04 Fakultät Energie-, Verfahrens- und Biotechnik
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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 Identifying and engineering bottlenecks of autotrophic isobutanol formation in recombinant C. ljungdahlii by systemic analysis(2021) Hermann, Maria; Teleki, Attila; Weitz, Sandra; Niess, Alexander; Freund, Andreas; Bengelsdorf, Frank Robert; Dürre, Peter; Takors, RalfClostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L-1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L-1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.Item Open Access Dynamische Modellierung und Simulation des Arzneimittelmetabolismus in humanen Leberzellen: Identifizierbarkeit, Robustheit und inter-individuelle Variabilität(2011) Bucher, Joachim; Reuss, Matthias (Prof. Dr.-Ing. Dr. h.c.)Die Effizienz in der heutigen Arzneimittelforschung und -entwicklung ist, bezogen auf das Verhältnis zwischen der Anzahl der potentiellen aktiven Wirkstoffe und der Anzahl der auf dem Markt vertriebenen Arzneimittel sehr niedrig. Diese Ineffizienz beruht darauf, dass mehr als die Hälfte der Wirkstoffe die klinischen Tests nicht besteht, was wiederum auf erhebliche toxische oder geringe pharmakokinetische Eigenschaften zurückzuführen ist. Untersuchungen zum Arzneimittelmetabolismus im Körper spielen eine große Rolle, da Arzneimittel toxische Eigenschaften annehmen können, wenn sie nicht effizient metabolisiert und aus dem Körper eliminiert werden. Die Leber trägt am meisten zum Arzneimittelmetabolismus bei, da Detoxifikationsenzyme, wie die Cytochrom-P450-Monooxygenasen (CYPs) und UDP-Glucuronosyltransferasen (UGTs), in großer Menge in den Leberzellen exprimiert sind und eine hohe inter-individuelle Variabilität aufweisen. Diese Variabilität darf jedoch nicht mit dem therapeutischen Fenster in Konflikt geraten. Daher erfordert diese inter-individuelle Variabilität eine breit gefächerte, auf Populations-Statistiken beruhende Risiko-Abschätzung der Toxizität in der pharmakologischen Forschung. Diese Arbeit, welche sich auf die dynamische Analyse des Arzneimittelmetabolismus in Hepatozyten konzentriert, setzt den Fokus auf die Modellierung des Netzwerkes in Phase I, II und III, in der Modellidentifikation anhand experimenteller Daten auf die Analyse der Parameter-Identifizierbarkeit, welche mit der Robustheit des Systems verknüpft ist, sowie auf die Vorhersage des individuellen Arzneimittelabbaus auf Basis von individuellen CYP- und UGT-Proteindaten. Die Untersuchung des Arzneimittelmetabolismus in dieser Arbeit zeigte, dass Modellierung und Simulation des Metabolismus und der Transportschritte in Leberzellen essentielle Elemente in der Vorhersage der Toxizität darstellen. Von Bedeutende sind vor allem die quantitative Bestimmung der Modellparameter, sowie die qualitative Beurteilung der Parameteridentifizierbarkeit anhand von Sensitivitäten und Korrelationen. Nur eine hochwertige Modellierung erlaubt die Vorhersage der pharmakokinetischen Eigenschaften des Arzneimittels in der Leber, die Prognose des inter-individuellen Ausmaßes und die statistische Analyse in der Bevölkerung. Diese Arbeit zeigte jedoch, dass dieses Isoenzym-System auf Basis von experimentellen Daten an hepatischen Mikrosomen kaum identifizierbar ist, da dieses System insensitive und nicht-identifizierbare Parameter aufweist. Daher zeigen die Ergebnisse dieser Arbeit, dass die detaillierte Untersuchung des Reaktionsnetzwerkes an rekombinanten Isoenzymen vielversprechender und bedeutender ist als an Mikrosomen, in dem Fall, dass mehrere Isoenzyme an dem Abbau eines Arzneimittels zu einem bestimmten Produkt beteiligt sind. Des Weiteren spielen Transportschritte eine wichtige Rolle in der Elimination und der Ausscheidung von Arzneimitteln aus den Leberzellen. Die Modellidentifikation anhand experimenteller Daten an primären humanen Hepatozyten zeigte in ähnlicher Weise zu der Modellverifikation an Lebermikrosomen insensitive und linear korrelierende Parameter in den Transportschritten. Eine Möglichkeit der Modellreduktion auf Basis einer lokalen Parametersensitivitätsanalyse konnte in dieser Arbeit erfolgreich aufgezeigt werden, welche zu sensitiven Parametern führte und damit zuverlässige Modellvorhersagen zulässt. Die Modellverifikation sollte jedoch in Zukunft durch die Implementierung von zusätzlichen Daten aus Untersuchungen an rekombinanten Transportsystemen ergänzt werden. Aufgrund der Tatsache, dass die Vorhersage der inter-individuellen Variabilität auf quantitativen Proteindaten beruht, sollten die zukünftige Verbesserung der quantitativen Bestimmung der CYP- und UGT-Enzyme, sowie die entsprechende Entwicklung von Methoden für die Transportproteine weiter verfolgt werden. Zukünftig sollte ebenso ein Fokus auf die Kopplung des Moduls des Arzneimittelmetabolismus mit den Modulen Zentralmetabolismus und Genregulation gelegt werden, was die holistische Simulation des Arzneimittelmetabolismus in Wechselwirkung mit den anderen Prozessen in der Zelle unter normalen und krankheitsbedingt gestörten Bedingungen erlaubt. Die Vorhersage der Reaktionswege, der Enzympartner und sogar der Modellparameter, sowie deren Verteilung in der Bevölkerung auf Basis von physikochemischen Eigenschaften von Substanzen, wird ebenso eine zentrale Rolle in der Zukunft spielen. Ein weiteres Potential des in dieser Arbeit vorgestellten Modellierungsansatzes liegt in der Implementierung in multi-kompartimentellen physiologisch basierten pharmakokinetischen Modellen. In diesem Rahmen könnten die individuellen in-silico-Vorhersagen verbessert und ein weiterer Fortschritt in der Entwicklung der personalisierten Medizin erreicht werden.Item Open Access A universal polyphosphate kinase : PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate(2020) Hildenbrand, Jennie C.; Teleki, Attila; Jendrossek, DieterPolyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP.Item Open Access Differential amino acid uptake and depletion in mono-cultures and co-cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a novel semi-synthetic medium(2022) Ulmer, Andreas; Erdemann, Florian; Mueller, Susanne; Loesch, Maren; Wildt, Sandy; Jensen, Maiken Lund; Gaspar, Paula; Zeidan, Ahmad A.; Takors, RalfThe mechanistic understanding of the physiology and interactions of microorganisms in starter cultures is critical for the targeted improvement of fermented milk products, such as yogurt, which is produced by Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus. However, the use of complex growth media or milk is a major challenge for quantifying metabolite production, consumption, and exchange in co-cultures. This study developed a synthetic medium that enables the establishment of defined culturing conditions and the application of flow cytometry for measuring species-specific biomass values. Time courses of amino acid concentrations in mono-cultures and co-cultures of L. bulgaricus ATCC BAA-365 with the proteinase-deficient S. thermophilus LMG 18311 and with a proteinase-positive S. thermophilus strain were determined. The analysis revealed that amino acid release rates in co-culture were not equivalent to the sum of amino acid release rates in mono-cultures. Data-driven and pH-dependent amino acid release models were developed and applied for comparison. Histidine displayed higher concentrations in co-cultures, whereas isoleucine and arginine were depleted. Amino acid measurements in co-cultures also confirmed that some amino acids, such as lysine, are produced and then consumed, thus being suitable candidates to investigate the inter-species interactions in the co-culture and contribute to the required knowledge for targeted shaping of yogurt qualities.Item Open Access Expressionsoptimierung und Produktion von Haloalkan-Dehalogenasen zur Umsetzung von 1,2,3-Trichlorpropan(2007) Fritz, Christina; Reuss, Matthias (Prof. Dr.-Ing. Dr. h. c. )Die vorliegende Arbeit befasst sich mit der Herstellung von Haloalkan Dehalogena-sen zur biokatalytischen Umsetzung des prochiralen 1,2,3 Trichlorpropan zu dem (R) oder (S) Enantiomer des 2,3-Dichlor-1-propanol. Die Bereitstellung dieser En-zyme schließt sowohl eine Optimierung der heterologen Proteinexpression im jewei-ligen Wirtsorganismus, als auch die Produktion und Aufreinigung des Biokatalysa-tors ein. Die von der Firma DOW Chemicals bereitgestellten Haloalkan Dehalogenasen un-terscheiden sich im Wesentlichen hinsichtlich ihrer ee-Werte bezüglich des Produkts 2,3-Dichlor-1-propanol und im Hinblick auf ihr Expressionsverhaltens in Escherichia coli. Für die aus einem Bodenscreening stammenden Haloalkan Dehalogenasen DSD4 und DSD49 wird auf Grundlage von experimentellen Ansätzen das intrazellu-läre Expressionsniveau dieser Proteine signifikant erhöht. Weiterhin werden auf Grundlage von modellgestützen Ansätzen Ursachen für die Bildung von "Inclusion bodies" während der heterologen Expression dieser Haloalkan Dehalogenasen her-ausgearbeitet. Der zweite Teil dieser Arbeit befasst sich mit der fermentativen Produktion von Ha-loalkan Dehalogenasen in E. coli über eine Hochzelldichte- Kultivierung im 30 l Maßstab und dem anschließenden Downstream-Prozess der Biokatalysatoren. Das Ziel dieses Arbeitsschwerpunkts liegt darin, den Biokatalysator im halbtechnischen Maßstab möglichst kostengünstig und innerhalb kurzer Zeit herzustellen. Für die Etablierung des Gesamtprozesses wird eine Haloalkan Dehalogenase aus Rhodo-coccus rhodochrous (DhaA) eingesetzt. DhaA weist gegenüber den DSD Varianten bezüglich des Produkts eine geringere Enantioselektivität auf, zeichnet sich aber bereits zu Beginn der Arbeit durch ein hohes Expressionsniveau in dem Wirtsorga-nismus E. coli aus. Innerhalb des Downstream- Prozesses wird die "High Gradient Magnetic Fishing"-Technik eingesetzt. Dieses Verfahren ermöglicht, wesentliche Schritte eines klassischen Chromatographie-Aufreinigungsverfahrens zu umgehen, was zu einer Kosten- und Zeitersparnis führt. Mit Hilfe der Magnettrenntechnik lässt sich der Downstream-Prozess für Haloalkan Dehlogenasen auf die drei Arbeits-schritte des Zellaufschlusses, der Affinitätsaufreinigung mittels HGMF und der Kon-ditionierung des Biokatalysators über eine Ultrafiltration reduzieren.Item Open Access Deciphering metabolic pathways in high-seeding-density fed-batch processes for monoclonal antibody production : a computational modeling perspective(2024) Bokelmann, Carolin; Ehsani, Alireza; Schaub, Jochen; Stiefel, FabianDue to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.Item Open Access S‐adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells(2020) Verhagen, Natascha; Teleki, Attila; Heinrich, Christoph; Schilling, Martin; Unsöld, Andreas; Takors, RalfThe improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S‐(5′‐adenosyl)-l‐methionine (SAM) and 5′‐deoxy‐5′‐(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti‐IL‐8‐producing CHO‐DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.Item Open Access Performing in spite of starvation : how Saccharomyces cerevisiae maintains robust growth when facing famine zones in industrial bioreactors(2022) Minden, Steven; Aniolek, Maria; Noorman, Henk; Takors, RalfIn fed‐batch operated industrial bioreactors, glucose‐limited feeding is commonly applied for optimal control of cell growth and product formation. Still, microbial cells such as yeasts and bacteria are frequently exposed to glucose starvation conditions in poorly mixed zones or far away from the feedstock inlet point. Despite its commonness, studies mimicking related stimuli are still underrepresented in scale‐up/scale‐down considerations. This may surprise as the transition from glucose limitation to starvation has the potential to provoke regulatory responses with negative consequences for production performance. In order to shed more light, we performed gene‐expression analysis of Saccharomyces cerevisiae grown in intermittently fed chemostat cultures to study the effect of limitation‐starvation transitions. The resulting glucose concentration gradient was representative for the commercial scale and compelled cells to tolerate about 76 s with sub‐optimal substrate supply. Special attention was paid to the adaptation status of the population by discriminating between first time and repeated entry into the starvation regime. Unprepared cells reacted with a transiently reduced growth rate governed by the general stress response. Yeasts adapted to the dynamic environment by increasing internal growth capacities at the cost of rising maintenance demands by 2.7%. Evidence was found that multiple protein kinase A (PKA) and Snf1‐mediated regulatory circuits were initiated and ramped down still keeping the cells in an adapted trade‐off between growth optimization and down‐regulation of stress response. From this finding, primary engineering guidelines are deduced to optimize both the production host's genetic background and the design of scale‐down experiments.Item Open Access Cell cycle control by optogenetically regulated cell cycle inhibitor protein p21(2023) Lataster, Levin; Huber, Hanna Mereth; Böttcher, Christina; Föller, Stefanie; Takors, Ralf; Radziwill, GeraldThe cell cycle is divided in four phases, the G1 phase for growth in cell size and increased protein biosynthesis, the S phase for the synthesis and replication of DNA, and the G2 phase for preparing the cell for the M phase, the phase of cell division. Cell cycle inhibitors control progression through the cell cycle. The cell cycle inhibitor p21 arrests cells in the G1 phase correlating with a prolonged protein production phase. This effect could be used to increase the production of biotherapeutic proteins. Here, we applied an optogenetic approach to control the function of p21. Optogenetics is an emerging field within synthetic biology and based on genetically encoded light-sensitive elements derived from plants, fungi or bacteria. Optogenetic tools can be used to control biological functions such as signaling pathways, metabolic pathways or gene expression via light with less side effects than when using chemical inducers. In this study, we designed and applied light switches to control the subcellular localization and thereby the function of p21via light. The stimulation of light-regulated p21 increased the number of cells arrested in the G1 phase correlating with the increased expression of a reporter protein. Implementation of this system could be used to optimize the production of biotherapeutic protein.