Molecular basis for ethyl acetate production and sulfonate transport in the yeast Saccharomyces cerevisiae

Flavor production is an essential property of brewer's yeast and a driving force for consumer's choice. Here, we have focused on identification of superior mutations responsible for low ethyl acetate in beer production. Ethyl acetate is a commonly used organic solvent that gives an undesirable solvent-like off-flavor in beer brewing. We have isolated a superior haploid strain (with a low ethyl acetate production) and an inferior haploid strain (with a high ethyl acetate production), which were used in pooled segregant whole genome sequence analysis experiments to identify the genomic areas linked to ethyl acetate production in Saccharomyces cerevisiae. In two strong quantitative trait loci (QTLs), appearing in both low and high ethyl acetate pools, we have identified the causative mutations responsible for 72% of its production in strains without ATF1. Overexpression of one of the causative genes, encoding for a putative mitochondrial enzyme, led to an increase with 85 mg/l ethyl acetate, without affecting other aroma compounds. Surprisingly, engineering of the mutations in the causative genes led to an increase in ethyl acetate in the presence of an active ATF1 gene. It was due to an increase in the ATF1-derived AATase activity, as isoamyl acetate, which is formed by the ATF1 gene, was also significantly increased. Isoamyl acetate is a 'banana' like ester, that provides an essential fruitiness in beer. Future industrial valorization will therefore identify whether reduction in the ethyl acetate levels or an increase in the isoamyl acetate production can be obtained by engineering of the mutations into commercial brewing strains. In addition to investigating engineering of low ethyl acetate production in S. cerevisiae, we have explored the potential usage of non-conventional yeast species to enhance the flavor enhancement in beer production. We first developed a novel methodology to facilitate quantification of phenolic compounds (4-vinyl guaiacol, 4-ethylguaiacol, and 4-ethylphenol) and their hydroxycinnamic acid precursors (trans-ferulic and p-coumaric acid), using HPLC coupled with simultaneous fluorescence and UV detection. This HPLC-UV/fluorescence method was used together with gas chromatography in screening for interesting flavor metabolite profiles (esters, alcohols, and phenolic compounds) in a collection of 17 different non-Saccharomyces species. All the Pichia kluyverii strains produced a high level of isoamyl acetate of 10 mg/L and above after