Browsing by Subject "Hypocrea jecorina"

Trichoderma reesei is an industrially widely utilized filamentous fungus currently used mainly for production of cellulases and xylanases. As T. reesei is a well-known, powerful and robust protein production organism, it is also being developed for heterologous expression of food protein and various enzymes by substituting the main cellulase coding sequence with the sequence of the produced protein. Transcription factor -based regulation of cellulase production in the fungus is well known, whereas thus far another member of Sordariomycetes, Neurospora crassa, has been the main model organism for noncoding RNA (ncRNA) studies in filamentous fungi. To find new ncRNA-derived regulatory mechanisms involved in cellulase expression in T. reesei, previously characterized potential micro-RNA-like RNAs (milRNAs) as well as other potential ncRNAs near cellulolytic pathway genes were deleted using CRISPR-Cas9. These candidates were based on existing total RNA expression data aligned with the Trichoderma reesei genome (Trire v2.0). The effects of these deletions on cellulase production, protease production, total protein production, and growth were analyzed by well-established methods. One purified deletion strain with unchanged growth in cellulase-inducing conditions (2% lactose) was established. Interestingly, this strain displayed low protein production (16%) together with abolished activity of cellulases (3%) and proteases (5%) compared to the parent strain. These findings warrant further studies on the nature of the deleted area, which could perhaps be an unannotated exon based on the RNA expression data. Another strain with a different deletion had abolished cellulase activity with otherwise largely unchanged properties, and all except for one of the analyzed deletion strains had significantly (p<0.05) diminished cellulase activity despite largely unchanged growth. Further investigation of the deleted areas is needed to elucidate the functions of these regions in more detail than was possible in the scope of this work. Seven potential noncoding RNAs were deleted in this work with interesting results that are likely to be useful in improvement of T. reesei production strains in the future.