Abstract
BackgroundFreezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing. However, the yeast mechanisms of tolerance and sensitivity to freeze or near-freeze stress are still poorly understood. More knowledge on this regard would improve their biotechnological potential. Glycerol, in particular intracellular glycerol, has been assigned as a cryoprotectant, also important for cold/near-freeze stress adaptation. The S. cerevisiae glycerol active transporter Stl1p plays an important role on the fast accumulation of glycerol. This gene is expressed under gluconeogenic conditions, under osmotic shock and stress, as well as under high temperatures.ResultsWe found that cells grown on STL1 induction medium (YPGE) and subjected to cold/near-freeze stress, displayed an extremely high expression of this gene, also visible at glycerol/H+ symporter activity level. Under the same conditions, the strains harbouring this transporter accumulated more than 400 mM glycerol, whereas the glycerol/H+ symporter mutant presented less than 1 mM. Consistently, the strains able to accumulate glycerol survive 25-50% more than the stl1Δ mutant.ConclusionsIn this work, we report the contribution of the glycerol/H+ symporter Stl1p for the accumulation and maintenance of glycerol intracellular levels, and consequently cell survival at cold/near-freeze and freeze temperatures. These findings have a high biotechnological impact, as they show that any S. cerevisiae strain already in use can become more resistant to cold/freeze-thaw stress just by simply adding glycerol to the broth. The combination of low temperatures with extracellular glycerol will induce the transporter Stl1p. This solution avoids the use of transgenic strains, in particular in food industry.
Highlights
Freezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing
Glucose repression over Stl1p is not overcome by low temperatures In a previous work [16], we showed that the glucose repression over STL1 expression [14,15,17] was overcome
We demonstrated here that disruption of the glycerol/H+ symporter gene STL1 led to a pronounced decrease in the levels of intracellular glycerol, with concomitant diminished survival to cold/freeze-stress
Summary
Freezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing. The S. cerevisiae glycerol active transporter Stl1p plays an important role on the fast accumulation of glycerol This gene is expressed under gluconeogenic conditions, under osmotic shock and stress, as well as under high temperatures. Freezing has become an important means of preservation and storage of strains used for many types of industrial and food processing, including the production of wine, cheese and bread. Near-freeze and freeze-thaw stress cause various types of damage to the cells, mainly due to the formation of intracellular ice crystals and dehydration during the freezing process, including effects upon the structure of the cell wall, the membrane, and the cellular organelles. Me2SO and trehalose are well-established cryoprotectants, while certain amino acids, such as proline, arginine and glutamate, have demonstrated a significant cryoprotective effect in
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