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 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 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 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 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 Rapid selection response to ethanol in S. eubayanus emulates the domestication process under brewing conditions

2020 ◽  
Author(s):  
Wladimir Mardones ◽  
Carlos A. Villarroel ◽  
Valentina Abarca ◽  
Kamila Urbina ◽  
Tomás A. Peña ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Time Course ◽  
De Novo ◽  
Rapid Evolution ◽  
Selection Response ◽  
Diauxic Shift ◽  
Lager Yeast ◽  
Altered Expression ◽  
Domestication Syndrome ◽  
Domestication Process

ABSTRACTAlthough the typical genomic and phenotypic changes that characterize the evolution of organisms under the human domestication syndrome represent textbook examples of rapid evolution, the molecular processes that underpin such changes are still poorly understood. Domesticated yeasts for brewing, where short generation times and large phenotypic and genomic plasticity were attained in a few generations under selection, are prime examples. To experimentally emulate the lager yeast domestication process, we created a genetically complex (panmictic) artificial population of multiple Saccharomyces eubayanus genotypes, one of the parents of lager yeast. Then we imposed a constant selection regime under a high ethanol concentration in 10 replicated populations during 260 generations (six months) and compared them with evolved controls exposed solely to glucose. Evolved populations exhibited a selection differential of 60% in growth rate in ethanol, mostly explained by the proliferation of a single lineage (CL248.1) that competitively displaced all other clones. Interestingly, the outcome does not require the entire time course of adaptation, as four lineages monopolized the culture at generation 120. Sequencing demonstrated that de novo genetic variants were produced in all evolved lines, including SNPs, aneuploidies, INDELs, and translocations. In addition, the evolved populations showed correlated responses resembling the domestication syndrome: genomic rearrangements, faster fermentation rates, lower production of phenolic-off flavors and lower volatile compound complexity. Expression profiling in beer wort revealed altered expression levels of genes related to methionine metabolism, flocculation, stress tolerance and diauxic shift, likely contributing to higher ethanol and fermentation stress tolerance in the evolved populations. Our study shows that experimental evolution can rebuild the brewing domestication process in “fast motion” in wild yeast, and also provides a powerful tool for studying the genetics of the adaptation process in complex populations.

scholarly journals Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation

Genes ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 17 ◽  
Author(s):  
Christian Diwo ◽  
Nediljko Budisa
Keyword(s):  
Genetic Code ◽  
Genetic Information ◽  
Information Transfer ◽  
De Novo ◽  
Genetic Isolation ◽  
Universal Genetic Code ◽  
Synthetic Life ◽  
Beneficial Traits ◽  
Genetic Pool ◽  
Almost All

The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern biodiversity. Today, the genetic modification techniques of synthetic biology allow for the design of specialized organisms and their employment as tools, creating an artificial biodiversity based on the same universal genetic code. As there is no natural barrier towards the proliferation of genetic information which confers an advantage for a certain species, the naturally evolved genetic pool could be irreversibly altered if modified genetic information is exchanged. We argue that an alien genetic code which is incompatible with nature is likely to assure the inhibition of all mechanisms of genetic information transfer in an open environment. The two conceivable routes to synthetic life are either de novo cellular design or the successive alienation of a complex biological organism through laboratory evolution. Here, we present the strategies that have been utilized to fundamentally alter the genetic code in its decoding rules or its molecular representation and anticipate future avenues in the pursuit of robust biocontainment.

scholarly journals Physiological state of reused brewing yeast

2013 ◽  
Vol 31 (No. 3) ◽  
pp. 264-269 ◽  
Author(s):  
E. Kordialik-Bogacka ◽  
A. Diowksz
Keyword(s):  
Yeast Strain ◽  
Significant Variation ◽  
Physiological Condition ◽  
Physiological State ◽  
Physiological Characteristics ◽  
Yeast Culture ◽  
Brewing Yeast ◽  
Flavour Compounds ◽  
The Impact ◽  
Is Strain

In brewing, yeast may be reused many times. A number of yeast repitchings differs significantly among the breweries. Adjusting the number of times a strain may be serially repitched is of great importance for quality and consistency of final products. The fermentative and physiological characteristics of the yeast culture used in successive laboratory scale fermentations were determined. Yeast physiological state was assessed by the measurement of the levels of intracellular carbohydrates. In our investigation there were not any detectable changes in yeast capacity to ferment. No significant variation in the production of flavour compounds was found either. However, intracellular glycogen and trehalose contents were dependant on the yeast strain, generation number and wort gravity. Nevertheless, an alteration in the yeast physiological condition during serial repitchings occurred in a different mode than in previous studies confirming that the impact of serial repitchings is strain and medium dependent to a large extent.  

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 Encapsulation of brewing yeast in alginate/chitosan matrix: lab-scale optimization of lager beer fermentation

2014 ◽  
Vol 28 (2) ◽  
pp. 277-284 ◽  
Author(s):  
Vessela Naydenova ◽  
Mariyana Badova ◽  
Stoyan Vassilev ◽  
Vasil Iliev ◽  
Maria Kaneva ◽  
...  
Keyword(s):  
Brewing Yeast ◽  
Beer Fermentation ◽  
Chitosan Matrix ◽  
Lager Beer ◽  
Scale Optimization

scholarly journals A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

2015 ◽  
Vol 81 (23) ◽  
pp. 8202-8214 ◽  
Author(s):  
Stijn Mertens ◽  
Jan Steensels ◽  
Veerle Saels ◽  
Gert De Rouck ◽  
Guido Aerts ◽  
...  
Keyword(s):  
Large Scale ◽  
Alcoholic Beverage ◽  
Pilot Scale ◽  
Temperature Tolerance ◽  
Large Set ◽  
Lager Yeast ◽  
Content Type ◽  
High Attenuation ◽  
Saccharomyces Pastorianus ◽  
Lager Beer

ABSTRACTLager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use ofSaccharomyces pastorianus, an interspecific hybrid betweenSaccharomyces cerevisiaeand the cold-tolerantSaccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains ofS. cerevisiae(six strains) andS. eubayanus(two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and referenceS. pastorianusyeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.

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