Irina Bolat
Irina Bolat
AnAlysis of the
hybrid genomes of
brewing yeAsts
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Irina Bolat
INVITATION
To attend the defence of
my PhD thesis:
Analysis of the hybrid
genomes of brewing yeasts
on Wednesday,
January 6th, 2015 at 15:00
in the Senaatzaal
of the Aula at TU Delft,
Mekelweg 6, Delft
Prior to the defence (14:30)
there will be
a presentation of the thesis
for non-experts
You are also invited to the
reception which follows,
starting 17:00,
in ‘t Keldertje,
Department of Biotechnology,
Julianalaan 67, Delft
Irina Bolat
bolatirina@yahoo.com
Paranymphs:
Barbara Kozak
Barbara.Urszula.Kozak@gmail.com
Daniel Solís Escalante
danielsoes@gmail.com
genomes of brewing yeasts
Proefschrift
ter verkrijging van de graad van doctor
aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus
prof. ir. K.Ch.A.M. Luyben,
voorzitter van het College voor Promoties,
in het openbaar te verdedigen op
woensdag, 6 januari 2016 om 15:00
door
Irina BOLAT
Dipl-Ing in Food Science and Engineering,
“Dun
ărea de Jos” University, Galaţi,
Copromotor: Dr. ir. J-M. Daran Composition of the doctoral committee: Rector Magnificus Chairperson
Prof. dr. J.T. Pronk Delft University of Technology, promotor Dr. ir. J-M. Daran Delft University of Technology, copromotor Independent members:
Prof. dr. E.J. Smid Wageningen University Prof. dr. G. Walker University of Abertay Dundee Prof. dr. W.R. Hagen Delft University of Technology
Dr. B. Gibson VTT Technical Research Centre, Espoo, Finland Dr. ir. J-M. Geertman Heineken Supply Chain, Zoeterwoude
The research described in this thesis was performed at the Industrial Microbiology Section, Department of Biotechnology, Delft University of Technology, the Netherlands and financed by Heineken Supply Chain via the R&I platform.
Samenvatting/Summary 9
Chapter 1 General introduction 19
Chapter 2 Chromosomal copy number variation in Saccharomyces
pastorianus is evidence for extensive genome dynamics in
industrial lager brewing strains
53
Chapter 3 amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae
89
Chapter 4 Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad-substrate-specificity 2-oxo-acid decarboxylases, in the brewing yeast
Saccharomyces pastorianus CBS1483
115
Chapter 5 Saccharomyces cerevisiae x Saccharomyces eubayanus
interspecific hybrid, the best of both worlds and beyond
141
Curriculum vitae 172
List of Publications 173
Born of the flowing water, Tenderly cared for by the Ninhursag,(…)
You are the one who handles the dough [and] with a big shovel, Mixing in a pit, the bappir with sweet aromatics,
Ninkasi, you are the one who handles the dough [and] with a big shovel, Mixing in a pit, the bappir with [date] - honey,
You are the one who bakes the bappir in the big oven, Puts in order the piles of hulled grains,
Ninkasi, you are the one who bakes the bappir in the big oven, Puts in order the piles of hulled grains,
You are the one who waters the malt set on the ground, The noble dogs keep away even the potentates,
Ninkasi, you are the one who waters the malt set on the ground, The noble dogs keep away even the potentates,
You are the one who soaks the malt in a jar, The waves rise, the waves fall.
Ninkasi, you are the one who soaks the malt in a jar, The waves rise, the waves fall.
You are the one who spreads the cooked mash on large reed mats, Coolness overcomes,
Ninkasi, you are the one who spreads the cooked mash on large reed mats, Coolness overcomes,
You are the one who holds with both hands the great sweet wort, Brewing [it] with honey [and] wine
Ninkasi, (...)(You the sweet wort to the vessel)
The fermenting vat, which makes a pleasant sound, You place appropriately on a large collector vat.
Ninkasi, the fermenting vat, which makes a pleasant sound, You place appropriately on a large collector vat. When you pour out the filtered beer of the collector vat,
It is [like] the onrush of Tigris and Euphrates.
Ninkasi, you are the one who pours out the filtered beer of the collector vat, It is [like] the onrush of Tigris and Euphrates.
The oldest recipe for brewing written by Sumerians as a poem dedicated to their beer goddess Ninkasi. Written down in 1800 BC, the hymn is in fact much older. Translation by Miguel Civil. (Joshua J. Mark, 2011. The hymn to Ninkasi, goddess of beer, Ancient Encyclopedia History)
SAMENVATTING
SUMMARY
of the PhD thesis
“Analysis of the hybrid
genomes of brewing yeasts”
SAMENVATTING
Eén van de best bewaarde geheimen van brouwers wordt vertegenwoordigd door de gebruikte gist in het brouwproces, vanwege de enorme impact die deze heeft op het specifieke smaakprofiel van het eindproduct. Dit vindt zijn oorsprong in de genetische aanleg van de gebruikte gist. Gebaseerd op het gedrag van de gistcellen aan het einde van de fermentatiestap in het brouwproces, kunnen er twee hoofdgroepen brouwgist worden onderscheiden: hooggistende Saccharomyces cerevisiae stammen (ale gist) en laaggistende Saccharomyces
pastorianus stammen (lager gist). Deze laatste hebben een complexe genetische architectuur
vanwege hun hybride genoom, bestaande uit de chromosomen van twee verschillende soorten: S. cerevisiae en S. eubayanus. De S. pastorianus giststammen zijn geclassificeerd in twee groepen, afgeleid van twee afzonderlijke hybridisaties tussen S. cerevisiae en S.
eubayanus stammen: de Saaz groep is ontstaan uit de samensmelting van een haploïde S. cerevisiae met een haploïde S. eubayanus; de Frohberg groep vindt zijn oorsprong in de fusie
van een diploïde S. cerevisiae met een haploïde S. eubayanus. De giststammen binnen de twee groepen hebben zowel verschillende genoomgroottes als duidelijk verschillende fysiologische eigenschappen. In dit proefschrift worden een aantal studies beschreven die uitgevoerd zijn met ondergistende (lager) giststammen, met CBS1483 uit de Frohberg groep als model. De oorsprong van lager giststammen wordt beschreven in Hoofdstuk 1. Hoewel al veel langer bekend was dat lager giststammen een hybride genoom hebben, was tot voor kort alleen het S. cerevisiae sub-genoom duidelijk geïdentificeerd. De non-cerevisiae tegenhanger is pas recent ontdekt en aangeduid als S. eubayanus. De complexiteit van het genoom van lager giststammen wordt verder vormgegeven door de brouwomstandigheden. Deze veroorzaken abnormale chromosoom kopieaantallen (aneuploïditeit), inter-chromosomale translocaties, geheel of gedeeltelijk verlies van chromosomen, chromosomale rearrangements met toenemende kopieaantallen en introgressie. Al deze veranderingen spelen een belangrijke rol in de uniekheid van ondergistende stammen. Dit wordt behandeld in Hoofdstuk 2, waar de totale genoomsequentie van zes ondergistende stammen duidelijk laat zien dat de hybride genomen van biergisten meer zijn dan alleen eenvoudige samenvoegingen van twee sub-genomen. De stam CBS1483, die gedurende dit proefschrift als studieobject werd gebruikt, is gesequenced met Illumina HiSeq2500 en 4 gepaarde libraries met verschillende insert groottes. Met deze methode kon de hoogste coverage van een lagergist genoom behaald worden (~270x) die tot nu toe gepubliceerd is. Tevens is het kopieaantal van de chromosomen in CBS1483 bepaald. Hierdoor werd het mogelijk om een genetische kaart samen te stellen, bestaande uit een totaal van 68 chromosomen en 35 verschillende chromosomale structuren. Chromosoom III vertoonde een zeer intrigerende structuur, als een uitzonderlijk chimeer chromosoom, zonder eenvoudige kopieën van één van de twee sub-genomen. De aneuploïditeit van CBS1483 werd benadrukt door de hoge variatie in kopieaantal van de 35 chromosomale structuren, uiteenlopend van 1 tot wel 5 kopieën.
Dergelijke buitengewone kopieaantallen werden bevestigd met qPCR en flow cytometrie. Al met al bleek het genoom van S. pastorianus CBS1483 te bestaan uit 56% van S. cerevisiae, 34% van S. eubayanus en 10% chimeer S. cerevisiae/S. eubayanus chromosomaal DNA, verdeeld over 68 chromosomen. Naast de identificatie van de chromosomale complexiteit werd nog een tweede bijzondere eigenschap vastgesteld in CBS1483 die zich uitte in allel variatie, waarbij kopieën van hetzelfde gen bestonden uit verschillende nucleotide sequenties. Om meer inzicht te krijgen in het genomische landschap van lager giststammen werden nog vijf Frohberg-type gisten gesequenced, resulterend in een complex beeld van de soort
S. pastorianus. Niet alleen varieerde het chromosoom kopieaantal significant tussen de
verschillende bestudeerde industriële lager stammen (van 49 tot 79), ook werd een duidelijke impact aangetoond op brouweigenschappen als de capaciteit om diacetyl te produceren en de flocculatie capaciteit.
Aneuploïditeit in lager gistcellen geeft overlevingsvoordelen, maar het onderhoud en de expressie van complete extra chromosomen zorgt ook voor een toegenomen energiebehoefte. Dit zou een verklaring kunnen zijn voor de hoge gevoeligheid voor antibiotica die aneuploïde stammen vertonen. Gezien het feit dat dergelijke remmers onderdeel uitmaken van de genetische modificatie strategie die gebruikt wordt bij de selectie van bepaalde eigenschappen, beperkt deze gevoeligheid van lager giststammen het aantal heterologe genen dat als selectiemarker gebruikt kan worden. Tegemoetkomend aan deze beperking, beschrijft Hoofdstuk 3 een nieuwe herbruikbare dominante markercassette amdSYM, succesvol gebruikt in zowel lager als ale giststammen. De amdSYM cassette, gevormd uit de
Ashbya gossypii TEF2 promotor en terminator en een codon-geoptimaliseerd aceetamidase
gen (Aspergillus nidulans amdS), zorgt ervoor dat de gisten aceetamide als enige stikstofbron kunnen gebruiken. Hergebruik van de amdSYM cassette zonder enige heterologe sequenties achter te laten in het genoom, werd eenvoudig mogelijk gemaakt door te groeien in aanwezigheid van fluoroaceetamide. Met behulp van deze techniek werden de volgende genen gedeleteerd: S. cerevisiae-type HXK1 in de Saccharomyces pastorianus lager giststam CBS1483, S. cerevisiae-type ARO80 in een Scottish Ale stam en S. eubayanus-type ARO80 in de nieuw ontdekte stam CBS12357. Geen van deze stammen bezit de mogelijkheid om aceetamide te gebruiken als enige stikstofbron, wat hen uitstekende kandidaten maakt voor de nieuwe marker. Een ander voordeel van deze marker is dat deze eindeloos hergebruikt kan worden, waardoor meerdere modificaties mogelijk gemaakt worden zonder de extra eiwitbelasting die de overexpressie van verschillende heterologe markers met zich mee zou brengen. De Euroscarf collectie bevat de nieuwe amdSYM cassette.
Er is nog maar weinig bekend over de regulering en de impact van de complexe genoomorganisatie van lager giststammen op de smaakproductie. In het kader hiervan wordt in Hoofdstuk 4 een case studie gepresenteerd over de bijdrage aan de smaakproductie van de S. cerevisiae en S. eubayanus subgenomen van de lager gist CBS1483. De studie concentreerde zich op ARO10, een 2 oxo-acid decarboxylase betrokken bij de productie van hogere alcoholen via de Ehrlich pathway, en de bijbehorende transcriptieregulator
ARO80. Beide genen bevinden zich op chromosoom IV, of een chromosoom met gelijke
grootte, met drie S. cerevisiae-type allelen (LgSc) en een S. eubayanus-type allel (LgSeub). De functionele analyse van de twee type allelen die de twee sub-genomen binnen de lager giststam CBS1483 vertegenwoordigen, werd uitgevoerd door beide allelen individueel tot expressie te brengen in een decarboxylase-negatieve laboratoriumstam van S. cerevisiae. Hierbij werden subtiele verschillen aangetoond in de substraatspecificiteit van de S. cerevisiae-achtige en S. eubayanus-cerevisiae-achtige isoenzymen van Aro10. Hoewel phenylpyruvaat het voorkeurssubstraat was voor beide, was de activiteit op ketoisovaleraat, een precursor voor isobutanol productie, tweemaal hoger voor SeubAro10. Transcript analyse toonde duidelijke verschillen aan in stikstofbron-afhankelijke regulatie van de twee allelen. Phenylalanine als stikstofbron was de sterkste inductor voor (Lg)ScARO10 in zowel de als controle gebruikte
S. cerevisiae stam als de lager giststam CBS1483. Het LgSeubARO10 allel in deze stam werd
anders gereguleerd dan het ScARO10 allel, met een hoge basale expressie indien gegroeid op ammonia en leucine als stikstofbron, en matige expressie op phenylalanine. Een andere interessante ontdekking was dat de ratio van de transcriptieniveau’s van LgScARO10 en LgSeubARO10 in phenylalanine-gegroeide cultures 3:1 was, wat overeenstemt met het aantal kopieën dat voor beide allelen bepaald werd. De herbruikbare dominante markercassette
amdSYM, beschreven in hoofdstuk 3, werd gebruikt om de enkele kopie van LgSeubARO80
uit CBS1483 te verwijderen. Dit onthulde een trans cross-regulatie tussen de twee type allelen, waarbij LgScerARO80 de regulatierol van zijn tegenhanger overneemt. De studie gaf een duidelijk gebrek in correlatie aan tussen de enzymatische activiteit van ARO10 en de genexpressie, wat er sterk op wijst dat post-transcriptionele regulatie een belangrijk rol speelt in aneuploïde lager giststammen.
Het gecompliceerde genoom van lager stammen vertegenwoordigt een sterk voorbeeld van de kracht van omgevingscondities op de moleculaire mechanismen die verantwoordelijk zijn voor bepaalde brouwkarakteristieken. Om de voordelen voortkomend uit het vroegere hybridisatiefenomeen tussen S. cerevisiae en S. eubayanus naar de S. pastorianus afstammeling te onderschrijven, werd een kunstmatige hybride geproduceerd en bestudeerd in Hoofdstuk
5. Met behulp van mass mating werd een hybride geconstrueerd tussen een haploïde S.
cerevisiae stam van de CEN.PK familie en een haploïde stram afgeleid van de S. eubayanus
type stam CBS12357. De genomen van de hybride stam en een van de ouders (S. eubayanus) werden gesequenced met behulp van Illumina technologie. Door de temperatuursinvloed tijdens fermentatie te bestuderen, werd duidelijk dat de nieuwe stam voor de meeste temperaturen de groeikarakteristieken had verworven van de best presterende ouder. Voor temperaturen tussen 20 en 30°C vertoonde de nieuwe stam betere prestaties dan de beste ouder. Het vermogen van giststammen om maltose en maltotriose te consumeren, is van het uiterste belang bij het produceren van bier, zowel voor stabiliteit als om economische redenen. In de Sc x Seub intersoort hybride, werd het onvermogen van S. eubayanus om maltotriose te consumeren gecompenseerd door de acquisitie van het S. cerevisiae genoom. De hybride vertoonde duidelijke diauxie bij gebruik van maltose en maltotriose, zoals ook
gezien in de S. pastorianus stam CBS1483. Hoewel de huidige data onvoldoende houvast biedt om de onderliggende mechanismen te identificeren die aan de basis liggen van de fysiologische verschillen tussen Saccharomyces soorten bij verschillende temperaturen, zou de verbeterde sequentie van S. eubayanus en de beschikbaarheid van de Sc x Seub hybride in de toekomst kunnen bijdragen een het ontcijferen van de multifactoriële en weinig begrepen moleculaire grondslag van koude tolerantie.
SUMMARY
One of the best guarded secrets of brewers is represented by the brewing yeast employed in beer fermentation, due to its profound impact upon the specific flavor profile of the final product. This is in turn imparted by its genetic make-up. Based on the behavior of the yeast cells at the end of the fermentation step of the brewing process, two main groups of brewing yeast can be distinguished: top fermenting yeast (ale yeast) Saccharomyces cerevisiae and bottom-fermenting yeast (lager yeast) Saccharomyces pastorianus. The latter has a complex genetic architecture due to its hybrid genome, comprising chromosomes from two different species: S. cerevisiae and S. eubayanus. The S. pastorianus brewing yeast strains have been classified in two groups derived from two distinct hybridization events between S. cerevisiae ale strains and S. eubayanus: the Saaz group resulted from the hybridization of haploid S.
cerevisiae with haploid S. eubayanus and the Frohberg group obtained from the fusion of
a diploid S. cerevisiae with a haploid S. eubayanus. The yeast strains within the two groups have different genome sizes as well as distinct physiological characteristics. This thesis presents a number of studies performed on bottom-fermenting (lager) brewing yeast strains, using as model the lager yeast CBS1483 from the Frohberg group.
The origin of lager brewing strains as well as their taxonomic classification is presented in
Chapter 1. Although lager brewing strains were long known to have a hybrid genome, only
the S. cerevisiae sub-genome was clearly identified, while the non-cerevisiae counterpart has only recently (has only been discovered recently) and designated as S. eubayanus. The complexity of the genome of lager brewing strains is further shaped by the brewing conditions. They trigger an unbalanced chromosome copy number (aneuploidy), inter-chromosomal translocations, complete or partial chromosome loss, chromosomal rearrangements with increased gene copy number and introgressions. All these changes play an important role in the unicity of lager brewing strains. This is addressed in Chapter 2 where the whole-genome sequence of six lager brewing strains clearly indicated that the hybrid genomes of brewing yeast strains are more than just simple pairings of two sub-genomes. The strain CBS1483, used as a study case throughout the thesis, was sequenced with Illumina HiSeq2500 and 4 paired libraries with different insert sizes. This method allowed the highest coverage of a lager genome (~270x) published to this day. The copy number of the chromosomes within CBS1483 was also identified, thus allowing the assembly of a genetic map, with a total count of 68 chromosomes and 35 different chromosomal structures. Chromosome III showed such an intriguing structure, standing out as a chimeric chromosome, with no plain copies of either of the subgenomes. The aneuploidy of CBS1483 was underlined by the high variation in copy number of the 35 chromosomal structures, ranging from 1 copy up to 5 copies. Such an exceptional chromosome copy number was also confirmed with qPCR and flow cytometry. Overall, the genome of S. pastorianus CBS1483 was composed of 56% of S. cerevisiae, 34% of
S. eubayanus and 10% of chimeric S. cerevisiae/S. eubayanus chromosomal DNA distributed
over 68 chromosomes. Alongside the identification of chromosome intricacy, another special feature was distinguished in CBS1483, represented by allelic variation, with copies of the
same gene displaying different nucleotide sequences. Further into understanding the genomic landscape of lager brewing strains, five more Frohberg-type yeasts were sequenced, revealing a complex picture of the S. pastorianus species. Not only the chromosome copy number significantly varied among the industrial lager strains studied (49-79), but it clearly impacted their brewing-related traits: the diacetyl production capacity, flocculation capacity.
Aneuploidy in lager yeast cells brings survival advantages but maintaining and expressing entire additional chromosomes also represents an energetic burden. This might also explain the high sensitivity exhibited by aneuploid strains to antibiotics. Considering that such inhibitors are part of the genetic engineering strategy involved in the selection of certain traits, this sensitivity of lager brewing strains restricts the number of heterologous genes that can be used as selectable markers. Addressing this constraint Chapter 3 describes a new recyclable dominant marker cassette amdSYM, successfully used in both lager and ale brewing strains. The amdSYM cassette, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), confers the yeasts the ability to use acetamide as sole nitrogen source. The recycling of the amdSYM cassette was easily performed by growth in the presence of fluoroacetamide, without leaving any heterologous sequences in the genome. With this technique the following genes were deleted: S. cerevisiae - HXK1 in the Saccharomyces pastorianus lager brewing strain CBS1483, S. cerevisiae - ARO80 in a Scottish Ale strain and S. eubayanus - ARO80 in newly discovered strain CBS12357. None of these strains have the capability to grow on acetamide as sole nitrogen source, which makes them good candidates for the new marker. Another advantage of this marker is the possibility to be re-used an unlimited number of times, thus enabling multiple modifications without the protein burden that would cause the overexpression of several heterologous markers. The Euroscarf collection hosts the new
amdSYM cassette.
The impact of the complex genome organization of lager brewing strains on flavour production and its regulation is poorly understood. In this respect a case study on the contribution of the S. cerevisiae and S. eubayanus subgenomes from lager brewing yeast CBS1483 upon aroma production is presented in Chapter 4. The study focused on ARO10, a 2 oxo-acid decarboxylase involved in production of higher alcohols via the Ehrlich pathway and its transcriptional regulator ARO80. Both genes are localized on chromosome IV, or a chromosome of similar size, with three S. cerevisiae-type alleles (LgSc) and one S.
eubayanus-type allele (LgSeub). The functional analysis of the two types of alleles reflecting
the two sub-genomes within lager brewing strain CBS1483 was performed by individual expression of each allele in a decarboxylase-negative laboratory strain of S. cerevisiae. Subtle differences were revealed in substrate specificity of the S. cerevisiae-like and S. eubayanus-like isoenzymes of Aro10. While phenylpyruvate was the preferred substrate for both, the activity towards ketoisovalerate, a precursor for isobutanol production, was 2-fold higher for LgSeubAro10. The transcript analysis revealed clear differences in nitrogen-source dependent regulation of the two alleles. Phenylalanine as nitrogen source was the strongest
inducer for (Lg)ScARO10 in both S. cerevisiae strain used as control and lager brewing strain CBS1483. The LgSeubARO10 allele in the brewing strain was regulated differently from the
LgScARO10 allele, showing a high basal expression when growing in ammonia and leucine
as nitrogen source and moderate in phenylalanine. Another interesting discovery was that the ratio of the transcript levels of LgScARO10 and LgSeubARO10 in phenylalanine-grown cultures was 3:1, which is consistent with the number of copies identified for each allele. The recyclable dominant marker cassette amdSYM described in Chapter 3, was used to delete the single copy of LgSeubARO80 from CBS1483. This disclosed a trans cross-regulation between the two types of alleles, with LgScARO80 taking over the regulatory role of its counterpart. The study clearly indicated a lack of correlation between the enzymatic activity of ARO10 and the gene expression, strongly suggesting that post-transcriptional regulation is very active in aneuploid lager brewing strains.
The intricate genome of lager strains represents a strong example of the power of the environmental conditions upon the molecular mechanisms, responsible for specific brewing traits. To endorse the advantages brought by the ancient hybridization phenomenon between
S. cerevisiae and S. eubayanus onto the new offspring S. pastorianus, an artificial hybrid
was produced and studied in Chapter 5. Using mass mating, a hybrid between a haploid S.
cerevisiae strain of the CEN.PK family and a haploid strain derived from the S. eubayanus
type strain CBS12357 was constructed. The genomes of the hybrid strain and one of the parents (S. eubayanus) were sequenced using Illumina technology. Studying the temperature response during fermentation, it was clear that the new strain acquired the growth characteristics of the best performing parent for most temperatures, and it outperformed the best parent for temperatures ranging from 20 to 30°C. The ability to consume maltose and maltotriose by the brewing yeast strains is of paramout importance in beer production, both for stability as well as economic reasons. In the Sc x Seub interspecific hybrid, the inability of
S. eubayanus to consume maltotriose was compensated by the acquisition of the S. cerevisiae
genome. Interestingly, the hybrid showed a pronounced diauxic utilization of maltose and maltotriose, also observed in the S. pastorianus strain CBS1483. While the present data are not sufficient to identify the underlying mechanisms that govern the physiological differences between the Saccharomyces species at different temperatures, the improved S. eubayanus sequence and the availability of the Sc x Seub hybrid should in the future contribute to deciphering the multifactorial and poorly understood molecular basis of cold tolerance.
CHAPTER 1
A BRIEF HISTORY OF BREWING
Beer brewing from germinated barley, together with wine and bread making, is among the oldest biotechnological achievements of humankind. As such, these ancient biotechnological processes helped pave the way from a nomadic lifestyle to more structured societies. The earliest chemical evidence of beer in the archaeological record was discovered in current Iran, nearby the Zagros Mountains. Calcium oxalate (beerstone) inside pottery vessels dating from 3400 - 3000 BC provide clear evidence for their use in brewing in ancient Mesopotamia (Michel, McGovern, 1992; Michel et al., 1993). As soon as the proto-cuneiform writing was invented, texts about beer production and consumption showed a well-developed knowledge and technology base for the brewing process (Figure 1, Figure 2) (Damerow, 2012). Although translations of the Sumerian administrative and literary texts (most notably the Hymn to the Sumerian goddess of beer, Ninkasi) are biased by the current terminology used for beer brewing, they leave no doubt on the ancient roots of modern beer brewing processes.
In Europe, the oldest evidence of beer production dates from 800 BC, in the form of an earthenware amphora discovered in Northern Bavaria that was shown to contain wheat beer residues. Celtic people that inhabited Bavaria fled to the British Isles after the Roman conquest of Central Europe, transferring the brewing knowledge to that part of Europe. The Celts may therefore be considered the ancestors of both German and English brewing culture (Holliland, 2012). As commercial and domestic brewing expanded, regulations were imposed starting as early as 1156 with a decree in the city of Augsburg (Holliland, 2012) stating that ‘the bad beer should be discarded’ and culminating in the Beer Purity Law. This law (Reinheitsgebot) was imposed in 1516 by the Bavarian Duke Wilhelm IV and stipulated that only barley, hops and water be used in Bavarian beers (Kunze, 1996). Later on, this law, which is regarded as the oldest food regulation, was applied to entire Germany and had a great positive impact on all breweries across Europe. In 1516, the nature and role of a fourth key ingredient of beer, the yeast, was still to be discovered.
Figure 1. Proto-cuneiform text
from Mesopotamia (ca. 3000 B.C.) showing calculations of ingredients for different beers (Nissen, 1990)
Figure 2. Impression on a lapis lazuli
cylinder seal from Queen Pu-abi’s tomb in the Royal Cemetery at Ur (ca. 2600-2500 B.C.) Top - couple sharing a pot of beer using long straws (Damerow, 2012).
1
The presence of yeast cells in beer was first observed by the Dutch scientist and tradesman Antonie van Leeuwenhoek in 1680, using his elegant self-made microscopes (Figure 3A). The important improvements brought by Giovanni Amici (1820) to the resolution of the microscope’s objectives opened the way to further studies concerning the involvement of yeast in alcoholic fermentation. Remarkable research was performed in 1837 by the French physicist Charles Cagniard-Latour, the algologist Friedrich Kützing and the German physiologist Theodor Schwann. They observed that yeasts are living organisms involved in the transformation of sugar into alcohol (Stewart & Russell, 1986; Barnett, 2003). This statement was later unequivocally proven by Louis Pasteur who showed, in 1860, that fermentation is a consequence of yeast metabolism (Pasteur, 1860).
Until the middle of the 16th century all beers were of the ale type. In ale fermentation,
the yeast characteristically converts an extract from malt (germinated barley) at relatively high temperatures (20-25º), followed by a short maturation period. After the prohibition of summer brewing imposed in 1553 in Bavaria by Duke Albrecht V (Holliland, 2012) a new type of beer started to be produced, known today as lager beer. The cold environment under which brewing was now performed resulted in the selection of new yeast strains that were capable of undergoing alcoholic fermentation at lower temperatures (8-15º), with long cold maturation periods ‘lagering’ (Kodama et al., 2005). Nowadays, lager beers dominate the beer market while ales and beers brewed by spontaneous fermentation (e.g. Lambic beer) complement the variety in commercially available beers.
A next crucial step in the brewing industry was represented by the novel technique of producing pure yeast cultures. The method was developed by Emil Christian Hansen (1883) in the Carlsberg Laboratory in Copenhagen (Polaina, 2002). Furthermore, he introduced a fed-batch system for yeast propagation which allowed increased biomass production (Boulton, Quain, 2001). This breakthrough, together with the development of the thermometer (G.D.Fahrenheit-1714, A. Celsius-1742) (Figure 3C), the saccharometer (Richardson-1788) for measuring sugar content of wort (Figure 3B), the steam engine (J.Watt-1765) and the refrigeration machine (C von Linde-1871), led to a rapid growth of the number of large, industrially operated breweries. (Kunze, 1996; van Hamersveld, 1996).
Figure 3. (A)Microscope invented by Antonie van Leeuwenhoek (Breig, 2006), (B)Saccharometer invented
by J. Richardson (http://www.olney-antiques.co.uk/product/9/35/saccharometer), (C)Thermometer with mercury invented by G.D. Fahrenheit http://www.bornrich.com/original-thermometer-invented-daniel-gabriel-fahrenheit-fetch-157000-london-auction.html.
THE OUTLINE OF THE BREWING PROCESS
Beer production comprises three main stages: a) wort production, b) fermentation and c) post-fermentation processing (Figure 4).
a) Wort production takes place in the brewhouse, where malt (germinated barley with 4-5% humidity) is grinded to a suitable size and mixed with water in a process called mashing. During mashing, the temperature is gradually increased (55ºC to 78ºC), with rest phases corresponding to the optimum temperature for the activity of the different enzymes present in malt to degrade its abundance of water-insoluble compounds (starch, glucans, proteins) to soluble, short chains molecules, that can be metabolised by the yeast cells during fermentation. The substances that go into solution are referred to as extract. The enzymatic activity is further improved by adjusting the pH of the mash to 5.5-5.6. To separate the insoluble materials, the mash is filtrated in a vessel with perforated floor, so-called lauter tun, that allows the aqueous solids-free extract, called wort, to be separated from the spent grains. To avoid extract loss and improve the brewhouse yield, the spent grains are washed with hot water. These are rich in sugar, proteins and inorganic material are sold as cattle food. The wort is collected in a wort kettle where it is boiled for 60-90 min. During this step, bitter and aromatic hop varieties are added. The high temperature induces the isomerisation of α-acids from hops, thereby giving beer its bitter taste. During wort boiling, other important processes occur: formation and precipitation of protein-polyphenol complexes, inactivation of all enzymes, wort sterilisation, increase in wort color by formation of melanoidins and oxidation of polyphenols. During boiling, the S-methylmethionine SMM is converted to dimethylsulfide (DMS) with an unpleasant smell and taste, and dimethylsulfoxide (DMSO). During this stage of the process, the evaporation rate of DMS must be high enough to reduce it below its flavour threshold value (50 – 60 µg·l-1 DMS) (Kunze, 1996). At the end of
boiling, the wort is transferred to a whirlpool, where large particles settle down in the shape of a cone in the middle of the vessel. This compact mass is called ‘coarse break’ or ‘hot trub’ and contains precipitated proteins, lipids and zinc. The clear wort is rapidly cooled down to 7-8ºC for lager beers and to 15-22ºC for ales (Goldammer, 1999). During wort cooling, protein-polyphenol complexes precipitate and form the ‘cold break’ or ‘cold trub’. The particles of this trub are small and remain in suspension for a long period of time. The extent of trub removal from wort has long been studied in relation with yeast metabolism in the fermentation step. A trub-rich wort causes a significant faster fermentation than clear worts because the trub contains unsaturated long-chain fatty acids and ergosterol, which cannot be synthesized by yeast under the anaerobic conditions of large-scale beer fermentation (Kühbeck et al., 2006). Moreover, zinc, one of the essential ions for yeast metabolism, is loosely bound to the trub particles, thus being easily released in the aqueous solution during fermentation (de Nicola, Walker, 2009). Another positive aspect brought by the presence of trub is related to its particulate characteristics that promote the formation of CO2 bubbles from the medium during fermentation, thus reducing the toxic effect of the aqueous species
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on yeast cells. The stirring effect produced by the rising bubbles keeps the yeast cells in suspension for a longer period of time, accelerating the fermentation process (Kühbeck et al., 2007). The downside is that turbid worts tend to have a poorer flavour quality and stability, beer filtration-characteristics and foam stability, although the differences compared with a completely clear wort are often minor.
b) Fermentation represents the most important process in beer production and takes place in the fermentation cellar: the cold wort is aerated and transferred to the fermentation vessels where it is inoculated (pitched) with yeast suspension. Yeast cells used for inoculation are collected from a previous fermentation. Their cellular membrane is sterol-depleted due to the anaerobic nature of the fermentation from which they were harvested that impairs sterol formation. After pitching the aerated wort with such cells, yeast survives on its glycogen reserve until the fluidity of the membrane is reinstated (Verbelen et al., 2009).
The reuse of yeast suspension harvested from a previous fermentation to a subsequent one is common practice in beer production and, upon each repitching, a brief aeration phase is required to restore the levels of lipids and sterols in the membrane. The build-up of these essential compounds enables multiplication of the yeast cells during the subsequent anaerobic fermentation phase and the amount of oxygen supplied at the beginning of fermentation determines the extent of yeast growth. As soon as oxygen is depleted, anaerobic fermentation commences, during which the yeast cells metabolise wort nutrients into ethanol, carbon dioxide and a series of secondary metabolites essential for beer flavour and stability (higher alcohols, esters, aldehydes, SO2). During fermentation, the temperature is allowed to rise to 10 - 11ºC for lager beers and to 18 - 25ºC for ales (Goldammer, 1999). Due to this difference in temperature, the process lasts longer (5-7 days) in case of bottom-fermentation (lager) than top-fermentation (ale) (3 days) and also the spectrum of flavour compounds differs considerably for these two beer types. Specific characteristics of ale and lager yeast strains are shown in Table 1. In particular for lager beers, a ‘diacetyl rest’ is applied when the majority of wort carbohydrates is consumed. To this end, the temperature is increased to 13-15 ºC to accelerate degradation of the undesirable flavour compound diacetyl, which has a pronounced buttery aroma. In case of ale beers this step is often omitted, as many brands prefer to keep this compound above its sensory threshold level as part of the aroma trademark of the beers. During this warmer phase of the process, other undesirable flavour compounds characteristic to “young/green” beer are also removed. These include acetaldehyde and sulfur compounds, whose removal further contributes to the maturation of the beer flavour.
General Introduction
Figure 4.
Schematic representation of the steps within the brewing process: the malt is milled and the resulted grist is mixed with water
(generally 1:3 ratio)
forming a mash.
This is heated according to a defined diagram followed by filtration in a lauter tun.
From here the sweet wort is collected and sent to the wort
copper for the boiling step,
while the spent grains are sold to farmers.
After boiling,
the precipitated proteins are being removed from wort in a whirlpool and the
clear wort is aerated and pitched with brewing yeast.
The fermentation starts and maturation continues in the same vessel,
followed by beer filtration and bottling.
Grist
Spent grains
Malt
Adjuncts:
barley, maize
Water
Yeast
Milling
Mashing
Lautering
Boiling
Clari
fication
Hops
Wort
cooling
WORT
MASH
SWEET
WORT
Beer
filtration
Bright beer tank
Bottling
Yeast storage tankBEER
Yeast re-pitcing
Fermentation
/Maturation
Oxygen1
Yeast growth in beer fermentation processes ceases when sterols are depleted. Yeast cells then sink to the bottom of the fermenter (lager) or rise to the surface (ale). The yeast biomass can then be harvested and stored at 4ºC, to be used for re-pitching of new fermentation batches later on. Beer conditioning starts with forced cooling of the beer, to 0ºC for lager beers and to 4ºC for ales, with the purpose of saturating the beer with carbon dioxide and to clarify it. Conditioning ensures colloidal stability of the final product by precipitation and removal of protein-tannin complexes and yeast sediment. In modern breweries, high-capacity cylindroconical tanks are used for both stages of fermentation as well as for conditioning. This ‘uni-tank’ system decreases costs and shortens the process time.
The concept of ‘high-gravity brewing’, in which fermentation is performed with very concentrated worts followed by dilution at a later stage, is becoming increasingly popular because it enables increased volumetric productivity. However, suitable yeast strains must be developed to sustain the stress factors brought by this method. A number of breweries employ continuous fermentation systems in which a group of vessels produce a continuous beer stream. This approach is mainly suitable for breweries that produce only a small number of brands, since the advantage of continuous brewing only applies during longer production runs.
There are two types of continuous-brewing systems: (i) those resembling chemostats (systems in which the medium is continuously added while the liquid culture is continuously removed); (ii) plug flow system (system with an elongated form, continuously fed with a mix of wort and yeast). For low or zero-alcohol beers, immobilised yeast reactors are employed, offering the possibility of a short contact between wort and yeast during primary fermentation, to remove wort flavour.
c) Post-fermentation processing comprises beer filtration, pasteurization and bottling during which the temperature is kept below 0°C and the sterile conditions impeccable.
THE ROLE OF YEAST IN BEER FERMENTATION
The efficiency of fermentation depends on the physiological properties of the yeast being inoculated as well as on external factors such as wort composition, wort turbidity, aeration, temperature and inoculum density (‘pitching rate’) (Guido et al., 2004).
The main role of brewing yeast is to ferment wort sugars to ethanol and carbon dioxide. Therefore, the correlation between the brewing yeast sugar substrate range and the wort sugar spectrum is of paramount importance. The composition of a typical all-malt wort is shown in
Table 2 and comprises glucose, fructose, sucrose, maltose, maltotriose and dextrins. With the
exception of dextrins, most S. pastorianus strains are capable of utilizing the entire range of carbohydrates present in wort. A number of physiological studies indicated that certain lager brewing strains lack the ability of utilizing maltotriose (Duval et al., 2010, Gibson et al., 2013). The consumption of these sugars occurs sequentially (Stewart, 2006). Sucrose is hydrolysed to glucose and fructose by invertase and disappears first from the wort (Meneses et al., 2002), then glucose and fructose and finally maltose and maltotriose. Glucose is the preferred substrate for fermentation and the presence of this sugar in the medium represses expression of plasma membrane transporters for the other sugar molecules (Hammond, 1993; Boulton, Quain, 2001). Therefore, only after more than 60% of the glucose in wort has been fermented, maltose is imported by the yeast cells (D’Amore et al., 1989). The length of the fermentation process is to a large extent determined by the rate of maltose fermentation, which is the most abundant sugar in wort. Unlike the facilitated diffusion of glucose and fructose, uptake of the α-glucosides maltose and maltotriose by yeast cells takes place via proton symport. Several genes have been reported to encode α-glucoside transporters in Saccharomyces yeast strains:
AGT1, MALx1genes, MPH2, MPH3 (maltose permease homolog) and MTT1 (MTY1). Of the encoded transporters, only Mtt1 has a higher affinity for maltotriose than for maltose (Dietvorst et al., 2005; Salema-Oom et al., 2005), while Agt1 has the highest affinity for maltose. At the beginning of fermentation, glucose represses the transcription of these genes and, moreover, inactivates α-glucoside transporters already present in the membrane (Boulton, Quain, 2001). In lager brewing strains, the AGT1 gene is truncated, with a premature stop codon in the sequence that disturbs its functionality (Vidgren et al., 2005; Nakao et al., 2009). In lager strains, MALx1 genes therefore play a dominant role in maltose uptake. The α-glucoside transporters also differ with respect to their temperature sensitivity. Activity of Agt1 is strongly temperature dependent, while Mtt1 has a comparatively low temperature dependence (Vidgren
et al., 2010). Many breweries employ adjuncts in the form of sucrose or maltose syrup to boost
the sugar content of worts. Addition of high concentrations of sucrose may lead to incomplete fermentation of wort, caused by a prolonged glucose repression of maltose and maltotriose transporters (Russell 1993). In areas where barley cultivation is not viable for climatic reasons, other cereals are used for beer production. The use of sorghum, maize and rice requires addition of β-amylase for fermentable sugar release, which is absent in their seeds. These alternative cereals contain high amounts of starch with limited solubilisation during mashing and the endosperm remains intact during malting. This causes the release of β-glucans in the mash and difficulties during filtration (Kunze, 2006; Ogbonna, 2011; Taylor et al., 2013).
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Table 1. Morphological and physiological differences between ale and lager brewing yeast strains Ale yeast Lager yeast Reference
History Before 3400 BC After 1500 AD Michel et al., 1993; Holliland, 2012
Taxonomic name Saccharomyces
cerevisiae
Saccharomyces pastorianus
van de Walt, 1970
Morphology
Branched chains of cells CBS 1171
Single/pairs of cells CBS 1538
Barnett, 1992
Fermentation
temperature 20-25ºC 8-15ºC Kodama et al., 2005 Optimum
temperature 30ºC 25ºC Reed, Nagodawithana, 1991
Max growth temperature
37ºC 34ºC Tornai-Lehoczki et al.,
1996
Briggs et al., 2004
Flocculation Weak Strong Walker , 1998
Hydrophobic Hydrophilic Rhymes, Smart, 2000
Low surface charge High surface charge Hammond, 1993 Mannose insensitive
phenotype
(Not inhibited by sugars)
NewFlo phenotype (Inhibited by mannose, sucrose, glucose, maltose but not by galactose)
Dengis et al., 1995; Boulton, Quain, 2001
Harvesting Top Bottom Kunze, 1996
Carbohydrate
utilization Melibiose not utilized Meliobiose fermented Walker, 1998; Briggs et al., 2004 Poor maltotriose
utilization for most strains
Good maltotriose utilization (Frohberg group) Stewart, 2006; Duval et al., 2010 Gibson et al., 2013 High temperature dependence of maltose transport (AGT1) Low temperature dependence of maltose transport (MTT1) Vidgren et al., 2010
Fructose uptake via
facilitated transport Fructose uptake via proton symport Briggs et al., 2004 Raffinose incompletely
utilized Raffinose completely utilized Kunze, 1996
Table 2. Sugar composition of an all-malt wort (Stewart, Russel, 1993)
Sugar type Wort composition, %
Glucose 10-15 Fructose 1-2 Sucrose 1-2 Maltose 50-60 Maltotriose 15-20 Dextrins 20-30
During fermentation, brewer’s yeast produces a wide range of flavour compounds, many of which are by-products of yeast nitrogen metabolism. The nitrogenous components present in wort are listed in Table 3. Due to the fact that proteolytic activity of brewers’ yeast cells is limited and free ammonia is only present in small amounts in wort (Kunze, 2006), the main source of nitrogen in wort is represented by free amino acids. The amino acids are not all directly incorporated into proteins. After entering the cell, certain amino acids (valine, leucine, isoleucine, methionine, phenylalanine) are transaminated, the amino group being donated to other carbon skeletons while free 2-oxo acids are generated (Fontana, Buiatti, 2008). These oxo-acids are further metabolised, yielding aldehydes and higher alcohols with great impact on beer flavour (Hazelwood et al., 2008). Nitrogen starvation of brewers’ yeast can occur when adjuncts are used in amounts that are not balanced with the available nitrogen in wort. The low amount of proteins characteristic to sorghum, maize and rice and the complete absence of nitrogen in sucrose syrups brings a deficit of α-amino nitrogen with direct impact on yeast cells performance. Under these circumstances yeast cells will synthesise de novo the essential aminoacids. Along these biosynthetic pathways a number of by products are formed that impart off-flavours to beer: vicinal diketones produced via the isoleucine-valine biosynthetic pathway (Krogerus, Gibson, 2013) while high concentrations of SO2 and H2S are registered when a deficit in the sulfur-containing amino acid methionine occurs (Fontana, Buiatti, 2008). Yeast metabolism and its viability depends on the (bio) availability of inorganic nutrients, including Zn, Ca, Na, K, Mg, Cu, Fe, Mn, SO42-, PO43-.
Moreover brewing yeasts required the organic growth factors biotin and panthotenic acid (Kunze, 2006). The uptake rate and utilization of these nutrients not only depends on their concentration in the wort, but also on their bio-availability (Aleksander et al., 2009). The availability or toxicity of metal ions for bewers yeast in wort exhibits synergistic effects. For example, low concentrations of manganese, an essential element for yeast, inhibits zinc availability (Helin, Slaughter, 1977).
Table 3. Nitrogen components in wort (Reed, Nagodawithana, 1991; Boulton, Quain, 2001) Nitrogen compound Wort composition, %
Aminoacids 30-40%
Polypeptides 30-40%
Protein 20%
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TAXONOMIC CLASSIFICATION OF BREWER’S YEAST
The ultimate goal of taxonomic studies is to classify organisms on the basis of their evolutionary relations (Price et al., 1978). However, the criteria originally used in yeast taxonomy relied heavily on morphological and physiological characteristics of different strains (van der Walt, 1970), with further attempts on more discriminative approaches like serology (Tsuchiya et al., 1974), structure of cell wall mannans (Ballou et al., 1974), DNA base composition (Yarrow, Nakase, 1975) and proton-magnetic resonance spectra of extracted mannans (Fukazawa et al., 1980). Later, DNA sequence similarity became increasingly important in studying the phylogenetic relationships and taxonomy of yeasts. Species from the genus Saccharomyces were historically classified in two groups: sensu stricto and sensu lato, with the former exhibiting closely related strains with high fermentative ability, displaying a uniform number and chromosome distribution (karyotype) while the species in
sensu lato are more heterogeneous (Figure 5) (Naumov et al., 1996a; Špírek et al., 2003).
All species initially cumulated under the sensu stricto group display 16 chromosomes upon karyotyping (Naumov et al., 2000c; Fischer et al., 2000) and chromosomal rearrangements were only been detected among closely related species, most probably due to recombinations between non-homologous regions of different chromosomes that might have been beneficial for the organism (Fischer et al., 2000). The exceptions are S. pastorianus with higher number of chromosomes and S. arboricola at the opposite end, with only 12-13 choromosomes.
Figure 5. Karyotypes corresponding to species within (A) Saccharomyces sensu stricto group (Nguyen
et al., 2000) and (B) Saccharomyces sensu lato group (Petersen et al., 1999) The species initially assigned to the sensu lato group, were recently reassigned to new genera (Naumovia, Kazachstania, Lachancea) (Kurtzman, 2003)
Kurtzman and Robmett (2003) studied the Saccharomyces complex based on multigene sequence analysis, leading to the assignment of 75 species to 14 clades. To classify yeast strains based on their evolutionary similarities, comparison at the genomic level proves to be the most suitable approach. In the DNA renaturation method (Seidler, Mandel, 1971), the extent of sequence identity in two strains is analysed by simply mixing isolated DNA of the two strains, briefly denaturating the DNA and later allowing to renaturate the two strands, followed by a spectrophotometric reading of the resulted sample. Using this approach, 24 species originally grouped in Saccharomyces sensu stricto (van der Walt, 1970; Yarrow , Nakase, 1975) were reduced to only 4 taxa due to the sequence homology displayed by many of the strains: S. cerevisiae (Sc), S. bayanus, S. pastorianus (S. carlsbergensis) and S.
kluyveri (Table 4 ) (Vaughan Martini, Kurtzman, 1985). The S. kluyveri was later shown to be
evolutionary distant from Saccharomyces sensu stricto (Vaughan-Martini et al., 1993; Ando
et al., 1996). Karyotyping (Sheehan et al., 1991), restriction analysis of mitochondrial DNA
(Guillamon et al., 1994), hybridization with probes from Ty1 retrotransposable element family (Naumov et al., 1998) and Ty2 element (Codon et al., 1998), PCR fingerprinting (de Barros Lopes et al., 1998), spore formation between species and their viability (Naumov, 2000a) showed clear polymorphisms between and within the four species. Later this allowed fast identification of species just by using specific PCR primers (de Melo Pereira et al., 2010; Muir et al., 2011). Saccharomyces bayanus was suggested to comprise two variants, displaying different karyotypes: S. bayanus var uvarum and S. bayanus var bayanus. The former one consists of yeast strains capable of fermenting melibiose and unable to grow above 37ºC (Naumov et al., 2000a, Pulvirenti et al., 2000). Moreover, they also display reduce homology at gene level (Casaregola et al., 2001) with semisterile hybrids (low viability ascospores) (Naumov et al., 2000c).
Another species, well-established in the Saccharomyces genra, is S. paradoxus shown to abundantly occur on plant leaves (Glushakova et al., 2007). While species corresponding to S. bayanus and S. pastorianus were predominantly isolated from winery and brewery environments (with few exceptions for S. bayanus), S. cerevisiae and S. paradoxus are readily isolated from similar natural habitats: broad-leaved trees, soil and insects (Table 4). This difference is mirrored in the mitochondrial DNA (mtDNA) of these strains, with conserved genetic organization in case of S. cerevisiae strains and high polymorphism of mtDNA corresponding to wine and brewers’ yeast strains (Codon et al., 1998; Foury et al., 1998). Three new species isolated in Brazil and Japan were designated as part of the Saccharomyces
genra: S. kudriavzevii (Kaneko, Banno, 1991), S. cariocanus (Morais et al., 1992) and S. mikatae (Yamada et al., 1993). Confirmation of these new strains involved a thorough
comparative analysis with the already known species (genetic crosses, molecular karyotyping, 18S rRNA sequence analysis) (Naumov et al., 2000c). S. kudriavzevii was recently discovered in oak bark, in Portugal. Unlike the strains from Japan, the Portugese strains were able to metabolise galactose (Sampaio JP, Gonçalves P, 2008) and showed gene sequences closer to the hybrid wine strains from Europe. Isolates from Taiwan (Naumov et al., 2013), confirmed
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the presence of the species in this new location, together with S. arboricola and omnipresent
S. cerevsiae. The taxonomic location of S. cariocanus is still controversial, with some authors
considering it as an independent species (Naumov et al., 2000c), while others (Liti et al., 2006) considering it still as part of the lineages of S. paradoxus due to the lack of aneuploidy in the viable spores resulted from S. cariocanus X S. paradoxus crosses.
S. arboricola is a recently described Saccharomyces species, discovered in the bark of the trees
in mainland China (Wang, Bai, 2008) and only one strain in Taiwan (Naumov et al., 2013). The genome of this new species was recently sequenced and phylogenetic analysis placed it between S. kudriavzevii and S. bayanus (Liti et al., 2013). The strains analysed within this species showed low growth and sensitivity to high temperatures, but good utilization of mannitol and biotin prototrophy.
The most recent and spectacular addition to the Saccharomyces group is represented by S. eubayanus (Seub). This species was initially discovered in Argentina in the galls of the Nothofagus trees in Patagonia. Its genome showed a 99.56% identity with the
non-cerevisiae part of the hybrid genome of S. pastorianus (Libkind et al., 2011) and proved to be
a cryotolerant strain (Gibson et al., 2013). Recently (Peris et al., 2014), a new S. eubayanus strain was discovered in North America (Wisconsin) being identified as an intraspecific hybrid of two Patagonian populations. At the same time, 3 lineages of S. eubayanus were identified in Far East Asia: Tibet, west and north-west China (Bing et al., 2014) with the Tibetan lineage considered as the real contributor to the lager brewing strains non-cerevisiae sub-genome due to a higher sequence similarity (99.82%) than the Patagonian strains. Low ascopore viability, was noted for S. eubayanus x S. bayanus var. uvarum hybrids (Naumov
et al., 2013).
Lager brewing strains are designated as Saccharomyces pastorianus. This name was first assigned by Reess in 1870 to alcoholic fermentation fungi with “sausage-shaped cells” (Hansen, 1895). In 1904, E.C. Hansen established the brewing yeast type I selected in Carlsberg breweries as being true S. pastorianus. Later studies performed on Hansen’s strain showed that it behaved like S. bayanus in terms of galactose and raffinose fermentation (van der Walt, 1965), seemingly refuting its classification as a separate species. Later tests involving single-chromosome transfer from S. pastorianus strains to kar1Δ S. cerevisiae mutants, followed by sequence homology checks at different loci (Nilsson-Tillgren et al., 1981; Holmberg, 1982; Pedersen, 1986), DNA reassociation tests of S. pastorianus strains with S. cerevisiae and S. bayanus species (Vaughan Martini, Martini, 1987), indicated the presence of both genomes in different proportions, thus reinstating S. pastorianus as a separate species.
In 1908, a new bottom-fermenting strain was selected by EC Hansen assigned as S.
carlsbergensis (Hansen, 1908). Due to the high similarity with the previously described S. pastorianus both strains correspond to the same species. According to the International
Code of Botanical Nomenclature the first name designated to a strain must be kept (Vaughan Martini, Martini, 1987) thus all lager brewing strains should be addressed as S. pastorianus. Nevertheless, many publications still use the name S. carlsbergensis when referring to these type of yeasts. The hybrid nature of lager strains and their chromosomal rearrangements were confirmed by a wide range of molecular techniques, including southern blotting with
S. cerevisiae probes (Tamai et al., 1998), comparative DNA sequence analysis via BLASTN,
BLASTX (Cliften et al., 2001), fluorescent amplified fragment length polymorphism (AFLP) on genomic DNA of S. pastorianus, S. cerevisiae and S. bayanus strains (Casaregola et al., 2001; de Barros Lopes et al., 2002).
Based on the existing sequences of S. cerevisiae (Goffeau et al., 1996) and S. bayanus var
uvarum (Kellis et al., 2003) a comprehensive study was performed by Rainieri et al. (2006),
on 35 strains assigned to S. bayanus/S. pastorianus that resulted in three groups, based on the contributing subgenomes: (a) S. cerevisiae/ S. bayanus lager genome, (b) S. cerevisiae/S.
uvarum/S. bayanus lager genome, (c) S. bayanus/ S. uvarum lager genome. Lager brewing
strains were found in the first group.
All the species currently recognized in the Saccharomyces genra are presented in Table 4. The first complete sequence of a lager brewing strain genome was published only in 2009 (Nakao et al.) for Weihenstephan 34/70. Since then, other research groups unravelled the sequences of a number of lager brewing strains (Hewitt et al., 2014; Walther et al., 2014; this thesis). Clear genetic evidence of two subgenomes, one similar to S. cerevisiae and the other to S. bayanus, further confirmed the hybrid nature of the genome of lager yeasts. The mitochondrial DNA was proved to be only from the non-cerevisiae parent, while the nucleic genome showed many translocations, deletions and insertions specific for cultured strains. Lager brewing strains were distributed in two groups by Dunn & Sherlock (2008) based on their origin and subsequent evolution: Group I comprising strains that arose via hybridization between a haploid S. cerevisiae spore with a haploid
S. bayanus spore, followed by massive losses of the cerevisiae subgenome (Saaz type strains),
and Group II with strains resulted from the fusion of a diploid S. cerevisiae with haploid S.
bayanus (Frohberg type strains). This last group kept important parts of both genomes with
only limited losses of genetic information from the S. bayanus part. Unlike the Saaz strains, the Frohberg strains have the ability to utilize maltotriose (Gibson et al., 2013) displaying a better fermentation performance. The lager brewing strain CBS1483 used throughout the research described in this thesis, was included in the second group.
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Table 4. Currently recognized species in the Saccharomyces genra
Species Identification Geographic range Yeast strains Reference Natural habitats
S. cerevisiae Soil,
broad-leaved trees, Drosophila
Europe (Finland, the Netherlands), Far East Asia, North America e.g. CBS1171 Naumov et al., 1992 Naumov, 1996b Sniegowski et al., 2002
S. paradoxus Oak exudates,
forest soil, peat, fruit body of mushrooms, Drosophila, fungal galls of some trees
Europe, Far East Asia, North America, Africa, Hawai
e.g.
CBS5829 Naumov et al., 1992Naumov et al., 1996a Sniegowski et al., 2002
S. kudriavzevii Decayed leaf
Oak bark Europe (Portugal), Asia (Japan, Taiwan, Malaysia) e.g. CBS12752s Kaneko, Banno, 1991 Naumov et al., 2000, 2013 Scannell et al., 2011 Sampaio, Gonçalves, 2008
S. mikatae Soil, decayed leaf Asia (Japan) e.g.
CBS8839s
Yamada et al., 1993 Naumov et al., 2000 Scannell et al., 2011
S. arboricola Bark tree Asia (China,
Taiwan) e.g. CBS10644s
Wang, Bai , 2008 Liti et al., 2013 Naumov et al., 2013
S. cariocanus Drosophila South America
(Brazil) e.g. CBS8841 Morais et al., 1992 Naumov et al., 2000 S. bayanus var.
uvarum Mesophylax adoperus,
Drosophila, mushrooms, hornbeam exudates Europe e.g. CBS7001s Naumov et al., 1992 Tornai-Lehoczki et al., 1996; Scannell et al., 2011 Naumov et al., 2013
S. eubayanus Galls on beach
trees South America (Patagonia) High land China North America e.g. CBS12357s Libkind et al., 2011 Peris et al., 2014 Bing et al., 2014 Industrial habitats S. cerevisiae Fermentation processes (high temperature)
Europe, Africa e.g.
CBS4054 Naumov et al., 1994Naumov, Naumova, 2011 S. pastorianus
(S. cerevisiae x S. eubayanus)
Breweries Europe e.g.
CBS1513, WS34/70s Vaughan Martini, Martini, 1987 Nakao et al., 2009 S. bayanus var bayanus (S. uvarum x S. eubayanus x S. cerevisiae) Brewing contaminant Wineries Pear/ Apple juice Europe e.g. NBRC1948 CBS424 Rainieri et al., 2006 Libkind et al., 2011 Naumov et al., 2013
The genome of the recently discovered cryotolerant species S. eubayanus showed even higher sequence identity with the non-cerevisiae portion of S. pastorianus than the genome of S. bayanus. The discovery of S. eubayanus and its genome sequence have therefore provided an invaluable, previously missing link in the phylogeny of lager brewing strains. The contribution of S. eubayanus rather than
S. bayanus to the complex hybrid genome of S. pastorianus has been extensively confirmed by
testing specific primer pairs designed on the sequence of S. eubayanus against the two species with negative results for S. bayanus (Pengelly, Wheals, 2012) and recently by whole-genome sequencing of a number of S. eubayanus strains from different origins (Bing et al., 2014). Numerous S. eubayanus strains have been detected in Patagonia with a diverse genetic composition. The S. eubayanus moiety within the S. pastorianus genome diverged from one such population, several thousand years ago (Peris et al. 2014). The genomes of lager strains continue to present intriguing genomic puzzles, not only with respect to their functionality and regulation, but also with respect to the question on how two species that have been discovered so far apart from the place where lager beers were first produced (Bavaria, Germany) might have mated. One attractive hypothesis is that the S. eubayanus species also occur in Europe, but that its niche is still waiting to be discovered. As shown for S. paradoxus (Glushakova et al., 2007), S. eubayanus may not be restricted to sugar rich environments but, for example, be an epiphytic species whose abundance follows seasonal dynamics, thereby necessitating year-long investigations for its successful isolation.
While growth and fermentation performance of S. eubayanus from Patagonia are relatively similar with those of the lager strains from the Saaz group, the ester profile of this new species is comparable with the Frohberg lager strains (Gibson et al., 2013).
Chimeric genomes containing subgenomes from species of the Saccharomyces complex have been frequently found in wine regions and breweries (Peris et al., 2012b) but also in human respiratory tract isolates (de Barros Lopes et al., 2002; Peris et al., 2012a) showing that interspecific hybridization among Saccharomyces species is more common than previously believed.
CHALLENGES IN ANALYSIS OF BREWING YEAST GENOMES
In-depth knowledge of the genome sequence of brewing strains is a prerequisite for understanding the genetic basis of their phenotype and the complex, multi-layered regulation of metabolism under industrially relevant conditions.
Industrial strains evolved to be polyploid or aneuploid, meaning that they harbour more than two sets of homologous chromosomes with, in the case of aneuploidy, different copy numbers for the individual chromosomes. Polyploidy and aneuploidy can occur via whole-genome duplications or by acquiring/losing single chromosomes. It has been observed that
1
in diploid cells of S. cerevisiae, adaptive mutations are 1.6 times more frequent than in an isogenic haploid population (Paquin, Adams, 1983). Ploidy thus contributes to genetic flexibility and, thereby, to the possibility to rapidly adapt to new environments (Stewart, 1981; Bond et al., 2004). Lager brewing yeasts are invariably aneuploids, with unique chromosomal rearrangements displayed by different strains (Hansen, Kielland-Brandt, 2003).
For a long time, quantitative analysis of overall ploidy has relied on the use of flow fluorocytometry (Hutter, Eipel, 1979), as well as colorimetric methods (Aigle et al., 1983). In such analyses, aneuploidy presented difficulties due to the impossibility of differentiating the copy number for each individual chromosome. Early methods employed the use of microarrays containing S. cerevisiae DNA, culminating in the competitive comparative genome hybridisation (CCGH) method that allowed the determination of the copy number of cerevisiae genes within the S. pastorianus hybrid relative to a haploid S. cerevisiae reference strain (Bond et al., 2004). This method could, however, only resolve half of the genome because the S. eubayanus part had not yet been identified or sequenced. Currently, the availability of whole genome sequences corresponding to S. cerevisiae, S. bayanus MCYC623 and
S. pastorianus Weihenstephan 34/70 allows the design of specific primers for each subgenome
within lager brewing strains. This in turn enables accurate analysis of chromosome copy number variation by real-time quantitative PCR method using either a control sample with a known ploidy (D’haene et al., 2010) or a reference gene within the analysed genome with an unambiguously defined copy number (Tadami et al., 2014). In recent studies performed on a meiotic segregant derived from WS34/70 (Ogata et al., 2011) as well as on different lager brewing strains (Tadami et al., 2014), custom DNA microarrays comprising both Sc- and
S. eub-type probes allowed the estimation of chromosomes copy number variation.
An additional challenge in analysing brewing yeast genomes is brought about by their hybrid nature, with chromosomes deriving from two species (Figure 6) with largely syntenic genomes (similar order of genetic loci on chromosomes): S. cerevisiae and S. eubayanus (Libkind et al., 2011).
The sequence of different lager strain (Nakao et al., 2009; Hewitt et al., 2014; Wendland et al., 2014), indicated the presence of more than 31 chromosomes. Three types of translocations were observed involving one or both subgenomes: Sc-Sc, Seub-Seub and Sc-Seub. It is well known that sequence divergence acts as a barrier against homologous recombination (Datta
et al., 1997). However, in lager brewing strains, the orthologous genes share a high similarity
of 78-88% (Hansen, Kielland-Brandt, 2003) displaying many identical short sequences that allow chromosomal translocations. At the same time, double strand breaks in the telomeric regions increased the chances of chromosomal recombination as well as the presence of transposon-related sequences (entire Ty elements or just terminal repeats) that were previously shown to be correlated with the localization of breakpoints (Dunham et al., 2002). The beneficial traits acquired by S. pastorianus are therefore the result of two factors: interspecies
