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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 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 Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates(2020) Frank, Celina; Teleki, Attila; Jendrossek, DieterAgrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data - in combination with in vivo results - suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification.Item Open Access Systembiologische Untersuchungen zur Optimierung mikrobieller Produzenten schwefelhaltiger Aminosäuren(2016) Teleki, Attila; Takors, Ralf (Prof. Dr.-Ing.)Die schwefelhaltige und essentielle Aminosäure L-Methionin besitzt vor allem aufgrund ihres Einsatzes zur Supplementierung von pflanzlichem Tierfutter eine enorme wirtschaftliche Bedeutung. Der globale Jahresbedarf wird auf ca. 1.000.000 Tonnen geschätzt und weist angesichts eines ansteigenden Fleischbedarfs, insbesondere in Schwellenländern, stabile jährliche Wachstumsraten von ca. 5-6% auf. Methionin wird derzeit nahezu ausschließlich durch chemische Synthese (Evonik-Verfahren) als DL-Razemat hergestellt. Ein moderner biotechnologischer Prozess würde eine nachhaltige Alternative zu bisher petrochemisch-basierten Verfahren darstellen und bietet das Potential einer erheblichen Kostensenkung, höherer Substratflexibilität sowie der Herstellung von enantiomer-reinem L-Methionin. Eine Überproduktion von L-Methionin stellt allerdings aufgrund der Komplexität zugrundeliegender Stoffwechselwege, einer strikten zellulären Regulation und eines vergleichsweise hohen energetischen Syntheseaufwands innerhalb gängiger mikrobieller Plattformen eine große Herausforderung dar. In der vorliegenden Arbeit wurde mittels systembiologischer Ansätze ein durch die Evonik Industries AG bereitgestellter Modellproduzent (E. coli SPOT01) auf Potentiale zu einer gezielten Methionin-Überproduktion untersucht. Rationale Stammoptimierungen basieren auf systematische genetische Modifikationen und setzen ein dynamisch-quantitatives Verständnis zugrundeliegender intrazellulärer Stoffwechselnetzwerke voraus (Systems Metabolic Engineering). Innerhalb zielgerichteter metabolischer Perturbationsanalysen können Kontrolleinflüsse beteiligter Enzyme auf die zu optimierenden Zielflüsse auf systemischer Ebene quantifiziert werden (Metabolic Control Analysis). Derartige Ansätze erfordern eine abgestimmte Anwendung intrazellulärer Metabolomanalysen, adäquater Perturbations- und Probennahmestrategien sowie mathematischer Modellierung. Hierzu wurde eine universelle LC-ESI-MS/MS-Methode entwickelt, die eine sensitive und selektive Quantifizierung eines breiten Spektrums niedermolekularer Metabolite des zellulären Stoffwechsels ermöglicht (Metabolic Profiling). In umfangreichen Transportkinetik-Studien konnte anschließend mit L-Serin ein alternatives und netzwerk-inhärentes Stimulussubstrat identifiziert werden, das eine direkte und zielgerichtete Perturbation des L-Methionin-Synthesenetzwerks ermöglicht. Unter Anwendung adaptierter Stimulusszenarien sowie semi-automatisierter Beprobungsstrategien konnten innerhalb produktionsrelevanter Kultivierungsbedingungen von E. coli SPOT01 aussagekräftige Konzentrationsdynamiken fokussierter Metabolitpools erzielt werden. Für die quantitative Auswertung resultierender Perturbationsprofile wurde ein modellbasierter Ansatz unter Verwendung einer nicht-mechanistischen LinLog-Kinetik sowie ein rein datengetriebener Ansatz unter Verwendung des PEC-Kriteriums (Pool Efflux Capacity) verfolgt. Insbesondere die PEC-Analyse stellte sich hierbei als zielführender Ansatz heraus, der aufgrund seiner hohen Robustheit auch bei messtechnischen Unsicherheiten valide Aussagen im Rahmen der Kontrollanalyse erlaubt. Innerhalb unterschiedlicher Perturbationsstudien konnten hierbei übereinstimmend die Cystathionin-β-Lyase (MetC), die Methioninsynthase (MetH/E) und das Methionin-Exportsystem (YjeH) als Enzyme mit hoher Flusskontrolle identifiziert werden. Aufgrund einer ausgeprägten Akkumulation des Vorläufermetabolits L-Homocystein und eines zunehmenden Übergewichts gegenüber L-Cystein, erfolgt die unspezifische Umsetzung des cytotoxischen Intermediats zum Akkumulationsprodukt Homolanthionin. Der unspezifische Abbau von Homolanthionin führt zur erneuten Bildung von L-Homocystein und stellt den Ausgangspunkt für eine alternative Syntheseroute des Haupt-Nebenprodukts L-Isoleucin. Dieses für die L-Methioninsynthese höchst unvorteilhafte Akkumulationsszenario (Kohlenstoff- und Energieverlust) konnte letztlich auf ein Ungleichgewicht in der Bereitstellung von L-Aspartat (TCA-Syntheseweg) und L-Serin (EMP-Syntheseweg) zurückgeführt werden. Unter Einsatz des vollmarkierten Stimulussubstrats [U13C]-L-Serin konnte der Informationsgehalt resultierender instationärer Datensätze weiter gesteigert werden. Die hohe Flusskontrolle durch die Methioninsynthase (MetH/E) konnte hierbei eindeutig auf eine limitierte Transmethylierungskapazität durch den C1-Stoffwechsel zurückgeführt werden. Ein Schlüsselenzym in diesem Zusammenhang stellt die Hydroxymethyltransferase (GlyA) dar, welche unter Umsetzung von L-Serin Methylgruppen für die finale Transmethylierung bereitstellt. Durch eine gezielte Erhöhung der enzymatischen Affinität von GlyA zu seinem Substrat L-Serin kann eine Kompensation des TCA/EMP-Ungleichgewicht unter einer gleichzeitigen Aufhebung des Akkumulationszyklus erfolgen.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 Micro‐aerobic production of isobutanol with engineered Pseudomonas putida(2021) Ankenbauer, Andreas; Nitschel, Robert; Teleki, Attila; Müller, Tobias; Favilli, Lorenzo; Blombach, Bastian; Takors, RalfPseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled‐up the production of isobutanol with P. putida from shake flask to fed‐batch cultivation in a 30 L bioreactor. The design of a two‐stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L-1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2‐ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro‐aerobic conditions during production doubled the integral glucose‐to‐isobutanol conversion yield to 60 mgisobutanol gglucose-1 while preventing undesired carbon loss as 2‐ketogluconic acid.Item Open Access Comparison of l‐tyrosine containing dipeptides reveals maximum ATP availability for l‐prolyl‐l‐tyrosine in CHO cells(2020) Verhagen, Natascha; Wijaya, Andy Wiranata; Teleki, Attila; Fadhlullah, Muhammad; Unsöld, Andreas; Schilling, Martin; Heinrich, Christoph; Takors, RalfIncreasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l‐tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l‐tyrosine (TYR, Y) containing dipeptides: glycyl‐l‐tyrosine (GY), l‐tyrosyl‐l‐valine (YV), and l‐prolyl‐l‐tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.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.