A Study On The Ethanol Tolerance Of A Self-flocculating Fusant Of Schizosaccharomyces Pombe And Saccharomyces Cerevisiae

The aim of this work is to investigate several aspects that may be involved in ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe mutant and Saccharomyces cerevisiae mutant (fusant SPSC). Investigation was initiated on any possible relationship between amino acid composition of plasma membrane and the tolerance of this strain to ethanol. When subjected to 20% (v/v) ethanol for 9 h at 30C, all cells died whereas 57% remained viable for the cells grown in the presence of three amino acids including isoleucine, methionine and phenylalanine, which were added at 1.0, 0.5 and 2.0 g/L respectively. Based on the analysis of amino acid composition of plasma membrane and the determination of membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown in medium containing three amino acids was due to the incorporation of the three amino acids into the plasma membrane of cells thus leading to enhanced ability of fusant SPSC to effectively counteract the fluidizing effect of ethanol on plasma membrane of cells subjected to ethanol stress.On the other hand, when grown with different supplemental fatty acids, such as palmitic, linoleic or linolemc acids, cells were enriched in the kind of added fatty acid in the plasma membranes, with the palmitic acid-enriched cells showing the highest ethanol tolerance, followed by linoleic and linolenic acid-enriched cells orderly. Measurement of plasma membrane permeability coefficients of cells subjected to 15% (v/v) ethanol at 30癈 led to the conclusion that increased ethanol tolerance of palmitic acid-enriched cells is closely associated with its ability to maintain a lower membrane permeability of cells under ethanol stress. Then experiments were performed to study ethanol-induced alterations in phospholipid fatty acid composition of plasma membrane of cells grown with exogenous fatty acid. The addition of palmitic acid (0.6 mmol/L) to the medium containing 3%-9% (v/v) ethanol resulted in a marked increase in the palmitic acid content and decreases in the content of myristoleic, palmitoleic, and oleic acids in the phospholipid fatty acid composition of plasma membrane, as compared with their contents in the absence of ethanol. On the other hand, the addition of both linoleic acid (0.6 mmol/L) and ethanol (3%-9%, v/v) to the basal medium resulted in a striking increase in oleic acid and corresponding decreases in myristoleic, palmitoleic and palmitic acids, while the linoleic acid content was slightly increased. Moreover, the addition of both linolenic acid (0.6 mmol/L) and ethanol (3%-9%, v/v) also resulted in a significant increase in oleic acid and corresponding decreases in myristoleic, palmitoleic and palmitic acids, however, the linolenic acid levels were decreased. All these alterations were confirmed to be the adaptive responses of cells to ethanol because cellsgrown with both fatty acid and ethanol acquired a pronounced increase in viability when they were subsequently exposed to high concentration of ethanol. Furthermore, it was observed that both palmitic acid and oleic acid were able to enhance the tolerance of fusant SPSC to ethanol by strengthening plasma membrane permeability barrier of cells under ethanol stress. This is a novel phenomenon because enhancement by unsaturated and saturated fatty acids on ethanol tolerance occurred to the same strain.Then investigation was conducted to examine the impact of alterations in phospholipid fatty acid composition of plasma membrane on plasma membrane ATPase activity and its relation to ethanol tolerance of this strain. It was found that although alterations in phospholipid fatty acid composition of plasma membrane had no effect on activities of plasma membrane ATPases of cells grown in the absence of ethanol (basal enzymes), they significantly affected the susceptibilities of enzymes to in vivo activation by ethanol: the maximal values for the activated enzymes in cells pregrown with 0.6 mm...