Main target specific enzymes and genes in yeast cells that may lead to lower ethanol yields. HXT—hexose transporter; Fps1p—aquaglyceroporin; GPD—glyceraldehyde-3-phosphate dehydrogenase; TPI1—triosephosphate isomerase; PDC—pyruvate decarboxylase; ADH—alcohol dehydrogenase and ALDH—Aldehyde dehydrogenase. GOXp—Glucose-oxidase—and Lact—lactonase—are expressions, in yeasts, of non-yeast genes.
One of the modifications that can be used to reduce ethanol production is linked to the overexpression of glycerol-3-phosphate dehydrogenase isozymes, namely through
Gpd1 and
Gpd2 genes
[113],
Figure 3. Glycerol production usually uses about 4% of grape juice carbon during fermentation by
S. cerevisiae, generally in the initial stages of biomass formation
[114]. Glycerol has two essential functions: to combat osmotic stress and to maintain the oxidation-reduction balance. The reaction behind glycerol formation is linked to the correction of redox balance within cells
[115]. The overexpression of these genes increases glycerol synthesis while decreasing ethanol synthesis
[116]. This increase in glycerol production can reach as much as 548%, while the reduction in ethanol can be of great significance, as shown in early works
[117]. Other results indicate an increase in glycerol production ranging from 109 to 275% and a reduction in ethanol from 3 to 24%, depending on the experimental medium (yeast extract, peptone, dextrose medium, yeast nitrogen base medium, synthetic medium, grape juice or synthetic Leu-free) or the overexpressed gene (
Gpd1 or
Gpd2)
[112]. However, other metabolites are also more produced and can cause changes in the quality of wine. Of those, succinate, acetate, acetaldehyde, acetoin, and 2,3-butanediol
[118][119],
Figure 3, must be referred to, as their presence above the sensory threshold may be detrimental to perceived wine quality
[112][120]. Mutant yeasts with modifications of GPD need further genetic modifications to avoid excessive production of these metabolites. One modification is the deletion of aldehyde dehydrogenases, namely the
Ald6 gene, that contribute to the formation of acetic acid
[121]. This modification decreased the formation of acetic acid, with increased glycerol production and lower ethanol yield
[119]. However, a subsequent problem arose, as the deletion of
Ald6 increased acetoin production, negatively affecting wine aroma. Ehsani and collaborators
[122] obtained a strain that produced lower ethanol levels (3%,
v/
v less) and higher glycerol production with a reduced impact on sensory parameters in the final wine.
Other changes in glycerol metabolism can be achieved by changing the expression of genes linked to Fps1p, an aquaglyceroporin channel that controls the intracellular glycerol concentration
[123][124][125]. The production of glycerol is controlled by a regulatory domain located in the N-terminal extension of Fps1p. This domain regulates glycerol transport; when removed, the channel becomes hyperactive. As a result, glycerol continuously leaks out of the cell, and the cell compensates for the loss by producing more glycerol
[124][126]. Varela et al.
[109] observed that the increased glycerol production, due to deleting the regulatory domain of Fps1p, lowered ethanol formation considerably (
Figure 3).
Another gene modification to reduce ethanol production can be deleting Pyruvate decarboxylase (PDC) genes. This enzyme catalyzes the decarboxylation of pyruvate to acetaldehyde and CO
2, being three genes known in
S. cerevisiae (
Pdc1,
Pdc5, and
Pdc6) up-regulated by the transcription factor
Pdc2p; however, only
Pdc1p and
Pdc5p are known to be active in yeast during fermentation
[127],
Figure 3. Modifying the PDC genes resulted in diverse outcomes. Deleting
Pdc1 resulted in lower activity and increased pyruvate levels, which are undesirable, considering microbial stability and balance of sulfur dioxide. Still, no reduction in ethanol, while deleting
Pdc2, led to a considerable decrease in ethanol levels and increased glycerol production while maintaining sufficient pyruvate decarboxylase activity to support glucose growth
[117]. Further changes in this set of genes were performed by Cuello et al.
[128], which resulted in a strain with reduced ethanol production without effect on other fermentation kinetics.
Another gene editing that can be used to reduce the production of ethanol is modifying the expression of triose-phosphate isomerase, encoded by the
Tpi1, which is the enzyme catalyzing the interconversion between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, the two products following the breakdown of fructose 1,6-bisphosphate
[111],
Figure 3. The lack of this gene resulted in high amounts of glycerol, with a reduced ethanol yield
[129]. However, the deletion of the
Tpi1 gene caused the inability of this strain to grow in a glucose medium, probably due to the reduced content of NADH, which is produced during the conversion of glyceraldehyde 3-phosphate into pyruvate
[130][131]. A complete loss of activity of the TPI1 enzyme seems like it would be more feasible, as a reduction in yeast growth with attendant fermentation problems is likely to occur. Still, a partial reduction in its activity could provide an opportunity for low alcohol-producing yeast. This partial reduction can be achieved by changing regulatory genes, like
Gcr1 and
Gcr2 (transcription factor for glycolytic genes), that can reduce the expression of
Tpi1 [132], with mutations in other genes, like
Reb1 (an essential gene that maps on chromosome),
Rap1 (Repressor Activator Protein), and
Grc1 (component of the minus-end located γ-tubulin ring complex) also able to reduce TPI1 activity
[133]. Avoiding glucose repression of respiration has also been a target for producing low-alcohol wines. This approach relied on the use of a chimeric glucose transporter, comprised of the amino-terminal part of HXT and the carboxy-terminal region of
Hxt7 (HXT1, 2, 3, 4, 6, and 7 are intramembrane transporter proteins known to be involved in the transport of glucose)
[134]. Using a yeast strain with deleted
Hxt1, 2, 3, 4, 6, and 7 resulted in a respiratory phenotype with low ethanol production
[135], even though results were achieved using low sugar concentrations medium (5%,
w/
v) compared to grape juice. This shifts the metabolism from the Pasteur effect (under low O
2 concentrations, yeasts conduct alcoholic fermentation forming ethanol and CO
2, or, under high O
2 concentrations, aerobic fermentation occurs with lower glucose consumption) to the Crabtree effect, where, in the presence of high sugar content (about 200 g/L), regardless of O
2 presence, fermentation can occur
[136].
A different approach is the expression, in yeasts, of non-yeast genes. Reducing the content of sugars in grape juice before fermentation can be achieved by glucose oxidase (GOXp), an oxygen-dependent dehydrogenase that catalyzes the first step of a two-step process associated with converting glucose to gluconic acid
[112]. Besides treating grape juice with GOX to reduce the sugar content, a strategy that has several limitations, even though it can reduce up to 40% of ethanol production
[137], the introduction of the gene encoding this enzyme has already been performed in
S. cerevisiae [138]. Microvinifications of Chardonnay juice samples resulted in wines containing 1.8–2.0% less alcohol, possibly due to the use of glucose to produce D-glucono-δ-lactone and gluconic acid by GOX (
Figure 3). Other authors overexpressed the gene
noxE from
Lactoccocus lactis, encoding H
2O-forming NADH oxidase,
NoxEp, that uses NADH, oxidizing it when oxygen is available
[139]. The introduction of this NADH oxidase can decrease the available intracellular NADH pool, affecting alcohol dehydrogenase (ADH), hence resulting in reduced ethanol formation
[32]. This resulted in using only half the available sugar but with paralleled changes in other metabolic pathways, increasing acetaldehyde production, impairing growth, and fermentation performance. All these genetic manipulation approaches to low ethanol-producing yeast, as well as others that might arise, will be dependent on the acceptability of both the industry and consumers of using such yeast, pushing current research on the search for non-GMO alternatives
[31].
2.2.2. Yeast Selection for Low Alcohol Production
One alternative to avoid GMO yeasts, designed for low-ethanol production, is to isolate those that naturally present that trait. However, this effort for
S. cerevisiae can be complex, as biochemical and physiological characteristics and the underlying genetics of this yeast have been pushed by natural selection to favor the yield of ethanol
[31][140]. This has resulted in a slight variation in this phenotype
[141], with currently available
S. cerevisiae wine yeasts resulting in similar ethanol production when fermenting the same must
[142]. Hence, the option would be selecting non-
Saccharomyces (NS) yeasts that preferentially consume sugars by respiration rather than fermentation
[136]. Therefore, evaluation of ethanol production variation among NS yeasts has been addressed. Using non-
Saccharomyces yeasts has garnered significant interest from the scientific community and winemakers, as the available data state (e.g.,
[143][144][145]). These yeasts can divert carbon or sugar metabolism into other pathways, thus avoiding ethanol production during fermentation
[143][144][145][146][147]. Several studies have shown significantly reduced wine ethanol levels when using these yeasts. For instance, Magyar and Toth
[148] identified
Saccharomyces uvarum,
Candida stellata, and
C. zemplinina strains with exciting properties. These strains produced, in laboratory fermentations, similar residual concentrations of sugars but with considerable chances in alcohol production, namely for
C. zemplinina, with approximately half the alcohol content that was recorded for
S. cerevisiae.
Another exciting work was performed by Gobbi et al.
[149], using
Zygosaccharomyces bailii,
Z. sapae,
Z. bisporus,
C. zemplinina,
C. stellata,
Hanseniaspora uvarum,
Saccharomycodes ludwigii,
Dekkera bruxellensis, and
S. cerevisiae, for fermentation tests with grape juices. Results showed significantly low ethanol production in
Z. bailii,
Z. sapae,
Z. bisporus, and
C. zemplinina, but more prominently when using
H. uvarum, confirming data observed by other authors
[150][151]. Low ethanol production has also been reported for strains of
Metschnikowia pulcherrima,
Schizosaccharomyces malidevorans and
C. stellata [146],
Torulaspora delbrueckii [150],
Pichia kudriavzevii and
Z. bailii [152].
Schizosaccharomyces pombe reduced 0.65% of ethanol in the fermentation of white Airén grapes
[153], and some
Saccharomyces species can also provide low ethanol-producing strains. A reduction of 0.7% was achieved with the use of
S. uvarum [154], with
S. kudriavzevii also presenting interesting results
[155]. However, some adverse effects have also been linked to the use of these alternative strains if partial aeration strategies during fermentation are applied to allow the use of sugar to be consumed via respiration rather than alcoholic fermentation, namely the formation of undesirable volatile compounds, including acetic acid
[152][156][157], even though positive effects on sensory characteristics also occur
[32].
Additionally, non-
Saccharomyces yeasts play a multifaceted role in wine fermentation, potentially enhancing its sensory profile and aromas and contributing to wine stability and complexity
[143][144][145][146][147][149][157][158][159][160][161][162]. Some non-
Saccharomyces possess antimicrobial properties, including the production of Killer factors (mycocins), which inhibit the growth of undesirable yeasts (
Zygosaccharomyces genus,
Brettanomyces bruxellensis, among others), providing an additional advantage
[163]. These “Killer factors”, such as CpKT1 and CpKT2 produced by
Candida pyralidae, have demonstrated effectiveness in controlling the population of undesired yeast strains, such as
B. bruxellensis, in winemaking conditions, without adversely affecting the fermentation processes of
S. cerevisiae or the tested lactic acid bacteria
[164]. Other “Killer factors”, such as KTCf20 and Pikt (produced by
Wickerhamomyces anomalus), Kwkt (produced by
Kluyveromyces wickerhamii), PMKT and PMKT2 (produced by
Pichia membranifaciens), have also demonstrated potential in controlling unwanted yeast strains in vinification environments
[165][166][167][168].
2.2.3. Co-Inoculations and Sequential Inoculations (Non-Saccharomyces and S. cerevisiae)
Considering some of the previously referred advantages and drawbacks of using different yeasts, two approaches to reducing the ethanol content in wine are co-inoculation of those yeasts or their sequential introduction in fermentation. The first approach (co-inoculation) involves concurrent inoculations of non-
Saccharomyces or other
Saccharomyces non-
cerevisiae yeasts at high cell concentration with
S. cerevisiae, and the second approach (sequential inoculation) consists of the start of fermentation with non-
Saccharomyces or other
Saccharomyces non-
cerevisiae yeasts, occurring for a given duration and inoculating
S. cerevisiae to take over and complete the fermentation
[169].
The critical factors affecting fermentation and oenological outcomes of this approach are the time leading to the inoculation of
S. cerevisiae (in sequential fermentations) and the ratio of
S. cerevisiae and other yeast
[160]. Besides ethanol changes, non-
Saccharomyces or other
Saccharomyces non-
cerevisiae yeasts are essential due to their contribution to wine aroma and flavor, with several yeasts already described as contributors to that profile. Padilla et al.
[160] and Ivit et al.
[144] point out several yeasts as having great oenological interest and used in co- or sequential inoculations, which will be briefly reviewed here.
One of the most important genera is
Hanseniaspora, which comprises at least ten species,
H. uvarum and
H. guilliermondii being the most common. Several
Hanseniaspora species have been tested in sequential or co-inoculated fermentation with
S. cerevisiae, with a recorded reduction in the ethanol content of wines. Reductions of around 1% were achieved using sequential inoculation of
H. uvarum and
S. cerevisiae in synthetic grape juice
[158], and, in white (Sauvignon blanc) and red (Pinotage) musts, reductions in ethanol were also achieved of around 1.3% and 0.8%, for white and red musts, respectively
[170]. Furthermore, three strains of
H. uvarum, in sequential or co-inoculated fermentations with
S. cerevisiae, resulted in lower ethanol concentration when compared to fermentations with the latter only
[171]. Also, in synthetic grape juice, a reduction in alcohol was recorded with sequential inoculation of
H. osmophyla and
S. cerevisiae [158] and white (Sauvignon blanc) and red (Pinotage) musts; the use of
H. opuntiae also resulted in less production of ethanol
[170]. However, some studies point out the increase in acetic acid when fermentations are performed using
Hanseniaspora yeast strains
[171].
Another important yeast already known to have essential winemaking traits
is Schizosaccharomyces pombe. Besides being able to moderate wine acidity by metabolizing malic acid, this strain enhances the color of red wine and reduces Ochratoxin A, biogenic amines, and ethyl carbamate
[145]. In addition, some
S. pombe strains used in sequential fermentation resulted in lower ethanol content
[153], even though a lack of reduction or even increase in alcohol has been reported when using
S. pombe, or even the presence of unsuitable aroma produced by the fermentative metabolism of
S. pombe [172].
An alternative non-
Saccharomyces yeast of great importance is
Metschnikowia pulcherrima. This non-
Saccharomyces yeast is commercially available from many suppliers and is known to improve several organoleptic characteristics of wines
[173]. Furthermore, the production of wines with lower ethanol in sequential fermentations with
S. cerevisiae has been reported in several works, either with grape juice
[174], synthetic grape must
[175], Chardonnay and Shiraz musts
[146], and in white grape must (mixture of Malvasia and Viura varieties)
[156]. A reduction of up to 1.6% in ethanol was recorded in Shiraz wines
[146], with a drop of alcohol further confirmed in later works
[176]. The use of
M. pulcherrima and
S. uvarum mixed inoculum, sequentially used with
S. cerevisiae, reduced 1.7%
v/
v of ethanol compared to wine fermented with
S. cerevisiae [176]. When immobilized, the sequential inoculation of
M. pulcherrima could also reduce ethanol content in synthetic or natural grape juice
[158]. However, results are linked to several conditions, namely aeration regimes, that must be carefully monitored
[156][177].
Lachancea thermotolerans (previously
Kluyveromyces thermotolerans) is a commercially available yeast that positively influences wine’s sensory profile and total acidity
[161]. Besides organoleptic advantages, reduction in ethanol content has been achieved in sequential or co-inoculation. A fermentation started with
L. thermotolerans and a sequential inoculation, after two days, with
S. cerevisiae, led to a reduction in ethanol up to 0.7%
v/
v [159][178][179]. Mixed or sequential fermentation with
L. thermotolerans and
S. cerevisiae also reduced alcohol production
[180]. Further works prove that sequential fermentations with
L. thermotolerans and
S. cerevisiae can reduce the ethanol content in must of Tempranillo grapes. Mixed fermentations of
S. pombe and
L. thermotolerans can lower the ethanol content in wine but may also increase the acetaldehyde content
[143]. The sensory threshold for acetaldehyde ranges from 100–125 mg/L. Typically, table wines have acetaldehyde levels below 75 mg/L immediately after fermentation. However, if the levels exceed 125 mg/L, it can result in unpleasant odors such as ‘over-ripe bruised apples’, ‘stuck ferment’ character, or ‘sherry’ and ‘nut-like’ characters.
Torulaspora delbrueckii was one of the first commercially accessible non-
Saccharomyces yeasts, as they had similar fermentation patterns as
S. cerevisiae and were able to enhance aroma composition and positively impacting properties for traditional methods of sparkling wine
[181]. Several studies have proven that its use in mixed or sequential fermentation r
esulted
in reduced ethanol. Most of these studies refer to reductions of 0.5% or below
[177][182][183][184][185][186]. Higher reductions of ethanol were recorded in other works, like 1% less alcohol using Chardonnay
[162] or less than 1.5% using chemically defined grape juice
[176]. However, to achieve higher levels of alcohol reduction,
T. delbru
eckiising must beT. delbrueckii used in regular fermentation
ss must be combined with high aeration processes
[187].
Another yeast commonly studied due to positive contributions during fermentations is
Starmerella bombicola (formerly known as
Candida stellata). Early works by Soden et al.
[188] with mixed and sequential fermentations with
C. stellata and
S. cerevisiae resulted in less alcohol than the mono-inoculated
S. cerevisiae control. Further works with sequential fermentations using
Starmerella bombicola and
S. cerevisiae in Chardonnay juice yielded lower ethanol concentrations when compared to
S. cerevisiae fermentations
[188]. Immobilizing
Starmerella bombicola is a practical approach to reducing the final ethanol content in grape must. Studies have shown that using this yeast species in the Trebbiano Toscano grape-must and the Verdicchio grape-must significantly reduce the final ethanol content
[158][189].