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Brewing up a storm: The genomes of lager yeasts and how they evolved
Biotechnology Advances 35 (2017) 512–519
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Research review paper
Brewing up a storm: The genomes of lager yeasts and how they evolved Chandre Monerawela, Ursula Bond ⁎ Department of Microbiology, School of Genetics and Microbiology, Trinity College Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 24 November 2016 Received in revised form 16 February 2017 Accepted 4 March 2017 Available online 8 March 2017 Keywords: Lager yeasts Biotechnology Genomes Evolution
a b s t r a c t Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500–600 years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts. © 2017 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Anthropogenic and human cultural influences on the evolution of S. pastorianus 1.2. The genomes of lager yeasts . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Whole genome sequencing . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Identification of the non-S. cerevisiae genome in lager yeasts . . . . . . . . . 1.5. More lager yeast genomes . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Recombination sites in Group I and II lager yeasts . . . . . . . . . . . . . . 1.7. Unique genetic features of lager yeast genomes . . . . . . . . . . . . . . . 1.7.1. Cryotolerance . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7.2. Gene copy number . . . . . . . . . . . . . . . . . . . . . . . . 1.7.3. Hybrid genes . . . . . . . . . . . . . . . . . . . . . . . . . . 2. On the origins of Group I and II lager yeasts . . . . . . . . . . . . . . . . . . . . 2.1. Origins of the S. eubayanus genome of lager yeast . . . . . . . . . . . . . . 2.2. Origins of the S. cerevisiae genome of lager yeast . . . . . . . . . . . . . . . 2.3. Sub-telomeric regions of lager yeast genomes . . . . . . . . . . . . . . . . 2.4. A model for the evolution of Group I and II lager yeasts. . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction 1.1. Anthropogenic and human cultural influences on the evolution of S. pastorianus ⁎ Corresponding author. E-mail address: [email protected] (U. Bond).
http://dx.doi.org/10.1016/j.biotechadv.2017.03.003 0734-9750/© 2017 Elsevier Inc. All rights reserved.
The history of the art of brewing is intimately associated with the social and cultural development of the human race, and has been
C. Monerawela, U. Bond / Biotechnology Advances 35 (2017) 512–519
influenced by human migration and the ancient customs and practices of brewing. Beer has been brewed for several millennia and most likely evolved independently in many societies during human development. Archaeological findings show evidence of brewing in China as early as 7000 BCE and in Mesopotamia and Egypt in the period 4000– 3500 BCE (Hornsey, 2012). Modern day brewing owes its origins to brewing practices in Medieval Europe. The temperate climate of Northern Europe and the British Isles provided ideal conditions for the cultivation of grains such as wheat, oats and barley, from which sugars such as maltose, the main ingredient in beer, could be extracted (Hornsey, 2003). Spontaneous fermentation by wild yeasts converted the sugars into alcohol. As brewing practices developed from a cottage industry to a skilled craft, the nature of the “substance” responsible for the fermentation remained elusive but was called yeast, a word deriving from the German word Gischt or the Dutch Gist, referring to the froth or foam on the top of the vat of beer at the end of the fermentation process. This froth was scooped off the top of the vat and used to “inoculate” the next batch of beer, thus continuing the propagation of the “ferment”. These yeasts are referred to as “Top Fermenters” as the yeast floated to the top of the vessel at the end of the fermentation and are associated with Ale production (Barnett, 1998). In Medieval times, the stability and purity of beer was a constant problem. As the brewing industry grew, laws were enacted to control the purity and the price of beer. The Reinheitsgebot, introduced into Bavaria in 1516, restricted brewing of beer between St. Michael's Day (September 29) and St. George's Day (April 23) and also restricted the ingredients of beer to barley, water and hops. The law did not mention yeast, which was considered a by-product of the process. To preserve the beer during the warm summer months, beer was stored in cool caves often packed with ice (Hornsey, 2012). This storing (lagering) process produced a more stable beer and over time it was noted that the yeasts rather than floating to the top of the vat, sank to the bottom at the end of the fermentation and thus were called “Bottom fermenters”. The shift to cold-temperature brewing, together with the practice of lagering, favoured the emergence of the “bottom fermenting” lager yeasts that we know of today. The bottom fermenters were given a taxonomical classification, Saccharomyces pastorianus by Max Reess in 1870. In 1890, while working as a mycologist at the Carlsberg Laboratory, Emil Christian Hansen developed techniques to separate and culture yeasts. One isolate, which he named “Unterhefe Nr. I” (bottom-fermenting yeast no. 1) was a particularly good fermenter and was named as Saccharomyces carsbergensis. Hansen subsequently identified several other yeast isolates from beer vats, and based on their fermentation characteristics and physiological properties, classified the isolates as separate species. One isolate was classified as Saccharomyces pastorianus, in deference to Reese (Barnett and Lichtenthaler, 2001). The Unterhefe Nr. I strain, which displayed very different physiological properties to the S. pastorianus strain was named Saccharomyces carlsbergensis while a third isolate Unterhefe Nr. II was designated as Saccharomyces monacensis. 1.2. The genomes of lager yeasts It would not be till the end of the 20th Century that an understanding of the composition of the genomes of lager yeasts would emerge. Genetic analysis of the yeast isolates originally identified by Hansen led to a reclassification of the original three species into a single species, designated S. pastorianus (Vaughan Martini and Martini, 1987). Using the technique of single chromosome transfer into kar1 mutants of Saccharomyces cerevisiae, Morten Kielland-Brand, at the Carlsberg Laboratory, demonstrated that certain regions of chromosomes from the lager yeasts recombined readily with S. cerevisiae chromosomes but other regions did not, leading to the hypothesis that sections of some chromosomes in the lager yeasts were S. cerevisiae-like while other
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sections were derived from a non-S. cerevisiae origin (Nilsson-Tillgren et al., 1986; Nilsson-Tillgren et al., 1981). Two different sets of chromosomes in S. pastorianus were later identified by Southern blot hybridisation. One set were defined as S. cerevisiae-like while the other was tentatively assigned as S. bayanus-like due to the conservation of the reciprocal translocation between chromosomes IV and II in S. bayanus and S. pastorianus (Tamai et al., 1998). Analysis of individual genes confirmed the presence of different gene alleles, with one allele being similar to S. cerevisiae and the other allele more similar to S. bayanus and S. uvarum (Hansen and Kielland-Brandt, 1994). At the end of the 20th century it was believed S. pastorianus was an allopolyploid interspecies hybrid between S. cerevisiae and most likely S. bayanus or a closely related species, containing at least three types of chromosomes; S. cerevisiae-like, S. bayanus-like and hybrid chromosomes consisting of part S. cerevisiae and part S. bayanus (Nilsson-Tillgren et al., 1981, 1986; Rainieri et al., 2006; Tamai et al., 1998). The composition of the lager yeast genomes was later assessed using the technique of competitive genomic hybridisation (CGH), which allowed for the estimation of the copy number of S. cerevisiaelike and S. bayanus-like genes and the genome locations where recombination between the homeologous chromosomes had occurred (Bond et al., 2004; Dunn and Sherlock, 2008). The analysis of the recombination sites and chromosome copy number indicated that the strains clearly segregated into two distinctive groups (Table 1). The Group I, or Saaz-type strains, included several strains isolated from breweries in the Czech Republic and Germany as well as strain CBS1513, a descent of the first pure culture of Unterhefe Nr. I, strain CBS1503, originally designated Unterhefe II (S. monacensis) and strain CBS1538, formerly designated S. pastorianus. The Group II, or Frohberg-type strains, included strains from Dutch, Danish and North American breweries. The Group I and II strains differ in their S. cerevisiae DNA content, with Group I strains having a lower S. cerevisiae content than Group II strains (Dunn and Sherlock, 2008).
Table 1 General recombination sites in Group I and II lager yeasts. Chr
ORF
Gene
II
YBR289W
SNF5
III
MATa
MAT
IV
YDR324C
UTP4
V
YER164W
CHD1
VII
YGL173Ca
XRN1
VII
YGR285C
ZUOI
VIII
YHR165Ca
PRP8
X
YJR009Ca
TDH2
XI
YKL203C
TOR2
XI
YKL080W
VMA5
XI
YKL045W
PRI2
XIII
YML074C-YML073C
Intergenic
XIII
YML051W
GAL80
XIII
YMR287C
DSS1
XIII
YMR302C
YME2
XIII
YMR306W
FKS3
XV
YOR092W
ECM3
XV
YOR109W
INP53
XV
YOR127W
RGA1
XV
YOR133W
EFT1
XVI
YPL240C
HSP82
XVI
YPL036W
PMA2
XVI
YPR160Wa
GPH1
XVI
YPR184W-YPR185W
Intergenic
XVI
YPR191W
QCR2
Group I CBS1513
Group I CBS1503
Group IIb
Shaded region, detected recombination site a Recombination site induced under heat shock stress (James, Usher, 2008) b Recombination sites found in at least two Group II S. pastorianus genomes (WS34/70 and CBS1260)
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1.3. Whole genome sequencing The first whole genome sequence of a lager yeast was obtained for the Group II strain Weihenstephan 34/70 (WS 34/70) (Nakao et al., 2009) and revealed a polyploid genome with an estimated 36 chromosomes (Table 2). Given the estimated tetraploid DNA content of the lager yeast genomes, the chromosome number appeared low and suggested the presence of multiple copies of near identical chromosomes. Analysis of the genome sequence confirmed the presence of three types of chromosomes, namely S. cerevisiae-like, S. bayanuslike and hybrid chromosomes, the latter resulting from homeologous recombination between parental chromosomes. S. cerevisiae-like genes shared an average 99.2% identity with the reference strain S. cerevisiae S-288C while the S. bayanus-like ORFs showed approximately 92.7% identity with the reference genome CBS 7001, the only S. bayanus genome available at the time. The sites of recombination between the hybrid chromosomes were identified at the nucleotide level and mostly coincided with the sites previously identified by CGH (Table 1). The copy number for S. bayanus ribosomal DNA (rDNA), encoded on chromosome XII, was found to be significantly lower than the corresponding S. cerevisiae rDNA copy number. Additionally, the mitochondrial genome displayed a higher sequence identity to the S. bayanus reference strain CBS 7001, indicating that the Group II lager yeasts have acquired their mitochondrial genome from the non-S. cerevisiae parent. 1.4. Identification of the non-S. cerevisiae genome in lager yeasts While S. bayanus was considered the most likely second parental species of the lager yeasts (Bond, 2009; Bond et al., 2004; Dunn and Sherlock, 2008; Nakao et al., 2009; Rainieri et al., 2006), the low sequence identity (92.5%) to the non-cerevisiae sub-genome suggested another source for the second parent. Further, the S. bayanus taxon appeared extremely complex containing both pure and hybrid strains, prompting the designation of at least two genetically diverse lineages, namely S. bayanus var. bayanus (CBS380) and S. bayanus var. uvarum or S. uvarum (CBS 7001 and type strain CBS395) (Rainieri et al., 2006). During a survey of yeast isolates in Patagonia, South America, Libkind et al. (2011) identified a new Saccharomyces species that they named S. eubayanus sp. nov. The species shares 99.5% identity with the non-S. cerevisiae portion of the lager yeast genome (Libkind et al., 2011). This and other studies also confirmed the pure nature of the lineage of S. uvarum and the hybrid nature of the type strain S. bayanus var. bayanus
(CBS380), which is mainly composed of 67% S. uvarum and 33% S. eubayanus-like sequences but also contained introgressions of S. cerevisiae (Libkind et al., 2011; Nguyen et al., 2011; Perez-Traves et al., 2014).
1.5. More lager yeast genomes Since the publication of the first genome sequence for the Group II lager yeast (WS 34/70), the genomes of several Group I and II lager yeast have been sequenced (Table 2) (De León-Medina et al., 2016; Hewitt et al., 2014; Kvasnicka et al., 2012; Okuno et al., 2016; van den Broek et al., 2015; Walther et al., 2014). The compiled genome sequences provide an insight into the relationship between the Group I and Group II yeasts. Surprisingly, estimated genome sizes and chromosome number for the same strain vary significantly from study to study (Table 2). The diverse range of estimates for genome sizes and chromosome numbers may reflect genuine differences in isolates held in different laboratories but more likely result from different methodologies used to assemble the genomes and to estimate gene copy number. Given the estimated minimum tetraploid DNA content of Group II strains, the expected minimum chromosome copy number should be 64 (haploid chromosome count 16 × 4). Chromosome copy number estimates based on read counts in whole genome sequencing rather than from the sum of nucleotides in assembled contigs appear to be more in line with the expected chromosome copy number as previously estimated by FACs and/or the weight of the DNA. Genome assembly models predict that the prototypic Group I lager yeast (S. carlsbergensis, CBS1513) contains 45–47 chromosomes, consisting of a haploid S. cerevisiae and a diploid S. eubayanus gene content, with significant aneuploidy (Fig. 1). Some chromosomes, for example XI and XII are entirely derived from the S. eubayanus parent while others, for example, chromosomes IX and X are derived from both S. cerevisiae and S. eubayanus. Hybrid chromosomes (III, VII, XIII, XV and XVI) are also present in this Group I strain (Fig. 1). The prototypic Group II strain, WS 34/ 70, is an allotetraploid (4n + 2), consisting of a diploid S. cerevisiae and diploid S. eubayanus gene content on 66 chromosomes (Fig. 1) (Walther et al., 2014; Wendland, 2014). The Group II strain contains both S. cerevisiae and S. eubayanus copies of chromosomes I, VI, IX, X, XI, XII and XIV or either type of chromosome II, III, IV, V, VII, VIII, XIII, and XV, in addition to hybrid chromosomes III, VII, X, XI, XIII, and XVI. Two translocations between S. eubayanus chromosomes II and IV and between VIII and XV are conserved in both Group I and II strains (Fig. 1).
Table 2 Whole genome sequencing of Group I and II lager yeasts. Group
Strain
Estimated genome size (Mb)
Estimated no. of chromosomes
Accession number
Reference
I
CBS 1513
ND 19.5 33.6 19.2 ND 34.1 17.2 14.4 25 22.9 22.5 50.5 56 53.5 ND 35.7 47.5 56.9 39.1 22.7 24.2
31 47 45 ND 31 47 ND ND 36 66 ND 71 77 75 48 55 68 79 49 32 N/A
SAMEA2240035 AZCJ01000000 SRX758305 BBYW01000000 SAMEA2240037 SRX758304 BBYV01000000 BBYX01000000 ABPO01000000 AZAA01000000 BBYY01000000 SRX758144 SRX758145 SRX758149 SAMEA2240036 SRX758306 SRX758140-143 Not deposited Not deposited LSMH01000000 ALJS01000000
Hewitt et al. (2014) Walther et al. (2014) van den Broek et al. (2015) Okuno et al. (2016) Hewitt et al. (2014) van den Broek et al. (2015) Okuno et al. (2016) Okuno et al. (2016) Nakao et al., 2009 Walther et al. (2014) Okuno et al. (2016) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) Hewitt et al. (2014) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) De León-Medina et al. (2016) Kvasnicka et al. (2012)
CBS 1503
II
CBS 1538 WS 34/70
A1 A1 + B11 A2 CBS 1260 CBS 1483 Spy1 Spy2 790 CCY48-91
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Fig. 1. Chromosome copy numbers and types in prototypic Group I (CBS1513) and II (WS 34/70) lager yeasts. S. cerevisiae chromosomes; red, S. eubayanus; blue, hybrid chromosomes red and blue. Intra-species translocations: S. eubayanus translocations between chromosomes II and IV; blue vertical stripes and between chromosomes VIII and XV; blue diagonal stripes. S. cerevisiae translocations between chromosome V and XI; red and white blocks. Inter-species translocations between VIII and XV; diagonal blue. CBS 1513 contains 47 chromosomes (3n-1) and WS 34/70 contains 66 chromosomes (4n + 2). Adapted from Nakao et al. (2009) and Wendland (2014).
1.6. Recombination sites in Group I and II lager yeasts Recombination between the parental chromosomes has occurred at specific chromosomal locations to create a unique set of hybrid chromosomes in lager yeasts. Most of the recombination sites are intragenic although a few intergenic sites are also observed (Table 1). Most of the recombination sites are non-reciprocal with the exception of a reciprocal translocation event at YJR009C/TDH2 on chromosome X (Fig. 1). Several themes emerge from a comparison of the recombination sites identified in the Group I and II strains. 1. Two recombination sites at YGL173C (XRN1) and YGR285C (ZUO1) are conserved between Group I and II strains for which genome sequences are available. A third recombination site at YPL0240C (HSP82) is present in Group I and II strains but appears to be absent in the Group I strain CBS1538 (Monerawela, unpublished). Previously, it was reported that the recombination site at the YCR041C (MAT locus) was also conserved between Group I and II lager yeasts, however a more detailed analysis of this region indicated that while recombination does occur at the MAT locus, the exact location is not conserved (Monerawela unpublished). Furthermore, the Group I strain CBS1538 does not contain a hybrid chromosome III. 2. In addition to the common sites, 4 additional recombination sites (YKL045W, YMR302C, YPR160W, YPR191W) are conserved in Group II strains. 3. Two distinctive patterns of recombination are found in the Group I strains as exemplified by strains CBS1503 and CBS1513 (Table 2). 4. Certain recombination sites are shared between one or more Group I and II strains, e.g. YHR165W. 5. Recombination sites unique to each strain have been identified.
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(It should be noted that data for some recombination sites may be missing from some genome data sets due to lack of sequence coverage). What molecular events could account for the observed recombination between homeologous chromosomes in the lager yeasts? Nakao et al., 2009 noted that the sequences flanking the recombination sites on the hybrid chromosomes share 81–96% sequence identity with sequences on the parental chromosomes. Recombination between sequences with this degree of diversity would be extremely rare (5.4 × 10− 8–2.2 × 10− 9). As similar percentage sequence identities can be observed throughout the genome, without resulting in recombination events, molecular event(s) other than shared sequence identity must define these specific recombination sites. One possibility is that the recombination sites represent fragile sites that are prone to double strand breaks (DSBs), as the rate of recombination is greatly increased by DSBs. What might induce such DSBs? Previous studies have shown that DSBs can be induced by exposure of yeasts to environmental stresses such as ionizing radiation, UV or can occur spontaneously due to replication fork collapse or during meiosis (Mehta and Haber, 2014). In a study designed to generate strains of lager yeasts tolerant to high gravity wort, James et al. (2008), exposed the Group II strain CMBS-33 to extreme high temperatures (50 °C, 15 min) to induce DSBs. The surviving cells were then fermented in high gravity wort (21°P) for 5 days. The selection procedure (high temperature and high gravity wort) was repeated several times and clonal isolates were selected. CGH analysis of the clones revealed that the genomes of several of the isolates had undergone recombination between the homeologous chromosomes at some of the previously identified recombination sites on the hybrid chromosomes (Table 1). This suggests that recombination can be induced at specific sites when yeasts are exposed to environmental stress. In fact, differences in chromosome profiles have been observed by pulse-field gel electrophoresis in single colony isolates at the end of fermentations, suggesting that chromosomes are dynamic and can be altered in a sub-population of cells during fermentation. An analysis of chromosome composition in yeast cultures, at the beginning and the end of fermentation, identified specific chromosomal regions with copy number alterations (James et al., 2008). In fermentations carried out in high specific gravity wort (20°P) and at 20 °C, rather than the normal 10°P and 13 °C, gene amplifications on chromosomes I and XII of the S. cerevisiae sub-genome were observed. The gene amplification on chromosome I was centered around the repetitive DUP240 locus while the amplification on chromosome XII was centered at the rRNA gene cluster. Gene copy number changes were also observed at the telomeres of several but not all chromosomes. Interestingly, gene copy number variations, most notably in telomeric regions, have been observed in a wide variety of industrial S. cerevisiae strains (Adamczyk et al., 2016; Deregowska et al., 2015; Dunn et al., 2012; Zhu et al., 2016). As lager yeasts encounter multiple stressful conditions such as anaerobiosis, high osmotic pressure, low temperature and high hydrostatic pressure during industrial fermentations, such environmental stressors may be the evolutionary driving force responsible for chromosomal recombination and amplification. 1.7. Unique genetic features of lager yeast genomes The happenstance of a hybridisation event between two species, S. cerevisiae and S. eubayanus resulted in the creation of a novel species with unique genetic characteristics. The unique physiological and metabolic properties, emanating from the complex polyploid genome, made this species the ideal yeasts for producing flavoursome beer. 1.7.1. Cryotolerance By far the most important genetic characteristic of S. eubayanus that has been passed onto lager yeasts is cryotolerance (Libkind et al., 2011; Peris et al., 2016). A comparison of the growth and fermentative capacities of 58 lager strains indicated that Group I strains, which contain a higher ratio of S. eubayanus to S. cerevisiae gene alleles grew better
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than Group II strains at 10 °C, although this did not result in a greater fermentation capacity (Gibson et al., 2013). Group II strains, which contain an approximately equal ratio of S. eubayanus to S. cerevisiae gene alleles displayed better growth profiles and fermented best at 22 °C. The Group II strains produce higher levels of flavour compounds than Group I strains, and production of higher alcohols and esters differ in Group I and II strains. Thus, the presence of the S. eubayanus genome greatly influences growth at lower temperatures and appears to contribute to the flavour profile of the resulting beer. To date, the genes responsible for cold tolerance have not been identified in S. pastorianus, however genetic studies on cold tolerance in other yeast species identified several genes, including ADH2, ADH3 encoding alcohol dehydrogenases and GUT2 encoding glycerol-3-phosphate dehydrogenase, as contributing to increased fitness at cold temperatures (Paget et al., 2014). 1.7.2. Gene copy number The polyploid genome of lager yeasts has the potential to uniquely influence the transcriptome, the proteome and ultimately the metabolic landscape of the lager yeast cell. Depending on the copy number of each parental chromosome, homeologous gene alleles from the S. cerevisiae and S. eubayanus sub-genomes can encode for multiple variant protein isoforms which may have significant effects on the cells metabolism through competition for substrate binding, and/or increased or decreased biochemical activities of the encoded proteins. The presence of multiple copies of gene alleles may also increase RNA transcripts levels within the cell, however, early transcriptome analyses of lager yeasts did not indicate a correlation between increased gene copy number and increased transcript levels (Bond et al., 2004). Nevertheless, gene copy number and/or the presence of homeologous gene alleles appears to influence the transcriptome as revealed from a comparison of the expression of S. cerevisiae gene transcripts in a haploid S. cerevisiae strain and two lager yeasts during fermentation (James et al., 2003; James et al., 2002). With the availability of high quality genome sequences for Group I and Group II lager yeasts and improved RNA sequencing technology, it would be a worthy pursuit to reexamine the role of gene copy number on transcript expression levels in the lager yeasts. 1.7.3. Hybrid genes In addition to homeologous alleles, the recombination events between the parental chromosomes, which mainly occur within intragenic regions, generated a set of hybrid genes truly unique to the lager yeast genomes (Table 1). These hybrid genes, composed of part S. cerevisiae and part S. eubayanus, encode for unique variant protein isoforms that may have different biochemical and biophysical activities than their parental proteins. To date, the only detailed study of a hybrid gene is that of YPR160W/GPH1 from the Group II strain CMBS-33. GPH1 encodes for the enzyme glycogen phosphorylase, which is involved in mobilising stored glycogen through conversion of glycogen to glucose-1-phosphate. The Group II strain CMBS-33 contains three hybrid alleles and one S. eubayanus allele. Analysis of the hybrid alleles revealed a stop codon within the ORFs leading to a loss of function. The sole S. eubayanus copy of GPH1 appears to be functional, as the lager yeast contains stored glycogen at levels similar to that observed in a haploid S. cerevisiae cell (Usher and Bond, 2009). Similarly, the introduction of a stop codon into the gene FLO1 as a result of a recombination event is also observed in a Japanese lager yeast strain, rendering the strain non-flocculent (Sato et al., 2002). 2. On the origins of Group I and II lager yeasts 2.1. Origins of the S. eubayanus genome of lager yeast S. eubayanus is a cold-tolerant wild yeast species found on Nothofagus and Araucaria araucana trees in Patagonia, Quercus trees in
Asia and with Cedrus species, Pinus taeda and Quercus rubra trees in North America (Peris et al., 2016; Peris et al., 2014; Rodriguez et al., 2014). Since domestic or wild isolates of S. eubayanus have not been identified in Europe, it was proposed that the species found its way to Europe as part of the trade route established by Conquistadors during the exploration of the New World. However, the recent discovery of wild isolates of S. eubayanus in Tibet, China, North America and New Zealand (Bing et al., 2014; Gayevskiy and Goddard, 2016; Peris et al., 2014) expands the possible geographical origins of the lager yeast parent. Genome sequencing and single nucleotide polymorphism (SNP) analysis of 12 genomic loci, identified the S. eubayanus strain from Tibet as sharing the greatest identity (99.77–99.82%) with the noncerevisiae portion of the lager yeast genome (Bing et al., 2014). However, a recent pan genome sequencing project of all currently known isolates of S. eubayanus, from diverse geographical locations, uncovers a more complicated genetic relationship amongst S. eubayanus isolates (Peris et al., 2016; Peris et al., 2014). Several of the isolates, including strains from Tibet and North America cluster together into a lineage referred to as Holartic. This group of strains display low diversity and are related to a second lineage primarily found in South America. A third lineage, containing a more diverse population is also identified in South America. The genetic diversity between S. eubayanus isolates appears to arise from admixture, outcrossing and incomplete lineage sorting. To date, no sole isolate appears to be the lowest common ancestor (LCA) of the non-cerevisiae parent of lager yeasts, however it appears that strains related to Tibetan isolates contributed 66% of the lager S. eubayanus sub-genome while the remainder of the sub-genome (34%) is most closely related to an isolate from North Carolina, U.S.A. (Peris et al., 2016). Thus, genetic variation within S. eubayanus isolates most likely accounts for the genetic variation observed in the S. eubayanus portion of the lager yeast genome. Based on this analysis the original hypothesis of S. eubayanus travelling to Europe from Patagonia was subsequently rivaled by an alternative hypothesis of a journey of S. eubayanus along the Silk Route from China or Tibet. Since the discovery of the New World post-dates historical records of lager brewing in Europe and predominately emanated from South-West Europe, a region not known for beer fermentations, the former hypothesis currently seems less likely. Rather, given the physical geographical proximity of Asia and Europe and the closer genetic relationship between Tibetan isolates and the lager yeasts, the latter hypothesis seems more favourable. As wild yeasts can be dispersed by environmental and animal means, alternative hypotheses cannot be ruled out at present. Whatever the means, the arrival of S. eubayanus in Europe and its subsequent hybridisation with S. cerevisiae would bring about the revolutionary shift in brewing practices to low temperature brewing through the introduction of cold-tolerance from S. eubayanus to the normally mesophilic S. cerevisiae. 2.2. Origins of the S. cerevisiae genome of lager yeast Prior to the availability of whole genome sequences for the wide variety of Saccharomyces cerevisiae strains and isolates, phylogenetic trees compiled from a comparison of intronic sequences or micro satellite gene loci identified Ale yeasts as the most likely ancestor of the S. cerevisiae sub-genome in lager yeasts (Dunn and Sherlock, 2008; Legras et al., 2007). In recent years, as a result of the concerted efforts of research groups around the world, genome sequences for greater than 1000 wild and domesticated S. cerevisiae isolates have become available (Bergström et al., 2014; Borneman et al., 2011; Borneman et al., 2016; Borneman et al., 2008; Gallone et al., 2016; Gonçalves et al., 2016; Liti et al., 2009; Strope et al., 2015; Wang et al., 2012; Zhu et al., 2016). Phylogenic analyses clearly segregates the different isolates/ strains into distinctive populations delineated by source (wine, cider, bread, beer, sake, clinical, laboratory, distilling etc.) or by geographic locations (Asia, North America, Africa, European etc.). Some isolates are classified as admixed mosaics, displaying ancestry from two or more
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populations. In addition to S. cerevisiae genes, several isolates were shown to contain non-S. cerevisiae gene sequences introgressed from other Saccharomyces stricto sensu species, mainly S. paradoxus or from other fungal species. In general, yeasts associated with brewing cluster together into a distinct sub-population (Gallone et al., 2016; Gonçalves et al., 2016). Furthermore, an extensive analysis of over 100 beer isolates revealed the segregation of beer yeasts into two distinct sub-groups, named, Beer 1 and 2 (Gallone et al., 2016). The Beer 1 subgroup contains isolates from The British Isles (Britain and Ireland), the USA and from Germany and Belgium. The USA isolates are phylogenetically related to the British isolates suggesting that early British settlers introduced these yeasts to the United States. The Beer 2 clade is more closely related to wine yeasts and contains approximately 20% of all brewing strains. In a separate study, Gonçalves et al. (2016) subdivided beer isolates into three clades, Wheat, English-Irish Ales and German Alt-Kolsch beers respectively. These data suggest that beer yeasts emerged as a result of at least two independent European domestication events. Using average-per-site nucleotide divergence and an estimated average mutation rate for beer yeasts, Gallone et al. (2016) suggests that the first domestication occurred between 1573 and 1604 CE and the second domestication event dated later between 1645 and 1671 CE. This timing coincides with the shift from cottage industry to the skilled craft industrialization of brewing. A comparison of the S. cerevisiae subgenomes of Group I and Group II lager yeasts with 90 yeasts strains including bread, beer, sake, wine, and wild yeasts from different geographical locations, placed the lager yeasts in the same clade as the beer yeast (Gonçalves et al., 2016). However, both lager yeast groups form a separate sub-clade within the beer clade and are clearly divided further into Group I and II lager yeasts within the sub-clade. Thus, lager yeasts appear to have arisen from Ale-like yeasts but may have been further domesticated following the hybridisation event with S. eubayanus. 2.3. Sub-telomeric regions of lager yeast genomes Genome analyses of S. cerevisiae strains and isolates revealed that a significant proportion of genetic diversity within Saccharomyces species is accounted for by changes within sub-telomeric regions of chromosomes (Bergström et al., 2014; Dunn et al., 2012; Gallone et al., 2016; James et al., 2008). Many unique genes, not found in the reference S. cerevisiae strain, S-288C, have been identified in the sub-telomeric regions in S. cerevisiae isolates (Bergström et al., 2014) and copy number variations (CNVs) resulting from gene amplification and deletions within the sub-telomeric regions of S. cerevisiae strains have been reported (Dunn et al., 2012). Sub-telomeric regions may be of specific importance in lager yeasts as several important gene families, such as the MAL genes required for maltose uptake and utilisation and the FLO genes required for the property of yeast flocculence, reside in these regions. In fact, recent genome analysis indicates that the MAL gene clusters are significantly amplified in beer isolates relative to other S. cerevisiae isolates (Gonçalves et al., 2016). Monerawela et al. (2015) compared the 32 sub-telomeric regions (right and left sub-telomeres of 16 chromosomes; RST, LST) of the Group II lager yeast chromosomes to the sub-telomeric regions of 32 S. cerevisiae strains from different industrial and geographic sources. Average nucleotide-per-site divergence rates placed the Ale yeasts Fosters O and B as the LCA of the Group II lager yeasts. A comparison of each of the 32 individual sub-telomeric regions in the Group II lager yeasts to the genomes of the 32 S. cerevisiae strains and to the Group I lager yeasts revealed that the RST of chromosome XIII in the Group II lager yeast was absent in the Group I strains and from 25 of the 32 S. cerevisiae strains examined. Second, sub-telomere regions of Group II chromosomes IV, VI, XI, XII, XV and XVI were absent in the Group I lager yeast. This loss of sub-telomeric regions accounts for some of the reduction in S. cerevisiae content noted between Group I and II lager yeasts. Several “lager-specific (LgS)” genes, residing at sub-telomeric locations were identified in the genome of the prototype Group II strain
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WS 34/70 (Nakao et al., 2009). Four of the genes, Lgs-FSY, Lgs-MCT, Lgs-AMD2, and Lgs-MEL, encoding for a fructose symporter, a monocarbohydrate transporter, an amidase, and a melibiase respectively, were subsequently assigned to the S. eubayanus sub-genome. Four other genes, Lgs-TYP, Lgs-AMD1, Lgs-TRR and Lgs-HYPO, encoding for a tyrosine permease, a second amidase, a transcriptional regulatory protein and a hypothetical open reading frame, respectively, are not present in the S. cerevisiae reference genome S-288C but PCR analysis confirmed the presence of the four LgS genes in nine other Group II production strains (Monerawela et al., 2015). Using RST XIII and LST IV as genome markers that distinguish Group I and II lager yeasts and the 4 Lg-S genes found on Group II strains, Monerawela et al. (2015) examined 78 S. cerevisiae strains to identify the most likely parents of the Group I and II yeasts. The pattern of presence or loss of the six genetic loci clearly distinguishes between S. cerevisiae strains and allows their assignment into specific sub-groups. All six DNA markers are found in all Group II lager yeast strains, as well as in a group of S. cerevisiae strains associated with stout brewing in the British Isles. This grouping also includes two S. cerevisiae strains used in the production of the alcoholic beverage “Toddy” from tender coconuts a South-West India. The Group I lager yeast strains have lost LST IV and RST XIII and the genes Lgs-TYP, Lgs-AMD1, which reside at the latter sub-telomere, but retain Lgs-TRR and Lgs-HYPO, which reside at LST XIV and RST XVI respectively. This same pattern of loss is also observed in most yeasts used for Ale production. All of the laboratory strains and most wine yeasts analysed have lost the four LgS genes as well as RST XIII but retain LST IV, and therefore are distinguishable from the Ale yeasts. Thus, the pattern of loss of “lager-specific” genes and specific sub-telomeric regions clearly clusters Group I and II lager yeasts with two distinctive groups of beer yeasts; Group I with Ale yeasts and Group II with stout yeasts and suggests that the S. cerevisiae sub-genomes in Group I and II lager yeasts are derived from different S. cerevisiae lineages. 2.4. A model for the evolution of Group I and II lager yeasts Based on the data accumulated thus far on the composition and structure of the Group I and Group II lager yeast chromosomes, several contrasting hypotheses have been proposed to account for the origin and evolution of the S. pastorianus species. The presence of shared recombination sites on hybrid chromosomes III, VII and XVI led Walther et al. (2014) to propose that Group I and II lager yeasts arose from a single hybridisation event between a diploid S. eubayanus strain and a common ancestral diploid S. cerevisiae strain. Following hybridisation, it was proposed, the Group I and II strains evolved into two distinctive groups through the significant loss of the S. cerevisiae genome content in the Group I strains and further recombination events leading to unique hybrid chromosomes in both groups. Since this initial proposition, several additional lager yeast genomes have been sequenced revealing a more complex relationship between the Group I and II lager yeasts. Firstly, the number of recombination sites conserved in all lager yeasts is less than previously estimated. Secondly, the Group I strains appear to be divided into two sub-groups each with its own unique set of recombination sites (Table 1). Thirdly, each lager yeast strain contains its own unique recombination sites, suggesting that recombination between the homeologous parental chromosomes is an ongoing process, most likely in a response to environmental stressors. The analysis of the sub-telomeric regions of Group I and II revealed a significant erosion of chromosome ends in Group I strains (Monerawela et al., 2015). The pattern of loss or retention of the sub-telomeric regions, and of a set of Lg-S genes encoded therein, reveals distinct relationships between Group I yeasts and S. cerevisiae strains used in Ale production and between Group II yeasts and S. cerevisiae strains used in Stout production. As it is highly unlikely that the same pattern of sub-telomeric region loss or retention would occur randomly in many different Ale and Stout strains, this suggests that the two groups of
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Fig. 2. Sequential hybridisation model for the evolution of Group I and II lager yeasts. The initial hybridisation event occurred between a haploid Ale S. cerevisiae isolate and a diploid S. eubayanus isolate to generate a progenitor lager yeast. A second hybridisation event between the progenitor strain and an S. cerevisiae isolate used in stout production generated the Group II lager yeasts. Both Group I and II lager yeasts underwent recombination between the parental chromosomes to generate hybrid chromosomes. The Group I strains CBS1513 and CBS1503 appear to have taken separate evolutionary pathways post hybridisation.
lager yeasts arose by separate independent hybridisation events between S. eubayanus and two distinct S. cerevisiae isolates. The recent discovery of two or three distinctive clades of beer yeasts lends support this hypothesis (Gallone et al., 2016; Gonçalves et al., 2016). Interestingly, the Group I and II lager yeasts do not cluster with any of the S. cerevisiae beer clades and instead form two distinct sub-clades within the beer groups. This may indicate that the true ancestor of the lager yeasts has yet to be discovered or alternatively that further evolution has occurred post hybridisation with S. eubayanus. Recent data from Okuno et al. (2016) suggests an evolutionary model that reconciles both the single and multiple hybridisation hypotheses. Analysing the genomes of 10 S. pastorianus strains, the research group identified two S. cerevisiae sub-genomes that can be distinguished by SNPs within the Group II yeasts, one of which is shared with the Group I strains. Thus, the most likely evolutionary path involved an initial hybridisation between a haploid S. cerevisiae strain and a diploid S. eubayanus strain leading to a progenitor S. pastorianus strain (Fig. 2). This progenitor lager yeast underwent recombination between the parental chromosomes at two loci on chromosome VII and reciprocal translocations between chromosomes II and IV and between VIII and XV. The parental S. cerevisiae strain was most likely a yeast isolate used in the production of Ales, which had already suffered significant chromosomal erosion at the telomeres, specifically at RST XIII and LST IV. The Group I lager yeasts underwent subsequent chromosomal recombination to produce two distinct sub-types. The adaptive/environmental pressures influencing this divergent evolution are currently unknown. A subsequent hybridisation between the progenitor lager yeast strain and a second distinct S. cerevisiae strain produced the Group II lineage (Fig. 2). The S. cerevisiae strain involved in the second hybridisation event contains additional genetic material at the ends of the chromosomes, specifically at RST XIII and LST IV. So far the only yeast strains identified that contain these regions are S. cerevisiae strains used in the production of Stout in the British Isles and two strains isolated
from South-West India used to make the alcoholic beverage Toddy from tender coconuts. It is tantalizing to speculate that yet another trade route (between India and Great Britain) may have played a role in the evolution of the lager yeasts, however we await further genome sequences, specifically of a broader range of Ale yeasts and from more geographical diverse locations, to uncover the true ancestors of the lager yeasts.
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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Brewing up a storm: The genomes of lager yeasts and how they evolved Chandre Monerawela, Ursula Bond ⁎ Department of Microbiology, School of Genetics and Microbiology, Trinity College Dublin, Ireland
a r t i c l e
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Article history: Received 24 November 2016 Received in revised form 16 February 2017 Accepted 4 March 2017 Available online 8 March 2017 Keywords: Lager yeasts Biotechnology Genomes Evolution
a b s t r a c t Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500–600 years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts. © 2017 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Anthropogenic and human cultural influences on the evolution of S. pastorianus 1.2. The genomes of lager yeasts . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Whole genome sequencing . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Identification of the non-S. cerevisiae genome in lager yeasts . . . . . . . . . 1.5. More lager yeast genomes . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Recombination sites in Group I and II lager yeasts . . . . . . . . . . . . . . 1.7. Unique genetic features of lager yeast genomes . . . . . . . . . . . . . . . 1.7.1. Cryotolerance . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7.2. Gene copy number . . . . . . . . . . . . . . . . . . . . . . . . 1.7.3. Hybrid genes . . . . . . . . . . . . . . . . . . . . . . . . . . 2. On the origins of Group I and II lager yeasts . . . . . . . . . . . . . . . . . . . . 2.1. Origins of the S. eubayanus genome of lager yeast . . . . . . . . . . . . . . 2.2. Origins of the S. cerevisiae genome of lager yeast . . . . . . . . . . . . . . . 2.3. Sub-telomeric regions of lager yeast genomes . . . . . . . . . . . . . . . . 2.4. A model for the evolution of Group I and II lager yeasts. . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction 1.1. Anthropogenic and human cultural influences on the evolution of S. pastorianus ⁎ Corresponding author. E-mail address: [email protected] (U. Bond).
http://dx.doi.org/10.1016/j.biotechadv.2017.03.003 0734-9750/© 2017 Elsevier Inc. All rights reserved.
The history of the art of brewing is intimately associated with the social and cultural development of the human race, and has been
C. Monerawela, U. Bond / Biotechnology Advances 35 (2017) 512–519
influenced by human migration and the ancient customs and practices of brewing. Beer has been brewed for several millennia and most likely evolved independently in many societies during human development. Archaeological findings show evidence of brewing in China as early as 7000 BCE and in Mesopotamia and Egypt in the period 4000– 3500 BCE (Hornsey, 2012). Modern day brewing owes its origins to brewing practices in Medieval Europe. The temperate climate of Northern Europe and the British Isles provided ideal conditions for the cultivation of grains such as wheat, oats and barley, from which sugars such as maltose, the main ingredient in beer, could be extracted (Hornsey, 2003). Spontaneous fermentation by wild yeasts converted the sugars into alcohol. As brewing practices developed from a cottage industry to a skilled craft, the nature of the “substance” responsible for the fermentation remained elusive but was called yeast, a word deriving from the German word Gischt or the Dutch Gist, referring to the froth or foam on the top of the vat of beer at the end of the fermentation process. This froth was scooped off the top of the vat and used to “inoculate” the next batch of beer, thus continuing the propagation of the “ferment”. These yeasts are referred to as “Top Fermenters” as the yeast floated to the top of the vessel at the end of the fermentation and are associated with Ale production (Barnett, 1998). In Medieval times, the stability and purity of beer was a constant problem. As the brewing industry grew, laws were enacted to control the purity and the price of beer. The Reinheitsgebot, introduced into Bavaria in 1516, restricted brewing of beer between St. Michael's Day (September 29) and St. George's Day (April 23) and also restricted the ingredients of beer to barley, water and hops. The law did not mention yeast, which was considered a by-product of the process. To preserve the beer during the warm summer months, beer was stored in cool caves often packed with ice (Hornsey, 2012). This storing (lagering) process produced a more stable beer and over time it was noted that the yeasts rather than floating to the top of the vat, sank to the bottom at the end of the fermentation and thus were called “Bottom fermenters”. The shift to cold-temperature brewing, together with the practice of lagering, favoured the emergence of the “bottom fermenting” lager yeasts that we know of today. The bottom fermenters were given a taxonomical classification, Saccharomyces pastorianus by Max Reess in 1870. In 1890, while working as a mycologist at the Carlsberg Laboratory, Emil Christian Hansen developed techniques to separate and culture yeasts. One isolate, which he named “Unterhefe Nr. I” (bottom-fermenting yeast no. 1) was a particularly good fermenter and was named as Saccharomyces carsbergensis. Hansen subsequently identified several other yeast isolates from beer vats, and based on their fermentation characteristics and physiological properties, classified the isolates as separate species. One isolate was classified as Saccharomyces pastorianus, in deference to Reese (Barnett and Lichtenthaler, 2001). The Unterhefe Nr. I strain, which displayed very different physiological properties to the S. pastorianus strain was named Saccharomyces carlsbergensis while a third isolate Unterhefe Nr. II was designated as Saccharomyces monacensis. 1.2. The genomes of lager yeasts It would not be till the end of the 20th Century that an understanding of the composition of the genomes of lager yeasts would emerge. Genetic analysis of the yeast isolates originally identified by Hansen led to a reclassification of the original three species into a single species, designated S. pastorianus (Vaughan Martini and Martini, 1987). Using the technique of single chromosome transfer into kar1 mutants of Saccharomyces cerevisiae, Morten Kielland-Brand, at the Carlsberg Laboratory, demonstrated that certain regions of chromosomes from the lager yeasts recombined readily with S. cerevisiae chromosomes but other regions did not, leading to the hypothesis that sections of some chromosomes in the lager yeasts were S. cerevisiae-like while other
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sections were derived from a non-S. cerevisiae origin (Nilsson-Tillgren et al., 1986; Nilsson-Tillgren et al., 1981). Two different sets of chromosomes in S. pastorianus were later identified by Southern blot hybridisation. One set were defined as S. cerevisiae-like while the other was tentatively assigned as S. bayanus-like due to the conservation of the reciprocal translocation between chromosomes IV and II in S. bayanus and S. pastorianus (Tamai et al., 1998). Analysis of individual genes confirmed the presence of different gene alleles, with one allele being similar to S. cerevisiae and the other allele more similar to S. bayanus and S. uvarum (Hansen and Kielland-Brandt, 1994). At the end of the 20th century it was believed S. pastorianus was an allopolyploid interspecies hybrid between S. cerevisiae and most likely S. bayanus or a closely related species, containing at least three types of chromosomes; S. cerevisiae-like, S. bayanus-like and hybrid chromosomes consisting of part S. cerevisiae and part S. bayanus (Nilsson-Tillgren et al., 1981, 1986; Rainieri et al., 2006; Tamai et al., 1998). The composition of the lager yeast genomes was later assessed using the technique of competitive genomic hybridisation (CGH), which allowed for the estimation of the copy number of S. cerevisiaelike and S. bayanus-like genes and the genome locations where recombination between the homeologous chromosomes had occurred (Bond et al., 2004; Dunn and Sherlock, 2008). The analysis of the recombination sites and chromosome copy number indicated that the strains clearly segregated into two distinctive groups (Table 1). The Group I, or Saaz-type strains, included several strains isolated from breweries in the Czech Republic and Germany as well as strain CBS1513, a descent of the first pure culture of Unterhefe Nr. I, strain CBS1503, originally designated Unterhefe II (S. monacensis) and strain CBS1538, formerly designated S. pastorianus. The Group II, or Frohberg-type strains, included strains from Dutch, Danish and North American breweries. The Group I and II strains differ in their S. cerevisiae DNA content, with Group I strains having a lower S. cerevisiae content than Group II strains (Dunn and Sherlock, 2008).
Table 1 General recombination sites in Group I and II lager yeasts. Chr
ORF
Gene
II
YBR289W
SNF5
III
MATa
MAT
IV
YDR324C
UTP4
V
YER164W
CHD1
VII
YGL173Ca
XRN1
VII
YGR285C
ZUOI
VIII
YHR165Ca
PRP8
X
YJR009Ca
TDH2
XI
YKL203C
TOR2
XI
YKL080W
VMA5
XI
YKL045W
PRI2
XIII
YML074C-YML073C
Intergenic
XIII
YML051W
GAL80
XIII
YMR287C
DSS1
XIII
YMR302C
YME2
XIII
YMR306W
FKS3
XV
YOR092W
ECM3
XV
YOR109W
INP53
XV
YOR127W
RGA1
XV
YOR133W
EFT1
XVI
YPL240C
HSP82
XVI
YPL036W
PMA2
XVI
YPR160Wa
GPH1
XVI
YPR184W-YPR185W
Intergenic
XVI
YPR191W
QCR2
Group I CBS1513
Group I CBS1503
Group IIb
Shaded region, detected recombination site a Recombination site induced under heat shock stress (James, Usher, 2008) b Recombination sites found in at least two Group II S. pastorianus genomes (WS34/70 and CBS1260)
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1.3. Whole genome sequencing The first whole genome sequence of a lager yeast was obtained for the Group II strain Weihenstephan 34/70 (WS 34/70) (Nakao et al., 2009) and revealed a polyploid genome with an estimated 36 chromosomes (Table 2). Given the estimated tetraploid DNA content of the lager yeast genomes, the chromosome number appeared low and suggested the presence of multiple copies of near identical chromosomes. Analysis of the genome sequence confirmed the presence of three types of chromosomes, namely S. cerevisiae-like, S. bayanuslike and hybrid chromosomes, the latter resulting from homeologous recombination between parental chromosomes. S. cerevisiae-like genes shared an average 99.2% identity with the reference strain S. cerevisiae S-288C while the S. bayanus-like ORFs showed approximately 92.7% identity with the reference genome CBS 7001, the only S. bayanus genome available at the time. The sites of recombination between the hybrid chromosomes were identified at the nucleotide level and mostly coincided with the sites previously identified by CGH (Table 1). The copy number for S. bayanus ribosomal DNA (rDNA), encoded on chromosome XII, was found to be significantly lower than the corresponding S. cerevisiae rDNA copy number. Additionally, the mitochondrial genome displayed a higher sequence identity to the S. bayanus reference strain CBS 7001, indicating that the Group II lager yeasts have acquired their mitochondrial genome from the non-S. cerevisiae parent. 1.4. Identification of the non-S. cerevisiae genome in lager yeasts While S. bayanus was considered the most likely second parental species of the lager yeasts (Bond, 2009; Bond et al., 2004; Dunn and Sherlock, 2008; Nakao et al., 2009; Rainieri et al., 2006), the low sequence identity (92.5%) to the non-cerevisiae sub-genome suggested another source for the second parent. Further, the S. bayanus taxon appeared extremely complex containing both pure and hybrid strains, prompting the designation of at least two genetically diverse lineages, namely S. bayanus var. bayanus (CBS380) and S. bayanus var. uvarum or S. uvarum (CBS 7001 and type strain CBS395) (Rainieri et al., 2006). During a survey of yeast isolates in Patagonia, South America, Libkind et al. (2011) identified a new Saccharomyces species that they named S. eubayanus sp. nov. The species shares 99.5% identity with the non-S. cerevisiae portion of the lager yeast genome (Libkind et al., 2011). This and other studies also confirmed the pure nature of the lineage of S. uvarum and the hybrid nature of the type strain S. bayanus var. bayanus
(CBS380), which is mainly composed of 67% S. uvarum and 33% S. eubayanus-like sequences but also contained introgressions of S. cerevisiae (Libkind et al., 2011; Nguyen et al., 2011; Perez-Traves et al., 2014).
1.5. More lager yeast genomes Since the publication of the first genome sequence for the Group II lager yeast (WS 34/70), the genomes of several Group I and II lager yeast have been sequenced (Table 2) (De León-Medina et al., 2016; Hewitt et al., 2014; Kvasnicka et al., 2012; Okuno et al., 2016; van den Broek et al., 2015; Walther et al., 2014). The compiled genome sequences provide an insight into the relationship between the Group I and Group II yeasts. Surprisingly, estimated genome sizes and chromosome number for the same strain vary significantly from study to study (Table 2). The diverse range of estimates for genome sizes and chromosome numbers may reflect genuine differences in isolates held in different laboratories but more likely result from different methodologies used to assemble the genomes and to estimate gene copy number. Given the estimated minimum tetraploid DNA content of Group II strains, the expected minimum chromosome copy number should be 64 (haploid chromosome count 16 × 4). Chromosome copy number estimates based on read counts in whole genome sequencing rather than from the sum of nucleotides in assembled contigs appear to be more in line with the expected chromosome copy number as previously estimated by FACs and/or the weight of the DNA. Genome assembly models predict that the prototypic Group I lager yeast (S. carlsbergensis, CBS1513) contains 45–47 chromosomes, consisting of a haploid S. cerevisiae and a diploid S. eubayanus gene content, with significant aneuploidy (Fig. 1). Some chromosomes, for example XI and XII are entirely derived from the S. eubayanus parent while others, for example, chromosomes IX and X are derived from both S. cerevisiae and S. eubayanus. Hybrid chromosomes (III, VII, XIII, XV and XVI) are also present in this Group I strain (Fig. 1). The prototypic Group II strain, WS 34/ 70, is an allotetraploid (4n + 2), consisting of a diploid S. cerevisiae and diploid S. eubayanus gene content on 66 chromosomes (Fig. 1) (Walther et al., 2014; Wendland, 2014). The Group II strain contains both S. cerevisiae and S. eubayanus copies of chromosomes I, VI, IX, X, XI, XII and XIV or either type of chromosome II, III, IV, V, VII, VIII, XIII, and XV, in addition to hybrid chromosomes III, VII, X, XI, XIII, and XVI. Two translocations between S. eubayanus chromosomes II and IV and between VIII and XV are conserved in both Group I and II strains (Fig. 1).
Table 2 Whole genome sequencing of Group I and II lager yeasts. Group
Strain
Estimated genome size (Mb)
Estimated no. of chromosomes
Accession number
Reference
I
CBS 1513
ND 19.5 33.6 19.2 ND 34.1 17.2 14.4 25 22.9 22.5 50.5 56 53.5 ND 35.7 47.5 56.9 39.1 22.7 24.2
31 47 45 ND 31 47 ND ND 36 66 ND 71 77 75 48 55 68 79 49 32 N/A
SAMEA2240035 AZCJ01000000 SRX758305 BBYW01000000 SAMEA2240037 SRX758304 BBYV01000000 BBYX01000000 ABPO01000000 AZAA01000000 BBYY01000000 SRX758144 SRX758145 SRX758149 SAMEA2240036 SRX758306 SRX758140-143 Not deposited Not deposited LSMH01000000 ALJS01000000
Hewitt et al. (2014) Walther et al. (2014) van den Broek et al. (2015) Okuno et al. (2016) Hewitt et al. (2014) van den Broek et al. (2015) Okuno et al. (2016) Okuno et al. (2016) Nakao et al., 2009 Walther et al. (2014) Okuno et al. (2016) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) Hewitt et al. (2014) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) van den Broek et al. (2015) De León-Medina et al. (2016) Kvasnicka et al. (2012)
CBS 1503
II
CBS 1538 WS 34/70
A1 A1 + B11 A2 CBS 1260 CBS 1483 Spy1 Spy2 790 CCY48-91
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Fig. 1. Chromosome copy numbers and types in prototypic Group I (CBS1513) and II (WS 34/70) lager yeasts. S. cerevisiae chromosomes; red, S. eubayanus; blue, hybrid chromosomes red and blue. Intra-species translocations: S. eubayanus translocations between chromosomes II and IV; blue vertical stripes and between chromosomes VIII and XV; blue diagonal stripes. S. cerevisiae translocations between chromosome V and XI; red and white blocks. Inter-species translocations between VIII and XV; diagonal blue. CBS 1513 contains 47 chromosomes (3n-1) and WS 34/70 contains 66 chromosomes (4n + 2). Adapted from Nakao et al. (2009) and Wendland (2014).
1.6. Recombination sites in Group I and II lager yeasts Recombination between the parental chromosomes has occurred at specific chromosomal locations to create a unique set of hybrid chromosomes in lager yeasts. Most of the recombination sites are intragenic although a few intergenic sites are also observed (Table 1). Most of the recombination sites are non-reciprocal with the exception of a reciprocal translocation event at YJR009C/TDH2 on chromosome X (Fig. 1). Several themes emerge from a comparison of the recombination sites identified in the Group I and II strains. 1. Two recombination sites at YGL173C (XRN1) and YGR285C (ZUO1) are conserved between Group I and II strains for which genome sequences are available. A third recombination site at YPL0240C (HSP82) is present in Group I and II strains but appears to be absent in the Group I strain CBS1538 (Monerawela, unpublished). Previously, it was reported that the recombination site at the YCR041C (MAT locus) was also conserved between Group I and II lager yeasts, however a more detailed analysis of this region indicated that while recombination does occur at the MAT locus, the exact location is not conserved (Monerawela unpublished). Furthermore, the Group I strain CBS1538 does not contain a hybrid chromosome III. 2. In addition to the common sites, 4 additional recombination sites (YKL045W, YMR302C, YPR160W, YPR191W) are conserved in Group II strains. 3. Two distinctive patterns of recombination are found in the Group I strains as exemplified by strains CBS1503 and CBS1513 (Table 2). 4. Certain recombination sites are shared between one or more Group I and II strains, e.g. YHR165W. 5. Recombination sites unique to each strain have been identified.
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(It should be noted that data for some recombination sites may be missing from some genome data sets due to lack of sequence coverage). What molecular events could account for the observed recombination between homeologous chromosomes in the lager yeasts? Nakao et al., 2009 noted that the sequences flanking the recombination sites on the hybrid chromosomes share 81–96% sequence identity with sequences on the parental chromosomes. Recombination between sequences with this degree of diversity would be extremely rare (5.4 × 10− 8–2.2 × 10− 9). As similar percentage sequence identities can be observed throughout the genome, without resulting in recombination events, molecular event(s) other than shared sequence identity must define these specific recombination sites. One possibility is that the recombination sites represent fragile sites that are prone to double strand breaks (DSBs), as the rate of recombination is greatly increased by DSBs. What might induce such DSBs? Previous studies have shown that DSBs can be induced by exposure of yeasts to environmental stresses such as ionizing radiation, UV or can occur spontaneously due to replication fork collapse or during meiosis (Mehta and Haber, 2014). In a study designed to generate strains of lager yeasts tolerant to high gravity wort, James et al. (2008), exposed the Group II strain CMBS-33 to extreme high temperatures (50 °C, 15 min) to induce DSBs. The surviving cells were then fermented in high gravity wort (21°P) for 5 days. The selection procedure (high temperature and high gravity wort) was repeated several times and clonal isolates were selected. CGH analysis of the clones revealed that the genomes of several of the isolates had undergone recombination between the homeologous chromosomes at some of the previously identified recombination sites on the hybrid chromosomes (Table 1). This suggests that recombination can be induced at specific sites when yeasts are exposed to environmental stress. In fact, differences in chromosome profiles have been observed by pulse-field gel electrophoresis in single colony isolates at the end of fermentations, suggesting that chromosomes are dynamic and can be altered in a sub-population of cells during fermentation. An analysis of chromosome composition in yeast cultures, at the beginning and the end of fermentation, identified specific chromosomal regions with copy number alterations (James et al., 2008). In fermentations carried out in high specific gravity wort (20°P) and at 20 °C, rather than the normal 10°P and 13 °C, gene amplifications on chromosomes I and XII of the S. cerevisiae sub-genome were observed. The gene amplification on chromosome I was centered around the repetitive DUP240 locus while the amplification on chromosome XII was centered at the rRNA gene cluster. Gene copy number changes were also observed at the telomeres of several but not all chromosomes. Interestingly, gene copy number variations, most notably in telomeric regions, have been observed in a wide variety of industrial S. cerevisiae strains (Adamczyk et al., 2016; Deregowska et al., 2015; Dunn et al., 2012; Zhu et al., 2016). As lager yeasts encounter multiple stressful conditions such as anaerobiosis, high osmotic pressure, low temperature and high hydrostatic pressure during industrial fermentations, such environmental stressors may be the evolutionary driving force responsible for chromosomal recombination and amplification. 1.7. Unique genetic features of lager yeast genomes The happenstance of a hybridisation event between two species, S. cerevisiae and S. eubayanus resulted in the creation of a novel species with unique genetic characteristics. The unique physiological and metabolic properties, emanating from the complex polyploid genome, made this species the ideal yeasts for producing flavoursome beer. 1.7.1. Cryotolerance By far the most important genetic characteristic of S. eubayanus that has been passed onto lager yeasts is cryotolerance (Libkind et al., 2011; Peris et al., 2016). A comparison of the growth and fermentative capacities of 58 lager strains indicated that Group I strains, which contain a higher ratio of S. eubayanus to S. cerevisiae gene alleles grew better
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than Group II strains at 10 °C, although this did not result in a greater fermentation capacity (Gibson et al., 2013). Group II strains, which contain an approximately equal ratio of S. eubayanus to S. cerevisiae gene alleles displayed better growth profiles and fermented best at 22 °C. The Group II strains produce higher levels of flavour compounds than Group I strains, and production of higher alcohols and esters differ in Group I and II strains. Thus, the presence of the S. eubayanus genome greatly influences growth at lower temperatures and appears to contribute to the flavour profile of the resulting beer. To date, the genes responsible for cold tolerance have not been identified in S. pastorianus, however genetic studies on cold tolerance in other yeast species identified several genes, including ADH2, ADH3 encoding alcohol dehydrogenases and GUT2 encoding glycerol-3-phosphate dehydrogenase, as contributing to increased fitness at cold temperatures (Paget et al., 2014). 1.7.2. Gene copy number The polyploid genome of lager yeasts has the potential to uniquely influence the transcriptome, the proteome and ultimately the metabolic landscape of the lager yeast cell. Depending on the copy number of each parental chromosome, homeologous gene alleles from the S. cerevisiae and S. eubayanus sub-genomes can encode for multiple variant protein isoforms which may have significant effects on the cells metabolism through competition for substrate binding, and/or increased or decreased biochemical activities of the encoded proteins. The presence of multiple copies of gene alleles may also increase RNA transcripts levels within the cell, however, early transcriptome analyses of lager yeasts did not indicate a correlation between increased gene copy number and increased transcript levels (Bond et al., 2004). Nevertheless, gene copy number and/or the presence of homeologous gene alleles appears to influence the transcriptome as revealed from a comparison of the expression of S. cerevisiae gene transcripts in a haploid S. cerevisiae strain and two lager yeasts during fermentation (James et al., 2003; James et al., 2002). With the availability of high quality genome sequences for Group I and Group II lager yeasts and improved RNA sequencing technology, it would be a worthy pursuit to reexamine the role of gene copy number on transcript expression levels in the lager yeasts. 1.7.3. Hybrid genes In addition to homeologous alleles, the recombination events between the parental chromosomes, which mainly occur within intragenic regions, generated a set of hybrid genes truly unique to the lager yeast genomes (Table 1). These hybrid genes, composed of part S. cerevisiae and part S. eubayanus, encode for unique variant protein isoforms that may have different biochemical and biophysical activities than their parental proteins. To date, the only detailed study of a hybrid gene is that of YPR160W/GPH1 from the Group II strain CMBS-33. GPH1 encodes for the enzyme glycogen phosphorylase, which is involved in mobilising stored glycogen through conversion of glycogen to glucose-1-phosphate. The Group II strain CMBS-33 contains three hybrid alleles and one S. eubayanus allele. Analysis of the hybrid alleles revealed a stop codon within the ORFs leading to a loss of function. The sole S. eubayanus copy of GPH1 appears to be functional, as the lager yeast contains stored glycogen at levels similar to that observed in a haploid S. cerevisiae cell (Usher and Bond, 2009). Similarly, the introduction of a stop codon into the gene FLO1 as a result of a recombination event is also observed in a Japanese lager yeast strain, rendering the strain non-flocculent (Sato et al., 2002). 2. On the origins of Group I and II lager yeasts 2.1. Origins of the S. eubayanus genome of lager yeast S. eubayanus is a cold-tolerant wild yeast species found on Nothofagus and Araucaria araucana trees in Patagonia, Quercus trees in
Asia and with Cedrus species, Pinus taeda and Quercus rubra trees in North America (Peris et al., 2016; Peris et al., 2014; Rodriguez et al., 2014). Since domestic or wild isolates of S. eubayanus have not been identified in Europe, it was proposed that the species found its way to Europe as part of the trade route established by Conquistadors during the exploration of the New World. However, the recent discovery of wild isolates of S. eubayanus in Tibet, China, North America and New Zealand (Bing et al., 2014; Gayevskiy and Goddard, 2016; Peris et al., 2014) expands the possible geographical origins of the lager yeast parent. Genome sequencing and single nucleotide polymorphism (SNP) analysis of 12 genomic loci, identified the S. eubayanus strain from Tibet as sharing the greatest identity (99.77–99.82%) with the noncerevisiae portion of the lager yeast genome (Bing et al., 2014). However, a recent pan genome sequencing project of all currently known isolates of S. eubayanus, from diverse geographical locations, uncovers a more complicated genetic relationship amongst S. eubayanus isolates (Peris et al., 2016; Peris et al., 2014). Several of the isolates, including strains from Tibet and North America cluster together into a lineage referred to as Holartic. This group of strains display low diversity and are related to a second lineage primarily found in South America. A third lineage, containing a more diverse population is also identified in South America. The genetic diversity between S. eubayanus isolates appears to arise from admixture, outcrossing and incomplete lineage sorting. To date, no sole isolate appears to be the lowest common ancestor (LCA) of the non-cerevisiae parent of lager yeasts, however it appears that strains related to Tibetan isolates contributed 66% of the lager S. eubayanus sub-genome while the remainder of the sub-genome (34%) is most closely related to an isolate from North Carolina, U.S.A. (Peris et al., 2016). Thus, genetic variation within S. eubayanus isolates most likely accounts for the genetic variation observed in the S. eubayanus portion of the lager yeast genome. Based on this analysis the original hypothesis of S. eubayanus travelling to Europe from Patagonia was subsequently rivaled by an alternative hypothesis of a journey of S. eubayanus along the Silk Route from China or Tibet. Since the discovery of the New World post-dates historical records of lager brewing in Europe and predominately emanated from South-West Europe, a region not known for beer fermentations, the former hypothesis currently seems less likely. Rather, given the physical geographical proximity of Asia and Europe and the closer genetic relationship between Tibetan isolates and the lager yeasts, the latter hypothesis seems more favourable. As wild yeasts can be dispersed by environmental and animal means, alternative hypotheses cannot be ruled out at present. Whatever the means, the arrival of S. eubayanus in Europe and its subsequent hybridisation with S. cerevisiae would bring about the revolutionary shift in brewing practices to low temperature brewing through the introduction of cold-tolerance from S. eubayanus to the normally mesophilic S. cerevisiae. 2.2. Origins of the S. cerevisiae genome of lager yeast Prior to the availability of whole genome sequences for the wide variety of Saccharomyces cerevisiae strains and isolates, phylogenetic trees compiled from a comparison of intronic sequences or micro satellite gene loci identified Ale yeasts as the most likely ancestor of the S. cerevisiae sub-genome in lager yeasts (Dunn and Sherlock, 2008; Legras et al., 2007). In recent years, as a result of the concerted efforts of research groups around the world, genome sequences for greater than 1000 wild and domesticated S. cerevisiae isolates have become available (Bergström et al., 2014; Borneman et al., 2011; Borneman et al., 2016; Borneman et al., 2008; Gallone et al., 2016; Gonçalves et al., 2016; Liti et al., 2009; Strope et al., 2015; Wang et al., 2012; Zhu et al., 2016). Phylogenic analyses clearly segregates the different isolates/ strains into distinctive populations delineated by source (wine, cider, bread, beer, sake, clinical, laboratory, distilling etc.) or by geographic locations (Asia, North America, Africa, European etc.). Some isolates are classified as admixed mosaics, displaying ancestry from two or more
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populations. In addition to S. cerevisiae genes, several isolates were shown to contain non-S. cerevisiae gene sequences introgressed from other Saccharomyces stricto sensu species, mainly S. paradoxus or from other fungal species. In general, yeasts associated with brewing cluster together into a distinct sub-population (Gallone et al., 2016; Gonçalves et al., 2016). Furthermore, an extensive analysis of over 100 beer isolates revealed the segregation of beer yeasts into two distinct sub-groups, named, Beer 1 and 2 (Gallone et al., 2016). The Beer 1 subgroup contains isolates from The British Isles (Britain and Ireland), the USA and from Germany and Belgium. The USA isolates are phylogenetically related to the British isolates suggesting that early British settlers introduced these yeasts to the United States. The Beer 2 clade is more closely related to wine yeasts and contains approximately 20% of all brewing strains. In a separate study, Gonçalves et al. (2016) subdivided beer isolates into three clades, Wheat, English-Irish Ales and German Alt-Kolsch beers respectively. These data suggest that beer yeasts emerged as a result of at least two independent European domestication events. Using average-per-site nucleotide divergence and an estimated average mutation rate for beer yeasts, Gallone et al. (2016) suggests that the first domestication occurred between 1573 and 1604 CE and the second domestication event dated later between 1645 and 1671 CE. This timing coincides with the shift from cottage industry to the skilled craft industrialization of brewing. A comparison of the S. cerevisiae subgenomes of Group I and Group II lager yeasts with 90 yeasts strains including bread, beer, sake, wine, and wild yeasts from different geographical locations, placed the lager yeasts in the same clade as the beer yeast (Gonçalves et al., 2016). However, both lager yeast groups form a separate sub-clade within the beer clade and are clearly divided further into Group I and II lager yeasts within the sub-clade. Thus, lager yeasts appear to have arisen from Ale-like yeasts but may have been further domesticated following the hybridisation event with S. eubayanus. 2.3. Sub-telomeric regions of lager yeast genomes Genome analyses of S. cerevisiae strains and isolates revealed that a significant proportion of genetic diversity within Saccharomyces species is accounted for by changes within sub-telomeric regions of chromosomes (Bergström et al., 2014; Dunn et al., 2012; Gallone et al., 2016; James et al., 2008). Many unique genes, not found in the reference S. cerevisiae strain, S-288C, have been identified in the sub-telomeric regions in S. cerevisiae isolates (Bergström et al., 2014) and copy number variations (CNVs) resulting from gene amplification and deletions within the sub-telomeric regions of S. cerevisiae strains have been reported (Dunn et al., 2012). Sub-telomeric regions may be of specific importance in lager yeasts as several important gene families, such as the MAL genes required for maltose uptake and utilisation and the FLO genes required for the property of yeast flocculence, reside in these regions. In fact, recent genome analysis indicates that the MAL gene clusters are significantly amplified in beer isolates relative to other S. cerevisiae isolates (Gonçalves et al., 2016). Monerawela et al. (2015) compared the 32 sub-telomeric regions (right and left sub-telomeres of 16 chromosomes; RST, LST) of the Group II lager yeast chromosomes to the sub-telomeric regions of 32 S. cerevisiae strains from different industrial and geographic sources. Average nucleotide-per-site divergence rates placed the Ale yeasts Fosters O and B as the LCA of the Group II lager yeasts. A comparison of each of the 32 individual sub-telomeric regions in the Group II lager yeasts to the genomes of the 32 S. cerevisiae strains and to the Group I lager yeasts revealed that the RST of chromosome XIII in the Group II lager yeast was absent in the Group I strains and from 25 of the 32 S. cerevisiae strains examined. Second, sub-telomere regions of Group II chromosomes IV, VI, XI, XII, XV and XVI were absent in the Group I lager yeast. This loss of sub-telomeric regions accounts for some of the reduction in S. cerevisiae content noted between Group I and II lager yeasts. Several “lager-specific (LgS)” genes, residing at sub-telomeric locations were identified in the genome of the prototype Group II strain
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WS 34/70 (Nakao et al., 2009). Four of the genes, Lgs-FSY, Lgs-MCT, Lgs-AMD2, and Lgs-MEL, encoding for a fructose symporter, a monocarbohydrate transporter, an amidase, and a melibiase respectively, were subsequently assigned to the S. eubayanus sub-genome. Four other genes, Lgs-TYP, Lgs-AMD1, Lgs-TRR and Lgs-HYPO, encoding for a tyrosine permease, a second amidase, a transcriptional regulatory protein and a hypothetical open reading frame, respectively, are not present in the S. cerevisiae reference genome S-288C but PCR analysis confirmed the presence of the four LgS genes in nine other Group II production strains (Monerawela et al., 2015). Using RST XIII and LST IV as genome markers that distinguish Group I and II lager yeasts and the 4 Lg-S genes found on Group II strains, Monerawela et al. (2015) examined 78 S. cerevisiae strains to identify the most likely parents of the Group I and II yeasts. The pattern of presence or loss of the six genetic loci clearly distinguishes between S. cerevisiae strains and allows their assignment into specific sub-groups. All six DNA markers are found in all Group II lager yeast strains, as well as in a group of S. cerevisiae strains associated with stout brewing in the British Isles. This grouping also includes two S. cerevisiae strains used in the production of the alcoholic beverage “Toddy” from tender coconuts a South-West India. The Group I lager yeast strains have lost LST IV and RST XIII and the genes Lgs-TYP, Lgs-AMD1, which reside at the latter sub-telomere, but retain Lgs-TRR and Lgs-HYPO, which reside at LST XIV and RST XVI respectively. This same pattern of loss is also observed in most yeasts used for Ale production. All of the laboratory strains and most wine yeasts analysed have lost the four LgS genes as well as RST XIII but retain LST IV, and therefore are distinguishable from the Ale yeasts. Thus, the pattern of loss of “lager-specific” genes and specific sub-telomeric regions clearly clusters Group I and II lager yeasts with two distinctive groups of beer yeasts; Group I with Ale yeasts and Group II with stout yeasts and suggests that the S. cerevisiae sub-genomes in Group I and II lager yeasts are derived from different S. cerevisiae lineages. 2.4. A model for the evolution of Group I and II lager yeasts Based on the data accumulated thus far on the composition and structure of the Group I and Group II lager yeast chromosomes, several contrasting hypotheses have been proposed to account for the origin and evolution of the S. pastorianus species. The presence of shared recombination sites on hybrid chromosomes III, VII and XVI led Walther et al. (2014) to propose that Group I and II lager yeasts arose from a single hybridisation event between a diploid S. eubayanus strain and a common ancestral diploid S. cerevisiae strain. Following hybridisation, it was proposed, the Group I and II strains evolved into two distinctive groups through the significant loss of the S. cerevisiae genome content in the Group I strains and further recombination events leading to unique hybrid chromosomes in both groups. Since this initial proposition, several additional lager yeast genomes have been sequenced revealing a more complex relationship between the Group I and II lager yeasts. Firstly, the number of recombination sites conserved in all lager yeasts is less than previously estimated. Secondly, the Group I strains appear to be divided into two sub-groups each with its own unique set of recombination sites (Table 1). Thirdly, each lager yeast strain contains its own unique recombination sites, suggesting that recombination between the homeologous parental chromosomes is an ongoing process, most likely in a response to environmental stressors. The analysis of the sub-telomeric regions of Group I and II revealed a significant erosion of chromosome ends in Group I strains (Monerawela et al., 2015). The pattern of loss or retention of the sub-telomeric regions, and of a set of Lg-S genes encoded therein, reveals distinct relationships between Group I yeasts and S. cerevisiae strains used in Ale production and between Group II yeasts and S. cerevisiae strains used in Stout production. As it is highly unlikely that the same pattern of sub-telomeric region loss or retention would occur randomly in many different Ale and Stout strains, this suggests that the two groups of
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Fig. 2. Sequential hybridisation model for the evolution of Group I and II lager yeasts. The initial hybridisation event occurred between a haploid Ale S. cerevisiae isolate and a diploid S. eubayanus isolate to generate a progenitor lager yeast. A second hybridisation event between the progenitor strain and an S. cerevisiae isolate used in stout production generated the Group II lager yeasts. Both Group I and II lager yeasts underwent recombination between the parental chromosomes to generate hybrid chromosomes. The Group I strains CBS1513 and CBS1503 appear to have taken separate evolutionary pathways post hybridisation.
lager yeasts arose by separate independent hybridisation events between S. eubayanus and two distinct S. cerevisiae isolates. The recent discovery of two or three distinctive clades of beer yeasts lends support this hypothesis (Gallone et al., 2016; Gonçalves et al., 2016). Interestingly, the Group I and II lager yeasts do not cluster with any of the S. cerevisiae beer clades and instead form two distinct sub-clades within the beer groups. This may indicate that the true ancestor of the lager yeasts has yet to be discovered or alternatively that further evolution has occurred post hybridisation with S. eubayanus. Recent data from Okuno et al. (2016) suggests an evolutionary model that reconciles both the single and multiple hybridisation hypotheses. Analysing the genomes of 10 S. pastorianus strains, the research group identified two S. cerevisiae sub-genomes that can be distinguished by SNPs within the Group II yeasts, one of which is shared with the Group I strains. Thus, the most likely evolutionary path involved an initial hybridisation between a haploid S. cerevisiae strain and a diploid S. eubayanus strain leading to a progenitor S. pastorianus strain (Fig. 2). This progenitor lager yeast underwent recombination between the parental chromosomes at two loci on chromosome VII and reciprocal translocations between chromosomes II and IV and between VIII and XV. The parental S. cerevisiae strain was most likely a yeast isolate used in the production of Ales, which had already suffered significant chromosomal erosion at the telomeres, specifically at RST XIII and LST IV. The Group I lager yeasts underwent subsequent chromosomal recombination to produce two distinct sub-types. The adaptive/environmental pressures influencing this divergent evolution are currently unknown. A subsequent hybridisation between the progenitor lager yeast strain and a second distinct S. cerevisiae strain produced the Group II lineage (Fig. 2). The S. cerevisiae strain involved in the second hybridisation event contains additional genetic material at the ends of the chromosomes, specifically at RST XIII and LST IV. So far the only yeast strains identified that contain these regions are S. cerevisiae strains used in the production of Stout in the British Isles and two strains isolated
from South-West India used to make the alcoholic beverage Toddy from tender coconuts. It is tantalizing to speculate that yet another trade route (between India and Great Britain) may have played a role in the evolution of the lager yeasts, however we await further genome sequences, specifically of a broader range of Ale yeasts and from more geographical diverse locations, to uncover the true ancestors of the lager yeasts.
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