Blazhenko O. The role of initial-stage glutathione metabolism genes in the defense against stress in the methylotrophic yeast Hansenula polymorpha

The thesis is devoted to the studying of initial stages of glutathione (GSH) metabolism and defence mechanisms against electrophilic and cadmium stress in the methylotrophic yeast H. polymorpha. GSH1/МЕТ1 and GGT1 genes of H. polymorpha encoding, respectively, S-adenosyl-L-methionine uroporphyrinogen III transmethylase and gamma-glutamyltranspeptidase (GGT) were identified by means of genetic engineering. It was established that HpGsh1р/Met1p activity is required for the synthesis of GSH biosynthesis precursor, cysteine. By using physiological and biochemical techniques it has been found for the first time in yeasts that GGT is involved in detoxification of electrophilic xenobiotics; however, GGT does not initiate utilization of GSH as an exogenous sulfur source in H. polymorpha. HPLC analysis revealed that H. polymorpha does not produce phytochelatins, but possesses GSH as the major cadmium ion chelator. Cadmium ions cause minor changes in the expression of H. polymorpha GSH2 gene, encoding the first enzyme in GSH biosynthesis, gamma-glutamylcysteine synthetase. Based on the results of atomic absorption analysis it was hypothesized that Cd-GSH complex regulates cadmium uptake in H. polymorpha, and GSH1/MET1 and MET4 genes are involved in maturation, while GGT1 gene is involved in metabolism of cellular Cd-GSH complex. A hypothetical scheme of cadmium and electrophilic xenobiotics detoxification in the methylotrophic yeast H. polymorpha was proposed.