Role of Bdh1 in metabolism of acetoin during wine fermentation.
Yeast can produce acetoin from pyruvate by three different pathways (33
). The major route is the condensation of active acetaldehyde (thiamine PPi
-acetaldehyde) with free acetaldehyde, catalyzed by the pyruvate decarboxylase (Fig. ). An alternative route involves the transformation of pyruvate and free acetaldehyde by a 2-acetolactate synthase into 2-acetolactate, which can be further converted into diacetyl by spontaneous nonenzymatic transformation. In the next step, diacetyl is reduced to acetoin, which in turn is reduced further to 2,3-BD. A final described route is the condensation of active acetaldehyde with acetyl coenzyme A to form diacetyl (5
), which is successively reduced to acetoin. Acetoin is converted into the end product 2,3-BD, which exists as optically active [(2R
)-2,3-BD and (2S
)-2,3-BD] and as meso
)-2,3-BD and (2S
Bdh1 is the main enzyme catalyzing the NADH-dependent reduction of acetoin into 2,3-BD in yeast (E. Gonzalez and J. Biosca, unpublished data). In addition, Bdh1 can use several other substrates in vitro, in particular diacetyl as the second best substrate after acetoin (12
To investigate more precisely the role of Bdh1 in the reduction of acetoin under wine fermentation conditions, we deleted BDH1 in a model wine yeast strain, V5, and studied the impact of BDH1 disruption during fermentation in MS medium (synthetic must) containing 200 g/liter glucose. The deletion of BDH1 did not affect either the growth or the fermentation rate compared to that of wild-type V5 (data not shown). Under these conditions, when all glucose was exhausted, V5 produced 553 mg/liter of 2,3-BD, as a mixture of about 77% active and 23% meso isomers (Table ). The bdh1 mutant, on the other hand, did not produce any detectable (2R,3R)-2,3-BD, and its residual production of 2,3-BD, consisting exclusively of the meso form, corresponded to ~15% of the total 2,3-BD production by wild-type V5. Moreover, while no acetoin was detected in the medium fermented by V5, V5 bdh1 produced ~400 mg/liter of this compound.
Production of acetoin, (2R,3R)-2,3-BD, (2S,3S)-2,3-BD, and meso-2,3-BD during alcoholic fermentation in MS medium containing 200 g/liter glucose
Overall, these data indicate that Bdh1 is responsible for ~85% of the total amount of 2,3-BD produced by yeast cells during wine fermentation, including the entire (2R,3R)-stereoisoform and ~40% of the meso form.
We additionally investigated the impact of BDH1 deletion on diacetyl formation. The diacetyl level produced by V5 bdh1 (Table ) increased by ~2-fold compared to that produced by native V5, indicating that Bdh1 also plays an important role in the reduction of diacetyl.
Identification of factors limiting the reduction of acetoin into 2,3-BD.
In the strains overproducing glycerol, the BDH reaction is limited above a certain level of glycerol production, resulting in a dramatic increase in the production of acetoin (4
). The accumulated level is increased further by the disruption of ALD6
, coding for the cytosolic acetaldehyde dehydrogenase (4
In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1
, coding for the native NADH-dependent Bdh1 and an engineered NADPH-dependent Bdh1 enzyme, respectively, in the strains V5 and V5 ald6
has the same apparent affinity for and performance efficiency with NADPH as Bdh1 has for NADH (6
The fermentation behavior of V5, V5 BDH1, and V5 BDH1221,222,223 during wine fermentation in MS medium was examined. BDH1 and BDH1221,222,223 overexpression did not affect growth compared to that of the reference strain, and no differences in the production of 2,3-BD were observed.
Next, we examined the impacts of BDH1 and BDH1221,222,223 overexpression on the levels of acetoin accumulated by yeast carrying an ALD6 disruption and producing high levels of glycerol. The strains V5 ald6, V5 ald6 BDH1, and V5 ald6 BDH1221,222,223 were transformed by the multicopy plasmid pVT100U-ZEO carrying GPD1 and studied in a preliminary experiment under alcoholic fermentation conditions in MS medium containing 50 g/liter glucose. The growth (Fig. ) and fermentation (data not shown) rates of the three strains V5 ald6 GPD1, V5 ald6 GPD1 BDH1, and V5 ald6 GPD1 BDH1221,222,223 were identical. Compared to the acetoin formation by strain V5 ald6 GPD1, which accumulates 1.5 g/liter acetoin, the acetoin formation by strains overproducing Bdh1 and Bdh1221,222,223 decreased (Table ; Fig. ). However, the overproduction of the NADPH-dependent enzyme resulted in more efficient conversion of acetoin into 2,3-BD than the overproduction of native NADH-dependent Bdh1. Indeed, under these conditions, V5 ald6 GPD1 BDH1 formed 37% less acetoin than the reference strain V5 ald6 GPD1, while V5 ald6 GPD1 BDH1221,222,223 produced 61% less acetoin than V5 ald6 GPD1 BDH1. In both cases, acetoin was reduced to 2,3-BD in a stoichiometric way. These results demonstrate that both the expression level of BDH1 and the NADH availability are limiting factors for the 2,3-BD pathway in glycerol-overproducing yeast. Nevertheless, the NADH availability is the most restricting parameter under these growth conditions.
FIG. 2. Growth and acetoin and 2,3-BD production profiles of modified wine yeast strains V5 ald6 GPD1 (black squares), V5 ald6 GPD1 BDH1 (black triangles), and V5 ald6 GPD1 BDH1221,222,223 (white triangles) in MS medium containing 50 g/liter glucose. Representative (more ...)
Production of acetoin and 2,3-BD during alcoholic fermentation in MS medium containing 50 g/liter glucose
Effects of BDH1 and BDH1221,222,223 overexpression on acetoin levels during wine fermentation.
In another step, we performed extensive characterization of the five strains V5, V5 ald6, V5 ald6 GPD1, V5 ald6 GPD1 BDH1, and V5 ald6 GPD1 BDH1221,222,223 in synthetic MS media containing 200 and 240 g/liter of glucose, corresponding to sugar levels commonly found in grape juice (Tables and ; Fig. ).
Impacts of genetic modifications on ethanol production and corresponding acetoin, acetate, and glycerol levels produced during alcoholic fermentationa
Metabolite and biomass levels and yields for strains V5, V5 ald6, V5 ald6 GPD1, V5 ald6 GPD1 BDH1, and V5 ald6 GPD1 BDH1221,222,223 in medium with 240 g/liter glucosea
FIG. 3. Fermentation performances of modified wine yeast strains V5 ald6 (white circles), V5 ald6 GPD1 (black squares), V5 ald6 GPD1 BDH1 (gray triangles), and V5 ald6 GPD1 BDH1221,222,223 (white triangles) in comparison to that of the corresponding reference (more ...)
Detailed results of one experiment in MS medium with 240 g/liter glucose are shown (Fig. ; Table ). The specific BDH activities in the different strains were determined at a time point corresponding to the release of 48 g/liter CO2 (~62 h of fermentation). The NADH-dependent BDH-specific activity in V5 ald6 GPD1 BDH1 was similar to the NADPH-dependent specific activity in V5 ald6 GPD1 BDH1221,222,223 (means ± standard deviations, 2.0 ± 0.06 and 1.8 ± 0.1 U/mg, respectively) and approximately 20-fold higher than the BDH-specific activities in V5 and V5 ald6 (0.1 ± 0.01 and 0.09 ± 0.01 U/mg, respectively).
As described previously (4
), the high-glycerol strain V5 ald6 GPD1
exhibited reduced growth (Fig. ) compared to that of V5. This result may be due to a toxic effect of acetaldehyde, which increased to 0.3 to 0.4 g/liter at the end of growth phase (data not shown), or to a net ATP loss resulting from the diversion of carbons toward glycerol (4
). The overexpression of BDH1
in V5 ald6 GPD1
did not further influence either growth or the CO2
production rate (Fig. ). Both gene modifications resulted in a considerable decrease of acetoin production (Fig. ; Table ). By the end of the first 60 h, corresponding to the midfermentation point for strains overproducing glycerol, acetoin was efficiently reduced to 2,3-BD by V5 ald6 GPD1 BDH1
, and the efficiency of this reaction in V5 ald6 GPD1 BDH1221,222,223
was further increased, as observed previously with MS medium containing 50 g/liter glucose. However, from midfermentation, these differences were attenuated, and the final acetoin concentrations for V5 ald6 GPD1 BDH1221,222,223
and V5 ald6 GPD1 BDH1
were very close (Fig. ). Similar effects in both MS medium with 200 g/liter glucose and MS medium with 240 g/liter glucose were observed (Table ). In both media, 83 to 90% of the acetoin produced from pyruvate was reduced into 2,3-BD in a stoichiometric manner, in contrast to the large accumulation of acetoin in the media and the low-level 2,3-BD production observed for V5 ald6 GPD1
(Table ; Fig. ).
As shown in Table , redox and carbon levels were balanced for all genetically modified strains. Apart from the effects on acetoin and 2,3-BD levels, BDH1 overexpression triggered, additionally, a decrease in the glycerol level by ~3 g/liter compared to that in V5 ald6 GPD1 (Table ), which can be explained by more restricted NADH availability for glycerol synthesis, in favor of 2,3-BD production. On the other hand, the overexpression of the NADPH-dependent Bdh1 restored the glycerol level to the one produced by V5 ald6 GPD1, suggesting that this enzyme uses NADPH rather than NADH in vivo. In addition, V5 ald6 GPD1 BDH1221,222,223 produced larger 2-ketoglutarate amounts than V5 ald6 GPD1 BDH1 (Fig. ; Table ). This finding also supports the NADPH specificity of this enzyme in vivo. A similar effect was observed for V5 ald6 compared to V5 (Fig. , Table ). Since Ald6 preferentially uses NADP, the deletion of ALD6 results in lower NADPH formation. In both cases, less NADPH will be available for the NADPH-dependent glutamate dehydrogenase Gdh1 reaction, responsible for 2-ketoglutarate conversion into glutamate, thus explaining the observed 2-ketoglutarate accumulation.
The overproduction of the NADH- and NADPH-dependent Bdh1 did not further influence the ethanol formation (Table ). Compared to that from the wild type, the ethanol yield was considerably reduced, by about 20%, this effect being essentially the result of glycerol overproduction. Depending on the initial glucose concentration, this strategy results in a decrease in the ethanol level of 2.1 to 2.8° (Table ).
Impacts on volatile aromatic compounds.
In the next step, we compared the effects of the genetic modifications on the production of some key aromatic compounds (Tables and ).
Higher-alcohol, ester, and diacetyl levels produced by genetically modified yeasts during alcoholic fermentation in MS medium containing 240 g/liter glucose
Aroma descriptors for analyzed flavor compounds listed in Table
The deletion of ALD6
, coding for the NADP-dependent cytosolic isoform of acetaldehyde dehydrogenase, induced the production of significantly larger amounts of isobutanol and isoamyl alcohol than those produced by the wild type V5 (Table ). These higher alcohols derive from the metabolism of valine and leucine (17
). Increased levels of these compounds may be explained by the slight transient increase of acetaldehyde and pyruvate in V5 ald6
(data not shown) (35
), which can favor the production of 2-acetolactate, an intermediary of valine and leucine synthesis. In addition, redox imbalances provoked by the disruption of ALD6
may contribute to these modifications. The deletion of ALD6
induced, additionally, an increase in the isoamyl acetate level, which may be a direct consequence of the higher isoamyl alcohol level produced.
In a general way, the overexpression of GPD1
led to a decreased level of higher alcohols compared to those produced by V5 ald6
(Table ). A likely explanation for this finding is the lower level of NADH availability for the NADH-dependent higher-alcohol production in this strain (17
), as NADH is preferentially used for glycerol synthesis. The production of isoamyl acetate in this strain was also reduced, which can be directly related to the lower level of isoamyl alcohol formation. In contrast, the production of diethyl succinate increased, probably as the direct consequence of the higher succinate levels formed by glycerol-overproducing yeast (4
The overproduction of Bdh1 or Bdh1221,222,223 had little effect on higher-alcohol and ester synthesis. The only effect was an additional decrease in the formation of isoamyl alcohol compared to that by the ald6 GPD1 strain, which may, again, be explained by a lower level of NADH availability for the NADH-dependent higher-alcohol production. In a similar way, the overproduction of NADPH-dependent Bdh1221,222,223 increased the production of isoamyl alcohol to levels similar to those of production by the ald6 GPD1 strain, as NADH became more available. Altogether, the various levels of all analyzed compounds remained in the range of concentrations found in wine (Table ), and no significant alteration in aroma traits compared to those obtained with the parental strain V5 can be attributed to the genetically modified strains V5 ald6 GPD1 BDH1 and V5 ald6 GPD1 BDH1221,222,223.
In the final stage, we investigated the consequences of BDH1
overexpression for diacetyl production by V5 ald6 GPD1
. The overexpression of GPD1
triggers high-level accumulation of diacetyl (Tables and ), as shown for a previously engineered glycerol-overproducing brewer's yeast (25
). This effect can be attributed to the higher levels of pyruvate and acetaldehyde production in this background (4
). The overexpression of the two Bdh1 forms decreased the diacetyl level by half. This result emphasizes the limitation of the diacetyl reduction reaction in a GPD1
background, due probably to the level of synthesis of Bdh1 and/or NADH availability, similar to that of the acetoin reaction.