Yeast genomics on food flavours

Abstract

The appearance and concentration of the fusel alcohol 3-methyl-1-butanol is important for the flavour of fermented foods. 3-Methyl-1-butanol is formed by yeast during the conversion of L-leucine. Identification of the enzymes and genes involved in the formation of 3-methyl-1-butanol is a major prerequisite to optimize and control the final food flavour. To identify genes involved in this metabolic route, cDNA microarrays were carried out to study yeast gene expression genome-wide. Crosshybridisation with probes from the ‘soy-yeast’ Zygosaccharomycs rouxii, responsible for flavour formation during soysauce fermentation, on Saccharomyces cerevisiae cDNA microarrays, revealed the presence of several Zr genes. However, this group (115 out of ~5000 Zr ORFs) was too small for studying the 3-methyl-1-butanol pathway. Conversely, arraydata of S. cerevisiae resulted in several clues concerning flavour formation. For example BAT2, encoding an enzyme responsible for the first step in the L-leucine breakdown, was strongly expressed in yeast grown on ethanol. In addition to the transcription data, deletion studies revealed that 3-methyl-1-butanol production stopped in S. cerevisiae lacking BAT2, indicating a major role for Bat2p. Furthermore, the microarrays showed that genes involved in branched chain amino acid biosynthesis, but also genes of amine biosynthesis, caboxylic acid- and organic acid metabolism, were altered in expression level. Interestingly, non-branched chain amino acids: aromatic amino acids, serine, arginine, glutamine, homoserine and lycine, superficially unrelated with branched chain amino acids, were also effected when L-leucine was used as nitrogen source. A subgroup of genes was significantly present, which contained regulatory elements for GCN4 and GLN3. GCN4 and GLN3 encoding transcription factors involved in nitrogen metabolism. BAP2 and BAP3, encoding permeases necessary for the uptake of L-leucine, PDX1 and YDL080c, encoding decarboxylases for 4-methyl-2-oxopentanoate and ADH3, ADH5, YMR 318c and YCR105w, encoding alcohol dehydrogenases were also upregulated in S. cerevisiae. Concerning flavour formation from L-leucine, besides 3-methyl-1-butanol, also 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutanoic acid and 3-methylbutyric acid emerged. The combination of metabolite formation and the expression data from cDNA microarrays thus provides strong indications of the existence of alternative pathways. Automated microtiter plates (MTP) fermentations with a pH or/and NaCl range, revealed the optimal fusel alcohol formation at pH 3.0 without NaCl. In addition to this metabolic screening, cDNA microarrays were carried out and showed a total 747 genes, which displayed significantly altered transcription activity at pH 3.0, compared to the standard pH 5.0. Further investigation on this dataset, by the web-tool SGD Gene Ontology (GO) Term Finder revealed that a significant part of these genes had a function in the metabolism of nicotinamides, vitamins, fatty acids, glutamate or carboxylic acids. From this group, 11 genes, BNA6, BNA2, BNA4, BNA3, YOR356W, RPE1, MDH3, CIT1, CIT2, KGD1 and KGD2, were appointed as promising targets for the development of improved production strains, based on their altered gene expression at pH 3.0 and literature research. Further experiments will establish the weight of these identified genes on the final taste of fermented foods