Technological Prospects of Saccharomyces eubayanus: Breakthroughs and Brewing Industry Applications

Abstract

This review explores the accumulated research and technological potential of Saccharomyces eubayanus, a cold-tolerant wild yeast first isolated in 1997 from the Andean-Patagonian forests of Argentina but formally described in 2011. S. eubayanus has garnered attention since it was identified as the missing parent of the lager-beer yeast S. pastorianus and because it demonstrated valuable fermentative skills and an unexpected large intraspecific genetic diversity. The article recapitulates the characterization of the fermentative capacity of the type strain, as well as its ability to produce distinctive aromatic profiles compared to conventional lager yeasts. We discuss how these features have driven the development of improved strains through experimental evolution and the generation of interspecific hybrids with S. cerevisiae exhibiting appropriate fermentation performance and a broad aromatic diversity. We also aim to address the applications of S. eubayanus in commercial brewing, especially in the craft beer industry, and highlight its potential to add value and/or regional identity to beer through novel flavor profiles. Finally, the review outlines the main challenges limiting large-scale implementation, emphasizing the importance of continued research into strain development and brewing strategies to fully harness the potential of this wild yeast species.

1. Introduction

Beer fermentation constitutes a millennia-long tradition that has endured throughout history, whose precise origins remain uncertain. Archaeological evidence suggests that early forms of brewing may have emerged thousands of years ago in civilizations distributed across different regions, including the Sumerian civilization in Mesopotamia (c. 4000–3100 BCE) [1] and the Natufian culture in the Levant (c. 12,000–9500 BCE) [2]. Today, the beer industry is a dynamic and rapidly evolving market, with a wide range of styles and flavors catering to a broad range of consumer preferences. Moreover, beer is the most popular fermented beverage worldwide, with an annual production of 188 billion liters in 2021 [3] and a global market size valued at approximately USD 850 billion, expected to exceed one trillion USD by 2030 [4].

Traditionally, two main yeast species have been used for beer brewing: Saccharomyces cerevisiae, associated with the production of ale beer and known as top-fermenting yeast due to its tendency to rise to the surface during fermentation; and Saccharomyces pastorianus, a bottom-fermenting yeast characterized by settling at the bottom of the tank, commonly used in lager beer production. Ale fermentation occurs between 18 °C and 25 °C, while lager beer production is developed at cooler temperatures, between 8 °C and 14 °C.

S. pastorianus is a naturally occurring hybrid between S. cerevisiae and Saccharomyces eubayanus [5] with an extensive chromosome aneuploidy, which has probably constituted a decisive factor shaping its adaptability and fermentation efficiency, thus gaining popularity as a beer fermenting organism [6]. Two groups of strains are identified within S. pastorianus based on genome organization: Group 1 (or Saaz) strains, which are approximately triploid (with haploid S. cerevisiae and diploid S. eubayanus chromosomal content); and Group 2 (or Frohberg) strains, approximately tetraploid (diploid to tetraploid S. cerevisiae and diploid S. eubayanus chromosome sets) [7,8].

S. pastorianus has been selected over centuries for its outstanding fermentation skills and reliable performance under brewing conditions, combining the fermentative capabilities and sugar utilization of the S. cerevisiae parent with the ability to ferment at low temperatures of the S. eubayanus parental strain. Human-driven selection has prioritized brewing-relevant traits in lager yeasts—such as efficient sugar utilization, cold tolerance, and the production of desirable aroma compounds—with this highly specialized niche promoting the selection of phenotypes that would be disadvantageous in natural settings. Indeed, the enhancement of sulfite production (an important antioxidant and flavor-stabilizing metabolite) and optimization in sugar utilization pathways were reported in distinct brewing-related hybrids [9]. Similarly, the severe selective pressures of the brewing environment with successive cycles of selection and re-pitching have profoundly shaped the S. eubayanus subgenome of lager-brewing yeasts, which exhibits a high degree of nonsynonymous substitutions in comparison to its wild counterpart, likely due to limited action exerted by purifying selection on this subgenome [10].

Although this domestication process has shaped the characteristic clean flavor profile and balanced aroma of lager beers, making lager-brewing the most popular malt fermentation technique for alcoholic beverage production [11], it has also led to a significant reduction in the genetic diversity within this yeast species. The comparatively higher number of commercial ale yeast strains, which result in beers with broader ranges of aromas and flavors [12], prompted intense research into alternative yeast species to introduce new flavors and enhance the complexity of lager beers. In this scenario, S. eubayanus, the cold-tolerant parent of the lager yeast, emerges as a promising candidate for diversifying lager-brewing since it can offer valuable phenotypic traits, currently absent in commercial strains.

S. eubayanus was described in 2011 and was found to share 99.6% genetic similarity with the non-S. cerevisiae subgenome of lager hybrid yeast, being the first Argentine yeast species to have its genome fully sequenced [5]. Recent advances in understanding its genetic and fermentative characteristics have established S. eubayanus as a key target for applied biotechnological research, particularly in the brewing industry. This review explores the technological characteristics of S. eubayanus, its potential to diversify beer flavors through novel strain development, and the latest advances and applications in the industry. By leveraging the exotic fermentative profile of beers brewed with S. eubayanus, it is possible to create innovative beers with distinctive aroma profiles, thus expanding the sensory attributes of beer brewing.

2. From Isolation to Novel Beer Strain Development

Following the identification of S. eubayanus as the missing progenitor of lager yeast, the increasing demand for novel products and innovative flavor profiles in the beer market has driven the exploration of its brewing potential. Figure 1 presents a historical timeline tracing the pivotal events from its description as a new species [5] to the present, highlighting the key scientific achievements that prompted both subsequent research and brewing industry applications. In the course of this work, we will revisit these events and expand the discussion regarding each innovation context.

2.1. Population Structure and Geographic Distribution of S. eubayanus

Since the discovery of S. eubayanus in Argentine Patagonia, efforts aimed at understanding its geographical distribution and ecological niche have increased exponentially. Initially, this species was found to be strongly associated with trees of the genus Nothofagus, particularly N. pumilio and N. antarctica, as well as with fungi from the genus Cyttaria. Subsequent broader sampling efforts, both across the Andean Patagonian region (in Argentina and Chile) and in other parts of the world, have led to the identification of this species in China, New Zealand, the United States, Canada, and Ireland [13,14,15,16,17,18,30,31].

These studies have also contributed to a better understanding of the species’ ecological niche. While the association with Nothofagus remains the most prominent—with around 50% of positive isolations obtained from N. pumilio and N. antarctica—S. eubayanus has also been isolated from other native Patagonian trees such as Araucaria araucana, as well as from exotic species such as oaks (Quercus spp.). Notably, the Saccharomyces genus is known to be closely associated with oak trees in Europe, which suggests that similar ecological interactions may occur in the Southern Hemisphere. With the increasing number of isolates and the application of molecular tools, such as Multilocus Sequence Analysis (MLSA), population genetic studies of S. eubayanus began to emerge. By 2014, two major populations had been identified: PA and PB. The PA population was restricted to Argentine Patagonia, whereas PB was found to have a broader, global distribution [19].

Further analyses based on whole-genome sequencing, combined with the inclusion of newly isolated Holarctic strains, allowed for a more detailed understanding of the species’ biogeography. Currently, two major populations are still recognized. The PA population includes two subpopulations, PA-1 and PA-2, both restricted to the northern region of Argentine Patagonia. The PB population, which displays the highest genetic diversity, consists of three subpopulations—PB-1, PB-2, and PB-3—distributed throughout Patagonia in Argentina and Chile. More recently, a fourth subpopulation was described, SoH, which is genetically closest to the Holarctic lineage, with isolates from China, the United States, and Ireland [16,17]. This Holarctic population shows the highest genomic similarity to the S. eubayanus subgenome of the lager-brewing hybrid S. pastorianus. Lastly, a distinct subpopulation, NoAm, has been identified exclusively in North America and exhibits an admixed genetic background derived from both the PA and PB populations [16].

To date, nearly 300 isolates of S. eubayanus have been reported worldwide (Figure 2). Due to its role as the wild parent of the lager hybrid that revolutionized brewing practices in Europe from the 16th century onward, extensive efforts have been made to isolate this species from natural environments within Europe. However, these attempts have so far been unsuccessful. Only two isolates of S. eubayanus have been reported in Ireland [18], and none have been found in continental Europe. Interestingly, another hybrid species associated with anthropogenic fermentations, such as wine and cider, and spoiled beer, S. bayanus, is present in Europe [32,33]. S. bayanus is a natural hybrid with parental contributions from both S. uvarum and S. eubayanus, with limited introgressions from S. cerevisiae. These introgressions are predominantly subtelomeric and often involve genes related to sugar metabolism [33,34]. Genomic analyses of S. pastorianus and S. bayanus strains have revealed that their S. eubayanus subgenomes likely originated from the same subpopulation [12]. A closer examination of S. bayanus isolates worldwide could, therefore, provide deeper insights into this phenomenon. Studying the origin of these hybrids is crucial not only to understand the fermentative potential of S. eubayanus in diverse environments but also to explore how different human cultures have historically shaped yeast species to such an extent.

2.2. Genetic and Phenotypic Insights into S. eubayanus Brewing Potential

S. eubayanus is a diploid species, characterized by a very low level of heterozygosity and a genome highly syntenic with that of S. cerevisiae, except for a few small inversions and two reciprocal translocations between chromosomes VIII and XV and chromosomes II and IV [13,20]. Early studies on its fermentative properties demonstrated that the S. eubayanus type strain (CBS 12357 ^T; see a list of the identification codes for S. eubayanus-type strain in different yeast culture collections in Table S1) exhibits a superior performance compared with most lager strains when cultured at 10 °C in synthetic media with 2% glucose or maltose [21]. Although this was not translated into good fermentation performance in beer wort at the same temperature, it achieved an efficiency equal to that of Saaz-group lager strains. Subsequent studies performed in brewing wort across different temperatures further confirmed S. eubayanus’s reduced fermentation ability compared to lager strains [20,22,23].

The weaker performance in beer wort of wild S. eubayanus strains compared to domesticated commercial strains is linked with their limited ability to utilize the most abundant sugars in wort, maltose, and maltotriose [15]. The sub-optimal utilization of maltose has been attributed to deficiencies in the transcriptional regulation of maltose metabolism genes. Such deficiencies were reported in two wild S. eubayanus strains (CDFM21L.1 and ABFM5L.1, Himalayan Holarctic clade) that were unable to consume maltose or maltotriose on diluted wort, despite the presence of functional transporters. The introduction of the S. cerevisiae regulatory gene ScMAL13 restored growth on both sugars, confirming regulatory deficiencies as the underlying cause [24]. Additionally, variation in glucose-induced inhibition of maltose utilization has been observed, through a rewiring mechanism by which cells switch to using a new carbon source after the preferred one is depleted (diauxic shift) [35]. In this study, Molinet et al. described that variations in diauxic shift among different S. eubayanus strains are influenced by differences in transcriptional activity, chromatin accessibility, glucose transporter expression, and transcription factor regulation. Under industrial conditions, this behavior was exacerbated, leading to elevated residual maltose in the final beer [21,36].

In the S. eubayanus-type strain, maltose consumption genes are organized within MAL loci located in subtelomeric regions, consisting of clusters of genes encoding a maltose transporter (MALT), an α-glucosidase enzyme (MALS), and a transcriptional regulator (MALR), as well as other fermentation-related genes [10]. Four open reading frames were identified in the S. eubayanus CBS 12357 ^T strain through the genetic analysis of maltose transport mechanisms (SeMALT1-4), with high sequence similarity to the maltose transporter MAL31 in S. cerevisiae. SeMALT2 and SeMALT4 appear to play primary roles, while SeMALT1 and SeMALT3 show reduced expression or possible loss of function [37].

While S. eubayanus maltose utilization is suboptimal under lager brewing conditions, maltotriose assimilation is entirely absent; all four transporters described are maltose-specific, and no wild S. eubayanus strain has been reported to metabolize maltotriose [18,24,37]. In S. cerevisiae, maltotriose uptake involves the broad-specificity α-glucoside transporter Agt1 (or Mal11). Unlike the ubiquitous Mal31-like family of transporters, Agt1 permease is less common in Saccharomyces species and displays a broad substrate recognition but with a comparatively lower affinity [38,39]. In lager-brewing yeasts, maltotriose utilization is believed to be a heterotic phenotype resulting from the interaction between the parental subgenomes. The permease function is mediated by SeAGT1 and SpMTT1 and regulatory genes provided by the S. cerevisiae parent, which highlights the crucial role of regulation in sugar metabolism [24,40]. Interestingly, in the S. pastorianus CBS 1483 Frohberg strain, no S. cerevisiae-derived maltotriose transporter genes have been identified, and maltotriose transport relies exclusively on the presence of the S. eubayanus-derived SeAGT1 [41].

The lack of maltotriose assimilation of wild S. eubayanus strains results in reduced attenuation levels and ethanol production in beer wort fermentations when compared to most lager strains [21,23,36,42]. However, this does not preclude its potential for beer brewing, as some widely used commercial brewing strains also lack this metabolic capacity; indeed, although rare among wild Saccharomyces yeasts, maltotriose consumption has been acquired through domestication-driven selection in numerous brewing strains [43].

In addition to efficient sugar utilization, yeast flocculation potential is particularly valuable in the brewing industry for ensuring efficient and low-cost beer clarification and enhancing fermentation performance. Flocculation is a calcium-dependent, reversible, and asexual mechanism whereby genetically similar cells aggregate into clumps and settle out of suspension, following the completion of sugar consumption. This adhesion phenomenon is a lectin-mediated mechanism that involves the interaction between flocculins (lectin-like proteins) on one yeast cell surface and mannose residues in the cell wall of adjacent cells. This process is strongly strain-dependent and, in S. cerevisiae, is described to be primarily governed by the FLO gene family, with a high rate of variability due to its sub-telomeric localization [44]. FLO genes are grouped into two main categories, according to their functionality: FLO1, FLO5, FLO9, and FLO10, encoding flocculins responsible for cell-to-cell adhesion; and FLO11, FIG2, and AGA1, involved in filamentation, invasive growth, cell-to-matrix adhesion, and mating [45]. For its part, Lg-FLO1 is a specific gene of S. pastorianus, mainly responsible for the flocculent phenotype of lager yeast [46].

Certain brewing yeast strains, including those employed in the production of German wheat beers, have been reported to lack flocculation capacity [47]. Similarly, and consistent with their wild origin, S. eubayanus strains also appear to lack this feature, although several FLO genes (FLO1, FLO5, FLO9, FLO10, and FLO11) were identified in the CBS 12357 ^T strain. These showed an organization similar to that in the non-flocculent S. cerevisiae S288c, in classical tandem repeat structures, but shorter than the corresponding genes found in the flocculent S. pastorianus strain CBS 1483 [41].

With regard to growth temperatures, Saccharomyces species exhibit a wide range and are broadly divided between cryotolerant and thermotolerant species, with mitochondrial DNA (mtDNA) playing a key role in shaping adaptation and evolution. In fact, the high structural and sequence similarity between mitochondrial genomes of S. eubayanus and S. pastorianus suggests that the mtDNA of lager yeast strains was provided by the S. eubayanus parent [10,48]. This likely exerted a fundamental role in shaping lager yeast adaptation to cold brewing conditions. The influence of mtDNA in providing competitive advantage under cooler brewing conditions has also been shown in synthetic hybrids of S. cerevisiae and S. uvarum—the sister species of S. eubayanus—in which the mitochondrial S. uvarum COX1 allele was demonstrated to confer both heat sensitivity and growth advantage at low temperatures [49]. Similarly, de novo hybrids carrying S. eubayanus mitochondria evidenced an enhanced growth rate at low temperatures compared to hybrids with S. cerevisiae mitotype, in synthetic medium with glucose and glycerol [50].

However, although increasing evidence indicates that cold-tolerance-related genes are primarily of mitochondrial origin, nuclear genomes have also shown to be crucial. Dominant cytonuclear incompatibilities between S. cerevisiae and both S. eubayanus and S. uvarum have been reported [49,50], and the evidence collected to date indicates that mtDNA effects result from complex genetic interactions with the nuclear genome, with a strong influence of mitochondrial genome on nuclear transcription, particularly in challenging environmental conditions [51]. Moreover, a recent study analyzing the impact on fitness and the gene expression of specific genes in eight Saccharomyces species indicates that S. eubayanus’s cold temperature tolerance would have evolved through mechanisms involving a nuclear gene—FAA1, critical for long-chain fatty acid metabolism [52].

In terms of yeast aromatic attributes, although ethanol, carbon dioxide and glycerol are the primary compounds produced by brewing yeasts during wort fermentation, they have a limited impact on the organoleptic profile of beer. Instead, the overall balance of beer flavor is largely determined by the concentration of various secondary metabolites derived from alcoholic fermentation. These molecules include carbonyl compounds, higher alcohols (fusel alcohols), esters, vicinal diketones, fatty acids, organic acids, and sulfur compounds [53,54]. Regarding secondary metabolism and the potential impact of S. eubayanus on the sensory profile of beer, Figure 3 provides an overview of the fermentation characteristics of the type strain, including maltose consumption, ethanol production, and the synthesis of esters and higher alcohols, based on data from various laboratory-scale studies (volumes ranging from 0.5 to 7 L). Ethanol levels for S. eubayanus were found around 3.8% v/v for standard-gravity worts (12 °P), values consistent with maltose consumption greater than 70% and no maltotriose fermentation; moreover, it turns out to be a strain that can withstand high-gravity worts (>15 °P), reaching average ethanol values of 6% v/v (except for Mardones et al. [55], but the fermentation time was shorter). Isobutanol and propanol were detected in all the reviewed studies that measured it, but below the perception threshold, which is positive since their presence at high concentrations is associated with an unpleasant solvent aroma. When determined, it was observed that S. eubayanus was able to produce the rest of the esters and higher alcohols analyzed (except ethyl octanoate) in all studies, and in 60% of them (on average), the production levels exceeded the sensory perception threshold for 2-phenylethyl acetate, 2-phenylethanol, amyl/isoamyl alcohol, and isoamyl acetate. These compounds positively influence the beer’s aroma and flavor, imparting notes of roses, honey, sweetness, fruitiness, banana, and perfume [54].

In addition, yeast can convert plant-derived precursors without odor into flavor-active compounds with a significant impact on the organoleptic attributes of the final beer. Among these are volatile phenols, a series of plant-derived decarboxylated hydroxycinnamic acids that impart spicy, medicinal, smoky, clove-like, and vanilla-like flavors to beer, referred to as ‘phenolic off-flavors’ (POFs). The flavor-active POFs most commonly detected in beer are 4-vinylguaiacol (4-VG), and 4-vinylphenol (4-VP) [59], and their production depends on the presence of functional copies of two subtelomeric genes: phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC1) [60]. In brewing, POF presence is considered a major drawback associated with contamination issues, although it is part of the essence of some beer styles, such as saison and hefeweizen. It was reported that 4-VG is a shared feature of the type strains of wild Saccharomyces species, including S. eubayanus [56], and perhaps the most characteristic aroma and flavor associated with beers brewed with this wild yeast is the clove and/or smoky flavor derived mainly from 4-VG and 4-VP. Indeed, all S. eubayanus strains characterized to date carry functional copies of these genes and have been reported to produce levels of 4-VG and 4-VP over the flavor perception threshold [36,61,62].

The fermentative potential of different S. eubayanus strains—other than the type strain—has also been evaluated. Eizaguirre et al. [15] performed micro-fermentations (50 mL) of 60 isolates from Argentine Patagonia using malt extract medium and found a differential behavior (in terms of attenuation and CO₂ production) between strains. In general, the fermentative performance of strains within PB population was improved compared to isolates from PA population; however, in terms of fermentation rate, the highest values corresponded to strains from PB-1 and PA-2. In further studies, Burini et al. [36] evaluated a candidate from each of these five geographically structured subpopulations in small-scale fermentations (150 mL) using all-malt wort. They found that the strain belonging to PB-3 subpopulation (the southernmost isolate, from Tierra del Fuego) showed the lowest fermentation parameters, with low maltose consumption and low ethanol production levels. Regarding flavor, differences were also found among subpopulations: The PA-1 and PB-1 strains produced the highest levels (Odour Activity Value ‘OAV’ > 1) of the fruity esters ethyl hexanoate and ethyl octanoate and of 2-phenylethanol (rose-like notes), the PB-3 and PB-2 strains exhibited the highest production of volatile phenols (4-VG and 4-VP) and a low contribution of fruity/floral aromas (OAV of esters and higher alcohols < 1), and the PA-2 strain was associated with high production of hydrogen sulfide (described as rotten eggs) with a negative impact on flavor.

Furthermore, isolates from Chile were analyzed in different studies that performed laboratory scale fermentations (50 mL–150 mL) in beer malt extract [17,25,61,62] and all-malt wort [55]. Nespolo et al. [17], Urbina et al. [61], and Vega-Macaya et al. [25] also observed fermentation differences (in terms of CO₂ production) when evaluating different strains of the species; some strains exhibited fermentative parameters comparable to those of the lager strain, while others, isolated further south, showed lower fermentation performance. Differences were also evident in terms of volatile compound production among different Chilean strains of S. eubayanus: Mardones et al. [55] found that some strains produce high levels of banana-like desirable acetate ester (3-methylbutyl acetate), while others revealed higher ethyl ester production (ethyl hexanoate and ethyl decanoate). Urbina and co-workers also demonstrated strain diversity by evaluating 12 aroma compounds and classifying S. eubayanus wild strains into four groups based on their differential aroma compound profiles; however, no associations could be made between these groupings and latitudinal, geographical, or genetic origin. It is noteworthy that all strains showed the production of volatile phenols (4-VG and/or 4-VP) when measured [36,61,62].

The high variability in fermentation performance and volatile compound production reported among S. eubayanus strains highlights its intraspecific phenotypic diversity, demonstrating the species potential for generating differential products, as well as the possibility for developing novel evolved strains and/or interspecific hybrids.

3. Development of Improved Strains Based on S. eubayanus

The strategies for obtaining improved S. eubayanus strains or novel S. eubayanus × S. cerevisiae hybrids with desirable traits for beer production described in this work are focused exclusively on approaches with general acceptance by consumers. This means that although some studies use genetic engineering techniques for this purpose, such approaches lack broad acceptance among both consumers and the brewing industry [63] and were, thus, not included in the scope of this review.

3.1. Evolved S. eubayanus Strains

Experimental evolution (EE) strategies constitute powerful tools to drive the adaptation of microbial populations to a desired environment and have been thoroughly applied for the obtention of improved beer brewing yeasts [26,27,64,65,66,67,68,69,70,71,72,73,74] as well as for understanding the evolutionary pathways involved in the adaptation of industrially relevant microorganisms [75,76]. EE techniques are designed to mimic Darwinian evolution in the laboratory, through the application of a constant selective pressure that generates gene diversification, followed by successive rounds of screening and phenotypic selection of fitter individuals. As occurs in natural evolution, these strategies rely on both the existing genetic diversity of the population and the spontaneous mutation rate of the organism, although mutation and recombination rates tend to be increased in these experimental conditions, to favor the emergence of the desired biological function in a limited number of generations [77,78].

In

Table 1. Evolved S. eubayanus strains obtained through experimental evolution strategies.

Publication	Original Strain	Developed Strain	Strategy	Improved Features
Eizaguirre et al., 2019 [42]	S. eubayanus CBS 12357 T	A62	EE on congress wort through 430 generations (62 passages)	Improved beer wort fermentation: threefold increase in fermentation rate with respect to the original strain (10 °C, 12 °P, 1.2 × 107 cell/mL) Improved sugar utilization: A62 consumed twice the amount of maltose as the original strain in the first 90 h of fermentation Sexual reproduction: loss of sporulation capacity Aroma profile: distinctive flavor and enhanced mouthfeel for the beer produced by A62, in comparison to CBS 12357 T
Brouwers et al., 2019 [26]	S. eubayanus CBS 12357 T	IMS0750	UV-mutagenesis and EE in chemostat culture on modified brewer’s wort for 125 generations (120 days)	Emergence of a functional maltotriose transporter named “SeMalt413” by recombination Maltotriose assimilation: 75% maltotriose utilization under fermentation conditions (16.6 °P, 15 °C, 5.106 cells/mL, 7 L). CBS 12357 T did not utilize any maltotriose. Improved maltose utilization: complete consumption within 200 h (330 h for CBS 12357 T) Aroma profile: 240% higher isoamylacetate concentration in beer brewed with IM0750 in comparison to CBS 12357 T. Similar final amounts of higher alcohols and other esters
Baker & Hittinger, 2019 [27]	S. eubayanus yHKS210 (admixed strain from Wisconsin, USA)	yHEB1505 and yHEB1506	EE on maltotriose synthetic medium through 2100 generations (100 passages) Selection of two single colony isolates	Evolution of a functional maltotriose transporter encoded by the chimeric “MALT434” gene Maltotriose assimilation: maltotriose utilization as a sole carbon source in growth assays performed in 96 well plates with synthetic medium + 2% maltotriose
Mardones et al., 2022 [80]	30 S. eubayanus strains from lineages PB-2 and PB-3	CL248.1 (evolved strain)	EE in YNB media with 2% glucose and 9% ethanol through 260 generations (~180 days)	Improved beer wort fermentation: 22.6% increase in CO2 loss for CLEt5.1 in beer wort fermentation compared to the original strain (12 °C, 12 °P, 1.8 × 107 cell/mL, 50 mL)
		CLEt5.1 (single colony isolate from CL248.1)		Maltotriose assimilation: 19.1% assimilation for CLEt5.1 in YNB + 2% maltotriose, although was unable to utilize it in beer wort. CL248.1 could not metabolize maltotriose at all
Vega-Macaya et al., 2025 [25]	9 S. eubayanus strains from Chilean Patagonia	CLAET815.1 (best evolved individual derived from CL815 evolved strain) ACS15, ACS19 and ACS25 (intra-specific hybrids)	EE in YNB media with 2% glucose and 9% ethanol through 250 generations Hybridization between CLAET815.1 and CLEtOH5.1 (a reported high fermentative strain [80]) through spore-to-spore mating	Improved beer wort fermentation: significant fitness improvement for CL815.1 evolved strain under lager conditions (12 °C, 12 °P, 1.8 × 107 cell/mL, 50 mL) versus original strain
				S. eubayanus intra-specific hybrids: hybrid vigor in lager type fermentations only after a single round of sexual reproduction. Significantly higher levels of CO2 produced by interspecies segregants compared to parental strains No maltotriose assimilation detected

, we have listed several EE attempts applied to wild S. eubayanus strains that, either alone or in combination with traditional random mutagenesis methods, are focused on overcoming one of the major challenges for industrial use: its inability to metabolize maltotriose [25,26,27,42]. Remarkably, two of these experiments—that were performed independently in different genetic backgrounds—led to the occurrence of chimeric, neo-functionalized genes encoding AGT1-like maltotriose transporters. These transporters enabled the evolved strains to utilize maltotriose in a synthetic medium with 2% maltotriose [26,27] and in high-gravity wort under pilot-scale (7 L) brewing conditions [26]. Both studies indicate that the novel function arose through recombination events between SeMALT genes that previously lacked maltotriose transport capacity. Recent research aimed at understanding the molecular basis underlying the acquisition of novel function in one of these chimeric proteins, Malt434 (derived from Baker and Hittinger research), suggests that maltotriose transport evolution requires rare, recurrent gene conversion events, which would constitute the only remotely plausible route for assembling all the critical residues into a single functional molecule [79]. The authors further argue that the evolution of new substrate specificity within this transporter family is a highly context-dependent and mechanistically complex process, difficult to predict or direct. Moreover, based on an extensive phylogenetic reconstruction of α-glucoside transporter evolution across 332 Saccharomycotina genomes, they suggest that specific MAL31-like maltose transporters have evolved from an ancestral generalist transporter and, thus, interpret the ability of Malt434 to transport maltotriose as a reemergence of an ancestral capability instead of a function innovation.

These findings emphasize the importance of genetic context in shaping evolutionary outcomes, which has also proven critical in other EE studies using S. eubayanus. For example, in a research study in which a pooled population of 30 different S. eubayanus strains were continuously exposed to high ethanol concentrations, a single strain consistently outcompeted the others under stressful conditions, demonstrating superior fitness [80]. In addition, a recent study that evaluated the fermentative performance in nine S. eubayanus strains, before and after evolution and under three distinct environmental conditions, obtained a single evolved genotype that exhibited the highest fitness gain across all tested media [25]. As expected, this strain was also the one with the worst initial fermentative skills, as lower-fitness genetic backgrounds have been reported to be more likely to benefit from adaptive mutations, whereas epistatic interactions in better-adapted genotypes tend to diminish the effect of such mutations [81]. The study of Vega-Macaya et al. [25] also demonstrated that integrating EE with intra-specific hybridization—by crossing two S. eubayanus evolved strains followed by meiotic segregation—further amplified phenotypic improvements beyond those obtained by evolution alone, in which attributes like aromatic diversification are not easily customized, given the inherent difficulty of selecting for this trait.

Finally, in an attempt to develop an improved S. eubayanus strain with added regional identity for local brewers in Argentine Patagonia, Eizaguirre [42] applied the EE approach to evolve CBS 12357 ^T strain in beer wort, with no additional selective pressure other than brewing conditions. The resulting evolved strain lost its sporulation ability, a trait commonly observed in domesticated yeasts, and exhibited a threefold increase in fermentation rate. Currently, complete genome assemblies and transcriptomic profiles of both the parental and evolved strains are being analyzed to identify genomic alterations or metabolic pathway changes that may underlie the evolved phenotype.

3.2. Laboratory-Made Interspecific Hybrids for Novel Lager Brewing

The unique hybridization and domestication history of lager yeast hybrids, coupled with the industrial standardization of lager beer production in the 19th century in Germany [8], resulted in significantly limited genetic diversity among commercial lager strains in comparison to ale strains. Hence, considering the key role attributed to interspecific hybridization for industrial yeast evolution and domestication, especially in beer brewing yeast [12], numerous attempts to generate de novo lager hybrids between wild S. eubayanus and different S. cerevisiae strains have been developed, which we aimed at compiling in Table 2. In most of these studies, interspecific hybridization was achieved through traditional breeding techniques, which typically allow for the creation of diploid hybrids with a high success rate and genetic stability, avoiding selection markers. These techniques involve mating spore cells derived from parent strains through spore-to-spore mating, by positioning spores next to each other on an agar plate using a micromanipulator; or mass mating, in which spores are mixed on solid or liquid growth media [82]. However, due to the low sporulation efficiency and viability typically exhibited by industrial ale strains—likely due to their aneuploidy—the rare mating breeding strategy was also applied [57,83,84]. The rare mating methodology exploits the spontaneous loss of the heterozygosity phenomenon at the mating type locus—an extremely low-frequency event—that allows yeast cells to mate with complementary sex types generating complex polyploid hybrid genomes [85]. Despite the low hybridization frequency expected when applying this strategy—reported to occur in 1 out of 10 million cells [57]—and the requirement of selection markers, it has been successfully applied, leading to the generation of hybrids with high ploidy levels.

Studies following this approach have shown a positive correlation of ploidy with fermentation performance, the production of flavor-active compounds, and stress tolerance [56,57,86]. Krogerus and co-workers [57] have specifically addressed this issue, generating allodiploid, allotriploid, and allotetraploid hybrids of S. eubayanus and S. cerevisiae, and demonstrated that even though all of them displayed better fermentative skills than parental strains in 15 °P wort, under intensified fermentative conditions—in 25 °P high-gravity wort with 5% ethanol—higher-ploidy hybrids showed a significantly improved fermentative performance than the allodiploid strain. The authors suggest that since higher ploidy may translate into a higher copy number of specific genes, this can also lead to an increased transcript level, thus conferring a series of improved fermentative and aromatic traits to the strains. A similar outcome was obtained for novel lager-type hybrids of S. cerevisiae ale strains and different cold-tolerant Saccharomyces species (including S. arboricola, S. eubayanus, S. mikatae, and S. uvarum). These allotriploid and allotetraploid hybrids also exhibited superior alcohol production in beer wort fermentations compared to their diploid counterparts, which were nonetheless capable of utilizing wort sugars that were not accessible to the non–S. cerevisiae parental species [56].

Although these observations were coherently based on the assumption that the increased genetic diversity of polyploid genomes would enhance the capacity of hybrids to adapt and tolerate stressful environments [87], contrasting evidence was recently published. When Zavaleta and co-workers [88] analyzed fermentative capacity in a set of de novo lager hybrids, they found that the main driving force of fitness was the lineage-specific inheritance of traits rather than the ploidy level. In this study, differences in fermentative capacity were primarily linked to variations in the hybrid parental lineages, with a particular influence of the S. cerevisiae parent. In addition, another study aimed at investigating how ploidy and genetic background affect heterosis in 960 S. cerevisiae hybrid strains under different stress conditions reported little—if any—influence of ploidy level in fitness gain across all tested media [89]. The authors suggest this could be related to deficiencies in osmotic regulation caused by increased cell size in highly polyploid strains, which would impart physiological limitations. Indeed, when analyzing hybrid strains showing best-parent heterosis, although results were variable and highly context-dependent, they report mainly negative correlations between ploidy and heterosis. Interestingly, among strains exhibiting best parent heterosis, domesticated parental strains appeared to be significantly underrepresented in comparison to strains isolated from natural environments, highlighting the role of wild strains in providing favorable genetic backgrounds for specific niche adaptation in hybrids.

In terms of sugar utilization in S. cerevisiae × S. eubayanus laboratory-made hybrids, positive heterosis—or fitness gain—has been extensively documented; indeed, all the studies listed in Table 2 report the obtention of at least one hybrid strain capable of efficient maltose consumption, with variable maltotriose assimilation rates. In most of these studies, improved fermentation rates and higher ethanol levels than those of parental strains were obtained when hybrids were evaluated in beer wort fermentations, highlighting the strong selective advantage of these species hybrids in the brewing environment. The first series of studies was conducted almost concurrently [20,22,23], and all developed hybrids showed faster fermentation rates in broader temperature ranges, more complete sugar utilization, and enhanced aroma profiles when compared to parent strains.

Table 2. List of research studies aimed at developing interspecific hybrids of S. eubayanus (S.eub) and S. cerevisiae (S.cer). For each study, results other than those listed as ‘Improved Features’ for the best performing hybrids were excluded from the table.

Reference	Strain	Parental Strains and Breeding Technique	Improved Features *
Helby et al., 2015 [20]	IMS0408	S.eub CBS 12357 T spores MATa S.cer IMK439 MATα ura3Δ::KanMX ** Mass mating	Ferm	↑ Fermentation rate in synthetic medium with glucose, maltose, and maltotriose (1.2 g/L·day at 20 °C), 10h faster vs. S.cer
			Mtt	Fast maltotriose consumption with low residual concentration (comparable to S.cer parent)
			Temp	Growth from 8 °C to 35 °C in anaerobic sequential batch reactors. ↑ Growth rate vs. best parent strain from 20 °C to 30 °C
Krogerus et al., 2015 [22]	H1-H4	S.eub CBS 12357 T lys− S.cer VTT- A81062 ura− (strongly flocculent)	Ferm	↑ Ethanol production (5.6% v/v vs. parents 4.2% S.cer, 4.5% S.eub) in beer wort fermentations (12 °P, 12 °C)
			Mtt	More efficient utilization of maltose and maltotriose compared to the best-performing parent
			Floc	Between 82–88% (71% S.cer, 15% S.eub)
		Mass mating	Phe	POF positive
			Flav	↓ Higher alcohols compared to the S.eub parent. Distinctive aroma profile: ↑ overall aroma-active esters
			Temp	Growth on YPM at 37 °C, like the S.cer parent. No growth at 40 °C, whereas S.eub grew at neither temperature
Mertens et al.,2015 [23]	31 new lager yeast hybrids	S.eub Y565 and Y567 (high spore viability) S.cer Y134, Y470, Y245 and Y397 (ale beer), Y184 (wine) Y243 (bread) Spore-to-spore mating and genome stabilization in industrial lager beer medium for 70 generations	Ferm	↑ Ethanol production: 28.8% higher vs. best-performing parent (H15: 6.20% v/v) in lager fermentation (12 °P, 16 °C, 150 mL)
			Mtt	↑ Maltose (>98.5%) and maltotriose (>59%) uptake in 7–8 days (50 L pilot scale, 12 °C, 12 °P), comparable to S.cer Y397 and Y134. S.past GSY501 only reached complete fermentation by day 16
			Phe	POF positive
			Flav	↑ Isoamyl acetate vs. S.past reference strains. ↑ Aromatic diversity but ↑ fusel alcohols levels
			Temp	↑ Growth range on YPD agar (4 °C to 37 °C) vs parental strains
Krogerus et al., 2016 [57]	A2 (diploid), B3 (triploid), C4 (tetraploid)	S.eub CBS 12357 T ura− or lys− S.cer VTT-A81062 lys− or ura− (strongly flocculent) Spore-to-spore mating for A2 diploid hybrid Rare mating for B3 triploid and C4 tetraploid hybrids	Ferm	↑ Ethanol production (6.9% v/v on average) vs. parents (6.6% S.cer, 5.7% S.eub) in 15 °P beer wort (15 ºC, 1.5 L). In 25 °P wort only B3 and C4 displayed hybrid vigor
			Mtt	Overall, 70% maltotriose uptake (comparable to S.cer parent)
			Floc	B3 (73.1%), C4 (58.6%) vs. S.cer (94%). A2 (6.2%), comparable to S.eub parent (2.6%)
			Phe	POF positive
			Flav	↑ Aroma esters but ↑ diacetyl vs. parents
			Temp	↑ Growth range on YPM agar (8 °C to 37 °C) vs. parental strains
Nikulin et al., 2017 [56]	H1	S.eub CBS 12357 T S.cer VTT-A81062 (strongly flocculent)	Ferm	↑ Maltose consumption (80% vs. 50% S.cer) and ethanol (6% v/v vs. 3.5% S.eub, 4.5% S.cer) in 12 °P beer wort (12 °C, 1.5 L)
			Mtt	↑ maltotriose uptake (60% vs. 50% S.cer)
		Characterization of H1 hybrid strain developed by Krogerus et al. [22]	Phe	POF positive
			Flav	↑ Aroma-active esters vs. both parent strains and ↓ higher alcohols vs. S.eub parent
Krogerus et al., 2017 [83]	H1: VTT-A1522, H2, H3, T1, T2	S.cer VTT-A81062 ura−, Mtt+, POF+ and WLP099 rho−, Mtt−, POF− S.eub CBS 12357 T lys−, Mtt−, POF+ Rare Mating. H1: A81062 × C12902 H2: WLP099 × C12902 H3: A81062 × WLP099 T1: H1 × WLP099 T2: T1 POF− meiotic segregant	Ferm	↑ Ethanol (6–7.3% v/v) produced by T2, H1, H3 and T1 vs. best S.cer parent (5.5%) in 15 °P beer wort (15 °C, 1.5 L)
			Mtt	T2, H1 and H3 consumed maltotriose efficiently (residual concentrations comparable to the Mtt+ parent A81062)
			Phe	POF negative phenotype for T2; others (H1, H2, H3 and T1) POF positive
			Flav	↓ Esters and ↑ higher alcohols in beer made with T2 vs. T1 beer
Eizaguirre, 2018 [42]	MC3(100gen), MA6(100gen)	S.cer WLP099 and WY1338 (high spore viability) S.eub CBS 12357 T Spore to spore mating followed by experimental evolution	Ferm	↑ Fermentation rates for evolved hybrids in beer wort (12.5 °P, 10 °C, 10 mL) vs. original hybrids. Similar fermentation rates to the reference W34/70 strain, but ↓ compared to the S.eub parent
			Mtt	Not maltotriose uptake
			Phe	POF positive
			Flav	↑ Ester profile vs. S.eub parent
Krogerus et al., 2021 [86]	A225 meiotic seggregants: A226 to A229, A227 meiotic segregants: A232 to A235	S.eub CBS 12357 T (high spore viability) S.cer VTT-A-81062 (Mtt+, POF+, strongly flocculent) Analysis of meiotic segregants derived from the previously obtained hybrid VTT-A15225 (A225) [83]	Ferm	↑ Fermentation rate and efficiency for A225 (15 °P beer wort,15 °C, 1.5 L) with ↑ethanol vs. parents (6.7% v/v vs. 5.7% S.cer, 4.9% S.eub). Variable performance for spore clones derived from A225, associated with ploidy (tetraploids > diploids)
			Mtt	Similar maltotriose uptake to that of the S.cer parent for all hybrids, except A226
			Floc	Strong flocculation for A225 and its spore clones A226 and A228 (similar to S.cer parent). Flocculation is not linked to ploidy
			Flav	↑ 3-methylbutyl acetate and ethyl hexanoate for spore clones A232-A235 vs. the best parent strain for both compounds
			Temp	Broad growth range, from 4 °C to 37 °C, with variability among strains
Turgeon et al., 2021 [84]	RB-1141, RB1186, RB115, RB2251, RB2403 ***	S.past W34/70 trp− meiotic segregants (domesticated S.eub sub-genome and mitochondria) S.cer RB-253, RB-48 and NCYC-1113 diploid meiotic segregants lys− or ura− Rare mating of mating competent meiotic segregants	Ferm	Maltose consumption >98% for all strains with similar ethanol production than the reference S.past W34/70 (average 7.3% v/v vs. 7.2% S.past) in 15 ºP beer wort (13 °C, 30 IBU, 80 mL)
			Mtt	86.4–90.2% maltotriose assimilation, comparable to S.past reference strains
			Floc	Flocculation by day 14 for most hybrids during 3 L wort fermentation (except RB-1186)
			Phe	POF negative
			Flav	Acceptable acetic acid and diacetyl and ↓ off-aromas compared to S.past control strains; profiles suitable for lager beer
			Temp	↑ Growth range on YEG medium (7 °C to 37 °C) vs. parental strains
Molinet et al., 2024 [90]	H3-A, H4-A, H6-A, H8-A	S.cer L270 L348 and L3 S.eub from different PB populations: CL710.1, CL216.1; CL450.1 ****	Ferm	↑ Fermentative capacity for evolved hybrids generated at 12 °C (S.eub mitochondria) in lager beer wort fermentations (12 °P, 12 °C, 50 mL) vs. ancestral lines, and similar to W34/70. ↑ 7.1% v/v ethanol for evolved single cell isolate H4 C1 vs. W34/70
			Mtt	Similar uptake to S.past reference strain W34/70 in best performing hybrids
		Spore to spore mating at 12 ºC or 20 °C followed by experimental evolution	Phe	The single cell isolate analyzed for POF phenotype (H3-4 C1) was POF positive
			Flav	H-4 C1 produced a unique fatty acid ethyl esters profile (herbal/spicy notes vs. W34/70 citrusy)
Zavaleta et al., 2024 [88]	47 de novo hybrids derived from 21 crosses	Seven S.cer strains with high fermentative capacity from different clades (beer, wine, sake and bioethanol) Six S.eub from different lineages Rare mating at 12 °C (to facilitate inheritance of S.eub mitochondria)	Ferm	↑ CO2 production in beer wort fermentations (12 ºP, 12 ºC, 50 mL) for hybrids derived from S.cer Beer lineage, similar to S.cer Beer parents
			Mtt	↑ maltotriose uptake (56.5% on average) for hybrids derived from S.cer Beer lineage vs. other hybrids
			Phe	POF positive
			Flav	Four distinctive aroma clusters were obtained. Hybrids from S.cer Beer lineage: ↑ ethyl acetate and isoamyl alcohol, ↓ off-flavors
			Temp	↑ Growth range (4 °C to 37 °C) with ↑ or similar fitness vs. parents
Murath et al., 2025 [28]	H5 stabilized isolates: H5_HT_L3i and H5_HT_L4i	S.cer BE011 (high sporulation rate and spore viability, desirable aroma compounds) S.eub DR1 and DR3 (Chile) Spore to spore mating followed by different stabilization procedures	Ferm	↑ Ethanol production for stabilized isolates derived from H5 (4.3% v/v vs. 5.2% S.cer, 4.1% S.eub) in lager type fermentation (12 °P, 14 °C, 150 mL). Similar CO2 production among all stabilized and non-stabilized hybrid strains
			Mtt	↑ Maltotriose uptake for isolates stabilized at high temperature (H5_HT_L3i and H5_HT_L4i) vs. non-stabilized H5_G0
			Phe	POF positive, with a subtle reduction in the amount produced by H5_HT_L4i
			Flav	Aromatic compound profile of isolates closer to S.eub (differences in higher alcohols and acetate esters), but shifts post-stabilization procedures

* Improved features for each hybrid strain compared to the corresponding parental strains are denoted as follows: fermentative performance (Ferm), maltotriose consumption (Mtt), flocculation ability (Floc), phenolic off-flavor phenotype (Phe, denoted as “POF negative” when the strain does not produce volatile phenols and “POF positive” when it does), flavor compound profile (Flav), and growth temperature range (Temp). Abbreviations and symbols: S.past (S. pastorianus), ↑ (increase), ↓ (decrease), vs. (versus). ** S. cerevisiae IMK439 strain was developed by Gonzalez-Ramos et al. [91]. *** RB: Collection of the Renaissance BioScience Corporation (RBSC), Canada. **** S. cerevisiae strains selected from a collection of strains isolated from wine-producing areas in Central Chile and previously described [92] and S. eubayanus strains belonging to PB population were described in Nespolo et al. [17].

Table 2. List of research studies aimed at developing interspecific hybrids of S. eubayanus (S.eub) and S. cerevisiae (S.cer). For each study, results other than those listed as ‘Improved Features’ for the best performing hybrids were excluded from the table.

Reference	Strain	Parental Strains and Breeding Technique	Improved Features *
Helby et al., 2015 [20]	IMS0408	S.eub CBS 12357 T spores MATa S.cer IMK439 MATα ura3Δ::KanMX ** Mass mating	Ferm	↑ Fermentation rate in synthetic medium with glucose, maltose, and maltotriose (1.2 g/L·day at 20 °C), 10h faster vs. S.cer
			Mtt	Fast maltotriose consumption with low residual concentration (comparable to S.cer parent)
			Temp	Growth from 8 °C to 35 °C in anaerobic sequential batch reactors. ↑ Growth rate vs. best parent strain from 20 °C to 30 °C
Krogerus et al., 2015 [22]	H1-H4	S.eub CBS 12357 T lys− S.cer VTT- A81062 ura− (strongly flocculent)	Ferm	↑ Ethanol production (5.6% v/v vs. parents 4.2% S.cer, 4.5% S.eub) in beer wort fermentations (12 °P, 12 °C)
			Mtt	More efficient utilization of maltose and maltotriose compared to the best-performing parent
			Floc	Between 82–88% (71% S.cer, 15% S.eub)
		Mass mating	Phe	POF positive
			Flav	↓ Higher alcohols compared to the S.eub parent. Distinctive aroma profile: ↑ overall aroma-active esters
			Temp	Growth on YPM at 37 °C, like the S.cer parent. No growth at 40 °C, whereas S.eub grew at neither temperature
Mertens et al.,2015 [23]	31 new lager yeast hybrids	S.eub Y565 and Y567 (high spore viability) S.cer Y134, Y470, Y245 and Y397 (ale beer), Y184 (wine) Y243 (bread) Spore-to-spore mating and genome stabilization in industrial lager beer medium for 70 generations	Ferm	↑ Ethanol production: 28.8% higher vs. best-performing parent (H15: 6.20% v/v) in lager fermentation (12 °P, 16 °C, 150 mL)
			Mtt	↑ Maltose (>98.5%) and maltotriose (>59%) uptake in 7–8 days (50 L pilot scale, 12 °C, 12 °P), comparable to S.cer Y397 and Y134. S.past GSY501 only reached complete fermentation by day 16
			Phe	POF positive
			Flav	↑ Isoamyl acetate vs. S.past reference strains. ↑ Aromatic diversity but ↑ fusel alcohols levels
			Temp	↑ Growth range on YPD agar (4 °C to 37 °C) vs parental strains
Krogerus et al., 2016 [57]	A2 (diploid), B3 (triploid), C4 (tetraploid)	S.eub CBS 12357 T ura− or lys− S.cer VTT-A81062 lys− or ura− (strongly flocculent) Spore-to-spore mating for A2 diploid hybrid Rare mating for B3 triploid and C4 tetraploid hybrids	Ferm	↑ Ethanol production (6.9% v/v on average) vs. parents (6.6% S.cer, 5.7% S.eub) in 15 °P beer wort (15 ºC, 1.5 L). In 25 °P wort only B3 and C4 displayed hybrid vigor
			Mtt	Overall, 70% maltotriose uptake (comparable to S.cer parent)
			Floc	B3 (73.1%), C4 (58.6%) vs. S.cer (94%). A2 (6.2%), comparable to S.eub parent (2.6%)
			Phe	POF positive
			Flav	↑ Aroma esters but ↑ diacetyl vs. parents
			Temp	↑ Growth range on YPM agar (8 °C to 37 °C) vs. parental strains
Nikulin et al., 2017 [56]	H1	S.eub CBS 12357 T S.cer VTT-A81062 (strongly flocculent)	Ferm	↑ Maltose consumption (80% vs. 50% S.cer) and ethanol (6% v/v vs. 3.5% S.eub, 4.5% S.cer) in 12 °P beer wort (12 °C, 1.5 L)
			Mtt	↑ maltotriose uptake (60% vs. 50% S.cer)
		Characterization of H1 hybrid strain developed by Krogerus et al. [22]	Phe	POF positive
			Flav	↑ Aroma-active esters vs. both parent strains and ↓ higher alcohols vs. S.eub parent
Krogerus et al., 2017 [83]	H1: VTT-A1522, H2, H3, T1, T2	S.cer VTT-A81062 ura−, Mtt+, POF+ and WLP099 rho−, Mtt−, POF− S.eub CBS 12357 T lys−, Mtt−, POF+ Rare Mating. H1: A81062 × C12902 H2: WLP099 × C12902 H3: A81062 × WLP099 T1: H1 × WLP099 T2: T1 POF− meiotic segregant	Ferm	↑ Ethanol (6–7.3% v/v) produced by T2, H1, H3 and T1 vs. best S.cer parent (5.5%) in 15 °P beer wort (15 °C, 1.5 L)
			Mtt	T2, H1 and H3 consumed maltotriose efficiently (residual concentrations comparable to the Mtt+ parent A81062)
			Phe	POF negative phenotype for T2; others (H1, H2, H3 and T1) POF positive
			Flav	↓ Esters and ↑ higher alcohols in beer made with T2 vs. T1 beer
Eizaguirre, 2018 [42]	MC3(100gen), MA6(100gen)	S.cer WLP099 and WY1338 (high spore viability) S.eub CBS 12357 T Spore to spore mating followed by experimental evolution	Ferm	↑ Fermentation rates for evolved hybrids in beer wort (12.5 °P, 10 °C, 10 mL) vs. original hybrids. Similar fermentation rates to the reference W34/70 strain, but ↓ compared to the S.eub parent
			Mtt	Not maltotriose uptake
			Phe	POF positive
			Flav	↑ Ester profile vs. S.eub parent
Krogerus et al., 2021 [86]	A225 meiotic seggregants: A226 to A229, A227 meiotic segregants: A232 to A235	S.eub CBS 12357 T (high spore viability) S.cer VTT-A-81062 (Mtt+, POF+, strongly flocculent) Analysis of meiotic segregants derived from the previously obtained hybrid VTT-A15225 (A225) [83]	Ferm	↑ Fermentation rate and efficiency for A225 (15 °P beer wort,15 °C, 1.5 L) with ↑ethanol vs. parents (6.7% v/v vs. 5.7% S.cer, 4.9% S.eub). Variable performance for spore clones derived from A225, associated with ploidy (tetraploids > diploids)
			Mtt	Similar maltotriose uptake to that of the S.cer parent for all hybrids, except A226
			Floc	Strong flocculation for A225 and its spore clones A226 and A228 (similar to S.cer parent). Flocculation is not linked to ploidy
			Flav	↑ 3-methylbutyl acetate and ethyl hexanoate for spore clones A232-A235 vs. the best parent strain for both compounds
			Temp	Broad growth range, from 4 °C to 37 °C, with variability among strains
Turgeon et al., 2021 [84]	RB-1141, RB1186, RB115, RB2251, RB2403 ***	S.past W34/70 trp− meiotic segregants (domesticated S.eub sub-genome and mitochondria) S.cer RB-253, RB-48 and NCYC-1113 diploid meiotic segregants lys− or ura− Rare mating of mating competent meiotic segregants	Ferm	Maltose consumption >98% for all strains with similar ethanol production than the reference S.past W34/70 (average 7.3% v/v vs. 7.2% S.past) in 15 ºP beer wort (13 °C, 30 IBU, 80 mL)
			Mtt	86.4–90.2% maltotriose assimilation, comparable to S.past reference strains
			Floc	Flocculation by day 14 for most hybrids during 3 L wort fermentation (except RB-1186)
			Phe	POF negative
			Flav	Acceptable acetic acid and diacetyl and ↓ off-aromas compared to S.past control strains; profiles suitable for lager beer
			Temp	↑ Growth range on YEG medium (7 °C to 37 °C) vs. parental strains
Molinet et al., 2024 [90]	H3-A, H4-A, H6-A, H8-A	S.cer L270 L348 and L3 S.eub from different PB populations: CL710.1, CL216.1; CL450.1 ****	Ferm	↑ Fermentative capacity for evolved hybrids generated at 12 °C (S.eub mitochondria) in lager beer wort fermentations (12 °P, 12 °C, 50 mL) vs. ancestral lines, and similar to W34/70. ↑ 7.1% v/v ethanol for evolved single cell isolate H4 C1 vs. W34/70
			Mtt	Similar uptake to S.past reference strain W34/70 in best performing hybrids
		Spore to spore mating at 12 ºC or 20 °C followed by experimental evolution	Phe	The single cell isolate analyzed for POF phenotype (H3-4 C1) was POF positive
			Flav	H-4 C1 produced a unique fatty acid ethyl esters profile (herbal/spicy notes vs. W34/70 citrusy)
Zavaleta et al., 2024 [88]	47 de novo hybrids derived from 21 crosses	Seven S.cer strains with high fermentative capacity from different clades (beer, wine, sake and bioethanol) Six S.eub from different lineages Rare mating at 12 °C (to facilitate inheritance of S.eub mitochondria)	Ferm	↑ CO2 production in beer wort fermentations (12 ºP, 12 ºC, 50 mL) for hybrids derived from S.cer Beer lineage, similar to S.cer Beer parents
			Mtt	↑ maltotriose uptake (56.5% on average) for hybrids derived from S.cer Beer lineage vs. other hybrids
			Phe	POF positive
			Flav	Four distinctive aroma clusters were obtained. Hybrids from S.cer Beer lineage: ↑ ethyl acetate and isoamyl alcohol, ↓ off-flavors
			Temp	↑ Growth range (4 °C to 37 °C) with ↑ or similar fitness vs. parents
Murath et al., 2025 [28]	H5 stabilized isolates: H5_HT_L3i and H5_HT_L4i	S.cer BE011 (high sporulation rate and spore viability, desirable aroma compounds) S.eub DR1 and DR3 (Chile) Spore to spore mating followed by different stabilization procedures	Ferm	↑ Ethanol production for stabilized isolates derived from H5 (4.3% v/v vs. 5.2% S.cer, 4.1% S.eub) in lager type fermentation (12 °P, 14 °C, 150 mL). Similar CO2 production among all stabilized and non-stabilized hybrid strains
			Mtt	↑ Maltotriose uptake for isolates stabilized at high temperature (H5_HT_L3i and H5_HT_L4i) vs. non-stabilized H5_G0
			Phe	POF positive, with a subtle reduction in the amount produced by H5_HT_L4i
			Flav	Aromatic compound profile of isolates closer to S.eub (differences in higher alcohols and acetate esters), but shifts post-stabilization procedures

* Improved features for each hybrid strain compared to the corresponding parental strains are denoted as follows: fermentative performance (Ferm), maltotriose consumption (Mtt), flocculation ability (Floc), phenolic off-flavor phenotype (Phe, denoted as “POF negative” when the strain does not produce volatile phenols and “POF positive” when it does), flavor compound profile (Flav), and growth temperature range (Temp). Abbreviations and symbols: S.past (S. pastorianus), ↑ (increase), ↓ (decrease), vs. (versus). ** S. cerevisiae IMK439 strain was developed by Gonzalez-Ramos et al. [91]. *** RB: Collection of the Renaissance BioScience Corporation (RBSC), Canada. **** S. cerevisiae strains selected from a collection of strains isolated from wine-producing areas in Central Chile and previously described [92] and S. eubayanus strains belonging to PB population were described in Nespolo et al. [17].

Although yeast flocculation potential is critical for the brewing industry, it is not the most frequently reported feature in studies addressing the development of de novo hybrids. Krogerus and co-workers have reported S. cerevisiae × S. eubayanus de novo hybrids with strong flocculation, comparable to that of the S. cerevisiae parent, and have dedicated part of their efforts to characterizing this trait. They have shown that non-flocculent hybrid phenotypes carry structural variations in their genomes, such as deletions in FLO5 and TIR2 genes, both codifying for proteins involved in cell-to-cell adhesion mechanisms, that could potentially explain the phenotype [86].

In terms of temperature growth range, heterosis has also been extensively reported, with hybrids exhibiting a broader range than either of their parental strains, growing from as low as 4 °C or 7 °C up to 35 °C or 37 °C. Concerning temperature tolerance in de novo S. cerevisiae × S. eubayanus hybrids, recent evidence suggests that mitotype—and, thus, a competitive advantage under cold brewing—would be primarily determined by the temperature in which the mating process takes place: When the hybridization procedure was conducted at a low temperature (12 °C), hybrids inherited S. eubayanus mitochondria, whereas hybrids obtained at 20 °C carried S. cerevisiae mitochondria [90]. Interestingly, when subjecting these hybrids—with no initial signs of hybrid vigor—to EE for 250 generations under beer wort fermentation conditions, the evolved lines showing the greatest fitness improvement were those carrying S. eubayanus mitochondria. The authors propose that species-specific mitotypes could play a role in sugar utilization and glucose repression mechanisms when adapting the hybrids to low-temperature brewing conditions, probably due to the interaction between mitochondrial and nuclear genomes. Hybrid fitness improvement was attributed to a complex interplay of mechanisms involving single nucleotide polymorphisms (SNPs), copy number variations (CNVs), and differential gene regulation in the genomes of the evolved lines.

Aside from the impact of mitochondrial origin in hybrids, the existence of unidentified nuclear genes with a direct influence on temperature tolerance regulation has been proposed, since a lack of correlation between mitotype and cryotolerance was also reported in a set of de novo lager strains [84]. These lager hybrids are distinctive because they carry a domesticated S. eubayanus genome derived from an industrial S. pastorianus strain. They were developed through the rare mating of allodiploid meiotic segregants of a lager strain with diploid S. cerevisiae ale strains. It is suggested that the S. eubayanus domesticated subgenome would provide a particular set of nuclear genes that, acting in a complementary manner with S. cerevisiae-derived genes, enable low-temperature efficient fermentation regardless of mitotype. Interestingly, these hybrids were also POF⁻ and displayed an unusual genomic composition, featuring an allotetraploid karyotype with a 3:1 S. cerevisiae to S. eubayanus ratio, in contrast to all currently characterized lager strains that harbor a diploid S. eubayanus genome and differ in their S. cerevisiae genome contribution [73].

Aromatic profile diversification in lager yeasts constitutes the main driver behind new strains development. In this regard, most of the studies presented in Table 2 report the obtention of hybrid strains with higher concentrations of desired aromatic esters than their parentals; a fruity aroma derived from isoamyl acetate, ethyl hexanoate, and ethyl octanoate compounds has been found in elevated levels in beers produced by de novo lager hybrids in comparison with their parental strains [22,23,56,57].

Aroma compound formation in de novo developed hybrids has been reported to depend on both the selection of the parental strains as well as the resulting genome architecture, including parental subgenome ratio and ploidy level. In this respect, the study conducted by Krogerus et al. [57] reports the obtention of polyploid hybrids with an increased production of desirable esters, likely as a result of increased gene copy number and transcript levels of a set of key genes involved in the synthesis of aroma compounds, particularly those encoding acetyl transferases ATF1 and ATF2 and acyl transferases EHT1 and EEB1. Furthermore, the study indicates that orthologous genes inherited from each parental strain contribute differentially to the final aroma profile. This was further reinforced in the study of Nikulin et al. [56], in which the combined expression and activity of parental genes in hybrid genomes also resulted in heterotic phenotypes in terms of aroma profile—i.e., the S. arboricola-derived hybrid produced the highest 3-methylbutyl acetate level (surpassing both parental strains), and a similar outcome was observed for ethyl hexanoate in the hybrid derived from S. mikatae. Aroma active compound amplification has also been explored beyond the previously reported values by the meiotic segregation of allotetraploid S. cerevisiae × S. eubayanus hybrids, and subsequent segregant sporulation, resulting in spore clones with a twofold increase in 3-methylbutyl acetate and ethyl hexanoate compared to the best parental strain [86].

It is noteworthy that the complex interplay between gene expression subgenomes may also lead to the generation of volatile compound combinations with undesirable aromatic characteristics. Of note are the strains developed by Mertens et al. [23], which produced beers with significantly higher concentrations of isoamyl acetate—associated with banana-like aromas—compared to reference strains, but with increased levels of fusel alcohols. Excessive fusel alcohol concentrations have a negative impact on the overall quality of the beer, contributing to the perception of off-flavors.

Another essential trait in lager strains is their POF⁻ phenotype, acquired throughout the domestication process in all S. pastorianus and in most S. cerevisiae ale strains, due to loss-of-function mutations in PAD1 and/or FDC1 genes. In contrast, wild strains retain functional copies of both genes [93], consistent with the protective roles attributed to phenolic compound production in natural environments. Similarly, bakery and bioethanol strains have been shown to retain the POF⁺ phenotype, since 4-VG production does not negatively affect the quality of the final product [94]. In a study involving the sequencing and analysis of numerous interspecific hybrids and introgressed Saccharomyces strains genomes, multiple mechanisms have been proposed for the lack of POF⁺ phenotype in lager strains. These include inheritance of a mutated PAD1 allele from the already domesticated S. cerevisiae ale parent, aneuploidies leading to the loss of functional PAD1/FDC1 genes from S. eubayanus, and chromosomal translocations in lager strain genomes [40].

In this context, when addressing the flavor customization of newly developed lager hybrids, a key feature is the inheritance of the undesired POF⁺ phenotype from wild S. eubayanus parental strains. To address this, an effectively applied method involved the induction of sporulation of an allotetraploid S. cerevisiae × S. eubayanus hybrid and mating of resulting spores with a third parental strain (POF⁻ S. cerevisiae ale yeast) to form a hybrid containing DNA from three parents. Further sporulation and selection of meiotic segregants led to the obtention of hybrid spore clones with non-functional PAD1/FDC1 genes [83]. Diderich et al. [95], in turn, obtained POF⁻ S. eubayanus mutants via UV mutagenesis of wild-type POF⁺ S. eubayanus strains and subsequent selection based on their sensitivity to cinnamic acid, an assay that allowed for the selection of strains responding negatively to weak acid stress. Further hybridization of the POF⁻ S. eubayanus strains with POF⁻ S. cerevisiae ale strains led to the obtention of hybrids with improved fitness in terms of maximum OD600 nm and ethanol production in synthetic medium supplemented with ferulic acid, with the ability to metabolize all sugars present in the medium (including maltotriose) and a lack of ferulic acid decarboxylation activity.

An additional aspect that is worth highlighting in terms of novel hybrid development is that the utilization of diploid parental strains has been shown to effectively result in fertile allotetraploid hybrids capable of producing viable spores. The sporulation of allotetraploid intermediates [84,86,90] or subsequent recombination of meiotic segregants, either with each other or with cells of different strains [83], was applied in cases in which a desirable trait combination was not observed in the first crossbreeding. These strategies allowed for the generation of genetic and phenotypic diversity in the progeny and led to further selection of meiotic segregants on the basis of the expected fitness advantage. However, a major drawback of the resulting spore clones is their genetic instability, often exhibiting elevated rates of structural rearrangements [96].

Genetic instability of hybrid genomes is an issue of relevance for the brewing industry, where yeast cultures are frequently reused multiple times. Indeed, after the development of the set of hybrids and meiotic segregants by Krogerus et al. [83], the researchers further applied a stabilization procedure to drive the adaptation of the strains to high ethanol levels [71]. By exposing some of the laboratory-made hybrids to 30 consecutive batch fermentations in media with 10% ethanol, they obtained strains with better fermentative skills in comparison to the non-stabilized ones, and beers resulting from 2-L-scale wort fermentations showed higher concentrations of desirable aroma-active compounds as well as low off-flavor levels. Later, Krogerus et al. [86] also reported phenotypic instability and heterogeneity in fermentation performances and aroma compound production in colony isolations derived from a meiotic segregant of a S. cerevisiae × S. eubayanus tetraploid hybrid, after subjecting it to 10 consecutive fermentations, when compared to the original strain. The isolates obtained after these fermentations displayed distinct chromosomal profiles, mainly attributed to CNVs such as the acquisition of two additional copies of S. eubayanus chromosome III or the loss of chromosome XII copies from both S. cerevisiae and S. eubayanus. More recently, a study addressing genetic stability of S. cerevisiae × S. eubayanus de novo hybrids, has also demonstrated that additional genetic and phenotypic diversity arises in subsequent mitotic cycles after the original hybridization event, and that the application of different stabilization regimes leads to marked differences in maltotriose consumption and aroma compounds production in the final strains [28]. These findings emphasize the importance of evaluating the genomic stability of newly developed hybrids when assessing their viability and scalability under industrially relevant conditions.

In addition to the development of de novo hybrid strains, the application of EE under simulated lager brewing conditions to hybrids has also attracted scientific research interest due to its potential to harness the existing genetic diversity of such genomes. These strategies have been reported to trigger diverse genetic modifications such as CNVs, SNPs, insertion/deletions, chromosomal recombinations, aneuploidies, and/or mitochondrial DNA loss [37,73,97]. It is worth mentioning that these intricate networks of modifications can give rise to both beneficial and detrimental traits in the progeny. For instance, Gorter de Vries et al. [73] evolved a previously developed hybrid strain [20] showing that both the SeSFL1 and ScMAL11 genes were affected through loss of heterozygosity mechanisms that resulted in the acquisition of a desired flocculating phenotype while simultaneously reducing the maltotriose utilization capacity previously described.

The understanding of S. eubayanus genetic and phenotypic characteristics, evolutive history and ecology, as well as its application for novel beer brewing strain development, highlights it as a valuable yeast resource and has promoted its utilization by industry.

4. S. eubayanus in the Brewing Industry

The relevance of S. eubayanus for beer brewing is reflected not only in the extensive body of academic research accumulated since its discovery, but also in its application at production scales in the craft and industrial brewing sectors, with the aim of developing innovative and differential products.

Table 3. S. eubayanus commercial products.

Fantasy Name	Brewery	Country, Locality	Base Style	Strain	Total Volume Produced (L)	Date
H41—Wild Lager	Heineken	Netherlands	Wild Lager	S. eubayanus CBS 12357 T	n.a.	2017–2018
H71—Forêts de Patagonie	Heineken	Netherlands	Wild Lager	S. eubayanus CBS 12357 T	n.a.	2018
H35—Blue Ridge Mountains	Heineken	Netherlands	Wild Lager	S. eubayanus yHRVM108	n.a.	2018
H32—Massif de L’Himalaya	Heineken	Netherlands	Wild Lager	S. eubayanus CDFM21L.1	n.a.	2018
Sauvage	Manush	Argentina, Bariloche	Summer Ale	Euby®	1550	2017
Madre Salvaje (Wild Mother)	Blest	Argentina, Bariloche	Pilsen	Euby®	1000	2018
Nativa (Native)	Diuka	Argentina, Bariloche	Pilsen	Euby®	1200	2018
Pan del Indio (Indian bread)	Berlina	Argentina, Bariloche	Amber	Euby®	1500	2018
Dorada Salvaje (Golden Wild)	Duham	Argentina, Bariloche	Dorada Pampeana	Euby®	7000	2018–2020–2021
IPA Salvaje (Wild IPA)	Duham	Argentina, Bariloche	IPA	Euby®	5000	2020–2021
Patagonia Belgian Lager Ale	Duham	Argentina, Bariloche	Belgian	Euby® and S. cerevisiae WLP500	1000	2022
Lager Salvaje/Amber Euby/Kölsch Euby/APA Euby	Bachmann	Argentina, Bariloche	Pilsen/Amber/Kölsch/APA	Euby®	15	2018
Eubakonna	Konna	Argentina, Bariloche	Pilsen	Euby®	500	2018
Wild Wild Yeast	Wesley	Argentina, Bariloche	Scotish	Euby®	5000	2018–2019
Wild Euby	Wesley/BrewSisters	Argentina, Bariloche	Amber	Euby®	300	2022
Paralelo 42—100% Patagónica (Parallel 42)	Awka	Argentina, El Bolson	Pilsen	Euby®	4750	2018–2019–2023–2024–2025
Paralelo 42 (Parallel 42)	Awka	Argentina, El Bolson	Pilsen	S. eubayanus A62 (evolved strain)	550	2023
Amber Salvaje (Wild Amber)	Antares	Argentina, Mar del Plata	Amber	Euby®	300	2021
Doppelbock Salvaje (Wild Doppelbock)	BrewSisters—Tibet	Argentina, Bariloche	Doppelbock	S. eubayanus A62 (evolved strain)	60	2021
Pilsen Eubayanus	Kuntsmann and Sayka	Chile, Los Ríos	Pilsen	S. eubayanus Et5.1	1000	2022
Lenga	Nothus	Chile, Valdivia	Saison	S. eubayanus Et5.1 and S. cerevisiae	500	2022
Salvaje Sur (Wild South)	Chaura	Argentina, El Hoyo	Blonde	Euby®	150	2023
Maibock	Esquel	Argentina, Esquel	Maibock	Euby®	300	2023
Maibock	Esquel	Argentina, Esquel	Maibock	Euby®	1200	2024
Euby	Biergarten Klein	Chile, Temuco		Hybrid strain ACS24	500	2024
Home sweet home	IPATEC	Argentina, Bariloche	Dubbel	S. eubayanus CBS 12357 T	25	2025

summarizes the commercial products brewed with S. eubayanus to date. The first commercial product was released by Heineken^® (Amsterdam, the Netherlands) in 2016 in several countries in a limited-edition named “Wild Lager H41” (strain CBS 12357 ^T, isolated in Bariloche, Patagonia at Latitude 41°). Later, three additional beers were launched involving S. eubayanus strains isolated from different locations: ‘Wild Lager H71 Patagonia’, ‘Wild Lager H32 Himalayas’, and ‘Wild Lager H35 Blue Ridge Mountains’. All Wild Lager series have been discontinued. In parallel, several Patagonian breweries in Argentina started using native strains of S. eubayanus for brewing, facilitated by the signing of commercial licenses with the Association of Craft Brewers of Bariloche and the Andean Region (ACAB) under the name ‘Wild Patagonia Project’. These efforts have enabled the production of beers with a strong regional identity, with 15 craft breweries developing distinctive products and around 30 hL of beer produced since the first commercial application in 2017 (Table 3). It also resulted in the development of the first 100% Patagonian beer, brewed in Awka brewery entirely from locally sourced ingredients (water of the Andes rivers, Patagonian hops and malts and S. eubayanus), which obtained a very good rating in the Acceptance testing carried out on 200 consumers (unpublished data) and is currently part of the brewery established portfolio of styles. In recent years, a strain of S. eubayanus was renamed Euby^® for communication purposes and started to gain adoption within the brewing community; also, the development of an identity style is in process.

In Chile, novel products have also been developed with local native strains (Table 3), as well as experimental products with S. cerevisiae × S. eubayanus hybrids developed by the USACH-iBio research group led by Dr. Cubillos [25]. In 2022, the Copa Cervezas de América announced its Experimental category, where beers based on S. eubayanus competed (Nothus brewery, Valdivia, Chile, awarded a gold medal for its Lenga beer brewed with a blend of S. eubayanus and S. cerevisiae, while the brewery Awka, Argentina, received a silver medal for a beer fermented only with Euby^®).

Since the isolation of pure yeast cultures by Emil Christian Hansen in 1883, the brewing industry has largely adopted the use of single-strain inoculum, enabling standardized and predictable fermentations. However, the resurgence of craft brewing and the continuous pursuit of novel aromas and flavors have renewed interest in mixed, spontaneous, and co-inoculated fermentations [98]. Notably, it is believed that the origin of S. pastorianus occurred in such environments during the 16th century in continental Europe, where multiple yeast species likely coexisted in the beers of the time [8]. From the long-standing Belgian traditions of Lambic and Gueuze beers production to modern Italian innovations, such as grape ales (grape must and beer wort blends), and global wild ales, mixed fermentations have flourished. While S. eubayanus exhibits distinctive sensory attributes (e.g., phenolic complexity and cold-tolerant ester production), its potential in such contexts remains largely underexplored. Moreover, recent evidence demonstrated enhanced antioxidant activity in wild ales [99] and herb-infused beers [100]. Further research employing S. eubayanus in co-inoculated fermentations, particularly in combination with fruits or spices and herbs, could elucidate its contribution to these properties, thereby expanding its potential applications for brewing.

Although working with wild yeasts results in exciting outcomes, their application in large-scale brewing can also be challenging. Indeed, Burini et al. [36] found that at productive volumes greater than 1000 L, fermentations with S. eubayanus took more than 20 days to complete (almost twice the time compared to the lab and semi-pilot scale fermentations), with lower attenuation values and higher residual maltose content in the final beer, and from various subsequent experiences, this behavior was observed from 150 L onwards. Variations in physical conditions can negatively influence yeast performance, particularly in wild strains that are not adapted to anthropogenic environments, as these changes often introduce substantial additional stress. Effectively managing native yeasts for brewing may require different approaches compared to conventional brewing yeasts, obtaining quality starter cultures, and adjustments in technical parameters such as inoculation rate, fermentation temperature, oxygenation strategy, and nutrient availability [101]. As a result, their application could necessitate significant modifications to existing process conditions; in fact, different strategies were applied in Argentine breweries to develop large-volume quality products with S. eubayanus in more efficient production times.

5. Conclusions and Future Directions

Strategies to improve S. eubayanus industrial potential and promote its adoption by the brewing market have been successfully applied. Major breakthroughs have been made with regard to the creation of novel strains with improved fermentative skills through EE strategies under strong selective pressures. However, phenotypes such as the absence of POF production or flocculation ability—which is governed by complex genetic architectures that involve multiple genes and regulatory networks—are more difficult to evolve. As the acquisition of these traits is not related to a direct fitness advantage under laboratory fermentative conditions, their selection through EE becomes more challenging, and the development of S. eubayanus strains that are both flocculent and POF⁻ is a research point with potential to be further addressed.

However, S. eubayanus-evolved strains capable of efficiently fermenting maltotriose constitute a valuable option for craft brewing markets, where the presence of phenolic notes may contribute to offering a distinguishable product. These strains are not available for brewers yet, and the industrial application of commercially available S. eubayanus strains involves the incorporation of alternative management strategies during the brewing process to obtain a beer product within a reasonable fermentation period and with properly balanced organoleptic attributes. Thus, obtaining strains well suited for lager beer production still represents a challenge for researchers, whose efforts are coming closer to achieving this goal.

In addition, the experiments with artificial hybrids of S. cerevisiae and S. eubayanus reviewed here highlight interspecific hybridization as a useful non-GMO strategy to confer fitness advantages in the highly specific niche of lager beer fermentation, combining phenotypic traits from both parentals. Although highly variable outcomes have been obtained, a common trend is the improvement in fermentation rate and/or higher ethanol yields compared to the parental strains and efficient maltotriose utilization, often comparable to that of the S. cerevisiae parental strain. In addition, regarding aroma-active compounds, all studies report significant modifications in hybrid profiles compared to the parental strains, with an overall increase in the production of desirable esters by de novo lager hybrids. While some of these are well-suited for lager beer production, others exhibit S. eubayanus-derived herbal, spicy, or phenolic notes, which are generally less desirable. Even though efforts have primarily been focused on reducing or eliminating the production of volatile phenols, the presence of phenolic notes—when properly balanced with the overall organoleptic profile of the final product—may represent an interesting trait for establishing novel lager beer styles. For these strains to be adopted by the brewing industry, several steps would still be required, such as genomic stability to ensure the maintenance of their fermentative properties while retaining consistent fermentative performance at both pilot and industrial scales, from kinetic parameters to flavor attributes.

From a scientific standpoint, the development of a hybrid strain that combines high aromatic compound production with low or absent phenolic off-flavors, strong flocculation capacity, and efficient wort sugar consumption still represents a challenge. To obtain strains with targeted fermentative properties, the selection of the appropriate hybridization strategy is critical, particularly in light of the specific genetic characteristics of the parents involved. In this regard, although spore-to-spore mating has been successfully applied for the development of interspecific hybrids, it requires yeast strains that retain the sporulation capacity and typically leads to diploid hybrid genomes. Instead, rare mating represents an alternative efficient strategy for generating polyploid hybrids of Saccharomyces species that lack sporulation capacity. These polyploid strains can be further induced to sporulate, and meiotic segregants with desired characteristics can be obtained.

To advance toward the industrial application of improved brewing yeasts—whether interspecific hybrids or evolved S. eubayanus strains—it is also essential to evaluate their specific propagation requirements and to develop effective inoculum strategies (such as liquid cultures or dry yeast) suitable for brewers.

The overall picture of beers produced with S. eubayanus underlines its application in the development of distinctive beers. Even more, the industrial application of S. eubayanus is also being explored for the production of a variety of fermented products, which include whisky, cider, wine, kombucha, and even bakery products [102,103,104,105,106,107,108]. These studies highlight the potential and versatility of this yeast species in diverse biotechnological processes. Moreover, the use of native yeast strains may favor the development of regionally distinctive products, particularly benefiting small-scale and craft producers who try to incorporate local identity and biodiversity into their fermentative processes.