Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

Abstract

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine's sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.

Viticultural and Biotechnological Strategies to Reduce Alcohol Content in Red Wines

“…On the other hand, an atractive option would be to develop engineered wine yeast with reduced ethanol yield. So far, one of the most promising strategies is to redirect the metabolic lux toward an increased production of glycerol instead ethanol [2,35]. However, this strategy has shown some unwanted efects like an overproduction of undesirable compounds from an organoleptic point of view [1].…”

“…This property has been used in recent years to modify the natural properties of yeasts by conducting adaptive laboratory evolution (ALE) experiments [2]. This approach mimics the natural evolution, by environmental or metabolic constraints, with the main purpose of obtaining improve yeast strains for several biotechnological applications and, of course, in winemaking processes [37][38][39][40].…”

“…These practices ensure an optimal phenolic maturity, which entails very mature grapes with high level of sugars [1,2]. Additionally, the timing of harvest is probably the single most important viticultural decision taken each season.…”

Synthetic genome engineering forging new frontiers for wine yeast

“…To date, using genetic engineering [20,43,44] and non-GM techniques, [45] such as classical breeding (hybridization), adaptive laboratory evolution, and mutagenesis, several non-GM and GM wine strains have been developed with increased robustness, fermentation performance, health-related properties, and/or sensory attributes. Examples include strains that produce wines with lower alcohol levels; [46][47][48][49][50][51][52][53][54][55] mutants that limit the production of unwanted hydrogen-sulfide off-flavors and volatile acidy; [56][57][58] and strains that produce desirable esters, [59,60] terpenes, [61] and thiols ( Figure 18). [62][63][64][65][66][67][68][69][70] Efforts are also underway to express the a-guaiene-2-oxidase from grapevine in S. cerevisiae, thereby equipping wine yeast to transform grapederived a-guaiene to the sought-after spicy aroma compound of Shiraz wine, rotundone.…”

Sequential fermentation using non-Saccharomycesyeasts for the reduction of alcohol content in wine

“…Several strategies that use genetically modified (GM) yeasts have been proposed for the production low-alcohol wine. Recently, Tilloy and co-workers [3] using evolution-based strategies together with breeding strategy showed that evolved or hybrid strains produced an ethanol reduction of 0.6-1.3% (vol/vol) in comparison with ancestral strain.…”