Cloning and characterisation of transactivating factors for the xylanolytic genes xyn1 and yxn2 of the industrially important filamentous fungus Trichoderma reesei

Trichoderma reesei is an industrial important filamentous fungus with the ability to produce a set of different extracellular hydrolases such as cellulases and xylanases. Besides this the secretion capacity of T. reesei (more than 40g/liter of one single enzyme) makes this fungus in general interesting for biotechnological applications (e.g. heterologous protein production). In the case of cellulases and xylanases the main applications of these enzymes are in food and feed industry, in pulp and paper industry and in textile industry. In many applications enzyme based technologies offer an environmental friendly process compared to chemical treatment procedures. For all this applications an indispensable prerequisite are tailor-made enzyme cocktails exactly matching the requirements of the respective production process. A typical example is the production of cellulase free xylanase mixtures for the pulp and paper industry which is technically difficult to achieve as T. reesei produces a set of both enzyme families which are furthermore to some extend co-regulated concerning their expression pattern. Genetic engineered strains would therefore be a useful tool to overcome this problems but simple gene knock out strategies failed as the number of genes to be deleted is too high. Therefore this project focused on the transcriptional regulation of the two major xylanases of T. reesei as a different approach to reach the goal of tailor- made enzyme mixtures for pulp and paper industry. T. reesei produces 2 major xylanases both of them following different ways of gene regulation. In this study we could demonstrate that xyn1 and xyn2 gene expression is based both on repression and induction. For the xyn1 gene a specific inducing element contacted by an XlnR like factor could be identified which is responsible for expression when the fungus is grown on xylan or on xylose (inducing carbon sources). In addition a more general element a CCAAT box was demonstrated to be involved in transcriptional activation of the xyn1 gene. Finally a the wide domain glucose repressor Cre1 could be shown to be responsible for the complete shut of of xyn1 expression under repressing conditions (e.g. when the fungus is grown on glucose). XlnR and the CCAAT binding proteins both responsible for the formation of the induction specific protein DNA complex on the xyn1 promoter contact DNA under all conditions tested (repressing, derepressing and inducing) whereas Cre1 protein only bind its target in the xyn1 promoter under repressing conditions. Therefore we conclude that switching between repression and derepression is maintained by Cre1 binding or being released from the xyn1 promoter. Some evidence was provided that when growing T. reesei on xylose or xylan additional protein/s is/are attached to the induction mediating protein/DNA complex. The additional factor does not directly contact DNA but targets the complex by protein/protein interaction with the XlnR like factor. Therefore we conclude that switching from a derepressed stage to an induced stage is elicited by an additional factor interacting with XlnR of T. reesei. For xyn2 regulation the picture appears rather different. No complete shut of of the whole system can be observed, but a week constitutive level of xyn2 is expressed all the time. Induction is again triggered by Xylan and by xylobiose but not by xylose. Also sophorose the best inducer of cellulases in T. reesei triggers the expression of xyn2. Our experiments could provide evidence that ACE2 an factor being involved in cellulase induction in T. reesei is also involved in xyn2 expression. In addition a novel specific repressing element in the xyn2 promoter could be identified. This element is responsible for repressing xyn2 expression to the previously mentioned constitutive level. Also a CCAAT box was determined functionally by being involved in the recruitment of the other factors contacting the xyn2 promoter. The project provided an insight into the transcriptional regulation of the two major xylanases of T. reesei. Interestingly no common elements or factors for xyn1 and xyn2 promoters (except the CCAAT box) could be identified and it could clearly be demonstrated that both genes follow different ways of regulation of expression. The regulatory elements and factors of xyn1 are not affected by the classical cellulase inducers and therefore the xyn1 promoter or elements of it are useful tools for the construction of recombinant strains producing tailor-made enzyme mixtures.