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

Abstract

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 48 hours of fermentation, while the three Brettanomyces strains were unique for production of the ethyl phenolic compounds 4-ethylguaiacol and 4-ethylphenol. Seven strains with potentially desirable flavor profiles were chosen for validation in sequential fermentations with inoculation of conventional brewing yeast to complete the alcoholic fermentation. The results show that the flavor production by non-conventional yeasts in sequential fermentations with S. cerevisiae is not additive, but appears to occur in a species/strain dependent manner. In conclusion, we have taken the first steps towards development of brewing protocol with bioflavoring of beer by non-conventional yeast species. The second major research line explored in this PhD, is based on the previous identification of the transceptor function of Sul1 and Sul2 in the MCB laboratory. These experiments had shown that the protein kinase A (PKA) nutrient sensing pathway was still activated by low levels of sulfite in a sul1Dsul2D strain, highlighting the presence of yet unidentified transporters or transceptors in the S. cerevisiae genome. In this thesis, we first assayed triple, quadruple, and quintuple deletion strains of Sul1 and Sul2 plus two homologs of SUL1 and SUL2 (namely YGR125W, YPR003C) and the putative MFS transporter gene SOA1 (YIL166C), which is highly upregulated under sulfur starvation, for growth with sulfite and high sulfate. The additional deletion of YIL166C in the sul1Dsul2Dyil166cD triple deletion strain was auxotrophic for sulfur-containing amino acids, even in the presence of high levels of sulfate (40 mM). We thus confirmed Yil166c to be the last remaining transporter of inorganic sulfur compounds. Single deletion of the Yil166c transporter led to lack of growth on range of organic sulfur compounds, including sulfonate compounds taurine and isethionate which are found in ecological niches were yeasts and fungi are frequently isolated. The transporter was therefore named Soa1 for Sulfonate transporter 1. The Soa1 gene product is remarkably conserved with transporter orthologs found in the three fungal domains and with multiple paralogs found in many fungi. S. uvarum, S. arboricola, and S. eubayanus also contained a paralog, Soa2, which complemented Soa1 for sulfonate transport. In conclusion, we have identified a novel family of inorganic sulfur, sulfonate, and sulfate ester transporters that previously had remained elusive.nrpages: 209status: publishe