UMR Sciences pour l'nologie, Microbiologie et Technologie des Fermentations, INRA, 2 Place Viala, F-34060 Montpellier Cedex 1, France
Correspondence
Sylvie Dequin
dequin{at}ensam.inra.fr
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ABSTRACT |
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INTRODUCTION |
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Two ACDHs were originally identified in S. cerevisiae: an Mg2+-activated, NADP+-dependent cytosolic enzyme (Seegmiller, 1953) and a glucose-repressed, NAD(P)+-dependent, mitochondrial enzyme activated by K+ and thiols (Jacobson & Bernofsky, 1974
). Five ACDH genes have now been identified in the genome sequence of S. cerevisiae S288C. According to the nomenclature of Navarro-Avino et al. (1999)
, the cytosolic ACDHs are encoded by ALD6 (YPL061w), ALD2 (YMR170c) and ALD3 (YMR169c), whereas the mitochondrial isoforms are encoded by ALD4 (YOR374w) and ALD5 (YER073w). Ald6p and Ald4p, which are Mg2+- and K+-dependent, respectively, are the major isoforms and have been shown to be involved in growth on glucose and on ethanol (Meaden et al., 1997
; Tessier et al., 1998
; Wang et al., 1998
). However, the behaviour of deletion mutants indicates that the cytosolic and mitochondrial isoforms may, in some conditions, be at least partially redundant (Boubekeur et al., 1999
, 2001
; Remize et al., 2000
). We reported previously that a double mutant, ald6
ald4
, produced acetate during anaerobic growth on glucose, indicating that other routes exist for the synthesis of cytosolic acetyl CoA (Remize et al., 2000
). The roles of the minor cytosolic and mitochondrial isoforms of ACDH in acetaldehyde metabolism have not been clearly defined. Ald2p and Ald3p are encoded by tandem reading frames, which display 92 % identity. ALD2 and ALD3 are induced by a variety of stresses (Navarro-Avino et al., 1999
; Norbeck & Blomberg, 2000
) and it has been suggested that these NAD+-dependent isoforms play a role in redox metabolism, particularly under conditions of osmotic stress (Norbeck & Blomberg, 1997
, 2000
; Akhtar et al., 1997
). However, the only detectable phenotype of the ald2
ald3
double mutation is a lower than normal growth rate on ethanol, suggesting a possible role for the corresponding isoforms in ethanol oxidation (Navarro-Avino et al., 1999
). Ald5p makes only a minor contribution to total ACDH activity (Navarro-Avino et al., 1999
). It has been suggested that this isoform is involved in respiratory metabolism, based on the low levels of cytochrome in an ald5
mutant (Kurita & Nishida, 1999
).
The objective of this work was to investigate the specific contribution of each Aldp isoform to acetate production during anaerobic glucose fermentation. This study, in addition to providing new insight into acetaldehyde metabolism, also has important implications for industrial yeast-based processes, because the acetate produced by the PDH bypass is the most abundant organic acid accumulating during the alcoholic fermentation of sugars. We constructed a set of single and multiple ald mutants in two S. cerevisiae strains with different genetic backgrounds (a laboratory and a wine-yeast-derived strain). We analysed the growth and acetate production of the deletion mutants during anaerobic fermentation, under standard (YPD 5 % glucose) or wine fermentation (MS 20 % glucose) conditions. Wine fermentation conditions typically involve multiple stresses (osmotic, acidic and ethanol stress, nitrogen limitation). We provide evidence that Ald6p, Ald5p and, depending on strain and culture conditions, Ald4p are required for acetate formation during anaerobic growth on glucose, whereas Ald2p and Ald3p are not. We also show that the mitochondrial PDH bypass can compensate for the absence of Ald6p and that this compensation involves the transcriptional activation of ALD4. We also report that mutants devoid of ACDH activity are viable, indicating that an unknown, alternative pathway produces acetate, and thus cytosolic acetyl-CoA, in the mutants.
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METHODS |
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Cells were first cultured in Erlenmeyer flasks containing 20 ml MS or YPD medium for 30 and 24 h, respectively, at 28 °C with shaking. Fermenters (0·25 l), filled to 80 % of their volume, were inoculated with cells from these precultures at a density of 1x106 cells ml1 and fermentation was carried out at 28 °C with continuous stirring (500 r.p.m.). The carbon dioxide produced by the fermentation reaction was evacuated from the fermenter via a thin glass tube that passed through the cork used to seal the fermenter. Fermentation experiments were performed in triplicate and one representative experiment is shown.
Construction of ald mutants in the V5 and CEN.PK strains.
Genes were deleted by the short flanking homology method using the loxP-kanMX4-loxP gene disruption cassette and the Cre-Lox recombination system, which allows marker recycling (Güldener et al., 1996). The deletion cassette was amplified from pUG6 (Güldener et al., 1996
) using the primer pairs listed in Table 2
for each ALD gene. The forward primer has 18 nt (bold type) complementary to the sequence of pUG6 and an extension of 3642 nt corresponding to the region upstream from the start codon of the ALD ORF. The reverse primer has 22 nt (bold type) complementary to pUG6 and an extension of 4041 nt, corresponding to the region immediately downstream from the stop codon of the ALD ORF. We transformed S. cerevisiae with the PCR products by the lithium acetate procedure (Schiestl & Gietz, 1989
). We generated multiple deletion mutants by excising the kanMX4 cassette using pSH47 as described by Güldener et al. (1996)
before carrying out the deletion procedure. We previously showed that the V5 strain, which was isolated as a meiotic segregant of a diploid industrial wine yeast strain, carries two copies each of ALD6 and ALD4 (Remize et al., 2000
). In this study, we found that V5 also possesses two copies of ALD5. Deletion of the two ALD6, ALD4 and ALD5 alleles was obtained directly or by two rounds of integrationexcision. The correct integration of the replacement cassettes was confirmed by PCR on genomic DNA using primers complementary to regions upstream and downstream from the ALD ORF, and by Southern blotting of chromosomes separated by PFGE (TAFE system; Beckman) using specific oligonucleotides (Table 2
) as probes. The CEN.PK ald0
strain was produced by mating strain CEN.PK2-1C ald6
ald4
ald2
ald3
and strain CEN.PK2-1D ald5
: : kanMX, sporulation of the resultant diploid and dissection of the ascospores. The haploid progeny was grown in the presence of acetate (0·5 %) and the spores were selected for G418 resistance. The presence of the ald5
: : kanMX allele was determined by PCR and Southern blot analysis of total genomic DNA. The deletion of each other ALD gene was checked by PCR.
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RNA extraction and Northern blot analyses.
Total RNA was isolated from 2x109 cells using Trizol reagent (Gibco-BRL, Life Technologies), as described previously (Remize et al., 2001). RNA samples (15 µg per lane) were separated by electrophoresis in 1 % agarose gels containing formaldehyde, blotted by capillary transfer onto Hybond-N+ membranes (Amersham) as described by Sambrook et al. (1989)
and cross-linked by exposure to low-wavelength UV radiation for 1 min. Membranes were prehybridized by incubation for 24 h at 50 °C in 0·5 M phosphate buffer (pH 7), 1 mM EDTA, 7 % SDS and 1 % BSA. Membranes were hybridized with 32P-labelled oligonucleotides (2x106 c.p.m. ml1) used as probes, in the same solution, at 50 °C for 18 h. The membranes were washed twice in 2·5 mM phosphate buffer (pH 7), 6x SSC, 0·25 % SDS for 5 min at room temperature and twice for 5 min at the hybridization temperature, and analysed with a PhosphorImager (Molecular Dynamics). Probes were prepared by labelling oligonucleotides (Table 2
) with [
-32P]ATP using T4 polynucleotide kinase (Promega) and purified using a silica-gel membrane (QIAquick Nucleotide Removal; Qiagen). The specificities of the oligonucleotides used to probe ALD genes were checked by hybridization to the chromosomes of the corresponding ald
mutants. S25 RNA was used as a loading control and was detected by probing the membrane with the oligonucleotide 5'- CCTCCGCTTATTGATATGCTTAAG-3'.
Crude cell extract preparation and Western blot analyses.
Yeast cells (1x109 cells) were harvested by centrifugation, washed in 9 g NaCl l1 and resuspended in 0·5 ml 100 mM phosphate buffer (pH 6). Cells were disrupted with glass beads and centrifuged. The supernatant was used as crude extract. The total protein concentration of the crude extract was determined by the method of Bradford (1976) using a Bio-Rad kit and BSA as the standard. HA-tagged proteins were analysed by SDS-PAGE followed by transfer onto Hybond-C Extra membranes (Amersham) as described by Sambrook et al. (1989)
. The gels used for SDS-PAGE contained 4·5 % acrylamide in the stacking gel and 10 % acrylamide in the resolving gel. Membranes were then incubated for 30 min at room temperature with 1 % BSA in 20 mM Tris/HCl, 150 mM NaCl and 0·05 % Tween 20 (pH 7·5) for blocking. The blots were incubated overnight at room temperature with the anti-HA mAb (Sigma) at a final dilution of 1 : 5000, washed and incubated for 30 min at room temperature with anti-mouse IgG alkaline phosphatase conjugate (Promega). The blots were washed and antibody binding was detected by incubation with Western Blue stabilized substrate for alkaline phosphatase (Promega), according to the manufacturer's instructions.
Analytical methods.
Optical density was measured at 660 nm. Acetic acid was determined by HPLC using an HPX-87H ion exclusion column (Bio-Rad) and by enzymic assays (Boehringer detection kit). Glucose was determined by a colorimetric method using 3,5-dinitrosalicylic acid as described by Miller (1959).
Cell extracts and enzyme assays.
Cells extracts were obtained as described previously (Remize et al., 2000). Enzyme activities were assayed immediately after the preparation of cell extracts. Protein concentration was determined by the method of Bradford (1976)
using a Bio-Rad kit and BSA as the standard. For the determination of total ACDH specific activity, the reaction was carried out as described by Postma et al. (1989)
except that we added MgCl2 (10 mM) and both NAD+ and NADP+ to the reaction mixture.
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RESULTS |
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DISCUSSION |
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It has been suggested that both Ald6p and Ald4p could contribute to the formation of acetate during fermentation, based on the slow growth on glucose of mutant ald6ald4
(Wang et al., 1998
). Results from expression studies and analysis of ald4 deletion mutants in V5 (MS and YPD medium) and CEN.PK (MS) support the idea that Ald4p is not involved in acetate formation. This is consistent with the fact that the K+ mitochondrial isoform is strongly repressed by glucose (Llorente & Nunez de Castro, 1977
). However, the role of Ald4p may depend both on strain genetic background and on medium composition since deletion of ALD4 slightly decreased the level of acetate formed in the CEN.PK strain when grown on YPD only. A possible explanation for the difference observed between MS 15 % glucose and YPD 5 % glucose medium may rely on a different level of glucose repression. It has been shown that the NAD+-linked activity found in cells grown on 0·5 % glucose could not be detected in cells grown on 10 % glucose (Llorente & Nunez de Castro, 1977
), but the level of repression as a function of glucose concentration has not been investigated. To test this hypothesis, we examined the impact of the deletion of ALD4 on YPD 5, 10 and 15 % glucose. We found that deletion of ALD4 in strain CEN.PK did not significantly affect the level of acetate produced at concentrations higher than 5 % (data not shown), supporting the view that Ald4p might be totally repressed on MS but not on YPD. The different behaviour of the ald4 mutant in the CEN.PK and V5 strains might be due to differences in the regulation of the corresponding isoforms or to other genetic variations. Major differences have been reported in the phenotype of ald4
mutants; growth on ethanol was abolished or impaired, depending on genetic background (Tessier et al., 1998
; Wang et al., 1998
). These differences have been explained by a partial compensation of the cytosolic ACDH for the lack of ALD4 only when the activity of the cytosolic enzyme is sufficient (Boubekeur et al., 2001
).
We showed during this work that Ald4p can compensate for the lack of Ald6p in yeast grown on glucose, and that this compensation requires the induction of ALD4 transcription. The mechanism underlying this induction remains to be elucidated. As K+-ACDH is subject to glucose repression, we cannot exclude that ALD4 is derepressed in ald6 mutants. This hypothesis is supported by the finding that a ald6
mutant exhibited an increase in K+-ACDH activity on 5 % glucose that was not observed on 0·5 % glucose (Tessier et al., 1999
).
The roles played by the products of the closely related ALD2 and ALD3 genes in acetaldehyde metabolism are unclear. The lower growth rate on ethanol of the ald2ald3
mutant suggested a possible role for these isoforms in ethanol oxidation (Navarro-Avino et al., 1999
). Other studies have suggested that these isoforms may be involved in redox metabolism, particularly under conditions of osmotic stress (Navarro-Avino et al., 1999
; Blomberg & Adler, 1989
). Our results show that Ald2p and Ald3p are not required for acetate production during growth on glucose, even under wine fermentation conditions, in which yeast cells are exposed to osmotic stress due to the large amount of sugar present. Very recently, these two proteins, which are more distantly related to ACDH (Navarro-Avino et al., 1999
; Meaden et al., 1997
), were shown to have a specialized function in coenzyme A biosynthesis, converting 3-aminopropanal to
-alanine (White et al., 2003
).
Role of the mitochondrial PDH bypass during anaerobic glucose fermentation
Overall, our data show that the cytosolic PDH bypass and a mitochondrial bypass may function simultaneously to produce acetate during fermentation (Fig. 6). Acetaldehyde produced from pyruvate is converted to acetate by Ald6p in the cytosol and by Ald5p in the mitochondria, and this results in the generation of NADPH reducing equivalents. Acetate formed in the mitochondria is then exported to the cytosol where it is converted to acetyl-CoA. When ALD6 is lost, acetate is formed by the mitochondrial enzymes. However, in this case overproduction of Ald4p is necessary to compensate efficiently for the loss of Ald6p.
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In the ald6 mutants, only the mitochondrial route is operative. Since redox balance has to be achieved in individual compartments, the compensation of the mitochondrial enzymes for the loss of ALD6 would result in redox imbalance. A first possibility to account for the expected imbalance is a relocalization of the Ald4p isoform in the cytosol. However, protein fractionation experiments allowed us to discard this hypothesis (data not shown), suggesting that the demand for reducing equivalents is handled in a different manner.
Alternative pathways for acetate production
The ald0 mutant, in which all five members of the ACDH family have been deleted, is viable. This mutant, and the ald6
ald4
ald5
mutant, both produced acetate, allowing acetyl-CoA production in the cytosol. The phenotype of a mutant devoid of PDC has demonstrated the absence of any other pathway for acetate production in the cytosol. Indeed, a pdc
mutant can only grow on minimal medium with glucose if the medium is supplemented with acetate (Pronk et al., 1996
). A currently unknown pathway, normally inoperative on glucose, may therefore be deregulated to produce acetate in mutants devoid of ACDH activity. There are several possible ways in which acetate may be formed in the ald6
ald4
ald5
and ald0
mutants. First, acetate may be produced by another dehydrogenase enzyme, capable of using acetaldehyde as a substrate. The ALD genes belong to the larger family of aldehyde dehydrogenases, which also includes MSC7, UGA2, PUT2, YMR110C, YKR096W and YIL151C. However, we detected no residual ACDH activity in the ald6
ald4
ald5
and ald0
mutants, suggesting that this hypothesis is incorrect. The conversion of acetaldehyde to acetate and ethanol is another possible source of acetate and such a conversion may be carried out by the dismutase activity which is a known secondary activity of alcohol dehydrogenases. Such activity has been described in various organisms, including humans, Drosophila melanogaster and Alcaligenes eutrophus (Svensson et al., 1999
; Winberg & McKinley-McKee, 1998
; Steinbüchel & Schlegel, 1984
), but has not yet been reported in yeast. Alternatively, acetate may be formed by hydrolysis of mitochondrial acetyl-CoA. S. cerevisiae has an acetyl-CoA hydrolase, which is glucose-repressed and which has been shown recently to be localized in the mitochondria (Buu et al., 2003
). Investigations are under way to examine these possibilities further.
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ACKNOWLEDGEMENTS |
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Received 19 December 2003;
revised 9 March 2004;
accepted 19 April 2004.