04 Fakultät Energie-, Verfahrens- und Biotechnik
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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 Untersuchungen zur Substratspezifität und Enantioselektivität mikrobieller Hydantoinasen(2000) Waniek, Thomas; Syldatk, Christoph (Prof. Dr.)Im Mittelpunkt der Untersuchungen stand die Hydantoinase aus Arthrobacter aurescens DSM 3745. Für die Standardumsetzung von 5-Benzylhydantoin wurden die spezifische Aktivität und die Enantioselektivität ermittelt und mit anderen Hydantoinen bzw. hydantoinanalogen Substanzen verglichen. Die Enantioselektivität der Umsetzung von 5-Benzylhydantoin zeigte weder eine Abhängigkeit von der Temperatur noch vom pH-Wert. Bei Hydantoinen mit Ethylgruppen wie 5-(Methylthioethyl)-hydantoin und 5-(Phenylethyl)-hydantoin konnte für diese Hydantoinase eine Umkehrung der Enantioselektivität festgestellt werden. Der gleiche Effekt konnte beim Austausch der Phenyl- gegen eine Trimethylsilygruppe gefunden werden. 5-(Trimethylsilyl)-hydantoin wurde D-spezifisch mit einer Enantioselektivität von E > 100 umgesetzt. Um den Einfluß einzelner Strukturelemente im Hydantoinring zu untersuchen, wurden verschiedene Hydantoinanaloga synthetisiert. 5-Benzyl-1,3-oxazolidin-2,4-dion, welches ein Sauerstoffatom anstelle der 1-NH-Gruppe besitzt, wurde von der Hydantoinase aus Arthrobacter aurescens DSM 3745 mit einer geringen Aktivität D-selektiv zu Carbamoylphenylmilchsäure umgesetzt. Benzylsuccinimid, welches eine Methylengruppe anstelle der 1-NH Gruppe trägt, wurde mit einer sehr geringen Aktivität zu D-3-Benzylbernsteinsäurehalbamid umgesetzt. 3-substituierte Hydantoinanaloga wurden nicht als Substrate akzeptiert. Benzylbernsteinsäurehalbamid zeigte auf Umsetzungen mit der Hydantoinase aus Arthrobacter aurescens DSM 3745 einen starken inhibitorischen Einfluß (Ki = 0.34 mM). Umsetzungen mit weiteren Hydantoinasen aus Bacillus thermoglucosidasius, Thermus species (beides rekombinante Enzyme aus Escherichia coli) und aus Arthrobacter crystallopoietes DSM 20117 verliefen ausschließlich D-selektiv oder D-spezifisch. Alle Hydantoinanaloga wurden an derselben Position hydrolysiert wie das unveränderte Hydantoin.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 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 Interloping frontiers of systems biology : mass spectrometry in bioprocesses optimization(2017) Sánchez-Kopper, Andrés; Takors, Ralf (Prof. Dr.-Ing.)System biology as the understanding and prediction of how intracellular machinery works, needs new technological support in order to observe cell metabolism at all its levels. From organism genome to its transcription to proteins and how these proteins regulate metabolite pools as the final response to determined stimuli, are needed to have a close look into specific metabolic states. Mass spectrometry is used here to obtain intracellular information at a metabolomics, peptidomics and proteomics level for specific questions concerning bioprocesses optimization. First, a ZIC-HILIC tandem mass spectrometric method was established to allow the intracellular quantitation of about 50 polar metabolites of the central carbon metabolism without extra derivatization steps, increasing detection limits using alkaline mobile phase, considering “wrong-way-around” ionization, as a basis to perform metabolic flux analysis, needed to evaluate metabolic engineering approaches over titer production. Also, culture performance boosting dipeptide uptake was for the first time revealed by intracellular dipeptide pools quantification in CHO cells, by means of high-resolution mass spectrometry, with which tracking its uptake rates, shows the presence of two possible mechanisms for dipeptide uptake in CHO-DP12 cells. Dipeptide metabolization inside the cell was additionally revealed, as amino acids coming from the dipeptides uptake are directly metabolized or expelled out of the cell proving that, in order to optimize mammalian cells bioprocesses, by means of dipeptide medium supplementation, there is a need to understand specific dipeptide uptake and metabolization. Furthermore, protein turnover of a reference molecular antibody, produced in CHO cells, was also chased by a novel methodology. Out of the traditional stable isotope labeling with amino acids (SILCA), production-like conditions were evaluated, the intracellular mAb degradation was for the first time partially quantified, as intracellular peptides produced by Anti-interleukin-8 monoclonal antibody proteolysis were identified by high-resolution mass spectrometry and the measurement of 13C- labeled L-lysine incorporation in the mAb fragments, during exponential cell growth, allowed the calculation of mAb specific degradation rates, where this approach would lead to quantifying how much product is lost by intracellular proteolysis and how this loss could be controlled under high productivity conditions. By means of mass spectrometry, considering the approaches disclosed in the present dissertation, improved bioprocesses optimization could be reached as we are able to quantitatively observe cell metabolism, from metabolomics to proteomics, allowing further understanding and prediction of biological systems.Item Open Access Biotransformationen an Derivaten ungewöhnlicher, cyclischer Aminosäuren(2003) Vielhauer, Oliver; Syldatk, Christoph (Prof. Dr.)Im Rahmen der vorliegenden Arbeit sollten Biotransformationsverfahren für die Darstellung von enantiomerenreinen Derivaten zweier cyclischer Aminosäuren entwickelt werden: (i) ß-Amino-cyclopropancarbonsäure (ß-ACC) und (ii) Pipecolinsäure (Pip, Piperidin-2-carbonsäure). Beide Stoffe sind als Synthesebausteine für pharmazeutisch wirksame Verbindungen von Interesse. Ausgehend von dem bisher nur racemisch zugänglichem ß-ACC-Syntheseintermediat N-tert-Butyloxycarbonyl-2-Azabicyclo[3.1.0]hex-3-en-6-carbonsäuremethylester [(rac)-1] wurden stereoselektive enzymatische Esterspaltungen und Umesterungen untersucht. Hydrolytische Biotransformationen erwiesen sich dabei als vorteilhafter. Mit dem aktivsten und selektivsten Enzym, der Lipase B aus Candida antarctica, wurde nach Optimierung der Reaktionsbedingungen ein präparatives Verfahren für die Racematspaltung von [1] entwickelt und angewandt. Dabei konnte eine Enantioselektivität der Reaktion von E = 34 erreicht werden. Die dargestellten Produkte [(-)-1] und (+)-N-tert-Butyloxycarbonyl-2-Azabicyclo[3.1.0]hex-3-en-6-carbonsäure dienten als Edukte für die Synthese von enantiomerenreinen cis- und trans-ß-ACC-Derivaten. Da bisher beschriebene biokatalytische Verfahren zur Darstellung von Pip-Derivaten durchweg Ausbeuten unter 50 % lieferten, lag der Schwerpunkt auf der Entwicklung eines Verfahrens, das prinzipiell Ausbeuten von 100 % enantiomerenreinem Produkt ermöglicht. Dazu sollte die spontane Racemisierung von N-p-Toluolsulfonyl-pipecolinaldehyd [(rac)-2] mit einer möglichst hochselektiven Biotransformation gekoppelt werden. Bei einem Screening erwies sich die Bioreduktion von [2] mit der Alkoholdehydrogenase aus Pferdeleber als hochaktiv und enantiospezifisch. Nach Kopplung mit einer enzymatischen NADH-Regenerierung wurde das resultierende Batch-System reaktionskinetisch charakterisiert und optimiert. In einer präparativen Umsetzung wurde enantiomerenreiner N-p-Toluolsulfonyl-D-pipecolinalkohol in 73 % Ausbeute gewonnen.Item Open Access Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression(2003) Ölschläger, Peter; Lange, Stefan; Schmitt, Jutta; Siemann-Herzberg, Martin; Reuss, Matthias; Schmid, Rolf D.In order to produce the atrazine-specific scFv K411B, it was expressed in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly: The expression level of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of the scFv with EGFP fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.Item Open Access Data‐driven in silico prediction of regulation heterogeneity and ATP demands of Escherichia coli in large‐scale bioreactors(2020) Zieringer, Julia; Wild, Moritz; Takors, RalfEscherichia coli exposed to industrial‐scale heterogeneous mixing conditions respond to external stress by initiating short‐term metabolic and long‐term strategic transcriptional programs. In native habitats, long‐term strategies allow survival in severe stress but are of limited use in large bioreactors, where microenvironmental conditions may change right after said programs are started. Related on/off switching of genes causes additional ATP burden that may reduce the cellular capacity for producing the desired product. Here, we present an agent‐based data‐driven model linked to computational fluid dynamics, finally allowing to predict additional ATP needs of Escherichia coli K12 W3110 exposed to realistic large‐scale bioreactor conditions. The complex model describes transcriptional up‐ and downregulation dynamics of about 600 genes starting from subminute range covering 28 h. The data‐based approach was extracted from comprehensive scale‐down experiments. Simulating mixing and mass transfer conditions in a 54 m3 stirred bioreactor, 120,000 E. coli cells were tracked while fluctuating between different zones of glucose availability. It was found that cellular ATP demands rise between 30% and 45% of growth decoupled maintenance needs, which may limit the production of ATP‐intensive product formation accordingly. Furthermore, spatial analysis of individual cell transcriptional patterns reveal very heterogeneous gene amplifications with hot spots of 50%-80% messenger RNA upregulation in the upper region of the bioreactor. The phenomenon reflects the time‐delayed regulatory response of the cells that propagate through the stirred tank. After 4.2 h, cells adapt to environmental changes but still have to bear an additional 6% ATP demand.Item Open Access Predicting by-product gradients of baker’s yeast production at industrial scale : a practical simulation approach(2020) Sarkizi Shams Hajian, Christopher; Haringa, Cees; Noorman, Henk; Takors, RalfScaling up bioprocesses is one of the most crucial steps in the commercialization of bioproducts. While it is known that concentration and shear rate gradients occur at larger scales, it is often too risky, if feasible at all, to conduct validation experiments at such scales. Using computational fluid dynamics equipped with mechanistic biochemical engineering knowledge of the process, it is possible to simulate such gradients. In this work, concentration profiles for the by-products of baker’s yeast production are investigated. By applying a mechanistic black-box model, concentration heterogeneities for oxygen, glucose, ethanol, and carbon dioxide are evaluated. The results suggest that, although at low concentrations, ethanol is consumed in more than 90% of the tank volume, which prevents cell starvation, even when glucose is virtually depleted. Moreover, long exposure to high dissolved carbon dioxide levels is predicted. Two biomass concentrations, i.e., 10 and 25 g/L, are considered where, in the former, ethanol production is solely because of overflow metabolism while, in the latter, 10% of the ethanol formation is due to dissolved oxygen limitation. This method facilitates the prediction of the living conditions of the microorganism and its utilization to address the limitations via change of strain or bioreactor design or operation conditions. The outcome can also be of value to design a representative scale-down reactor to facilitate strain studies.Item Open Access Streamlining the analysis of dynamic 13C-labeling patterns for the metabolic engineering of corynebacterium glutamicum as L-histidine production host(2020) Feith, André; Schwentner, Andreas; Teleki, Attila; Favilli, Lorenzo; Blombach, Bastian; Takors, RalfToday’s possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicum l-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.