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Applied and Environmental Microbiology, February 2008, p. 702-707, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01805-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Effects of ADH2 Overexpression in Saccharomyces bayanus during Alcoholic Fermentation

Oscar Maestre,¹ Teresa García-Martínez,¹ Rafael A. Peinado,² and Juan C. Mauricio^1*

Department of Microbiology, University of Córdoba, Edificio Severo Ochoa, Campus Universitario de Rabanales, 14014 Cordóba, Spain,¹ Department of Agricultural Chemistry, University of Córdoba, Edificio Marie Curie, Campus Universitario de Rabanales, 14014 Córdoba, Spain²

Received 3 August 2007/ Accepted 18 November 2007

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The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

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Saccharomyces cerevisiae possesses at least five genes (ADH1 to ADH5) that encode alcohol dehydrogenase isoenzymes involved in ethanol metabolism. The isoenzymes alcohol dehydrogenase I (ADH I), III, IV, and V reduce acetaldehyde to ethanol during alcoholic fermentation. In contrast, ADH II (EC 1.1.1.1) is glucose repressed and catalyzes the reverse reaction (i.e., the oxidation of ethanol to acetaldehyde). Therefore, when glucose in the fermentation medium is depleted, ADH II is the first enzyme in the use of ethanol (11). S. cerevisiae mutants possessing no ADH activity are unable to grow on ethanol as their sole carbon source, so they tend to accumulate large amounts of glycerol as a result (56). Although ADH1 and ADH2 share 89% sequence similarity, their respective expression products, ADH I and ADH II, differ in affinity for the substrate. Thus, the K_m value for ADH II for ethanol is 10 times lower than those for the other ADHs (37). Some authors have studied the presence of various transcription factors for the activation (derepression) of the gene ADH2 (10, 13, 14, 40, 43, 48, 53, 55).

Once alcoholic fermentation completes and fermentable sugars are depleted, some S. cerevisiae strains can switch from a fermentative metabolism to an oxidative (respiratory) metabolism and form a biofilm known as "flor" on the wine surface (24). Biological aging is carried out by such yeast strains (flor yeasts) (5). The molecular basis for the film formation has been the subject of recent study (19, 30, 52). During biological aging of wine, flor yeasts grow in a medium containing a high ethanol concentration and produce substantial amounts of acetaldehyde (2). The isoenzyme ADH II plays a key role in this process (6, 17, 24).

Overexpression of the ADH2 gene during fermentation can be expected to create conditions of a futile cycle and affect cellular demands for cofactors and also the redox status of the yeast cells.

In order to evaluate the impact of overexpression of the gene ADH2 in the V5 wine strain of Saccharomyces bayanus (MATa ura3) during alcoholic fermentation, we cloned the ADH2 gene from S. cerevisiae in a multicopy expression plasmid (pVT100-U) under control of the constitutive promoter ADH1. Overexpression was confirmed by enzymatic analysis and ADH II presence by proteomic analysis of the V5-pVT100-U-ADH2 strain after 48 h of cell growth in a glucose-containing medium and compared to that in the V5-pVT100-U strain used as a control. Once constitutive overexpression of gene ADH2 was confirmed, the physiological behavior of the recombinant during alcoholic fermentation of glucose toward ethanol was studied. This involved examining cell growth, glucose and ammonium uptake, the proteomic profile, and the intracellular concentrations of redox coenzymes (nicotinamide adenine dinucleotides) and the metabolome (specifically, the contents in volatiles and amino acids accumulating in the medium).

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Microorganisms and fermentation conditions.
Cloning tests were conducted with Escherichia coli DH5. The microbe was cultured according to the method of Sambrook et al. (39). The haploid model strain V5 of S. bayanus (MATa ura3) used in the present study was derived from the industrial strain W6, which was isolated from a Champagne wine; this strain is similar to industrial wine yeast strains with regard to its fermentation capabilities (26). The V5 yeast strain was maintained and grown on yeast-peptone-dextrose (YPD) medium containing 1% yeast extract, 2% peptone, and 5% glucose. The transformant yeast strains (viz. pVT100-U and V5-pVT100-U-ADH2) were stored in YNB medium containing 2% glucose and 0.5% ammonium sulfate but no amino acids. The fermentation medium, which consisted of YNB medium without amino acids containing 10% glucose, was adjusted to pH 3.5 in order to mimic wine-making conditions. Fermentations were performed by inoculating of 5 x 10⁶ cells per ml in fermentor flasks (250 ml) furnished with a rubber stopper through which a micropipette tip was inserted. All runs were performed under continuous stirring (175 rpm) at 28°C for 48 h.

DNA manipulation, cloning techniques, and transformation methods.
Restriction and modification enzymes were used according to the manufacturer's instructions (Promega, Madison, WI). Plasmids were prepared in accordance with standard protocols (39). Purified oligonucleotides for PCR were synthesized by Sigma-Genosys (United Kingdom). E. coli was transformed by using the CaCl₂-RbCl₂ method (18), and S. bayanus was transformed by following the lithium acetate procedure (42).

Plasmid construction.
The ADH2 gene was cloned downstream of the ADH1 promoter from S. cerevisiae and was amplified from the plasmid pMW5-ADH2 by PCR (E. T. Young, Department of Biochemistry, University of Washington, Seattle), using oligonucleotides bordering the expression cassette (forward primer, 5'-CGTCTGCAGAATGTCTATTCCAGAAACTCAA-3'; reverse primer, 5'-ATGGATCCCGCTTATTTAGAAGTGTCAACAACG-3'), into which BamHI and PstI sites were introduced. The PCR fragment was digested by BamHI and PstI and cloned into the BamHI and PstI sites of the pVT100-U plasmid (51) in order to obtain the pVT100-U-ADH2 vector. Primers were designed with the software Oligo 6.83 for Macintosh (Molecular Biology Insights, Plymouth, MN) to process the sequence of the gene ADH2 from S. cerevisiae (GenBank J01314) with an open reading frame size of 1,047 pb.

Protein extraction.
Yeast cell extracts were obtained by cellular lysis and protein solubilization, using the procedures of Khoudoli et al. (21) and Mathesius et al. (23) with slight modifications. After centrifugation of a suspended cell culture at 4°C at 12,000 x g for 5 min, the supernatant was discarded, and the pellet washed twice in bidistilled water. Each sample was then suspended in 160 µl of sterile water containing 5 mg of dithiothreitol (DTT) and broken using 0.2 to 0.3 g of glass beads up to 0.6 mm in diameter. The sample was denatured with 40 µl of buffer 1 (5% sodium dodecyl sulfate [SDS] in 0.5 M Tris-HCl [pH 7.5]), shaken, and incubated at 95°C for 5 min, after which it was supplied with 500 µl of buffer 2 (2 M thiourea, 7 M urea, 4% [wt/vol] CHAPS{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 1% [wt/vol] DTT, and 2% [vol/vol] Biolytes 3-10 from Bio-Rad) and placed in an orbital shaker at room temperature for 2 h. After centrifugation at 4°C at 12,000 x g for 20 min, the debris was removed, and the supernatant was collected and either stored at –80°C or used to clean up the extracts.

Protein extracts were cleaned up with 10% (wt/vol) trichloroacetic acid diluted in cold acetone containing 0.07% (wt/vol) DTT. The protein extract and trichloroacetic acid solution were used in identical volumes (125 µl). Proteins were allowed to precipitate at –20°C overnight, after which the protein pellet was collected by centrifugation at 4°C at 12,000 x g for 15 min and suspended in cold acetone containing 0.07% (wt/vol) DTT. After recentrifugation, the resulting pellet was dissolved in buffer 2. The protein concentration in each cell extract was determined by the method of Bradford (9), using the RC-DC protein test from Bio-Rad with bovine gamma globulin as standard (Sigma).

Samples were collected in liquid nitrogen and stored at –80°C until isoelectrofocusing (IEF).

Two-dimensional and gel analysis.
The first dimension was determined by using IPG strips (17 cm, 3-10 linear pH gradient) from Bio-Rad. The strips were hydrated with 150 µg of protein in 300 µl of buffer 2. IEF was done in a Protean IEF cell from Bio-Rad, using a constant current of 50 µA per strip up to 40,000 V·h. The strips were stored frozen at –20°C or immediately equilibrated for the next electrophoresis run by using the methods of Görg et al. (16) and Boucherie et al. (8).

The second SDS-polyacrylamide gel electrophoresis (PAGE) dimension was determined by previously soaking the strips in equilibration buffer I (1.5 M Tris-HCl [pH 8.8], 6 M urea, 2% SDS, 20% glycerol, 2% DTT) for 10 min and buffer II (1.5 M Tris-HCl [pH 8.8], 6 M urea, 2% SDS, 20% glycerol, 2.5% iodoacetamide) for another 10 min.

SDS-PAGE was performed in 13% polyacrylamide gels by using a Protean Plus Dodeca Cell. A constant current of 60 mA per gel was applied until the dye (bromophenol blue) front reached the end portion.

Preparative gels were stained with CBB G-250 (Merck, Darmstadt, Germany) according to the method of Mathesius et al. (23). Gel images were captured with a GS-800 densitometer from Bio-Rad and analyzed qualitatively by using the software PDQuest 7.4 from Bio-Rad.

In-gel digestion of proteins and simple preparation for MS analysis.
Proteins were identified by the Proteomics Service of the University of Córdoba. Spots of the target proteins were automatically extracted by using an Investigator Propia Station with a micropipette tip trimmed to fit the spot dimensions and then automatically digested by using a Proteineer DP protein digestion station from Bruker-Daltonics (Bremen, Germany). Digestion was performed according to the protocol of Shevchenko et al. (44) with slight modifications. The resulting digests were collected, vacuum dried, and stored frozen for subsequent mass spectrometry (MS) analysis.

MALDI PMF, LIFT TOF/TOF acquisition, and database searching.
Protein spots in the two-dimensional gels were identified by using a method based on the mass spectrum for the protein (viz. PMF). Peptide mass fingerprint (PMF) spectra were recorded on a Bruker Ultraflex time-of-flight (TOF)/TOF matrix-assisted laser desorption ionization (MALDI) mass spectrometer from Bruker-Daltonics, using the positive ion reflector mode (47). Whenever possible, mass measurements were made automatically by using fuzzy logic-based software. Each spectrum was internally calibrated against mass signals for trypsin autolysis ions in order to reach a typical mass measurement accuracy of ±25 ppm. Precursor ions for fragment ion analysis in the TOF/TOF mode were selected in accordance with their intensity and resolution by using a timed ion gate. The measured tryptic peptide masses, together with the corresponding MS/MS spectra, were transferred through the software MS BioTools (Bruker-Daltonics) as inputs to search the NCBInr database using the software MASCOT (Matrix Science, London, United Kingdom). Protein spots were identified in the Swiss-Prot database and the TrEMBL base for the S. cerevisiae yeast proteome (www.expasy.ch/sprot).

Analytical methods.
Yeast cells were directly counted under a light microscope in a Thoma chamber. The in vitro enzyme activities of ADH I and II were determined according to the method of Mauricio et al. (24). Coenzymes were determined by a high-pressure liquid chromatography-UV method as described by Theobald et al. (50) and Mailinger et al. (22). Glucose and ammonium ion were quantified by using the corresponding enzyme tests from Boehringer-Mannheim (Mannheim, Germany).

Major volatile compounds and polyols were quantified on a model 6890 gas chromatograph from Agilent Technologies (Palo Alto, CA), using the method of Peinado et al. (29). Quantitation was based on the response factors obtained for standard solutions of each compound.

Minor volatile compounds were determined by capillary column gas chromatography-MS after continuous extraction of 100 ml of sample with 100 ml of Freon-11 for 24 h. Samples were adjusted to pH 3.5 and supplied with 5 ml of a 30-mg/liter solution of 2-octanol as an internal standard. The Freon extracts containing the volatile compounds were concentrated to 0.2 ml in a Kuderna-Danish microconcentrator, and 1.5-µl aliquots were injected into an HP-6890 gas chromatograph equipped with an HP MS 5972A mass detector from Agilent Technologies (Palo Alto, CA). An HP-Innowax fused silica capillary column (60 m long by 0.32 mm [inner diameter], 0.25-µm film thickness) was used for this purpose. The temperature program was as follows: an initial temperature of 40°C, held for 10 min, followed by a 1°C/min ramp to 180°C, held for 35 min. Helium at a constant flow rate of 0.9 ml/min was used as carrier gas, and a 30:1 split ratio used at the injection port. The mass detector was used at a voltage of 1612 V in the scan mode in order to span the mass range from 39 to 300 atomic mass units.

Retention times, spectral libraries from Wiley, and pure chemical compounds obtained from Merck, Sigma-Aldrich, Riedel de Haën, and Fluka were used for identification, confirmation, and preparation of standard solutions of the volatile compounds studied. Each compound was quantified from its response factor, which was obtained from standard solutions of known concentration subjected to the same treatment as the samples, using the target and qualifier ions for each compound selected by a Hewlett-Packard Chemstation (Palo Alto, CA).

Free amino acids were determined according to the method of Botella et al. (7), using an absorbance at 254 nm of their dansyl derivatives (49); the derivatives were previously separated by high-pressure liquid chromatography on a reversed-phase column (15 by 0.4 cm) packed with Spherisorb ODS resin of 5-µm particle size that was supplied by Tracer Analitica (Barcelona, Spain). L-Norleucine at 5 mM was used as an internal standard. Free amino acids were identified by comparing their retention times to those for the standards (Sigma-Aldrich, Madrid, Spain) and quantified by using calibration curves constructed from solutions containing known variable concentrations of amino acids that were analyzed under the same conditions as the samples.

Statistical processing.
The results given are the averages of three independent experiments. ADH activities and compounds exhibiting significant differences between the two transformed yeast strains (viz. pVT100-U and pVT100-U-ADH2) were identified by one-way analysis of variance using the statistical software package Statgraphics Plus v.2 (STSC, Inc., Rockville, MD).

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Effect of overexpression of the gene ADH2 on the enzymatic activity of ADH I and II, proteome, cell growth, glucose and ammonium uptake, and intracellular redox potential.
Overexpression of the isoenzyme ADH II in the V5 strain of S. bayanus after 48 h of fermentation in a glucose-containing medium was confirmed by in vitro analysis of the activity of ADH II (Table 1), and also the location and identification of ADH II were confirmed by proteomic analysis (Fig. 1).

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TABLE 1. Specific activity of ADH I and II in the pVT100-U and pVT100-U-ADH2 transformants after 48 h of growth in YNB medium without amino acids containing 10% glucose

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FIG. 1. Two-dimensional CBB-stained gels for the V5-pVT100-U and V5-pVT100-U-ADH2 transformants after 48 h of growth in YNB medium without amino acids containing 10% glucose. Proteins were separated on 17-cm, pH 3-10 linear gradient IPG gel strips (IEF) and 13% polyacrylamide gels (SDS-PAGE). The gel area shows spots identified as ADH II, ADH I, and enolases.

Table 2 shows the number of yeast cells of the two studied strains, as well as the glucose and ammonium concentrations, in the medium after 48 h of fermentation. The V5-pVT100-U-ADH2 strain, which overexpresses the gene ADH2, grew to a somewhat lesser extent but used more glucose than did the control strain (V5-pVT100-U); therefore, glycolytic flux in the former strain seemingly increased under the influence of ADH2 overexpression, a finding that is consistent with the slight activation of ADH I observed in such a strain (Table 1). In contrast, the control strain exhibited greater cell growth and ammonium uptake.

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TABLE 2. Number of cells and concentrations of glucose and ammonium ion in the medium for the V5-pVT100-U and V5-pVT100-U-ADH2 transformants after 48 h of growth in YNB medium without amino acids containing 10% glucose

Overexpression of ADH II had no effect on the proteomic profile of transformant V5-pVT100-U-ADH2 (data not shown) but altered the intracellular redox potential (Table 3). Nevertheless, a dramatic increase in NAD⁺ was observed, which suggests the occurrence of many reduction reactions. On the other hand, the intracellular concentration of NADPH was lower in the V5-pVT100-U-ADH2 transformant than in the control strain, which suggests more extensive biosynthesis in the latter and is consistent with its increased biomass production (Tables 2 and 3).

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TABLE 3. Intracellular concentrations of nicotinamide adenine dinucleotides for the V5-pVT100-U and V5-pVT100-U-ADH2 transformants after 48 h of growth in YNB medium without amino acids containing 10% glucose

Effect of overexpression of the gene ADH2 on the yeast metabolome.
There were no significant differences in the following volatile compounds and polyols resulting from the overexpression of the gene ADH2 on the yeast metabolome: ethanol, acetaldehyde, glycerol, isoamyl alcohols, 2-phenylethanol, ethyl propanoate, 2,3-butanedione, ethyl 3-OH-butanoate, isobutanoic acid, butanoic acid, 2- and 3-methylbutanoic acid, furfuryl alcohol, hexanoic acid, and octanoic acid. However, overexpression led to a decreased concentration of some higher alcohols (isopropanol, 2,3-butanediol, methionol, and 3-ethoxypropanol), esters (ethyl acetate, isoamyl acetate, ethyl hexanoate, and monoethyl succinate), and pantolactone; in addition, overexpression indirectly increased the concentrations of acetoin, acetic acid, and decanoic acid (Table 4). Both transformants exhibited de novo synthesis and release of the amino acids L-proline, L-leucine, and L-tyrosine. However, the V5-pVT100-U-ADH2 transformant released 10 times more L-proline than did the control strain (Table 4).

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TABLE 4. Concentrations of volatile compounds, polyols, and free amino acids released to the fermentation medium by the V5-pVT100-U and V5-pVT100-U-ADH2 transformants after 48 h of growth in YNB medium without amino acids containing 10% glucose

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Alcoholic fermentation is characterized by an equilibrated redox balance. Any change in the fermentation conditions causes such a balance to readjust (35), and ADH modulation mechanisms play a prominent role in the re-adjustment (54). Glucose-repressed ADH II is crucial for yeasts growing on ethanol as the sole carbon source (particularly for flor yeasts) (24). The present study was conducted in order to examine the effect of overexpression of the gene ADH2 on a wine yeast strain during alcoholic fermentation.

Based on our results, the V5-pVT100-U-ADH2 transformant grew less than the control strain (V5-pVT100-U). Smits et al. (45) previously observed an identical phenomenon in transformants containing genes that encode glycolytic enzymes and ascribed it to a potential protein burden effect (46).

Acetaldehyde is produced by S. cerevisiae metabolism during the course of various natural processes such as alcoholic fermentation and biological aging of wines (24). During alcoholic fermentation, acetaldehyde forms as an intermediate product of pyruvate metabolism by the effect of glycolytic enzymes in the yeasts; in addition, acetaldehyde is a precursor of acetate and acetoin and also of ethanol (34). This aldehyde is a well-known inhibitor for a wide range of metabolic activities and is even more toxic than ethanol (3, 4, 35, 36, 54). The amounts of acetaldehyde, ethanol, and glycerol—three key compounds of alcoholic fermentation—released to the medium were similar for the two transformants studied here. Previously, Schaaff et al. (41) and Smits et al. (45) found the intracellular concentrations of metabolites in upper glycolysis not to be influenced by the overexpression of lower glycolysis enzymes, even though an entire sequence of glycolytic enzymes was overproduced. This suggests that the regulatory network is rigid enough to stabilize such an important pathway as glycolysis toward changes in enzyme levels. The glycolytic flux was only increased under conditions of increased ATP demand by the yeast cells (45). However, we found that metabolites other than those involved in glycolysis (viz. acetic acid, acetoin, and L-proline) accumulate in the medium containing the recombinant strain V5-pVT100-U-ADH2. Genetic engineering strategies aimed at modifying glycerol accumulation in wines have exposed the difficulty of altering yeast metabolism in the desired direction without introducing unwanted side effects (15, 26, 32).

During alcoholic fermentation, acetic acid is produced by S. cerevisiae as an intermediate of the pyruvate dehydrogenase bypass, which is an alternative via the pyruvate dehydrogenase reaction for converting pyruvic acid into to acetyl coenzyme A (27; J. M. Cherry, C. Ball, K. Dolinski, S. Dwight, M. Harris, J. C. Matese, G. Sherlock, G. Binkley, H. Jin, S. Weng, and D. Botstein, Saccharomyces genome database [http://genome-www.stanford.edu/Saccharomyces/]). Acetic acid may play a key physiological role in maintaining the redox balance (31, 38). The transformant strain V5-pVT100-U-ADH2 produced increased concentrations of this acid, probably as a consequence of its increased acetaldehyde production, as previously suggested by Roustan and Sablayrolles (35), who found gradually raising the acetaldehyde concentration in the fermentation medium resulted in gradually increased acetic acid contents.

Acetoin is the key compound in the biosynthesis of 2,3-butanediol and diacetyl. These three compounds represent three levels of oxidation in one four-carbon skeleton (12). One crucial reaction in the utilization of pyruvate during alcoholic fermentation is its decarboxylation to hydroxyethyl-thiamine PP_i, which is called the acetaldehyde-TPP complex (active acetaldehyde), by thiamine PP_i (TPP). Depending on the particular substrate for active acetaldehyde, the following sequences are possible (33): pathway A, active acetaldehyde + pyruvate acetolactate acetoin; pathway B, active acetaldehyde + acetyl coenzyme A diacetyl acetoin; and pathway C, active acetaldehyde + acetaldehyde acetoin.

Our results suggest that pathway C may be the most favorable. This is also consistent with the increased acetoin production observed upon addition of acetaldehyde to yeast extracts (33). The production of acetoin in response to an increase in acetaldehyde concentration has also been observed in other situations involving acetaldehyde accumulation. Roustan and Sablayrolles (35) found the addition of acetoin to the fermentation medium to be a highly efficient way of boosting fermentation kinetics by keeping the residual acetaldehyde concentration at an optimal level over long periods. According to these authors (36), the production of acetoin and its subsequent reduction to a diol provides an important mechanism for maintaining the redox balance. This synthetic pathway is also useful because it facilitates a detoxification of acetaldehyde (28, 35).

The previous results suggest that yeasts readjust a number of mechanisms to maintain their redox balance by synthesizing and releasing some compounds such as L-proline (25). Also, yeasts regulate the production of toxic compounds by triggering detoxification mechanisms such as that involving the production of acetoin and acetate. Therefore, overexpression of gene ADH2 may lead to the accumulation of acetaldehyde and NADH; this may disrupt the redox balance and result in accumulation of a toxic compound, which is bound to elicit a defensive response from the yeasts via the above-described mechanisms. Figure 2 shows a scheme consistent with the biosynthesis of some metabolites related to the maintenance of the redox balance and triggering of the acetaldehyde detoxification mechanism as a result of ADH2 overexpression. In addition, overexpression of ADH2 stimulates, in an indirect manner, glucose consumption without increasing the net ethanol concentration; also, as noted earlier, overexpression of the gene boosts the formation of acetoin and acetic acid and the de novo synthesis of L-proline. The overproduction of these compounds may occur at the expense of biomass production and of other biosynthetic pathways such as the formation of pantolactone, some higher alcohols, and various esters, all of which are a consequence of a compensatory effect in the pVT100-U-ADH2 strain. Thus, the metabolomic analysis of the pVT100-U-ADH2 transformant revealed that overexpression of the gene ADH2 led to an altered production of some secondary metabolites of fermentation, including acids, amino acids, esters, lactones, and higher alcohols, many of which are active wine odorants. These results were obtained for a laboratory yeast strain grown in a synthetic medium. Further tests involving the use of industrial wine strains during the fermentation of grape juice are needed to assess the impact of these changes on the sensory properties of wine.

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FIG. 2. Biosynthesis of various compounds associated with the maintenance of the intracellular redox potential and the acetaldehyde detoxification mechanism (1, 20, 25, 31, 33). The thicker arrows denote the most favorable steps for the transformant strain pVT100-U-ADH2.

 
This study was cofunded by Spain's Ministry of Education and Science (project AGL2005-01232/ALI) and FEDER.

We thank B. Blondin and S. Dequin of the UMR 1083 Sciences pour l'Oenologie, INRA, Montpellier, France, for kindly supplying S. bayanus strain V5 and the multicopy plasmid pVT100-U.

 
* Corresponding author. Mailing address: Departamento de Microbiología, Universidad de Córdoba, Edificio Severo Ochoa, Campus Universitario de Rabanales, 14014 Córdoba, Spain. Phone: 34 957218640. Fax: 34 957218650. E-mail: mi1gamaj{at}uco.es

Published ahead of print on 7 December 2007.

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Applied and Environmental Microbiology, February 2008, p. 702-707, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01805-07
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