Genes in Saccharomyces cerevisiae important in flavour production
There are a number of genes in yeast that play a pivotal role in yeast metabolism and the associated production of flavour compounds (Verstrepen et al. 2003b; Swiegers et al. 2006; Saerens, S. et al. 2008b). Different strains of yeast have been shown to produce different levels of metabolites under identical growth conditions, and it is genetic variation that is thought to account for these differences (Swiegers et al. 2006). While it is very difficult to source identical raw materials for each brew or wine fermentation, it is thought that a better understanding of how variation in amounts of amino acid content, metal ion levels and so on affect gene expression may lead to the ability to make a more consistent product (Bromberg et al. 1997; Younis and Stewart 1999; Pretorius and Bauer 2002; Verstrepen et al. 2003c; Swiegers et al. 2006).
Genes involved in the Ehrlich Pathway
There are multiple genes able to catalyse each step of the pathway, and the major enzyme responsible for each step depends on the amino acid being degraded (Hazelwood et al. 2008). The initial reversible transamination step is catalysed by BAT1 and BAT2 (branched-chain amino acids), or ARO8 and ARO9 (aromatic amino acids) (Eden et al. 2001; Vuralhan, Z et al. 2005), although both ARO8 and ARO9 both appear to have a broader substrate specificity than originally reported. ARO9 in particular is up-regulated in glucose-limited chemostats with phenylalanine, methionine and leucine as the sole nitrogen source (compared to yeast grown with a nitrogen source not involved in the Ehrlich pathway) (Godard et al. 2007).
The decarboxylation step is irreversible and commits the 2-oxo acid product of transamination to the Ehrlich pathway. There are four genes thought to play a role in the decarboxylation step: the pyruvate decarboxylases encoded by PDC1, PDC5 and PDC6, and ARO10 (Ter Schure et al. 1998; Vuralhan, Z et al. 2005). Previously it was thought that THI3 encoded an enzyme with decarboxylase activity, but recently it has been suggested that it plays a regulatory role instead (Vuralhan, Z. et al. 2003; Vuralhan, Z et al. 2005).
The fate of the resulting aldehyde is thought to depend on the redox state of the cell. While the oxidation of the aldehyde consumes NAD(P)+ and produces NAD(P)H, the reduction consumes NADH, yielding NAD+. It has been demonstrated that any one of Adh1p, Adh2p, Adh3p, Adh4p, Adh5p or Sfa1p can catalyse the formation of fusel alcohols, and Ypr1p and Gre2p have been shown to have activity towards 2-methylbutyralaldehyde and isovaleraldehyde respectively (Dickinson et al. 2003; Hazelwood et al. 2008). S. cerevisiae also harbours seven putative aryl alcohol dehydrogenases, but these do not appear to play a role in the pathway (Dickinson et al. 2003). It is thought that the aldehyde dehydrogenases, Ald4p, Ald5p and Ald6p are responsible for the synthesis of fusel acids, with Ald6p playing the major role (Saint-Prix et al. 2004; Hazelwood et al. 2008).
The alcohol acetyl transferases
There are two enzymes responsible for acetate ester synthesis in S. cerevisiae; the alcohol acetyl transferases, Atf1p and Atf2p (Mason and Dufour 2000). Acetate esters may be formed by a spontaneous chemical reaction, but their rate of accumulation is too high for this to account for their levels in wine and beer. In 1964 Nordstrom demonstrated that they are formed by the action of an “ester synthase” (Nordstrom 1964), but it wasn’t until 1994 that ATF1 was identified (Fujii et al. 1994), while ATF2 was isolated in 1998 (Nagasawa et al. 1998). Atf1p plays the major role in acetate ester synthesis, accounting for 80% of isoamyl acetate synthesis. Atf2p accounts for the majority of the remaining isoamyl acetate, however the ATF1/ATF2 double mutant still retains some AATase activity (Verstrepen et al. 2003c). At the deduced amino acid level, the two genes are 36.9% identical and both contain a heptapeptide sequence, WRLICLP, which is unique to these proteins in the S. cerevisiae genome (Mason and Dufour 2000; Van Laere et al. 2008). Both also contain the HXXXD motif present in the enzyme superfamily that includes the plant alcohol acyltranferases. However, neither contains the DFGWG motif that is highly conserved in the BAHD enzyme family of plant enzymes (D'Auria 2006).
ATF1
Construction of an ATF1::GFP fusion construct by Verstrepen and co-workers led them to conclude that Atf1p localises to the lipid particles, along with many other proteins, most of which are involved in lipid metabolism (Athenstaedt et al. 1999; Verstrepen et al. 2004). ATF1 expression is profoundly affected by environmental conditions. The presence of oxygen directly represses ATF1 expression, as does the presence of unsaturated fatty acids (both properties shared by the D9 fatty acid desaturase gene, OLE1) (Fujiwara et al. 1998; Mason and Dufour 2000; Verstrepen et al. 2003a). Interestingly, the lower the melting temperature of the fatty acid, the stronger their repressive effect on the transcription of ATF1 (Fujiwara et al. 1999). ATF1 transcription can also be effected by factors as diverse as carbon and nitrogen availability, temperature and zinc availability (Mason and Dufour 2000; Verstrepen et al. 2003a; Verstrepen et al. 2003b). Given that it is the expression of the alcohol acetyl transferase genes, rather than substrate availability, that plays the major role in acetate ester synthesis, any factor that effects transcription also effects final ester concentrations (Verstrepen et al. 2003c).
ATF2
It was recently found that ATF2 plays a role in sterol acetylation, a process necessary for the export of sterols and steroids from the yeast cell. Briefly, ATF2 was found to be responsible for the non-specific acetylation of sterols and steroids. These acetylated compounds are then deacetylated according to the substrate specificity of the deacetylase Say1p, with the acetylated compounds then being exported out of the cell in what is presumed to be a vesicle mediated process (Tiwari et al. 2007). This is in accordance with the earlier findings of Cauet and co-workers, who found that a strain of S. cerevisiae lacking ATF2 was unable to acetylate pregnenolone, and that this led to an observable toxic effect (Cauet et al. 1999). Atf2p is an integral membrane protein of the ER that contains at least two transmembrane domains with both termini oriented towards the lumenal compartment (Tiwari et al. 2007). A mean hydrophobicity index of –0.36 and sub-cellular fractionation data suggests that Atf2p is a mainly soluble protein (Mason and Dufour 2000).
Physiological role of Atf1p and Atf2p
A bioinformatic analysis of AATases from various yeast species found that while Saccharomyces sensu stricto yeasts have two genes encoding AATases, more distantly related yeasts have only one orthologue. In those yeasts with just one AATase gene, the protein sequence was found to be most similar to ATF2. It is thought that the presence of two AATase genes may be due to a whole genome duplication (WGD) event thought to have occurred during the evolution of ascomycetous fungi (Wolfe 2004; Van Laere et al. 2008). Van Laere et al. suggest that in S. cerevisiae, ATF2 retained the initial function of AATase’s pre WGD, while ATF1 developed a new, specific function, most likely involved in anaerobic lipid metabolism (Van Laere et al. 2008). While these authors do not mention the work of Tiwari and co-workers, it seems likely that function of ATF2 type AATases is to acetylate sterols and steroids for detoxification (Tiwari et al. 2007). The precise physiological role of ATF1, and indeed acetate ester synthesis remains unclear. However, the findings of Delneri et al., that the heterozygous diploid ATF1 mutant is haploinsufficent in glucose limited, ammonium limited and phosphate limited conditions (Delneri et al. 2008), suggest that ATF1 has a physiological role beyond the synthesis of acetate esters.
IAH1
IAH1 is an esterase that breaks down acetate esters (Fukuda et al. 1996). Originally named EST2, the protein was found to lack the GSXSG consensus motif of serine type esterases and lipases. However, it was assumed that the similar AXSXG pentapeptide sequence was the active site (Fukuda et al. 1996). This observation, as well as the fact that the enzyme was completely inhibited by diisopropyl fluorophosphate and partly inhibited by phenylmethylsulfonyl fluoride, led to the conclusion that Iah1p was a serine type carboxylesterase (Fukuda et al. 2000). Using various overexpression constructs and the DIAH1 and DATF1 strains, it was found that isoamyl acetate production in a sake brew was a result of the balance of expression of these two genes. The authors found that overexpression of ATF1 was more significant in isoamyl acetate ester production than IAH1 levels, however (Fukuda et al. 1998).
