We show that the deletion of ALD6
efficiently reduces acetate production both by wild-type industrial wine yeasts and by engineered strains in which the carbon flux has been strongly shifted towards glycerol. Interestingly, the production of glycerol was increased as a result of the ALD6
deletion, and this effect was particularly clear when ALD6
was deleted from GPD1
strains. The high glycerol production, which in turn results in the production of NAD+
, is surprising, since Ald6p preferably uses NADPH (11
); the deletion of ALD6
was therefore expected to result in an NADPH shortage. We show here that the redistribution of carbon in the GPD1 ald6
strains involves mainly acetaldehyde accumulation and increased flux through the acetoin-butanediol pathway (Fig. ), consistent with the limitation of both alcohol dehydrogenase and ACDH reactions. Since the synthesis of acetaldehyde and acetoin from glucose results in a net production of 2 mol NADH and the synthesis of 2,3-butanediol generates 1 mol NADH, glycerol production might be increased to balance this NADH surplus. Indeed, the amount of NADH generated by the surplus of acetaldehyde, acetoin, and 2,3-butanediol in GPD1 ald6
compared to GPD1
strains is similar to the amount of NAD+
generated by the excess of glycerol (data not shown).
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 (more ...)
An inverse correlation was clearly shown between the ethanol and glycerol yields of the strains (Fig. ). The ethanol yield of the ald6 GPD1 strains was the lowest, 15 to 20% lower than that of the wild type.
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. .
overexpression per se largely affects central metabolism, in particular at the acetaldehyde node (15
), additional changes are triggered by the ALD6
deletion. The production of acetaldehyde of ald6 GPD1
strains was higher than that of GPD1
strains (up to 300 mg/liter instead of 50 to 150 mg/liter) but remained in the upper range of the concentrations found in wine (28
). The concentration of 2,3-butanediol can reach 1.3 g/liter in some wines and is not expected to appreciably affect the sensory qualities of wine. In contrast, acetoin levels higher than the detection threshold (150 mg/liter) are undesirable in table wines (29
). The reduction of acetoin to 2,3-butanediol is catalyzed by the butanediol dehydrogenase Bdh1p, which uses NADH as a preferred cofactor (9
). We show in this study that this reaction was less efficient in ald6 GPD1
strains than in GPD1
strains, resulting in the accumulation of considerable amounts of acetoin. Furthermore, the efficiency of this reaction seems to be directly correlated to the amount of glycerol produced. Since the production of 2,3-butanediol from acetoin consumes 1 mol of NADH, acetoin accumulation may therefore be due to the low availability of NADH in ald6 GPD1
strains, as they produce more glycerol than GPD1
strains. Alternatively, it is possible that butanediol dehydrogenase becomes rate limiting in ald6 GPD1
In summary, these results highlight the great potential of yeast strains overexpressing GPD1 and producing ALD6 for producing low-alcohol wines. An alcohol content at least 2% (vol/vol) lower might be expected in wines produced using a yeast strain overproducing around 20 g/liter glycerol. However, accumulation of acetoin limits the extent to which carbon flux can be diverted to glycerol. Further improvement of these strains requires new efforts to minimize the formation of undesirable compounds, in particular at the acetaldehyde branch point.