As the species of the genus Saccharomyces —and particularly S. In particular, in the beer industry, the goal of the use of inoculated yeast is to increase the fermentation efficiency, to develop new beers, and especially to enhance the sensory complexity of the beer that is produced. Brewing yeast are mainly classified as the top-fermenting or ale yeast of S. These two yeast species can be differentiated in terms of their temperature of fermentation, sugar assimilation, genomic organization, evolutionary domestication, and phylogenesis.
In general, brewing yeast requires high water activity, and in high sugar-containing wort they can overcome this stress condition through the overproduction of osmolytes, such as glycerol and trehalose, to protect their cell membranes.
These osmolytes can replace water, to restore cell volume and osmotic pressure, and thus to allow regular yeast metabolism. For their pH requirements, yeast cells grow well between pH 4.
During nutrient transport, yeast cells acidify their environment through a combination of proton secretion, direct secretion of organic acids, and CO 2 dissolution. Oxygen is required as a growth factor for the biosynthesis of their membrane fatty acids and sterols. As well as oxygen, yeast cells require the macronutrients i. The micronutrients required by yeast cells i. Generally, the malt wort provides growth factors, although in certain cases it can be necessary to supplement the wort with commercial yeast growth factors, as a mix of yeast extract, ammonium sulfate, and minerals e.
These are derived from the hydrolysis of the starch by malt amylases during malting. However, S. Carbohydrate uptake during wort fermentation adapted from Ref. The uptake of nutrients by yeast depends on the nutrient type, the yeast species, and the fermentation conditions.
Generally, glucose is transported through the cell membrane into the cell by facilitated diffusion, and maltose by active transport.
A high glucose concentration in the wort can suppress the assimilation of maltose and other sugars i. As a source of nitrogen, brewing yeast require assimilable organic e.
Again, high levels of ammonium ions in the wort can suppress the uptake of amino acids i. Amino acid uptake occurs through two transport systems: general amino acid permease GAP and specific transporters for the different amino acids. The dissimilation of amino acids i.
Over time, there was gradual domestication and selection of yeasts [ 3 ]. Moreover, there are two counteracting forces that act on yeast selection: yeast research needs to homogenize biological systems and to refer different yeast strains to species, while brewers need to differentiate and select yeast strains based on their fermentation characteristics. Ale strains of S. However, the strains of S. Brewers have selected yeast strains over the centuries for the stability of their traits, and this has resulted in low spore viability and yeast that are deficient in sexual recombination.
Brewing strains belonging to S. The origin of these strains has only recently been discovered through comparative genomic analysis, which revealed that S. The production of lager beers started in central Europe around the end of the fourteenth century.
Recently, it was shown that on the basis of the sequence of different isolates of lager beer strains, S. One of these is associated with breweries in Denmark i. The other lineage is from Germany i. Furthermore, the present-day S. In European wild environments, S. Cold-tolerant yeast hybrids adapted well through serial passages during lager production, and these have become the dominant strains. Indeed, S. Source and selection of brewing yeast species adapted from Ref. Other species can contribute to wort fermentation and beer quality, including wild strains and species in open and uncontrolled fermentations.
Brettanomyces bruxellensis is characteristic of the fermentation of Belgian lambic and geuze beers. Recently, genomic differences were reported for the B. This study revealed that 20 genes in the spoilage strain genomes have been deleted in the brewing strains, many of which are involved in carbon and nitrogen metabolism. DNA fingerprinting has revealed that brewing strains have a unique profile, which means that they can be distinguish from spoilage strains. Thus, as for Saccharomyces strains, the selection undergone by Brettanomyces spp.
Generally, at the end of the boiling, the wort contains all of the nutrients that are required for yeast growth and fermentation. Thus, from a microbiological perspective, the main aspects to consider are not whether the wort is suitable for yeast growth, but rather what is the balance of the flavor compounds that will be produced by the yeast. Mathematical models have been developed to predict the final concentrations of some of these volatile compounds from the known quantities of their precursor s in the wort [ 6 ].
However, the application of these methods requires particularly deep knowledge of the wort composition, while brewers usually conduct very basic measurements in their evaluation of wort quality. For example, there are indications that small changes in the spectrum of the wort amino acid composition can result in dramatic changes in the final beer aroma. The most important metabolites synthesized by yeast and related to beer quality are sulfur compounds, organic and fatty acids, carbonyl compounds, higher alcohols, and esters.
Sulfur compounds, such as hydrogen sulfide, methional, and dimethyl sulfide DMS , are active flavor components of the beer that is generated during mashing and fermentation. During fermentation, through the metabolizing of amino acids and vitamins, and through the use of the inorganic components of the wort e.
Sulfur compounds impart specific flavors to the beer, which have been defined as onion, rotted vegetables, or cabbage flavors, among others. While the over accumulation of these sulfur compounds is often undesirable, under specific circumstances, sufficient sulfite levels are necessary in the beer, to maintain flavor stability.
In particular, it has been shown that the disruption of the genes coding for methionine sulfoxide reductase abolished formation of DMS from DMSO in both S. Methionine has a key role in the production of sulfur compounds by yeast. Indeed, when there are sufficient levels of methionine in the wort, this can cause inhibition of sulfite uptake, and the production of hydrogen sulfide [ 9 ]. Other organic acids, such as citrate and pyroglutamate, derive directly from the wort, and the yeast do not affect their concentrations in the beer.
Overall, more than organic acids have been identified in beers. Fatty acids are of particular interest here, because of their involvement in the synthesis of esters. Yeast can incorporate saturated fatty acids and unsaturated fatty acids UFAs from the wort, or they can synthesize these from acetyl-CoA.
However, the lack of sufficient oxygen in the later phases of fermentation makes the synthesis of UFAs impossible, and as a consequence, medium-chain fatty acids MCFAs are released into the medium [ 10 ]. These MCFAs are powerful detergents, and they can influence yeast vitality, beer taste, and foam stability.
The presence of aldehydes and vicinal diketones is considered undesirable for beer quality. In some circumstances, such as when there is excessive wort oxygenation and high pitching rates, aldehydes can accumulate in concentrations above the flavor threshold [ 11 ]. The vicinal diketones are important off-flavors of lager beers, which include diacetyl. Diacetyl has a strong aroma of toffee and butterscotch, and at concentrations above 0.
During conditioning, diacetyl is assimilated by yeast, and thus reduced to acetoin and butandiol, which have much lower impact on beer quality.
Traditionally, the rate-determining step of diacetyl accumulation in beer has been considered as the spontaneous decarboxylation of acetolactate, with yeast assimilation left with a marginal role.
However, the physiological conditions of yeast are essential for diacetyl production and the time necessary for its reduction.
High concentrations of valine and isoleucine in the wort inhibit vicinal diketone production by yeast. High assimilation rates have been observed at higher fermentation temperatures and when yeast is grown under aerobic de-repressed conditions. On the contrary, at higher pitching rates, the elevated concentrations of vicinal diketones produced by yeast require longer standing times [ 12 ]. Isoamyl alcohol, n-propanol, isobutanol, 2-phenyl-ethanol, and triptothol are important flavor and aroma components in terms of their concentrations.
On the contrary, above these concentrations, these compounds can have unpleasant heavy solvent-like odors. The formation of higher alcohols by brewing yeast involves different complex pathways, and a lot of progress has been made in the determination of the roles of the key genes involved in their biosynthesis [ 13 ]. The predominant idea for many years was that the higher alcohols are produced via the Ehrlich pathway.
While this pathway can correctly explain the relationships between leucine and the corresponding isoamyl alcohol, it fails to explain why some fusel alcohols e. Indeed, in complex media such as the wort, most higher alcohols are formed following the glycolytic pathway. By fermenting the wort sugars, yeast not only produce ethanol, but also a number of long-chain alpha-acids that can subsequently be transformed into amino acids such as aspartate and glutamate.
Finally, the choice of yeast strain can have great impact on higher alcohol production, and ale strains are considered to be higher producers than lager strains [ 13 ]. Esters are chemical compounds derived from a carboxylic acid and an alcohol, and they are of major industrial interest because they have very low thresholds and define the fruity aroma of the beer. Two main classes of esters are of particular interest for brewers: acetate esters and MCFA esters. Acetate esters have concentrations above threshold levels in most lager beers e.
It is generally believed that the acetyl-CoA that is necessary for formation of acetate esters derives from oxidation of acetaldehyde. Among the MCFA esters, ethyl hexanoate i. Different studies have focused on the manipulation of fermentation conditions and the wort composition in ways that favor the availability of these factors and that lead to increased production of higher alcohols and esters.
It is generally accepted that any condition that stimulates yeast growth will increase the production of higher alcohols and their acetate esters during fermentation. In this respect, increasing fermentation temperatures leads to an accumulation of acetate esters, with no significant differences in the levels of the MCFA esters. This phenomenon is dependent on increased alcohol acetyltransferase activity and stimulation of higher alcohol synthesis, which results from greater amino acid turnover.
In addition, it has been suggested that higher fermentation temperatures increase the synthesis of a specific permease, Bap2p, that is, involved in import of the branched-chain amino acids valine, leucine, and isoleucine, which are known precursors of the higher alcohols [ 15 ]. Oxygen has an ambiguous role here. Indeed, oxygenation of the wort provides for better yeast growth, and consequently increased higher alcohol production. However, it is well-known that oxygenation leads to lower levels of esters in the beer.
Oxygen acts in two different ways. First, availability of oxygen allows the biosynthesis of UFAs that is required to sustain yeast growth during fermentation. Second, oxygen inhibits transcription of the alcohol-acetyltransferase-encoding gene ATF1 , and consequently it reduces synthesis of acetate esters [ 13 ]. Thus, correct management of wort oxygenation at the time of pitching is essential to produce quality beers.
Another fermentation parameter that can affect ester synthesis is the hydrostatic pressure on the yeast cells. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis , which was originally termed Unterhefe No. Hansen and has been used in production in since Here, we sequenced S. Lager yeasts are descendants from hybrids formed between a S. Accordingly, the S. Based on the sequence scaffolds, synteny to the S.
We present evidence for genome and chromosome evolution within S. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. This inferred that this strain is basically triploid with a diploid S. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. Based on conserved translocations between the parental genomes in S.
Starting from the early ages of agriculture and the domestication of barley, fermented beverages played an important role in the emerging societies.
Beer has been known for millennia dating back at least to the Sumerians BC. Fermented beverages provided not only nutrition but were basically the only sources of uncontaminated clean liquids and thus of medicinal value.
Although there is a plethora of microorganisms within the Saccharomyces complex that can be found in natural fermentations, Saccharomyces cerevisiae has been the predominant species in certain types of fermentations, e. Today, however, most beer volume is generated with lager beers. Lager brewing was initiated in Bavaria in the 15th century Libkind et al. The German Reinheitsgebot from regulated that beer should only be made of water, malt, and hops without any other ingredients—of course at that time S.
In the 19th century, lager beer gained so much popularity that keeping up production required a break with tradition. Supported by the invention of refrigeration, lager beer was then also produced in the summer months, which traditionally had been considered the off-season. However, beer spoilage of lager beers became increasingly frequent over summer due to contamination with wild yeasts. This led to the scientific investigation of this problem by Louis Pasteur and Emil Chr.
Hansen verified that wort became infected by wild yeasts and therefore devised a method to isolate pure cultures of yeast strains Hansen One of these strains, Unterhefe No. Jacobsen and later entered the CBS strain collection in Lager yeasts are interspecies hybrids between S. Previous analyses of lager yeast strains indicated that different isolates contain different gene or chromosome sets Hansen and Kielland-Brandt ; Fujii et al.
Using polymerase chain reaction PCR -restriction fragment length polymorphism, two types of lager yeasts could be distinguished.
On the one hand there were lager strains currently used in production that showed almost a complete set of both of the parental genomes, and on the other a set of lager yeast strains, including S. By means of array-based comparative genomic hybridization array-CGH , this partition into two groups was further refined. This indicated that regional distribution matches the gene content and suggested that group I corresponds to the Saaz type, whereas group 2 is represented by the Frohberg type.
It was also suggested that two independent hybridization events generated the two types of lager yeast Dunn and Sherlock The origin of the non- cerevisiae parent in lager yeast has long been debated.
Recently, the isolation of S. Throughout this paper, we refer to the non- cerevisiae part of lager yeast genomes as S. Here we report the genome sequence and analysis of the first pure culture lager yeast production strain S. Strains were inoculated with an initial OD i. Industrial brewing conditions are produced by small-scale fermentations in tall tube cylinders with mL of volume. Yeast strains were propagated in granmalt prior to pitching with an OD of 0.
Stirring of the fermentation cyclinders was set to rpm. The fermentation performance was followed by online measuring of CO 2 loss and wort density using an Anton Paar DMA 35 densitometer measuring gravity i.
The end of fermentation was reached when the sugar concentration did not decrease further for 2 d. All fermentations were conducted in biological triplicates. A volume of mL was used for flavor analysis. Samples of mL were removed at the end of fermentation for analysis of aroma compounds.
Alcohols and esters were measured by solvent extraction with carbon disulfide. Genome sequencing of S. A fragment library and the additional 8-kb paired-end library were constructed with Rapid Library Prep Kit. An initial number of , reads and , paired end reads of an 8-kb library were assembled into contigs and further combined into 78 scaffolds. Assembly into whole chromosomes was based on synteny to S. Primers are listed in Supporting Information , Table S1. The S.
For the visualization of chromosomal rearrangements in a Circos plot, a pairwise comparison of S. The information about all chromosomal rearrangements was then synthesized in a tabular matrix which can be represented in a circular plot using the Circos software package Krzywinski et al.
Ploidy analysis was visualized using a violin plot. Then its output was parsed with SAMtools Li et al. A running average of mapped reads per window was calculated.
The violin plot shows the distribution of the log2 ratios of copy number variation across each chromosome. Ploidy analysis was confirmed using FACS. We compared growth and fermentation characteristics of two group I strains, S. PK laboratory yeast strain.
This profile was best matched by S. At greater temperatures, however, S. The Weihenstephan lager yeast showed intermediate growth rates at the upper and lower end of the temperature range Figure 1.
This finding indicates that S. Currently, however, greater fermentation temperatures are applied in industry. Under these conditions the group II lager yeast was fastest in fermentation and wort attenuation. Among the group I lager yeasts, S. These results indicate that group II lager yeasts are better adapted to greater temperature fermentation conditions than group I yeasts. Two main postfermentation parameters of industrial importance are the percentage of surviving cells and the ratio of petite cells among them.
Survival rates of group I and II lager yeasts at the end of fermentation were similar. Growth comparisons with lager yeasts. The following strains were used: Group I lager yeast S. Malt-based fermentations and volatile compound analysis with lager yeast strains. A Representative fermentation kinetics of S. Lager yeast strains provide a clean taste to beers associated with rather low levels of aroma alcohols and esters compared to more fruity ale and wine yeasts.
We sequenced the S. More than 10 6 reads were generated and assembled into a A draft genome of the Weihenstephan has recently been generated Nakao et al. Due to the large number of sequence contigs, we resequenced this strain using Illumina MiSeq v2 to obtain similar high level coverage and quality as for the S. To this end 10 7 reads derived from bp paired-end reads and an 8-kb mate-pair library were used and assembled in the 23 Mb genome resulting in a total coverage of 55x based on high quality reads Table 1.
The Weihenstephan lager yeast contains essentially two complete parental genomes. To generate an overview of which parts of the parental genomes were lost, we partitioned the scaffolds into their S.
This is straightforward as scaffolds derived from the S. The two sets of scaffolds were then aligned to the SC genome sequence. To generate a genome overview and visualize both parental genomes of S. It became apparent that S. Our results are consistent with previous data obtained by PCR-restriction fragment length polymorphism mapping or by array-CGH Rainieri et al.
Next to loss of complete S. In contrast, there was only two position of LOH for the S. In these cases sequences that were lost were replenished by orthologous regions from the other parental genome, which resulted in homozygous sequences derived from only one parental genome.
In the S. High functional homology was found between the transferred S. Despite the functional homology, the transferred chromosome had a structure that was substantially different from that of standard S. It did not recombine with S. Furthermore, restriction endonuclease analysis showed that the variant chromosome has a nucleotide sequence in the HIS4 region different from that of S.
The S. Furthermore, restriction endonuclease analysis showed that the variant chromosome has a nucleotide sequenc. From the journal. Carlsberg Research Communications Denmark. Bibliographic information.
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