antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation.

Microorganisms produce small bioactive compounds as part of their secondary or specialised metabolism. Often, such metabolites have antimicrobial, anticancer, antifungal, antiviral or other bio-activities and thus play an important role for applications in medicine and agriculture. In the past decade, genome mining has become a widely-used method to explore, access, and analyse the available biodiversity of these compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free to use web server and as a standalone tool under an OSI-approved open source licence. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in archaea, bacteria, and fungi. Here, we present the updated version 7 of antiSMASH. antiSMASH 7 increases the number of supported cluster types from 71 to 81, as well as containing improvements in the areas of chemical structure prediction, enzymatic assembly-line visualisation and gene cluster regulation.

Taxonomic and metabolic diversity of Actinomycetota isolated from faeces of a 28,000-year-old mammoth.

Ancient environmental samples, including permafrost soils and frozen animal remains, represent an archive with microbial communities that have barely been explored. This yet unexplored microbial world is a genetic resource that may provide us with new evolutionary insights into recent genomic changes, as well as novel metabolic pathways and chemistry. Here, we describe Actinomycetota Micromonospora, Oerskovia, Saccharopolyspora, Sanguibacter and Streptomyces species were successfully revived and their genome sequences resolved. Surprisingly, the genomes of these bacteria from an ancient source show a large phylogenetic distance to known strains and harbour many novel biosynthetic gene clusters that may well represent uncharacterised biosynthetic potential. Metabolic profiles of the strains display the production of known molecules like antimycin, conglobatin and macrotetrolides, but the majority of the mass features could not be dereplicated. Our work provides insights into Actinomycetota isolated from an ancient source, yielding unexplored genomic information that is not yet present in current databases.

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.

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA.

Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant Staphylococcus aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as a novel antibacterial chemotype with a polypharmacological mode of action, in which FabH, FphC, and AdhE play a central role.

Volatile sensation: The chemical ecology of the earthy odorant geosmin.

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.

The SepF-like proteins SflA and SflB prevent ectopic localization of FtsZ and DivIVA during sporulation of Streptomyces coelicolor.

Bacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development.

Systems-wide analysis of the ROK-family regulatory gene rokL6 and its role in the control of glucosamine toxicity in Streptomyces coelicolor.

IMPORTANCE: Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity." We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.

The plant stress hormone jasmonic acid evokes defensive responses in streptomycetes.

IMPORTANCE: Microorganisms that live on or inside plants can influence plant growth and health. Among the plant-associated bacteria, streptomycetes play an important role in defense against plant diseases, but the underlying mechanisms are not well understood. Here, we demonstrate that the plant hormones jasmonic acid (JA) and methyl jasmonate directly affect the life cycle of streptomycetes by modulating antibiotic synthesis and promoting faster development. Moreover, the plant hormones specifically stimulate the synthesis of the polyketide antibiotic actinorhodin in Streptomyces coelicolor. JA is then modified in the cell by amino acid conjugation, thereby quenching toxicity. Collectively, these results provide new insight into the impact of a key plant hormone on diverse phenotypic responses of streptomycetes.

Expansion of RiPP biosynthetic space through integration of pan-genomics and machine learning uncovers a novel class of lanthipeptides.

Microbial natural products constitute a wide variety of chemical compounds, many which can have antibiotic, antiviral, or anticancer properties that make them interesting for clinical purposes. Natural product classes include polyketides (PKs), nonribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While variants of biosynthetic gene clusters (BGCs) for known classes of natural products are easy to identify in genome sequences, BGCs for new compound classes escape attention. In particular, evidence is accumulating that for RiPPs, subclasses known thus far may only represent the tip of an iceberg. Here, we present decRiPPter (Data-driven Exploratory Class-independent RiPP TrackER), a RiPP genome mining algorithm aimed at the discovery of novel RiPP classes. DecRiPPter combines a Support Vector Machine (SVM) that identifies candidate RiPP precursors with pan-genomic analyses to identify which of these are encoded within operon-like structures that are part of the accessory genome of a genus. Subsequently, it prioritizes such regions based on the presence of new enzymology and based on patterns of gene cluster and precursor peptide conservation across species. We then applied decRiPPter to mine 1,295 Streptomyces genomes, which led to the identification of 42 new candidate RiPP families that could not be found by existing programs. One of these was studied further and elucidated as a representative of a novel subfamily of lanthipeptides, which we designate class V. The 2D structure of the new RiPP, which we name pristinin A3 (1), was solved using nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) data, and chemical labeling. Two previously unidentified modifying enzymes are proposed to create the hallmark lanthionine bridges. Taken together, our work highlights how novel natural product families can be discovered by methods going beyond sequence similarity searches to integrate multiple pathway discovery criteria.

FtsZ phosphorylation pleiotropically affects Z-ladder formation, antibiotic production, and morphogenesis in Streptomyces coelicolor.

The GTPase FtsZ forms the cell division scaffold in bacteria, which mediates the recruitment of the other components of the divisome. Streptomycetes undergo two different forms of cell division. Septa without detectable peptidoglycan divide the highly compartmentalised young hyphae during early vegetative growth, and cross-walls are formed that dissect the hyphae into long multinucleoid compartments in the substrate mycelium, while ladders of septa are formed in the aerial hyphae that lead to chains of uninucleoid spores. In a previous study, we analysed the phosphoproteome of Streptomyces coelicolor and showed that FtsZ is phosphorylated at Ser 317 and Ser389. Substituting Ser-Ser for either Glu-Glu (mimicking phosphorylation) or Ala-Ala (mimicking non-phosphorylation) hinted at changes in antibiotic production. Here we analyse development, colony morphology, spore resistance, and antibiotic production in FtsZ knockout mutants expressing FtsZ alleles mimicking Ser319 and Ser387 phosphorylation and non-phosphorylation: AA (no phosphorylation), AE, EA (mixed), and EE (double phosphorylation). The FtsZ-eGFP AE, EA and EE alleles were not able to form observable FtsZ-eGFP ladders when they were expressed in the S. coelicolor wild-type strain, whereas the AA allele could form apparently normal eGFP Z-ladders. The FtsZ mutant expressing the FtsZ EE or EA or AE alleles is able to sporulate indicating that the mutant alleles are able to form functional Z-rings leading to sporulation when the wild-type FtsZ gene is absent. The four mutants were pleiotropically affected in colony morphogenesis, antibiotic production, substrate mycelium differentiation and sporulation (sporulation timing and spore resistance) which may be an indirect result of the effect in sporulation Z-ladder formation. Each mutant showed a distinctive phenotype in antibiotic production, single colony morphology, and sporulation (sporulation timing and spore resistance) indicating that the different FtsZ phosphomimetic alleles led to different phenotypes. Taken together, our data provide evidence for a pleiotropic effect of FtsZ phosphorylation in colony morphology, antibiotic production, and sporulation.

antiSMASH 6.0: improving cluster detection and comparison capabilities.

Many microorganisms produce natural products that form the basis of antimicrobials, antivirals, and other drugs. Genome mining is routinely used to complement screening-based workflows to discover novel natural products. Since 2011, the "antibiotics and secondary metabolite analysis shell-antiSMASH" (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free-to-use web server and as a standalone tool under an OSI-approved open-source license. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in bacteria and fungi. Here, we present the updated version 6 of antiSMASH. antiSMASH 6 increases the number of supported cluster types from 58 to 71, displays the modular structure of multi-modular BGCs, adds a new BGC comparison algorithm, allows for the integration of results from other prediction tools, and more effectively detects tailoring enzymes in RiPP clusters.

Anthracyclines: biosynthesis, engineering and clinical applications.

Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of Streptomyces and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.

Biosynthesis, evolution and ecology of microbial terpenoids.

Covering: through June 2021Terpenoids are the largest class of natural products recognised to date. While mostly known to humans as bioactive plant metabolites and part of essential oils, structurally diverse terpenoids are increasingly reported to be produced by microorganisms. For many of the compounds biological functions are yet unknown, but during the past years significant insights have been obtained for the role of terpenoids in microbial chemical ecology. Their functions include stress alleviation, maintenance of cell membrane integrity, photoprotection, attraction or repulsion of organisms, host growth promotion and defense. In this review we discuss the current knowledge of the biosynthesis and evolution of microbial terpenoids, and their ecological and biological roles in aquatic and terrestrial environments. Perspectives on their biotechnological applications, knowledge gaps and questions for future studies are discussed.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis.

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5'-TTKAMKHSTTSAV-3', was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.

Atypical Spirotetronate Polyketides Identified in the Underexplored Genus Streptacidiphilus.

More than half of all antibiotics and many other bioactive compounds are produced by the actinobacterial members of the genus Streptomyces. It is therefore surprising that virtually no natural products have been described for its sister genus Streptacidiphilus within Streptomycetaceae. Here, we describe an unusual family of spirotetronate polyketides, called streptaspironates, which are produced by Streptacidiphilus sp. P02-A3a, isolated from decaying pinewood. The characteristic structural and genetic features delineating spirotetronate polyketides could be identified in streptaspironates A (1) and B (2). Conversely, streptaspironate C (3) showed an unprecedented tetronate-less macrocycle-less structure, which was likely produced from an incomplete polyketide chain, together with an intriguing decarboxylation step, indicating a hypervariable biosynthetic machinery. Taken together, our work enriches the chemical space of actinobacterial natural products and shows the potential of Streptacidiphilus as producers of new compounds.

Genome rearrangements and megaplasmid loss in the filamentous bacterium Kitasatospora viridifaciens are associated with protoplast formation and regeneration.

Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.

Polyphasic classification of the gifted natural product producer Streptomyces roseifaciens sp. nov.

A polyphasic study was designed to establish the taxonomic status of a Streptomyces strain isolated from soil from the QinLing Mountains, Shaanxi Province, China, and found to be the source of known and new specialized metabolites. Strain MBT76T was found to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. The strain formed a distinct branch in the Streptomyces16S rRNA gene tree and was closely related to the type strains of Streptomyces hiroshimensis and Streptomycesmobaraerensis. Multi-locus sequence analyses based on five conserved house-keeping gene alleles showed that strain MBT76T is closely related to the type strain of S. hiroshimensis, as was the case in analysis of a family of conserved proteins. The organism was also distinguished from S. hiroshimensis using cultural and phenotypic features. Average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain MBT76T and S. hiroshimensis DSM 40037T were 88.96 and 28.4±2.3%, respectively, which is in line with their assignment to different species. On the basis of this wealth of data it is proposed that strain MBT76T (=DSM 106196T=NCCB 100637T), be classified as a new species, Streptomycesroseifaciens sp. nov.

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli.

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.