Deciphering novel mechanisms of bacterial secondary metabolite biosynthetic pathways

Bacterial secondary metabolites display enormous structural diversity with astonishing biological activities. This thesis confers new insights into the biosynthesis of the myxobacterial secondary metabolites tubulysins by both in vitro and in vivo experiments, along with the identification and characterization of new structural variants. The cryptic biosynthetic pathway of the aurachins in Stigmatella aurantiaca Sg a15 was elucidated. In vitro experiments revealed an unparalleled priming mechanism for the anthranilate starter unit. In vivo gene inactivation experiments resulted in the identification of the tailoring functionalities responsible for the successive transformations from aurachin D to aurachin A. Another topic of this thesis was the investigation of the biosynthesis of 2-heptyl-4(1H)-quinolone (HHQ), an important quorum sensing molecule in Pseudomonas aeruginosa. In vitro reconstitution of HHQ biosynthesis by employing recombinant PqsD protein has shed light on this enzyme and its substrates, leading to the development of an in vitro test system for the identification of HHQ biosynthesis inhibitors, a promising alternative target in P. aeruginosa therapy. Exploiting the newly sequenced genome of S. aurantiaca Sg a15 a genome mining approach, combining targeted gene inactivation with a statistics based comparative secondary metabolite profile analysis, was adapted and employed. This culminated in the identification and characterization of the rhizopodin biosynthetic gene cluster.Von Bakterien produzierte Sekundärmetabolite weisen enorme strukturelle Diversität und erstaunliche biologische Aktivitäten auf. Diese Arbeit gewährt neue Einblicke in die Biosynthese der myxobakteriellen Sekundärmetabolitfamilie der Tubulysine durch in vitro und in vivo Experimente zusammen mit der Identifizierung und Charakterisierung neuer struktureller Varianten. Die komplizierte Biosynthese der Aurachine in Stigmatella aurantiaca Sg a15 wurde aufgeklärt. In vitro Experimente legten einen einmaligen Mechanismus zur Bereitstellung der Anthranilsäure Startereinheit offen und in vivo Inaktivierungsexperimente resultierten in der Identifizierung der modifizierenden Enzyme, die für die schrittweise Umwandlung von Aurachin D zu Aurachin A verantwortlich sind. Ein weiterer Gegenstand dieser Arbeit war die Untersuchung der Biosynthese von 2 Heptyl-4(1H)-chinolin (HHQ), ein wichtiges Quorum sensing Molekül in Pseudomonas aeruginosa. Die Rekonstruktion der HHQ-Biosynthese in vitro mittels rekombinantem PqsD Protein gab Aufschluss über dieses Enzym und seine Substrate und führte zur Entwicklung eines in vitro Testsystems zur Identifizierung von Inhibitoren der Biosynthese von HHQ, ein vielverspechendes alternatives Angriffsziel für die Therapie von Infektionen mit P. aeruginosa. Das kürzlich sequenzierte Genom von S. aurantiaca Sg a15 wurde für einen „Genome mining“ Ansatz genutzt. Dieser setzt sich zusammen aus der gezielten Inaktivierung bestimmter Gene und einer statistikgestützten vergleichenden Analyse der Sekundärmetabolitprofile. Dadurch konnte der Biosynthesegencluster von Rhizopodin identifiziert und charakterisiert werden

Kendomycin Cytotoxicity against Bacterial, Fungal, and Mammalian Cells Is Due to Cation Chelation

Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.Peer reviewe

The retrospective analysis of Antarctic tracking data project

The Retrospective Analysis of Antarctic Tracking Data (RAATD) is a Scientific Committee for Antarctic Research project led jointly by the Expert Groups on Birds and Marine Mammals and Antarctic Biodiversity Informatics, and endorsed by the Commission for the Conservation of Antarctic Marine Living Resources. RAATD consolidated tracking data for multiple species of Antarctic meso- and top-predators to identify Areas of Ecological Significance. These datasets and accompanying syntheses provide a greater understanding of fundamental ecosystem processes in the Southern Ocean, support modelling of predator distributions under future climate scenarios and create inputs that can be incorporated into decision making processes by management authorities. In this data paper, we present the compiled tracking data from research groups that have worked in the Antarctic since the 1990s. The data are publicly available through biodiversity.aq and the Ocean Biogeographic Information System. The archive includes tracking data from over 70 contributors across 12 national Antarctic programs, and includes data from 17 predator species, 4060 individual animals, and over 2.9 million observed locations

The retrospective analysis of Antarctic tracking data project

The Retrospective Analysis of Antarctic Tracking Data (RAATD) is a Scientific Committee for Antarctic Research project led jointly by the Expert Groups on Birds and Marine Mammals and Antarctic Biodiversity Informatics, and endorsed by the Commission for the Conservation of Antarctic Marine Living Resources. RAATD consolidated tracking data for multiple species of Antarctic meso- and top-predators to identify Areas of Ecological Significance. These datasets and accompanying syntheses provide a greater understanding of fundamental ecosystem processes in the Southern Ocean, support modelling of predator distributions under future climate scenarios and create inputs that can be incorporated into decision making processes by management authorities. In this data paper, we present the compiled tracking data from research groups that have worked in the Antarctic since the 1990s. The data are publicly available through biodiversity.aq and the Ocean Biogeographic Information System. The archive includes tracking data from over 70 contributors across 12 national Antarctic programs, and includes data from 17 predator species, 4060 individual animals, and over 2.9 million observed locations.Supplementary Figure S1: Filtered location data (black) and tag deployment locations (red) for each species. Maps are Lambert Azimuthal projections extending from 90° S to 20° S.Supplementary Table S1: Names and coordinates of the major study sites in the Southern Ocean and on the Antarctic Continent where tracking devices were deployed on the selected species (indicated by their 4-letter codes in the last column).Online Table 1: Description of fields (column names) in the metadata and data files.Supranational committees and organisations including the Scientific Committee on Antarctic Research Life Science Group and BirdLife International. National institutions and foundations, including but not limited to Argentina (Dirección Nacional del Antártico), Australia (Australian Antarctic program; Australian Research Council; Sea World Research and Rescue Foundation Inc., IMOS is a national collaborative research infrastructure, supported by the Australian Government and operated by a consortium of institutions as an unincorporated joint venture, with the University of Tasmania as Lead Agent), Belgium (Belgian Science Policy Office, EU Lifewatch ERIC), Brazil (Brazilian Antarctic Programme; Brazilian National Research Council (CNPq/MCTI) and CAPES), France (Agence Nationale de la Recherche; Centre National d’Etudes Spatiales; Centre National de la Recherche Scientifique; the French Foundation for Research on Biodiversity (FRB; www.fondationbiodiversite.fr) in the context of the CESAB project “RAATD”; Fondation Total; Institut Paul-Emile Victor; Programme Zone Atelier de Recherches sur l’Environnement Antarctique et Subantarctique; Terres Australes et Antarctiques Françaises), Germany (Deutsche Forschungsgemeinschaft, Hanse-Wissenschaftskolleg - Institute for Advanced Study), Italy (Italian National Antarctic Research Program; Ministry for Education University and Research), Japan (Japanese Antarctic Research Expedition; JSPS Kakenhi grant), Monaco (Fondation Prince Albert II de Monaco), New Zealand (Ministry for Primary Industries - BRAG; Pew Charitable Trusts), Norway (Norwegian Antarctic Research Expeditions; Norwegian Research Council), Portugal (Foundation for Science and Technology), South Africa (Department of Environmental Affairs; National Research Foundation; South African National Antarctic Programme), UK (Darwin Plus; Ecosystems Programme at the British Antarctic Survey; Natural Environment Research Council; WWF), and USA (U.S. AMLR Program of NOAA Fisheries; US Office of Polar Programs).http://www.nature.com/sdataam2021Mammal Research Institut

Aufklärung neuartiger Mechanismen in Biosynthesewegen bakterieller Sekundärmetabolite

Bacterial secondary metabolites display enormous structural diversity with astonishing biological activities. This thesis confers new insights into the biosynthesis of the myxobacterial secondary metabolites tubulysins by both in vitro and in vivo experiments, along with the identification and characterization of new structural variants. The cryptic biosynthetic pathway of the aurachins in Stigmatella aurantiaca Sg a15 was elucidated. In vitro experiments revealed an unparalleled priming mechanism for the anthranilate starter unit. In vivo gene inactivation experiments resulted in the identification of the tailoring functionalities responsible for the successive transformations from aurachin D to aurachin A. Another topic of this thesis was the investigation of the biosynthesis of 2-heptyl-4(1H)-quinolone (HHQ), an important quorum sensing molecule in Pseudomonas aeruginosa. In vitro reconstitution of HHQ biosynthesis by employing recombinant PqsD protein has shed light on this enzyme and its substrates, leading to the development of an in vitro test system for the identification of HHQ biosynthesis inhibitors, a promising alternative target in P. aeruginosa therapy. Exploiting the newly sequenced genome of S. aurantiaca Sg a15 a genome mining approach, combining targeted gene inactivation with a statistics based comparative secondary metabolite profile analysis, was adapted and employed. This culminated in the identification and characterization of the rhizopodin biosynthetic gene cluster.Von Bakterien produzierte Sekundärmetabolite weisen enorme strukturelle Diversität und erstaunliche biologische Aktivitäten auf. Diese Arbeit gewährt neue Einblicke in die Biosynthese der myxobakteriellen Sekundärmetabolitfamilie der Tubulysine durch in vitro und in vivo Experimente zusammen mit der Identifizierung und Charakterisierung neuer struktureller Varianten. Die komplizierte Biosynthese der Aurachine in Stigmatella aurantiaca Sg a15 wurde aufgeklärt. In vitro Experimente legten einen einmaligen Mechanismus zur Bereitstellung der Anthranilsäure Startereinheit offen und in vivo Inaktivierungsexperimente resultierten in der Identifizierung der modifizierenden Enzyme, die für die schrittweise Umwandlung von Aurachin D zu Aurachin A verantwortlich sind. Ein weiterer Gegenstand dieser Arbeit war die Untersuchung der Biosynthese von 2 Heptyl-4(1H)-chinolin (HHQ), ein wichtiges Quorum sensing Molekül in Pseudomonas aeruginosa. Die Rekonstruktion der HHQ-Biosynthese in vitro mittels rekombinantem PqsD Protein gab Aufschluss über dieses Enzym und seine Substrate und führte zur Entwicklung eines in vitro Testsystems zur Identifizierung von Inhibitoren der Biosynthese von HHQ, ein vielverspechendes alternatives Angriffsziel für die Therapie von Infektionen mit P. aeruginosa. Das kürzlich sequenzierte Genom von S. aurantiaca Sg a15 wurde für einen „Genome mining“ Ansatz genutzt. Dieser setzt sich zusammen aus der gezielten Inaktivierung bestimmter Gene und einer statistikgestützten vergleichenden Analyse der Sekundärmetabolitprofile. Dadurch konnte der Biosynthesegencluster von Rhizopodin identifiziert und charakterisiert werden

Discovery of the rhizopodin biosynthetic gene cluster in Stigmatella aurantiaca Sg a15 by genome mining

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript

A semipinacol rearrangement directed by an enzymatic system featuring dual-function FAD-dependent monooxygenase

Nature has invented ingenious ways to biosynthesize biologically active small molecules that have been applied ever since to benefit human life in various ways. During the underlying biosynthetic processes, highly elaborate chemical reactions are often catalyzed by enzymatic systems, thereby enabling transformations under physiological conditions that would require harsh conditions or are hardly possible without enzymatic catalysis. Consequently, understanding novel biochemical transformations is of importance to eventually apply the knowledge gained to generate molecules of interest

Deliberations on Natural Products and Future Directions in the Pharmaceutical Industry

Natural Products are molecular “special equipment” that impart survival benefits on their producers in nature. Due to their evolved functions to modulate biology these privileged metabolites are substantially represented in the drug market and are continuing to contribute to the discovery of innovative medicines such as the recently approved semi-synthetic derivative of the bacterial alkaloid staurosporin in oncology indications. The innovation of low molecular weight compounds in modern drug discovery is built on rapid progress in chemical, molecular biological, pharmacological and data sciences which together provide a rich understanding of disease-driving molecular interactions and how to modulate them. NPs investigated in these pharmaceutical research areas create new perspectives on their chemical and biological features and thereby new chances to advance medical research. New methods in analytical chemistry linked with searchable NP-databases solved the issue of reisolation and enabled targeted and efficient access to novel molecules from nature. Cheminformatics delivers high resolution descriptions of NPs and explores the substructures that systematically map NP-chemical space by sp3-enriched fragments. Whole genome sequencing has revealed the existence of collocated gene clusters that form larger functional entities together with proximate resistance factors thus avoiding self-inhibition of the encoded metabolites. The analysis of bacterial and fungal genes provides tantalizing glimpses of new compound-target pairs of therapeutic value. Furthermore, a dedicated investigation of structurally unique, selectively active NPs in chemical biology demonstrates their extraordinary power as shuttles between new biological target spaces of pharmaceutical relevance

Genetic Engineering of Chromobacterium Vaccinii DSM 25150 for Improved Production of FR900359

The depsipeptide FR900359 has been first described in literature in 1988 (Fujioka et al, 1988) to be isolated from a methanol extract of the whole plant of Ardisia crenata. FR900359 can be isolated from the leaves of A. crenata, but the very low quantities and the complex matrix prevent access to sufficient amounts of FR900359 to enable drug development efforts and potential commercial manufacturing. Almost two decades later, it has been discovered that FR900359 is in fact produced by a strictly obligate bacterial endosymbiont, Candidatus Burkholderia crenata, of the plant Ardisia crenata (Carlier et al, 2016). This study identified also the DNA sequence of the biosynthetic gene cluster (BGC) of FR900359. In order to identify alternative and scalable methods for production of FR900359, a genome mining effort on bacterial genomes from both public sequence databases and genome sequences generated from internal efforts at Novartis was initiated. Translated amino acid sequences of the FR900359‑BGC from Candidatus B. crenata were used as query sequence. While the query of public sequence databases did not return highly similar sequences, a gene cluster with very high homology in translated amino acid sequence and identical prediction of protein functions was discovered in the genome of Chromobacterium vaccinii DSM 25150, which had been sequenced internally at Novartis. Here we describe the genetic engineering of Chromobacterium vaccinii DSM 25150 resulting in mutants that exhibit improved production of FR900359 and improved characteristics concerning downstream processing and purification.</p

Chemical Constituents from Gouania longipetala and Glyphaea brevis

Five compounds were isolated altogether from the two medicinal plants. Glycerol monovalerate (1), palmarumycin BG1 (2), and de-O-methyllasiodiplodin (3) were isolated from G. longipetala. Additionally, epicatechin (4) and its’ dimer procyanidin B2 (5) were isolated from the stem bark of G. brevis. Their structures were elucidated by spectroscopic experiments. 1 displayed moderate antibacterial activities with the least MIC of 125 µg/mL against E. faecalis. In addition, 1 reduced DPPH with an IC50 value of 102.34 µg/mL