Harnessing yeast metabolism of aromatic amino acids for fermented beverage bioflavouring and bioproduction.

Aromatic amino acid metabolism in yeast is an important source of secondary compounds that influence the aroma and flavour of alcoholic beverages and foods. Examples are the higher alcohol 2-phenylethanol, and its acetate ester, 2-phenylethyl acetate, which impart desirable floral aromas in wine, beer and baker's products. Beyond this well-known influence on the organoleptic properties of alcoholic beverages and foods, there is a growing interest in understanding and modulating yeast aromatic amino acid metabolism. The tryptophan derivatives melatonin and serotonin have bioactive properties and exert positive effects on human health, and aromatic amino acids are also the precursors of products of industrial interest, such as nutraceuticals, fragrances, and opium-derived drugs. This mini-review presents current knowledge on the formation of compounds from aromatic amino acids by Saccharomyces cerevisiae, from genetic and environmental influences on their flavour impacts in alcoholic beverages to their potential as bioactive compounds, and the use of yeast as microbial factories for the production of commercially relevant aromatic compounds.

Inactivating Mutations in Irc7p Are Common in Wine Yeasts, Attenuating Carbon-Sulfur ß-Lyase Activity and Volatile Sulfur Compound Production.

During alcoholic fermentation of grape sugars, wine yeasts produce a range of secondary metabolites that play an important role in the aroma profile of wines. In this study, we have explored the ability of a large number of wine yeast strains to modulate wine aroma composition, focusing on the release of the "fruity" thiols 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP) from their respective cysteinylated nonvolatile precursors. The role of the yeast gene IRC7 in thiol release has been well established, and it has been shown that a 38-bp deletion found in many wine strains cause them to express a truncated version of Irc7p that does not possess cysteine-S-conjugate ß-lyase activity. In our data, we find that IRC7 allele length alone does not fully explain the capacity of a strain to release thiols. Screening of a large number of strains coupled with analysis of genomic sequence data allowed us to identify several previously undescribed single-nucleotide polymorphisms (SNPs) in IRC7 that, when coupled with allele length, more robustly explain the ability of a particular yeast strain to release thiols from their cysteinylated precursors. We also demonstrate that allelic variation of IRC7 not only affects the release of thiols but modulates the formation of negative volatile sulfur compounds from the amino acid cysteine. The results of this study provide winemakers with an improved understanding of the genetic determinants that affect wine aroma and flavor, which can be used to guide the choice of yeast strains that are fit for purpose.IMPORTANCE Volatile sulfur compounds contribute to wine aromas that may be considered pleasant, such as "tropical," "passionfruit," and "guava," as well as aromas that are considered undesirable, such as "rotten eggs," "onions," and "sewer." During fermentation, wine yeasts release some of these compounds from odorless precursor molecules, a process that is most efficient when performed by yeasts that express active forms of the protein Irc7p. We show that most wine yeasts carry mutations that reduce activity of this protein, affecting the formation of volatile sulfur compounds that impart both pleasant and unpleasant aromas. The results provide winemakers with guidance on the choice of yeasts that can emphasize or deemphasize this particular contribution to wine quality.

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

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.

Hydroxycinnamoyl Glucose and Tartrate Esters and Their Role in the Formation of Ethylphenols in Wine.

Synthesized p-coumaroyl and feruloyl l-tartrate esters were submitted to Brettanomyces bruxellensis strains AWRI 1499, AWRI 1608, and AWRI 1613 to assess their role as precursors to ethylphenols in wine. No evolution of ethylphenols was observed. Additionally, p-coumaroyl and feruloyl glucose were synthesized and submitted to B. bruxellensis AWRI 1499, which yielded both 4-ethylphenol and 4-ethylguaiacol. Unexpected chemical transformations of the hydroxycinnamoyl glucose esters during preparation were investigated to prevent these in subsequent synthetic attempts. Photoisomerization gave an isomeric mixture containing the trans-esters and undesired cis-esters, and acyl migration resulted in a mixture of the desired 1-O-ß-ester and two additional migrated forms, the 2-O-α- and 6-O-α-esters. Theoretical studies indicated that the photoisomerization was facilitated by deprotonation of the phenol, and acyl migration is favored during acidic, nonaqueous handling. Preliminary LC-MS/MS studies observed the migrated hydroxycinnamoyl glucose esters in wine and allowed for identification of feruloyl glucose in red wine for the first time.

Genomic insights into the evolution of industrial yeast species Brettanomyces bruxellensis.

Brettanomyces bruxellensis, like its wine yeast counterpart Saccharomyces cerevisiae, is intrinsically linked with industrial fermentations. In wine, B. bruxellensis is generally considered to contribute negative influences on wine quality, whereas for some styles of beer, it is an essential contributor. More recently, it has shown some potential for bioethanol production. Our relatively poor understanding of B. bruxellensis biology, at least when compared with S. cerevisiae, is partly due to a lack of laboratory tools. As it is a nonmodel organism, efforts to develop methods for sporulation and transformation have been sporadic and largely unsuccessful. Recent genome sequencing efforts are now providing B. bruxellensis researchers unprecedented access to gene catalogues, the possibility of performing transcriptomic studies and new insights into evolutionary drivers. This review summarises these findings, emphasises the rich data sets already available yet largely unexplored and looks over the horizon at what might be learnt soon through comprehensive population genomics of B. bruxellensis and related species.

Novel wine yeast with mutations in YAP1 that produce less acetic acid during fermentation.

Acetic acid, a byproduct formed during yeast alcoholic fermentation, is the main component of volatile acidity (VA). When present in high concentrations in wine, acetic acid imparts an undesirable 'vinegary' character that results in a significant reduction in quality and sales. Previously, it has been shown that saké yeast strains resistant to the antifungal cerulenin produce significantly lower levels of VA. In this study, we used a classical mutagenesis method to isolate a series of cerulenin-resistant strains, derived from a commercial diploid wine yeast. Four of the selected strains showed a consistent low-VA production phenotype after small-scale fermentation of different white and red grape musts. Specific mutations in YAP1, a gene encoding a transcription factor required for oxidative stress tolerance, were found in three of the four low-VA strains. When integrated into the genome of a haploid wine strain, the mutated YAP1 alleles partially reproduced the low-VA production phenotype of the diploid cerulenin-resistant strains, suggesting that YAP1 might play a role in (regulating) acetic acid production during fermentation. This study offers prospects for the development of low-VA wine yeast starter strains that could assist winemakers in their effort to consistently produce wine to definable quality specifications.

Flavour-active wine yeasts.

The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can influence wine style. This review explores recent progress towards understanding the range of 'flavour phenotypes' that wine yeast exhibit, and how this knowledge has been used to develop novel flavour-active yeasts. In addition, emerging opportunities to augment these phenotypes by engineering yeast to produce so-called grape varietal compounds, such as monoterpenoids, will be discussed.

The winemaker's bug: From ancient wisdom to opening new vistas with frontier yeast science.

The past three decades have seen a global wine glut. So far, well-intended but wasteful and expensive market-intervention has failed to drag the wine industry out of a chronic annual oversupply of roughly 15%. Can yeast research succeed where these approaches have failed by providing a means of improving wine quality, thereby making wine more appealing to consumers? To molecular biologists Saccharomyces cerevisiae is as intriguing as it is tractable. A simple unicellular eukaryote, it is an ideal model organism, enabling scientists to shed new light on some of the biggest scientific challenges such as the biology of cancer and aging. It is amenable to almost any modification that modern biology can throw at a cell, making it an ideal host for genetic manipulation, whether by the application of traditional or modern genetic techniques. To the winemaker, this yeast is integral to crafting wonderful, complex wines from simple, sugar-rich grape juice. Thus any improvements that we can make to wine, yeast fermentation performance or the sensory properties it imparts to wine will benefit winemakers and consumers. With this in mind, the application of frontier technologies, particularly the burgeoning fields of systems and synthetic biology, have much to offer in their pursuit of "novel" yeast strains to produce high quality wine. This paper discusses the nexus between yeast research and winemaking. It also addresses how winemakers and scientists face up to the challenges of consumer perceptions and opinions regarding the intervention of science and technology; the greater this intervention, the stronger the criticism that wine is no longer "natural." How can wine researchers respond to the growing number of wine commentators and consumers who feel that scientific endeavors favor wine quantity over quality and "technical sophistication, fermentation reliability and product consistency" over "artisanal variation"? This paper seeks to present yeast research in a new light and a new context, and it raises important questions about the direction of yeast research, its contribution to science and the future of winemaking.

Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine.

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.