Novel wine yeast with ARO4 and TYR1 mutations that overproduce ‘floral’ aroma compounds 2-phenylethanol and 2-phenylethyl acetate

Abstract

It is well established that the choice of yeast used to perform wine fermentation significantly influences the sensory attributes of wines; different yeast species and strains impart different profiles of esters, volatile fatty acids, higher alcohols, and volatile sulphur compounds. Indeed, choice of yeast remains one of the simplest means by which winemakers can modulate the sensory characteristics of wine. Consequently, there are more than 100 commercially available Saccharomyces cerevisiae wine yeast strains available, mostly derived by isolation from vineyards and successful fermentations. Nevertheless, some desirable characteristics such as 'rose' and 'floral' aromas in wine are not present amongst existing strains. Such aromas can be conferred from the higher alcohol 2-phenylethanol (2-PE) and its acetate ester, 2-phenylethyl acetate (2-PEA). These metabolites of the aromatic amino acid phenylalanine are present at concentrations below their aroma detection thresholds in many wines, so their contribution to wine style is often minimal. To increase the concentration of phenylalanine metabolites, natural and chemically mutagenised populations of a S. cerevisiae wine strain, AWRI796, were exposed to toxic analogues of phenylalanine. Resistant colonies were found to overproduce 2-PE and 2-PEA by up to 20-fold, which resulted in a significant increase in 'floral' aroma in pilot-scale white wines. Genome sequencing of these newly developed strains revealed mutations in two genes of the biosynthetic pathway of aromatic amino acids, ARO4 and TYR1, which were demonstrated to be responsible for the 2-PE overproduction phenotype.

Overproduction of hydroxytyrosol in Saccharomyces cerevisiae by heterologous overexpression of the Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase

“…Our findings establish the basis to continue optimizing cultivation media and yeast strains that are able to synthesize HT from glucose, without spiking precursors. Yeast strains capable of overproducing higher alcohols directly from glucose have already been reported (Cordente et al, 2018). A recent study has also shown that certain mutant forms of HpaBC exhibit greater activity and specificity to produce hydroxytyrosol from tyrosol (Chen et al, 2019).…”

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages

“…The fluorinated phenylalanine analogs p -fluoro-DL-phenylalanine and o -fluoro-DL-phenylalanine have also been used for selection of ARO4 and TYR1 mutants that produced up to 20-fold more 2-phenylethanol and 2-phenylethyl acetate than the wild-type wine yeast (Cordente et al . 2018). The isolated ARO4 mutants contained mutations in specific amino acid residues that release Aro4 from suppression by tyrosine (Hartmann et al.…”

“…2003) leading to higher levels of the three aromatic amino acids intracellularly, whereas mutants in TYR1 accumulate phenylalanine extracellularly and show reduced intracellular tyrosine formation, indicating a lower activity of the prephenate dehydrogenase (Cordente et al. 2018). The TYR1 mutants showed the highest increase in 2-phenylethanol levels, suggesting that the flux from prephenate to phenylpyruvate and the release of feedback inhibition on Aro4 by lower intracellular tyrosine levels are determining factors for the production of 2-phenylethanol.…”

Sensorial and Volatile Analysis of Wines Made from Partially Dehydrated Grapes: An Ontario Case Study

“…Importantly, grape dehydration impacts amino acid catabolism (due to water stress), which can increase the concentration of higher alcohols [1]. Higher alcohols are important compounds in wine, and 2-phenylethanol is the most desirable phenolderived higher alcohol [33], as it can impart a rose-like aroma, while other higher alcohols have nondesirable aromas at high concentrations [39,40]. Higher alcohols are also precursors to desirable ester production [40].…”

“…Higher alcohols are important compounds in wine, and 2-phenylethanol is the most desirable phenolderived higher alcohol [33], as it can impart a rose-like aroma, while other higher alcohols have nondesirable aromas at high concentrations [39,40]. Higher alcohols are also precursors to desirable ester production [40]. is class of compounds commonly accounts for about 50% of the aromatic constituents in wine, excluding ethanol [33].…”