Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats such as rule-based BGC detection, sequence and annotation quality and cluster boundary prediction, which all have to be considered while planning for, performing and analyzing the results of genome mining studies

The secondary metabolite bioinformatics portal:Computational tools to facilitate synthetic biology of secondary metabolite production

AbstractNatural products are among the most important sources of lead molecules for drug discovery. With the development of affordable whole-genome sequencing technologies and other ‘omics tools, the field of natural products research is currently undergoing a shift in paradigms. While, for decades, mainly analytical and chemical methods gave access to this group of compounds, nowadays genomics-based methods offer complementary approaches to find, identify and characterize such molecules. This paradigm shift also resulted in a high demand for computational tools to assist researchers in their daily work. In this context, this review gives a summary of tools and databases that currently are available to mine, identify and characterize natural product biosynthesis pathways and their producers based on ‘omics data. A web portal called Secondary Metabolite Bioinformatics Portal (SMBP at http://www.secondarymetabolites.org) is introduced to provide a one-stop catalog and links to these bioinformatics resources. In addition, an outlook is presented how the existing tools and those to be developed will influence synthetic biology approaches in the natural products field

Draft Genome Sequence of the Antitrypanosomally Active Sponge-Associated Bacterium Actinokineospora sp. Strain EG49

The marine sponge-associated bacterium Actinokineospora sp. strain EG49 produces the antitrypanosomal angucycline-like compound actinosporin A. The draft genome of Actinokineospora sp. EG49 has a size of 7.5 megabases and a GC content of 72.8% and contains 6,629 protein-coding sequences (CDS). antiSMASH predicted 996 genes residing in 36 secondary metabolite gene clusters

Genome-guided bioprospecting for novel antibiotic lead compounds.

Antimicrobial resistance continues to pose a threat to health and wellbeing. Unmitigated, it is predicted to be the leading cause of death by 2050. Hence, the sustained development of novel antibiotics is crucial. As over 60% of licensed antibiotics are based on scaffolds derived from less than 1% of all known bacterial species, bacterial secondary metabolites constitute an untapped source of novel antibiotics. The aim of this project therefore was to expand the chemical space of bacteria-derived antibiotic lead compounds, using genomics approach. To that end, a topsoil sample was collected from the rhizosphere in which antibiosis occurs naturally. Using starvation stress, sixty-five isolates were recovered from the sample, out of which four were selected based on morphology and designated A13BB, A23BA, A13AA and A23AA. A13BB was identified by 16S rRNA gene sequence comparison as a Pseudomonas spp. and the other three isolates as Hafnia/Obesumbacterium spp. A database search showed that species belonging to these genera have genomes larger than the 3 Mb size above which an increasing proportion of a bacterial genome is dedicated to secondary metabolism. Given their ecological origin, expected genome size and ability to withstand starvation stress, these four isolates were presumed to harbour antibiotic-encoding gene clusters. Isolates A13BB and A23BA were therefore selected for genome mining in the first instance. Illumina and GridION/MinION sequencing data were obtained for both isolates and assembled into high-quality genomes. Isolates' identities were confirmed by FastANI analysis as strains of P. fragi and H. alvei, with 4.94 and 4.77 Mb genomes, respectively. Assembled genomes were mined with antiSMASH. Amongst other secondary metabolite biosynthetic gene clusters (smBGCs) detected, the β-lactone smBGCs in both genomes were selected for activation as their end products bear the hallmarks of an 'ideal antibiotic' that can inhibit several bacteria-specific enzymes simultaneously. Analysis of these smBGCs revealed genes encoding two core enzymes: 2-isopropylmalate synthase (2-IPMS) and acyl CoA ligase homologues. In the biosynthetic pathway, 2-IPMS catalyses the condensation of acetyl CoA with the degradation product of valine or isoleucine to form 2-IPM. 2-IPM is isomerised to 3-IPM which then forms the β-lactone warhead through reactions catalysed by acyl CoA ligase. It was speculated that the β-lactone compound is biosynthesised to efficiently rid the organism of potentially harmful metabolic intermediates as it grows on poor carbon and nitrogen sources. Strain fermentation was therefore performed with 10.8 mM acetate as the main carbon source, and 5 mM L-valine or L-isoleucine as the nitrogen source. Fermentation extracts were analysed by LC-MS with at least thirty-seven metabolite ions detected. Many of these ions have masses in the range m/z 230-750, which is an ideal mass range for antibiotic molecules. As β-lactone compounds are difficult to identify in crude extracts, especially when utilising single-stage mass spectrometry, reactivity-guided screening of extracts with cysteine thiol probe was performed as the probe forms UV- and MS-visible adducts with β-lactone compounds. However, complete dimerization of probe at a faster-than-expected rate in extract matrices hindered successful screening. This meant that it was not possible to determine if any crude extract components were β-lactone compounds without further analysis. Measures to limit or eliminate probe dimerization are proposed, together with molecular networking strategies that can afford global visualisation and rapid dereplication of extract components, using tandem mass spectrometry fragmentation patterns of parent ions. This project provides an original and robust workflow that serves as a strong starting point in the isolation of novel β-lactone compounds from crude extracts, followed by structural optimisation and bioactivity profiling. The hitherto unrecognised potential of β-lactone natural compounds as 'ideal antibiotics' is highlighted, and several structural optimisation strategies required to harness this potential are proposed. The genomes assembled here, and associated data have been deposited in the repositories of the International Nucleotide Sequence Database Collaboration for repurposing by other researchers. Likewise, the hidden metabolic and biosynthetic potentials of P. fragi and H. alvei species uncovered by RASTtk and antiSMASH analyses have been catalogued and placed in the public domain, with many of these attributes reported for the first time

Draft Genome Sequence of <i>Photobacterium halotolerans</i> S2753, Producer of Bioactive Secondary Metabolites

We report here the whole draft genome sequence of marine isolate Photobacterium halotolerans S2753, which produces the known antibiotic holomycin and also ngercheumicins and solonamides A and B, which interfere with virulence of methicillin-resistant Staphylococcus aureus strains by interacting with the quorum-sensing system

An Integrated Metabolomic and Genomic Mining Workflow to Uncover the Biosynthetic Potential of Bacteria

Microorganisms are a rich source of bioactives; however, chemical identification is a major bottleneck. Strategies that can prioritize the most prolific microbial strains and novel compounds are of great interest. Here, we present an integrated approach to evaluate the biosynthetic richness in bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds, including the thiomarinols that were reported from P. luteoviolacea here for the first time. By comparative genomics, we identified the biosynthetic cluster responsible for the production of the antibiotic indolmycin, which could not be predicted with standard methods. In conclusion, we present an efficient, integrative strategy for elucidating the chemical richness of a given set of bacteria and link the chemistry to biosynthetic genes. IMPORTANCE We here combine chemical analysis and genomics to probe for new bioactive secondary metabolites based on their pattern of distribution within bacterial species. We demonstrate the usefulness of this combined approach in a group of marine Gram-negative bacteria closely related to Pseudoalteromonas luteoviolacea, which is a species known to produce a broad spectrum of chemicals. The approach allowed us to identify new antibiotics and their associated biosynthetic pathways. Combining chemical analysis and genetics is an efficient “mining” workflow for identifying diverse pharmaceutical candidates in a broad range of microorganisms and therefore of great use in bioprospecting

plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters

Plant specialized metabolites are chemically highly diverse, play key roles in host-microbe interactions, have important nutritional value in crops and are frequently applied as medicines. It has recently become clear that plant biosynthetic pathway-encoding genes are sometimes densely clustered in specific genomic loci: Biosynthetic gene clusters (BGCs). Here, we introduce plantiSMASH, a versatile online analysis platform that automates the identification of candidate plant BGCs. Moreover, it allows integration of transcriptomic data to prioritize candidate BGCs based on the coexpression patterns of predicted biosynthetic enzyme-coding genes, and facilitates comparative genomic analysis to study the evolutionary conservation of each cluster. Applied on 48 high-quality plant genomes, plantiSMASH identifies a rich diversity of candidate plant BGCs. These results will guide further experimental exploration of the nature and dynamics of gene clustering in plant metabolism. Moreover, spurred by the continuing decrease in costs of plant genome sequencing, they will allow genome mining technologies to be applied to plant natural product discovery.</p

Exploiting gene regulation as an approach to identify, analyze and utilize the biosynthetic pathways of the glycopeptide ristomycin A and the zincophore [S,S]-EDDS in Amycolatopsis japonicum

The microbial secondary metabolism is a rich source for valuable products that have found their way into various clinical and industrial applications. A particularly productive bacterial genus for the discovery of natural products is Amycolatopsis. The most frequently reported type of secondary metabolites produced by this genus, are glycopeptide antibiotics like balhimycin or the medically relevant vancomycin. In contrast to most other members of the Amycolatopsis genus, Amycolatopsis japonicum was never described to produce any product with antibacterial activity. This strain however is known to synthesize the chelating agent ethylenediamine-disuccinate ([S,S]-EDDS), a biodegradable EDTA isomer in response to zinc deficiency. This zinc responsive repression of [S,S]-EDDS production indicates that it contributes to zinc uptake and that it belongs to the rarely described physiological group of the zincophores. Combining excellent chelating properties with the accessibility to biodegradation, [S,S]-EDDS is considered as a sustainable chelating agent, possessing the potential to replace EDTA and other environmentally threatening chelating agents in various applications. In this study, two distinct molecular genetic strategies were developed and implemented to activate the biosynthesis of the glycopeptide antibiotic ristomycin A or to identify the [S,S]-EDDS biosynthetic genes in Amycolatopsis japonicum, respectively. Genetic evaluation of the Amycolatopsis antibiotic biosynthetic potential indicated that A. japonicum might has the capability to produce a glycopeptide antibiotic. Since the biosynthesis of the predicted glycopeptide was not inducible by variations in culture conditions, a molecular genetic approach was employed to activate its production. Heterologous expression of the characterized pathway specific activator Bbr, naturally inducing the balhimycin biosynthesis in A. balhimycina, also induced the synthesis of a bioactive substance by A. japonicum. The bioactivity could be assigned to the production of ristomycin A, a highly glycosylated peptide antibiotic which is used as compound in diagnostic kits to detect widespread hereditary coagulation disorders. Full sequencing of the A. japonicum genome and its computational analysis led to the identification of the corresponding biosynthetic gene cluster which is directing the biosynthesis of ristomycin A. Such computational genome analyses by various bioinformatic tools are nowadays standardized applied strategies to identify secondary metabolite gene clusters. These approaches however failed in the identification of the [S,S]-EDDS biosynthetic genes. This required the development of a new approach which relies on the assumption that the zinc repressed biosynthesis of [S,S]-EDDS is regulated by a zinc responsive regulatory element. Therefore, the major zinc responsive transcriptional regulator of A. japonicum (Zur) was characterized in detail. Zur regulates the expression of the high affinity zinc uptake system ZnuABC by binding to a specific DNA binding sequence. The screening of the A. japonicum genome for further Zur regulated genes by using this deduced Zur binding sequence led to the identification of the operon aesA-D. Extensive transcriptional analyses and band shift assays revealed that aesA-D is zinc responsively regulated by Zur and involved in [S,S]-EDDS biosynthesis, as shown by inactivation studies. The [S,S]-EDDS biosynthesis was uncoupled from zinc repression by deleting zur. This mutant sets the stage to establish a sustainable [S,S]-EDDS production process without limits formerly imposed by zinc repression. The strategy to awake predicted silent gene clusters by using a characterized regulator as well as the strategy to identify new biosynthetic genes by characterizing an environmental signal-sensing regulator enabled the isolation of novel biosynthetic pathways in A. japonicum. Both approaches follow the joint concept to exploit knowledge of regulatory pathways and have the prospect to be generally applicable in order to guide future detection of new natural products.Der mikrobielle Sekundärmetabolismus ist eine reichhaltige Quelle für Naturstoffe, von denen viele klinische beziehungsweise industrielle Anwendung gefunden haben. Die Gattung Amycolatopsis ist für die Synthese vieler Naturstoffe bekannt. Beispielsweise werden viele Glykopeptid-Antibiotika, wie das klinisch relevante Vancomycin oder das Balhimycin, von Stämmen dieser Gattung produziert. Im Gegensatz dazu wurde der Stamm Amycolatopsis japonicum nie als Produzent einer biologisch aktiven Substanz beschrieben. Dieser Stamm produziert jedoch unter Zinkmangelbedingungen das EDTA-Isomer Ethylendiamindisuccinat ([S,S]-EDDS). Diese zinkabhängige [S,S]-EDDS Produktion lässt darauf schließen, dass [S,S]-EDDS ein Zinkophor ist, das an der Zinkaufnahme beteiligt ist. [S,S]-EDDS weist Komplexbildungseigenschaften auf, die mit denen von EDTA vergleichbar sind. Im Gegensatz zu EDTA ist [S,S]-EDDS jedoch biologisch abbaubar. Die weite industrielle Anwendung von EDTA in Kombination mit dessen Unzugänglichkeit für biologische Abbauprozesse führt zu einer umweltgefährdenden EDTA-Persistenz in aquatischen Lebensräumen. Der Naturstoff [S,S]-EDDS ist deshalb ein nachhaltiger EDTA Ersatz mit einem verbesserten ökologischen Fingerabdruck. In dieser Arbeit wurden zwei molekulargenetische Strategien entwickelt, um die Biosynthese des Glykopeptid-Antibiotikums Ristomycin A zu aktivieren und um die [S,S]-EDDS-Biosynthese-Gene in A. japonicum zu identifizieren. Untersuchungen des genetischen Potenzials der Gattung Amycolatopsis ließen vermuten, dass auch A. japonicum die Fähigkeit besitzt, ein Glykopeptid-Antibiotikum zu synthetisieren. Um dieses nicht exprimierte, sogenannte „stille Gencluster“ zu aktivieren, wurde ein molekulargenetischer Ansatz verwendet, bei dem der Biosynthese-spezifische Aktivator Bbr heterolog in A. japonicum exprimiert wurde. Bbr reguliert die Balhimycin-Biosynthese in Amycolatopsis balhimycina. In A. japonicum induzierte dessen Expression die Produktion von Ristomycin A, was durch HPLC-DAD, MS, MS/MS, HR-MS, und NMR-Analysen bestätigt werden konnte. Ristomycn A ist ein vielfach glykosyliertes Heptapeptid, das als Hauptwirkstoff in Diagnoseverfahren zur Bestimmung von angeborenen und weitverbreiteten Blutgerinnungsstörungen verwendet wird. Die Sequenzierung des A. japonicum Genoms und dessen computergestützte Auswertung führten zur Identifizierung des Biosynthese-Genclusters, das für die Synthese von Ristomycin A verantwortlich ist. Solche computergestützten Genomanalysen mittels verschiedenster bioinformatischen Plattformen werden heutzutage standardmäßig zur Identifizierung von Sekundärmetabolit-Gencluster angewandt, die bekannten Synthesemechanismen zugeordnet werden können. Allerdings konnten die [S,S]-EDDS-Biosynthese-Gene mit diesen Tools nicht entdeckt werden, was auf einen bislang nicht bekannten Biosynthesemechanismus hindeutet. Um diesen zu identifizieren, wurde ein neuer Ansatz entwickelt, der auf der Annahme beruht, dass die Zink-reprimierte [S,S]-EDDS-Biosynthese durch einen Zink-sensitiven Regulator gewährleistet wird. Die bakterielle Zink-Homöostase wird meistens durch den globalen Zink-spezifische Transkriptionsregulator Zur reguliert. Das Zur Protein von A. japonicum wurde identifiziert und detailliert charakterisiert. Es konnten gezeigt werden, dass ZurAj die Transkription des hoch affinen Zinkaufnahmesystems ZnuABCAj durch seine Zink-abhängige Bindung an spezifische DNA Bindesequenzen reguliert. Diese Zur-Bindesequenzen wurden verwendet, um das A. japonicum Genom nach weiteren, ZurAj regulierten, Genen zu durchsuchen. Dies führte zur Auffindung des aesA-D Operons. Umfangreiche Transkriptions-Untersuchungen ergaben, dass aesA-D Zink-abhängig von ZurAj reguliert wird. Die Beteiligung von aesA-D an der [S,S]-EDDS konnte durch Inaktivierungsversuche nachgewiesen werden. Zusätzlich führte die Deletion des Zinkregulators ZurAj (A. japonicum Δzur) dazu, dass auch in Gegenwart von hohen Zink-Konzentrationen [S,S]-EDDS in hohen Mengen produziert wird. A. japonicum Δzur ist eine erfolgversprechende Ausgangsbasis, um einen nachhaltigen und wirtschaftlich verwertbaren [S,S]-EDDS Produktionsprozess zu entwickeln, der keiner Limitierung durch negative Einflüsse von Zink unterliegt. Die Strategie, ein vorhergesagtes, stilles Gencluster durch die Expression eines spezifischen Regulators zu aktivieren, sowie auch die Strategie, neue Biosynthese-Gene durch die Charakterisierung eines globalen Regulators, der spezifische Umweltsignale wahrnimmt, zu identifizieren, ermöglichte die Charakterisierung neuer Naturstoffsynthesewege in A. japonicum. Beide Ansätze nutzen Erkenntnisse über regulatorische Mechanismen und besitzen das Potenzial zukünftig angewendet zu werden, um neue Naturstoffe und neue Synthesewege zu identifizieren

antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software