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

Additional file 3: of BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

Table S3. Genotyping experiment. De-multiplexed NGS read counts of the BRCA genotyping NGS experiments. (a) Genotyping using barcodes without a protection group, (b) frequencies of NNN protection groups, (c) genotyping using barcodes with 5′CCA protection group (XLSX 74 kb

Additional file 6: of BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

Table S6. Wnt experiment. De-multiplexed NGS read counts of the differentiation (Wnt/β-catenin) experiment. (a) Replicate 1, (b) replicate 2, (c) replicate 3 (XLSX 851 kb

Additional file 5: of BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

Table S5. Growth media experiment. De-multiplexed NGS read counts of the media comparison experiment. (a) Replicate 1 and (b) replicate 2 (XLSX 662 kb

Additional file 7: of BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

Figure S1. Supporting evidence regarding barcode assembly, gBART-Seq, and protection groups, related to main Figs. 1 and 2. Figure S2. Supporting evidence for RNA quantification experiments, related to main Fig. 3. Figure S3. Using spike-ins for read normalizing, related to main Figs. 3, 4, and 5. Figure S4. Supporting evidence of the growth media comparison experiment, related to Fig. 4. Figure S5. Supporting evidence of Wnt/β-catenin pathway manipulation, related to Fig. 5 (PDF 20474 kb

Additional file 1: of BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis

Table S1. Barcodes. Forward and reverse barcode sets used for BART-Seq experiments (a) barcodes v1, (b) NNN barcodes, (c) barcodes v2 (XLSX 20 kb