Universität Stuttgart
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Item Open Access Construction of robust Escherichia coli strains for large-scale production(2022) Ziegler, Martin; Takors, Ralf (Prof. Dr.-Ing.)The biotechnical production of many fine chemicals, proteins or pharmaceuticals depends on large-scale microbial cultivations. Due to limited mixing, heterogeneities in process relevant parameters such as nutrient concentrations arise in such fermentations. Escherichia coli (E. coli) is a model organism frequently used in the biotechnological industry. If E. coli is cultivated under heterogeneous conditions, biological reactions of the microorganism result in reduced process performance. Since large-scale fermentations are not economically feasible in academic settings, scale-down reactors that mimic aforementioned heterogeneities are used to investigate heterogenous fermentations. Previous studies in scale-down reactors unraveled that, depending on the process strategy, the unstable supply of a limiting primary carbon or nitrogen source such as glucose or ammonium is one of the underlying causes of process performance loss. Low concentrations of glucose or ammonium elicit the stringent response as a biological starvation reaction which comprises extensive transcriptional reactions. In the first project that contributes to this thesis, the regulatory and transcriptional reactions of the strains E. coli MG1655 and E. coli SR to repeated exposure to ammonium starvation zones were examined in a scale-down reactor. The scale-down reactor followed a two-compartment approach and consisted of a stirred tank reactor and a plug-flow reactor simulating passage through a starvation zone. E. coli SR is a strain with modulated stringent response. It was observed that short-term starvation stimuli do not trigger this regulatory program in E. coli SR and the transcriptional reaction was noticeably reduced. Long-term adaptation of the strain to repeated cycles of limitation and starvation also clearly differed from E. coli MG1655. Despite lack of the stringent response, E. coli SR showed no deficits in the assimilation of the limiting ammonium or in biomass yield on ammonium. In the second project of this thesis, a series of deletion strains with robust phenotype against glucose starvation zones were constructed. Candidate genes were identified and successively removed from the genome of E. coli MG1655 by Recombineering. The fundamental growth parameters of the strains were determined in shaking flask fermentations and no noticeable differences compared to E. coli MG1655 were found. Chemostat cultivations in a scale-down reactor with glucose as the limiting nutrient source revealed that the final strain of the deletion series, E. coli RM214, had a significantly lower maintenance coefficient under heterogeneous conditions than E. coli MG1655. Moreover, in an exemplary heterologous protein productionscenario E. coli RM214 rhaB- pJOE4056.2_tetA proved to be more robust to heterogeneities and showed a significantly higher product yield than E. coli MG1655 rhaB- pJOE4056.2_tetA. In the third project of this thesis, the production of pyruvate in E. coli MG1655 by inhibition of pyruvate dehydrogenase through CRISPR interference was investigated. A central goal was to achieve the stable production in nitrogen-limited conditions. For this, different target sequences in the operon pdhR-aceEF-lpd were tested and the strains cultivated in shaking flask fermentations. All tested target sequences were generally suitable to trigger the accumulation of pyruvate. Combined CRISPR interference against two target sequences did not lead to an increased pyruvate yield in most cases. In addition, the strains E. coli MG1655 pdCas9 psgRNA_aceE_234 and E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 were characterized in two phase fermentations in lab-scale reactors. The initial phase was an unlimited exponential growth phase and was followed by an ammonium-limited production phase. E. coli MG1655 pdCas9 psgRNA_aceE_234 only produced pyruvate during the exponential phase, and reuptake of pyruvate occurred in the second phase. In contrast, E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 stably produced pyruvate during the exponential and the ammonium-limited phase and is a potential chassis strain for the growth-decoupled production of pyruvate derived bioproducts. The overarching research issues of the projects were the characterization of strains in heterogeneous conditions and the development of new strategies to improve their performance. The collected data leads me to conclude that the construction of robust microbial strains for large-scale applications is both expedient and feasible. Tailored genetic modifications are the method of choice to achieve this goal. Furthermore, suitable genetic constructs offer promising possibilities for the stable growth-decoupled production of chemicals in nitrogen-limited conditions.Item Open Access Resolving heterogeneities in single and multiphase bioreactor systems - Predictive modelling tools towards successful scale-up(2020) Kuschel, Maike; Takors, Ralf (Prof. Dr.-Ing.)Item Open Access Quantification of metabolic interactions between microorganisms(2024) Ulmer, Andreas; Takors, Ralf (Prof. Dr.-Ing.)Item Open Access Algorithms for the global mapping of RNA-RNA interactomes(2024) Schäfer, Richard A.; Voß, Björn (Prof. Dr.)RNA-RNA intra- and intermolecular interactions are fundamental for numerous biological processes. While there are reasonable approaches to map RNA secondary structures genome-wide, understanding how different RNAs interact to carry out their regulatory functions requires mapping of intermolecular base pairs. RNA-RNA interaction prediction algorithms alone are not capable to consider all biological factors, thus, they suffer from low accuracy. Recently, different strategies to detect RNA-RNA duplexes in living cells, so called direct duplex detection (DDD) methods, have been developed. Common to all is the psoralen-mediated in vivo RNA crosslinking followed by RNA Proximity Ligation to join the two interacting RNA strands. Sequencing of the RNA via classical RNA-Seq and subsequent specialised bioinformatic analyses, which results in the prediction of intra- and intermolecular RNA-RNA interactions. Existing approaches adapt standard RNA-seq analysis pipelines but often neglect inherent features of RNA-RNA interactions that are useful for filtering and statistical assessment. In this work, RNAnue is presented, a general pipeline for the inference of RNA-RNA interactions from DDD experiments that takes into account hybridisation potential and statistical significance to improve prediction accuracy. RNAnue was applied to data from different DDD studies, and the results were compared to those of the original methods. This showed that RNAnue performs better in terms of prediction quantity and quality.Item Open Access Metabolic compartmentalization in 13C metabolic flux analysis of Chinese hamster ovary cells(2023) Wijaya, Andy Wiranata; Takors, Ralf (Prof. Dr.-Ing.)Chinese hamster ovary cells is currently a prime focus in mAb production, comprising over 70% of the total approved biologics. The coming patent expiration of blockbuster drugs triggers new market competition for alternative biosimilars. These alternatives create price competition, hence, biosimilar development has been driven by the need to reduce the manufacturing cost. Reducing the manufacturing cost could be achieved by the optimization of the cell lines or cell culture media. For this purpose, OMICS plays an important role, especially to understand cellular metabolism. OMICS technologies provide insights into the cellular capabilities (blueprint) to the actual activities (metabolome/fluxome). This thesis utilized flux analysis to study the metabolism of CHO cells, especially with the use of Carbon-13 (13C Metabolic Flux Analysis). The combination of isotopic non-stationary 13C MFA, compartmented metabolic model, and the sub-cellular metabolome profile allow the quantification of the in vivo mitochondrial shuttles and identification of the sub-cellular fluxes (e.g. isoenzymes). The latter was identified as the bottleneck for improving IgG1 production.Item Open Access Interaction between CO2/HCO3- and the iron homeostasis in Corynebacterium glutamicum(2020) Müller, Felix; Takors, Ralf (Prof. Dr.-Ing.)This work demonstrates, that the chemical iron reduction through phenolic compounds is accelerated in the presence of HCO3-. By that, the intracellular Fe2+ concentration was increased and growth was stimulated in C. glutamicum. Phenolic compounds, which were tested as the reductant in this study, are wide spread in nature and the same habitats can be characterised by high CO2/HCO3- contents. Hence, it can be concluded that the abiotic effect described here impacts other biological as well as geochemical systems.Item Open Access Knowledge-based optimization of cell culture production media(2021) Verhagen, Natascha; Takors, Ralf (Prof. Dr.-Ing)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 Development of a novel Escherichia coli production platform with uncoupled stringent response(2021) Kahlig, Annette Margarete; Takors, Ralf (Prof. Dr.-Ing.)Aerobe Kultivierungsprozesse im großskaligen Industriemaßstab werden oftmals durch gegebene technische Grenzen der Fermentationsanlagen limitiert. Die vorliegende Arbeit präsentiert die genetisch modifizierte Mutante E. coli HGT (‘high glucose throughput’) mit einer ausbalancierten Stringenten Kontrolle, welche eine erhöhte Stresstoleranz unter nährstoffreduzierten Bedingungen zusammen mit einer verstärkten Glukoseaufnahme- und Produktbildungsrate in ruhenden Zellen ermöglicht.Item Open Access Micro-aerobic production of isobutanol with Pseudomonas putida and investigation of its resilience to substrate limitations(2022) Ankenbauer, Andreas; Takors, Ralf (Prof. Dr.-Ing.)The transition from an economy based on fossil resources to a regenerative economy is imminent yet uncertain. Despite an awareness of the scarcity of fossil fuels and the increasing natural disasters due to climate change, the world’s renewable energy supply still constitutes less than 20% of the total energy supply. To reduce humankind’s carbon footprint, next-generation biofuels, such as ethanol or isobutanol, have the potential to revolutionize the transportation energy demand. These biofuels can be produced from renewable feedstock in large bioreactors using microorganisms. However, inhomogeneities, such as carbon or oxygen gradients in large-scale bioreactors, often limit the efficiency of such industrial bioprocesses. The soil bacterium Pseudomonas putida has the potential to supersede existing industrial workhorses like Escherichia coli or Corynebacterium glutamicum due to its natural tolerance towards alcohols, its versatile repertoire of stress responses, and its affinity to a broad range of substrates. This thesis aims to investigate the capability of isobutanol production in P. putida KT2440 and to explore its performance during large-scale stress.