Screening system for isolating regulatory genes of cellulase expression in Trichoderma reesei: Master's thesis

Trichoderma reesei is asexual, multicellular, soft rot, filamentous soil fungus. It can secrete extremely high amount of cellulolytic enzymes. The cellulolytic system of this fungus includes two cellobiohydrolases, five endoglucanases and two ƒÒ-glucosidases capable of enzymatic conversion of cellulose into glucose. Cellulase genes are abundantly expressed under inducing conditions, the promoter for cellobiohydrolase I being the strongest known in this fungus. Cellulase production is regulated at the transcriptional level and regulation is mainly dependent on the available carbon sources. The aim of this work was to create a screening system for the isolation of transcription factor genes regulating the promoter of the major cellulase gene cbh1 in filamentous fungus Trichoderma reesei. The system utilised pyr4 gene as a selectable marker for the isolation of regulatory factors. The pyr4 gene codes for orotidine 5' -monophosphate decarboxylase enzyme, which is involved in uridine biosynthesis. This gene product produces toxic compound in presence of fluoroorotic acid (FaA) and ultimately expression of pyr4 in presence of FaA leads to inviability of the cells. On the other hand, pyr4 negative strain is uridine auxotroph so requires uridine for its growth but pyr4 positive strain can grow in the absence of uridine. Therefore, pyr4 gene can act as a suitable marker for selection of both positive and negative regulatory factors. For the isolation of cbh1 regulating factors, a Trichoderma reesei strain expressing pyr4 gene under the control of cbh1 promoter has been constructed in this study. A pyr4 negative mutant of the strain Rut-C30 was transformed with the constructed pyr4 expression cassette and transformants that are able to grow in the absence of uridine under cellulase inducing conditions were obtained. The integration of expression cassette containing pyr4 gene with the selected transform ants were confirmed by southern and northern analysis. For the isolation of positive and negative regulatory factors of cbh1 promoter, constructed pyr4 expressing Rut-C30 was transformed with episomal genomic DNA library in telomere vector. For activator isolation, transformants were screened for growth on sorbitol as neutral carbon source and in the absence of uridine. 24 transformants were selected on hygromycin and without uridine on sorbitol medium indicating pyr4 activity and activation of the cbh1 promoter. For repressor isolation, transformants were screened for growth on lactose media in presence of FOA and uridine. 60 transformants were selected under these conditions as candidates harbouring a repressor gene from the genomic library. Furthermore, the rescue of the plasmid transformed into the fungus, DNA isolation from the selected purified transformants and sequencing of the insert to identify the genes revealed by the activator and repressor screening are still needed in future.