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 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 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 Mimicking CHO large‐scale effects in the single multicompartment bioreactor : a new approach to access scale‐up behavior(2024) Gaugler, Lena; Hofmann, Sebastian; Schlüter, Michael; Takors, RalfDuring the scale‐up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed‐batch cultivations in the single multicompartment bioreactor (SMCB), a new scale‐down reactor system that offers new access to study large‐scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4-1.5 W m-3, large‐scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set‐up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large‐scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale‐up behavior and to predict cell line‐specific drawbacks at an early stage of process development.Item Open Access Comprehensive analysis of C. glutamicum anaplerotic deletion mutants under defined d-glucose conditions(2021) Kappelmann, Jannick; Klein, Bianca; Papenfuß, Mathias; Lange, Julian; Blombach, Bastian; Takors, Ralf; Wiechert, Wolfgang; Polen, Tino; Noack, StephanWild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.Item Open Access Revisiting basics of kLa dependency on aeration in bubble columns : a is surprisingly stable(2023) Wild, Moritz; Mast, Yannic; Takors, RalfA comprehensive experimental characterization of a small‐scale bubble column bioreactor (60 mL) is presented. Bubble size distribution (BSD), gas holdup, and kLa were determined for different types of liquids, relevant fermentation conditions and superficial gas velocities uG. The specific interfacial area a and liquid mass transfer coefficient kL have been identified independent of each other to unravel their individual impact on kLa. Results show that increasing uG leads to larger bubbles and higher gas holdup. As both parameters influence a in opposite ways, no increase of a with uG is found. Furthermore, kL increases with increasing bubble size outlining that improved oxygen transfer is not the result of higher a but of risen kL instead. The results build the foundation for further simulative investigations.Item Open Access Compartment‐specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell‐specific productivity for IgG1 producing CHO cells after MTA treatment(2021) Wijaya, Andy Wiranata; Verhagen, Natascha; Teleki, Attila; Takors, RalfIncreasing cell‐specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), a chemical degradation product of S‐(5′‐adenosyl)‐ʟ‐methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1‐producing CHO cells by 50%. Compartment‐specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell‐cycle arrest and increased cell volumes. Carbon fluxes into the pentose‐phosphate pathway increased 22 fold in MTA‐treated cells compared to that in non‐MTA‐treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α‐ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA‐treated cells improved three fold compared to that in non‐MTA‐treated cells, which can be regarded as a major factor for explaining the boosted CSPs.Item Open Access Synthetic mutualism in engineered E. coli mutant strains as functional basis for microbial production consortia(2022) Müller, Tobias; Schick, Simon; Beck, Jonathan; Sprenger, Georg; Takors, RalfIn nature, microorganisms often reside in symbiotic co‐existence providing nutrition, stability, and protection for each partner by applying “division of labor.” This principle may also be used for the overproduction of targeted compounds in bioprocesses. It requires the engineering of a synthetic co‐culture with distributed tasks for each partner. Thereby, the competition on precursors, redox cofactors, and energy - which occurs in a single host - is prevented. Current applications often focus on unidirectional interactions, that is, the product of partner A is used for the completion of biosynthesis by partner B. Here, we present a synthetically engineered Escherichia coli co‐culture of two engineered mutant strains marked by the essential interaction of the partners which is achieved by implemented auxotrophies. The tryptophan auxotrophic strain E. coli ANT‐3, only requiring small amounts of the aromatic amino acid, provides the auxotrophic anthranilate for the tryptophan producer E. coli TRP‐3. The latter produces a surplus of tryptophan which is used to showcase the suitability of the co‐culture to access related products in future applications. Co‐culture characterization revealed that the microbial consortium is remarkably functionally stable for a broad range of inoculation ratios. The range of robust and functional interaction may even be extended by proper glucose feeding which was shown in a two‐compartment bioreactor setting with filtrate exchange. This system even enables the use of the co‐culture in a parallel two‐level temperature setting which opens the door to access temperature sensitive products via heterologous production in E. coli in a continuous manner.
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