Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Abstract

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the ⌬ 9 position. We expressed two sunflower (Helianthus annuus) oleate ⌬ 12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2⌬ 9,12 , the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp ؉ or Trp ؊ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.Tolerance to freezing is an essential trait influencing the viability and leavening capacity of baker's yeast in frozen dough (4, 42). The so-called frozen-dough technology has been widely accepted by consumers and bakers due to several advantages, which include supplying oven-fresh bakery products and improving labor conditions. However, no appropriate industrial strain with a high tolerance to freezing is available, and it is unlikely that the classical breeding program could significantly improve this trait. Freezing is a complex and multifaceted stress, in which different stressors and stress responses appear to play important roles. Cells exposed to subzero temperatures are injured by the formation of ice crystals and crystal growth during frozen storage (33). At a slow freezing rate, cells are exposed to hyperosmotic solutions and equilibrate by movement of water across the membranes (65). Finally, during the thawing process, cells can suffer biochemical damage by oxidative stress (16). It is not surprising, therefore, that tolerance to freezing involves several mechanisms working in concert.Biological membranes are the first barrier that separates cells from their environment and are a primary target for damage during environmental stress. Sudden changes in envi...

Glutathione Protects Lactobacillus sanfranciscensis against Freeze-Thawing, Freeze-Drying, and Cold Treatment

“…However, exposure of L. sanfranciscensis GSH ϩ and GSH Ϫ cells to 4°C for 7 days led to a significant increase in SOD activities in both cells (Table 1). These results suggest that oxidative stress is a contributory factor for chilling injury, consistent with previously reported studies of several microorganisms (8,12,37) and plants (21).…”

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…The multicopy overexpression in S. cerevisiae of either FAD2-1 or FAD2-3, encoding two different desaturases from sunflower, increased the dienoic fatty acid content, particularly 18:2⌬ (9, 12), plus the unsaturation index, yeast membrane fluidity, and freezing tolerance. For example, about 90% of the FAD2-3-overproducing cells survived for 35 days when frozen at Ϫ20°C, in contrast to roughly 50% wild-type cell survival (297). Given the numerous examples of cryoresistence improvements in baker's yeast, it will be interesting to see whether combined approaches might result in additive effects.…”

Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

“…The cho1⌬ mutant, which is defective in phosphatidylserine synthesis, exhibited impaired tryptophan uptake (23). Fluidization of membrane lipids by increased unsaturation of fatty acids also caused tryptophan requirement (24). However, little is known about how Tat2p is missorted to the vacuole by changes in the lipid microenvironment.…”