scholarly journals Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

2020 ◽  
Author(s):  
Martina Catallo ◽  
Fabrizio Iattici ◽  
Cinzia Randazzo ◽  
Cinzia Caggia ◽  
Kristoffer Krogerus ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Yeast Culture ◽  
Culture Collections ◽  
Lager Yeast ◽  
Ethyl Hexanoate ◽  
Brewing Yeast ◽  
Saccharomyces Bayanus ◽  
Lager Beer ◽  
Saccharomyces Eubayanus

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomyces cerevisiae ale x Saccharomyces eubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus x Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae x S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20°C, one triple S. cerevisiae x S. eubayanus x S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2L wort fermentation trials at 12°C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for design of de novo lager beer hybrids and may help to diversify aroma profiles in lager beers.

scholarly journals Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

2021 ◽  
Vol 9 (3) ◽  
pp. 514
Author(s):  
Martina Catallo ◽  
Fabrizio Iattici ◽  
Cinzia L. Randazzo ◽  
Cinzia Caggia ◽  
Kristoffer Krogerus ◽  
...  
Keyword(s):  
De Novo ◽  
Yeast Culture ◽  
Culture Collections ◽  
Lager Yeast ◽  
Ethyl Hexanoate ◽  
Brewing Yeast ◽  
Single Organism ◽  
Saccharomyces Bayanus ◽  
Lager Beer ◽  
Beneficial Traits

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomycescerevisiae ale × Saccharomyceseubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus × Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae × S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20 °C, one triple S. cerevisiae × S. eubayanus × S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2 L wort fermentation trials at 12 °C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for designing de novo lager beer hybrids and may help to diversify aroma profiles in lager beer.

scholarly journals Enhanced Wort Fermentation with De Novo Lager Hybrids Adapted to High-Ethanol Environments

2017 ◽  
Vol 84 (4) ◽  
Author(s):  
Kristoffer Krogerus ◽  
Sami Holmström ◽  
Brian Gibson
Keyword(s):  
Adaptive Evolution ◽  
Ethanol Tolerance ◽  
De Novo ◽  
Fermentation Performance ◽  
Lager Yeast ◽  
Brewing Yeast ◽  
Ploidy Levels ◽  
Ester Formation ◽  
Lager Beer ◽  
High Ethanol

ABSTRACT Interspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2-liter-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance than those derived from diploid strains. Interestingly, it was not only the hybrid strains, but also the Saccharomyces cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred at both the chromosome and single nucleotide levels in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation. IMPORTANCE Recent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here, we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolated and characterized a number of adapted variants which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variant strains were produced without any genetic modification and are suitable for industrial lager beer fermentations.

scholarly journals Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid

2021 ◽  
Vol 2 ◽  
Author(s):  
Kristoffer Krogerus ◽  
Frederico Magalhães ◽  
Sandra Castillo ◽  
Gopal Peddinti ◽  
Virve Vidgren ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Fermentation Performance ◽  
Hybrid Strain ◽  
Lager Yeast ◽  
Brewing Yeast ◽  
Acetate Ester ◽  
Copy Numbers ◽  
Saccharomyces Eubayanus

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

scholarly journals Enhanced wort fermentation with de novo lager hybrids adapted to high ethanol environments

2017 ◽  
Author(s):  
Kristoffer Krogerus ◽  
Sami Holmström ◽  
Brian Gibson
Keyword(s):  
Adaptive Evolution ◽  
Ethanol Tolerance ◽  
De Novo ◽  
Fermentation Performance ◽  
Lager Yeast ◽  
Brewing Yeast ◽  
Ploidy Levels ◽  
Ester Formation ◽  
Lager Beer ◽  
High Ethanol

AbstractInterspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2L-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance. Interestingly, it was not only the hybrid strains, but also the S. cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred both at chromosome and single nucleotide level in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation.ImportanceRecent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolate and characterize a number of adapted variants, which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variants strains were produced without any genetic modification, and are suitable for industrial lager beer fermentations.

scholarly journals Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid

2021 ◽  
Author(s):  
Kristoffer Krogerus ◽  
Frederico Magalhaes ◽  
Sandra Castillo ◽  
Gopal Peddinti ◽  
Virve Vidgren ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Fermentation Performance ◽  
Hybrid Strain ◽  
Lager Yeast ◽  
Brewing Yeast ◽  
Acetate Ester ◽  
Copy Numbers ◽  
Saccharomyces Eubayanus

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae x Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15 C, 15 Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

scholarly journals Overexpression of RAD51 Enables PCR-Based Gene Targeting in Lager Yeast

2019 ◽  
Vol 7 (7) ◽  
pp. 192 ◽  
Author(s):  
Beatrice Bernardi ◽  
Yeseren Kayacan ◽  
Madina Akan ◽  
Jürgen Wendland
Keyword(s):  
Gene Targeting ◽  
Gene Function ◽  
Genetic Alterations ◽  
Selection Marker ◽  
Lager Yeast ◽  
Diagnostic Pcr ◽  
Local Selection ◽  
Lager Beer ◽  
Low Efficiency ◽  
Saccharomyces Eubayanus

Lager beer fermentations rely on specific polyploid hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus falling into the two groups of S. carlsbergensis/Saaz-type and S. pastorianus/Frohberg-type. These strains provide a terroir to lager beer as they have long traditional associations and local selection histories with specific breweries. Lager yeasts share, based on their common origin, several phenotypes. One of them is low transformability, hampering the gene function analyses required for proof-of-concept strain improvements. PCR-based gene targeting is a standard tool for manipulating S. cerevisiae and other ascomycetes. However, low transformability paired with the low efficiency of homologous recombination practically disable targeted gene function analyses in lager yeast strains. For genetic manipulations in lager yeasts, we employed a yeast transformation protocol based on lithium-acetate/PEG incubation combined with electroporation. We first introduced freely replicating CEN/ARS plasmids carrying ScRAD51 driven by a strong heterologous promoter into lager yeast. RAD51 overexpression in the Weihenstephan 34/70 lager yeast was necessary and sufficient in our hands for gene targeting using short-flanking homology regions of 50 bp added to a selection marker by PCR. We successfully targeted two independent loci, ScADE2/YOR128C and ScHSP104/YLL026W, and confirmed correct integration by diagnostic PCR. With these modifications, genetic alterations of lager yeasts can be achieved efficiently and the RAD51-containing episomal plasmid can be removed after successful strain construction.

scholarly journals Creation and Characterization of Industrially Applicable de Novo Lager Yeast Hybrids with a Unique Genomic Architecture

2020 ◽  
Author(s):  
Zachari Turgeon ◽  
Thomas Sierocinski ◽  
Cedric A. Brimacombe ◽  
Yiqiong Jin ◽  
Brittany Goldhawke ◽  
...  
Keyword(s):  
De Novo ◽  
Phenotypic Diversity ◽  
Breeding Strategy ◽  
Lager Yeast ◽  
Genomic Architecture ◽  
Group I ◽  
Saccharomyces Pastorianus ◽  
Lager Beer ◽  
Group Ii ◽  
Beer Production

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus. Lager strains are classified into two major groups based largely on genomic composition: Group I and Group II. Group I strains are allotriploid, whereas Group II are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off-flavours and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus. The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production. IMPORTANCE All lager beer is produced using two related lager yeast types: Group I and Group II, which are highly similar resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (Group III). We anticipate that these novel lager strains will be of great industrial relevance, and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

scholarly journals Potential for Lager Beer Production from Saccharomyces cerevisiae Strains Isolated from the Vineyard Environment

Processes ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1628
Author(s):  
Massimo Iorizzo ◽  
Francesco Letizia ◽  
Gianluca Albanese ◽  
Francesca Coppola ◽  
Angelita Gambuti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pilot Scale ◽  
Brewing Industry ◽  
Saccharomyces Pastorianus ◽  
Lager Beer ◽  
Efficient Fermentation ◽  
Saccharomyces Eubayanus ◽  
Beer Production ◽  
New Strategies

Saccharomyces pastorianus, genetic hybrids of Saccharomyces cerevisiae and the Saccharomyces eubayanus, is one of the most widely used lager yeasts in the brewing industry. In recent years, new strategies have been adopted and new lines of research have been outlined to create and expand the pool of lager brewing starters. The vineyard microbiome has received significant attention in the past few years due to many opportunities in terms of biotechnological applications in the winemaking processes. However, the characterization of S. cerevisiae strains isolated from winery environments as an approach to selecting starters for beer production has not been fully investigated, and little is currently available. Four wild cryotolerant S. cerevisiae strains isolated from vineyard environments were evaluated as potential starters for lager beer production at laboratory scale using a model beer wort (MBW). In all tests, the industrial lager brewing S. pastorianus Weihenstephan 34/70 was used as a reference strain. The results obtained, although preliminary, showed some good properties of these strains, such as antioxidant activity, flocculation capacity, efficient fermentation at 15 °C and low diacetyl production. Further studies will be carried out using these S. cerevisiae strains as starters for lager beer production on a pilot scale in order to verify the chemical and sensory characteristics of the beers produced.

scholarly journals Stress Resistance and Adhesive Properties of Commercial Flor and Wine Strains, and Environmental Isolates of Saccharomyces cerevisiae

Fermentation ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 188
Author(s):  
Michail A. Eldarov ◽  
Daria A. Avdanina ◽  
Elena Ivanova ◽  
Maksim Y. Shalamitskiy ◽  
Tatiana N. Tanashchuk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Resistance ◽  
Rapid Screening ◽  
Clear Correlation ◽  
Yeast Culture ◽  
Environmental Isolates ◽  
Adhesive Properties ◽  
Experimental Conditions ◽  
Culture Collections ◽  
Flor Yeast

Flor strains of Saccharomyces cerevisiae represent a special group of yeasts used for producing biologically aged wines. We analyzed the collection of commercial wine and flor yeast strains, as well as environmental strains isolated from the surface of grapes growing in vineyards, for resistance to abiotic stresses, adhesive properties, and the ability to form a floating flor. The degree of resistance of commercial strains to ethanol, acetaldehyde, and hydrogen peroxide was generally not higher than that of environmental isolates, some of which had high resistance to the tested stress agents. The relatively low degree of stress resistance of flor strains can be explained both by the peculiarities of their adaptive mechanisms and by differences in the nature of their exposure to various types of stress in the course of biological wine aging and under the experimental conditions we used. The hydrophobicity and adhesive properties of cells were determined by the efficiency of adsorption to polystyrene and the distribution of cells between the aqueous and organic phases. Flor strains were distinguished by a higher degree of hydrophobicity of the cell surface and an increased ability to adhere to polystyrene. A clear correlation between biofilm formation and adhesive properties was also observed for environmental yeast isolates. The overall results of this study indicate that relatively simple tests for cell hydrophobicity can be used for the rapid screening of new candidate flor strains in yeast culture collections and among environmental isolates.

scholarly journals The impact of commercially available ale and lager yeast strains on the fermentative diversity of beers

2020 ◽  
Author(s):  
Diego Bonatto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lager Yeast ◽  
Brewing Yeast ◽  
Beer Fermentation ◽  
Yeast Strains ◽  
Saccharomyces Pastorianus ◽  
Commercial Yeast ◽  
Main Component ◽  
The Impact ◽  
Attenuation Profile

AbstractYeasts from the species Saccharomyces cerevisiae (ale yeast) and Saccharomyces pastorianus (lager yeast) are the main component of beer fermentation. It is known that different beer categories depend on the use of specific ale or lager strains, where the yeast imprint its distinctive fermentative profile to the beer. Despite this, there are no studies reporting how diverse, rich, and homogeneous the beer categories are in terms of commercially available brewing yeast strains. In this work, the diversity, richness, and evenness of different beer categories and commercial yeast strains available for brewing were evaluated by applying quantitative concepts of ecology analysis in a sample of 121,528 beer recipes. For this purpose, the frequency of ale or lager and dry or liquid yeast formulations usage was accessed and its influence in the fermentation temperature, attenuation profile, and number of recipes for a beer category were analyzed. The results indicated that many beer categories are preferentially fermented with dry yeast strains formulations instead of liquid yeasts, despite considering the high number of available liquid yeast formulations. Moreover, ale dry strains are preferentially used for lager brewing. The preferential use of specific yeast formulations drives the diversity, richness, and evenness of a beer category, showing that many yeast strains are potentially and industrially underexplored.

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