Trichoderma reesei: Neue Einblicke in seine Genregulation und seine Anwendung als Zellfabrik

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersDer filamentöse Ascomyzet Trichoderma reesei ist vor allem für seine großartigen Eigenschaften in der industriellen Produktion von Proteinen bekannt. Trichoderma reesei wird vor allem für die industrielle Produktion von Zellulasen, Hemizellulasen und anderen biopolymer-abbauenden Enzymen eingesetzt. Daher wurden in der vorliegenden Arbeit die folgenden Themen behandelt, um weitere Einblicke in die Regulation der Genexpression und den möglichen Einsatz von Trichoderma reesei als Zellfabrik zu erhalten. Die Regulation der Zellulase- und Hemizellulase-codierenden Gene wird von verschiedenen unterschiedlichen Transkriptionsfaktoren beeinflusst. Um die Induktionsmuster der Proteinexpression zu verstehen ist die Untersuchung von Protein-DNA-Interaktionen in vivo von größter Bedeutung. Daher verbesserten wir die traditionelle Methode des "in vivo footprinting", um eine reproduzierbare, stabile und zuverlässige Methode für die Identifikation von Protein-DNA-Interaktionen zu erhalten. In Kombination mit der neuen computerunterstützten, automatisierten Datenanalyse gewährleistet dieser Ansatz eine objektive Auswertung von Hochdurchsatzprojekten. Eine einzige Punktmutation im Transaktivator Xyr1 führt zu einer Deregulation der Xylanaseexpression, während die Induktion der Zellulaseexpression weiterhin durch Sophorose induzierbar bleibt. CD-Analysen von Xyr1 und Xyr1A824V zeigen eine Änderung der Sekundärstruktur und einen Verlust der Strukturänderung als Reaktion auf die Gegenwart bestimmter Zuckermoleküle nahe. Diese Ergebnisse legen den Schluss nahe, dass Xyr1 eine sogenannte "nuclear Rezeptor" - ähnliche Domäne besitzt. Trichoderma reesei ist ein wichtiger industrieller Produzent von hydrolytischen Enzymen. Daher wäre auch ein Einsatz als Zellfabrik denkbar. Daher wurde zum ersten Mal die heterologe Expression einer bakteriellen Zwei-Enzymkaskade in Trichoderma reesei durchgeführt. Das Ziel war die Produktion von N-Acetylneuraminsäure, einem wertvollen Rohstoff für die pharmazeutische Industrie, durch Fermentation auf kolloidalem Chitin, einem billigen und ergiebigen Rohstoff.The filamentous ascomycete Trichoderma reesei is known for its high protein production and secretion capabilities. In industry Trichoderma reesei has been used extensively for the production of cellulases, hemicellulases, and other biopolymer-degrading enzymes. Therefore, the following topics were addressed in order to obtain further insights into gene regulation and the potential use of Trichoderma reesei as a cell factory. The regulation of cellulase- and hemicellulase-encoding genes is subject to a number of different transcription factors. In order to understand the induction patterns responsible the study of protein-DNA interactions in vivo is highly relevant. Therefore we improved traditional in vivo footprinting to obtain a reproducible, stable and reliable method to identify protein-DNA interaction events. The combination with automated data analysis ensures objective evaluation of high-throughput projects. A single point mutation in Xyr1 was shown to cause strong deregulation of xylanase expression, while cellulase expression remained inducible by sophorose. CD analysis of the wild-type Xyr1 and Xyr1A824V suggests a change in secondary structure as well as loss of structural response to the carbohydrate present. This points towards the presence of a nuclear receptor-like domain in Xyr1. Trichoderma reesei is an important industrial producer of hydrolytic enzymes, which suggests the potential use of Trichoderma reesei as cell factory. For the first time the heterologous expression of a bacterial two-enzyme cascade was attempted, which resulted in the production of N-acetyl neuraminic acid, a valuable chemical for the pharmaceutical industry, through fermentation of colloidal chitin, a cheap and abundant carbon source.9

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from <i>Trichoderma reesei</i>

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity

Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst

Abstract Background Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material. Results In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy. Conclusions This study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts.</p