Yeast Saccharomyces cerevisiae as a tool in cloning and analysis of fungal genes: Applications for biomass hydrolysis and utilisation: Dissertation

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 beta -1,3-1,4-glucanase enzyme was cloned and studied. The enzyme was produced in insect cells, and analysis of the degradation products of -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 other hand, was shown to be an activator of cellulase expression. However, it is certainly not the only one, since its deletion did not result in a cellulase-negative phenotype. Additionally, a sugar permease-encoding gene was isolated from T. reesei by complementation. The yeast strain used as a host was deleted for the major hexose transporter genes (hxt1-7, gal2), and additionally engineered for xylose utilisation. The T. reesei permease complemented the growth defect of the mutant strain on xylose-maltose medium. However, adaptive mutation(s) were needed in the host to enable growth on xylose of the permease-expressing strain. The same, engineered yeast strain was used as a host for the native S. cerevisiae hexose transporter genes HXT1, HXT2, HXT4 and HXT7, and the kinetics of xylose transport were studied. The affinities of the permeases for xylose varied, Km values of 190-900 mM were detected. Interesting differences were obtained in the levels of inhibition by the presence of glucose. The single-Hxt strains exhibited a biphasic growth mode on xylose media, where an initially very slow growth was followed by exponential growth after a lag of several days.