Transcriptional analysis of Trichoderma reesei under conditions inducing cellulase and hemicellulase production, and identification of factors influencing protein production: Dissertation

Utilisation of non-edible, renewable lignocellulosic biomass for the production of second generation biofuels and chemicals is hindered especially by the high price of enzymes needed for biomass degradation. Filamentous fungi are natural producers of enzymes active against plant cell wall polymers. Especially the ascomycota fungus Trichoderma reesei is widely utilised in the industry for the production of cellulases and hemicellulases. However, the efficiency of enzyme production needs to be further improved in order to ensure economical production of biobased products. Several environmental factors affect protein production by filamentous fungi. Cellulase and hemicellulase genes of T. reesei are activated by inducer molecules derived from different substrates. The need for cooperation of different hydrolytic enzymes for the total degradation of plant cell wall material has led to coordinated expression of these genes. However, the extent and timing of induction can vary between different genes and especially the hemicellulase genes are differentially induced by various substrates. The direct regulation of cellulase and hemicellulase genes by transcriptional regulators has been widely studied and several activators and repressors of these genes have been characterized in detail. However, little is still known concerning the exact regulatory pathways and mechanisms utilised by the fungus for the accurate timing and composition of the hydrolytic enzymes produced. In this study, a genome-wide transcriptional analysis of T. reesei gene expression at different ambient pH conditions was conducted in order to identify genes affected by extracellular pH. The role of a T. reesei orthologue for the characterized pH regulator, PacC, in the expression of cellulase and hemicellulase genes was also studied. An extensive induction experiment together with transcriptional profiling was then utilised to study the effects of several different substrates on the expression of genes encoding carbohydrate active enzymes (CAZy). In addition, transcriptomics data was utilised for the identification of novel candidate regulators affecting cellulase and xylanase production by T. reesei. Transcriptional profiling identified pH as an important determinant of T. reesei gene expression. Ambient pH was also found to affect the expression of several cellulase and hemicellulase genes and more information on the role of a PacC orthologue in the expression of cellulase and hemicellulase genes was gained. A profiling study utilising different substrates as inducers together with a thorough annotation of the T. reesei CAZy genes revealed the expression patterns of novel candidate genes possibly involved in the degradation of different types of cellulosic and hemicellulosic substrates. In addition, a phylogenetic analysis indicated that functional diversification of the carbohydrate active enzymes of T. reesei is a rather common phenomenon and is reflected in the differential regulation of the corresponding genes. A transcription factor gene named ace3 was identified from the profiling data and was shown to be essential for cellulase production and for the expression of cellulase genes. Over-expression of ace3 led to improved production of cellulase and xylanase activities. Several other candidate regulators were also identified as interesting subjects for more detailed studies. Overall, the use of genome-wide methods increased understanding concerning the genome organisation of T. reesei and its possible evolutionary benefits, and enabled identification of co-regulated genomic regions possibly involved in enzyme production.