Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids

Abstract

The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop ␣-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-␣-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-␣-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-␣-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-␣-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-␣-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-␣-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-␣-acids across the plasma membrane. Furthermore, iso-␣-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators.The hop plant, Humulus lupulus L., belongs to the Cannabaceae family (36). Hop cones have an exceptionally high content of secondary metabolites, the hop acids, which account for up to 25% of their dry weight (13). The hop acids consist of two related groups of compounds, the ␣-acids and the ␤-acids. Three major types of ␣-acids (humulone, cohumulone, and adhumulone) are water extracted from the resin and isomerized to six different iso-␣-acid isomers (cis,trans-humulone, cis,trans-cohumulone, and cis,trans-adhumulone, Fig.

Application of Non-Saccharomyces Yeasts Isolated from Kombucha in the Production of Alcohol-Free Beer

“…The resistance against iso-a-acids and their induced weak organic acid stress were studied for the Saccharomyces species but it has barely been investigated for non-Saccharomyces species [28][29][30]. All investigated strains were able to grow in wort with 0, 50 and 100 IBU (international bitterness units).…”

Stress responses in brewing yeast.

“…Concentration of ~35 mg/l inhibits most beer-spoiling lactic acid (G+) bacteria while inhibition of yeast growth occurs at ~ 0.5 g/l of the acids, i.e. > 10 times more than the concentrations inhibiting bacterial growth [33]. These concentrations of alpha-bitter acids are not attained even at high hopping.…”

“…Alfa-hořké kyseliny fungují jako protonofory narušující transmembránovou protonmotorickou sílu. V koncentraci ~ 35 mg/l inhibují většinu mléčných bakterií kazících pivo, zatímco růst kvasinek je ovlivněn až při koncentraci ~ 0,5 g/l, tedy více než 10 x vyšší nežli je koncentrace inhibující bakteriální růst [33]. Takových …”

“…Alpha-bitter acids are protonophores that abolish the transmembrane protonmotive Pivovarské kvasinky a reakce na stres getiku a výživu. Tolerance kvasinek k alfa-hořkým kyselinám zahrnuje, mimo jiné, aktivní vylučování těchto látek z buněk PDR pumpami [33]. Během stárnutí význam tohoto mechanizmu klesá, protože po diauxické změně aktivita H + -ATPázy [35] i PDR pump ustává [13].…”

“…Moreover, it has been shown that protonophores activate the H + -ATPase of the yeast plasma membrane [34,Sigler et al, unpublished results], a yeast enzyme crucial for cell energetics and nutrition. Tolerance of yeast to iso-alpha-acids involves, apart from other mechanisms, active export from the cells by PDR pumps [33]. During aging the importance of this mechanism decreases as after diauxic shift the activity of both the H + ATPase [35] and the PDR pumps drops [13].…”