Effektoren der DNA Zugänglichkeit in Trichoderma reesei

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersDer filamentöse Pilz Trichoderma reesei ist von Natur aus fähig pflanzliche Biomasse abzubauen. Er sondert eine Reihe von hydrolytischen Enzymen in seine Umgebung ab, die das pflanzliche Material, das zum größten Teil aus Zellulose und Hemizellulose besteht, in niedermolekulare Kohlehydrate zerlegen. Die Abbauprodukte stehen nun dem Pilz zur Nahrungsaufnahme zur Verfügung. Je nachdem, welche Kohlehydrate in der Umgebung vorhanden sind, werden die regulatorischen Schaltkreise des Pilzes daran angepasst. Damit gibt es auch ein bevorzugtes Substrat für die Zellulasen- und Hemizellulasenproduktion. Beispielsweise übt der Einfachzucker D-Glukose, der leicht zu verstoffwechseln ist, eine Katabolitrepression auf die hydrolytischen Enzyme aus. Die Aufnahme solcher Einfachzucker wird priorisiert und die Energie wird vor allem in Biomassebildung und Selbsterhaltung gesteckt. Das stellt besonders für die industrielle Enzymproduktion eine große Herausforderung dar. Um dieses unerwünschte Phänomen zu umgehen, wurden Stämme wie Rut-C30 durch zufällige Mutagenese auf erhöhte Zellulaseproduktion erstellt. Die meisten Industriesstämme haben einen ähnlichen genetischen Hintergrund wie die Mutante Rut-C30. Zu diesen genetischen Merkmalen zählen die erhöhte Zellulaseproduktion und die fehlende Katabolitrepression auf D-Glukose. Es ist jedoch noch unklar, welches genetische Merkmal nun für den Rut-C30-Phänotyp konkret ausschlaggebend ist, da durch die zufällige Mutagenese eine Reihe von Mutationen aufgetreten sind. Die Kontrolle der Expression der hydrolytischen Enzyme erfolgt zu einem großen Teil durch den Transaktivator Xyr1 und das Katabolit-Repressor-Protein Cre1. Beide Transkriptionsfaktoren agieren in einer Abhängigkeit von bestimmten Zuckern. Bisher ist viel über das Zusammenspiel von Cre1 und Xyr1 und deren Einfluss auf die Zellulase- und Xylanaseexpression bekannt. Es ist jedoch zu bedenken, dass der Pilz (wie jeder andere Eukaryot) seine DNA mittels Histonproteine verpackt und dadurch die lokale Genstruktur eine andere Zugänglichkeit für bestimmte Faktoren bekommt. Dies führt zu einem zusätzlichen Aspekt in der Genregulation, der berücksichtigt werden muss und auch gezielt genutzt werden kann. Diese Dissertation untersucht die verschiedenen Einflüsse auf die DNA-Zugänglichkeit und die weiteren Folgerungen für die Zellulase- und Xylanaseexpression. Die DNA-Zugänglichkeit kann durch Änderungen in der Chromatinstruktur und durch das Bindeverhalten von bestimmten Transkriptionsfaktoren, wie zum Beispiel, Cre1 verändert werden. Zur Wirkungsweise der jeweiligen Einflüsse wurden Transkriptionsanalysen und Chromatinzugänglichkeiten bestimmt. Sowohl D-Xylose als auch ¿-Sophorose, induzieren in Trichoderma die Expression der Xylanasen. In beiden Fällen trägt die Chromatinstruktur zur Induktion bei, sei es im Wildtypstamm aber auch in der Mutante Rut-C30. Im Gegensatz zum Wildtypstamm, reagiert die Mutante Rut-C30 bei ¿-Sophorose immer mit einer Chromatinöffnung. Um zwei ähnliche Induktionsprozesse durch zwei unterschiedliche Zucker genauer zu untersuchen, wie es für die Xylanase XYNII der Fall ist, wurden in vivo Footprints gemacht. Diese stellen Veränderungen in der Proteinbesetzung an der DNA dar. Als Ergebnis wurden Unterschiede in Protein-DNA-Wechselwirkungen zwischen den Induktionsmodellen gefunden, was auf eine vermutlich unterschiedliche Signaltransduktion zurückgeht. Nicht nur die Xylanaseinduktion ist vom Chromatin beeinflusst, die Chromatinstruktur spielt auch im ¿Upstream¿-Genbereich des Transaktivators Xyr1 eine wichtige Rolle. Auf ¿-Sophorose, wurden höhere xyr1 Transkripte und eine gleichzeitig erhöhte Chromatinzugänglichkeit gemessen. Im Gegensatz zu xyr1 und den Xylanasen, zeigten beide Zellulase-kodierende Gene cbh1 und cbh2 keine Chromatinöffnung bei ¿-Sophorose auf. Im letzten Teil der Arbeit wurde auf die partielle Deletion von Cre1 in der Mutante Rut-C30 eingegangen. Daraus ergibt sich, wie bereits bekannt, die fehlende Katabolitrepression auf D-Glukose in Rut-C30. Mechanistisch gesehen, ist die verkürzte Version von Cre1 (hier Cre1-96 genannt) bisher einer vollständigen Abwesenheit des Repressors Cre1 gleichgesetzt worden. Die Transkript- und Chromatinanalysen zeigten jedoch, dass sich Cre1-96 von einer vollständigen Deletion von Cre1 unterscheidet. Im Gegensatz zur vollständigen Deletion, erhöht Cre1-96 die Zellulaseaktivität, indem es eine offenere Chromatinstruktur in den ¿Upstream¿-Bereichen der Zellulasegenen (cbh1 und cbh2) und des Transaktivators Xyr1 verursacht, was mit erhöhtem Transkripten der jeweiligen Gene korreliert. Weiters reguliert Cre1-96 einen potentiell neuen Transkriptionfaktor, der womöglich auf die Umstruktuierung des Chromatins Einfluss nimmt.The filamentous fungus Trichoderma reesei is a natural degrader of plant-based biomass. It secretes various hydrolytic enzymes, which act on the plant cell wall¿s main components, cellulose and hemicellulose. Thereby, the fungus has access to low molecular sugars as nutrients derived from complex polysaccharides. In response to different (sugar) stimuli, T. reesei adapts its regulatory circuits and thus the secreted, enzymatic profile. In the presence of D-glucose, an easily-to-metabolize sugar, T. reesei undergoes carbon catabolite repression (CCR) of its hydrolytic enzymes. The uptake of such sugars is prioritized and the energy is put into maintance and biomass gain of the fungus. Especially industry was facing here a main bottleneck in cellulase and hemicellulase production. To circumvent the CCR, strain improvement strategies employed random mutagenesis and screenings to create the mutant Rut-C30. The nowadays used industrial T. reesei strains are derived from the mutant Rut-C30. This means that they have a partly similar genetic background. The most important characteristics of Rut-C30 are the release of CCR and the increased amount of cellulolytic enzymes. It is still not clear, which exact genetic trait is responsible for the hypercellolytic Rut-C30 phenotype. The production of the hydrolytic enzymes is regulated to a great extent by the transactivator Xyr1 and the catabolite repressor protein Cre1. Both transcription factors act in a carbon source dependent manner. So far, a lot is known about the interplay between Cre1 and Xyr1 and the impact on cellulase and xylanase expression. However, it has to be considered that the fungal (as any other eukaryotic) DNA is condensed by histones, leading to the formation of nucleosomal arrays along the DNA. By that, the access is modulated for DNA approaching factors (e.g. transcription factors, chromatin remodelers). Together, the binding of transcription factors and the DNA accessibility regulate gene expression. Gathered knowledge about both, can be used for further strain improvements. This thesis revealed that the DNA accessibility has an impact on cellulase and xylanase expression. Additionally, the effectors of DNA accessibility are either a change in chromatin or the binding of transcription factors, such as Cre1. Transcriptional analysis and chromatin studies showed that the both inducers, D-xylose and ¿-sophorose, are involved in a chromatin-related induction mechanism of xylanases in the wild-type strain and the mutant Rut-C30. In contrast to the wild-type, an chromatin opening is always observed on ¿-sophorose in Rut-C30. To distinguish the induction processes by two different inducers, as it is the case for the xylanase XYNII on ¿-sophorose and D-xylose, changes in protein-binding to specific DNA-binding sites were monitored by in vivo footprinting. This result showed that differences in protein-DNA interactions are inducer dependent and the signalling might be different too. In addition to the xylanase-encoding genes, the DNA accessibility was also investigated in the upstream regulatory region of xyr1 and of both main cellulases cbh1 and cbh2. In case of xyr1, an increased DNA accessibility was found to be a result of an opening in chromatin and led to higher xyr1 transcript levels upon induction by ¿-sophorose. In contrast to xyr1, the cbh1 and cbh2 upstream regulatory regions did not show any chromatin opening in the presence of the inducer ¿-sophorose. Finally, the thesis focuses on the partial deletion of Cre1 in Rut-C30. Mechanistically, the partial deletion was equated with a full deletion of Cre1. Transcriptional and chromatin analyses showed that the truncated version of Cre1 (Cre1-96) outcompetes a full deletion of Cre1 in cellulolytic performance. Additionally, Cre1-96 contributes to a more accessible chromatin in the upstream regulatory regions of cbh1, cbh2 and xyr1 than the full deletion, which results in higher transcript levels of those genes. Last but not least, Cre1-96 influences a helicase-like transcription factor (encoded by htf1), which might be involved in chromatin remodelling.5

Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator

Abstract Background The filamentous fungus Trichoderma reesei (T. reesei) is a natural producer of cellulolytic and xylanolytic enzymes and is therefore industrially used. Many industries require high amounts of enzymes, in particular cellulases. Strain improvement strategies by random mutagenesis yielded the industrial ancestor strain Rut-C30. A key property of Rut-C30 is the partial release from carbon catabolite repression caused by a truncation of the repressor Cre1 (Cre1-96). In the T. reesei wild-type strain a full cre1 deletion leads to pleiotropic effects and strong growth impairment, while the truncated cre1-96 enhances cellulolytic activity without the effect of growth deficiencies. However, it is still unclear which function Cre1-96 has in Rut-C30. Results In this study, we deleted and constitutively expressed cre1-96 in Rut-C30. We found that the presence of Cre1-96 in Rut-C30 is crucial for its cellulolytic and xylanolytic performance under inducing conditions. In the case of the constitutively expressed Cre1-96, the cellulase activity could further be improved approximately twofold. The deletion of cre1-96 led to growth deficiencies and morphological abnormalities. An in silico domain prediction revealed that Cre1-96 has all necessary properties that a classic transactivator needs. Consequently, we investigated the cellular localization of Cre1-96 by fluorescence microscopy using an eYFP-tag. Cre1-96 is localized in the fungal nuclei under both, inducing and repressing conditions. Furthermore, chromatin immunoprecipitation revealed an enrichment of Cre1-96 in the upstream regulatory region of the main transactivator of cellulases and xylanases, Xyr1. Interestingly, transcript levels of cre1-96 show the same patterns as the ones of xyr1 under inducing conditions. Conclusions The findings suggest that the truncation turns Cre1 into an activating regulator, which primarily exerts its role by approaching the upstream regulatory region of xyr1. The conversion of repressor proteins to potential activators in other biotechnologically used filamentous fungi can be applied to increase their enzyme production capacities

Additional file 1: of Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei

Deletion and over-expression of xpp1 (A) Deletion of xpp1 in the parent strain QM6aΔtmus53 (Δtmus53) by homologous recombination with the plasmid pCD-Δxpp1 yielding an xpp1 deletion strain (Δtmus53Δxpp1) is represented schematically. Position of the xpp1 locus on scaffold 16 is indicated at the top. Thin black arrows indicate the approximate positions of the primers used for genotype analysis via PCR (5f2, xpp1-5fwd2 and Pkr, Ppki_5rev). Black-rimmed, light gray arrow represents the xpp1 gene; hatched boxes represent regions for homologous recombination; thick, black arrow represents the hph gene; gray arrow indicate the homologous recombination event; gray, dotted lines represent genomic DNA sequence; solid, black lines represent plasmid DNA sequence. (B) Correct and exclusive integration of the hygromycin cassette in the xpp1 locus was verified by Southern Blot analysis. The obtained signals correspond to the expected fragment sizes after digestion with PstI (3,410 bp and 4,622 bp) using the hph coding region as probe. (C) Ectopic insertions of xpp1 expression cassettes in the over-expression strain were determined by Southern Blot analysis using the xpp1 coding region as probe. L, 1-kb DNA ladder; P, parent strain (QM6aΔtmus53); Δ, xpp1 deletion strain; O, xpp1 over-expression strain

Additional file 2: of Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei

Impact of Xpp1 on growth of T. reesei. (A) T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) D-glucose (squares, solid lines) or lactose (triangles, dashed lines), or CMC (circles, dotted lines) for 18, 24, 30, 38, and 50 hours. The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. (B) T. reesei QM6aΔtmus53 (left lane) and the xpp1 deletion strain (right lane) were pre-grown on MA medium plates containing glycerol. Equal pieces of overgrown agar were transferred to MA medium plates containing 1% (w/v) D-glucose (G) or xylan (XN) or lactose (L) or CMC. Pictures were taken after 48 hours growth at 30°C in darkness

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei

Abstract Background Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. Results Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. Conclusions The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status