The inheritance of mtDNA in lager brewing strains

In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Two-dimensional protein map of an "ale"-brewing yeast strain: proteome dynamics during fermentation

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.     

Role of bottom-fermenting brewer's yeast KEX2 in high temperature resistance and poor proliferation at low temperatures

Variants of bottom-fermenting brewer's yeast that grew at high temperatures and showed poor proliferation and fermentation at low temperatures were isolated. Similar variants of laboratory yeast were also isolated and found to be incapable of mating. The KEX2 gene was cloned by complementation. It was shown to be responsible for these traits, because a KEX2 disruptant of Saccharomyces cerevisiae (S. cerevisiae) laboratory yeast grew poorly at low temperatures and was resistant to high temperatures. In addition, a Saccharomyces bayanus (S. bayanus)-type KEX2 (Sb-KEX2) disruptant of bottom-fermenting brewer's yeast grew poorly at low temperatures and was resistant to high temperatures. The KEX2 gene product plays an important role in proliferation of yeast at low temperatures, which is an important trait of bottom-fermenting brewer's yeast. These findings advance our understanding of the proliferation of yeast at low temperatures, especially that of bottom-fermenting brewer's yeast.     

Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts

AIMS: The aim of the present study is to identify genes and proteins whose expression is induced in lager brewing yeast during the lag phase and early exponential growth. METHODS AND RESULTS: Two-dimensional gel electrophoresis was used to identify proteins induced during the lag and early exponential phase of lager brewing yeast in minimal medium. The identified, early-induced proteins were Ade17p, Eno2p, Ilv5gp, Sam1p, Rps21p and Ssa2p. For most of these proteins, the patterns of induction differed from those of the corresponding genes. However, the genes had similar early expression patterns in minimal medium as observed during lager brewing conditions. The expression of previously identified early-induced genes in Saccharomyces cerevisiae grown in minimal medium, ADO1, ALD6, ASC1, ERG4, GPP1, RPL25, SSB1 and YKL056C, was also early induced in lager yeast under brewing conditions. CONCLUSIONS: The results indicate that the above-mentioned genes in general are induced during the lag phase and early exponential growth in Saccharomyces yeasts. The processes in which these genes take part are likely to play an important role during growth initiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge regarding the early growth phase of lager brewing yeast was obtained. Further, the universality of the identified expression patterns suggests new methodologies for optimization and control of growth initiation during brewing fermentations.     

Deciphering the hybridisation history leading to the Lager lineage based on the mosaic genomes of Saccharomyces bayanus strains NBRC1948 and CBS380

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380(T) and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380(T) harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380(T) and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380(T) or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Two-dimensional protein map of an “ale”-brewing yeast strain: proteome dynamics during fermentation

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.     

The impact of media composition and petite mutation on the longevity of a polyploid brewing yeast strain

Ageing in Saccharomyces cerevisiae is a finite phenomenon, determined by replicative, rather than chronological lifespan. Yeast physiological condition is known to influence industrial fermentation performance, however, until recently cellular senescence has not been considered as a brewing yeast stress factor. A polyploid lager yeast (BB11) and a brewery isolate, exhibiting petite mutation were analysed for longevity. It was observed that mitochondrial deficiency induced a reduction in lifespan. In addition, replicative capacity was perceived to be dependent on environmental conditions.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

Effects of Lys and His supplementations on the regulation of nitrogen metabolism in lager yeast

Significant positive correlations between wort fermentability and the assimilation of Lys and His under normal-gravity and high-gravity conditions indicated that Lys and His were the key amino acids for lager yeast during beer brewing. In order to obtain insight into the roles of Lys and His in nitrogen regulation, the influences of Lys, His and their mixture supplementations on the fermentation performance and nitrogen metabolism in lager yeast during high-gravity fermentation were further investigated in the present study. Results showed that Lys and His supplementations improved yeast growth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lys supplementation up-regulated Ssy1p–Ptr3p–Ssy5p (SPS)-regulated genes (LYP1, HIP1, BAP2 and AGP1) dramatically compared to nitrogen catabolite repression (NCR)-sensitive genes (GAP1 and MEP2), whereas His supplementation activated SPS-regulated genes slightly in exponential phase, and repressed NCR-sensitive genes significantly throughout the fermentation. Lys and His supplementations increased the consumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover, Lys and His supplementations exhibited similar effects on the fermentation performance, and were more effective than their mixture supplementation when the same dose was kept. These results demonstrate that both Lys and His are important amino acids for yeast nitrogen metabolism and fermentation performance.     

Variability of HXT2 at the protein and gene level among the Saccharomyces sensu stricto group

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.     

Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene.

The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.     

Kinetics of citrate uptake in growing cells of Leuconostoc spp.

Abstract Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus , first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus . Enological strains classified as S., bayanus , S. chevalieri , and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus . Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.     

Engineering of yeast Put4 permease and its application to lager yeast for efficient proline assimilation

The Saccharomyces cerevisiae Put4 permease is significant for the transport of proline, alanine, and glycine. Put4p downregulation is counteracted by npi1 mutation that affects the cellular ubiquitination function. Here we describe mutant Put4 permeases, in which up to nine lysine residues in the cytoplasmic N-terminal domain have been replaced by arginine. The steady-state protein level of the mutant permease Put4-20p (Lys9, Lys34, Lys35, Lys60, Lys68, Lys71, Lys93, Lys105, Lys107 --> Arg) was largely higher compared to that of the wild-type Put4p, indicating that the N-terminal lysines can undergo ubiquitination and the subsequent degradation steps. Proline is the only amino acid that yeast assimilates with difficulty under standard brewing conditions. A lager yeast strain provided with Put4-20p was able to assimilate proline efficiently during beer fermentations. These results suggest possible industrial applications of the mutant Put4 permeases in improved fermentation systems for beer and other alcoholic beverages based on proline-rich fermentable sources.     

Molecular analysis of alpha-galactosidase MEL genes from Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the alpha-galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1-99.3%), comparable with interspecific homology (94.8-100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae (MELj) was 76.2-81.7%, suggesting certain species specificity. On this evidence, the alpha-galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.