Inheritance of brewing-relevant phenotypes in constructed Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids
© The Author(s) 2017
Received: 11 January 2017
Accepted: 9 April 2017
Published: 21 April 2017
Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity.
Here we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn’t produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose.
Our study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.
KeywordsYeast Beer Rare mating Lipid Fatty acid Phenolic off-flavour Aroma
Yeast hybrids have been extensively used for centuries in the brewing and winemaking industries . Lager yeast in particular, a natural interspecies hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, is used for the majority of global industrial beer production. It possesses a range of desirable phenotypes that are relevant for the production of lager beer: cold tolerance, efficient use of wort sugars, and low formation of undesirable off-flavours. Recent studies have revealed that generating new lager yeast hybrids is a powerful strain-development tool, as hybrid strains have exhibited various improved traits including faster fermentation rates, more complete sugar use, and increases in aroma compound production [2–4]. Hybridization enables the combination and enhancement of phenotypic features from two different parent strains . In order to further improve the potential of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from the hybrid. This could be achieved through sporulation, meiotic recombination and further mating of the hybrid. However, interspecies yeast hybrids, such as lager yeast, tend to be sterile, and therefore their sporulation efficiencies and spore viabilities are usually poor [6–9]. Studies have revealed that allotetraploid hybrids are usually not constrained by sterility [6, 8], and these tend to be capable of producing viable diploid spores. Hence, allotetraploid interspecific hybrids may undergo meiosis, during which recombination may give rise to crossovers and gene conversions . This in turn causes phenotypic variation, as traits may get strengthened, weakened or even removed .
Many strains of Saccharomyces produce vinyl phenols (POF; phenolic off-flavours) from hydroxycinnamic acids, and these phenolic compounds are considered undesirable in lager beer. The most well-studied of these vinyl phenols is 4-vinyl guaiacol, which is formed from ferulic acid. The ability of brewing yeast to produce volatile phenols has been attributed to the adjacent PAD1 and FDC1 genes, both of which are essential for the POF+ phenotype . Wild yeast strains, such as the S. eubayanus strains that are available for de novo lager yeast creation, tend to have functional PAD1 and FDC1 genes, while domesticated POF− brewing yeast have nonsense or frameshift mutations in these genes, rendering them non-functional [13–15]. As the functional genes from S. eubayanus are passed on to any lager hybrids created from it, these hybrids have all been afflicted with the POF+ phenotype [2–4]. However, if any non-functional alleles of PAD1 or FDC1 are present in the hybrid genome, it may be possible to remove the POF+ phenotype through meiotic recombination as demonstrated in studies with intraspecific S. cerevisiae hybrids [13, 16].
Besides the ability to produce clean flavour profiles (i.e. no off-aromas such as 4-vinyl guaiacol), one of the main traits of lager yeast is its ability to stay metabolically active and ferment in the cold . Recent studies have revealed that the S. eubayanus parent strain contributes this cold tolerance to lager yeast [4, 17]. However, unlike the POF+ phenotype, the mechanisms that contribute to cold tolerance in brewing yeast are not fully understood. It has been revealed that low temperature affects protein translation and folding efficiencies, mRNA stabilities and the product activity and expression of central metabolic genes [18–24]. Recent studies have also suggested that differences in the lipid composition of the plasma membrane play a vital role in temperature tolerance [25–27]. The fluidity of the plasma membrane is affected by its lipid composition and the temperature [26, 28], and a decrease in membrane fluidity caused by a low fermentation temperature can, in turn, result in impaired transporter function and lower nutrient uptake [27, 29]. Relatively high levels of ergosterol and low levels of unsaturated fatty acids could, for example, result in a greater tendency of cell membranes to freeze at lower temperatures, thereby reducing functionality [26, 30, 31]. The lipid composition of yeast has also been shown to influence ethanol tolerance . Similarly to cold tolerance, ethanol tolerance has been shown to be dependent on unsaturated fatty acid and ergosterol concentrations [31, 33]. They have been hypothesized to function by maintaining optimum membrane thickness and fluidity by counteracting the fluidizing effects of ethanol. Here, we wanted to investigate what lipid species correlated with good fermentation performance at low temperatures and high alcohol levels, by examining how the lipidomes of brewing yeasts react to changes in temperature and ethanol concentration.
In this study we wanted to both demonstrate the possibility of using an allotetraploid interspecific hybrid as an intermediate to create a POF− lager hybrid with DNA from three parent strains, and use these hybrids to elucidate relationships between different lipid species and tolerance towards low temperatures and high ethanol concentrations. This was accomplished by first rare mating a set of three parent strains, all with different desirable properties, to produce five different hybrid strains. Their fermentation kinetics and lipid compositions were compared in small-scale fermentations performed at low temperatures and in the presence of ethanol. Lipids were analysed using both a targeted GC/MS and non-targeted UPLC/MS approach. Using multivariate analysis, it was revealed that high levels of unsaturated fatty acids and especially phospholipids containing palmitoleic and oleic acid were associated with good fermentation performance at low temperatures and high ethanol concentrations. Furthermore, ergosterol was associated with good fermentation performance at high ethanol concentrations, but had a negative effect at low temperatures. These observations were supported with growth data of laboratory strains lacking OLE1 and ERG4 genes in media containing ethanol and oleic acid. The potential application of these strains in industrial fermentations was finally demonstrated in 2-L wort fermentations. These revealed that Hybrid T2, a meiotic segregant of Hybrid T1 (containing DNA from all three parent strains), reached the highest ethanol concentration, consumed the most maltotriose, and did not produce any 4-vinyl guaiacol, therefore making it a suitable candidate for industrial lager beer fermentations.
Generation of inter- and intra-specific hybrids
Yeast strains used in the study
VTT-A81062. Maltotriose fermentation, POF+
VTT culture collection
WLP099. No maltotriose fermentation, POF−
White Labs Inc
VTT-C12902. No maltotriose fermentation, POF+
VTT culture collection
P1 × P3 interspecific hybrid (VTT-A15225)
Created in this study
P2 × P3 interspecific hybrid
Created in this study
P1 × P2 intraspecific hybrid
Created in this study
H1 × P2 interspecific triple hybrid
Created in this study
Meiotic segregant of T1
Created in this study
BY4741 MAT a; his3∆1; leu2∆0; met15∆0; ura3∆0
BY4741 MAT a; his3∆1; leu2∆0; met15∆0; ura3∆0; ole1-m2:kanMX
BY4741 MAT a; his3∆1; leu2∆0; met15∆0; ura3∆0; YGL012w::kanMX4
The tetraploid nature of H1 allowed it to form viable spores (47% viability) despite being an interspecific hybrid, and these spores could subsequently be mated with P2 to form the triple hybrid T1 (Fig. 1). PCR was again used to confirm that the strain contained DNA from all three parent strains (Additional file 1: Figure S1). Whole genome sequencing of the strains also revealed that Hybrid T1 contained a higher ratio of S. cerevisiae-derived to S. eubayanus-derived chromosomes (approximately 3:1; Additional file 1: Figure S3E). Additionally, the strain appeared to contain a chimeric chromosome consisting of approximately 355 kbp of the S. cerevisiae P1-derived chromosome II and 437 kbp of the S. eubayanus P3-derived chromosome IV, apparently formed during sporulation of Hybrid H1. Like Hybrid H1, Hybrid T1 was also able to form viable spores (38% viability). The spore clones of T1 were screened for the POF phenotype in media containing ferulic acid, and out of 12 spores clones that were assayed, only 3 were POF−. The best growing of these was given the name Hybrid T2, i.e. a POF− meiotic segregant of Hybrid T1. Whole genome sequencing of the strains revealed that Hybrid T2 contained at least one copy of the S. cerevisiae-derived chromosomes, but had lost several chromosomes derived from S. eubayanus (Additional file 1: Figure S3F).
Effects of temperature and ethanol on fermentation rate and lipid composition
Modelled (A, μ, λ) and measured (dry mass) fermentation and growth parameters of the fermentation assays that were performed at different temperatures (10 and 20 °C) and supplemented ethanol levels (0 and 8% (v/v) EtOH)
Strain and condition
μ (°brix h−1)
Dry mass (g L−1)
10 °C, 0% EtOH
H1 (P1 × P3)
H2 (P2 × P3)
H3 (P1 × P2)
T1 (H1 × P2)
T2 (T1 segregant)
20 °C, 0% EtOH
H1 (P1 × P3)
H2 (P2 × P3)
H3 (P1 × P2)
T1 (H1 × P2)
T2 (T1 segregant)
10 °C, 8% EtOH
H1 (P1 × P3)
H2 (P2 × P3)
H3 (P1 × P2)
T1 (H1 × P2)
T2 (T1 segregant)
20 °C, 8% EtOH
H1 (P1 × P3)
H2 (P2 × P3)
H3 (P1 × P2)
T1 (H1 × P2)
T2 (T1 segregant)
Fermentations were not as efficient in the growth media supplemented with 8% (v/v) ethanol. At 10 °C, it was only S. eubayanus P3 and Hybrid H1 which managed to reach the same maximum fermentation level (A) as in the unsupplemented growth media. However, at 20 °C P3 both fermented and grew the worst of the 8 strains, suggesting it is ethanol-tolerant at 10 °C but not 20 °C. S. cerevisiae P1 on the other hand, performed relatively well in the presence of ethanol at 20 °C, but not at 10 °C. The interspecific hybrid between the two, Hybrid H1, was able to ferment in the presence of ethanol at both 10 and 20 °C, and even outperforming P1 with regards to maximum fermentation rate (μ) at 20 °C. The two triple hybrids T1 and T2, as well as the S. cerevisiae P2 (the genome of which dominates in T1 and T2), performed poorly in the presence of ethanol at both 10 and 20 °C. Hybrid T2 did not grow or ferment at 10 °C and with 8% supplemented ethanol, and samples of it at this condition were thus excluded from subsequent lipid analysis.
The 10 most significant Variable Importance in Projection (VIP) scores and regressions coefficients of the three PLS models presented in Fig. 6
10 °C, 0% supplemented EtOH
10 °C, 8% supplemented EtOH
20 °C, 8% supplemented EtOH
Supplementation of oleic acid enhances growth in the presence of ethanol
Fermentations in wort confirm POF− phenotype
The beers produced with the 8 brewing strains also varied considerably in concentrations of aroma-active compounds (Fig. 8b). The most ester-rich beers were produced with Hybrids H1, H3 and T1, and these contained as high or higher concentrations of most esters compared to either of the parent strains. Comparing the aroma profiles of the beers made with Hybrids T1 and T2, it is revealed that the meiotic segregant T2 produced lower concentrations of most esters, while its beer contained higher concentrations of most higher alcohols. This would suggest lower activities or expression of alcohol acetyl transferases in Hybrid T2. Of the 8 brewing strains, the POF− S. cerevisiae P2 and Hybrid T2 strains were the only ones that did not produce any detectable amounts of 4-vinyl guaiacol (detection limit 0.2 mg L−1), thus confirming their POF− phenotype (Fig. 8c). All other strains produced 4-vinyl guaiacol in concentrations above the flavour threshold of 0.3–0.5 mg L−1 . Yeasts produce 4-vinyl guaiacol from ferulic acid with the aid of the PAD1- and FDC1-encoded enzymes. By comparing the sequences of these two genes in the 8 brewing strains, we found that Hybrid T2 only carried the PAD1 allele that was derived from S. cerevisiae P2 (Fig. 8e; Additional file 1: Table S3). This particular allele, contained a possible loss-of-function SNP at position 638 (A>G, resulting in an amino acid substitution of aspartate to glycine). The other strains, including Hybrid T1, which Hybrid T2 was derived from, carried either or both of the functional PAD1 alleles derived from S. cerevisiae P1 or S. eubayanus P3. While the alleles of FDC1 in the parent strains contained different SNPs (Additional file 1: Table S3), none of them appeared to cause loss-of-function.
Interspecific hybridization has been shown to be a promising tool for increasing the diversity of lager yeasts available to the brewing industry. However, de novo lager yeast hybrids have so far inadvertently possessed a POF+ phenotype; a trait which they inherit from the S. eubayanus parent. In this study, we demonstrate the possibility of using an allotetraploid interspecific hybrid as an intermediate to create a POF− lager hybrid with DNA from three parent strains, and we use a set of brewing yeast hybrids to reveal that unsaturated fatty acids and ergosterol are associated with good fermentation performance at low temperatures and high ethanol concentrations.
Sporulation efficiency and spore viability tend to be poor in interspecies yeast hybrids, which limits the possibility of introducing variation to such a hybrid through meiotic recombination and chromosomal cross-over. The mechanisms contributing to hybrid sterility are not completely understood, but studies suggests that large sequence divergence, abnormal chromosome segregation and reciprocal gene loss contribute to it . However, hybrid fertility can be recovered through a number of ways, one of which is genome doubling. Studies have revealed that doubling the genome content of sterile allodiploid hybrids result in allotetraploids capable of producing viable spores [6, 8]. Here, the allotetraploid interspecies hybrids H1 and T1 showed spore viabilities of 47 and 38%, respectively, and sporulation allowed us to not only cross Hybrid H1 with a third parent strain (P2), but also remove the POF+ phenotype from Hybrid T1. As was revealed from the genome sequences of Hybrids T1 and T2, chromosome losses may occur during spore formation (e.g. the S. eubayanus-derived chromosome XIII carrying PAD1 and FDC1), suggesting that genetically diverse strains can be obtained. Hence, the use of fertile intermediates allows for the construction of complex interspecific hybrid strains, which can be screened and selected to contain desirable traits. While not investigated here, one may expect that meiotic segregants of interspecies hybrids vary considerably phenotypically as a result of orthologous gene segregation and the creation of unique biochemical pathways and regulatory mechanisms [37, 38]. We believe this would be particularly beneficial for generating novel and diverse lager yeast strains, as there currently exist limited genetic and phenotypic diversity between natural lager yeast hybrids [1, 39]. As the stability test of Hybrid T2 revealed that some genetic changes occurred after 10 successive batch cultures in relatively non-stressful media, it is vital that any hybrids are stabilised and phenotypically reassessed before they are viable candidates for industrial beer fermentations. Previous studies have shown that sufficient stabilisation can be achieved by growing the hybrids for 30–70 generations under fermentative conditions in high-sugar media [4, 40]. The inherent instability of interspecific yeast hybrids could also be exploited for further strain development through adaptive evolution, as was recently demonstrated for biofuel applications .
Phenolic off-flavours are undesirable in many beer styles, and in lager beer especially their presence is considered a flaw . Hybridization and subsequent sporulation has been proposed as one technique of removing the POF+ phenotype from crossed S. cerevisiae strains . Gallone et al.  have also shown that a POF− hybrid can be constructed when both parent strains carry PAD1 or FDC1 alleles containing loss-of-function mutations. S. eubayanus however, contains functional alleles of both PAD1 and FDC1, so any hybrids made from it will initially be POF+ as well. Here we obtained a POF− interspecies hybrid through the use of a fertile tetraploid intermediate, and demonstrated that it didn’t produce 4-vinyl guaiacol in wort fermentations and only contained PAD1 and FDC1 alleles derived from the POF− S. cerevisiae P2 parent. No nonsense or frameshift mutations were detected in the PAD1 and FDC1 alleles of P2, but a SNP at position 638 (A>G) of PAD1 results in an Asp213Gly amino acid substitution, possibly affecting the functionality of the enzyme. This same SNP (PAD1: 638 A>G) was present in the POF− K1V-1116 strain that was studied by Mukai et al.  and the POF− strains Beer024, Beer033, Beer088, Wine001, Wine009 and Spirits002 that were studied by Gallone et al. . However, it may be possible that this SNP is not the cause for the POF− phenotype in P2 and T2, as well as the POF− strains from the previously mentioned studies, and rather another unknown mechanism is responsible.
Among the defining characteristics of lager yeast is their ability to grow and ferment at low temperatures and high ethanol concentrations. Unlike the POF+ phenotype, the mechanisms that contribute to cold and ethanol tolerance in brewing yeast are more complex and not fully understood. Differences in the lipid composition of the plasma membrane have however been shown to play a vital role in determining both temperature and ethanol tolerance [25–27, 33]. Here, we observed variations in cellular lipid composition both between yeast strains and between environmental conditions. When temperatures were lowered or ethanol content was increased, the degree of unsaturation tended to increase. Similar conclusions have also been reached in recent studies in wine-making conditions [25, 26, 32]. It is assumed that these changes in response to a lowered temperature or an increase in ethanol concentration are vital for maintaining membrane fluidity and functionality [25, 33, 42]. The PLS models that were constructed between the lipid dataset and fermentation parameters at the different conditions revealed that yeast strains which performed well at low temperatures and/or high ethanol concentrations were associated with increased concentrations of phospholipids and triacylglycerides containing unsaturated fatty acids, such as palmitoleic acid (C16:1) and oleic acid (C18:1). Ergosterol was also shown to be positively correlated with ethanol tolerance, particularly at 20 °C. Studies have revealed that ergosterol plays an important role in maintaining membrane rigidity and protecting against ethanol toxicity when ethanol concentrations are increased [30, 31]. Results from microplate cultivations of ole1∆ and erg4∆ knockout strains supplemented with oleic acid and ethanol were in agreement with these observations. Redón et al.  also observed that wine fermentations at low temperatures proceeded faster when unsaturated fatty acids such as palmitoleic and linolenic acid were supplemented to the must. Despite the PLS models revealing that lipid composition does seem to influence cold and ethanol tolerance in brewing yeast, we feel that the lipidomics data generated here did not reveal any obvious patterns between the two. It is thus plausible that a combination of other factors, e.g. protein translation and folding efficiencies, mRNA stabilities and the product activity and expression of central metabolic genes [18–24], also contribute to determining cold and ethanol tolerance, and suggest that this topic should be addressed in future studies.
Recent studies have revealed that the creation of de novo lager yeast hybrids has the potential to considerably increase the genetic and phenotypic diversity of lager yeast strains available to the brewing industry. During interspecific hybridization, one is typically limited to combining traits from two different strains. Here we demonstrated the possibility of constructing complex yeast hybrids, through the use of a fertile allotetraploid intermediate, that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains. The presence of unsaturated fatty acids and ergosterol in particular, were shown to benefit growth in the presence of ethanol and at lower temperatures.
Yeast strains and hybrid generation
The yeast strains used in the study are listed in Table 1. The interspecific yeast hybrids H1 (P1 × P2) and H2 (P2 × P3) and intraspecific yeast hybrid H3 (P1 × P2) were generated using rare mating according to the method described in Krogerus et al. . Prior to mating, a uracil auxotroph (ura-) of P1 was selected on 5-fluoroorotic acid (5-FOA) agar , a respiratory-deficient mutant (rho-) of P2 was selected on YPDG agar containing 3% glycerol and 0.1% glucose, and a lysine auxotroph (lys-) of P3 was selected on α-aminoadipic (α-AA) agar  to allow for the selection of hybrids on minimal selection agar medium (0.67% Yeast Nitrogen Base without amino acids, 3% glycerol, 3% ethanol and 2% agar). The interspecific hybrid H1 was further mated with P2 (rho-) to form the interspecific triple hybrid T1 [(P1 × P3) × P2]. Prior to mating, a lysine auxotroph (lys-) of H1 was first selected as described above, after which ascospores of it were generated on 1% potassium acetate agar. Spore viability was calculated based on the amount of colonies formed from the dissection of up to 20 tetrads. Ascus walls were digested with Zymolyase 100T, after which spores of H1 (lys-) were mixed with P2 (rho-). Hybrid T1 was selected on minimal selection agar. Hybrid T2, a meiotic segregant of T1, was created by generating ascospores of Hybrid T1 on 1% potassium acetate agar and then dissecting the spores on YPD agar. All spore dissections were carried out using the Singer MSM 400 dissecting microscope (Singer Instruments, UK). The viable spore clones were then screened for the POF phenotype in a small-scale assay. 1 ml of YPM supplemented with 100 mg L−1 of trans-ferulic acid was inoculated with a colony of the spore clones, and they were allowed to incubate for 48 h at 25 °C. The POF phenotype was determined sensorially by examining for the presence (POF+) or absence (POF−) of the distinct clove-like aroma of 4-vinyl guaiacol. Hybrid T2 was a spore clone of T1 which did not produce 4-vinyl guaiacol in this assay. An overview of these brewing strains and their creation is presented in Fig. 1.
The hybrid status of isolates was confirmed by PCR as described in Krogerus et al. . Briefly, the rDNA ITS region was amplified using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and amplicons were digested using the HaeIII restriction enzyme (New England BioLabs, USA) as described previously . Amplification of the S. eubayanus-specific FSY1 gene (amplicon size 228 bp) and the S. cerevisiae-specific MEX67 gene (amplicon size 150 bp) was also performed using the primers SeubF3 (5′-GTCCCTGTACCAATTTAATATTGCGC-3′), SeubR2 (5′-TTTCACATCTCTTAGTCTTTTCC-AGACG-3′), ScerF2 (5′-GCGCTTTACATTCAGATCCCGAG-3′), and ScerR2 (5′-TAAGTTGGTTGTCAGCAAGATTG-3′) as described by Muir et al.  and Pengelly and Wheals . Additionally, the hybrid statuses of the intraspecific hybrid H3 and the triple hybrids T1 and T2 were confirmed by amplifying interdelta sequences using the delta12 (5′-TCAACAATGGAATCCCAAC-3′) and delta21 (5′-CATCTTAACACCGTATATGA-3′) primers as described by Legras and Karst .
Flow cytometry was also performed on the brewing yeast strains to estimate ploidy essentially as described by Haase and Reed . Cells were grown overnight in YPD medium (1% yeast extract, 2% peptone, 2% glucose), and approximately 1 × 107 cells were washed with 1 mL of 50 mM citrate buffer. Cells were then fixed with cold 70% ethanol, and incubated at room temperature for 1 h. Cells were then washed with 50 mM citrate buffer (pH 7.2), resuspended in 50 mM citrate buffer containing 0.25 mg mL−1 RNAse A and incubated overnight at 37 °C. 1 mg mL−1 of Proteinase K was then added, and cells were incubated for 1 h at 50 °C. Cells were then stained with SYTOX Green (2 μM; Life Technologies, USA), and their DNA content was determined using a FACSAria IIu cytometer (Becton–Dickinson, USA). DNA contents were estimated by comparing fluorescence intensities with those of S. cerevisiae haploid (CEN.PK113-1A) and diploid (CEN.PK) reference strains. Measurements were performed on duplicate independent yeast cultures, and 100,000 events were collected per sample during flow cytometry.
Whole genome sequences of the brewing strains P1 and P3 have been published previously [3, 50]. The other 6 brewing strains were sequenced by Biomedicum Genomics (Helsinki, Finland). In brief, an Illumina NexteraXT pair-end 150 bp library was prepared for each hybrid and sequencing was carried out with a NextSeq500 instrument. Pair-end reads from the NextSeq500 sequencing were quality-analysed with FastQC  and trimmed and filtered with Skewer . Alignment, re-alignment and variant analysis was carried out using SpeedSeq  and its FreeBayes SNP prediction and CNVnator copy number variation prediction modules [54, 55]. Reads of S. cerevisiae P2 were aligned to that of S. cerevisiae S288c , while reads of hybrid strains were aligned to concatenated reference sequences of strains P1 and P3 [3, 50] as described previously . SNPs predicted by FreeBayes with less than five left and right aligning reads were discarded. Prior to SpeedSeq variant analysis, alignments were filtered to a minimum MAPQ of 50 with SAMtools . Quality of alignments was assessed with QualiMap . The median coverage over 10,000 bp windows was calculated with BEDTools  and visualized with R (http://www.r-project.org/). The coverage of S. cerevisiae P2 and the 5 hybrid strains (H1-H3 and T1-T2) across the S. cerevisiae and S. eubayanus reference genomes are displayed in Additional file 1: Figure S3.
The fermentation kinetics and lipid compositions of the 8 brewing strains at two different temperatures (10 and 20 °C) and two different initial ethanol concentrations [0 and 8% (v/v)] were assayed in 100 mL shake-flask fermentations. The strains were grown in media containing 1% yeast extract, 2% peptone, 8% maltose, and up to 8% ethanol. Prior to the assay, the strains were pre-cultivated in media containing 1% yeast extract, 2% peptone and 4% maltose for 24 h at 20 °C. The OD600 of the pre-cultivations was measured, and the growth assays were started by inoculating 100 mL of media to a starting OD600 of 0.01 in triplicate flasks. Flasks were capped and then incubated at either 10 or 20 °C with light agitation (80 RPM) for up to 33 days. Fermentation was monitored (up to twice daily) by drawing 100 μL samples and measuring the refractive index (°brix) with a Quick-Brix 90 digital refractometer (Mettler-Toledo AG, Switzerland). Samples were drawn for lipid analysis at the end of the exponential fermentation phase. 15 mL samples of fermentation media were centrifuged for 5 min at 9000×g, after which the yeast pellet was washed twice in 15 mL of ice-cold deionized water. The washed yeast pellet was then transferred to a cryotube, and flash-frozen in liquid nitrogen. The samples were stored at −80 °C prior to lipid analysis. After fermentations were complete, the biomass concentration determined by drawing and centrifuging 30 mL samples of the fermentation media (10 min at 9000×g), washing the yeast pellets gained from centrifugation twice with 25 mL deionized H2O and then suspending the washed yeast in a total of 6 mL deionized H2O. The suspension was then transferred to a pre-weighed porcelain crucible, and was dried overnight at 105 °C and allowed to cool in a desiccator before the change of mass was measured. Fermentation curves for the fermentations were modelled based on the decrease in °brix over time using the ‘grofit’-package for R . The fermentation parameters were determined using the logistic model in ‘grofit’.
Prior to lipid extraction, the frozen cell samples were freeze-dried at −55 °C overnight (Martin Christ Alpha 1-4 LDplus, Germany). For fatty acid (free and bound) and sterol analysis, 10 mg of freeze-dried sample was rehydrated into 200 µL of 0.9% sodium chloride solution and spiked with heptadecanoic acid (FFA 17:0; 5.36 µg) and glyceryl triheptadecanoate [TG(17:0/17:0/17:0); 21.11 µg]. Lipids were extracted with chloroform:methanol (2:1 v/v; 800 µL) by homogenizing the samples with grinding balls in a Retsch mixer mill MM400 homogenizer (Retsch GmbH, Haan, Germany) at 20 Hz for 2 min. After 30 min standing at room temperature, the samples were centrifuged at 10,620×g for 5 min and the lower layer was separated into glass tubes and evaporated to dryness under nitrogen flow.
The evaporation residues from lipid extractions were dissolved into petroleum ether (b.p. 40–60 °C; 700 µL). Fatty acids were transesterified with sodium methoxide (NaOMe; 0.5 M; 250 µL) in dry methanol by boiling at 45 °C for 5 min. The mixture was acidified with 15% sodium hydrogen sulphate (NaHSO4; 500 µL). The petroleum ether phase containing the fatty acid methyl esters (FAME) as well as free fatty acids (FFA) was collected into an Eppendorf tube and centrifuged (10,620×g; 5 min). Half of the petroleum ether layer was separated into a GC vial and evaporated, after which the residue was dissolved into hexane (100 µL) and taken into a vial insert. FAMEs were analysed on an Agilent 7890A GC combined with an Agilent 5975C mass selective detector controlled by MSD ChemStation software (Agilent Technologies Inc., Santa Clara, CA, USA). The column was an Agilent FFAP silica capillary column (25 m × 0.2 mm × 0.3 µm). Helium was used as carrier gas and the samples were injected in splitless mode. The oven temperature programme was from 70 °C (2 min) to 240 °C at a rate of 15 °C min−1, total run time was 39 min. The temperatures of the injector and MS source were 260 and 230 °C, respectively. The samples (1 µL) were injected by a Gerstel MPS injection system (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) and the data were collected in EI mode (70 eV) at a mass range of m/z 40–600.
The other half of the petroleum ether layer was transferred to a GC vial and evaporated into dryness for the determination of free fatty acid and sterols. The residue was dissolved into 50 µL of DCM and derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA; 40 µL) and trimethylchlorosilane (TMCS; 10 µL) by incubating at 80 °C for 20 min. The samples (1 µL aliquots) were injected in splitless mode at 300 °C, and analysed on an Agilent DB-5MS column (30 m × 0.2 mm × 0.25 µm). The oven temperature programme was from 50 °C (1.5 min) to 325 °C at a rate of 10 °C min−1, total run time was 49 min.
For lipidomics analyses, approx. 5 mg of freeze-dried cell samples were rehydrated in 50 µL of 0.9% sodium chloride in Eppendorf tubes, mixed with 400 µL chloroform:methanol (2:1) and spiked with 10 µL of an internal standard mixture 1 [IS1; containing LysoPC(17:0), MG(17:0), DG(17:0/17:0), TG(17:0/17:0/17:0), PG(17:0/17:0), Cer(d18:1/17:0), PC(17:0/17:0), PE(17:0/17:0), CE(19:0), CA(14:0), C12(β)-d-GluCer and C8-l-threo-LacCer] (Avanti Polar Lipids, Alabaster, AL, USA; Larodan Fine Chemicals AB, Malmö, Sweden; Nu-Chek Prep, Inc., Elysian, MN, USA) at concentration levels of 0.4–3.2 µg/sample. The samples were homogenized with grinding balls in a Retsch mixer mill MM400 homogenizer at 25 Hz for 2 min and after 30 min standing were centrifuged at 10,620×g for 3 min. Before UPLC-MS analysis, a 20 µL aliquot of a labelled lipid standard mixture [IS2; containing LysoPC(16:0-D3), PC(16:0/16:0-D6) and TG(16:0/16:0/16:0-13C3)] (Avanti Polar Lipids, Alabaster, AL, USA) was added into the separated lipid extracts.
Lipid extracts were analyzed on a Waters Q-TOF Premier mass spectrometer combined with an Acquity Ultra Performance LC™ (UPLC) under the control of MassLynx software (v 4.1; Waters Inc., Milford, MA, USA). The column (at 50 °C) was an Acquity UPLC™ BEH C18 (2.1 × 100 mm with 1.7 μm particles). The solvent system included (A) ultrapure water (1% 1 M NH4Ac, 0.1% HCOOH) and (B) LC/MS grade acetonitrile/isopropanol (1:1, 1% 1 M NH4Ac, 0.1% HCOOH). In ESI− mode, the same solvent system but without acid was used. The gradient started from 65% A/35% B, reached 80% B in 2 min, 100% B in 7 min and remained there for the next 7 min. There was a 5 min re-equilibration step before the next run. The flow rate was 0.400 mL min−1 and the injected amount 1.0 μL (Acquity Sample Organizer at 10 °C). The ESI source was at 120 °C and the capillary voltage 3.0 and 2.5 kV in positive and negative mode, respectively. N2 was used as desolvation gas (800 L h−1) at 270 °C.
The data were collected at a mass range of m/z 300–1200 with a scan duration of 0.2 s. Reserpine was used as the lock spray reference compound in ESI+ mode and leucine enkephaline in ESI− mode. Data processing was carried out with MZmine software (version 2.20)  enabling peak integration and alignment of the peaks. An internal spectral MS/MS library was used for identification of the compounds.
Quantification of lipid subspecies was based on peak heights of internal standards. All monoacyl lipids except cholesterol esters, such as monoacylglycerols and monoacylglycerophospholipids, were normalized with LysoPC(17:0), all diacyl lipids except ethanolamine phospholipids were normalized with PC(17:0/17:0), all ceramides with Cer(d18:1/17:0), all diacyl ethanolamine phospholipids with PE(17:0/17:0), and TGs and sterylesters were normalized with TG(17:0/17:0/17:0) and CE(19:0), respectively. Other (unidentified) molecular species were normalized with LysoPC(17:0) for retention time <300 s, PC(17:0/17:0) for retention time between 300 and 410 s, and TG(17:0/17:0/17:0) for higher retention times.
To assess the roles of oleic acid (C18:1) on cold and ethanol tolerance in yeast, microcultures were carried out in media containing various concentrations of supplemented oleic acid (0 or 0.8 mM) and ethanol (1, 5 or 10% v/v). This concentration of oleic acid was chosen based on values found previously in literature . The microcultures were carried out in 100-well honeycomb microtiter plates at 15 and 20 °C (with continuous shaking), and their growth dynamics were monitored with a Bioscreen C MBR incubator and plate reader (Oy Growth Curves Ab, Finland). The wells of the microtiter plates were filled with 300 µL of YPDt medium (1% yeast extract, 2% peptone, 2% glucose, and 1% Tergitol NP-40) supplemented with oleic acid (0 or 0.8 mM) and ethanol (1, 5 or 10% v/v). Oleic acid was added to the media from a 100 × stock solution (80 mM oleic acid) prepared in 50% ethanol and 35% Tergitol NP-40. Precultures of the laboratory strains WT, ole1∆, and erg4∆ (Table 1) were started in 10 mL YPD medium (1% yeast extract, 2% peptone, and 2% glucose) and incubated at 25 °C with shaking at 120 rpm. The cultures were centrifuged and the yeast pellets were washed once with sterile deionized water. The yeast was then resuspended in YPDt medium to an OD600 value of 10. The microcultures were started by inoculating the microtiter plates with 3 µL of cell suspension per well (for an initial OD600 value of 0.1) and placing the plates in the Bioscreen C MBR. The optical density of the microcultures at 600 nm was automatically read every 30 min. Four replicates were performed for each strain in each medium. Growth curves for the microcultures were modelled based on the OD600 values over time using the ‘grofit’-package for R .
Fermentation and analysis
The set of eight brewing strains were characterized in fermentations performed in a 15 °Plato high gravity wort at 15 °C. Yeast was propagated essentially as described previously , with the use of a ‘Generation 0’ fermentation prior to the actual experimental fermentations. The experimental fermentations were carried out in duplicate, in 2-L cylindroconical stainless steel fermenting vessels, containing 1.5 L of wort medium. The 15 °Plato wort (69 g maltose, 17.4 g maltotriose, 15.1 g glucose, and 5.0 g fructose per litre) was produced at the VTT Pilot Brewery from barley malt. Yeast was inoculated at a rate of 15 × 106 viable cells mL−1. The wort was oxygenated to 15 mg L−1 prior to pitching (Oxygen Indicator Model 26073 and Sensor 21158, Orbisphere Laboratories, Switzerland). The fermentations were carried out at 15 °C until an apparent attenuation of 80% (corresponding to approx 7% ABV) was reached, until no change in residual extract was observed for 24 h, or for a maximum of 14 days.
Wort samples were drawn regularly from the fermentation vessels aseptically, and placed directly on ice, after which the yeast was separated from the fermenting wort by centrifugation (9000×g, 10 min, 1 °C). Samples for yeast-derived flavour compounds, fermentable sugars and 4-vinyl guaiacol analysis were drawn from the beer when fermentations were ended. Yeast viability was measured from the yeast that was collected at the end of the fermentations using a Nucleocounter® YC-100™ (ChemoMetec, Denmark).
The alcohol level (% v/v) and pH of samples was determined from the centrifuged and degassed fermentation samples using an Anton Paar Density Meter DMA 5000 M with Alcolyzer Beer ME and pH ME modules (Anton Paar GmbH, Austria). The yeast dry mass content of the samples (i.e. yeast in suspension) was determined by washing the yeast pellets gained from centrifugation twice with 25 mL deionized H2O and then suspending the washed yeast in a total of 6 mL deionized H2O. The suspension was then transferred to a pre-weighed porcelain crucible, and was dried overnight at 105 °C and allowed to cool in a desiccator before the change of mass was measured. The measured pH values and suspended dry mass are presented in Additional file 1: Figure S4.
Concentrations of fermentable sugars (glucose, fructose, maltose and maltotriose) were measured by HPLC using a Waters 2695 Separation Module and Waters System Interphase Module liquid chromatograph coupled with a Waters 2414 differential refractometer (Waters Co., Milford, MA, USA). An Aminex HPX-87H Organic Acid Analysis Column (300 × 7.8 mm, Bio-Rad, USA) was equilibrated with 5 mM H2SO4 (Titrisol, Merck, Germany) in water at 55 °C and samples were eluted with 5 mM H2SO4 in water at a 0.3 mL min−1 flow rate.
Yeast-derived flavour compounds were determined by headspace gas chromatography with flame ionization detector (HS-GC-FID) analysis. 4 mL samples were filtered (0.45 µm), incubated at 60 °C for 30 min and then 1 mL of gas phase was injected (split mode; 225 °C; split flow of 30 mL min−1) into a gas chromatograph equipped with an FID detector and headspace autosampler (Agilent 7890 Series; Palo Alto, CA, USA). Analytes were separated on a HP-5 capillary column (50 m × 320 µm × 1.05 µm column, Agilent, USA). The carrier gas was helium (constant flow of 1.4 mL min−1). The temperature program was 50 °C for 3 min, 10 °C min−1 to 100 °C, 5 °C min−1 to 140 °C, 15 °C min−1 to 260 °C and then isothermal for 1 min. Compounds were identified by comparison with authentic standards and were quantified using standard curves. 1-Butanol was used as internal standard.
4-Vinyl guaiacol was analyzed using HPLC-PAD based on methods described by Coghe et al.  and McMurrough et al. . The chromatography was carried out using a Waters Alliance HPLC system consisting of a Waters e2695 Separations Module equipped with a XTerra® MS C18 column (5 µm, 4.6 × 150 mm) and a Waters 2996 Photodiode Array Detector. The mobile phase consisted of H2O/CH3OH/H3PO4 (64:35:1, v/v) and flow rate was 0.5 mL min−1. The diode array detector was used at 190–400 nm. 4-Vinyl guaiacol was quantified at 260 nm using standard curves of the pure compound (0.3–30 mg L−1).
Stability of Hybrid T2
The karyotype stability of Hybrid T2 was evaluated after 10 successive batch cultures (corresponding to approximately 65 cell generations) in two different media. Media 1 consisted of 1% yeast extract, 2% peptone, 1% maltose and 1% maltotriose, while Media 2 consisted of 1% yeast extract, 2% peptone, 2% maltose and 14% sorbitol. Media 2 was used to mimic the osmotic stress occurring during high-gravity wort fermentations. Hybrid T2 was first grown overnight in YPM at 25 °C. This culture was used to inoculate 1 mL of Media 1 or Media 2 in duplicate to a starting OD600 of 0.1. The cultures were allowed to grow for 7 days at 18 °C, after which they were used to inoculate a fresh 1 mL aliquot of Media 1 or Media 2 to a starting OD600 of 0.1. This was repeated for a total of 10 successive cultures (10 weeks). After this, 20 μL aliquots of the cultures were spread out on YPM agar, and a total of 8 colonies were randomly selected and isolated for further analysis (2 isolates per duplicate per media).
The karyotype stability of the 10-week isolates was assessed by determining their DNA content by flow cytometry as described above, and their karyotypes by pulsed-field gel electrophoresis (PFGE). PFGE was carried out essentially as described previously . Sample plugs were prepared with the CHEF Genomic DNA Plug Kit for Yeast (Bio-Rad) according to the manufacturer’s instructions with minor modifications. Instead of lyticase treatment, the plugs were treated with 0.1 mg mL−1 Zymolyase 100T in buffer containing 1 mM dithiothreitol. The sample plugs were loaded into the wells of a 1.0% pulse field certified agarose (Bio-Rad) gel. PFGE was performed at 14 °C in 0.5 × TBE buffer [89 mM Tris, 89 mM boric acid, 2 mM EDTA (pH 8)]. A CHEF Mapper XA pulsed field electrophoresis system (Bio-Rad) was used with the following settings: 6 V cm−1 in a 120° angle, pulse length increasing linearly from 26 to 228 s, and total running time of 40 h. A commercial chromosome marker preparation from S. cerevisiae strain YNN295 (Bio-Rad) was used for molecular mass calibration. After electrophoresis, the gels were stained with ethidium bromide and scanned with Gel Doc XR+ imaging system (Bio-Rad).
Data and statistical analyses were performed with R (http://www.r-project.org/). The distributions of the lipidomic data were estimated by histograms and the Shapiro–Wilk test, and the lipidomic data was consequently log10-transformed to correct for skewed distributions. The change in lipid composition compared to the control cultivation at 20 °C and 0% supplemented EtOH was tested by Student’s t test (two-tailed, unpaired, and unequal variances). To control for multiple testing, the p values were further adjusted for Benjamini–Hochberg false discovery rate (FDR). Strain-specific differences in fatty acid, squalene and ergosterol concentrations were tested with one-way ANOVA with Tukey’s post hoc test. Multivariate analysis was performed with Partial Least Squares (PLS) and PLS-Discriminant Analysis (PLS-DA) using the ‘ropls’ package in R . PLS-DA was initially performed on the lipid data of all samples in order to determine whether the lipid compositions of the yeast in low temperatures or high alcohol levels could be distinguished from those at control conditions (20 °C, 0% supplemented EtOH). PLS models were constructed from the fermentation and lipid data obtained at the different temperatures and supplemented ethanol levels in order to elucidate which lipid species contributed positively and negatively to fermentation performance at those conditions. The Y response variable of the models was the maximum fermentation level divided by the lag time (A and λ from Table 2, respectively), while the X predictor variables were the combined dataset of the compositions of fatty acids, squalene and ergosterol obtained from GC/MS analysis and the compositions of the 60 lipid species obtained from UPLC/MS lipidomics analysis. PLS(-DA) models were cross-validated (Q 2 > 0.5 was considered significant ), and the significance of the Q 2 value was tested with 200 random permutations of the X dataset.
partial least squares
partial least squares discriminant analysis
saturated fatty acid
single nucleotide polymorphism
unsaturated fatty acid
Conceived and designed the experiments: KK TSL BG. Performed the experiments: KK. Analyzed the data: KK SC TSL. Supervised the work: BG. Wrote the manuscript: KK TSL BG. All authors read and approved the final manuscript.
We thank Annika Wilhelmson for her support throughout, Ulla Lahtinen, Anna-Liisa Ruskeepää, and Airi Hyrkäs for assistance with lipid analysis, Eero Mattila for wort preparation and other assistance in the VTT Pilot Brewery, and Aila Siltala for skilled technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
The Illumina reads generated in this study have been submitted to NCBI-SRA under BioProject number PRJNA357993 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).
This work was supported by the Alfred Kordelin Foundation, Svenska Kulturfonden - The Swedish Cultural Foundation in Finland, PBL Brewing Laboratory, and the Academy of Finland (Academy Project 276480).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Gibson B, Liti G. Saccharomyces pastorianus: genomic insights inspiring innovation for industry. Yeast. 2015;32:17–27.Google Scholar
- Krogerus K, Magalhães F, Vidgren V, Gibson B. New lager yeast strains generated by interspecific hybridization. J Ind Microbiol Biotechnol. 2015;42:769–78.View ArticleGoogle Scholar
- Krogerus K, Arvas M, De Chiara M, Magalhães F, Mattinen L, Oja M, et al. Ploidy influences the functional attributes of de novo lager yeast hybrids. Appl Microbiol Biotechnol. 2016;100:7203–22.View ArticleGoogle Scholar
- Mertens S, Steensels J, Saels V, De Rouck G, Aerts G, Verstrepen KJ. A large set of newly created interspecific Saccharomyces hybrids increases aromatic diversity in lager beers. Appl Environ Microbiol. 2015;81:8202–14.View ArticleGoogle Scholar
- Krogerus K, Magalhães F, Vidgren V, Gibson B. Novel brewing yeast hybrids: creation and application. Appl Microbiol Biotechnol. 2017;101:65–78.View ArticleGoogle Scholar
- Greig D, Borts RH, Louis EJ, Travisano M. Epistasis and hybrid sterility in Saccharomyces. Proc Biol Sci. 2002;269:1167–71.View ArticleGoogle Scholar
- Marinoni G, Manuel M, Petersen RF, Hvidtfeldt J, Sulo P, Piskur J. Horizontal transfer of genetic material among Saccharomyces yeasts horizontal transfer of genetic material among saccharomyces yeasts. J Bacteriol. 1999;181:6488–96.Google Scholar
- Sebastiani F, Barberio C, Casalone E, Cavalieri D, Polsinelli M. Crosses between Saccharomyces cerevisiae and Saccharomyces bayanus generate fertile hybrids. Res Microbiol. 2002;153:53–8.View ArticleGoogle Scholar
- Morales L, Dujon B. Evolutionary role of interspecies hybridization and genetic exchanges in yeasts. Microbiol Mol Biol Rev. 2012;76:721–39.View ArticleGoogle Scholar
- Mancera E, Bourgon R, Brozzi A, Huber W, Steinmetz LM. High-resolution mapping of meiotic crossovers and non-crossovers in yeast. Nature. 2008;454:479–85.View ArticleGoogle Scholar
- Marullo P, Aigle M, Bely M, Masneuf-Pomarède I, Durrens P, Dubourdieu D, et al. Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains. FEMS Yeast Res. 2007;7:941–52.View ArticleGoogle Scholar
- Mukai N, Masaki K, Fujii T, Kawamukai M, Iefuji H. PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. J Biosci Bioeng. 2010;109:564–9.View ArticleGoogle Scholar
- Gallone B, Steensels J, Baele G, Maere S, Verstrepen KJ, Prahl T, et al. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts. Cell. 2016;166(1397–1410):e16.Google Scholar
- Mukai N, Masaki K, Fujii T, Iefuji H. Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production. J Biosci Bioeng. 2014;118:50–5.View ArticleGoogle Scholar
- Gonçalves M, Pontes A, Almeida P, Barbosa R, Serra M, Libkind D, et al. Distinct domestication trajectories in top-fermenting beer yeasts and wine yeasts. Curr Biol. 2016;26:2750–61.View ArticleGoogle Scholar
- Tubb RS, Searle BA, Goodey AR, Brown AJP. Rare mating and transformation for construction of novel brewing yeasts. In: Proceedings of 18th Congress European Brewery Convention. 1981; p. 487–96.
- Gibson BR, Storgårds E, Krogerus K, Vidgren V. Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus. Yeast. 2013;30:255–66.View ArticleGoogle Scholar
- Aguilera J, Randez-Gil F, Prieto JA. Cold response in Saccharomyces cerevisiae: new functions for old mechanisms. FEMS Microbiol Rev. 2007;31:327–41.View ArticleGoogle Scholar
- Deed RC, Deed NK, Gardner RC. Transcriptional response of Saccharomyces cerevisiae to low temperature during wine fermentation. Antonie Van Leeuwenhoek. 2015;107:1029–48.View ArticleGoogle Scholar
- López-Malo M, Querol A, Guillamon JM. Metabolomic comparison of Saccharomyces cerevisiae and the cryotolerant species S. bayanus var. uvarum and S. kudriavzevii during wine fermentation at low temperature. PLoS ONE. 2013;8:e60135.View ArticleGoogle Scholar
- Paget CM, Schwartz JM, Delneri D. Environmental systems biology of cold-tolerant phenotype in Saccharomyces species adapted to grow at different temperatures. Mol Ecol. 2014;23:5241–57.View ArticleGoogle Scholar
- Sahara T, Goda T, Ohgiya S. Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. J Biol Chem. 2002;277:50015–21.View ArticleGoogle Scholar
- Schade B, Jansen G, Whiteway M, Entian KD, Thomas DY, Goethe-university JW, et al. Cold adaptation in budding yeast. Mol Biol Cell. 2004;15:5492–502.View ArticleGoogle Scholar
- Tai SL, Daran-Lapujade P, Walsh MC, Pronk JT, Daran J-M. Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis. Mol Biol Cell. 2007;18:5100–12.View ArticleGoogle Scholar
- Henderson CM, Zeno WF, Lerno LA, Longo ML, Block DE. Fermentation temperature modulates phosphatidylethanolamine and phosphatidylinositol levels in the cell membrane of Saccharomyces cerevisiae. Appl Environ Microbiol. 2013;79:5345–56.View ArticleGoogle Scholar
- Redón M, Guillamón JM, Mas A, Rozès N. Effect of growth temperature on yeast lipid composition and alcoholic fermentation at low temperature. Eur Food Res Technol. 2011;232:517–27.View ArticleGoogle Scholar
- Vicent I, Navarro A, Mulet JM, Sharma S, Serrano R. Uptake of inorganic phosphate is a limiting factor for Saccharomyces cerevisiae during growth at low temperatures. FEMS Yeast Res. 2015;15:1–13.View ArticleGoogle Scholar
- Beltran G, Novo M, Guillamón JM, Mas A, Rozès N. Effect of fermentation temperature and culture media on the yeast lipid composition and wine volatile compounds. Int J Food Microbiol. 2008;121:169–77.View ArticleGoogle Scholar
- Abe F, Horikoshi K. Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae. Mol Cell Biol. 2000;20:8093–102.View ArticleGoogle Scholar
- Abe F, Hiraki T. Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae. Biochim Biophys Acta Biomembr. 2009;1788:743–52.View ArticleGoogle Scholar
- Vanegas JM, Contreras MF, Faller R, Longo ML. Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes. Biophys J. 2012;102:507–16.View ArticleGoogle Scholar
- Henderson CM, Lozada-Contreras M, Jiranek V, Longo ML, Block DE. Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains. Appl Environ Microbiol. 2013;79:91–104.View ArticleGoogle Scholar
- You KM, Rosenfield C, Knipple DC. Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content. Appl Environ Microbiol. 2003;69:1499.View ArticleGoogle Scholar
- Triba MN, Le Moyec L, Amathieu R, Goossens C, Bouchemal N, Nahon P, et al. PLS/OPLS models in metabolomics: the impact of permutation of dataset rows on the K-fold cross-validation quality parameters. Mol BioSyst. 2014;11:13–9.View ArticleGoogle Scholar
- Redón M, Guillamón JM, Mas A, Rozès N. Effect of lipid supplementation upon Saccharomyces cerevisiae lipid composition and fermentation performance at low temperature. Eur Food Res Technol. 2009;228:833–40.View ArticleGoogle Scholar
- Vanbeneden N, Gils F, Delvaux F, Delvaux FR. Formation of 4-vinyl and 4-ethyl derivatives from hydroxycinnamic acids: occurrence of volatile phenolic flavour compounds in beer and distribution of Pad1-activity among brewing yeasts. Food Chem. 2008;107:221–30.View ArticleGoogle Scholar
- Landry CR, Lemos B, Rifkin SA, Dickinson WJ, Hartl DL. Genetic properties influencing the evolvability of gene expression. Science. 2007;317:118–21.View ArticleGoogle Scholar
- Tirosh I, Reikhav S, Levy AA, Barkai N. A yeast hybrid provides insight into the evolution of gene expression regulation. Science. 2009;324:659–62.View ArticleGoogle Scholar
- Dunn B, Sherlock G. Reconstruction of the genome origins and evolution of the hybrid lager yeast Saccharomyces pastorianus. Genome Res. 2008;18:1610–23.View ArticleGoogle Scholar
- Pérez-Través L, Lopes CA, Barrio E, Querol A. Stabilization process in Saccharomyces intra and interspecific hybrids in fermentative conditions. Int Microbiol. 2014;17:213–24.Google Scholar
- Peris D, Moriarty RV, Alexander WG, Baker E, Sylvester K, Sardi M, et al. Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production. Biotechnol Biofuels. 2017;154:78.View ArticleGoogle Scholar
- Alexandre H, Rousseaux I, Charpentier C. Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata. FEMS Microbiol Lett. 1994;124:17–22.View ArticleGoogle Scholar
- Boeke JD, Trueheart J, Natsoulis G, Fink GR. 5-Fluoroorotic acid as a selective agent in yeast molecular genetic. Methods Enzymol. 1987;154:164–75.View ArticleGoogle Scholar
- Zaret KS, Sherman F. α-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants. J Bacteriol. 1985;162:579–83.Google Scholar
- Pham T, Wimalasena T, Box WG, Koivuranta K, Storgårds E, Smart KA, et al. Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery “wild” yeast contaminants. J Inst Brew. 2011;117:556–68.View ArticleGoogle Scholar
- Muir A, Harrison E, Wheals A. A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces. FEMS Yeast Res. 2011;11:552–63.View ArticleGoogle Scholar
- Pengelly RJ, Wheals AE. Rapid identification of Saccharomyces eubayanus and its hybrids. FEMS Yeast Res. 2013;13:156–61.View ArticleGoogle Scholar
- Legras JL, Karst F. Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation. FEMS Microbiol Lett. 2003;221:249–55.View ArticleGoogle Scholar
- Haase SB, Reed SI. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle. 2014;1:117–21.View ArticleGoogle Scholar
- Baker E, Wang B, Bellora N, Peris D, Hulfachor AB, Koshalek JA, et al. The genome sequence of Saccharomyces eubayanus and the domestication of lager-brewing yeasts. Mol Biol Evol. 2015;32:2818–31.View ArticleGoogle Scholar
- Andrews S. FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. 2010.
- Jiang H, Lei R, Ding S-W, Zhu S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinform. 2014;15:182.View ArticleGoogle Scholar
- Chiang C, Layer RM, Faust GG, Lindberg MR, Rose DB, Garrison EP, et al. SpeedSeq: ultra-fast personal genome analysis and interpretation. Nat Methods. 2015;12:1–5.View ArticleGoogle Scholar
- Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv Prepr. arXiv1207.3907 2012;9. http://arxiv.org/abs/1207.3907.
- Abyzov A, Urban AE, Snyder M, Gerstein M. CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. Genome Res. 2011;21:974–84.View ArticleGoogle Scholar
- Engel SR, Dietrich FS, Fisk DG, Binkley G, Balakrishnan R, Costanzo MC, et al. The reference genome sequence of Saccharomyces cerevisiae: then and now. G3. 2014;4:389–98.View ArticleGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map FORMAT AND SAMtools. Bioinformatics. 2009;25:2078–9.View ArticleGoogle Scholar
- García-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, Götz S, Tarazona S, et al. Qualimap: evaluating next-generation sequencing alignment data. Bioinformatics. 2012;28:2678–9.View ArticleGoogle Scholar
- Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–2.View ArticleGoogle Scholar
- Kahm M, Hasenbrink G, Lichtenberg-frate H, Ludwig J, Kschischo M. Grofit: fitting biological growth curves. J Stat Softw. 2010;33:1–21.View ArticleGoogle Scholar
- Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinform. 2010;11:395.View ArticleGoogle Scholar
- Coghe S, Benoot K, Delvaux F, Vanderhaegen B, Delvaux FR. Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae. J Agric Food Chem. 2004;52:602–8.View ArticleGoogle Scholar
- McMurrough I, Madigan D, Donnelly D, Hurley J, Doyle A-M, Hennigan G, et al. Control of ferulic acid and 4-vinyl guaiacol in brewing. J Inst Brew. 1996;102:327–32.View ArticleGoogle Scholar
- Thévenot EA, Roux A, Xu Y, Ezan E, Junot C. Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses. J Proteome Res. 2015;14:3322–35.View ArticleGoogle Scholar
- Meilgaard MC. Prediction of flavor differences between beers from their chemical composition. J Agric Food Chem. 1982;30:1009–17.View ArticleGoogle Scholar
- Engan S. Organoleptic threshold values of some alcohols and esters in beer. J Inst Brew. 1972;78:33–6.View ArticleGoogle Scholar