Metabolic and genomic profiling of actinobacteria strains : from new natural products to biosynthetic pathways

Marine actinomycetes are known to be a promising source for new natural products with putative application as therapeutic agents. Thus, the exploitation of novel discovered actinomycetes strains remains in the focus of NP research. The presented thesis deals on the one hand with the isolation and structural characterization of new NPs produced by streptomycetes species. On the other hand, the construction of a novel biosensor concept for the detection of secondary metabolite production is discussed. New spiroindimicins E and F were isolated from the Streptomyces sp. MP131-18 and structures were confirmed by NMR. Additionally, two new lagunapyrones D and E were identified by characteristic MS/MS fragmentation. The genome of MP131-18 has been sequenced and the gene cluster, responsible for the synthesis of bisindole compounds has been identified and connected to lynamicins/spiroindimicin production. Furthermore, four new alpiniamides B-D have been isolated from the Streptomyces sp. IB 2014/11-12. The sequenced genome enabled the identification of the gene cluster responsible for alpiniamide production. The predicted biosynthetic pathway was confirmed by feeding experiments and gene deletions in a heterologous host. Finally, a contribution to the construction of a repressor-based biosensor, which detects the products of awakened silent gene clusters in streptomycetes, has been made. This concept was successfully applied to the activated coelimycin gene cluster.  Marine Aktinomyceten stellen eine beliebte Quelle neuer Naturstoffe dar, welche unter Umständen Anwendung als therapeutische Arzneimittel finden. Daher steht die Untersuchung neuer Aktinomyceten Stämme im Fokus der Naturstoffforschung. Die dargelegte Arbeit handelt von der Isolierung und strukturellen Charakterisierung neuer Naturstoffe, welche von Streptomyceten produziert wurden. Desweiteren, wird die Konstruktion eines neuen Biosensors dargestellt, welcher die Produktion von Sekundärmetaboliten detektieren soll. Es wurden zwei neue spiroindimicine E und F aus der Streptomyces sp. MP131-18 isoliert und die chemische Struktur mittels NMR bestätigt. Das Genom von MP131-18 wurde sequenziert und der Gencluster, welcher verantwortlich ist für die Synthese der Bisindole-Substanzen identifiziert und den Lynamicinen/Spiroindmicinen zugeordnet. Weiterhin, wurden vier neue Alpiniamide B-D aus dem Stamm Streptomyces sp. IB 2014/11-12 isoliert. Das sequenzierte Genom ermöglichte die Identifizierung des Genclusters, welcher für die Alpiniamide Produktion verantwortlich ist. Die vorgeschlagene Biosyntheseroute wurde bestätigt durch Fütterungsexperimente und Gendeletierungen in einem Hostorganismus. Abschließend wurde ein Beitrag zur Konstruktion eines Repressor-basierender Biosensors geleistet, welcher die Aktivierung von zuvor inaktiven Gencluster in Streptomyceten detektieren soll. Dieses Konzept wurde erfolgreich an dem aktivierten Coelimycin Gencluster angewendet

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992)

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method

New Kendomycin Derivative Isolated from Streptomyces sp. Cl 58-27

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools

New Alpiniamides From Streptomyces sp. IB2014/011-12 Assembled by an Unusual Hybrid Non-ribosomal Peptide Synthetase Trans-AT Polyketide Synthase Enzyme

The environment of Lake Baikal is a well-known source of microbial diversity. The strain Streptomyces sp. IB2014/011-12, isolated from samples collected at Lake Baikal, was found to exhibit potent activity against Gram-positive bacteria. Here, we report isolation and characterization of linear polyketide alpiniamide A (1) and its new derivatives B–D (2–5). The structures of alpiniamides A–D were established and their relative configuration was determined by combination of partial Murata’s method and ROESY experiment. The absolute configuration of alpiniamide A was established through Mosher’s method. The gene cluster, responsible for the biosynthesis of alpiniamides (alp) has been identified by genome mining and gene deletion experiments. The successful expression of the cloned alp gene cluster in a heterologous host supports these findings. Analysis of the architecture of the alp gene cluster and the feeding of labeled precursors elucidated the alpiniamide biosynthetic pathway. The biosynthesis of alpiniamides is an example of a rather simple polyketide assembly line generating unusual chemical diversity through the combination of domain/module skipping and double bond migration events

Miramides A–D : Identification of Detoxin-like Depsipeptides after Heterologous Expression of a Hybrid NRPS-PKS Gene Cluster from Streptomyces mirabilis Lu17588

Natural products derived from plants, fungi or bacteria have been used for years in the medicine, agriculture and food industries as they exhibit a variety of beneficial properties, such as antibiotic, antifungal, anticancer, herbicidal and immunosuppressive activities. Compared to synthetic compounds, natural products possess a greater chemical diversity, which is a reason why they are profitable templates for developing pharmaceutical drug candidates and ongoing research on them is inevitable. Performing heterologous expression with unknown gene clusters is the preferred method to activate gene clusters that are not expressed in the wild-type strain under laboratory conditions; thus, this method offers a way to discover new interesting metabolites. Here, we report the gene cluster assembly of a hybrid NRPS-PKS gene cluster from Streptomyces mirabilis Lu17588, which was heterologously expressed in Streptomyces albus Del14. Four new compounds were produced by the obtained strain, which were named miramides A–D. Isolation and structure elucidation revealed similarity of the isolated compounds to the known depsipeptides rimosamides/detoxins

An arrhythmogenic metabolite in atrial fibrillation

Abstract Background Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. Methods and results Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. Conclusion Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF

New Kendomycin Derivative Isolated from <i>Streptomyces</i> sp. Cl 58-27

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992)

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method

New Kendomycin Derivative Isolated from sp. Cl 58-27.

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools

Targeted Genome Mining-From Compound Discovery to Biosynthetic Pathway Elucidation.

Natural products are an important source of novel investigational compounds in drug discovery. Especially in the field of antibiotics, Actinobacteria have been proven to be a reliable source for lead structures. The discovery of these natural products with activity- and structure-guided screenings has been impeded by the constant rediscovery of previously identified compounds. Additionally, a large discrepancy between produced natural products and biosynthetic potential in Actinobacteria, including representatives of the order Pseudonocardiales, has been revealed using genome sequencing. To turn this genomic potential into novel natural products, we used an approach including the in-silico pre-selection of unique biosynthetic gene clusters followed by their systematic heterologous expression. As a proof of concept, fifteen Saccharothrixespanaensis genomic library clones covering predicted biosynthetic gene clusters were chosen for expression in two heterologous hosts, Streptomyceslividans and Streptomycesalbus. As a result, two novel natural products, an unusual angucyclinone pentangumycin and a new type II polyketide synthase shunt product SEK90, were identified. After purification and structure elucidation, the biosynthetic pathways leading to the formation of pentangumycin and SEK90 were deduced using mutational analysis of the biosynthetic gene cluster and feeding experiments with 13C-labelled precursors