Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress

Abstract

Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.

Stl1 transporter mediating the uptake of glycerol is not a weak point of Saccharomyces kudriavzevii's low osmotolerance

“…Our results of a higher STL1 expression in the presence of a nonfermentable source of carbon are in agreement with a previous study of various Saccharomyces species in which the induction of various genes involved in glycerol production and transport was studied under microvinification conditions in synthetic must supplemented with various solutes (Perez‐Torrado et al . ). To verify the function of SkSTL1 's product, we both deleted the gene in the S. kudriavzevii genome and cloned it into vectors suitable for expression in S. cerevisiae .…”

“…This osmosensitivity is probably not due to a diminished ability to produce the necessary amount of glycerol upon osmotic stress, as S. kudriavzevii cells produce a substantial amount of glycerol even without stress, and this amount increases upon stressful conditions (Perez‐Torrado et al . ; Zemancikova et al . ).…”

“…Plasmids for the expression of SkSTL1 were pSkSTL1 (an YEp352 derivative with SkSTL1 expressed from the ScNHA1 promoter (Perez‐Torrado et al . )) and pSkSTL1‐GFP (a pGRU1 derivative with SkSTL1 expressed from the ScNHA1 promoter and tagged with the GFP sequence at its 3’ end) generated by homologous recombination (with the use of oligonucleotides listed in Table and genomic S. kudriavzevii DNA as a template for STL1 amplification). For intracellular pH measurement, the cells were transformed with a pHl‐U plasmid containing the pHluorin gene (Maresova et al .…”

“…Saccharomyces kudriavzevii also possesses an STL ‐like gene, and it has been shown that its expression is induced under osmotic stress in microvinification experiments in synthetic must, and additional experiments showed indirectly that the uptake of glycerol might be the preferred strategy for S. kudriavzevii to compensate for an increase in the external osmotic pressure (Perez‐Torrado et al . ). Nevertheless, S. kudriavzevii is highly osmosensitive compared to other Saccharomyces species (Zemancikova et al .…”

How to adapt winemaking practices to modified grape composition under climate change conditions

“…Interestingly, other species belonging to the Saccharomyces group (S. uvarum and S. kudriavzevii) differ in the metabolism for glycerol production and transport, NADH balance, and acetic acid production (Masneuf-Pomarède et al, 2010;López-Malo et al, 2013;Pérez-Torrado et al, 2016). These species can be used as an original genetic material to get non-GM strains showing a partial reduction in ethanol content.…”