Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes

The baker ́s yeast, Saccharomyces cerevisiae has been employed by man for centuries in manufacturing of bread, beer, and wine. In science, it has become a useful tool as well. In this work, methods were developed in order to study the molecular biology of the cellulolytic filamentous fungus Trichoderma reesei with the aid of yeast. Cellulose is the most abundant carbon source in nature, and its enzymatic degradation is essential for carbon turnover. In addition, cellulose is used as a raw material in microbial processes. In this work, a previously unknown cellulase-encoding gene was cloned by expression in yeast and detection of hydrolysis halos on substrate plates. This EGV enzyme consists of an exceptionally small core domain, a cellulose-binding domain, and a linker region connecting the two. EGV belongs to family GH45 of glycosyl hydrolases. Additionally, a gene encoding a β-1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of β-glucan by NMR showed that it was a laminarinase (EC 3.2.1.6). A yeast-based cloning method for positively acting regulatory proteins was set up, and two regulatory genes of the T. reesei cellulases, ace1 and ace2, were isolated. The isolation was based on the ability of the encoded proteins to activate expression of a reporter gene, which was linked to the full-length promoter of the major cellulase gene cbh1 in yeast. No homologs of the new regulatory proteins were detected outside the Mycota. The DNA-binding properties of the regulatory proteins were studied both in vitro and in vivo in yeast. Deletion of the ace1 gene resulted in slower radial growth of the fungus on cellulose-containing plates. However, although isolated as an activator, ACEI was later shown to act as a repressor of hydrolase expression. ACEII, on the