The aim of this study was to determine whether the transcription profiles of the various strains during fermentation could be reconciled with the volatile aroma compound production of these strains, and whether this comparative analysis could be used to predict the impact of individual gene expression levels on aroma compounds and profiles.
The data generated by the overexpression of four of the genes whose expression was statistically most significantly linked to the production of aroma profiles suggest that this approach has been successful. Indeed, overexpression of the selected genes had a far reaching impact on the aroma profiles produced by the fermenting yeast, and this impact was generally well aligned with the impact predicted from the comparative omics analysis. Indeed, the data aligned better than we, considering the significant challenges when approaching complex systems, had expected. Our data show that the metabolic changes observed upon overexpression of three of the four genes, AAD10, AAD14 and BAT1, were very significantly aligned with the changes that were predicted from the alignment of transcriptome and metabolome data alone. The predictions, as can be seen from the alignment of predicted vs. observed changes in metabolite levels in a qualitative manner, indeed proved fairly reliable. The model was able to assign positive and negative influences on a particular compound with relative accuracy. Although the extent/magnitude of the increase/decrease is not always well aligned with model values, the absolute direction of the change holds true in most cases. An absolute alignment would not be expected, since the level of expression in a plasmid-based system can not be adjusted to the differences of expression observed between the different strains. In the case of AAD10, only the influence of the overexpression on decanoic acid was not in line with the projection. Predictions for AAD14 and BAT1 were well matched with the observed changes in metabolite profiles. Predicted and real changes did not match satisfactorily in only one case, ACS1. Nevertheless, even in this case, eight out of the thirteen compounds evolved in the predicted direction. It should also be noted that the expression of this gene had generally a less severe impact on changes in the aroma profile than those of the other three genes.
Considering the complexity of the system, the rate of success achieved in this study can be considered as highly significant. To our knowledge, this is the first report to exploit such an intra- and interstrain comparative approach to identify genes that play a significant role in a complex metabolic network.
While we were clearly able to identify genes with significant impact on aroma compound production in a specific industrial environment, and which in some cases had not been previously directly linked to these pathways, the data do not allow a firm conclusion on the exact metabolic role of these genes. Indeed, the vast number of significant changes to metabolite levels makes it difficult to identify the specific 'point of influence' of any overexpressed gene in a given pathway.
On the whole though, our analysis shows that the cross-comparison of gene expression data with metabolite levels has the potential to identify points of interest on a genomic scale. This also opens new possibilities to design improved yeast enhancement strategies for optimized aroma production and fermentation performance.
Other genes of interest
Many other genes showed significant variation in expression between different strains and/or time points, as well as high loadings on PLS models and strong negative or positive correlations with specific aroma compounds. These genes encode enzymes that either are known to participate in aroma compound production, or have activities (either experimentally proven or suggested through sequence alignments) that could suggest such roles. Here we discuss some of the most relevant of these enzymes, which fall into several categories, either according to their place in a specific metabolic pathway such as the metabolisms of branched chain amino acids or of aromatic amino acids, or based on their specific activity such as dehydrogenases (in particular aldehyde and alcohol dehydrogenases) and acetyl transferases.
Of the enzymes involved in branched chain amino acid metabolism, BAT1
has been discussed above. Other genes that encode enzymes in this pathway and that were identified in our study for their strong statistical link between expression levels and the production of specific compounds include LEU2
, encoding a beta-isopropylmalate dehydrogenase that catalyzes the third step in the leucine biosynthesis pathway, and, to a lesser degree, LEU1
, which encodes an isopropylmalate isomerase [22
]. Both of these genes showed a significant statistical correlation with compounds such as isobutanol. Of the genes involved in the metabolism of isoleucine and valine (Ilv), only ILV5
, which encodes an acetohydroxyacid reductoisomerase involved in branched-chain amino acid biosynthesis [24
], showed a very strong positive correlation with almost all of the compounds analysed here, and, interestingly, a negative correlation with ethanol, suggesting that this gene could be an interesting target for metabolic engineering.
While BAT1 expression showed a significant positive correlation with a large number of the volatile compounds measured in our study, the cytosolic isoform (BAT2) of this enzyme showed no significant correlations with any of these aroma compounds. Although this isoform is supposedly highly expressed during stationary phase and repressed during the logarithmic phase, BAT2 expression levels in our study were found to stay constant, if not to decrease slightly upon entry into stationary phase in comparison to the exponential phase at day 2. In addition, BAT2 expression levels were generally considerably lower throughout fermentation when compared to BAT1.
Of the genes involved in aromatic amino acid metabolism, three, ARO1
, which encodes a pentafunctional arom protein, ARO7
, which encodes a chorismate mutase responsible for the conversion of chorismate to prephenate and ARO8
, which codes for an aromatic aminotransferase showed statistically significant correlations between expression levels and metabolite production [25
]. All three genes showed a modest positive correlation (r2
= 0.7) with 2-phenyl ethanol and mild negative correlations with all the other compounds. Only octanoic acid showed a very strong (r2
= 0.82) negative correlation with ARO8
expression at day 2 of fermentation. Despite its seemingly crucial role, ARO10
, which encodes a phenylpyruvate decarboxylase corresponding to the first specific step in the Ehrlich pathway did not show any noteworthy correlations between its expression and any of the volatile compounds in our study [27
]. Of course the possibility of translational or post-translational control of activity cannot be excluded.
Several specific enzyme activities were also overrepresented in our list. Such enzymes include many dehydrogensases. Aldehyde and alcohol dehydrogenases such as those encoded by ALD5
showed a substantial decline in expression levels between days 2 and 5 of fermentation, while others (such as ALD3
) increased during this time. The distinct expression patterns during fermentation reflects the different regulatory mechanisms governing the expression of these genes (i.e. expression of ALD3
is glucose-repressed and stress-induced) and suggests that the different ALD gene products have specific roles during different stages of fermentation [28
(mitochondrial), and ALD3
(cytoplasmic) encode aldehyde dehydrogenases involved in the conversion of acetaldehyde to acetate [29
encodes a mitochondrial aldehyde dehydrogenase (utilizing NADP+ or NAD+) that is required for growth on ethanol and conversion of acetaldehyde to acetate [29
]. Expression of ALD4
is also glucose repressed, and increases 2–4 -fold from day 2 to 5 of fermentation. ALD4
expression shows a very strong correlation to the amount of hexyl acetate (R2
= 0.82) produced by the fermenting yeast, as well as to ethyl acetate (0.77), isoamyl alcohol (0.91) and isoamyl acetate (0.85).
encodes a constitutively expressed cytosolic aldehyde dehydrogenase (utilizes NADP+ as the preferred coenzyme) and is required for conversion of acetaldehyde to acetate [30
]. Not surprisingly, ALD6
expression showed a very strong positive correlation to the levels of acetic acid produced by the fermenting cells (0.92). Also, expression was very strongly inversely correlated to ethanol production (R2
= 0.81). Interestingly, fairly strong positive correlations were also evident for 2-phenyl ethanol (R2
= 0.79) and 2-phenyl ethyl acetate (R2
encodes an NADPH-dependent cinnamyl alcohol dehydrogenase family member with broad substrate specificity [31
]. Expression was correlated very strongly with isobutanol levels (0.81), isobutyric acid (0.86), propionic acid (0.81), acetic acid (0.87) and 2-phenyl ethanol (0.92). ADH4
on the other hand showed only modest correlations with the above-mentioned, or any other aroma compounds for that matter.
With respect to the aryl alcohol dehydrogenase family of genes, the transcripts for AAD3
showed the greatest variation in expression, both on an intra- and interstrain level. Expression of AAD10
, for example, was increased more than twofold in most of the strains at day 5 relative to day 2 of fermentation. No distinct physiological role has been established for the products of these genes [7
], but it is reasonable to suspect that the consistent increase in their respective transcript levels during the course of fermentation could be associated with the increase in one or several of the long chain alcohols or their acid counterparts as fermentation progresses (tables , ).
This hypothesis is supported by the data generated through the overexpression of these genes. Indeed, overexpression yielded changes to the aroma profile that were very similar to those predicted from the alignment of transcriptome and metabolome data sets. The expression of AAD10 showed weak yet significant positive correlations with a number of the aroma compounds. Expression of AAD14 between different strains and time points was also highly variable. Highest expression levels were noted for the DV10 strain, and significant positive correlations with ethyl acetate (0.67) and ethyl caprate (0.74) were observed for this gene.
Acetyl transferases are another family of enzymes of relevance to aroma compound metabolism [32
]. However, neither ATF1
, the two most prominent alcohol acetyl transferases, showed statistically strong correlations between expression levels and metabolite production. EEB1
, on the other hand, which encodes an acyl-coenzymeA:ethanol O-acyltransferase and is responsible for the major part of medium-chain fatty acid ethyl ester biosynthesis during fermentation [33
], showed weak negative correlations with ethanol and other higher alcohols, and a strong positive correlation for 2-phenylethyl acetate (0.9) as well as octanoic acid (0.78). It is tempting to speculate that Eeb1p may thus be largely responsible for the acetylation of 2-phenyl ethanol to produce 2-phenylethyl acetate.
encodes an acyl-coenzymeA:ethanol O-acyltransferase that plays a role in medium-chain fatty acid ethyl ester biosynthesis, but also contains a known esterase activity [33
expression increased somewhat as fermentation progressed and inter-strain expression at both day 2 and 5 of fermentation varied significantly. Interestingly, EHT1
expression showed a fairly strong inverse correlated with 2-phenylethyl acetate (R2
= 0.74) and octanoic acid (R2
= 0.75), as well as a weaker yet significant inverse correlation with decanoic acid (R2
= 0.59). This could indicate that the esterase activity of Eht1p could predominate under certain conditions.
encodes a putative acyltransferase with similarity to both Eeb1p and Eht1p, and may have a minor role in medium-chain fatty acid ethyl ester biosynthesis [33
]. Expression was positively correlated with ethyl acetate (0.74), ethyl caprylate (0.85) and isoamyl acetate (0.78).
In addition to these relatively well studied acetyltransferases, the mRNA levels of the AYT1
gene, encoding a transferase of unknown substrate specificity, also showed considerable variation at different fermentative stages [34