ABSTRACT The clinical deployment of antibiotics is undermined by antimicrobial resistance. Without new agents to treat antibiotic‐resistant bacterial infections, mortality rates are predicted to reach 10 million people per year by 2050. Most antibiotics are derived from natural products (NPs) produced by bacteria; however, this resource was abandoned by industry because of high rediscovery rates. We are amid a natural product renaissance fuelled by inexpensive access to genome sequencing and sophisticated bioinformatic tools, which have highlighted that most of the biosynthetic pathways for NPs are not expressed in the laboratory. Here, we engineered the expression of a silent biosynthetic gene cluster harboured by an environmental isolate of Streptomyces albidoflavus . Using a bioinformatics‐guided approach, we isolated and structurally characterised a novel glycopeptide antibiotic (GPA) named biffamycin A, which is the smallest GPA known and harbours unprecedented 5‐chloro‐4‐methoxy tryptophan and 3‐hydroxy(α‐D‐mannoysl)‐D‐lysine moieties. Biffamycin A possesses antimycobacterial and antistaphylococcal bioactivity, including against methicillin‐ and vancomycin‐resistant Staphylococcus aureus .

ABSTRACT Cereal crops rely on a wide array of specialized metabolites to defend themselves against microbial pathogens. Here, using a combination of genome-wide analysis, heterologous pathway reconstruction, structural elucidation and in planta validation, we identify two pathogen-induced diterpenoid pathways in wheat that produce diterpenoids: the new glycosylated diterpenes aspisoside A and aspisoside B, and the diterpene alcohols scutenol A and scutenol B. The pathogen-responsive nature of these pathways, together with their antimicrobial activity are consistent with a likely defensive role in wheat. These compounds are produced by two biosynthetic gene clusters, each encoding a discrete pathway, so revealing organizational separation of wheat diterpenoid-based chemical defenses. The aspisoside-producing gene cluster is syntenic with diterpenoid phytoalexin-producing clusters in rice and barley, for momilactone and hordedane biosynthesis, respectively, yet gives rise to structurally distinct phytoalexins in wheat. The scutenol cluster is syntenic with currently uncharacterized predicted biosynthetic gene clusters in barley, oat, and Brachypodium . These findings establish diterpene glycosides as a previously unrecognized component of wheat defense chemistry and provide new insights into the chemical diversification of defense-related biosynthetic gene clusters within the Poaceae.

Streptomyces bacteria have complex life cycles involving hyphal growth, sporulation and the production of diverse specialised metabolites, including antibiotics. In this study, we investigated the role of the highly conserved orphan response regulator OrrA in Streptomyces venezuelae NRRL B-65442. We show that S. venezuelae ∆orrA mutants are defective in sporulation and used chromatin immunoprecipitation followed by sequencing to identify five OrrA binding sites in vivo. Tandem-mass-tag proteomics revealed that OrrA directly activates two of these putative target genes, wblA and vnz_04640, a finding consistent with previous work on OrrA in the distantly related Streptomyces coelicolor. We also demonstrate that deleting wblA blocks sporulation and that overexpressing wblA restores sporulation in the ∆orrA mutant. Additionally, chloramphenicol biosynthesis is upregulated in both the ∆orrA and ∆wblA mutants compared with the wild-type strain. Taken together, these results indicate that the primary function of OrrA is to regulate WblA production and that reduced intracellular WblA levels underlie the phenotypes observed in the ∆orrA mutant.

Abstract The domestication of Streptomyces species for antibiotic production involves long-term, iterative mutagenesis and selection, yet the genomic changes driving enhanced production remain unclear. Analysis of five strains from an industrial lineage of Streptomyces clavuligerus using comparative genomics, transcriptomics and phenotypic profiling for dynamic genome architectures with plasmid integration events and chromosomal rearrangements, alongside the accumulation of mutations affecting metabolic pathways and global gene regulation. These changes increased precursor supply and reprogrammed transcription leading to enhanced clavulanic acid production but reduced catabolic flexibility. Complementation experiments confirmed the functional impacts of specific mutations. These findings reveal that artificial selection shapes genome evolution in industrial strains, balancing production gains with metabolic trade-offs. This work will likely inform rational design of Streptomyces strains for improved natural product production in industry while highlighting the constraints imposed by domestication on metabolic versatility. More broadly it shows that many of the evolutionary processes in industrial strain improvement programmes mirror those at play during natural selection.

Embleya is a genus within the family Streptomycetaceae, a group of Actinobacteria with an outstanding capacity for the production of specialized metabolites, and a strikingly complex life cycle. In this work, we sequenced the complete genome of the new species Embleya australiensis MST-111070 and validated the assembly using optical mapping. The genome of E. australiensis MST-111070 consists of a 7.1 Mb linear chromosome and three additional replicons (EEC, Embleya extrachromosomal element), including a 4.2 Mb linear replicon, EEC1, significantly larger than all previously described secondary replicons from bacteria. EEC1 is typified by its similar composition to the chromosome in terms of G+C content, codon usage and gene functions. It also carries terminal inverted repeats identical to the chromosome. EEC1 is enriched in biosynthetic gene clusters (BGCs), including the only copy of the BGCs for the spore pigment and the surfactant peptide SapB, metabolites essential for the organism's life cycle. EEC1 contains an origin of replication with at least some chromosomal properties, and its replication is likely to depend on functions provided by chromosomally located genes. Further comparison of Embleya spp. genomes suggests that EEC1-like replicons are conserved across the genus, in contrast to other known large linear extrachromosomal replicons (megaplasmids) in the family Streptomycetaceae. EEC1 is thus a hallmark of the Embleya genus and is central to its evolution within the Streptomycetaceae family. We propose EEC1 as a secondary chromosome and the largest secondary replicon reported in bacteria to date.

The MtrAB two-component system is a master regulator of antibiotic biosynthesis in Streptomyces species. MtrA is also required for sporulation under certain growth conditions, which means that on some growth media, ∆mtrA mutant colonies do not produce aerial hyphae or spores. These mutants are referred to as (conditionally) bald because they lack the hairy appearance of wild-type colonies. Here, we report that Streptomyces venezuelae NRRL B-65442 ∆mtrA is bald on R2YE agar but sporulates normally on MYM (Maltose Yeast Extract Medium) agar, and we demonstrate that this is caused by the presence of 10.2% sucrose in R2YE agar. Consistent with this, we found that adding 10.2% sucrose to MYM agar also inhibits sporulation of the ∆mtrA mutant. Proteomics combined with DNA binding studies revealed that MtrA directly activates the expression of the key developmental regulator genes bldM and whiI on R2YE but not MYM agar. BldM and WhiI work together to activate genes required for aerial hyphae production and sporulation. Crucially, over-expression of bldM-whiI in the ∆mtrA mutant restored normal sporulation in the presence of 10.2% sucrose. We hypothesized that MtrAB must sense and respond to osmotic stress, and consistent with this, we found that ∆mtrA and ∆mtrB mutants are bald on MYM agar containing 0.5 M NaCl. MtrA also directly activates biosynthesis of the compatible solute and osmoprotectant ectoine on growth media containing 10.2% sucrose. We propose a model in which high concentrations of sucrose or salt induce hyperosmotic stress in Streptomyces species, and this activates MtrAB. The response regulator MtrA then directly activates expression of the ectABCD operon to switch on ectoine biosynthesis and expression of bldM and whiI to trigger entry into sporulation.

Formicamycins and their biosynthetic precursors, the fasamycins, form part of the phenylnaphthacenoid family of polyketide natural products. A recent atroposelective total synthesis of formicamycin H brought into question our original stereochemical assignment of the axially chiral linkage between C-6 and C-7. To address this, we obtained an X-ray crystal structure for formicamycin H that unambiguously confirmed our original assignment as the Sa atropisomer. X-ray structures for multiple additional fasamycins and formicamycins confirmed that this is common to all congeners. However, these studies identified a compounded error made by us whereby several structures previously reported as para-methoxy were found to have ortho-methoxy groups on the hanging E-ring. To address this for congeners that did not crystallize or gave nondiffracting crystals, we turned to the surprisingly underutilized 1,n-ADEQUATE NMR experiment. In total, we generated X-ray structures for 15 phenylnaphthacenoid metabolites and by combining these results report the corrected structures for three formicamycins, six fasamycins, and three biosynthetic lactone intermediates, noting that several revised fasamycin structures now match previously reported naphthacemycins. Our results highlight the utility of 1,n-ADEQUATE experiments for regiochemical determination in polysubstituted aromatic molecules. Moreover, our investigations uncovered a potential deracemization step during biosynthesis of the formicamycin framework.

Streptomyces specialized metabolites account for over half of all clinically used antibiotics, as well as numerous antifungal, anticancer, and immunosuppressant agents. Two-component systems, which are widespread in bacteria, are key regulators of antibiotic production in Streptomyces species, yet their activating signals remain poorly understood. CutRS was the first two-component system identified in the genus Streptomyces, and deletion of cutRS in Streptomyces coelicolor was shown to enhance antibiotic production, although its CutR regulon does not include biosynthetic genes. Here, we used Streptomyces venezuelae NRRL B-65442 to further investigate CutRS function. We show that deletion of cutRS increases growth rate and a reversal of the glucose-mediated carbon catabolite repression typically observed in Streptomyces species. We also demonstrate that CutR DNA binding is glucose-dependent, but CutR does not directly regulate genes involved in growth, antibiotic biosynthesis, or glucose metabolism. The only CutR targets conserved in both S. coelicolor and S. venezuelae are the foldase genes htrA3 and htrB, which are involved in the protein secretion stress response. Consistent with this, we show that CutS homologs all contain two conserved cysteine residues in their extracellular sensor domains and that changing these residues to serine constitutively activates S. venezuelae CutRS. We propose that failure of a disulfide bond to form between these cysteine residues indicates secretion stress and leads to activation of the CutRS system and the secretion stress response.IMPORTANCEStreptomyces bacteria are the primary source of clinically useful antibiotics. While many two-component systems have been linked to antibiotic biosynthesis in Streptomyces species, few have been well characterized. Here, we characterize a secretion stress-sensing two-component system called CutRS and propose a model for how the sensor kinase detects extracellular protein misfolding via two highly conserved cysteine residues. Importantly, we also show that deletion of cutRS triggers antibiotic overproduction in the presence of glucose. Since glucose normally represses antibiotic biosynthesis in Streptomyces species through carbon catabolite repression, this finding reveals a simple genetic route to bypass this barrier. This has significant implications for antibiotic discovery pipelines and industrial production, where glucose-rich media are preferred for cost and scalability. Our results position CutRS as a key target for future strain-improvement strategies.

Bacteria and fungi produce a wide range of specialised metabolites, including volatile organic compounds (VOCs) that can act as signals or act directly to inhibit niche-competing microbes. Despite their ecological importance, most VOCs involved as signalling compounds remain uncharacterised. We have previously screened a collection of Actinobacteria strains sourced from Western Australia for their ability in vitro to suppress the growth of plant fungal pathogens. Here we explored the potential of four of the most active strains to produce antifungal metabolites by growing the strains on a range of nutrient-containing media. A casein-based (CYPS) culture medium was found to induce the production of antifungal compounds with high activity against Sclerotinia sclerotiorum, a major necrotrophic fungal pathogen of crops such as canola. We further observed that VOCs were produced that influenced pH and affected the bacterium-fungus interaction. The presence of Sclerotinia induced further VOC production in the Actinobacteria. Solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) analysis identified 2,4,6-trimethylpyridine, a compound not identified previously from Actinobacteria, which showed antifungal activity against different isolates of S. sclerotiorum and increased the pH of the medium. Overall, this study showed that Actinobacteria or their volatile products have the potential to be used in the protection of crops against S. sclerotiorum.

Translocating unfolded polypeptides across membranes is essential in all domains of life and in bacteria requires the conserved Sec machinery and ATP. Bacterial Sec substrates fold outside the cell and often use DsbA-catalysed disulfide bond formation between cysteines to ensure correct folding. Extracellular protein misfolding triggers a stress response that involves production of dual function HtrA-family chaperone/proteases. In Gram-negative bacteria this is called the envelope stress response and in Gram-positive bacteria the secretion stress response, but the exact signals sensed by bacteria to trigger these stress responses are not well understood. In Streptomyces bacteria the secretion stress response is mediated by the CutRS and CssRS two-component systems which control the levels of four conserved HtrA-family chaperones. Here we show that the CutS sensor kinase contains two conserved cysteine residues in its extracellular sensor domain that control CutS activity. CssS also has two conserved and invariant cysteines in its sensor domain, and we propose that CutS and CssS detect the extracellular redox state and work together to ensure secreted proteins fold correctly in the fluctuating soil environment. Further analysis of ~12,800 genomes indicated that 98.9% of strains across all bacterial classes have at least one sensor kinase with two or more extracellular cysteine residues, suggesting that extracellular redox sensing by two-component systems is widespread in bacteria. ### Competing Interest Statement The authors have declared no competing interest.

Streptomyces formicae KY5 was isolated from a Tetraponera penzigi plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus, and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including Lomentospora prolificans. S. formicae is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.

Streptomyces bacteria make diverse specialized metabolites that form the basis of ~55% of clinically used antibiotics. Despite this, only 3% of their encoded specialized metabolites have been matched to molecules, and understanding how their biosynthesis is controlled is essential to fully exploit their potential. Here, we use Streptomyces formicae and the formicamycin biosynthetic pathway as a model to understand the complex regulation of specialized metabolism. We analyzed all three pathway-specific regulators and found that biosynthesis is subject to negative feedback and redox control via two MarR-family proteins, while activation of the pathway is dependent on a cytoplasmic two-component system. Like many Streptomyces antibiotics, formicamycins are only produced in solid culture, and biosynthesis is switched off in aerated liquid cultures. Here, we demonstrate that a redox-sensitive repressor named ForJ senses oxygen via a single cysteine residue that is required to repress formicamycin biosynthesis in liquid cultures.IMPORTANCEAntimicrobial resistance presents a significant threat to human health. Streptomyces bacteria are a promising source of novel antimicrobials; however, encouraging production of these molecules under laboratory conditions remains a challenge because we have limited understanding of the signals that control their production. Here, we use the formicamycin producer, Streptomyces formicae, as a model to further understand how antibiotic production is regulated in response to various signals. We show that three regulatory elements work together to coordinate formicamycin biosynthesis in response to intracellular signals, redox stress, and formicamycin accumulation. We also show that by making the regulators "blind" to these signals, we can induce high-level production of formicamycins in industrially relevant conditions, which facilitates their development as new antimicrobials.

Pseudonocardia species comprise a genus of filamentous, sporulating bacteria belonging to the phylum Actinomycetota, formerly Actinobacteria. They are found in marine and freshwater sediments and soils and associated with marine animals, insects, and plants. To date, they have mostly been studied because of their mutually beneficial symbiosis with fungus- growing ants in the tribe Attini. They have also attracted interest due to their biosynthetic capabilities, including the production of variably glycosylated polyenes and other novel antifungal compounds, and for their capacity to grow on a variety of hydrocarbons. The majority of clinically used antibiotics are derived from the specialised metabolites of filamentous actinomycete bacteria and most of these come from the genus Streptomyces. However, in the quest for novel chemistry there is increasing interest in studying other filamentous actinomycete genera, including Pseudonocardia. Here we outline the biological properties, genome size and structure and key features of the genus Pseudonocardia, namely their specialised metabolites and ecological roles.

Abstract The Gram-negative ESKAPE pathogen Pseudomonas aeruginosa is currently listed top of the priority one pathogens by the WHO that require urgent research and development of new antimicrobials. Currently, microbiologists and clinicians face an ever-growing challenge in treating MDR bacterial infections, for this, and other pathogens, using conventional antibiotics. With a limited number of antibiotics currently in clinical trials and a general stagnation in the discovery of promising antimicrobials, new drug targets are desperately needed, and recent investigations include targeting bacterial virulence factors. Targeting bacterial virulence factors should reduce the pathogenesis of an organism, slow its infection progression and prevent vital virulence processes such as biofilm formation as well as potentially increasing a strain’s susceptibility to antibiotics. However, given the incredible diversity of bacteria, virulence mechanisms vary massively, meaning that identifying a broad-spectrum virulence target presents a major challenge. This important point has brought researchers to a fascinating group of enzymes that are ubiquitous in nature, and possess a plethora of biological roles, including acting as virulence factors. The macrophage infectivity potentiators (Mips) belong to the ubiquitous FK506 binding protein (FKBPs) family of peptidyl-prolyl cis/trans isomerases (PPIases). FKBPs are found in all bacteria serving as general house-keeping proteins involved in protein folding and chaperoning. However, a small subset has been identified as virulence proteins. Mips have been characterized in several Gram-negative pathogens including Legionella pneumophila and Burkholderia pseudomallei and were shown to be essential for invasion of macrophages, both in vitro and in murine-infection models, and for other virulence determinants. Pseudomonas aeruginosa also possess three Mip virulence factors, PaMip1, PaMip2 and PaMip3, which are required for the full virulence of P. aeruginosa in in vivo and ex vivo models. Virulence can be addressed in a pharmacological manner using bacterial natural products.

Isoflavones are a group of phenolic compounds mostly restricted to plants of the legume family, where they mediate important interactions with plant-associated microbes, including in defense from pathogens and in nodulation. Their well-studied health promoting attributes have made them a prime target for metabolic engineering, both for bioproduction of isoflavones as high-value molecules, and in biofortification of food crops. A key gene in their biosynthesis, isoflavone synthase, was identified in legumes over two decades ago, but little is known about formation of isoflavones outside of this family. Here we identify a specialized wheat-specific isoflavone synthase, TaCYP71F53, which catalyzes a different reaction from the leguminous isoflavone synthases, thus revealing an alternative path to isoflavonoid biosynthesis and providing a non-transgenic route for engineering isoflavone production in wheat. TaCYP71F53 forms part of a biosynthetic gene cluster that produces a naringenin-derived O -methylated isoflavone, 5-hydroxy-2′,4′,7-trimethoxyisoflavone, triticein. Pathogen-induced production and in vitro antimicrobial activity of triticein suggest a defense-related role for this molecule in wheat. Genomic and metabolic analyses of wheat ancestral grasses further show that the triticein gene cluster was introduced into domesticated emmer wheat through natural hybridization ~9000 years ago, and encodes a pathogen-responsive metabolic pathway that is conserved in modern bread wheat varieties.

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 lessfrequent 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.

Formicamycins and their biosynthetic intermediates the fasamycins are polyketide antibiotics produced by Streptomyces formicae KY5 from a pathway encoded by the for biosynthetic gene cluster. In this work the ability of Streptomyces coelicolor M1146 and the ability of Saccharopolyspora erythraea Δery to heterologously express the for biosynthetic gene cluster were assessed. This led to the identification of eight new glycosylated fasamycins modified at different phenolic groups with either a monosaccharide (glucose, galactose, or glucuronic acid) or a disaccharide comprised of a proximal hexose (either glucose or galactose), with a terminal pentose (arabinose) moiety. In contrast to the respective aglycones, minimal inhibitory screening assays showed these glycosylated congeners lacked antibacterial activity.

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor. However, despite this early work, the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 300-fold, explaining the increase in actinorhodin production. However, while ChIP-seq identified 85 CutR binding sites in S. coelicolor none of these are in the actinorhodin biosynthetic gene cluster, meaning the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved HtrA-family foldases: HtrA3 and HtrB, and a putative VKOR enzyme, which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. Thus, we tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins, its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.

Obafluorin is a Pseudomonas fluorescens antibacterial natural product that inhibits threonyl-tRNA synthetase (ThrRS). It acts as a broad-spectrum antibiotic against a range of clinically relevant pathogens and comprises a strained β-lactone ring decorated with catechol and 4-nitro-benzyl moieties. The catechol moiety is widespread in nature and its role in the coordination of ferric iron has been well-characterised in siderophores and Trojan horse antibiotics. Here we use a combination of mutasynthesis, bioassays, enzyme assays and metal binding studies to delineate the role of the catechol moiety in the bioactivity of obafluorin. We use P. fluorescens biosynthetic mutants to generate obafluorin analogues with modified catechol moieties. We demonstrate that an intact catechol is required for both antibacterial activity and inhibition of the ThrRS molecular target. Although recent work showed that the obafluorin catechol coordinates Zn2+ in the ThrRS active site, we find that obafluorin is a weak Zn2+ binder in vitro, contrasting with a strong, specific 1 : 1 interaction with Fe3+. We use bioassays with siderophore transporter mutants to probe the role of the obafluorin catechol in Fe3+-mediated uptake. Surprisingly, obafluorin does not behave as a Trojan horse antibiotic but instead exhibits increased antibacterial activity in the presence of Fe3+. We further demonstrate that Fe3+ binding prevents the hydrolytic breakdown of the β-lactone ring, revealing a hitherto unreported function for the catechol moiety in natural product bioactivity.

Many, if not all, plants and animals form mutually beneficial symbioses (mutualisms) with microbes and a subset of these mutualisms are defensive, in which the host provides food and housing in return for defence against disease. These symbioses typically involve antibiotic-producing bacteria, the best known of which are filamentous actinomycetes in the genera Streptomyces and Pseudonocardia and unicellular species in the genus Pseudomonas. Such mutualisms are likely to be widespread in nature, but they are best characterised in insects, which provide experimentally tractable models for studying symbiosis and microbiome formation because they typically host less complex microbial communities. Here, we examine the mutualisms formed between insects and antibiotic-producing bacteria using well-characterised examples, including digger wasps and their endosymbiotic Streptomyces species, attine ants and their mutualist Pseudonocardia species and Paederus beetles with their pederin-producing Pseudomonas species. We also discuss how searching such symbiotic niches can give insights into the evolution and functions of microbial specialised metabolites and provide new platforms for antibiotic discovery.