Trichoderma reesei as an expression system for homologous production of individual cellulases

Cellulases are a group of enzymes that can synergistically catalyze hydrolysis of cellulose into glucose, which is an essential process for conversion of huge amounts of dormant cellulosic biomass into fermentable sugar, one of the most potent alternative energy sources of the new world. Since purification is difficult and time-consuming, production of cellulases individually is more favorable for these applications that may require specific combination of different enzyme components. In order to evaluate the filamentous fungus Trichoderma reesei as an expression system for production of individual cellulases, Endoglucanase I (EG1/Cel7B), Endoglucanase III (EG3/Cel12A) and Cellobiohydrolase I (CBH1/Cel7A) were homologously expressed in the cellulase-negative mutant strain delta-xyr1 using two alternative promoters (tef1 and cdna1) on glucose medium. In this thesis we show that individual cellulase components (EG1, EG3 and CBH1) could be successfully overexpressed in active form in a cellulase negative T.reesei background under noninducing conditions for the first time in the literature. We also show that cdna1 promoter resulted in higher expression levels of EG1 and EG3. Additionally, T.reesei was established and partially optimized as an expression system which can be employed for future applications

A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on d-glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background

Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes