Synthesis of new allyl palladium complexes bearing purine-based NHC ligands with antiproliferative and proapoptotic activity on human ovarian cancer cell lines

A series of new palladium allyl complexes bearing purine-based carbenes derived from caffeine, theophylline and theobromine have been prepared and characterized by NMR spectroscopy, and elemental analysis and in two cases by single crystal X-ray diffraction. The cytotoxic and proapoptotic activities of compounds have been determined in vitro on human ovarian cancer A2780 and SKOV-3 cell lines. These experiments have shown that the palladium-allyl fragment induces a general cytotoxicity, but the choice of the supporting ligands is of paramount importance for achieving the best results. In particular complexes 4c, 4d and 5d exhibit a higher antiproliferative effect (IC50: 0.09, 0.81 and 0.85 μM respectively) than cisplatin (IC50: 1.5 μM) on A2780 cells, and 4d (IC50: 1.7 μM vs. 5.94 μM) on SKOV-3 cell line. Moreover in many cases it has been proved that the cytotoxicity of our complexes is associated with the induction of apoptosis.A series of new palladium allyl complexes bearing purine-based carbenes derived from caffeine, theophylline and theobromine have been prepared and characterized by NMR spectroscopy, and elemental analysis and in two cases by single crystal X-ray diffraction. The cytotoxic and proapoptotic activities of compounds have been determined in vitro on human ovarian cancer A2780 and SKOV-3 cell lines. These experiments have shown that the palladium-allyl fragment induces a general cytotoxicity, but the choice of the supporting ligands is of paramount importance for achieving the best results. In particular complexes 4c, 4d and 5d exhibit a higher antiproliferative effect (IC50: 0.09, 0.81 and 0.85 μM respectively) than cisplatin (IC50: 1.5 μM) on A2780 cells, and 4d (IC50: 1.7 μM vs. 5.94 μM) on SKOV-3 cell line. Moreover in many cases it has been proved that the cytotoxicity of our complexes is associated with the induction of apoptosis

Systems Biology for Power-to-Gas Applications with Methanothermobacter spp.

Die Dissertation ist gesperrt bis zum 11. November 2024 !Der wachsende Bedarf an Energieerzeugung bei gleichzeitigem Anstieg der Treibhausgaskonzentration in der Atmosphäre erfordert neue Lösungen für den Energieund Abfallwirtschaftssektor. Biotechnologische Ansätze, wie der Einsatz lebender Mikroben oder Enzyme, könnte hierfür als Lösung dienen. Mikroben sind attraktive Biokatalysatoren sowohl für die Speicherung von erneuerbarer elektrischer Energie als auch für die Umwandlung von Abfallströmen in höherwertige Chemikalien. Die Power-to-Gas-Plattform wandelt überschüssige erneuerbare elektrische Energie durch Elektrolyse von Wasser, in molekularen Wasserstoff und Sauerstoff um. Die Energie kann dann als Wasserstoffgas gespeichert oder in andere Produkte, wie z. B. Methan, umgewandelt werden. Methan, der Hauptbestandteil von Erdgas, kann verbrannt und in der Erdgasinfrastruktur verwendet werden. Hydrogenotrophe Methanogene sind gasfermentierende Mikroben, die als Biokatalysatoren eingesetzt werden können, um dieses Wasserstoffgas über die Methanogenese in Methan (und Biomasse) umzuwandeln. Kohlendioxid aus der Atmosphäre oder aus Verbrennungsprozessen mit nicht fossilen Brennstoffen sollte idealerweise als Kohlenstoffquelle für diese Mikroben genutzt werden. Die Firma Electrochaea GmbH hat dieses Verfahren unter Verwendung einer thermophilen Methanothermobacter sp. kommerzialisiert. Die Produktion von Methan durch hydrogenotrophe Methanogenese besteht aus einem Methylzweig, der Kohlendioxid bindet und reduziert, und einem Carbonylzweig, der die Biomasseproduktion einleitet. Darüber hinaus erzeugt die hydrogenotrophe Methanogenese eine geringe ATP-zu-Methan-Ausbeute, so dass diese Mikroben an der thermodynamischen Grenze des Lebens leben. Einige dieser thermophilen Mikroben wurden zur Untersuchung der hydrogenotrophen Methanogenese eingesetzt. Zu den meisten anderen Stoffwechselwegen in diesen Mikroben fehlt es jedoch an Wissen. Es wäre von Vorteil, ihre Stoffwechselvorgänge besser zu verstehen, um diese Mikroben für biotechnologische Anwendungen voll nutzbar zu machen. In der ersten Hälfte dieser Dissertation habe ich den Stoffwechsel von drei Methanothermobacter spp. verglichen: 1) Methanothermobacter thermautotrophicus ΔH; 2) Methanothermobacter thermautotrophicus Z-245; und 3) Methanothermobacter marburgensis Marburg. Zur Untersuchung dieser Mikroben habe ich Systembiologie eingesetzt, die Transkriptomics und Proteomics, und Stoffwechselmodellierung auf Genomebene in Verbindung mit kontinuierlichen Bioreaktorstudien umfasst. Ich kultivierte auch M. thermautotrophicus ΔH pMVS1111A:PhmtB-fdhZ-245, einen Stamm, der kürzlich in unserer Gruppe genetisch verändert wurde, um auf Formiat zu wachsen. Ich habe neue Erkenntnisse über die unterschiedlichen Mechanismen der anabolen Formiatproduktion in den drei Methanothermobacter spp. gewonnen. Auserdem untersuchte ich die Auswirkungen auf das Wachstum der Mikrobe und die Energiekosten der Formiat-Dehydrogenase-Kassette, die für das Wachstum auf Formiat erforderlich ist. In der zweiten Hälfte dieser Dissertation untersuchte ich das acetogene Bakterium Clostridium ljungdahlii. Acetogene Bakterien wandeln über den Wood-Ljungdahl-Weg (WLP) neben molekularem Wasserstoff und Kohlendioxid auch Kohlenmonoxid in Biomasse, Acetat und Ethanol um. Wie die Methanogenese besteht auch der WLP aus einem Methyl- und einem Carbonylzweig. Während der Carbonylzweig zwischen den beiden Stoffwechselwegen konserviert ist, ist der Methylzweig nicht konserviert. Auserdem leben Acetogene wie Methanogene an der thermodynamischen Grenze des Lebens. Zudem gibt es keine Netto-ATP-Ausbeute vom WLP aus Kohlendioxid und Wasserstoff. Daher ist das Verständnis des Energiestoffwechsels von Acetogenen besonders wichtig für das autotrophe Wachstum. Auserdem ist die Rolle des energieerhaltenden RNF-Komplexes entscheidend. Trotz energetischer Einschränkungen hat das Unternehmen LanzaTech die Nutzung von Acetogenen für die autotrophe Ethanolproduktion (über den WLP) bereits kommerzialisiert. LanzaTech veredelt Abfallkohlenstoff (Kohlenmonoxid und Kohlendioxid), um Ethanol zu produzieren, das als (Bio-)Kraftstoff verwendet werden kann. In dieser Dissertation führte ich computergestützte Analysen an CRISPR-Systemen durch, die in unserem Labor entwickelt und eingesetzt wurden, um die Rolle von Genen zu untersuchen und den Kohlenstofffluss im zentralen Kohlenstoffstoffwechsel von C. ljungdahlii umzulenken. Ich habe In-silico-Tools implementiert, um das Potenzial eines neuartigen CRISPR-Cas9-basierten Base-Editing-Systems und die Bedeutung des rseC, eines potenziellen Transkriptionsregulators für den RNF-Komplex, in acetogenen Bakterien abzuschätzen.The growing demand for power production with the concurrent rise in atmospheric greenhouse gas concentrations requires new solutions for the energy and waste management sectors. Biotechnological approaches, such as the implementation of living microbes or enzymes, are emerging as solutions. Microbes are attractive biocatalysts for both the storage of renewable electric power and the conversion of waste streams into valorized products. The power-to-gas platform converts excess renewable electric power by electrolyzing water to produce molecular hydrogen and oxygen. The energy can be stored as the hydrogen gas or further converted into other products, such as methane. Methane, which is the main component in natural gas, can be combusted and used in the natural gas infrastructure. Hydrogenotrophic methanogens are gas-fermenting microbes that can be used as biocatalysts to convert this hydrogen gas into methane (and biomass) via methanogenesis. Carbon dioxide from the atmosphere or from combustion processes with non-fossil fuels should ideally be utilized as the carbon source for these microbes. The company Electrochaea GmbH has commercialized this process using a thermophilic Methanothermobacter sp. Methane production via hydrogenotrophic methanogenesis is comprised of a methyl branch that fixes and reduces carbon dioxide and a carbonyl branch that initiates biomass production. Furthermore, hydrogenotrophic methanogenesis produces a low ATP-to-methane yield, and thus, these microbes are often nominated to live at the thermodynamic limit of life. A few of these thermophiles have been used to study hydrogenotrophic methanogenesis. However, knowledge of these microbes is lacking in most other metabolic pathways. It would be beneficial to understand their metabolisms better to fully harness these microbes for biotechnological applications. In the first half of this dissertation, I compare the metabolism of three Methanothermobacter spp.: 1) Methanothermobacter thermautotrophicus ΔH; 2) Methanothermobacter thermautotrophicus Z-245; and 3) Methanothermobacter marburgensis Marburg. I employed systems biology, including transcriptomics and proteomics, and genome-scale metabolic modeling together with continuous bioreactor runs to investigate these microbes. I also cultivated M. thermautotrophicus ΔH pMVS1111A:PhmtB-fdhZ-245, which is a strain that was recently genetically modified in our group to grow on formate. I made new insights regarding the different mechanisms of anabolic formate production in the three Methanothermobacter spp. Further, I studied the effect on the growth of the microbe and energy costs from the formate dehydrogenase cassette, which is required for growth on formate. In the second half of this dissertation, I investigated the model acetogen Clostridium ljungdahlii. Acetogenic bacteria convert carbon monoxide in addition to molecular hydrogen and carbon dioxide into biomass, acetate, and ethanol via the Wood-Ljungdahl pathway (WLP). Similar to methanogenesis, the WLP consists of a methyl and a carbonyl branch. While the carbonyl branch is conserved between the two pathways, the methyl branch is not. Furthermore, like methanogens, acetogens live at the thermodynamic limit of life. There is no net ATP yield during the WLP from carbon dioxide and molecular hydrogen. Thus, understanding the energy metabolism of acetogens is particularly important for autotrophic growth, and the role of the energy-conserving RNF-complex is crucial. Despite energetic limitations, the company LanzaTech has already commercialized the use of acetogens for autotrophic ethanol production (via the WLP). LanzaTech upgrades waste carbon (carbon monoxide and carbon dioxide) gas streams to produce the ethanol, which can be used as a (bio)fuel. In this dissertation, I performed computational analyses on CRISPR systems that were developed and employed in our lab to investigate the roles of genes and redirect carbon flux in the central carbon metabolism of C. ljungdahlii. I implemented in-silico tools for estimating the potential of a novel CRISPR-Cas9-based base-editing system, and the prominence and importance within acetogenic bacteria of the rseC, that is a potential transcriptional regulator for the RNF-complex

Genetic Evidence Reveals the Indispensable Role of the rseC Gene for Autotrophy and the Importance of a Functional Electron Balance for Nitrate Reduction in Clostridium ljungdahlii

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: (1) the RNF complex-encoding gene cluster rnfCDGEAB; (2) the putative RNF regulator gene rseC; and (3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy, and in addition, contribute to understand the connection of nitrate reduction to energy metabolism

Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination

Acetogenic bacteria are rising in popularity as chassis microbes for biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general

Enhancing CO2-valorization using Clostridium autoethanogenum for sustainable fuel and chemicals production

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H-2 revealed that captured carbon (460 +/- 80 mmol/gDCW/day) had a significant distribution to ethanol (54 +/- 3 C-mol% with a 2.4 +/- 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H-2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 +/- 0.4 g/L ethanol with a 66 +/- 2 C-mol% distribution, and 540 +/- 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H-2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change