@phdthesis{Dehm2020,
  author    = {Dehm, Daniel},
  title     = {Development of concepts for the genomic mining of novel secondary metabolites in symbiotic cyanobacteria},
  doi       = {10.25932/publishup-47834},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-478342},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VIII, 122, XII},
  year      = {2020},
  abstract  = {Naturstoffe sind seit der goldenen {\"A}ra der Antibiotika von immer gr{\"o}ßerem Interesse, sowohl f{\"u}r die Grundlagenforschung als auch die Angewandten Wissenschaften, da sie die Hauptquelle f{\"u}r neuartige Pharmazeutika mit starken antibiotischen, anti-entz{\"u}ndlichen und Antitumor-Aktivit{\"a}ten darstellen. Neben den technologischen Fortschritten im Bereich der Hochdurchsatz Genomsequenzierung und dem verbesserten Verst{\"a}ndnis des modularen Aufbaus der Biosynthesewege von Sekund{\"a}rmetaboliten, kam es auch zu einem Wechsel vom labor-gest{\"u}tzten Screening aktiver Zellextrakte hin zum Algorithmen-basierten in silico Screening nach neuen Naturstoff-Biosyntheseclustern. Obwohl die steigende Zahl verf{\"u}gbarer Genomsequenzen zeigte, dass nicht-ribosomale Peptid-Synthetasen (NRPS), Polyketid-Synthasen (PKS), und ribosomal synthetisierte und posttranslational modifizierte Peptide (RiPPs) ubiquit{\"a}r in allen Sparten des Lebens gefunden werden k{\"o}nnen, so zeigen einige Phyla wie Actinobakterien oder Cyanobakterien eine besonders hohe Dichte an Sekund{\"a}rmetabolitclustern. Der fakultativ symbiotische, N2-fixierende Modellorganismus N. punctiforme PCC73102 ist ein terrestrisches typ-IV Cyanobakterium, welches nicht nur einen besonders hohen Anteil seines Genoms der Produktion von Sekund{\"a}rmetaboliten widmet, sondern zus{\"a}tzlich noch genetisch modifizierbar ist. Eine AntiSMASH Analyse des Genoms zeigte, dass N. punctiforme insgesamt sechzehn potentielle Sekund{\"a}rmetabolitcluster besitzt, von denen aber bis heute nur zweien ein spezifisches Produkt zugewiesen werden konnte. Das macht N. punctiforme zu einem perfekten Testorganismus f{\"u}r die Entwicklung eines neuartigen kombinatorischen Genomic Mining Ansatzes zur Detektion von bislang unbeschriebenen Naturstoffen. Der neuartige Ansatz, der im Rahmen dieser Studie entwickelt wurde, stellt eine Kombination aus Genomic Mining, unabh{\"a}ngigen Monitoring-Techniken sowie modifizierten Kultivierungsbedingungen dar und f{\"u}hrte nicht nur zu neuen Erkenntnissen im Bereich cyanobakterieller Naturstoffsynthese, sondern letztlich auch zur Entdeckung eines neuen, von N. punctiforme produzierten, Naturstoffs. Die Herstellung und Untersuchung einer Reporterstamm Bibliothek, bestehend aus je einem CFP-produzierenden Transkriptionsreporter f{\"u}r jedes der sechzehn Sekund{\"a}rmetabolitcluster von N. punctiforme, zeigte, dass im Gegensatz zur Erwartung nicht alle Biosynthesecluster f{\"u}r die man kein Produkt nachweisen kann auch nicht exprimiert werden. Stattdessen konnten klar definierbare Expressionsmuster beschrieben werden, was deutlich machte, dass die Naturstoffproduktion einer engen Regulation unterliegt und nur ein kleiner Teil der Biosynthesecluster unter Standardbedingungen tats{\"a}chlich still sind. Dar{\"u}ber hinaus f{\"u}hrte die Erh{\"o}hung der Lichtintensit{\"a}t sowie der Kohlenstoffdioxid-Verf{\"u}gbarkeit zusammen mit der Kultivierung von N. punctiforme zu extrem hohen Zelldichten zu einer starken Erh{\"o}hung der gesamten metabolischen Aktivit{\"a}t des Organismus. N{\"a}here Untersuchungen der Zellextrakte dieser hoch-dichte Kultivierungen f{\"u}hrten letztlich zur Entdeckung einer neuartigen Gruppe von Microviridinen mit verl{\"a}ngerter Peptidsequenz, welche Microviridin N3-N9 genannt wurden. Sowohl die Kultivierung der Transkriptionsreporter als auch die RTqPCR-basierte Untersuchung der Transkriptionslevel der verschiedenen Biosynthesecluster zeigten, dass die hoch-Zelldichte Kultivierung von N. punctiforme zu einer Aktivierung von 50\% der vorhandenen Sekund{\"a}rmetabolitcluster f{\"u}hrt. Im Gegensatz zu dieser sehr breit-gef{\"a}cherten Aktivierung, f{\"u}hrt die Co-Kultivierung von N. punctiforme in chemischen oder physischen Kontakt zu einer N-gehungerten Wirtspflanze (Blasia pusilla) zu einer sehr spezifischen Aktivierung der RIPP4 und RiPP3 Biosynthesecluster. Obwohl dieser Effekt mittels verschiedener unabh{\"a}ngiger Methoden best{\"a}tigt werden konnte und trotz intensiver Analysebem{\"u}hungen, konnte jedoch keinem der beiden Cluster ein Produkt zugeordnet werden. Diese Studie stellt die erste weitreichende, systematische Analyse eines cyanobakteriellen Sekund{\"a}rmetaboloms durch einen kombinatorischen Ansatz aus Genomic Mining und unabh{\"a}ngigen Monitoring-Techniken dar und kann als neue strategische Herangehensweise f{\"u}r die Untersuchung anderer Organismen hinsichtlich ihrer Sekund{\"a}rmetabolit-Produktion dienen. Obwohl es bereits gut beschriebene einzelne Sekund{\"a}rmetabolite gibt, wie beispielweise den Zelldifferenzierungsfaktor PatS in Anabaena sp. PCC7120, so ist der Grad an Regulation der in dieser Studie gezeigt werden konnte bislang beispiellos und die Entschl{\"u}sselung dieser Mechanismen k{\"o}nnte die Entdeckung neuer Naturstoffe stark beschleunigen. Daneben lassen die Ergebnisse aber auch darauf schließen, dass die Induktion der Biosynthesewege nicht das eigentliche Problem darstellt, sondern vielmehr die verl{\"a}ssliche Detektion deren Produkte. Die Erarbeitung neuer Analytik-Strategien k{\"o}nnte somit auch einen deutlichen Einfluss auf die Geschwindigkeit der Entdeckung neuer Naturstoffe haben.},
  language  = {en}
}
@phdthesis{Krumbholz2021,
  author    = {Krumbholz, Julia},
  title     = {Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme},
  doi       = {10.25932/publishup-54024},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-540240},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xxiii, 187},
  year      = {2021},
  abstract  = {Specialized metabolites, so-called natural products, are produced by a variety of different organisms, including bacteria and fungi. Due to their wide range of different biological activities, including pharmaceutical relevant properties, microbial natural products are an important source for drug development. They are encoded by biosynthetic gene clusters (BGCs), which are a group of locally clustered genes. By screening genomic data for genes encoding typical core biosynthetic enzymes, modern bioinformatical approaches are able to predict a wide range of BGCs. To date, only a small fraction of the predicted BGCs have their associated products identified. The phylum of the cyanobacteria has been shown to be a prolific, but largely untapped source for natural products. Especially multicellular cyanobacterial genera, like Nostoc, harbor a high amount of BGCs in their genomes. A main goal of this study was to develop new concepts for the discovery of natural products in cyanobacteria. Due to its diverse setup of orphan BGCs and its amenability to genetic manipulation, Nostoc punctiforme PCC 73102 (N. punctiforme) appeared to be a promising candidate to be established as a model organism for natural product discovery in cyanobacteria. By utilizing a combination of genome-mining, bioactivity-screening, variations of culture conditions, as well as metabolic engineering, not only two new polyketides were discovered, but also first-time insights into the regulation of the specialized metabolism in N. punctiforme were gained during this study. The cultivation of N. punctiforme to very high densities by utilizing increasing light intensities and CO2 levels, led to an enhanced metabolite production, causing rather complex metabolite extracts. By utilizing a library of CFP reporter mutant strains, each strain reporting for one of the predicted BGCs, it was shown that eight out of 15 BGCs were upregulated under high density (HD) cultivation conditions. Furthermore, it could be demonstrated that the supernatant of an HD culture can increase the expression of four of the influenced BGCs, even under conventional cultivation conditions. This led to the hypothesis that a chemical mediator encoded by one of the affected BGCs is accumulating in the HD supernatant and is able to increase the expression of other BGCs as part of a cell-density dependent regulatory circuit. To identify which of the BGCs could be a main trigger of the presumed regulatory circuit, it was tried to activate four BGCs (pks1, pks2, ripp3, ripp4) selectively by overexpression of putative pathway-specific regulatory genes that were found inside the gene clusters. Transcriptional analysis of the mutants revealed that only the mutant strain targeting the pks1 BGC, called AraC_PKS1, was able to upregulate the expression of its associated BGC. From an RNA sequencing study of the AraC_PKS1 mutant strain, it was discovered that beside pks1, the orphan BGCs ripp3 and ripp4 were also upregulated in the mutant strain. Furthermore, it was observed that secondary metabolite production in the AraC_PKS1 mutant strain is further enhanced under high-light and high-CO2 cultivation conditions. The increased production of the pks1 regulator NvlA also had an impact on other regulatory factors, including sigma factors and the RNA chaperone Hfq. Analysis of the AraC_PKS1 cell and supernatant extracts led to the discovery of two novel polyketides, nostoclide and nostovalerolactone, both encoded by the pks1 BGC. Addition of the polyketides to N. punctiforme WT demonstrated that the pks1-derived compounds are able to partly reproduce the effects on secondary metabolite production found in the AraC_PKS1 mutant strain. This indicates that both compounds are acting as extracellular signaling factors as part of a regulatory network. Since not all transcriptional effects that were found in the AraC_PKS1 mutant strain could be reproduced by the pks1 products, it can be assumed that the regulator NvlA has a global effect and is not exclusively specific to the pks1 pathway. This study was the first to use a putative pathway specific regulator for the specific activation of BGC expression in cyanobacteria. This strategy did not only lead to the detection of two novel polyketides, it also gave first-time insights into the regulatory mechanism of the specialized metabolism in N. punctiforme. This study illustrates that understanding regulatory pathways can aid in the discovery of novel natural products. The findings of this study can guide the design of new screening strategies for bioactive compounds in cyanobacteria and help to develop high-titer production platforms for cyanobacterial natural products.},
  language  = {en}
}
@phdthesis{ReynaGonzalez2017,
  author    = {Reyna Gonz{\´a}lez, Emmanuel},
  title     = {Engineering of the microviridin post-translational modification enzymes for the production of synthetic protease inhibitors},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-406979},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XI, 91, CI},
  year      = {2017},
  abstract  = {Natural products and their derivatives have always been a source of drug leads. In particular, bacterial compounds have played an important role in drug development, for example in the field of antibiotics. A decrease in the discovery of novel leads from natural sources and the hope of finding new leads through the generation of large libraries of drug-like compounds by combinatorial chemistry aimed at specific molecular targets drove the pharmaceutical companies away from research on natural products. However, recent technological advances in genetics, bioinformatics and analytical chemistry have revived the interest in natural products. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of natural products generated by the action of post-translationally modifying enzymes on precursor peptides translated from mRNA by ribosomes. The great substrate promiscuity exhibited by many of the enzymes from RiPP biosynthetic pathways have led to the generation of hundreds of novel synthetic and semisynthetic variants, including variants carrying non-canonical amino acids (ncAAs). The microviridins are a family of RiPPs characterized by their atypical tricyclic structure composed of lactone and lactam rings, and their activity as serine protease inhibitors. The generalities of their biosynthetic pathway have already been described, however, the lack of information on details such as the protease responsible for cleaving off the leader peptide from the cyclic core peptide has impeded the fast and cheap production of novel microviridin variants. In the present work, knowledge on leader peptide activation of enzymes from other RiPP families has been extrapolated to the microviridin family, making it possible to bypass the need of a leader peptide. This feature allowed for the exploitation of the microviridin biosynthetic machinery for the production of novel variants through the establishment of an efficient one-pot in vitro platform. The relevance of this chemoenzymatic approach has been exemplified by the synthesis of novel potent serine protease inhibitors from both rationally-designed peptide libraries and bioinformatically predicted microviridins. Additionally, new structure-activity relationships (SARs) could be inferred by screening microviridin intermediates. The significance of this technique was further demonstrated by the simple incorporation of ncAAs into the microviridin scaffold.},
  language  = {en}
}