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.

FIG 1

Glycerol concentration (bars) and ethanol yield (Y [g/g glucose consumed]) (black diamonds) (A and B), and glycerol concentration (bars) and residual glucose after 15 (white triangles) and 30 (black triangles) days of fermentation (C and D) for evolved populations (dark gray) and isolates (evolved strains) (light gray) from the independent lineages a (A and C) and b (B and D). Fermentations were carried out in 300 ml MS210 containing 260 g/liter glucose at 28°C in triplicate.

FIG 2

Selective advantage of the evolved strains. Viability of EC1118, K300.2(a), and K300.1(b) during culture in YPD plus 8% glucose and 2.4 M KCl at 28°C. Sugar exhaustion is observed after 100 h.

FIG 3

Fermentation performance (A) and cell population (B) for the ancestral strain EC1118 (black) and the evolved strains K300.2(a) and K300.1(b) (dark and light gray) on MS210 medium containing 260 g/liter glucose at 28°C. dCO2/dt, first derivate of CO₂ produced with respect to time (t).

FIG 4

By-product yields (Y [g/g glucose consumed]) for strains EC1118 and K300.1(b). Metabolites were measured after 30 days of fermentation in 300 ml of MS210 containing 260 g/liter glucose at 16, 20, 24, 28, 32, and 34°C.

FIG 5

Kinetics of wine fermentation on Syrah for EC1118, K300.1(b), and H2. Nitrogen (72 mg/liter) was added in the form of 25 g/hl DAP and 20 g/hl FermaidE at 45 g/liter of CO₂ release (time point indicated by an arrow). dCO2/dt, first derivate of CO₂ produced with respect to time (t).