T. reesei xylanase expression: Different inducer recognition

Filamentous ascomycetes of the genus Trichoderma mainly act as saprophytes thereby degrading a wide variety of bio-polymeric substrates such as cellulose and hemicelluloses, predominant components of plant material. Hemicellulose as a collective term summarizes a variety of heteropolysaccharides composed either of a backbone formed by xylose (xylans) or mannose and glucose (mannans, glucomannans) with additional side chain substitutes such as arabinose, galactose, and acetic or glucuronic acid. Hemicelluloses are largely water insoluble hence their hydrolysis sets a challenge for saprophytes. Complete degradation of hemicelluloses requires a large number of extracellular enzymes working in synergistic action to allow hydrolysis to smaller oligosaccharides and finally to the respective monomers. In research on regulation of production of hemicellulolytic enzymes in filamentous fungi, Trichoderma has - together with Aspergillus - the leading role. Significant progress has been made during the last few years in the identification and characterisation of in cis-acting elements and in trans-acting factors regulating xylanase expression. In Trichoderma, the general regulator of hydrolase formation (Xyr1) seems to be directly modulated in its mode of action by additional narrow domain transcription factors such as Ace1 and Ace2. Even though the binding-elements are highly similar, DNA-contact of these two factors is dedicated to only either one of the promoters of the two major xylanolytic genes (i.e. Ace1 with pxyn1 and Ace2 with pxyn2). First investigations revealed a spectrum of different mechanisms modifying the Xyr1-dependent transcriptosomes of xyn1 and xyn2 respectively, including phosphorylation, competition, homo- and hetero- dimerisation and recruiting of additional regulatory proteins. At the current state of investigations deduced models for the assembling and modification of both transcriptosomes exist, but those working hypotheses still miss substantial experimental proof. To further elucidate this complex interplay of regulatory proteins the following main questions are addressed in this research project: i) is Ace1 modified to achieve its active form and/or what is the role of the Ace1 interacting protein (Aip) in this repressor complex? ii) Does the Ace1/Aip heterodimer bind to Xyr1 or is it simply competing out the second Xyr1 of the induction-specific protein-DNA complex? iii) is homodimerisation of Xyr1 the precondition for xyn1 induction or is the mechanism based upon competition between a second Xyr1 and the Ace1/Aip repressor complex? iv) does Ace2 form a heterodimer with Xyr1? V) What are the modifications and interaction partners of the Ace2/Xyr1 - DNA complex enabling the various steps of xyn2 transcriptional regulation? vi) compare the assembly of the xyn2 transcriptosome in an ace2 deletion strain (having lost several facets of transcriptional regulation but still expressing xyn2) with the WT. vii) compare the assembly of the sophorose dependent with the xylobiose-dependent transcriptosome in the xyn2 promoter

The filamentous ascomycete Trichoderma reesei (T. reesei) is of significant industrial importance due to its production of cellulose- and hemicellulose-degrading enzymes and its considerable secretory capacity. The enzymes produced by T. reesei are widely used in a number of processes in the pulp and paper, food and feed, and textile industries. Furthermore, these enzymes play a key role in the production of second-generation biofuels. In order to improve T. reesei`s native enzyme production and to promote efforts towards the expression of heterologous proteins as well as the production of important building blocks for the chemical industry, a detailed understanding of the regulatory processes governing the expression of hydrolytic enzymes is essential. Therefore, significant progress has been made in recent years to shed light on the regulatory mechanisms controlling the expression of hydrolytic enzymes, for example the identification of activating factors and their effects on transcriptional regulation. Studies further showed that the activating factors examined so far seem to bind their respective cis acting elements regardless of the culture conditions. This suggests the involvement of specific modifications, such as phosphorylation or dimerization processes that these factors have to undergo in order to mediate substrate-specific induction. With that in mind this project was dedicated to elucidate the inducer specificity of xylanase expression. The insights obtained through this work helped to significantly elaborate on the existing model of xylanase gene expression, which in turn will help to improve the production of customized enzyme cocktails for specific applications, and the efforts currently undertaken towards the cost-effective production of cellulosic bioethanol.