The thesis discribes results of genetic, molecular-biological and ultrastuctural analysis of the mutants of H. polymorpha with deleted НрHXS1, НрHXT1, НрMIG1 and НрMIG2 genes. We identified in the methylotrophic yeast H. polymorpha a novel hexose transporter homo-logue, HXS1 (HeXose Sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier HXT1 (HeXose Transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the S. cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is non-functional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence(R203K) con-verts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essen-tial for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite in-activation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1delta deletion strain. Our combined re-sults suggest that in the Crabtree-negative yeast H. polymorpha the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake af-fects the strength catabolite repression. Peroxisomes in the methanol-utilizing yeast H. polymorpha are dispensable for growth on rich carbon sources, such as glucose and sucrose. These substrates and ethanol trigger both, the repression of peroxisomal enzymes at the transcriptional level and rapid and selective degradation of methanol-induced peroxisomes via a process termed pexophagy. How the signal transduction proceeds from the substrate-effectors to its different downstream targets, e.g. transcriptional rep-ressors and autophagy machinery, remains largely unknown. In S. cerevisiae, the glucose-regulated transcriptional repressor Mig1 interacts with the gen-eral repressor complex Tup1-Ssn6 to confer repression of many target genes. In this report we demonstrate that deficiency in the putative H. polymorpha homologues of Mig1 (HpMig1 and HpMig2), as well as HpTup1, partially and differentially affects the repression of peroxisomal al-cohol oxidase by sugars and ethanol. As reported earlier, deficiency in HpTup1 leads to impair-ment of glucose- or ethanol induced macropexophagy. In H. polymorpha mig1mig2 double dele-tion cells, macropexophagy was also largely impaired, whereas micropexophagy became a domi-nant mode of autophagic degradation. Our findings suggest that homologues of the elements of the S. cerevisiae main repression pathway have pleiotropic functions in H. polymorpha. H. polymorpha mutant EAO2 defective in glucose repression was utilized in our study as the host for expression of recombinant secreted form of A. niger GO under control of alcohol oxidase promotor (PMOX). We demonstrated that wilde type producer yielded GO only in methanol me-dium, while EAO2-G or its derivative EAO172-G produced GO also on glucose. GO synthesis in EAO2 producer was repressed in the presence of sucrose or ethanol. Our data suggest that GO synthesis in mutants impaired in glucose repression is efficient in the absence of methanol and strictly regulated by carbon sources, similarly to synthesis of native AO. It could be envisaged that glucose-induced expression of other recombinant proteins, including those of biotechnologi-cal importance, under PMOX in EAO mutants, or in constructed by us gcr1 hxs1 strain may be comparable or exceed that in methanol-grown wild-type cells. These results may add in further improvement of H. polymorpha-based expression platform for recombinant proteins. Key words: methylotrophic yeast, catabolite regulation, glucose repression, glucose trans-port, glucose sensing.
