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

Abstract

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.

Guttation capsules containing hydrogen peroxide: an evolutionarily conserved NADPH oxidase gains a role in wars between related fungi

“…The open reading frames of genes nox1 (OPB44254), nox2 (OPB36463) and nor1 (OPB40972) were retrieved from the genome of Tgui (NCBI GenBank: LVVK00000000.1). A plasmid with a hygromycin resistance cassette (Derntl et al ., ), a complementary plasmid encoding geneticin resistance (Seiboth et al ., ) and an overexpression plasmid harbouring the T. reesei cdna1 promoter (Uzbas et al ., ) were constructed and directly used for polyethylene glycol (PEG)‐mediated protoplast transformation as described in in Supporting Information S6. Transformants were screened by PCR with a Phire Plant Direct PCR kit (Thermo Scientific, USA) and subjected to three rounds of single‐spore purification.…”

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion

“…This swo1 expression plasmid (p_swo1oe) contains the hygromycin B phosphotransferase ( hph ) expression cassette as fungal selection marker and 930 bp of the T. reesei cdna1 promoter region [49] to drive swo1 expression. DNA fragments were purified using the QIAquick gel extraction kit (QIAGEN GmbH, Hilden, Germany).…”

“…To largely eliminate the otherwise huge cellulase and hemicellulase background in the secretome of T. reesei , a mutant QM9414 strain was used in which, as shown in earlier work, the hydrolytic enzyme production was strongly downregulated due to xyr1 transcriptional regulator gene knockout [49]. Using the target protein thus produced and purified from culture supernatant, a detailed functional characterization of SWO1 was performed.…”

The neutral metallopeptidase NMP1 of Trichoderma guizhouense is required for mycotrophy and self‐defence

“…For overexpression of nmp1 in T. guizouense NJAU4743 under a constitutive promoter, we first amplified the marker cassette (conferring geneticin resistance) from the plasmid pPki‐Gen (Seiboth et al ., ) using the primers Infusion‐Gen‐fw and Infusion‐Gen‐rev, and inserted it into the BamHI site of the linearized plasmid pUC19 to yield pUC10‐Gen, using the In‐Fusion HD cloning kit (Clontech, CA, USA). The coding region of nmp1 was then amplified with primers nmp1 ‐fw and nmp1 ‐rev from cDNA (for preparation vide supra ), the T. reesei cdna1 promoter (Nakari‐Setälä and Penttilä, ) with the primer pair Promoter‐fw and Promoter‐rev from pPcdna1‐cel7b (Uzbas et al ., ), and 500 bp of the nmp1 ‐terminator amplified from WT genomic DNA with primer Terminator‐fw and Terminator‐rev. These three PCR products were purified and ligated together using recombinational cloning in yeast (Colot et al ., ) using strain FY834.…”