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.

Chemistry and biology of specialized metabolites produced by Actinomadura.

Covering: up to the end of 2022In recent years rare Actinobacteria have become increasingly recognised as a rich source of novel bioactive metabolites. Actinomadura are Gram-positive bacteria that occupy a wide range of ecological niches. This review highlights about 230 secondary metabolites produced by Actinomadura spp., reported until the end of 2022, including their bioactivities and selected biosynthetic pathways. Notably, the bioactive compounds produced by Actinomadura spp. demonstrate a wide range of activities, including antimicrobial, antitumor and anticoccidial effects, highlighting their potential in various fields.

Aerobic Denitrification Promoting by Actinomycetes Coculture: Investigating Performance, Carbon Source Metabolic Characteristic, and Raw Water Restoration.

The coculture theory that promotes denitrification relies on effectively utilizing the resources of low-efficiency denitrification microbes. Here, the strains Streptomyces sp. PYX97 and Streptomyces sp. TSJ96 were isolated and showed lower denitrification capacity when cultured individually. However, the coculture of strains PYX97 and TSJ96 enhanced nitrogen removal (removed 96.40% of total nitrogen) and organic carbon reduction (removed 92.13% of dissolved organic carbon) under aerobic conditions. Nitrogen balance analysis indicated that coculturing enhanced the efficiency of nitrate converted into gaseous nitrogen reaching 70.42%. Meanwhile, the coculturing promoted the cell metabolism capacity and carbon source metabolic activity. The coculture strains PYX97 and TSJ96 thrived in conditions of C/N = 10, alkalescence, and 150 rpm shaking speed. The coculturing reduced total nitrogen and CODMn in the raw water treatment by 83.32 and 84.21%, respectively. During this treatment, the cell metabolic activity and cell density increased in the coculture strains PYX97 and TSJ96 reactor. Moreover, the coculture strains could utilize aromatic protein and soluble microbial products during aerobic denitrification processes in raw water treatment. This study suggests that coculturing inefficient actinomycete strains could be a promising approach for treating polluted water bodies.

Actinobacteria as Microbial Cell Factories and Biocatalysts in The Synthesis of Chiral Intermediates and Bioactive Molecules; Insights and Applications.

Actinobacteria are one of the most intriguing bacterial phyla in terms of chemical diversity and bioactivities of their reported biomolecules and natural products, including various types of chiral molecules. Actinobacterial genera such as Detzia, Mycobacterium, and Streptomyces are among the microbial sources targeted for selective reactions such as asymmetric biocatalysis catalyzed by whole cells or enzymes induced in their cell niche. Remarkably, stereoselective reactions catalyzed by actinobacterial whole cells or their enzymes include stereoselective oxidation, stereoselective reduction, kinetic resolution, asymmetric hydrolysis, and selective transamination, among others. Species of actinobacteria function with high chemo-, regio-, and enantio-selectivity under benign conditions, which could help current industrial processing. Numerous selective enzymes were either isolated from actinobacteria or expressed from actinobacteria in other microbes and hence exploited in the production of pure organic compounds difficult to obtain chemically. In addition, different species of actinobacteria, especially Streptomyces species, function as natural producers of chiral molecules of therapeutic importance. Herein, we discuss some of the most outstanding contributions of actinobacteria to asymmetric biocatalysis, which are important in the organic and/or pharmaceutical industries. In addition, we highlight the role of actinobacteria as microbial cell factories for chiral natural products with insights into their various biological potentialities.

A unique sigma/anti-sigma system in the actinomycete Actinoplanes missouriensis.

Bacteria of the genus Actinoplanes form sporangia that contain dormant sporangiospores which, upon contact with water, release motile spores (zoospores) through a process called sporangium dehiscence. Here, we set out to study the molecular mechanisms behind sporangium dehiscence in Actinoplanes missouriensis and discover a sigma/anti-sigma system with unique features. Protein σSsdA contains a functional sigma factor domain and an anti-sigma factor antagonist domain, while protein SipA contains an anti-sigma factor domain and an anti-sigma factor antagonist domain. Remarkably, the two proteins interact with each other via the anti-sigma factor antagonist domain of σSsdA and the anti-sigma factor domain of SipA. Although it remains unclear whether the SipA/σSsdA system plays direct roles in sporangium dehiscence, the system seems to modulate oxidative stress responses in zoospores. In addition, we identify a two-component regulatory system (RsdK-RsdR) that represses initiation of sporangium dehiscence.

[Advances in the biosynthesis of cyclodipeptide type natural products derived from actinomycetes].

Cyclodipeptide (CDP) composed of two amino acids is the simplest cyclic peptide. These two amino acids form a typical diketopiperazine (DKP) ring by linking each other with peptide bonds. This characteristic stable ring skeleton is the foundation of CDP to display extensive and excellent bioactivities, which is beneficial for CDPs' pharmaceutical research and development. The natural CDP products are well isolated from actinomycetes. These bacteria can synthesize DKP backbones with nonribosomal peptide synthetase (NRPS) or cyclodipeptide synthase (CDPS). Moreover, actinomycetes could produce a variety of CDPs through different enzymatic modification. The presence of these abundant and diversified catalysis indicates that actinomycetes are promising microbial resource for exploring CDPs. This review summarized the pathways for DKP backbones biosynthesis and their post-modification mechanism in actinomycetes. The aim of this review was to accelerate the genome mining of CDPs and their isolation, purification and structure identification, and to facilitate revealing the biosynthesis mechanism of novel CDPs as well as their synthetic biology design.

Antibiotics from rare actinomycetes, beyond the genus Streptomyces.

Throughout the golden age of antibiotic discovery, Streptomyces have been unsurpassed for their ability to produce bioactive metabolites. Yet, this success has been hampered by rediscovery. As we enter a new stage of biodiscovery, omics data and existing scientific repositories can enable informed choices on the biodiversity that may yield novel antibiotics. Here, we focus on the chemical potential of rare actinomycetes, defined as bacteria within the order Actinomycetales, but not belonging to the genus Streptomyces. They are named as such due to their less-frequent isolation under standard laboratory practices, yet there is increasing evidence to suggest these biologically diverse genera harbour considerable biosynthetic and chemical diversity. In this review, we focus on examples of successful isolation and genera that have been the focus of more concentrated biodiscovery efforts, we survey the representation of rare actinomycete taxa, compared with Streptomyces, across natural product data repositories in addition to its biosynthetic potential. This is followed by an overview of clinically useful drugs produced by rare actinomycetes and considerations for future biodiscovery efforts. There is much to learn about these underexplored taxa, and mounting evidence suggests that they are a fruitful avenue for the discovery of novel antimicrobials.