04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
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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 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 Optimizing mass transfer in multiphase fermentation : the role of drag models and physical conditions(2023) Mast, Yannic; Wild, Moritz; Takors, RalfDetailed knowledge of the flow characteristics, bubble movement, and mass transfer is a prerequisite for the proper design of multiphase bioreactors. Often, mechanistic spatiotemporal models and computational fluid dynamics, which intrinsically require computationally demanding analysis of local interfacial forces, are applied. Typically, such approaches use volumetric mass-transfer coefficient (kLa) models, which have demonstrated their predictive power in water systems. However, are the related results transferrable to multiphase fermentations with different physicochemical properties? This is crucial for the proper design of biotechnological processes. Accordingly, this study investigated a given set of mass transfer data to characterize the fermentation conditions. To prevent time-consuming simulations, computational efforts were reduced using a force balance stationary 0-dimension model. Therefore, a competing set of drag models covering different mechanistic assumptions could be evaluated. The simplified approach of disregarding fluid movement provided reliable results and outlined the need to identify the liquid diffusion coefficients in fermentation media. To predict the rising bubble velocities uB, the models considering the Morton number (Mo) showed superiority. The mass transfer coefficient kL was best described using the well-known Higbie approach. Taken together, the gas hold-up, specific surface area, and integral mass transfer could be accurately predicted.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.Item Open Access Electron availability in CO2, CO and H2 mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii(2020) Hermann, Maria; Teleki, Attila; Weitz, Sandra; Niess, Alexander; Freund, Andreas; Bengelsdorf, Frank R.; Takors, RalfAcetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO2 footprint by converting industrial emissions containing CO2, CO and H2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO2 + H2, CO and CO + CO2 + H2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD+ provider for CO2 + H2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fdred) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fdred formation when CO is used as substrate, whereas Fdred is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO2 + H2. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.Item Open Access A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes(2011) Bucher, Joachim; Riedmaier, Stephan; Schnabel, Anke; Marcus, Katrin; Vacun, Gabriele; Weiss, Thomas S.; Thasler, Wolfgang E.; Nüssler, Andreas K.; Zanger, Ulrich M.; Reuss, MatthiasBackground The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models. Results In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics. Conclusions A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.