beverages

Review Conducting Wine Symphonics with the Aid of Yeast Genomics

Isak S. Pretorius

Chancellery, Macquarie University, Sydney, NSW 2109, Australia; [email protected]; Tel.: +61-2-9850-8645

Academic Editors: Graeme Walker and Graham G. Stewart Received: 17 November 2016; Accepted: 12 December 2016; Published: 19 December 2016

Abstract: A perfectly balanced wine can be said to create a symphony in the mouth. To achieve the sublime, both in wine and music, requires imagination and skilled orchestration of artistic craftmanship. For wine, inventiveness starts in the vineyard. Similar to a composer of music, the grapegrower produces grapes through a multitude of specifications to achieve a quality result. Different Vitis vinifera grape varieties allow the creation of wine of different genres. Akin to a conductor of music, the winemaker decides what genre to create and considers resources required to realise the grape’s potential. A primary consideration is the yeast: whether to inoculate the grape juice or leave it ‘wild’; whether to inoculate with a specific strain of Saccharomyces or a combination of Saccharomyces strains; or whether to proceed with a non-Saccharomyces species? Whilst the various Saccharomyces and non-Saccharomyces yeasts perform their role during fermentation, the performance is not over until the ‘fat lady’ (S. cerevisiae) has sung (i.e., the grape sugar has been fermented to specified dryness and alcoholic fermentation is complete). Is the wine harmonious or discordant? Will the consumer demand an encore and make a repeat purchase? Understanding consumer needs lets winemakers orchestrate different symphonies (i.e., wine styles) using single- or multi-species ferments. Some consumers will choose the sounds of a philharmonic orchestra comprising a great range of diverse instrumentalists (as is the case with wine created from spontaneous fermentation); some will prefer to listen to a smaller ensemble (analogous to wine produced by a selected group of non-Saccharomyces and Saccharomyces yeast); and others will favour the well-known and reliable superstar soprano (i.e., S. cerevisiae). But what if a digital music synthesizer—such as a synthetic yeast—becomes available that can produce any music genre with the purest of sounds by the touch of a few buttons? Will synthesisers spoil the character of the music and lead to the loss of the much-lauded romantic mystique? Or will music synthesisers support composers and conductors to create novel compositions and even higher quality performances that will thrill audiences? This article explores these and other relevant questions in the context of winemaking and the role that yeast and its genomics play in the betterment of wine quality.

Keywords: alcohol; aroma; bioengineering; flavour; synthetic genomics; taste; wine; yeast

1. Blending the Art of Bach and the Craft of Bacchus Music and wine are found in every known culture—past and present—varying markedly between places and times, and shaping civilizations in very significant ways the world over. Music might have been present in the ancestral population prior to the dispersal of humans around the world 50,000 years ago, while the history of winemaking stretches back to almost 7000 BC (Figure1). Over the centuries, the histories and spirit of music and wine merged to meld art and craft, and greatly enrich the way we live today.

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Figure 1. The spread of producing wine from Vitis vinifera noble grape varieties from Asia minor Figure 1. The spread of producing wine from Vitis vinifera noble grape varieties from Asia minor (around 6500 BC) to other parts of the world. (Adapted from [1]). (aroundFigure 6500 1. BC) The to spread other of parts producing of the world.wine from (Adapted Vitis vinifera from noble [1]). grape varieties from Asia minor (around 6500 BC) to other parts of the world. (Adapted from [1]). The art of composing and conducting music, and the craftsmanship of grapegrowing and The art of composing and conducting music, and the craftsmanship of grapegrowing and winemaking,The art are of composingboth considered and conducting expressions music, of humanand the creativitycraftsmanship and ofimagination—drawing grapegrowing and winemaking,metaphoricwinemaking, parallels are are both bothbetween considered considered music expressions andexpressions wine is ofnaturalof humanhuman (Figure creativity 2). Beethoven and and imagination—drawing imagination—drawing once said “music is metaphoricthemetaphoric wine parallelswhich parallels inspires between between one musicto music new and andgenerative wine wine is naturalprocesses”, natural (Figure (Figure and 2). 2went ).Beethoven Beethoven on to once describe once said said“musichimself “music is as is the“Bacchusthe wine wine whichwho which presses inspires inspires out one gloriousone to to new new wine generativegenerative for humankind processes”, processes”, and makesand and went them went on spiritually onto todescribe describe drunken”. himself himself as We as “Bacchuswill“Bacchus never who know presseswho whether presses out glorious out Beethoven glorious wine would wine for humankindfor have humankind agreed and with and makes a makes view them that,them spiritually for spiritually example, drunken”. drunken”. Bach’s artform We will neverwaswill know more never whetherof knowa musical whether Beethoven expression Beethoven would and would have application agreed have agreed withof his with a imagination, view a view that, that, for feelings,for example, example, thoughts Bach’s Bach’s artform andartform ideas was morewhilewas of aBacchus’ more musical of acraft expression musical relied expression more and application heavily and application on ofa learned his of imagination, his skill imagination, and feelings,experience feelings, thoughts in thoughts the creation and and ideas ideas of whilethe Bacchus’tangiblewhile craft productBacchus’ relied of craft morewine. relied What heavily more we on doheavily aknow, learned on however, a skilllearned and is skillthat experience bothand experience the sound in the ofin creation music the creation and of the of taste tangible the of tangible product of wine. What we do know, however, is that both the sound of music and the taste of productwine provides of wine. us What pleasure we do in a know, crossmodal however, sensory is that fashion. both Scientific the sound evidenceof music is and now the emergingtaste of that wine wine provides us pleasure in a crossmodal sensory fashion. Scientific evidence is now emerging that providesshows usthat pleasure regular inwine a crossmodal drinkers are sensory able to fashion. reliably Scientific match certain evidence wines is with now particular emerging thatpieces shows of musicshows [2–4]. that regular wine drinkers are able to reliably match certain wines with particular pieces of that regularmusic [2–4]. wine drinkers are able to reliably match certain wines with particular pieces of music [2–4].

FigureFigureFigure 2. 2.A A2. metaphoric Ametaphoric metaphoric parallel parallel parallel between between between musicmusic music andand wine as as expressionsexpressions expressions of of ofhuman human human creativity creativity creativity and and and imagination.imagination.imagination. The The The grapegrower grapegrower grapegrower composescomposes composes thethe notes notes of of wine wine inin in thethe the vineyardvineyard vineyard while while while the the the winemaker winemaker winemaker interpretsinterpretsinterprets and and conductsand conducts conductsthe the the original original original composition composition composition with with with his his instruments of of of choice choice choice in in inthe the the winery winery winery with with with the the the sole aim to meet the expectations of his audience in a specifically‐targetted consumer market. solesole aim aim to to meet meet the the expectations expectations of of his his audience audience in in aa specifically-targettedspecifically‐targetted consumer consumer market. market.

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Akin to a composer who intertwines elements to create an original piece of music—a melody, underlyingBeverages harmony 2016, 2, 36 or chord progression, and sometimes lyrics—a grapegrower composes3 of the 28 notes of a wine in the vineyard. By applying their craft to what nature and the land bestow on their vineyards,Akin viticulturists to a composer nurture who intertwines their vines elements to produce to create grapes an original as sheet piece music of music—a for various melody, types and stylesunderlying of wines. harmony While honouring or chord progression, what the and composer sometimes has lyrics—a written, grapegrower the conductor composeshas the the notes liberty to of a wine in the vineyard. By applying their craft to what nature and the land bestow on their creatively orchestrate the original composition to his instruments of choice and to rearrange the tempo vineyards, viticulturists nurture their vines to produce grapes as sheet music for various types and winemaker to satiatestyles the of musical wines. While appetite honouring of his audience. what the Incomposer the winery, has written, the the conductorhonours has thethe scoreliberty arranged to by thecreatively grapegrower orchestrate in the the vineyard, original composition and uniquely to his interprets instruments grape of choice compositions and to rearrange by conducting the differenttempo arrangements to satiate the ofmusical selected appetite pre-fermentation of his audience. practices, In the winery, yeast the strains, winemaker fermentation honours the conditions,score post-fermentationarranged by the technologies, grapegrower packaging in the vineyard, materials, and as uniquely well as interprets marketing grape and retailingcompositions strategies by to achieveconducting their own different vision. arrangements In smaller operational of selected pre settings,‐fermentation the interconnected practices, yeast choices strains, madefermentation at each step in the from-grape-to-glassconditions, post‐fermentationvalue chain technologies, might be simplerpackaging because materials,maestro as well vintners as marketingcan be and both retailing the composer in thestrategies vineyard to and achieve the conductor their ownin vision. the winery. In smaller operational settings, the interconnected choices made at each step in the from‐grape‐to‐glass value chain might be simpler because maestro vintners can Good music and good wine please different senses in similar ways. Pulling the cork from a be both the composer in the vineyard and the conductor in the winery. bottle of wineGood in music a pleasant and good social wine setting please different evokes senses a similar in similar experience ways. Pulling and appreciation the cork from asa bottle attending a concertof wine and in listening a pleasant to social a familiar setting evokes piece ofa similar music, experience even when and thereappreciation is all the as attending uncertainty a concert of no two performancesand listening ever to being a familiar quite piece the same of music, (Figure even3). when Our individualthere is all preferencesthe uncertainty for of certain no two music genresperformances and wine styles ever oftenbeing evolvequite the over same time. (Figure Musicians 3). Our individual and winemakers preferences know for allcertain too wellmusic that, to keep theirgenres audiences and wine styles and consumers often evolve engaged over time. and Musicians entertained, and winemakers their art and know craft all too also well need that, to to evolve with time.keep their Some audiences of these and changes consumers will engaged be due to andmarket-pull entertained,forces their art and and others craft also will need be catlysed to evolve by the with time. Some of these changes will be due to market‐pull forces and others will be catlysed by the forces of technology-push (Figure4). The result of these two sets of dynamic, trendsetting forces often forces of technology‐push (Figure 4). The result of these two sets of dynamic, trendsetting forces often convertconvert today’s today’sinnovation innovationinto tomorrow’s into tomorrow’stradition tradition. However,. However, sophisticated sophisticated audiences audiences and consumersand usuallyconsumers embrace usually the excitement embrace the of excitement new trends ofwithout new trends losing without their losing appreciation their appreciation for the charm for the of the timelesscharmclassics of the. timeless classics.

FigureFigure 3. The 3. differentThe different aspects aspects of of the the sensorial sensorial quality quality of of wine. wine. Listening Listening to music to music and anddrinking drinking wine wine in pleasant social settings are manifestly sensuous experiences. Four senses are involved in defining in pleasant social settings are manifestly sensuous experiences. Four senses are involved in defining the organoleptic quality of wine: sight, smell, taste and touch. Wine jargon is used to describe what a the organolepticwine drinker quality senses of(as wine: summarised sight, smell, by [5]): taste appearance and touch. (sight—e.g., Wine jargon cloudy, is hazy, used deposit to describe in the what a wine drinkerbottom of senses the glass, (as summarised depth of colour, by hue, [5]): mousse),appearance nose(sight—e.g., (smell—e.g., cloudy, aroma and hazy, bouquet) deposit and in palate the bottom of the(taste glass, and depth touch—flavour of colour, hue, and mouth mousse),‐feel).nose The(smell—e.g., term aroma is aroma normally and used bouquet) to describe and thepalate smell(taste of and touch—flavoura young, fresh and wine; mouth-feel). primary aromas The term originatearoma duringis normally fermentation—typically used to describe youthful the smellwith upfront of a young, fresh wine;fresh fruit primary notes. aromas Bouquet originateis the term duringfor an older fermentation—typically wine, less fresh but more youthful complex; with secondary upfront aromas fresh fruit notes.stemBouquet fromis oak the maturation term for anand older tertiary wine, aromas less originate fresh but during more complex;bottle ageing—developed secondary aromas fruit stem from oakshowing maturation more age, and with tertiary stewed or aromas dried fruit originate and other during smells bottle also seeking ageing—developed attention. The term fruit flavour showing refers to sweetness, acidity, bitterness, saltiness and the taste of umami. Mouth‐feel relates to the body more age, with stewed or dried fruit and other smells also seeking attention. The term flavour refers and texture of wine as influenced by factors such as alcohol strength (sensation of warmth) and to sweetness, acidity, bitterness, saltiness and the taste of umami. Mouth-feel relates to the body and tannins (drying sensation). The structure of a wine includes acidity, sweetness, bitterness texture(occasionally), of wine as influenced tannin (in red by factorswine), alcohol, such as palate alcohol weight strength and (sensationlength, mouth of warmth)‐feel, mousse and (in tannins (dryingsparkling sensation). wine), The as structurewell as theof intensity a wine includesof fruit aroma acidity, and sweetness, flavour, and bitterness complexity (occasionally), (diversity and tannin (in redlayers wine), of alcohol,flavour). palate weight and length, mouth-feel, mousse (in sparkling wine), as well as the intensity of fruit aroma and flavour, and complexity (diversity and layers of flavour). Beverages 2016, 2, 36 4 of 28 Beverages 2016, 2, 36 4 of 28

FigureFigure 4. 4. TheThe valuevalue chain chain inin wine wine production. production. The The traditional traditional productionproduction-driven‐driven viewview of of the the supplysupply chain chain hashas been been largely largely replaced replaced by by a a more more contemporary contemporary marketmarket-driven‐driven approachapproach in in a a demanddemand-chain‐chain scenario.scenario. This paradigm shift shift in in the the wine wine industry industry has has placed placed its its product product at at the the centre centre of of an an arena arena between between the the forcesforces of market-pullmarket‐pull andandtechnology-push technology‐push, where, where tradition tradition and and innovation innovation must must co-exist co‐exist [6]. [6]. Modern-day Modern‐ dayconsumers consumers demand demand wines wines that that are are enjoyable enjoyable in in all all sensory sensory aspects, aspects, healthful healthful and and producedproduced in an an environmentallyenvironmentally sustainable sustainable manner manner and and which which measure measure up up to to the the standards standards of of wine’s indefinable indefinable mystique.mystique. In In a a globalised globalised economy economy in in which which quality quality is is defined defined as as ‘sustainable ‘sustainable customer customer and and consumer consumer satisfaction’,satisfaction’, it it is is a struggle struggle to deliver wines that meet these expectations at competitive quality/price ratiosratios while while remaining remaining profitable. profitable. In In a a fiercely fiercely competitive competitive marketplace marketplace with with ever ever-changing‐changing consumer consumer preferences, producers have to be constantly on the lookout lookout for for ways ways to to anticipate anticipate new new market market trends trends andand to to be be able able to to deliver products that meet the expectations of consumers in a timely fashion.

Great musicians and vintners are able to accurately interpret the dynamics of these Great musicians and vintners are able to accurately interpret the dynamics of these interconnected interconnected factors in a timely fashion, and to correctly anticipate what the future preferences of factors in a timely fashion, and to correctly anticipate what the future preferences of their target their target audiences and consumer markets might be. With wine, accurate market anticipation and audiences and consumer markets might be. With wine, accurate market anticipation and fleet-footed fleet‐footed agility require masterful orchestration of a harmonious blend of an artist’s intuition, a agility require masterful orchestration of a harmonious blend of an artist’s intuition, a craftsman’s craftsman’s skill and a scientist’s logic—and that makes for crowd‐thrilling entertainment, standing skill and a scientist’s logic—and that makes for crowd-thrilling entertainment, standing ovations and ovations and encore sales in the global marketplace. encore sales in the global marketplace. In this context, this article seeks to shine the spotlight on the fermentation performance of classic In this context, this article seeks to shine the spotlight on the fermentation performance of classic and trendy yeasts in the winery, and to trumpet the sound of future music from the research and trendy yeasts in the winery, and to trumpet the sound of future music from the research laboratory. laboratory. 2. Translating Grape Compositions into Enchanting and Distinctive Wine Genres 2. Translating Grape Compositions into Enchanting and Distinctive Wine Genres Listening to a philharmonic orchestra’s performance, an audience values the quality and harmony of a multitudeListening to of pleasinga philharmonic sounds, orchestra’s as well as feelingperformance, inspired an by audience the whole values experience. the quality This is and not harmonydissimilar of to a themultitude expectations of pleasing and preferences sounds, as well of wine as feeling drinkers inspired who often by the use whole the terms experience.quality Thisand isvalue not whendissimilar they to refer the to expectations a particular and wine preferences (Figure5). of To wine the consumer drinkers who who often compares use the different terms quality wines andfor purchase,value when the they term referquality to a particularrelates to thewine ‘intrinsic’ (Figure 5). quality To the of consumer the wine, who meaning compares how thedifferent wine winesgratifies for on purchase, appearance, the term the nose quality and relates the palate, to the as ‘intrinsic’ well as the quality perceived of the value wine, [7]. meaning When consumers how the wineuse the gratifies term value on ,appearance, they usually the refer nose to bothand thethe intrinsicpalate, as value welland as thetheimage perceivedof a wine value in [7]. relation When to consumersthe price. The use image the term of a value product, they depends usually on refer how to aboth wine the is intrinsic marketed, value the and origin the and image regionality of a wine of in a relationwine, how to environmentally-soundthe price. The image of thea product winery’s depends practices on are, how how a wine many is medals marketed, have the been origin awarded, and regionalityhow high sommeliers of a wine, how and environmentally other influencers‐sound rate a the wine, winery’s and price. practices Consumers are, how will many consider medals a have wine beenhigh inawarded, value if how the product high sommeliers is sensorially and pleasing other influencers and recognisable, rate a wine, and perceived and price. as Consumers high in image will at considera competitive a wine price high [7 in]. value if the product is sensorially pleasing and recognisable, and perceived as high in image at a competitive price [7].

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Figure 5. The quality tree of grape cultivars and varietal wines (adapted from [8]). In most consumer Figure 5. The quality tree of grape cultivars and varietal wines (adapted from [8]). In most consumer markets, the ‘Big Six’ varieties—Cabernet Sauvignon, Merlot, Pinot Noir, Chardonnay, Riesling and markets, the ‘Big Six’ varieties—Cabernet Sauvignon, Merlot, Pinot Noir, Chardonnay, Riesling and Sauvignon Blanc—are regarded as ‘noble’ varieties from which the most sought‐after varietal wines Sauvignon Blanc—are regarded as ‘noble’ varieties from which the most sought-after varietal wines are made. One level down are the so‐called ‘classic’ grape cultivars which include the red varieties, are made. One level down are the so-called ‘classic’ grape cultivars which include the red varieties, Cabernet Franc, Nebbiolo, Sangiovese, Syrah (or Shiraz), Tempranillo and Touriga Naçional and the Cabernet Franc, Nebbiolo, Sangiovese, Syrah (or Shiraz), Tempranillo and Touriga Naçional and the white varieties, Chenin Blanc, Gewürztraminer, Semillon and Viognier. The category below the latter white varieties, Chenin Blanc, Gewürztraminer, Semillon and Viognier. The category below the latter cultivars are generally referred to as ‘interesting’ varieties and there is a long list of them. The image cultivars are generally referred to as ‘interesting’ varieties and there is a long list of them. The image and brand‐strength of some of these so‐called ‘interesting’ cultivars are strongly linked to specific and brand-strength of some of these so-called ‘interesting’ cultivars are strongly linked to specific wine wine producing countries and regions, for example, Italian Barbera, Chilean Carmenére, Argentinian producing countries and regions, for example, Italian Barbera, Chilean Carmenére, Argentinian Malbec, Malbec, South African Pinotage, Uruguayan Tannat and Californian Zinfandel. South African Pinotage, Uruguayan Tannat and Californian Zinfandel.

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SimilarSimilar to music enthusiasts—young and and old, old, male male and and female—who female—who vary vary widely widely in in their their preferencespreferences forfor differentdifferent music genres, wine consumers’ consumers’ senses senses are are not not uniformly uniformly sensitive sensitive or or attuned attuned toto thethe subtlesubtle assortment of changing changing sensations sensations evoked evoked by by different different wine wine types types and and styles styles (Figure (Figure 6).6 ). SomeSome ofof thethe diversitydiversity in sensory perception and and preferences preferences for for different different wine wine products products among among individualsindividuals and populations is is cultural—some cultural—some genetic genetic and and some some learned—and learned—and some some preferences preferences are are alsoalso influencedinfluenced byby gender, age and fashion [5]. [5].

FigureFigure 6. TheThe general general range range of of wine wine types types (adapted (adapted from from [9]). [ ‘Natural’9]). ‘Natural’ wine winegenerally generally refers refersto a type to a typeof wine of wine that thatcontains contains alcohol alcohol that thatformed formed during during yeast yeast fermentation fermentation while while ‘fortified’ ‘fortified’ wine wine refers refers to towine wine types types that that contain contain alcohol alcohol that that formed formed during during yeast yeast fermentation fermentation as well as as well added as added alcohol. alcohol. The Thecategory category of ‘natural’ of ‘natural’ wine wine includes includes red, red, rosé rosé and and white white wine wine that that can can either either be be‘still’ ‘still’ or or ‘sparkling’. ‘sparkling’. ‘Fortified’‘Fortified’ wine wine typestypes includeinclude sherry,sherry, portport andand severalseveral formats of herbal wine, such such as as vermouth. vermouth.

ForFor thethe past 35 years, the global global wine wine industry industry has has been been producing producing approximately approximately 15% 15% in in excess excess ofof annualannual consumptionconsumption and struggles to to hold hold on on to to its its 11% 11% share share of of the the global global market market for for alcoholic alcoholic drinksdrinks [[8].8]. By By volume volume beer beer occupies occupies about about 80% 80% of ofthe the alcoholic alcoholic drinks drinks market market with with spirits spirits making making up upthe the other other 9%. 9%. The The production production value value of the of ~32 the billion ~32 billion bottles bottles (or equivalents) (or equivalents) of wine of sold wine every sold year every is roughly US$100 billion, representing about a quarter of the total value of the global market for year is roughly US$100 billion, representing about a quarter of the total value of the global market alcoholic drinks [8]. The widening gap between wine production and consumption poses a real threat for alcoholic drinks [8]. The widening gap between wine production and consumption poses a real to the profitability of many wine businesses in all wine producing countries. Unsurprisingly, this threat to the profitability of many wine businesses in all wine producing countries. Unsurprisingly, market pressure drives vintners to strive for a deeper understanding of, and insights into, the this market pressure drives vintners to strive for a deeper understanding of, and insights into, the expectations and preferences of individuals and populations in their targeted markets so that they expectations and preferences of individuals and populations in their targeted markets so that they can can deliver desirable and distinctive wines accordingly. deliver desirable and distinctive wines accordingly. It is impossible to orchestrate appealing music from a badly composed piece—similarly, it is It is impossible to orchestrate appealing music from a badly composed piece—similarly, it is impossible to make good wine from badly grown grapes. So, the process of winemaking really starts impossible to make good wine from badly grown grapes. So, the process of winemaking really starts in the vineyard with optimally ripened grapes on the vine. The grapes, as they are harvested, contain in the vineyard with optimally ripened grapes on the vine. The grapes, as they are harvested, contain the potential of the wine but the winemaker has a critical role in realising that potential in full. the potential of the wine but the winemaker has a critical role in realising that potential in full. At each step of the winemaking process, the winemaker is required to make well‐informed, At each step of the winemaking process, the winemaker is required to make well-informed, insightful decisions as to what technique, technology and practice will best guide the process to fulfill insightfulthe potential decisions of the asgrapes to what and technique, achieve a technologypredetermined and outcome. practice willThere best are guide five thebasic process stages: to fulfill(i) harvesting, the potential crushing of the and grapes pressing; and achieve (ii) fermentation; a predetermined (iii) clarification, outcome. There fining are and five stabilisation; basic stages: (i)(iv) harvesting, maturation crushing and ageing; and and pressing; (v) bottling, (ii) fermentation; labelling and packaging (iii) clarification, (Figure fining 7). However, and stabilisation; there are (iv)variations maturation and deviations and ageing; along and the (v) way bottling, that labellingenable winemakers and packaging to put (Figure their own7). However, individual there stamp are on the production chain, thereby making their wine unique (for further reading see [10–12]).

Beverages 2016, 2, 36 7 of 28 variations and deviations along the way that enable winemakers to put their own individual stamp on theBeverages production 2016, 2, chain,36 thereby making their wine unique (for further reading see [10–12]). 7 of 28

Figure 7. The main steps in the production of red and white wine (adapted from [13]). Some steps, Figure 7. The main steps in the production of red and white wine (adapted from [13]). Some steps, and and the sequence thereof, differ between the production of red and white wine. the sequence thereof, differ between the production of red and white wine. Grape harvesting. Determining when to harvest the grapes is the first step and decision‐making pointGrape in the harvesting winemaking. Determining process [12]. when This to harvestdecision the is heavily grapes isinfluenced the first stepby the and weather decision-making and it pointrequires in the a winemaking touch of science process (i.e., [chemical12]. This analysis) decision and is heavily old‐fashioned influenced tasting by the of grapes. weather The and minute it requires a touchgrapes of are science picked (i.e., by hand chemical or mechanically analysis) and harvested, old-fashioned will largely tasting determine of grapes. the Theacidity, minute sweetness, grapes are pickedalcoholic by hand strength, or mechanically tannin texture, harvested, astringency will and largely flavour determine of the wine the [10]. acidity, Once sweetness, the grapes alcoholic are strength,delivered tannin at the texture, winery, astringency they are sorted and into flavour bunches, of the and wine rotten [10 or]. Onceunder the‐ripe grapes grapes are are delivered removed. at the winery,Grape they crushing are sorted and into wine bunches, pressing. andThe rottennext steps or under-ripe involve destemming, grapes are crushing removed. and pressing of the sorted grapes [12]. The traditional stomping of grapes by workers’ feet has mostly been replaced by mechanical crushing and pressing of grapes into ‘must’. White wine production requires grape

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Grape crushing and wine pressing. The next steps involve destemming, crushing and pressing of the sorted grapes [12]. The traditional stomping of grapes by workers’ feet has mostly been replaced by mechanical crushing and pressing of grapes into ‘must’. White wine production requires grape skins, seeds, and stems to be removed immediately after crushing to prevent unwanted colour and tannins from leaching into the wine. Whereas the production of red wine requires that, following crushing, the grape skins remain in the juice during the vinification (fermentation) process. During maceration—the time period during which the skins and grape juice are allowed to interact—polyphenolic material consisting of anthocyanins (water-soluble pigments), tannins and flavour-active compounds are leached from the grape skins, seeds and stems into the must, giving red wine its colour, structure and aroma [11]. With longer maceration, the onset of spontaneous fermentation is delayed by cooling the must—a process called ‘cold soak’ or ‘cold maceration’. Short periods (usually no more than a couple of days) of maceration and cold-soaking are also done for a limited range of white wine styles, but these practices are mostly applied in red wine making. Depending on the grape variety and intended style of red wine to be made, the length of maceration can be extended for a period of several weeks and is one of the most influential controls available to the winemaker to shape the style of the end-product. In addition, the way the wine is pressed, can also significantly influence the style and quality of the end-product. The practice of wine pressing has evolved from the traditional foot-stomping of grapes to basket-presses and to modern-day bladder-presses (batch or continuous mechanical presses) by which an airbag fills up with air and gently squeezes the grapes against the inner wall of a perforated cylinder. Generally, the wine will first be ‘racked’ so that clear ‘free-run wine’ separates from the solids before the pomace (grape skins, pulp, seeds and stems) is pressed. ‘Press wine’ is usually inferior in quality to ‘free-run wine’ and much more astringent [11]. At the crushing or pressing stage, the winemaker might make a number of additions to the juice (for white wine) or to the must (for red wine) to adjust its composition prior to fermentation. These additions might include acid (‘acidification’ in hot climate regions), sugar (‘chaptalisation’ in cold climate regions), sulfur dioxide (as an anti-microbial and anti-oxidisation agent) and ascorbic acid (as an anti-oxidant in white winemaking). Alcoholic and malolactic fermentation. The alcoholic fermentation is conducted by ambient yeast (‘spontaneous’ fermentation) or added yeast (‘inoculated’ fermentation). During this process, the main grape sugars, glucose and fructose, are assimilated and converted to ethyl alcohol (ethanol) and carbon dioxide (CO2) by the anaerobic metabolism (glycolysis) of yeast cells. One glucose molecule generates two ethanol and two carbon dioxide molecules, i.e., C6H12O6→2C2H5OH + 2CO2. The yeast-driven alcoholic fermentation is similar for white and red wine except for the fact that fermentation of red wine occurs in the presence of grape solids and at a slightly higher temperature (18–30 ◦C). The temperature maintained in the fermentation mass is the main factor that determines the duration of fermentation—even more so than the initial sugar concentration, yeast type, aeration of the must and the quantity of micro-nutrients in the juice or must [10]. Typically, red wine fermentations are complete within a week whereas white wines, which are often fermented at much lower temperatures (12–18 ◦C), might take several weeks to ferment to dryness (that is, when all the sugars have been consumed). Depending on what style of wine a winemaker is striving to achieve, the alcoholic fermentation is usually undertaken in oak barrels, large oak vats, open-top concrete vessels and stainless steel tanks [8]. Irrespective of the type of fermentation vessel, the large amount of grape skins, stems and seeds in red must, interacting with effervescing carbon dioxide from the fermentation, produces a solid mass (known as the ‘cap’) which floats to the top. To maintain contact between the red grape solids and the juice, the ‘cap’ is regularly mixed through the juice. ‘Cap management’ can be done manually (‘punch-down’), mechanically (using rotary tanks) or by pump-over methods (pumping the liquid from the bottom of the vessel over the ‘cap’). Malolactic bacteria drive the secondary fermentation known as malolactic fermentation. During this post-alcoholic fermentation process, malic acid is converted into lactic acid (which has lower acidity) and carbon dioxide: COOH-CHOH-CH2-COOH→COOH-CHOH-CH3 + CO2. Malolactic fermentation is uniformly done for all red wine except for the lightest-bodied red wine. Beverages 2016, 2, 36 9 of 28

By contrast, malolactic fermentation is uncommon in white wine making, except for the production of certain styles of Chardonnay and sparkling wines. In addition to its role in the deacidification of wine, malolactic fermentation can also add to the aroma, flavour and complexity of wine, as well as rendering the wine microbiologically stable (lowering the risk of gas production in bottled wine). Wine clarification and stabilisation. Once the fermentation stage is complete, clarification begins by removing suspended and insoluble material (referred to as ‘lees’) from young wines [10]. The ‘lees’ include excess tartrates, pectins and gums, some proteins and small numbers of microbial cells (including dead yeast cells). Clarification and stabilisation of wine can be achieved by simply holding the wine in storage until the larger particles settle (a process known as ‘settling’ or ‘débourbage’). After the initial ‘settling’, the fine ‘lees’ remaining in the vessel of the unfinished wine can be gently stirred (a process known as ‘batonnage’) before the final ‘débourbage’, followed by siphoning or ‘racking’ of the clear upper layer from the compact layer of solids at the bottom of the vessel [8]. This process can be accelerated by physical means, such as cold filtration, centrifugation and flotation, or by the much cheaper chemical process known as ‘fining’. Refrigeration and cold filtration will reduce the concentration of tartrates in young wines supersaturated in tartrates to levels below concentrations that would subsequently form crystals in the bottle [11]. The purpose of adding fining agent preparations (e.g., clarifying enzyme preparations and the volcanic clay bentonite) to wine is to soften or reduce its astringency and/or bitterness; remove proteins capable of haze formation; or reduce colour by the adsorption and precipitation of polymeric phenols and tannins. The fining agent reacts with wine components either chemically or physically, to form a new complex that can separate from the wine. The degree to how thoroughly the wine is clarified and stabilised will depend on the style target of the winemaker: ‘everyday’ wines are usually more thoroughly clarified and stabilised whereas there is a market for ‘unfiltered’ wines—some consumers consider these to be of better quality as they accept that a sediment can slowly form in bottles and they believe that the less stable constituents contribute positively to the ageing process [11]. Wine maturation and ageing. The period of time during which wine is handled and stored in the winery is commonly referred to as ‘maturation’ (sometimes in contact with wood, e.g., in oak barrels for wooded wine styles, and sometimes in stainless steel tanks for zesty white wines), while the period of time that wine is stored in the bottle is known as ‘ageing’ [8,11]. Before blending and bottling, most wines are usually stored for several months in tanks or wooden cooperage. During oak barrel maturation (which is more commonly used for red wine than white wine), wine undergoes a series of complex transformations, including controlled oxidisation which can result in smoother and rounder wines because of the polymerisation of tannins. Oak-induced transformations can significantly change the aroma, composition and quality of the end-product by imparting a range of flavours including vanilla, oakiness and toasty smokiness [7]. By contrast, ‘ageing’ of bottled wine only involves a subtle evolution of the wine. Contrary to popular opinion, age in a wine is not necessarily a virtue—only a relatively small subset of wine types benefit from extended bottle ageing [11]. The majority of wines produced today are meant to be drunk within a couple of years of bottling, whether sealed under cork closures, screw caps or glass stoppers. Growing the grapes, making the wine, packaging and labelling of the end-product are only half of the story—there is still many a slip between the cup and the lip. If the marketing, retailing or selling of wine is not done well, that is potentially akin to being on centre stage with no audience to applaud the performance. Moreover, experienced vintners and marketeers of wine also know that even with perfectly executed well-considered strategies to promote and sell wine, things can still be turned upside down by unexpected and sudden changes in the marketplace. For example, on 17 November 1991, when American television host, Morley Safer, of the popular news program, Sixty Minutes, held up a glass of red wine, looked into the camera and declared that “the answer to France’s low rate of heart disease [despite their unusually high consumption of cheese and high-fat, high-cholesterol meats] may lie in this inviting glass” the so-called French Paradox was born. Within one year, consumption of red wine increased more than 40% in the US alone. Another example is the 2004 film titled Sideways. Beverages 2016, 2, 36 10 of 28

When the main character, Miles (played by Paul Giamatti) was asked by an attractive waitress, Maya (played by Virginia Madsen) “Why are you so into Pinot?”, his 60-s response about how Pinot Noir vines only grow in “specific tucked-away corners of the world” and how dependent they are on the patient nurturing of a select group of growers to fully express the grapes’ flavours, which are “the most haunting and brilliant and thrilling and subtle and ancient on this planet”, American wine consumers fell inBeverages love with 2016, 2 this, 36 grape cultivar. Before this movie, Pinot Noir was considered a hard-to-grow10 of grape 28 and a tough-to-sell wine. The Sideways Effect had an instantaneous impact on the sales of Pinot Noir. considered a hard‐to‐grow grape and a tough‐to‐sell wine. The Sideways Effect had an instantaneous Thisimpact resulted on in the a massivesales of Pinot and rapid Noir. expansion This resulted of Pinot in a massive Noir plantings, and rapid and expansion a corresponding of Pinot declineNoir in Merlotplantings, sales and and a corresponding value also as a decline result in of Merlot this character sales and who value disliked also as a Merlot result of wine. this character who dislikedThe unforeseen Merlot wine. impact of these two examples, the French Paradox and the Sideways Effect, provides valuableThe lessons unforeseen to vintners impact and of marketeers these two alike—always examples, the expectFrench theParadox unexpected and the in Sideways the marketplace Effect, andprovides be prepared valuable to face lessons the music to vintners of shifting and marketeers consumer preferences.alike—always expect the unexpected in the marketplace and be prepared to face the music of shifting consumer preferences. 3. Playing Microbiological Musical Chairs in the Fermentation Vat 3. Playing Microbiological Musical Chairs in the Fermentation Vat The recruitment of an optimal arrangement of talented musicians playing different instruments in an orchestraThe isrecruitment a complex of task, an optimal which demands arrangement a great of talented deal of skill.musicians The fermentation playing different of grape instruments must into winein is an an orchestra equally is complex a complex ecological task, which and demands biochemical a great process deal of involving skill. The fermentation the sequential of grape development must of microbialinto wine species, is an equally as affected complex by prevailing ecological conditions and biochemical in vineyards process and involving wineries the [13 –sequential15]. Grapes anddevelopment grape musts naturallyof microbial contain species, a mixture as affected of fungi,by prevailing yeasts, conditions bacteria, mycoviruses in vineyards and wineries bacteriophages [13– 15]. Grapes and grape musts naturally contain a mixture of fungi, yeasts, bacteria, mycoviruses and (Figure8). Grape must is relatively complete in nutrient content; however, it can support the growth of bacteriophages (Figure 8). Grape must is relatively complete in nutrient content; however, it can only a limited number of microbial species. The type and numerical presence of indigenous microbes support the growth of only a limited number of microbial species. The type and numerical presence depend on factors such as the method of grape harvest (hand-picked or mechanical), grape temperature, of indigenous microbes depend on factors such as the method of grape harvest (hand‐picked or transportmechanical), from vineyard grape temperature, to winery (distance/time, transport from initialvineyard grape to winery temperature, (distance/time, air temperature, initial grape sulfite addition),temperature, condition air temperature, of grapes (time, sulfite temperature, addition), condition sulfite addition) of grapes and (time, must temperature, pre-treatment sulfite (cellar hygiene,addition) aeration, and enzymemust pre treatment,‐treatment sulfite(cellar addition,hygiene, aeration, clarification enzyme method, treatment, temperature, sulfite inoculationaddition, withclarification yeast starter method, cultures) temperature, [9]. inoculation with yeast starter cultures) [9].

FigureFigure 8. Teleomorphs, 8. Teleomorphs, anamorphs anamorphs and and synonyms synonyms ofof somesome of the non non-‐SaccharomycesSaccharomyces yeastsyeasts in inthe the AscomycetousAscomycetous genera genera reported reported to to have have been been found found onon grapesgrapes and in in wine wine fermentations fermentations (adapted (adapted from [13]). from [13]). Depending on the abundance and robustness of the microbial species in grape must, these organisms might or might not survive and/or play an active role in fermentation under the particular oenological conditions applied by the winemaker. Similar to a conductor choosing different instrumentalists required to perform different roles in the orchestra, the winemaker chooses fermentation conditions and practices that determine which yeasts and bacteria will be allowed to

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Depending on the abundance and robustness of the microbial species in grape must, these organisms might or might not survive and/or play an active role in fermentation under the particular oenological conditions applied by the winemaker. Similar to a conductor choosing different instrumentalists required to perform different roles in the orchestra, the winemaker chooses fermentation conditions and practices that determine which yeasts and bacteria will be allowed to help shape the wine for a targetted style. In essence, the objective of a winemaker is to apply [in addition to the natural selective pressures that exist in grape must (e.g., osmotic pressure exerted by high sugar concentration)] fermentation conditions (e.g., low pH) and practices (e.g., sulfite additions) that would eliminate spoilage bacteria (e.g., acetic acid bacteria and certain spoilage species of lactic acid bacteria) and yeasts (e.g., Brettanomyces, also known as Dekkera). The selectivity of fermenting must is further bolstered once anaerobic conditions are established in the vat or tank; certain nutrients become depleted and the increasing concentration of ethanol starts to eliminate alcohol-sensitive microbial species. Such conditions invariably favour yeast species with the most efficient fermentative metabolism that are able to withstand low-pH, high-sulfite and high-ethanol environments. With its Crabtree-positive carbon metabolism, Saccharomyces cerevisiae is the yeast best equipped to take advantage of these restrictive winemaking conditions—it is able to generate energy under fermentative conditions and limit the growth of competing microorganisms by producing inhibiting metabolites such as ethanol and carbon dioxide. The prevalence of S. cerevisiae (indigenous or inoculated) in wine ferments is therefore expected and desired [13]. Unfortunately, an undesirable yeast, Brettanomyces bruxellensis, can also survive the harsh conditions prevailing during wine fermentation as well as during wine maturation, particularly in oak barrels for extended periods [16]. If present, this spoilage yeast converts hydroxycinnamic acids into odourous ethylphenols, producing ‘metallic’, ‘medicinal’ and ‘barnyard’ aromas in wine. The most practical way for winemakers to gain the upper hand against Brettanomyces contamination is to: employ sound cellar hygiene practices; inoculate their grape musts with fast-fermenting S. cerevisiae strains; and maintain an optimal concentration of free sulfite in wine that would, on one hand, eliminate Brettanomyces contaminants while on the other, allow S. cerevisiae to rapidly complete the alcoholic fermentation [16]. And, if malolactic fermentation is desired, this would also permit Oenococcus oeni to convert malic acid into lactic acid. Inoculating grape must with a robust S. cerevisiae strain and applying an optimal combination of low pH and a high concentration of free sulfite (SO2) would not only restrict Brettanomyces contaminants but would also eliminate undesirable acetic and lactic bacteria. Such winemaking practices would ensure rapid, reliable and trouble-free (non-‘sluggish’ and non-‘stuck’) fermentations as well as consistent, predictable and desirable wine flavours and end-product quality (e.g., no volatile acidity or presence of other unwanted metabolites). The only down-side of the widely-used practice of inoculating grape must with a strong-fermenting S. cerevisiae strain is that desirable non-Saccharomyces yeasts originating from the grapes have limited opportunity to positively contribute to the complexity of wine [9]. Some winemakers prefer to conduct spontaneous fermentations (comprising mixed and sequential dominance of non-Saccharomyces and Saccharomyces yeasts). They are prepared to take the risk of contamination of their grape must by spoilage yeasts because they consider indigenous non-Saccharomyces yeasts as essential to the authenticity of their wines by imparting desired and distinct superior regional characteristics [13]. In this context, the phrase ‘non-Saccharomyces yeasts’ has emerged as a loose colloquial term now widely used in the wine industry for all yeast species found in wine other than S. cerevisiae, with the proviso that this only includes yeasts with a positive role in the production of wine. During the initial phases of fermentation, non-Saccharomyces yeasts are both present and active in all ferments whether inoculated or not (Figure9). These non- Saccharomyces yeasts can be classed into three groups: (i) yeasts that are mostly aerobic, such as species of Candida, Cryptococcus, Debaryomyces, Pichia and Rhodoturula; (ii) apiculate yeasts with low fermentative activity, such as Hanseniaspora uvarum (Kloeckera apiculata), Hanseniaspora guilliermondii (Kloeckera Beverages 2016, 2, 36 12 of 28 apis) and Hanseniaspora occidentalis (Kloeckera javanica); and (iii) yeasts with a fermentative metabolism, for example Kluyveromyces marxianus (Candida kefyr), Metschnikowia pulcherrima (Candida pulcherrima), TorulasporaBeverages 2016 delbrueckii, 2, 36 (Candida colliculosa), and Zygosaccharomyces bailii [13]. 12 of 28

FigureFigure 9. 9.The The sequential sequential participation of of various various yeast yeast species species during during the fermentation the fermentation of grape ofmust. grape The selective pressures prevailing in fermenting grape must (e.g., sugar‐induced osmotic pressure), must. The selective pressures prevailing in fermenting grape must (e.g., sugar-induced osmotic fermentation conditions (e.g., temperature and pH) and winemaking practices (e.g., sulfite additions) pressure), fermentation conditions (e.g., temperature and pH) and winemaking practices (e.g., sulfite inhibit and/or eliminate non‐Saccharomyces yeast species during the course of fermentation. The additions) inhibit and/or eliminate non-Saccharomyces yeast species during the course of fermentation. selective nature of grape must becomes more pronounced once anaerobic conditions are established, The selective nature of grape must becomes more pronounced once anaerobic conditions are nutrients become depleted and rising alcohol levels start to constrain the survival of ethanol‐sensitive established, nutrients become depleted and rising alcohol levels start to constrain the survival of yeasts. ethanol-sensitive yeasts. During fermentation, some of these yeasts are metabolically active until an ethanol concentration ofDuring 3% to 4% fermentation, is reached while some others of these remain yeasts present are metabolically for longer until active S. cerevisiae until an finally ethanol exert concentration absolute of 3%dominance to 4% is reachedto successfully while complete others remain the alcoholic present fermentation. for longer until ApartS. from cerevisiae their effectfinally on exert the growth absolute dominanceof bacteria to and successfully Saccharomyces complete yeasts, the some alcoholic of the fermentation.flavour‐active non Apart‐Saccharomyces from their effect yeasts on can the have growth a of bacteriasignificant and impactSaccharomyces on the absoluteyeasts, and some relative of the concentration flavour-active of aroma non-Saccharomyces compounds andyeasts the quality can have of a significantthe end‐ impactproduct on [13]. the absolute and relative concentration of aroma compounds and the quality of the end-product [13]. 4. Auditioning Saccharomyces and Non‐Saccharomyces Yeasts for Different Wine Roles 4. AuditioningConductorsSaccharomyces of orchestrasand always Non- seekSaccharomyces to find giftedYeasts performers for Different and new Wineways to Roles stage novel, authentic and entertaining performances. Similarly, many trendsetting winemakers are always on Conductors of orchestras always seek to find gifted performers and new ways to stage novel, the lookout for novel strains in their vineyards’ treasure trove of ambient yeasts. Fortunately, these authentic and entertaining performances. Similarly, many trendsetting winemakers are always trailblazers are no longer faced with a binary choice between spontaneous fermentation and ferments on theinoculated lookout with for a novel fast‐fermenting strains in S. their cerevisiae vineyards’ wine strain. treasure Over trove the past of ambientdecade or yeasts. so, a third Fortunately, option thesehas trailblazers emerged with are noinoculated longer facedmixed with‐species a binary and mixed choice‐strain between ferments spontaneous whereby winemakers fermentation can and fermentskeep spoilage inoculated microbes with at a bay fast-fermenting and still take advantageS. cerevisiae of thewine positive strain. contributions Over the pastof flavour decade‐active or so, a thirdnon‐Saccharomyces option has emerged and non‐ withcerevisiae inoculated yeasts [17,18]. mixed-species Along with and the mixed-strain availability of ferments more than whereby 250 winemakersdifferent cancommercial keep spoilage wine strains microbes of atS. baycerevisiae and, still some take yeast advantage manufacturing of the positive companies contributions are now of flavour-activeoffering non non-‐cerevisiaeSaccharomyces and nonand‐Saccharomyces non-cerevisiae yeastsyeasts for [deliberate17,18]. Along inoculation with the of availability wine ferments. of more thanPositive 250 different results commercial have been wine reported strains with of S. cerevisiaeTorulaspora, some delbrueckii yeast, manufacturing Pichia kluyveri companies, Lachancea are nowthermotolerans offering non- andcerevisiae Candida/Metschnikowiaand non-Saccharomyces pulcherrimayeasts [13]. for deliberate inoculation of wine ferments. PositiveAs results described have beenbefore reported [13], winemakers with Torulaspora can now delbrueckii conduct their, Pichia distinctive kluyveri, ‘wineLachancea symphonies’ thermotolerans by andchoosingCandida/Metschnikowia and inoculating pulcherrima their ferments[13 ].with different combinations of these newly‐available non‐ cerevisiae and non‐Saccharomyces yeasts. In doing so, well‐informed winemakers also realise that nothing in the fermentation vessel is over until the fat lady—S. cerevisiae—has sung and fermented

Beverages 2016, 2, 36 13 of 28

As described before [13], winemakers can now conduct their distinctive ‘wine symphonies’ by choosing and inoculating their ferments with different combinations of these newly-available non-Beveragescerevisiae 2016, and2, 36 non- Saccharomyces yeasts. In doing so, well-informed winemakers also realise13 of 28 that nothing in the fermentation vessel is over until the fat lady—S. cerevisiae—has sung and fermented grape sugars to ‘dryness’ and successfully completed the alcoholic fermentation. Embracing the grape sugars to ‘dryness’ and successfully completed the alcoholic fermentation. Embracing the music music orchestrated in the winery, consumers have a wide spectrum of genres to choose from. Some orchestrated in the winery, consumers have a wide spectrum of genres to choose from. Some audiences audiences might prefer the pleasing ‘city‐hall‐filling’ sounds of a large philharmonic orchestra mightcomprising prefer the a pleasinggreat range ‘city-hall-filling’ of diverse instrumentalists, sounds of a large as philharmonic is the case orchestrawith wine comprising created from a great rangespontaneous of diverse fermentation. instrumentalists, Other as audiences is the case might with seek wine out created the notes from spontaneousgenerated by fermentation.a smaller Otherensemble audiences of yeasts might inoculated seek out in the multi notes‐species generated or multi by a‐strain smaller ferments, ensemble while of yeastsothers inoculatedwill keep in multi-speciespurchasing ortheir multi-strain tickets to be ferments, entertained while by othersthe well will‐known keep lyrics purchasing of the reliable their tickets superstar to be soprano, entertained byS. the cerevisiae well-known (Figure lyrics 10). of the reliable superstar soprano, S. cerevisiae (Figure 10).

FigureFigure 10. 10. TheThe diverse array array of of approaches approaches to toyeast yeast fermentation fermentation in the in production the production of wine. of In wine. Inspontaneous spontaneous ferments, ferments, winemakers winemakers depend depend entirely entirely on on ambient ambient yeast yeast originating originating from from the thevineyard vineyard andand winery winery to to ferment ferment grape grape sugars sugars to ‘dryness’. With With ‘inoculated’ ‘inoculated’ ferments, ferments, winemakers winemakers can can choose choose to conduct the yeast ferment with mixed species (which can include species from either Saccharomyces to conduct the yeast ferment with mixed species (which can include species from either Saccharomyces or non‐Saccharomyces species, or both), mixed strains (different strains of S. cerevisiae), or with a single or non-Saccharomyces species, or both), mixed strains (different strains of S. cerevisiae), or with a single strain (a selected S. cerevisiae strain) to craft a specific wine style. strain (a selected S. cerevisiae strain) to craft a specific wine style. 5. Fine‐Tuning Saccharomyces cerevisiae Strains for Different Wine Performances 5. Fine-Tuning Saccharomyces cerevisiae Strains for Different Wine Performances In an orchestra, every musician must know their role in performing a particular music compositionIn an orchestra, and they every are musician responsible must for know fine‐ theirtuning role their in performing own instruments a particular before music the conductor composition andsteps they up are to the responsible lectern to for commence fine-tuning the performance. their own instruments In inoculated before ferments, the conductor winemakers steps know up that to the lecternthe primary to commence role of S. the cerevisiae performance. is to catalyse In inoculated the rapid, ferments, complete winemakers and efficient knowconversion that theof grape primary rolesugars of S. cerevisiae(mainly glucoseis to catalyse and fructose) the rapid, to ethanol, complete carbon and efficientdioxide conversionand other desirable of grape wine sugars aroma (mainly‐ glucoseshaping and metabolites fructose) to(e.g., ethanol, acids, alcohols, carbon dioxide carbonyls, and esters, other terpenes, desirable thiols) wine without aroma-shaping the development metabolites (e.g.,of off acids,‐flavours alcohols, (e.g., hydrogen carbonyls, sulfide) esters, (Figures terpenes, 11 and thiols) 12). without The most the exquisite development performance of off-flavours comes (e.g.,only hydrogen from a precisely sulfide)‐tuned (Figures instrument, 11 and and12). so The it is most also with exquisite inoculated performance yeast. Strain comes development only from a precisely-tunedapproaches usually instrument, start with and the so isolation it is also and with selection inoculated of variants yeast. Strainaccording development to their robustness, approaches usuallyfermentation start with performance the isolation and and sensory selection attributes of variants (Figure according 13). Once the to their best robustness,candidate strains fermentation have performancebeen identified, and yeast sensory researchers attributes employ (Figure hybridisation, 13). Once the mutagenesis, best candidate genetic strains engineering have been or genome identified, yeastengineering researchers techniques employ to hybridisation, genetically improve mutagenesis, the oenological genetic properties engineering of selected or genome strains engineering (Figure 14). Improved mutants and hybrids are routinely used in commercial winemaking whereas genetically modified (GM) strains generated through genetic and genome engineering are largely reserved for research purposes at this stage [6,9,19–24]. Researchers are investigating non‐GM

Beverages 2016, 2, 36 14 of 28 techniques to genetically improve the oenological properties of selected strains (Figure 14). Improved mutants and hybrids are routinely used in commercial winemaking whereas genetically modified (GM) strainsBeverages generated 2016, 2, through36 genetic and genome engineering are largely reserved for research purposes14 of 28 at this stage [6,9,19–24]. Researchers are investigating non-GM strategies, including hybridisation, strategies,Beverages 2016 including, 2, 36 hybridisation, mutagenesis and adaptive laboratory evolution, to circumvent14 of 28 mutagenesis and adaptive laboratory evolution, to circumvent the current anti-GM constraints from the current anti‐GM constraints from consumers facing comestible food products generated through consumersstrategies, facing including comestible hybridisation, food products mutagenesis generated and adaptive through laboratory bioengineering evolution, strategies to circumvent [25]. bioengineering strategies [25]. the current anti‐GM constraints from consumers facing comestible food products generated through bioengineering strategies [25].

FigureFigure 11. The 11. The conversion conversion of grapeof grape juice juice to to wine wine by by the the actionaction of the yeast, yeast, SaccharomycesSaccharomyces cerevisiae cerevisiae (adapted from [22]). A wine yeast converts grape nutrients into ethanol, carbon dioxide and (adaptedFigure from 11. [The22]). conversion A wine yeast of grape converts juice grapeto wine nutrients by the action into ethanol,of the yeast, carbon Saccharomyces dioxide and cerevisiae secondary secondary metabolites through its glycolytic metabolism. Some grape‐derived compounds, such as metabolites(adapted through from [22]). its glycolytic A wine yeast metabolism. converts Somegrape grape-derivednutrients into ethanol, compounds, carbon such dioxide as acids and and secondaryacids and metabolitespolyphenols, through remain its largely glycolytic untouched metabolism. by the Some yeast grape cells‐derived while compounds,other compounds such as are polyphenols, remain largely untouched by the yeast cells while other compounds are wholly generated acidswholly and generated polyphenols, as part remain of the largely fermenting untouched yeast cells’by the metabolism. yeast cells whileA number other ofcompounds the compounds are as part of the fermenting yeast cells’ metabolism. A number of the compounds synthesised or modified whollysynthesised generated or modified as part by of yeast the fermenting impart aromas yeast that cells’ can metabolism. diminish or A enhance number the of qualitythe compounds of wine. by yeastsynthesised impart aromasor modified that by can yeast diminish impart aromas or enhance that can the diminish quality of or wine.enhance the quality of wine.

Figure 12. The derivation and synthesis of flavour‐active compounds from sugar, amino acids and Figure 12. The derivation and synthesis of flavour‐active compounds from sugar, amino acids and Figuresulfur 12. Themetabolism derivation by wine and yeast synthesis (adapted of flavour-active from [7]). Different compounds strains of fromwine yeast sugar, possess amino different acids and sulfur metabolism by wine yeast (adapted from [7]). Different strains of wine yeast possess different sulfurabilities metabolism to form by and wine modulate yeast (adaptedchemical compounds from [7]). Different that affect strains wine sensory of wine properties. yeast possess different abilities to form and modulate chemical compounds that affect wine sensory properties. abilities to form and modulate chemical compounds that affect wine sensory properties.

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FigureFigure 13. Different 13. Different strains strains of Saccharomyces of Saccharomyces cerevisiae cerevisiaeproduce produce secondary secondary flavour-active flavour‐active metabolites andFigure impartand impart13. different Different different aromas strains aromas to of wine. Saccharomyces to wine. Not Not all wineallcerevisiae wine yeast yeast produce strains strains secondary are are equal. equal. flavour Juice Juice from‐fromactive the metabolites same same grapes grapes and impart different aromas to wine. Not all wine yeast strains are equal. Juice from the same grapes will deliverwill deliver quite quite different different wines, wines, depending depending on on the the choice choice of ofS. S. cerevisiae cerevisiaestrain. strain. Because there there are are will deliver quite different wines, depending on the choice of S. cerevisiae strain. Because there are huge differenceshuge differences in consumer in consumer preferences, preferences, shaped shaped by by such such variables variables as as ethnicity, ethnicity, gendergender and age, age, it it is is hugeimportant differences for inwinemakers consumer preferences,to know which shaped yeast by suchto choose variables if they as ethnicity, want to gendertailor theirand age, wines it is for important for winemakers to know which yeast to choose if they want to tailor their wines for particular importantparticular for markets. winemakers We are to onlyknow just which beginning yeast toto chooseunderstand if they the want genetics to tailor underpinning their wines the for way markets. We are only just beginning to understand the genetics underpinning the way different wine particulardifferent markets. wine yeast We ‘shape’ are only wines. just beginning to understand the genetics underpinning the way yeastdifferent ‘shape’ wine wines. yeast ‘shape’ wines.

Figure 14. Different strain development techniques used to optimise certain winemaking properties FigureFigureof 14. selected 14.Different Different wine strain yeasts. strain development These development techniques techniques techniques include used geneticused to to optimisemodification optimise certaincertain (GM) winemaking and non‐GM properties properties techniques. of selectedof Theyselected wine are wine yeasts. applied yeasts. These to These study techniques techniques and improve include include yeast genetic genetic attributes modificationmodification relating (GM) (GM) to fermentationand and non non-GM‐GM techniques.performance, techniques. TheyTheyrobustness, are are applied applied spoilage to to study study‐control, and and processing improve improve yeastefficiency,yeast attributes product relating relating wholesomeness to to fermentation fermentation and sensory performance, performance, quality. It robustness,robustness,continues spoilage-control, spoilageto boggle‐control, the minds processingprocessing of many efficiency, efficiency, researchers product product why wholesomenessGM wholesomeness medicine has and been andsensory accepted sensory quality. widely, quality. It It continuescontinueswhereas to to GM boggle boggle food the theproducts mindsminds (including of many researcherswine) researchers have whynot, why despiteGM GM medicine medicinethe numerous has has been beenstudies accepted accepted demonstrating widely, widely, whereaswhereasthat GM such GM food products food products products are no (including more(including unsafe wine) wine) to eat havehave or drink not, despite than any the the other numerous numerous food product. studies studies Thisdemonstrating demonstrating scientific and thatthat suchcultural such products products quagmire are are no becomes no more more unsafe even unsafe more to to eat eat intriguing or or drinkdrink than when any any one other other considers, food food product. product.for example, This This scientific the scientific more and than and culturalcultural1000 quagmire transposons quagmire becomes becomes that ‘jump even even and more more bounce’ intriguing intriguing all over when the grapevine one one considers, considers, genome—genetically for for example, example, the modifying the more more than thanthe 1000 transposons that ‘jump and bounce’ all over the grapevine genome—genetically modifying the 1000 transposonsVitis vinifera thatgenome ‘jump in andevery bounce’ vineyard all across over thethe grapevinecommercial genome—genetically wine‐producing world modifying without any the Vitis vinifera genome in every vineyard across the commercial wine‐producing world without any Vitis viniferainterventiongenome by viticulturists. in every vineyard across the commercial wine-producing world without any intervention by viticulturists. intervention by viticulturists.

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5.1. Toning Down Disharmonious Off-Flavours in Wine Under-prepared musicians, faulty instruments and discordant notes can spoil the entire performance of an orchestra and cause long-term reputational damage with audiences. Likewise, unbalanced wines or wines containing unwanted flavours can damage a brand and deter consumers Beverages 2016, 2, 36 16 of 28 from repeat-sales. Just5.1. like Toning not Down all violinistsDisharmonious are Off Yehudi‐Flavours Menuhin,in Wine not all strains of S. cerevisiae are superstar performers.Under Most‐prepared commercially-available musicians, faulty instruments wine strains and of discordantS. cerevisiae notesare can able spoil to catalysethe entire the rapid, completeperformance and efficient of an conversion orchestra and of cause grape long sugars‐term reputational to ethanol anddamage carbon with dioxideaudiences. but Likewise, not all of these strains produceunbalanced optimal wines or concentrations wines containing of unwanted desirable flavours secondary can damage metabolites, a brand and deter in some consumers instances even producefrom off-flavours repeat‐sales. under certain fermentation conditions. The genetic variation among wine strains Just like not all violinists are Yehudi Menuhin, not all strains of S. cerevisiae are superstar of S. cerevisiae is well documented [26–32]. performers. Most commercially‐available wine strains of S. cerevisiae are able to catalyse the rapid, Breedingcomplete of and new efficient strains conversion has been of quitegrape sugars effective to ethanol in providing and carbon winemakers dioxide but not with all better of these options to improvestrains fermentation produce optimal performance concentrations and the of desirable profile of secondary aroma compoundsmetabolites, and of in wines some [ 33instances–35]. There are severaleven examples produce of off commercialised‐flavours under certain hybrid fermentation strains (e.g., conditions. NT116, The VIN13, genetic variation VL3, etc.) among originating wine from mating andstrains cross-breeding of S. cerevisiae is [well36]. documented Similarly, interspecific[26–32]. hybridisation between commercial S. cerevisiae Breeding of new strains has been quite effective in providing winemakers with better options to strains and closely-related species of the Saccharomyces sensu stricto complex (S. arboricolus, S. cariocanus, improve fermentation performance and the profile of aroma compounds of wines [33–35]. There are S. eubayanusseveral, S. examples kudriavzevii of commercialised, S. mikatae, S. hybrid paradoxus strains, S.(e.g., uvarum NT116,and VIN13,Naumovozyma VL3, etc.) originating castellii) from has also been used tomating introduce and aroma cross‐breeding and flavour [36]. diversity Similarly, to interspecific wines as a meanshybridisation of improving between stylecommercial and increasing productS. differentiation cerevisiae strains and [32– closely35]. ‐related species of the Saccharomyces sensu stricto complex (S. arboricolus, ThereS. cariocanus are also, S. eubayanus examples, S. kudriavzevii where mutagenesis, S. mikatae, S. paradoxus was effectively, S. uvarum and applied Naumovozyma to improve castellii) existing has also been used to introduce aroma and flavour diversity to wines as a means of improving style commercialand increasing wine strains product so thatdifferentiation no longer [32–35]. can they form off-flavours (Figure 15). The development of low-H2S andThere low-volatile are also examples acidity (VA)where mutants mutagenesis of the was Maurivin effectively PDM applied strain to isimprove a case existing in point [37–39]. The low-Hcommercial2S variants wine of strains PDM so were that no generated longer can by they mutating form off‐ theflavoursMET10 (Figureand 15).MET5 The developmentgenes, which encode the catalyticof lowα‐H- and2S andβ -subunitslow‐volatile of acidity the sulfite (VA) mutants reductase of the enzyme. Maurivin These PDM mutationsstrain is a case significantly in point [37– decreased or eliminated39]. The thelow‐H capacity2S variants to of produce PDM were unwanted generated by ‘reductive’ mutating the characters MET10 and MET5 (typified genes, by which cabbage-like encode the catalytic α‐ and β‐subunits of the sulfite reductase enzyme. These mutations significantly aromas)decreased during the or fermentationeliminated the ofcapacity grape muststo produce low unwanted in nitrogen ‘reductive’ [37,38]. Furthercharacters improvement (typified by of these strains wascabbage achieved‐like aromas) by generating during the mutantsfermentation that of producedgrape musts considerably low in nitrogen less [37,38]. acetic Further acid (the main componentimprovement of volatile of these acidity strains and was colloquially achieved by referred generating to asmutants VA by that winemakers) produced considerably [39,40]. When less present in concentrationsacetic acid (the above main the component sensory of threshold, volatile acidity acetic and colloquially acid imparts referred an undesirable to as VA by winemakers) vinegary character. These low-VA[39,40]. strainsWhen present had either in concentrations mutations in above the YAP1 the (involvedsensory threshold, in oxidative acetic stress) acid imparts or AAF1 an(involved undesirable vinegary character. These low‐VA strains had either mutations in the YAP1 (involved in in fermentationoxidative stress) stress or response) AAF1 (involved gene. in fermentation stress response) gene.

Figure 15. The construction of wine strains of Saccharomyces cerevisiae that produce low or no Figure 15. The construction of wine strains of Saccharomyces cerevisiae that produce low or no concentrations of hydrogen sulfide which can spoil the aroma and quality of wine. Low‐H2S mutants concentrationsare now commercially of hydrogen available sulfide to which winemakers. can spoil the aroma and quality of wine. Low-H2S mutants are now commercially available to winemakers. Beverages 2016, 2, 36 17 of 28 Beverages 2016, 2, 36 17 of 28

5.2.5.2. Striking Striking the the Right Right Chord Chord between between Alcohol Alcohol Strength Strength and and Fruitiness Fruitiness in in Wine Wine LikeLike a conductorconductor striving striving to to harmonise harmonise all theall soundsthe sounds from allfrom instruments all instruments playing playing in an orchestra, in an orchestra,a winemaker’s a winemaker’s goal is to goal produce is to well-balancedproduce well‐balanced wines where wines the where alcoholic the alcoholic strength, acidity,strength, sweetness, acidity, sweetness,fruitiness andfruitiness tannin and structure tannin complementstructure complement each other each so that other no so single that componentno single component dominates dominateson the palate. on the To palate. strike To the strike right the chemical right chemical balance balance in wine in is wine no small is no feat. small For feat. example, For example, many manyconsumers consumers prefer prefer rich rich and and fruitier fruitier styles styles of wineof wine made made from from mature, mature, ripe ripe grapes. grapes. Such Such wines wines areare free free from from undesirable undesirable unripe, unripe, vegetable vegetable flavours flavours but but they they also also contain contain much much higher higher levels levels of of alcoholalcohol than than wines wines made made from from early early-harvested‐harvested grapes. grapes. Depending Depending on on the the particular particular style, style, too too high high a a concentrationconcentration of of ethanol ethanol can can be be perceived perceived as as ‘hotness’ ‘hotness’ on on the the palate, palate, making making overly overly-alcoholic‐alcoholic wines wines appearappear unbalanced unbalanced [41–43]. [41–43]. InIn an an attempt attempt to to generate generate non non-GM‐GM low low-ethanol‐ethanol wine wine strains strains for for the the production production of of consumer consumer demanddemand-driven‐driven fruity fruity wines wines with with reduced reduced alcoholic alcoholic strength, strength, an an adaptive adaptive laboratory laboratory evolution evolution approachapproach was was used used [44–46]. [44–46]. This This strategy strategy was was reasonably reasonably successful successful in in that that the the resulting resulting mutants mutants producedproduced significantly significantly higher higher concentrations concentrations of of glycerol glycerol at at the the expense expense of of ethanol ethanol biosynthesis. biosynthesis. OverOver the the past past two two decades, decades, several several genetic genetic engineering engineering strategies strategies have have also also been been explored explored to to generategenerate wine wine strains strains capable capable of ofpartially partially diverting diverting carbon carbon flow flow away away from from ethanol ethanol (Figure (Figure 16). The 16). mostThe mostpromising promising results results were were achieved achieved by over by over-expression‐expression of either of either one one of ofthe the two two paralogue paralogue genes, genes, GPD1GPD1 andand GPD2GPD2, ,encoding encoding glycerol glycerol-3-phosphate‐3‐phosphate dehydrogenase dehydrogenase isozymes isozymes [47–53]. [47–53]. For For example, example, multiplemultiple copies copies of of GPD1GPD1 genegene constructs constructs were were successfully successfully integrated integrated into the into genome the genome of two of wine two strainswine strains and these and bioengineered these bioengineered strains were strains able were to lower able to the lower ethanol the content ethanol from content 15.6% from (v/ 15.6%v) to 13.2%(v/v) ( tov/v 13.2%) and 15.6% (v/v) and(v/v) 15.6% to 12% (v (/vv/v)) to in 12% Chardonnay (v/v) in Chardonnayand Cabernet and Sauvignon Cabernet wines, Sauvignon respectively wines, [53].respectively Unfortunately, [53]. Unfortunately, both strains produced both strains unacceptable produced unacceptableconcentrations concentrations of acetaldehyde of acetaldehydeand acetoin, impartingand acetoin, negative imparting ‘bruised negative apple’ ‘bruised aromas apple’ to the aromas resulting to thewine. resulting To redirect wine. the To metabolic redirect the flow metabolic away fromflow acetaldehyde, away from acetaldehyde, acetoin and acetic acetoin acid and towards acetic the acid sensorially towards neutral the sensorially metabolite neutral 2,3‐butanediol, metabolite the2,3-butanediol, BDH1 gene—encoding the BDH1 gene—encoding butanediol dehydrogenase—was butanediol dehydrogenase—was overexpressed overexpressed in these inhigh these‐ glycerol/lowhigh-glycerol/low-ethanol‐ethanol GM stains. GM stains. This resulted This resulted in a insignificant a significant decrease decrease in inthe the formation formation of of acetaldehyde,acetaldehyde, acetic acetic acid acid and and acetoin; acetoin; however, however, unexpectedly unexpectedly the the overexpression overexpression of of BDH1BDH1 alsoalso interferedinterfered with with the the formation formation of of glycerol glycerol and and ethanol, ethanol, most most likely likely driven driven by by alterations alterations in in the the redox redox balancebalance of of the the yeast’s yeast’s metabolism metabolism [53]. [53].

FigureFigure 16. 16. The constructionconstruction of of low-alcohol low‐alcohol wine wine strains strains of Saccharomyces of Saccharomyces cerevisiae cerevisiae(adapted (adapted from [ 22from,23]). [22,23]).

The desire to tone down the decibels of ethanol in fruity wines made from well‐ripened grapes remains high on the priority list of vintners; however, the development of an ‘ideal’ low‐alcohol wine

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The desire to tone down the decibels of ethanol in fruity wines made from well-ripened grapes remains high on the priority list of vintners; however, the development of an ‘ideal’ low-alcohol wine yeast that can achieve an optimal balance between alcoholic strength and fruitiness can at best be described as a ‘work in progress’.

5.3. PerformingBeverages 2016 Effortlessly, 2, 36 in Solo Malolactic and Malo-Ethanolic Fermentations 18 of 28

Thereyeast is that no doubt can achieve that it an would optimal demand balance muchbetween more alcoholic effort strength to direct and a largefruitiness philharmonic can at best be orchestra than, say,described a string as quartet—the a ‘work in progress’. less participants, the less effort is required. This intuitive assumption can be extended to winemaking—the less microbes involved, the less complex the process. This was 5.3. Performing Effortlessly in Solo Malolactic and Malo‐Ethanolic Fermentations the objective when researchers developed yeast strains capable of conducting both the alcoholic and malolactic fermentationThere is no doubt [54,55 that]. it would demand much more effort to direct a large philharmonic Normally,orchestra malolactic than, say, a fermentationstring quartet—the is driven less participants, by fastidious the less lactic effort acid is required. bacteria, This such intuitive as O. oeni, assumption can be extended to winemaking—the less microbes involved, the less complex the following alcoholic fermentation conducted by S. cerevisiae yeast. During malolactic fermentation, process. This was the objective when researchers developed yeast strains capable of conducting both grape-derivedthe alcoholic malic and acid malolactic is deacidified fermentation (decarboxylated) [54,55]. to lactic acid. It often happens that malolactic fermentationNormally, is ‘sluggish’ malolactic or gets fermentation ‘stuck’ because is driven of by inhibitory fastidious conditionslactic acid bacteria, ranging such from as O. low oeni pH,, high alcohol contentfollowing and alcoholic poor fermentation nutrient availability. conducted Thereby S. cerevisiae is also the yeast. potential During formalolactic some bacteriafermentation, to produce biogenicgrape amines‐derived that canmalic impose acid is health deacidified risks to(decarboxylated) consumers of to fermented lactic acid. products. It often Ahappens wine yeastthat strain malolactic fermentation is ‘sluggish’ or gets ‘stuck’ because of inhibitory conditions ranging from low that is able to convert grape sugars into ethanol, carbon dioxide and flavour-active metabolites as pH, high alcohol content and poor nutrient availability. There is also the potential for some bacteria well as sharp-tastingto produce biogenic malic amines acid that into can soft-tasting impose health lactic risks acid to consumers would therefore of fermented simplify products. and A improve wine the reliabilityyeast of thestrain winemaking that is able process.to convert grape sugars into ethanol, carbon dioxide and flavour‐active A widely-usedmetabolites as winewell as strain sharp‐ oftastingS. cerevisiae malic acid, equipped into soft‐tasting with lactic the Schizosaccharomyces acid would therefore simplify pombe malate transporterand improve gene (mae1 the reliability) and the ofO. the oeni winemakingmalolactic process. enzyme gene (mleA), was engineered, thoroughly tested, clearedA widely by the‐used American wine strain and of Canadian S. cerevisiae regulatory, equipped with authorities the Schizosaccharomyces and commercialised pombe malate under the transporter gene (mae1) and the O. oeni malolactic enzyme gene (mleA), was engineered, thoroughly name ML01 [54,55]. These two genes were placed under the control of the strong constitutive regulatory tested, cleared by the American and Canadian regulatory authorities and commercialised under the sequencesname of theML01S. cerevisiae[54,55]. These PGK1 twopromoter genes were and placed terminator under and the integratedcontrol of the into strong the genome constitutive of the host strain. ML01regulatory is capable sequences of performing of the S. cerevisiae malolactic PGK1 fermentationpromoter and interminator parallel and with integrated alcoholic into fermentation the (Figure 17genome). In fermentation of the host strain. trials, ML01 it wasis capable shown of thatperforming 5 g/L malolactic of malic acid fermentation was decarboxylated in parallel with to lactic acid withinalcoholic five days,fermentation without (Figure negative 17). In impacts fermentation on the trials, sensory it was aspects shown onthat wine. 5 g/L Furtherof malic acid analyses was of the phenotype,decarboxylated genotype, to transcriptome lactic acid within and five proteome days, without revealed negative that impacts ML01 on was the ‘substantially sensory aspects equivalent’ on wine. Further analyses of the phenotype, genotype, transcriptome and proteome revealed that ML01 to its parentalwas ‘substantially industrial equivalent’ wine yeast. to its parental industrial wine yeast.

Figure 17. The construction of malolactic and maloethanolic wine strains of Saccharomyces cerevisiae Figure 17. The construction of malolactic and maloethanolic wine strains of Saccharomyces cerevisiae which eliminates the need for malolactic bacteria to drive the conversion of malic acid into lactic acid. which eliminatesA genetically the modified need for malolactic malolactic wine bacteria yeast strain, to drive ML01, the was conversion commercialised of malic and made acid available into lactic acid. A geneticallyfor commercial modified wine malolactic production wine in a yeast few countries. strain, ML01, was commercialised and made available for commercial wine production in a few countries.

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A second variant also became available with the integration of the S. pombe malate transporter gene ( mae1) and malic enzyme gene ( mae2) into the genome of the the engineered engineered strain. strain. With With this this strain, strain, malate is decarboxylated to pyruvate, which is thenthen convertedconverted toto ethanol.ethanol. In 2005, thethe ML01ML01 malolactic malolactic strain strain became became the the first first commercialised commercialised GM GM wine wine yeast yeast released released to the to market.the market. However, However, the prevailing the prevailing anti-GM anti sentiments‐GM sentiments from consumers from consumers have prevented have theprevented widespread the usewidespread of this strain use of in this commercial strain in winecommercial production. wine production.

5.4. Safeguarding against Unhealthy Notes of Ethyl Carbamate in Wine Production A second GM wine yeast,yeast, ECMo01, obtained clearance from the American and Canadian regulatory bodies in 2006 [[56].56]. ECMo01 ECMo01 was was constructed constructed to to reduce reduce the the risk of ethyl carbamate production during fermentation. This This potentially potentially carcinogenic carcinogenic compound compound is is the the product of urea reacting with ethanol. Fortunately, ethyl carbamate is ordinarily produced at such low levels (if at all) all) in winemakingwinemaking that that it it is is normally normally not not a concern.a concern. Nonetheless, Nonetheless, it is it present is present in some in some fortified fortified wines wines from certainfrom certain wine-producing wine‐producing regions. regions. ECMo01 has has an an extra extra copy copy of of the the S. cerevisiaeS. cerevisiae DUR1,2 DUR1,2 genegene under under the control the control of the of yeast the PGK1 yeast PGK1regulatoryregulatory sequences sequences (Figure (Figure 18). This 18). gene This codes gene codes for urea for ureaamidolyase, amidolyase, which which converts converts urea ureainto intoammonia ammonia and carbon and carbon dioxide, dioxide, thereby thereby removing removing substrate substrate for ethyl for ethyl carbamate carbamate production. production. The Theammonia ammonia formed formed by this by reaction this reaction is consumed is consumed as a preferred as a preferred nitrogen nitrogen source source by the by yeast the yeastcells. cells.ECMo01 ECMo01 has been has reported been reported to reduce to reduce ethyl ethylcarbamate carbamate in Chardonnay in Chardonnay wine wineby almost by almost 90%, 90%,and andanalyses analyses of ECMo01’s of ECMo01’s phenotype phenotype and and transcriptome transcriptome also also revealed revealed that that the the ECMo01 ECMo01 yeast yeast is ‘substantially‘substantially equivalent’equivalent’ toto itsits parental strainstrain [[56].56]. However, although this ECMo01 is a cis (or ‘self’) cloned yeast (it carries no foreign DNA and, therefore, is not transgenic), it suffered the same fate as ML01in thethe marketmarket place place in in terms terms of of reluctance reluctance to to use use wine wine yeasts yeasts in commercial in commercial wine wine production production that arethat considered are considered to be to genetically-modified be genetically‐modified organisms organisms (GMOs). (GMOs).

Figure 18. The construction of a wine strain of Saccharomyces cerevisiae that reduces the levels of ethyl carbamate formation during wine fermentation (adapted from [[22,23]).22,23]). A genetically modifiedmodified wine yeast strain, Eco01, Eco01, was was commercialised commercialised and and made made available available for for commercial commercial wine wine production production in a infew a fewcountries. countries.

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5.5.5.5. Directing Directing a aSymphony Symphony of of Aromas Aromas and and Flavoursome Flavoursome Tastes Tastes in in a a Wine Wine Glass Glass TheThe combination combination and and harmony harmony of of complex complex notes notes produced produced by by a asymphony symphony orchestra orchestra determine determine thethe quality quality of of their their performance. performance. This This can can also also be be said said for for the the complex complex mixture mixture of of chemical chemical compoundscompounds that that define define a awine’s wine’s appearance, appearance, aroma, aroma, flavour flavour and and mouth mouth-feel‐feel properties properties (Figure (Figure 19). 19 ). TheThe sensorially sensorially-active‐active chemical chemical compounds compounds originate originate from from the the grapes, grapes, microbes microbes and and oak oak (when (when used). used).

FigureFigure 19. 19. TheThe chemical chemical composition composition of wine. of wine. Wine Wine consists consists of ca. 86% of ca. water, 86% ca. water, 13% alcohols ca. 13% (mainly alcohols ethanol)(mainly and ethanol) ca. 1% and secondary ca. 1% secondary metabolites. metabolites. The latter The contain latter the contain compounds the compounds that shape that the shape aroma the andaroma flavour and of flavour wine. of wine.

The grape‐derived compounds give wines their basic structure and provide their varietal The grape-derived compounds give wines their basic structure and provide their varietal distinction (Figure 20). Examples of flavour‐active grape‐derived compounds are aliphatics, benzene‐ distinction (Figure 20). Examples of flavour-active grape-derived compounds are aliphatics, derivatives, esters (cis‐rose oxide, ethyl acetate, ethyl butyrate, ethyl formate, ethyl hexanoate, ethyl benzene-derivatives, esters (cis-rose oxide, ethyl acetate, ethyl butyrate, ethyl formate, ethyl propionate, furaneol, isoamyl acetate, isobutyl acetate, methyl cinnamate), lactones (sotolon), hexanoate, ethyl propionate, furaneol, isoamyl acetate, isobutyl acetate, methyl cinnamate), lactones norisprenoids (e.g., β‐demascenone, β‐ionone, 1,1,6‐trimetyl‐1,2‐dihydronaphtalene), phenols, (sotolon), norisprenoids (e.g., β-demascenone, β-ionone, 1,1,6-trimetyl-1,2-dihydronaphtalene), pyrazines (e.g., 3‐isobutyl‐2metoxypyrazine), sesquiterpenes (e.g., rotundone), monoterpenes (e.g., phenols, pyrazines (e.g., 3-isobutyl-2metoxypyrazine), sesquiterpenes (e.g., rotundone), monoterpenes linalool, geraniol, hotrienol, nerol, citronellol, cis‐rose oxide and α‐terpineol) and thiols (e.g., linalool, geraniol, hotrienol, nerol, citronellol, cis-rose oxide and α-terpineol) and thiols (e.g., 4‐mercapto‐4‐metyl‐pentan‐2‐one, 3‐mercaptohexan‐ol, 3‐mercaptohexyl acetate, etc.) [7]. Each (e.g., 4-mercapto-4-metyl-pentan-2-one, 3-mercaptohexan-ol, 3-mercaptohexyl acetate, etc.) [7]. of these compounds impart, individually or in combination with others, specific characters to wines Each of these compounds impart, individually or in combination with others, specific characters [8]. For instance, the ‘bell pepper’ character of Cabernet Sauvignon stems from 3‐isobutyl‐2‐ to wines [8]. For instance, the ‘bell pepper’ character of Cabernet Sauvignon stems from methoxypyrazine while the strawberry‐like characters of Pinot Noir are driven by a combination of 3-isobutyl-2-methoxypyrazine while the strawberry-like characters of Pinot Noir are driven by a several esters, including ethyl acetate, ethyl butyrate, ethyl formate, ethyl hexanoate, furaneol and combination of several esters, including ethyl acetate, ethyl butyrate, ethyl formate, ethyl hexanoate, methyl cinnamate [8]. However, some esters can also act in pairs or individually as aroma‐defining furaneol and methyl cinnamate [8]. However, some esters can also act in pairs or individually as compounds. For example, the fig‐like character of Semillon is the result of two esters, ethyl propanate aroma-defining compounds. For example, the fig-like character of Semillon is the result of two and isobutyl acetate [8]. An example of an ester that acts individually is cis‐rose oxide that determines esters, ethyl propanate and isobutyl acetate [8]. An example of an ester that acts individually is the well‐known lychee‐like flavour of Gewürztraminer. When Gamay grapes undergo carbonic cis-rose oxide that determines the well-known lychee-like flavour of Gewürztraminer. When Gamay maceration, isoamyl acetate imparts pronounced banana‐like aromas in the resulting wine [8]. grapes undergo carbonic maceration, isoamyl acetate imparts pronounced banana-like aromas in Terpenes are another class of compounds that determine characteristic grape flavours and aromas; the resulting wine [8]. Terpenes are another class of compounds that determine characteristic grape for example, the sesquiterpene rotundone is responsible for the spicy and peppery notes in Shiraz flavours and aromas; for example, the sesquiterpene rotundone is responsible for the spicy and [57,58] and the monoterpene geraniol defines the ‘grapey’ flavour of Muscat grapes. The lactone, peppery notes in Shiraz [57,58] and the monoterpene geraniol defines the ‘grapey’ flavour of Muscat sotolon, is responsible for the honey‐like characters in botrytised wines and 1,6 trimethyl‐1,2‐ grapes. The lactone, sotolon, is responsible for the honey-like characters in botrytised wines and dihydronaphthalene drives the kerosene‐like characters in Riesling. Thiols play a major role in the 1,6 trimethyl-1,2-dihydronaphthalene drives the kerosene-like characters in Riesling. Thiols play a fragrance of Sauvignon Blanc: 4‐mercapto‐4‐methylpentan‐2‐one (4MMP) imparts grassy and box‐ major role in the fragrance of Sauvignon Blanc: 4-mercapto-4-methylpentan-2-one (4MMP) imparts tree flavours while 3‐mercaptohexan‐ol (3MH) and 3‐mercaptohexyl acetate (3MHA) are responsible for tropical fruit flavours similar to those typifying the smell of gooseberries and passion‐fruit [59– 69].

Beverages 2016, 2, 36 21 of 28 grassy and box-tree flavours while 3-mercaptohexan-ol (3MH) and 3-mercaptohexyl acetate (3MHA) areBeverages responsible 2016, 2, 36 for tropical fruit flavours similar to those typifying the smell of gooseberries21 of 28 and passion-fruit [59–69].

FigureFigure 20. 20.The The main main classes classes of of grape-derived grape‐derived aromas that that determine determine the the recognisable recognisable and and distinctive distinctive characteristicscharacteristics of of varietal varietal wines wines (adapted (adapted fromfrom [[8]).8]).

The interaction of yeasts with these grape‐derived compounds and the de novo synthesis of its own secondary metabolites (esters, fusel alcohols, carbonyls, volatile fatty acids, sulfur compounds, etc.) give wine its ‘vinous’ character [65,70–72]. During the alcoholic fermentation, several grape‐

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The interaction of yeasts with these grape-derived compounds and the de novo synthesis of its own secondary metabolites (esters, fusel alcohols, carbonyls, volatile fatty acids, sulfur compounds,Beverages 2016, 2 etc.), 36 give wine its ‘vinous’ character [65,70–72]. During the alcoholic fermentation,22 of 28 several grape-derived compounds are biotransformed and released from grape-derived precursors. Forderived example, compounds non-volatile are biotransformed cysteinylated grape and released thiols (Cys-4MMP from grape and‐derived Cys-3MH) precursors. can be For released example, from non‐volatile cysteinylated grape thiols (Cys‐4MMP and Cys‐3MH) can be released from cysteine cysteine (4MMP and 3MH) and converted into the more potent volatile compound, 3MHA [58–68]. (4MMP and 3MH) and converted into the more potent volatile compound, 3MHA [58‒68]. Wine yeast Wine yeast strains can be engineered to enhance their capacity and capability to modulate these strains can be engineered to enhance their capacity and capability to modulate these grape‐derived grape-derived thiol precursors [65] (Figure 21). During malolactic fermentation, malolactic bacteria thiol precursors [65] (Figure 21). During malolactic fermentation, malolactic bacteria deacidifies wine deacidifies wine by converting malic acid into lactic acid, and produces additional metabolites, such by converting malic acid into lactic acid, and produces additional metabolites, such as diacetyl which as diacetyl which imparts a buttery flavour to wine [7]. In addition, when wine comes in contact imparts a buttery flavour to wine [7]. In addition, when wine comes in contact with oak during with oak during fermentation and/or maturation, a series of complex chemical transformations can fermentation and/or maturation, a series of complex chemical transformations can result in result in significant changes in the aroma and flavour profile of the end-product [73]. Toasted oak significant changes in the aroma and flavour profile of the end‐product [73]. Toasted oak staves are staves are often used in oak barrel manufacturing and the related potent aroma compounds (vanillin often used in oak barrel manufacturing and the related potent aroma compounds (vanillin and andvanillin vanillin-derivates,‐derivates, volatile volatile phenols phenols and lactones) and lactones) can strongly can strongly influence influence the aroma the and aroma style and of stylewine. of wine.When When yeast yeastfermentation fermentation is conducted is conducted in oak barrels, in oak barrels,some of somethese oak of these‐derived oak-derived compounds compounds migrate migratefrom the from wood the to wood the fermenting to the fermenting grape must, grape which must, in turn, which can in be turn, transformed can be transformed into highly into aromatic highly aromaticcompounds compounds [73]. For instance, [73]. For vanillin instance, can vanillin be reduced can beby reducedyeast to vanillic by yeast alcohol, to vanillic which alcohol, has a much which haslower a much sensory lower threshold sensory thresholdthan vanillin. than vanillin.Similarly, Similarly, furfuryl furfuryl alcohol alcoholand furfuryl and furfuryl mercaptan mercaptan (2‐ (2-furanmethanethiol)furanmethanethiol) can can be be formed formed from from oak oak-derived‐derived furfural. furfural. The The accumulation accumulation of of oak oak-derived‐derived compoundscompounds in in wine wine during during barrel barrel storage storage increase increase the intensitythe intensity of sensory of sensory descriptors descriptors such as such coconut, as spicecoconut, and spice vanilla. and vanilla.

FigureFigure 21. 21. TheThe construction construction of of a wine a wine strain strain of Saccharomyces of Saccharomyces cerevisiae cerevisiae that enhancesthat enhances the typical the thiol typical‐ thiol-drivendriven aromas aromas in Sauvignon in Sauvignon Blanc Blanc and other and otherwine types wine types(adapted (adapted from [22,23]). from [22 ,23]).

Vintners use a large number of variations of viticultural and oenological practices to obtain the optimalVintners absolute use and a large relative number concentration of variations of these of viticultural grape‐, oak and‐ and oenological microbial‐ practicesderived chemical to obtain thecompounds optimal absolute to shapeand the relativearoma andconcentration flavour profile of these of their grape-, wines oak- according and microbial-derived to the preferences chemicalof their compoundstargeted consumer to shape markets—a the aroma harmonious and flavour profilesymphony of their in a wineswine glass. according to the preferences of their targeted consumer markets—a harmonious symphony in a wine glass. 6. Orchestrating Future Music in the Research Laboratory No‐one knows where music came from but we do know that the earliest human ‘music‐makers’ used whatever was available to create musical instruments with which they could create rhythms for telling their stories. Their traditional poetry with rhythm eventually evolved into songs as they invented and improved their musical instruments. The invention of these first musical instruments

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6. Orchestrating Future Music in the Research Laboratory No-one knows where music came from but we do know that the earliest human ‘music-makers’ used whatever was available to create musical instruments with which they could create rhythms for telling their stories. Their traditional poetry with rhythm eventually evolved into songs as they invented and improved their musical instruments. The invention of these first musical instruments came about accidentally, much like the early inventions in winemaking. As civilisations became more sophisticated, inventions became more deliberate until innovations became continuous. The evolution of the earliest forms of, say, the harpsichord, clavichord, piano and organ to today’s electronic keyboards, digital pianos and synthesisers is not dissimilar from the progression of yeast strain development—from the passive use of ambient yeasts in spontaneous fermentations to the application of hybrids, mutants and bioengineered strains with specialised winemaking properties. It is tempting to speculate that the invention of the first electronic music synthesiser might have been perceived by musicians and music enthusiasts as much of a threat to the future of, for example, classical music played by a philharmonic orchestra, as a yeast with ‘synthetic’ DNA would be perceived by some vintners and consumers to pose to the future of winemaking. Over the years, those in the music industry have come to realise that composers, conductors and musicians are benefitting from the availability of synthesisers in writing their music, preparing for their performances and practicing their beloved art. They know that technology, in general, and digital music synthesisers, more specifically, are not a threat but a great help in their effort to improve their art form. Over the past decades, the wine industry benefitted enormously from a wide range of technological inventions; however, while the prospect of ‘synthetically-tailored’ yeast strains might be music to the ears of some avant-garde scientists, such news might send shivers down the spine of many winemakers and wine drinkers. It is for this reason that the first notes of future music emanating from trailblazing Synthetic Biology laboratories about ‘synthetic’ yeasts be crystal clear and dealt with sensitively. Those who research in the emerging field of synthetic genomics have to work relentlessly to engage all stakeholders, including fellow scientists, industry practitioners, policymakers, regulators, commentators and consumers [24]. It is important for researchers to explain that the development of ‘synthetic’ yeast models in the laboratory would help yeast researchers to further expand their understanding of the biological fundamentals, limitations and potential of existing wine yeasts—much like what the physicist Richard Feynman meant when he famously said “What I cannot create, I do not understand”. This ‘philosophy’ is what inspired a consortium of a dozen laboratories from five countries—the US, UK, Australia, China and Singapore—to embark on a collaborative project aimed at the synthesis of the entire genome of a laboratory S. cerevisiae strain (S288c) by early 2018. The goals of this large international project—the Synthetic Yeast Genome Project (referred to as the Yeast 2.0 or Sc2.0 project)—include answering a broad spectrum of “profound questions about fundamental properties of chromosomes, genome organisation, gene content, function of RNA splicing, the extent to which small RNAs play a role in yeast biology, the distinction between prokaryotes and eukaryotes, and questions relating to genome structure and evolution” while recognising that “the eventual ‘synthetic yeast’ being designed and refined could ultimately play an important practical role” (www.syntheticyeast.org). The early learnings from the Yeast 2.0 project are already starting to forge new frontiers for wine yeast development [24]. The technology used in the Yeast 2.0 project on a laboratory yeast strain has indeed spilled over into wine yeast research with the development of a synthetic raspberry wine yeast model—the first wine yeast containing a ‘synthetic DNA circuit’. This wine strain of S. cerevisiae was developed as an instrument for the unravelling of metabolic flavour pathways relevant to winemaking [74]. The primary objective of this project was not to apply the ‘synthetic’ raspberry yeast in commercial winemaking; rather, the goal was to use the ‘synthetic’ yeast as a study model to uncover the biochemical pathways leading to the formation of the raspberry ketone, 4-[4-hydroxyphenyl]butane-2-one. A ‘DNA circuit’ consisting of four chemically-synthesised, codon-optimised genes were expressed in a Beverages 2016, 2, 36 24 of 28

S. cerevisiae wine strain, AWRI1631. The synthetic copies were derived from the following genes: RtPAL from an oleaginous yeast, Rhodosporidium toruloides; AtC4H from the well-studied model plant, Arabidopsis thaliana; Pc4CL2 from parsley, Petroselinum crispum; and RpBAS from rhubarb, Rheum palmatum. The wine yeast containing the synthetic version of these genes was used to produce experimentalBeverages 2016, 2 Chardonnay,, 36 which smelled and tasted like raspberries (Figure 22). This ‘synthetic’24 wine of 28 yeast offers prospects for the development of non-GM wine yeast starter strains with a capability to intensifya capability the raspberryto intensify notes the raspberry in both red notes and in white both wine. red and If successful, white wine. this If couldsuccessful, assist this winemakers could assist in theirwinemakers effort to in consistently their effort produce to consistently wine to produce definable wine market to definable specifications market and specifications styles. and styles.

FigureFigure 22. 22.The The construction construction of of a a ‘synthetic’ ‘synthetic’ wine wine strain strain of of SaccharomycesSaccharomyces cerevisiae cerevisiaecapable capable of of producing producing raspberryraspberry aroma aroma in in Chardonnay Chardonnay (adapted (adapted from from [24]). [24]). This is This the firstis the wine first yeast wine containing yeast containing a ‘synthetic a DNA‘synthetic circuit’ DNA coding circuit’ for raspberrycoding for ketone raspberry formation. ketone formation.

TheThe prospect prospect of ‘synthetically-tailored’of ‘synthetically‐tailored’ Yeast Yeast 2.0 strains 2.0 isstrains much moreis much real thanmore most real stakeholders than most instakeholders the wine industry in the wine realise—the industryYeast realise—the 2.0 project Yeast is already 2.0 project 75% is complete already 75% and complete on track toand equip on track the firstto equip yeast cellthe withfirst yeast 16 chemically-synthesised cell with 16 chemically chromosomes‐synthesised (~12 chromosomes Mb carrying (~12 ~6000 Mb genes). carrying This ~6,000 yeast willgenes). become This the yeast first will eukaryotic become cell the to first cross eukaryotic the horizon cell of into silicocross design the horizon of complex of in cells silico through design de of novocomplex synthesis, cells through reshuffling de novo and editing synthesis, of yeast reshuffling genomes. and As editing with most of yeast disruptive genomes. innovations, As with most the ‘techno’disruptive sound innovations, of Yeast 2.0 the future ‘techno’ music sound is bound of Yeast to unsettle2.0 future and music cause is discordantbound to unsettle notes within and cause the globaldiscordant wine notes industry. within the global wine industry. ThereThere is is no no question question that that the the future future music music of of scientific scientific research research and and breakthrough breakthrough discoveries discoveries in in leading-edgeleading‐edge yeast yeast laboratories laboratories will will continue continue to to play play on; on; however, however, the the question question is is whether whether there there will will bebe receptive receptive and and appreciative appreciative audiences audiences to to applaud applaud and and call call for for encore encore performances. performances. Acknowledgements: The author acknowledges the ongoing support from all members of the international Acknowledgments: The author acknowledges the ongoing support from all members of the international Synthetic Yeast Genome Project (Sc2.0) consortium, and the research contributions of his team at Macquarie Synthetic Yeast Genome Project (Sc2.0) consortium, and the research contributions of his team at Macquarie University:University: Thomas Thomas Williams, Williams, Heinrich Heinrich Kroukamp, Kroukamp, Hugh Goold,Hugh NatalieGoold, CurachNatalie and Curach Ian Paulsen and andIan collaboratorsPaulsen and atcollaborators The Australian at WineThe Australian Research Institute: Wine Research Anthony Borneman,Institute: Anthony Dariusz KutynaBorneman, and DanielDariusz Johnson. Kutyna I amand grateful Daniel toJohnson. Rae Blair, I am Tori grateful Hocking to Rae and Blair, Erin Tori Semon Hocking for helping and Erin to Semon proofread for helping the manuscript, to proofread and the to manuscript, Bill Hope and and Theto Bill Drawing Hope and Book The Studios Drawing for creating Book Studios the artwork for creating used inthe the artwork diagrams. used in the diagrams.

Conflicts of Interest: The author declares that he does not have any competing interests. The Synthetic Biology initiative at Macquarie University is financially supported by an internal start‐up grant from the University, and external grants from Bioplatforms Australia, the New South Wales (NSW) Chief Scientist and Engineer, and the NSW Government’s Department of Primary Industries.

References

1. Chambers, P.J.; Pretorius, I.S. Fermenting knowledge: The history of winemaking, science and yeast research. EMBO Rep. 2010, 11, 914–920.

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Conflicts of Interest: The author declares that he does not have any competing interests. The Synthetic Biology initiative at Macquarie University is financially supported by an internal start-up grant from the University, and external grants from Bioplatforms Australia, the New South Wales (NSW) Chief Scientist and Engineer, and the NSW Government’s Department of Primary Industries.

References

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