Glycerol overproduction by engineered saccharomyces cerevisiae wine yeast strains leads to substantial changes in By-product formation and to a stimulation of fermentation rate in stationary phase

Abstract

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.

FIG. 1

Glycerol and ethanol production by S. cerevisiae strains overexpressing GPD1 on MS medium. The fermentation of wild-type (□) and engineered strains (■) was performed on MS medium in 1.2-liter fermentors at 28°C with agitation with commercial wine yeast strains (A and C) and nonindustrial yeast strains (B and D). Wine yeast strains and laboratory strains (L1, L2) were transformed by the pVT100-U-ZEO-GPD1 plasmid. The V5 GPD1 strain contains the pVT100-U-GPD1 plasmid (23). The strain V5 GPD1L (V5L) contains the pVT100-U-ZEO-GPD1 ΔURA3 plasmid. Glycerol and ethanol were determined after complete sugar exhaustion, except for L1 and L2 strains, which were unable to complete fermentation. The residual sugar content in the corresponding fermented medium was 35 and 9 g/liter (L1 and L1 GPD1) and 0.6 and 30 g/liter (L2 and L2 GPD1).

FIG. 2

By-products formed by engineered V5, industrial, and laboratory strains. Strains and growth conditions are as described in the legend to Fig. 1. The detectable level for acetoin was 40 mg/liter. V5L, V5 GPD1L.

FIG. 3

Fermentation kinetics of the engineered (•) and control R strains (○) on MS medium. dCO₂/dt, CO₂ production rate (grams of CO₂ produced per liter per hour).