Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

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1 Yeast Yeast 2012; 29: Published online in Wiley Online Library (wileyonlinelibrary.com).2914 Research Article Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance Rosa Garcia Sanchez, Natalia Solodovnikova and Jürgen Wendland* Carlsberg Laboratory, Yeast Genetics, Copenhagen, Denmark *Correspondence to: J. Wendland, Carlsberg Laboratory, Yeast Genetics, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Present address: Carlsberg Research Centre, Applied Research in Yeast and Ingredients, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Received: 2 May 2012 Accepted: 1 July 2012 Abstract Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18 Plato at C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright 2012 John Wiley & Sons, Ltd. Keywords: stress resistance; fermentation; mating type; mitochondria; brewer s yeast Introduction Lager brewing yeast, also known as Saccharomyces carlsbergensis, is a hybrid generated by fusion of S. cerevisiae with S. bayanus-type yeasts (Nilsson-Tillgren et al., 1981). The S. bayanus species has long been sought after and one contemporary representative was found recently in South America and named S. eubayanus (Libkind et al., 2011). In contrast ale, distiller s, and wine yeasts are predominantly strains of S. cerevisiae (Azumi and Goto-Yamamoto, 2001). Lager yeast has been adapted to low-temperature fermentations and, not surprisingly within the Saccharomyces sensu stricto group, S. bayanus is more cold-tolerant than Copyright 2012 John Wiley & Sons, Ltd. S. cerevisiae (Giudici et al., 1998). Interestingly, lager brewing yeast strains were found to harbour predominantly S. bayanus-type mitochondrial DNA, suggesting a selective advantage under low-temperature lager beer fermentation conditions (Rainieri et al., 2008). Maltose and maltotriose account for 80% of the total amount of fermentable sugars in wort. Maltose transporters with different temperature optima have been identified in ale and lager yeasts; this provides some molecular evidence in favour of lager yeasts in fermentations at lower temperatures (Vidgren et al., 2010). Industrial fermentation conditions impose several different stresses upon yeast cells, from pitching into aerated wort until cropping of yeast cells at

2 344 R. Garcia Sanchez et al. the end of fermentation (reviewed in Gibson et al., 2007). Factors that could be considered are high osmolarity/sugar content of the wort in the beginning, oxygen limitation during fermentation, oxidative stress, nutrient limitation and ethanol stress in the end of fermentation, particularly during secondary fermentation/maturation. Prior to their re-use, yeast cells may be stored in storage tanks at low temperature. These conditions are chosen to maximize the efficiency of fermentation but may result in decreased yeast viability and an increased amount of respiratory-deficient petite cells at the end of fermentation. Yeast possesses a general stress response to deal with challenging environments (Gasch et al., 2000). This response requires the zinc-finger transcription factors Msn2/4 that bind to stressresponse elements in target promoters, thereby activating gene expression (Martinez-Pastor et al., 1996). Contrary to the idea that a robust stress response may result in increased fermentation performance, Watanabe et al. (2011) showed that in sake yeasts dysfunctional MSN2 and/or MSN4 actually allowed for a higher initial rate of fermentation. This suggests that other parameters intrinsic to yeast physiology play an important role in the stress response. Examples of that could be the ability to detoxify inhibitors from the medium, ability to accumulate storage carbohydrate/ compatible solutes or the membrane composition of the yeast cell (Gibson et al., 2007). Since the exploration of the sexual cycle of yeast by Ojvind Winge (1935), yeast has become a model system for eukaryote genetics, molecular and cell biology (Botstein and Fink, 1988, 2011). This awesome power of yeast genetics, however, reaches some limits with allotetraploid lager yeast. As with other hybrids, e.g. the mule generated from horse and donkey, lager yeast have a crippled sexual reproduction potential. Sporulation frequency in lager yeast is very low and the occurrence of four-spored asci even rarer, and the viability of spores is an additional impediment (Hansen and Kielland-Brandt, 2003). Nevertheless, classical breeding is possible (Gjermansen and Sigsgaard, 1981). Yet, starting from an alloploid strain, its spores can be aneuploid as well. Such spores can give rise to new yeast lines that may be mutagenized and selected for specific traits. Subsequently, the ability to mate will be required to reconstitute a lager yeast strain. This explains why lager yeast breeding is much more challenging than classical genetics with S. cerevisiae. Furthermore, the uncertainty of the genome composition of spore clones constitutes a black box that adds an additional level of complexity. Nevertheless, lager yeast breeding depends on suitable spore clones. Diploid spore clones allow for the selection of recessive mutants upon mutagenesis, thereby opening possibilities to generate novel traits in lager yeasts (Gjermansen, 1983). Due to the decision not to use genetically modified recombinant yeast strains for production, classical genetics and selection procedures have to be employed to generate diversity in yeasts used for the brewing industry (Pretorius, 2000). We are therefore interested to further develop lager yeast breeding by introducing new traits or molecular markers. In this study we show: (a) that breeding lager yeast spore clones with S. cerevisiae laboratory yeast strains or ale yeasts enhances the stress-resistance profile and the fermentation performance of the new hybrids; and (b) that assessment of mating type and other molecular markers by PCR can be used to select suitable spore clones. This study, therefore, provides an advance to classical yeast breeding employed in the beer and wine industry. Materials and methods Strains and cultivation conditions Strains used in this study are listed in Table 1. All strains were stored in 15% glycerol at 80 C. BY strains were obtained from EUROSCARF (Frankfurt, Germany). Yeast strains were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose). For auxotrophic selection, synthetic medium (YNB with ammonium sulphate; Bio 101 W Systems) supplemented with CSM lacking uracil and adenine (Bio 101 W Systems) and 2% glucose was used. Propagation of yeast strains was done at either room temperature or 30 C. For solid medium, 20 g/l agar-agar was added. Sporulation media contained 1% potassium acetate, 0.1% yeast extract and 0.05% glucose. Sporulation plates were incubated at 22 C for up to 2 weeks. For petite cell quantification, YP glycerol (1% yeast extract, 2% peptone, 2% glycerol) plates

3 Yeast breeding 345 Table 1. Yeast strains used in this study Strain Genotype/description Source BY4741 MATa; his3δ1; Δleu2; Δmet15; Δura3 EUROSCARF BY4742 MATa; his3δ1; Δleu2; Δlys2; Δura3 EUROSCARF BY4743 MATa/a; his3δ1/his3δ1; Δleu2/Δleu2; Δmet15/MET15; Δura3/Δura3; LYS2/Δlys2 EUROSCARF Lager yeast S. carlsbergensis MATa/a Carlsberg Research Centre Wine yeast S. cerevisiae Carlsberg Research Centre WS34/70 Weihenstephan lager yeast Carlsberg Research Centre CG1161 MATa;Δhis4, Δleu2,Δade2 lager yeast spore clone Carlsberg Research Centre CG1162 MATa; Δhis4, Δleu2, Δade2 lager yeast spore clone Carlsberg Research Centre Ale yeast S. cerevisiae MATa/a Carlsberg Research Centre BC1 BY4741 CG1162 This study BC2 BY4742 CG1161 This study were used. YP galactose (1% yeast extract, 2% peptone, 2% galactose) plates supplemented with 50 mg/ml X-a-gal were used to screen for yeast strains with a-galactosidase activity or melibiose utilization. For stress sensitivity assays on plates, yeast strains were grown overnight in YPD, harvested, washed with sterile water and diluted in water. This dilution was used for the first spot on the plate. The cell suspension was serially diluted 10-fold for subsequent spots. Aliquots of 7 ml/spot were applied. For fermentation experiments, either YP maltose containing 1% yeast extract, 2% peptone and 25% maltose or a wort-like medium was used with lager, ale and lager ale hybrid yeast strains. The wort-like medium consisted of 180 g/l granulated malt (GranMalt Granules GM100, GranMalt AG, Freising-Attaching, Germany) supplemented with 10 g/l yeast extract. Molecular biology techniques PCR was carried out on whole cells as described previously (Walther and Wendland, 2008). Primers were synthesized by biomers ( or by Integrated DNA Technologies (eu.idtdna. com). Amplification of the COX2 gene was done with primers according to Belloch et al. (2000). Sequences of COX2 from S. cerevisiae, S. pastorianus and S. bayanus were compared and aligned with the DNASTAR. Nucleotide polymorphisms of the COX2 sequences between S. pastorianus/ S. bayanus and S. cerevisiae establish restriction fragment length polymorphisms when using PvuII as a diagnostic restriction enzyme. Dissecting mated yeast cells by micromanipulation Cells of two strains to be mated were mixed on the same spot on a YPD plate and then incubated at 22 C until zygote formation was observed. Microscopic observation over time indicated that zygotes were formed after h of incubation. Cells presumed to be zygotes were micromanipulated using a Singer Instruments MSM300 on new YPD plates and then incubated at 22 C. Colonies were then restreaked to isolate colonies derived from single cells. The hybrid nature of these colonies was checked by PCR. Microscopy Microscopy was done using an AxioImager (Zeiss, Germany) equipped with a MicroMax1024 CCD camera (Princeton Instruments, USA) driven by Metamorph software (Molecular Devices, USA). Analysis of the fermentation performance Cell inocula for the fermentations were propagated for 3 days in test tubes, using 5 ml on the same medium used for fermentation experiments. Media were either YP maltose or granulated malt, depending on the strain requirements. Inocula were propagated at 22 C on a rotational shaker wheel to provide aeration. Fermentations were done at C in 250 ml measuring cylinders filled with 200 ml medium. The cells were pitched to a final optical density of OD 600nm = 0.4 for YP maltose and 0.2 for granulated malt medium. Fermentations were stirred at 190 rpm using a magnetic

4 346 R. Garcia Sanchez et al. stirrer. Fermentation progress was monitored by weighing the cylinders to measure the CO 2 loss, and by measuring the sugar content (gravity in Plato), using a DMA 35 Anton Paar densitometer. Fermentations were regarded as finished when 0 Plato were reached for the YP maltose medium or when maximal attenuation was reached for granulated malt medium. At the end of fermentations, aliquots of the cultures were used to determine the total amount of cells, survival rate, amount of petite cells (amount of viable cells on YPD amount of viable cells on YP glycerol) and for repitching experiments. All experiments were carried out using biological duplicates and technical triplicates. Vitality was evaluated by comparing total cell counts vs colony-forming units (CFU). Results Analysis of stress-resistance profiles of lager yeast strains Lager yeast is adapted to low-temperature fermentation conditions. We used several lager yeast strains from different origins to compare their sensitivity to high-temperature, high-salt and ethanol stress (Figure 1A). S. cerevisiae strains, either laboratory yeast or wine yeast, were much more robust under these conditions than all lager yeasts, although within lager yeast strains different levels of sensitivity against high ethanol concentrations were observed. This is of interest, as lager yeasts may experience high osmotic stress at the beginning of fermentation and ethanol stress at the end Figure 1. Lager yeasts are more sensitive to high-temperature and high-salt stress than S. cerevisiae strains. Yeast strains were grown overnight in YPD with 2% glucose, washed with water and adjusted to an OD 600nm = 0.08, followed by 1:10 serial dilutions that were spotted on YPD plates supplemented with 10 12% ethanol or 1 M NaCl where indicated. Plates were incubated at either 30 Cor37 C (A, B). Representative pictures were taken after 5 days of growth. S. cerevisiae strains: BY laboratory yeast strains and a wine yeast strain. Lager yeast strains are commonly referred to as S. carlsbergensis. CG strains are lager yeast spore clones and BC strains are derived from crosses of BY and CG strains

5 Yeast breeding 347 of fermentation (Gibson et al., 2007). Lager yeast hybrids are very poor sporulaters. To facilitate lager yeast breeding, we previously generated lager yeast spore clones with distinct mating behaviour and his4, leu2, ade2 auxotrophies (Gjermansen, unpublished). We made use of two of these strains that mated either as MATa (CG1161) and MATa (CG1162) and mated them to the corresponding BY4742 (MATa, ura3) or BY4741 (MATa, ura3) laboratory strains. Selection on SD ade ura plates was used to identify the backcross strains BC1 (BY4741 CG1162) and BC2 (BY4742 CG1161). The stress profile of BC1 and BC2 strains was compared with the parental strains and a lager yeast production strain (Figure 1A, B). We found that a single backcross generated F1 hybrids that were clearly improved in their resistance profile over lager yeasts. To further characterize these new hybrids, we performed bench-top fermentations using high-gravity maltose medium (25ºPlato) at 22 C. We were especially interested in higher temperature fermentation performance instead of fermentations under lager brewing conditions (10 15 C), as we hypothesized that an increase in S. cerevisiae genome content would also improve the performance at higher temperatures, e.g. with respect to total time of fermentation. Fermentation curves of these F1 hybrids, i.e. sugar consumption and weight loss generated by CO 2 production, were compared to a S. carlsbergensis lager yeast strain (Figure 2). It was evident that under high-gravity brewing conditions and at elevated temperature the new hybrids fermented maltose faster than lager yeast. This feature was stable upon repitching (not shown). At the end of fermentation, yeast cells were cropped and their robustness was analysed by quantifying the percentage of cell survival and the rate of petite cell formation (Figure 3). Interestingly, BC1 and BC2 provided different results. The amount of viable cells at the end of fermentation was higher in BC2 than in BC1, yet both strains had an improved rate of viable cells compared to the lager yeast strain (Figure 3A). The faster fermentation of the BC strains could thus be a consequence of the increased vitality/ viability throughout fermentation. The amount of petite cells at the end of fermentation showed an average of ca. 4% petite cells for the lager yeast strain. This is within the normally observed range in lager yeast. BC1 performed less well in this respect, with an elevated count for petites. Remarkably, cells of strain BC2 cropped at the end of fermentation contained only a very small fraction of petites (Figure 3B). Fermentation performance and analysis of parameters at the end of fermentation thus clearly showed the possibility for improvement of lager brewing yeast upon breeding with S. cerevisiae. Figure 2. Lager yeasts show an increased resistance to oxidative stress. Representative fermentation kinetics of the BC1 and BC2 hybrid strains in comparison with a lager yeast strain. High gravity fermentation was done with YP maltose as carbon source adjusted to 25 Plato. Fermentation temperature was 22 C. Data are averaged (n > 4). The plot contains reduction of sugar content as well as increase in weight loss (g/l) over time Figure 3. End-of-fermentation analysis of BC-hybrids The amount of viable cells (CFUs, compared to total cell count) was indicated as survival rate (A) and the percentage of petite cells of CFUs after end-fermentation was determined. Petite cells, i.e. cells that could not use glycerol as carbon source, were determined by replica-plating from YPD plates on YP glycerol plates. Data are averaged from two independent experiments. SEM is shown (n > 6)

6 348 R. Garcia Sanchez et al. Mating-type genotype in lager yeast spore clones may not correspond to mating behaviour This encouraging result of strain improvement based on breeding lager yeast spore clones with S. cerevisiae promoted a more detailed study. Lager yeast is a hybrid between two Saccharomyces species. Usually, one parent is of S. cerevisiae origin while the other is closely related to S. bayanus (Libkind et al., 2011). Based on this hybrid nature lager, yeasts generally sporulate very poorly. Added to this is the uncertainty of the chromosomal content of spore clones ( 2n or allodiploid). To disclose the MAT genotype and the actual mating behaviour of spore clones, we used PCR with MATa- and MATa-specific primers (Huxley et al., 1990). This generated the somewhat surprising result that often lager spore clones were heterozygous at the MAT locus and yet showed a specific mating behaviour with tester strains (Figure 4a). This method does not distinguish copy numbers of MAT loci; thus, at present we do not know whether an imbalance at MAT or the presence of cerevisiae/non-cerevisiae MAT-loci generates this non-canonical mating ability. In our limited study we found more a- than a-maters in lager yeast spore clones of different origin. From a breeding perspective, we favour a strain in which mating behaviour is matched by MAT locus composition (Figure 4a). In contrast to lager yeasts, ale and distiller s yeasts are generally non-hybrid strains of S. cerevisiae. Thus, to generate a more robust lager yeast strain, we aimed at crossing lager yeast with an ale yeast to combine the properties of two experienced wort-fermenting strains. To this end we also generated spore clones of an ale yeast strain (MATa/ MATa) and analysed their MAT locus composition (Figure 4b). Also in this case we identified strains with both mating types (which could simply have been diploid cells) and selected only strains with consistent mating-type and mating behaviour for further breeding (see Table 1 for list of strains). Mating properties of the ale isolates were stable and were confirmed by PCR and with mating to tester strains BY4741 (MATa) and BY4742 (MATa) (not shown). Lager vs ale yeast hybrids were generated by mixing the strains and using micromanipulation to isolate zygotes (Figure 5A). Of the isolated cells, < 50% formed colonies. These were further analysed by PCR to identify strains that indeed carried both mating-type loci (Figure 5B). Due to the appearance of odd-looking cells, not all Figure 4. Assessment of yeast strain mating type by colony PCR. Cells from the indicated strain were used for colony-pcr, using three primers to amplify either a MATa- ormata-specific band. Equal amounts of these PCR reactions were separated on agarose gels. Ethidium bromide-stained gel images are shown (A, B). Strains from (A) were further used in mating reactions against haploid tester strains. The respective results are shown as mating phenotype. The ale yeast spore clones were derived from four-spored asci. Strains with both MAT loci are regarded as diploids. Whenever a single band was observed in the mating type PCR, it corresponded with the mating phenotype

7 Yeast breeding 349 Figure 5. Generation of lager ale hybrids via micromanipulation and mating type PCR. (A) Cells of opposite mating type of lager and ale yeast spore clones were mixed and cells resembling zygotes were isolated via micromanipulation. (B) Derived colonies of these zygotes were analysed by mating type PCR. Only strains containing both MATa and MATa bands are hybrids. Here the identification of seven of the 13 lager ale hybrids is shown. Strains with single bands are, therefore, ale yeast strains. (C) The same colonies as in (B) were assayed for the S. bayanus ORF YBR033W, using specific primers that do not amplify the S. cerevisiae YBR033W homologue of the cells originally manipulated represented true hybrids. Lager yeasts generally can utilize melibiose (galactose-glucose disaccharide), due to the presence of MEL genes. This feature was analysed by growth with the chromogenic substrate X-a-gal, which is converted to a blue dye in the presence of a secreted a-galactosidase (not shown). The MEL + phenotype combined with growth at 37 C can corroborate the generation of lager ale hybrids. We employed a growth assay at 37 C to eliminate lager yeast cells and then verified the presence of the lager yeast S. bayanus copy of YBR033W, using specific primers (Figure 5C; Torriani et al., 2004). This, in combination with the MAT locus PCR, unambiguously identified 13 lager ale hybrids (C600 C612). These new hybrids were tested for stress resistance against high temperature and high salt concentration (Figure 6). The parental strains segregated in phenotype according to lager and ale yeasts. Ale yeasts and their spore clones were generally more resistant to higher temperature and salt stress than lager yeasts and their derivatives. As seen with the previous backcrosses, BC1 and BC2 also in the lager ale yeast hybrids, the resistance pattern of all of the hybrids resembled that of the ale yeast parent (Figure 6A, B). Thus, similarly to the initial experiment, the lager ale hybrids were also more robust and stress-resistant than lager yeast strains. The fermentation properties of these lager ale hybrids were then tested using granulated malt (18 Plato) in 200 ml fermentations at 18 C and 25 C (Figure 7A D). Most of the strains performed between the ranges of their lager or ale yeast

8 350 R. Garcia Sanchez et al. Figure 6. Stress profile of lager ale hybrids compared with the parental strains. The novel lager ale hybrids and their parental strains were grown and spotted as described in Figure 1. Stress conditions tested were high temperature (37 C, A) and high salt (B). Pictures were taken after 5 days of growth

9 Yeast breeding 351 Figure 7. Analysis of fermentation performance of lager ale yeast hybrids. Representative fermentation profiles are shown of lager yeast, ale yeast and two hybrids thereof, C601 and C612, which showed most pronounced characteristics. Fermentations were done using high-gravity granulated malt at 18 C (A, B) and 25 C (C, D). Sugar consumption is indicated by the decrease in Plato over time (A, C) and weight loss by CO 2 release is indicated in (B, D) (g/l). (E) The amounts of viable cells for each strain were quantified parents. Lager yeast strains could utilize more total sugar of the granulated malt and reached a lower Plato value at the end of fermentation. Most hybrids displayed fermentation performances of the lager type, while fewer performed more similarly to the ale type. As an example, strain C601

10 352 R. Garcia Sanchez et al. showed a typical ale yeast fermentation curve, while another, C612, showed a lager yeast fermentation profile. Interestingly, C612 could consistently utilize more sugars from the granulated malt than its lager yeast parent and also shortened the total fermentation time (Figure 7, Table 2). To analyse whether the increased robustness of the lager ale hybrids also resulted in better survival at the end of fermentation, we quantified the total amount of cells and the viable colonyforming cells after fermentation at 18 C was completed (Figure 7E). This indicated that the lager ale hybrids, similar to the ale parental strain, generally had an increased total cell count and also an increase in the fraction of viable cells compared to the lager yeast parental strain. However, we did not identify a strain that was improved in these properties compared to the ale yeast parent. During hybridization we expected that the hybrids would maintain only one type of mitochondrial DNA (uniparental inheritance). Therefore, we used restriction length polymorphisms of ale and lager COX2 genes to determine the mito-dna inheritance in our lager ale hybrids (Figure 8A). Ten of these strains carried ale mito-dna, two lager mito-dna and one strain was found to be petite, which corresponds to the colony morphology of this strain and its inability to utilize glycerol as carbon source. Within our set of hybrids, two strains, C604 and C605, were derived from the same parental spore clones. Thus, these strains are isogenic and differ only in their mito-dna. The fermentation profiles of both strains, using 18 Plato/18 C as fermentation conditions, were very similar (Figure 8B). Furthermore, our best performing isolate, C612, was also found to contain ale-type mito-dna. Table 2. End-of-fermentation performance of yeast strains Strain Final Plato at 18 C Final weight loss (g) at 18 C Final Plato at 25 C Final weight loss (g) at 25 C Lager yeast Ale yeast C C Figure 8. Analysis of mitochondrial DNA inheritance in lager ale yeast hybrids. Mitochondrial DNA typing of lager ale hybrids (C600 C612) and their parental lager and ale strains was based on PCR amplification of COX2 alleles and subsequent digestion with the restriction enzyme PvuII. For each strain, the left band corresponds to the uncleaved PCR product, while the right band shows the PvuII cleavage pattern. The PvuII restriction pattern for the lager yeast COX2 contains two bands, while those for S. cerevisiae and ale yeast COX2 show three bands

11 Yeast breeding 353 Discussion Lager yeast, Saccharomyces carlsbergensis (Hansen), is a natural hybrid between S. cerevisiae and another Saccharomyces species, represented by a strain closely related to S. eubayanus (Dunn and Sherlock, 2008; Libkind et al., 2011). The two parts in the lager yeast genome can be easily distinguished, based on the very high sequence identity of the lager yeast cerevisiae part to S. cerevisiae (> 95%) and the lower identity of the lager yeast non-cerevisiae part (< 90%) to S. cerevisiae (Nakao et al., 2009). Lager beers, which represent the largest part of the beer market, are traditionally fermented at lower temperatures using bottomfermenting lager yeast strains, while ale yeasts ferment at higher temperatures and are top-fermenting. Ale yeasts, similarly to wine and distiller s yeasts, are mostly S. cerevisiae strains. Before the onset of purifying lager yeast strains and using pure cultures for beer brewing, lager yeasts were propagated over hundreds of years. This selected for lager yeast strains able to ferment efficiently at lower temperatures and able to survive severe fermentation conditions, e.g. high osmotic pressure, high alcohol content and low temperature. Not surprisingly, lager yeasts are cold-adapted and do not grow at 37 C (Cardinali and Martini, 1994). The cold adaptation of lager yeast may have been inherited from the non-cerevisiae part of the genome (Sato et al., 2002). However, we found that generally lager yeast strains are more sensitive to high ethanol and high salt concentrations than S. cerevisiae strains. We therefore wanted to make use of the increased stress resistance of S. cerevisiae and chose an ale yeast as breeding partner for lager yeast to increase the stress resistance in the novel lager ale hybrids. Initially, we used mating assays based on cross-complementation of auxotrophies to select for hybrids. Sato et al. (2002) described a procedure of mass-mating S. cerevisiae with lager yeast, then screening cells that grew at high temperature (removing lager yeast cells) for the ability to utilize X-a-gal, thus identifying novel hybrids. However, their goal was to improve low-temperature fermentation performance. Our approach of making use of natural isolates enabled us to select for mated strains under non-restrictive conditions, e.g. selection of growth at a certain temperature or based upon a certain sugar utilization pattern. This approach can also be applied in the breeding of industrial yeast strains, e.g. with wine yeasts or yeast strains used in bioethanol production, where phenotypic characteristics of breeding partners may be more even. We show that using molecular markers, e.g. MAT locus distribution, selection of hybrids can be facilitated. All of our F1 lager ale hybrids showed a stress response profile similar to S. cerevisiae, while fermentation profiles were either parental or a mixture of lager and ale yeast profiles. A relevant parameter for fermentation performance is the number of respiratory-deficient petite cells accumulated during fermentation, particularly when re-using (repitching) yeasts for consecutive fermentations. These cells contribute to off-flavour production and reduced fermentation (Gibson et al., 2008). Remarkably, one of the BC strains generated in our study was significantly reduced in petite formation and also showed a higher survival rate at the end of fermentation. Combined with a faster attenuation of high-gravity wort under the fermentation conditions used, our study clearly demonstrates the potential of strain improvement by constructing lager ale hybrids. Breeding of lager yeast using conventional classical genetics is important, as genetically modified strains are not used in production. Therefore, tools need to be developed to characterize breeding partners in sufficient ways to minimize strain-handling procedures. However, lager yeast hybrids are very poorly sporulating strains. The underlying incompatibility leading to hybrid sterility was addressed in recent studies. The S. bayanus AEP2 gene was shown to be incompatible with S. cerevisiae mitochondria, leading to a sporulation defect in lager-type hybrids (Lee et al., 2008). Conversely, the same group also identified the S. cerevisiae AIM22 gene to be incompatible with S. bayanus mitochondria (Chou et al., 2010). Mitochondrial inheritance can therefore be important for yeast breeding. Even though we have generated only a dozen lager ale hybrids, we have noticed a bias towards S. cerevisiae mitochondrial DNA inheritance. Similar results of preferential inheritance of S. cerevisiae mitochondrial DNA were reported previously (Lee et al., 2008), which may be due to faster replication of Sc-MT-DNA over Sb-MT-DNA (Piskur et al., 1998). Another pressing issue is the generation of spore clones from allotetraploid lager yeast that display a stable mating type. We identified lager yeast spore

12 354 R. Garcia Sanchez et al. clones that mated as either a or a cells yet showed a/a genotype at the MAT locus. The molecular mechanisms allowing these lager yeast spore clones to escape cell-type control are currently not understood the simplest explanation could be non-functional MAT alleles or a copy number effect unbalancing the ratios of MATa1 to MATa proteins (Schmidlin et al., 2008). It may also be interesting to analyse the effects of cerevisiae vs non-cerevisiae Rap1 on the expression of mating type genes on lager yeast, as a temperaturesensitive rap1 mutant has been shown to produce bi-maters at the semi-permissive temperature (Giesman et al., 1991). Breeding with MATa and MATa spore clones generates zygotes that are MATa/a as expected. The sporulation frequency of the lager ale hybrids varied between lager type (< 5%) or ale type (> 80%) or was intermediate. The spore germination frequency was generally low (0 5%) and only two strains (C602 and C610) produced a large number of viable spores. This generates some difficulties when using these strains for further breeding. However, further breeding may be necessary, as, for example, flavour profiles may change considerably in lager ale hybrids compared to the lager parent. This is due to the different flavour profiles of ale yeast strains compared to lager yeast strains. Upon breeding we can expect some mixed or intermediary flavour profiles in the hybrids. This may not be acceptable when conservation of a lager beer flavour profile is wished for. In conclusion, our experiments show that lager yeast strains can be improved by crossing of lager yeast spore clones with S. cerevisiae strains, which results in dominantly increased stress resistance of the F1 hybrids, in increased attenuation of sugars in certain hybrids and in increased survival of cells at the end of fermentation. The molecular tools implemented here, e.g. use of mating-type PCR and characterization of mitochondrial inheritance, can facilitate mating and promote yeast breeding of industrial yeast strains. Acknowledgements The authors wish to thank Claes Gjermansen and Carlsberg Applied Research for the construction of lager yeast spore clones used in this study, and Andrea Walther and Klaus Lengeler for reviewing and discussions. References Azumi M, Goto-Yamamoto N AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering. Yeast 18: Belloch C, Querol A, Garcia MD, Barrio E Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome c oxidase II gene. Int J Syst Evol Microbiol 50: Botstein D, Fink GR Yeast: an experimental organism for modern biology. Science 240: Botstein D, Fink GR Yeast: an experimental organism for 21st century biology. 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