Neuartige multifunktionelle Liganden basierend auf 4H-Imidazolen und deren Metallkomplexe

Ziel der hier vorliegenden Arbeit war es, die Klasse der 4H-Imidazole 1 als multifunktionelle Liganden für Metallkomplexe zu etablieren. Dazu wurden zunächst bekannte 4H-Imidazole in Komplexierungsreaktionen getestet sowie leicht zugängliche Bis-4H-imidazole hinsichtlich einer selektiven Komplexierung untersucht. Um neuartige funktionalisierte 4H-Imidazole zu synthetisieren, wurden bekannte Synthesewege für den Aufbau von 4H-Imidazolen optimiert und für eine größere Anzahl von Edukten etabliert. Sterisch anspruchsvolle 4H-Imidazole wurden synthetisiert um den Einfluss der Ar2-Reste auf das Chromophor und die Komplexbildungseigenschaften zu untersuchen. Ausgehend von entsprechend funktionalisierten Edukten wurde anschließend die Möglichkeit des Aufbaus unsymmetrischer Bis-4H-imidazole getestet. Funktionalisierte 4H-Imidazol-Pd(II)- und bipy/terpy-Ru(II)-Komplexe wurden hinsichtlich eines Aufbaus einer zweiten Ligandsphäre studiert. Schließlich wurden 4H-imidazolbasierte Bi- und Terpyridine aufgebaut und zu Metallkomplexen umgesetzt

A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts

Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production

Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids. Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ± 5 mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ± 17.7 mg/L in shake flasks, and 1232 ± 64 mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium

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Genetic engineering of Aspergillus niger for organic acid production

Die industrielle Biotechnologie eröffnet neue Perspektiven und Chancen für die Herstellung von Produkten aus nachwachsenden Rohstoffen. Wissensbasiertes Engineering von industriellen Mikroorganismen wird dabei immer wichtiger für die Stammverbesserung. Moderne genetische Methoden erlauben die Rekonstruktion und Modifikation von komplexen Stoffwechselwegen, um metabolische Muster, die Produktbildung oder die Robustheit der Zellen zu modifizieren. Die Entwicklung geeigneter genetischer Methoden für das Metabolic Engineering von industriellen Stämmen ist für weitere Fortschritte in der mikrobiellen Biotechnologie sehr wichtig. Diese Arbeit konzentriert sich auf die Entwicklung einer genetischen Tool-Box für den filamentösen Pilz Aspergillus niger, der ein effizienter Produzent organischer Säuren ist. A. niger hat das Potential, einer der wichtigsten Wirtsorganismen für die Herstellung bio-basierter Chemikalien zu werden. Der erste Teil dieser Arbeit beschäftigt sich mit der Charakterisierung neuer konstitutiver Promotoren von A. niger. Diese Promotoren ermöglichen verschiedene Stärken der Genexpression und bilden eine hervorragende Grundlage für Metabolic Engineering. Der Einsatz dieser Promotoren im Stoffwechselweg zur Herstellung von Itaconsäure in A. niger führt zu neuen Stämmen, die diese Säure im Kulturmedium akkumulieren. Die Konzentration der Itaconsäure korreliert dabei mit der Stärke der Promotoren, was die Anwendbarkeit der Promotoren zeigt. Im zweiten Teil dieser Arbeit wurden die beiden Schlüsselenzyme für die Itaconsäurebiosynthese gezielt in den Mitochondrien exprimiert, was zu einem maßgeblichen Anstieg der Produktion von Itaconsäure geführt hat. Diese Daten unterstreichen die Bedeutung der Enzym-Expression im richtigen Zellkompartiment, um Produktionsprozesse zu optimieren. ^Schlußendlich wurden im Laufe dieser Arbeit noch auf einem modifizierten AMA1 Fragment basierende Vektoren entwickelt, die eine transiente Transformation der Pilze ermöglichen.Industrial biotechnology is opening new perspectives and opportunities for the production of bio-based products from renewable resources. The rational engineering of industrial microorganisms becomes increasingly important for strain improvement. Advanced genetic techniques allow the reconstruction of complex networks of metabolic pathways and their modification to alter cell behavior, metabolic patterns and product formation. The development and implementation of appropriate genetic tools for genetic and metabolic engineering of industrial strains is essential for further progress in microbial biotechnology. This work focuses on development of a genetic tool-box for the filamentous fungus Aspergillus niger being an efficient organic acid producer. A. niger has a great potential to become one of the most powerful host organisms for the production of bio-based chemicals. The first part of this work aimed at the characterization of novel constitutive promoters of A. niger providing various expression levels of target genes as a powerful tool for metabolic engineering. Applying these promotors to the metabolic pathway for the production of itaconic acid in A. niger resulted in novel strains able to release itaconic acid into the culture medium. Itaconic acid concentrations correlated to the strength of the applied promoters, thus proving their functionality. In the second part of this work, the key enzymes of this metabolic pathway, cis-aconitate decarboxylase and aconitase were targeted into mitochondria which resulted in a substantial increase in the production of itaconic acid. This data highlights the importance of enzyme expression in the correct cellular compartment to establish an adequate flux of metabolites and optimize production processes. Finally, autonomously replicating transient vectors containing a modified AMA1 fragment as an origin of replication for fungi were developed.eingereicht von Marzena BlumhoffAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2013OeBB(VLID)193090