Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes

Abstract

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.

FIG. 1.

Southern blot analysis of chromosomes of wild-type (WT) and ald6 GPD VL1, K1M, and BC strains. Chromosomes were separated by pulsed-field electrophoresis. Membranes were hybridized with an ALD6-specific oligonucleotide probe.

FIG. 2.

Growth and fermentation rate of the wine yeast strains VL1, K1M, and BC (•) and the corresponding engineered ald6 (○), GPD1 (▴), and ald6 GPD1 (▵) strains. Fermentation experiments were performed on MS medium at a 1.1-liter scale under conditions simulating wine fermentation. One representative of three independent fermentation experiments is shown. OD, optical density.

FIG. 3.

By-product concentrations in fermentations with the strains K1M (•), K1M GPD1 (▴), K1M ald6 (○), and K1M ald6 GPD1 (▵) at various time points. Fermentation conditions are described in the legend of Fig. 2. OD, optical density.

FIG. 4.

Glycerol (•), acetoin (⧫), and 2,3-butanediol (▵) production by the strains K1M, K1M GPD1, and K1M ald6 GPD1 grown on MS medium containing 140 (a), 170 (b), 200 (c), and 250 (d) g/liter glucose.

FIG. 5.

Pathways for acetaldehyde metabolism in Saccharomyces cerevisiae. The main isoforms active during glucose fermentation are indicated. Pdc1 and Pdc5, pyruvate decarboxylase; Adh1, alcohol dehydrogenase; Ald6, Ald5, acetaldehyde dehydrogenase; Acs1, acetyl coenzyme A (CoA) synthase; Bdh1, butanediol dehydrogenase; TPP, thiamine pyrophosphate.

FIG. 6.

Ethanol yield as a function of glycerol yield. Strains K1M (•), K1M GPD1 (▴), K1M ald6 (○), and K1M ald6GPD1 (▵) were cultivated under fermentation conditions described in the legend of Fig. 2.