Glutathione (GSH), a tripeptide containing a sulfhydryl group, is a highly distinctive amino acid derivative with several important roles involved in the cellular defense to oxidative stress and to heavy metals. We have investigated the GSH cellular content and the cadmium resistance in the DL-1, CBS 4732, and NCYC 495 strains of the metylotrophic yeast Hansenula polymorpha. The DL-1 strain with the highest resistance to cadmium was shown to contain the highest cellular content of GSH compared to the other strains. The NCYC 495 strain with the lowest tolerance to cadmium showed the lowest GSH level. Moreover, enhanced cadmium tolerance was achieved in a GSH-deficient mutant strain by overexpressing the HpGSH2 gene, a functional homologue of ScGSH1 gene encoding gamma-glutamylcysteine synthetase, the first enzyme of GSH biosynthetic pathway. The biosorption capacity of cadmium in the H. polymorpha strains was proportional to the cellular level of GSH, supporting a strong correlation between the cellular GSH content and the cadmium tolerance. To investigate the cadmium-induced regulation of HpGSH2 expression, we have constructed a P_HpGSH2-GOD gene fusion, in which the HpGSH2 promoter was fused to Aspergillus niger glucose oxidase (GOD). The analysis of GOD expression in the transformants harboring the P_HpGSH2-GOD cassette suggested that the induction of HpGSH2 expression was dependent on cadmium concentrations but reversely affected by the extent of cadmium tolerance.

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