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

Biosynthetic gene cluster identification in plasmids and characterization of plasmids from animal-associated microbiota

Individual bacteria in complex microbial communities can acquire and accumulate new traits. These traits are reflective of their environment, being niche-specific. A major player in trait sharing is horizontal gene transfer (HGT). Plasmids, extrachromosomal DNA molecules, have a role in HGT and can change the host’s phenotype. Considering the transformative role of plasmids in bacterial lifestyle, we investigated the prevalence, distribution and products of biosynthetic gene clusters (BGCs) present in plasmids. Sequences available on the National Center for Biotechnology Information (NCBI) database (n=101 416) were run through two bioinformatic pipelines for BGC detection that apply different approaches, deepBGC and antiSMASH (antibiotics and secondary metabolites analysis shell). The highest percentage of plasmids with BGCs was detected in Actinobacteria but, apart from Chlamidiae and Tenericutes, all phyla had BGCs in their plasmids, with predictions varying according to the software used. The BGCs identified comprised a range of classes, indicating that plasmid encoded BGCs could be leveraged for the discovery of new molecules. In order to apply that concept to real-life examples, plasmids were isolated from animal-associated microbial communities and characterized. Plasmids from Escherichia coli isolated from wild birds (n=36) were screened for phenotypes of interest in human and animal health. Seven isolates displayed plasmid-encoded antibiotic resistance. Taxonomic identification of the hosts of plasmids isolated from bovid-associated microbiomes (n=38) was determined via 16S rRNA gene, and placed the majority of the isolated in the phylum Firmicutes, apart from a single Klebsiella pneumoniae isolate. Twelve plasmids were sequenced. Three plasmids from different hosts (pRAM-12, pRAM-19-2 and pRAM-30-2) shared 100% nucleotide sequence and a gene cluster for the bacteriocin cloacin. Two of those hosts shared not one, but two plasmids, pRAM-19-1 and pRAM-30-1, despite being in different phyla. This highlights the intimacy of gene sharing and the importance of HGT. pRAM-28 and pRAM-21 shared a plasmid that harbors the BGC for the bacteriocin aureocin A70, the only four peptide bacteriocin known to date. Additional analysis revealed two putative novel lanthipeptide gene clusters in pRAM-2. These results suggest that the plasmidome is a neglected source of secondary metabolites with the potential for molecule discovery. Furthermore, it can be leveraged to study genetic exchange in a community and how plasmid-encoded featured can mediate interactions in a microbiome

Mining Selected Metagenomes/metatranscriptomes for Biosynthetic Gene Clusters and Antimicrobial Resistance Genes

Antimicrobial resistance is one of the serious global challenges in the current century. The fact that resistance genes transfer between bacteria, coupled with the fact that the world is connected through complex dynamics. Studying microbial behavior and understanding the different factors coffering microbial resistance to a broad spectrum of the available drug classes, parallel with a comprehensive analysis of the natural microbial products as the primary source of the novel antibiotics, might shed some light on solutions for this problem. Microbial environments harbor a wide range of secondary metabolites (SM) with different functional groups. SMs are not directly involved in vital microbial processes such as reproduction, growth, and development. However, these organic compounds, which exist in many different chemical structures, carry out a broad range of functions. Some bioactive SMs are widely used in drug development of various therapeutic classes such as antibacterial, anticancer, immunosuppressant, diabetic, and cholesterol-lowering agents. These bioactive compounds’ metabolic pathways are encoded by co-localized genes collectively called Biosynthetic Gene Clusters (BGCs). The majority of the discovered bioactive natural products are from microbial strains that are cultivatable. However, the advancement in sequencing techniques, bioinformatics, and metagenomics opened unlimited opportunities to reach and study the uncultivatable microbial communities, which represent the more significant fraction of the underexplored microbial ecology. In this study, selected samples of seven selected metatranscriptomic/metagenomic datasets were subjected to assembly, taxonomic assignment to the reads, and assembled contigs. The aim of this study is two-fold. Firstly, the assembled contigs were then investigated by two primary distinct computational methods, namely antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) and deep-learning (deepBGC) methods. A comparative study was performed to determine the biosynthetic gene clusters (BGCs) present in each of the included samples and compare their taxonomic differences. Secondly, the assembled contigs were also analyzed to determine the antimicrobial resistance (AMR) genes present in each sample by using the Resistance Gene Identifier (RGI) algorithm, which is a part of the Comprehensive Antibiotic Resistance Database (CARD). A total of 65 samples from the seven selected v metagenomic and metatranscriptomic datasets were investigated by antiSMASH, deepBGC pipelines, and CARD in the present study. The different classes of detected BGCs and their corresponding microbial taxa and the antimicrobial resistance gene families and their corresponding resistance mechanisms against specific drug classes were reported. In the current study, we reported that the datasets with a large extent of variability (i.e. sex, age and illness state) due to the nature of their environments, such as host microbiome samples of patients in two ecosystems (COVID-19 & Atopic Dermatitis), gave the most variable number of BGC classes detected by antiSMASH, where 19 different classes detected in skin microbiome of AD patients and 16 different classes detected in gut microbiome of COVID-19 patients. On the other hand and due to the selection pressure on the microbial ecosystems by the wide use of antibiotics, gut microbiome of COVID-19 patients’ and water sewage samples had more than 70% of the detected AMR gene families where gut microbiome of COVID-19 patients’ sample alone reported to had more than 50% of AMR genes detected by CARD. In conclusion, ecological characteristics and microbial diversity in terms of composition and relative abundance dramatically affect the dynamics of secondary metabolites’ production and transferring antimicrobial resistance genes between bacteria. Microbial strains with higher biosynthetic and antimicrobial resistance potentials were enriched in environments with a rich microbial diversity such as host microbiome (i.e., COVID-19 patients), with patterns of abundance of biosynthetic gene clusters and AMR genes fluctuating by taxonomy

antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline

Secondary metabolites produced by bacteria and fungi are an important source of antimicrobials and other bioactive compounds. In recent years, genome mining has seen broad applications in identifying and characterizing new compounds as well as in metabolic engineering. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' (https://antismash.secondarymetabolites.org) has assisted researchers in this, both as a web server and a standalone tool. It has established itself as the most widely used tool for identifying and analysing biosynthetic gene clusters (BGCs) in bacterial and fungal genome sequences. Here, we present an entirely redesigned and extended version 5 of antiSMASH. antiSMASH 5 adds detection rules for clusters encoding the biosynthesis of acyl-amino acids, β-lactones, fungal RiPPs, RaS-RiPPs, polybrominated diphenyl ethers, C-nucleosides, PPY-like ketones and lipolanthines. For type II polyketide synthase-encoding gene clusters, antiSMASH 5 now offers more detailed predictions. The HTML output visualization has been redesigned to improve the navigation and visual representation of annotations. We have again improved the runtime of analysis steps, making it possible to deliver comprehensive annotations for bacterial genomes within a few minutes. A new output file in the standard JavaScript object notation (JSON) format is aimed at downstream tools that process antiSMASH results programmatically.</p

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

Integrating perspectives in actinomycete research: an ActinoBase review of 2020-21

Last year ActinoBase, a Wiki-style initiative supported by the UK Microbiology Society, published a review highlighting the research of particular interest to the actinomycete community. Here, we present the second ActinoBase review showcasing selected reports published in 2020 and early 2021, integrating perspectives in the actinomycete field. Actinomycetes are well-known for their unsurpassed ability to produce specialised metabolites, of which many are used as therapeutic agents with antibacterial, antifungal, or immunosuppressive activities. Much research is carried out to understand the purpose of these metabolites in the environment, either within communities or in host interactions. Moreover, many efforts have been placed in developing computational tools to handle big data, simplify experimental design, and find new biosynthetic gene cluster prioritisation strategies. Alongside, synthetic biology has provided advances in tools to elucidate the biosynthesis of these metabolites. Additionally, there are still mysteries to be uncovered in understanding the fundamentals of filamentous actinomycetes' developmental cycle and regulation of their metabolism. This review focuses on research using integrative methodologies and approaches to understand the bigger picture of actinomycete biology, covering four research areas: i) technology and methodology; ii) specialised metabolites; iii) development and regulation; and iv) ecology and host interactions

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

Advances in actinomycete research: an ActinoBase review of 2019

The actinomycetes are Gram-positive bacteria belonging to the order Actinomycetales within the phylum Actinobacteria. They include members with significant economic and medical importance, for example filamentous actinomycetes such as Streptomyces species, which have a propensity to produce a plethora of bioactive secondary metabolites and form symbioses with higher organisms, such as plants and insects. Studying these bacteria is challenging, but also fascinating and very rewarding. As a Microbiology Society initiative, members of the actinomycete research community have been developing a Wikipedia-style resource, called ActinoBase, the purpose of which is to aid in the study of these filamentous bacteria. This review will highlight 10 publications from 2019 that have been of special interest to the ActinoBase community, covering 4 major components of actinomycete research: (i) development and regulation; (ii) specialized metabolites; (iii) ecology and host interactions; and (iv) technology and methodology

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