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Item Open Access Investigation of the impact of different scale-up dependent stimuli on metabolism and population heterogeneity in Corynebacterium glutamicum(2024) Eilingsfeld, Adrian; Takors, Ralf (Prof. Dr.-Ing.)This thesis investigates the impact of elevated carbon dioxide levels on population heterogeneity in Corynebacterium glutamicum, a widely used industrial production host. Through a series of experiments involving cultivation at varying CO2 partial pressures, flow cytometry, and analysis of DNA content, the research reveals that increased CO2 exerts significant selection pressure, affecting growth rates and cell aggregation tendencies. Key findings indicate that higher growth rates speed up DNA replication levels, while elevated CO2 levels slow them down. The results contribute to understanding how CO2 influences population dynamics, providing insights for optimizing industrial bioprocesses and support Corynebacterium glutamicum as a robust production strain.Item Open Access Gold nanoparticle-mediated DNA origami nanoarchitectures(2024) Peil, Andreas; Na Liu, Laura (Prof. Dr.)Since its origin in the 1980s, DNA (deoxyribonucleic acid) nanotechnology has established itself as a captivating nanofabrication technique with ever increasing impact that combines aspects from physics, chemistry, and biology to construct artificial nanosystems by means of molecular self assembly. Within the field of DNA nanotechnology, the DNA origami technique represents one of the most versatile fabrication tools to craft functional two-dimensional (2D) and three-dimensional (3D) nanostructures from the bottom up. These structures offer precisely tailored geometries along with programmable functions, featuring positional addressability with sub-5 nm resolution and exceptional spatiotemporal accuracy. This thesis discusses strategies to employ the DNA origami technique to assemble intricate hybrid nanosystems with synergistically integrated gold nanoparticles (AuNPs). The AuNPs take over different roles; they grant (i) structural and (ii) functional features and enable the (iii) optical monitoring of the systems. This approach allows the fabrication of nanostructures piece by piece to explore their structural and functional properties at the nanoscale in detail. The first publication covers different strategies for the hierarchical assembly of topological DNA origami structures using a AuNP-templated self-assembly approach. The assembly of [2], [3], and [4]catenanes with interconnecting AuNPs is elucidated. The AuNPs can be controllably released to disconnect the individual rings, leaving only the mechanical bond of the catenane chain. In the second publication, a dynamic AuNP-DNA origami gear system is presented that is designed to emulate a planetary gearset with precise spatiotemporal control over its rotation dynamics. The AuNPs serve three crucial tasks. They (i) structurally link the origami ring modules, (ii) mediate the rotation and (iii) enable the real time optical tracking of the rotation via fluorescence spectroscopy. The system enables tightly orchestrated and programmable bidirectional rotations. In the third publication, reconfigurable chiral metastructures comprising multiple plasmonic particles that are accurately positioned in a helical manner around a DNA origami template are discussed. The implementation of a DNA ‘swingarm strategy’ enables the simultaneous and efficient relocation of multiple closely spaced AuNPs over large distances to precisely tune the chiroptical response of the system. The presented publications illustrate the beneficial synergies between DNA origami systems and rationally integrated AuNPs with the aim to advance and expand the application spectrum of these hybrid nanosystems within their scientific disciplines.Item Open Access Einfluss der Protein Arginin Methyltransferase 6 auf die Adipogenese(2024) Gerstner, Mirjam; Lausen, Jörn (Prof. Dr.)Transkriptionsfaktoren und Histon-modifizierende Enzyme sind zentrale Regulatoren der Genexpression sowie der Differenzierung. Die Adipogenese wird durch ein Netzwerk an verschiedenen Transkriptionsfaktoren reguliert. Bei der Differenzierung von MSCs legen Transkriptionsfaktoren fest, in welchen Zelltyp die Zellen differenzieren. Eine wesentliche Rolle in der Adipozyten-Differenzierung spielen Pparγ und C/ebpα. Diese Transkriptionsfaktoren bilden während der Adipogenese eine Vorwärtsschleife zur gegenseitigen Verstärkung aus. Pparγ interagiert mit epigenetischen Cofaktoren um C/ebpα und das nachgeschaltete adipozytäre Genexpressionsprogramm zu aktivieren und zu etablieren. Für das Verständnis der Adipogenese, bei normaler Differenzierung sowie bei Krankheit, ist die Kenntnis der epigenetischen Cofaktoren und Signalwege, die mit Pparγ in Verbindung stehen, von zentraler Bedeutung. In der vorliegenden Arbeit wurde gezeigt, dass Prmt6 und Pparγ in Vorläuferzellen gemeinsam an die Promotorregionen von C/ebpα und Pparγ binden. Dies führt zu einer reduzierten Expression dieser Gene, welche auch durch die katalytische Fähigkeit zur Induktion von H3R2me2a, beeinflusst wird. Durch Induktion Adipogenese, verlässt die Protein Arginin Methyltransferase 6 die Promotoren von C/ebpα und Pparγ und das adipozytäre Genexpressionsprogramm wird aktiviert. Durch die einen CRISPR/Cas9 vermittelten Knockout von Prmt6, sowie der Hemmung von Prmt6 durch den Inhibitor SGC6870, konnte der negative Effekt von Prmt6 auf die Adipogenese gezeigt werden. Der Verlust von Prmt6 wirkte fördernd auf die Adipogenese. Wohingegen eine Überexpression von Prmt6 die Differenzierung in Adipozyten hemmte. Dieses Wissen eröffnet damit die Möglichkeit einer epigenetischen Manipulation der Differenzierung für therapeutische Zwecke. Darüber hinaus wurde die Beteiligung des für die Osteogenese relevanten Transkriptionsfaktors Runx2 an diesem Corepressorkomplex an den Loci nachgewiesen. Daher liegt die Vermutung nahe, dass dieser Komplex essenziell zur Linienentscheidung zwischen Adipogenese und Osteogenese beiträgt. Diese Daten liefern detaillierte Informationen über den molekularen Mechanismus, der die Pparγ-C/ebpα Vorwärtsschleife steuert. Sie tragen somit zu unserem Verständnis der Adipogenese bei normaler und krankhaft veränderter Adipogenese bei.Item Open Access From stress to acclimation : a systems biology look on the life of Saccharomyces cerevisiae in industrial bioreactors(2024) Minden, Steven; Takors, Ralf (Prof. Dr.-Ing.)Carbon limitation is a fundamental feeding strategy in commercial fermentations guaranteeing efficient substrate-to-product conversion. However, industrial reaction volumes often prevent a microbe from performing optimally. One common source of interference is insufficient mixing resulting in the formation of concentration gradients. For instance, faster microbial consumption versus convective supply depletes the highly diluted limiting substrate locally. The industrial workhorse Saccharomyces cerevisiae (S. cerevisiae) naturally possesses adaptive mechanisms to cope with substrate depletion. Whether triggered response mechanisms benefit strain performance is doubtful, given that enough substrate is present in an industrial carbonlimited process on average. On the contrary, unnecessary or futile adaptation mechanisms often cause unexpected microbial behavior on large scales. Exploring and elucidating this behavior is the focal point of this thesis. The presented case study employs a stimulus-response approach mimicking a baker’s yeast fermentation snapshot featuring non-ideal starvation zones. In brief, glucose-limited chemostats with two-minute intervals of stopped feeding induce transitions between limitation and starvation. Metabolomic and transcriptomic measurements enable a systems biology analysis of either non-adapted or stimulus-adapted yeasts. One part of this study investigates the haploid laboratory strain CEN.PK113-7D under aerobic conditions. Another part reports gene expression dynamics of the diploid industrial strain Ethanol RedTM under anaerobic conditions. Both strains display robust growth under the tested conditions at the cost of tactic and strategic investments. The laboratory yeast responds to a 110 μmol·L-1 glucose gradient with a modified energy and redox homeostasis. Non-adapted cells perceive this stimulus as a threat, as evidenced by a futile triggering of the environmental stress response causing transient growth rate reduction and increased maintenance demand. Complete adaptation evokes a distinct ‘bioreactor phenotype’ characterized by increased growth capacities and repressed stress response. Results obtained with Ethanol RedTM confirm this stress defense-growth trade-off to be a conserved implication in bioprocesses with fluctuating carbon supply. Altogether, the findings presented in this thesis contribute to a fundamental understanding of how S. cerevisiae operates in heterogeneous commercial-scale fermentations. Finally, the gained knowledge reveals optimization targets for both strain engineering and bioprocess development.Item Open Access Introduction of novel artificial pathways in Escherichia coli with fructose 6-phosphate aldolase (FSA)(2024) Guitart Font, Emma; Sprenger, Georg A. (Prof. Dr.)Fructose 6-phosphate aldolase (FSA) enzymes are known to catalyse aldol and retroaldol reactions. These reactions have been shown in vitro by Schürmann and Sprenger (2001), Schürmann et al. (2002), Garrabou et al. (2009), Castillo et al. (2010), Sánchez-Moreno et al. (2012a and 2012b), among other groups. However, it was unclear whether these reactions would be possible in vivo. Since the discovery of FSA, encoded by the genes fsaA and fsaB in the Escherichia coli chromosome in 2001 (Schürmann and Sprenger, 2001), its true physiological function has not been reported yet (Samland and Sprenger, 2014). Due to the weak expression of the native promoters of fsaA and fsaB, the only enzymatic data reported so far are from recombinant FSA. To evaluate whether the cleavage of fructose 6-phosphate (F6P) in glycolysis, and the formation of arabinose 5-phosphate (A5P) in the synthesis of 2-keto-3-deoxymanno-octulosonic acid (KDO) could also be catalysed in vivo by FSA, E. coli mutant strains with numerous mutations in metabolic pathways were constructed. These mutant strains had blockades in central carbon metabolism or anabolism. Thus, these mutations impacted pleiotropic genes and, therefore, growth. Afterwards, it was examined if the activity of recombinant FSA in these mutant strains could restore deficiencies in growth. With the reactions catalysed by FSA, the blocked pathways were not restored, but new artificial pathways emerged. Through the F6P bypass a new route for the production of dihydroxyacetone (DHA) and glycerol was opened. Furthermore, a new approach for the provision of A5P was established.Item Open Access Improving optical measurements, online monitoring, growth modeling, and automated control in microalgae production of Phaeodactylum tricornutum(2024) Yeh, Yen-Cheng; Tovar, Günter E. M. (Prof. Dr.)