Saccharomyces cerevisiae is the most important industrial yeast and the main microorganism employed in bioethanol production. Industrial yeast strains during fermentation are exposed to various stresses such as osmotic shock, oxidative stress and toxicity of secondary metabolites which lead to the loss of biological products. At relatively low concentrations, ethanol inhibits cell division, decreases cell volume and specific growth rate, while high ethanol concentrations reduce cell vitality and increase cell death. Therefore successful fermentation requires yeast strains resistant to high concentrations of ethanol. Studies have shown increased expression of a number of genes involved in the detoxification of reactive oxygen species (ROS) including thioredoxin, catalase and glutathione reductase in response to an oxidant challenge. This increased synthesis of antioxidants forms the basis of the adaptive response in which treatment with low amounts of oxidant induces resistance to subsequent and otherwise lethal doses. In this study, the rice metallothionein encoding gene isoform OsMTI-1b in the yeast S. cerevisiae in order to enhance resistance against H 2 O 2 . This gene was first cloned in GPD shuttle expression vector and then transferred to S. cerevisiae . However, the engineered strain showed little resistance to oxidative stress. To enhance expression, solubility and stability of OsMTI-1b in yeast cells, the sequence encoding glutathione S-transferase (GST) was placed at the beginning of OsMTI-1b . Then the GPD-GST-OsMTI-1b construct was transferred to yeast cells. After confirming the GST-OsMTI-1b protein expression by Western blot, the culture media were supplemented with different concentrations of H 2 O 2 , cadmium and ethanol. The results revealed better growth and higher final biomass concentrations of yeast cells expressing OsMTI-1b fused with GST as compared to the control strain (the strain expressing only empty plasmid) in the presence of 0.9 mM cadmium, 10% ethanol, and 3 mM H 2 O 2 . Determination of cadmium concentration in the culture medium at T0 (immediately after the addition of cadmium to the medium) and T1 (five hours after the addition of cadmium) showed lower cadmium concentrations in the culture medium for the engineered strain which suggests metal accumulation in the yeast cells containing the GST-OsMTI-1b and the cadmium-binding ability of this protein. Keyword : Saccharomyces cerevisiae. Rice, Metallothionein, Cloning, Oxidative Stress, Ethanol, Cadmium.
