Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast

Beer is a fermented beverage with a history as old as human civilization. Ales and lagers are by far the most common beers; however, diversification is becoming increasingly important in the brewing market and the brewers are continuously interested in improving and extending the range of products, especially in the craft brewery sector. Fermentation is one of the widest spaces for innovation in the brewing process. Besides Saccharomyces cerevisiae ale and Saccharomyces pastorianus lager strains conventionally used in macro-breweries, there is an increasing demand for novel yeast starter cultures tailored for producing beer styles with diversified aroma profiles. Recently, four genetic engineering-free approaches expanded the genetic background and the phenotypic biodiversity of brewing yeasts and allowed novel costumed-designed starter cultures to be developed: (1) the research for new performant S. cerevisiae yeasts from fermented foods alternative to beer; (2) the creation of synthetic hybrids between S. cerevisiae and Saccharomyces non-cerevisiae in order to mimic lager yeasts; (3) the exploitation of evolutionary engineering approaches; (4) the usage of non-Saccharomyces yeasts. Here, we summarized the pro and contra of these approaches and provided an overview on the most recent advances on how brewing yeast genome evolved and domestication took place. The resulting correlation maps between genotypes and relevant brewing phenotypes can assist and further improve the search for novel craft beer starter yeasts, enhancing the portfolio of diversified products offered to the final customer.

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Designing New Yeasts for Craft Brewing: When Natural Biodiversity Meets Biotechnology

10.20944/preprints201911.0265.v1 ◽

2019 ◽

Author(s):

Fabrizio Iattici ◽

Martina Catallo ◽

Lisa Solieri

Keyword(s):

Production Efficiency ◽

Raw Materials ◽

Starter Cultures ◽

Yeast Genome ◽

Evolutionary Engineering ◽

Brewing Yeast ◽

Saccharomyces Pastorianus ◽

Wide Range ◽

Sequential Fermentation ◽

Saccharomyces Eubayanus

Beer is a fermented beverage with a history as old as human civilization and its productive process has been spread all around the world becoming unique in every country and iconic of entire populations. Ales and lagers are by far the most common beers; however, the combination of raw materials, manufacture techniques and aroma profiles are almost infinite, so it is not surprising to notice that there is a large amount of different beer styles, each of them with unique characteristics. Nowadays, diversification is becoming increasingly important in the brewing market and the brewers are continuously interested in improving and extending the already wide range of products, especially in craft brewery. One of the major components that can have a deep impact on the final product is yeast, since it is able to convert carbohydrates in wort, especially maltose and maltotriose, into ethanol, carbon dioxide and other minor aroma-active compounds. Saccharomyces cerevisiae (top‐fermenting yeasts used to produce ales) and Saccharomyces pastorianus (cryotolerant bottom‐fermenting hybrids between S. cerevisiae and Saccharomyces eubayanus responsible for the fermentation of lagers) are most used in breweries. However, an increasing number of different yeast starter cultures are commercially available, to improve the production efficiency also at relative low temperatures and to obtain desirable and diversified aroma profiles avoiding undesired compounds. Four main genetic engineering-free trends are becoming popular in craft brewing yeast development: 1) the research for novel reservoirs as source of new performant S. cerevisiae yeasts; 2) the creation of synthetic hybrids between S. cerevisiae and Saccharomyces non-cerevisiae in order to mimic lager yeasts by expanding their genetic background; 3) the exploitation of evolutionary engineering approaches; 4) the usage of non-Saccharomyces yeasts either in co-coculture or in sequential fermentation with S. cerevisiae. In the present work we summarized pro and contra of these approaches and provided an overview on the most recent advances on how brewing yeast genome evolved and domestication took place. Finally, we delineated how the correlations maps between genotypes and relevant brewing phenotypes can assist and further improve the search for novel craft beer starter yeasts.

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Lager-brewing yeasts in the era of modern genetics

FEMS Yeast Research ◽

10.1093/femsyr/foz063 ◽

2019 ◽

Vol 19 (7) ◽

Cited By ~ 7

Author(s):

Arthur R Gorter de Vries ◽

Jack T Pronk ◽

Jean-Marc G Daran

Keyword(s):

Genome Editing ◽

Industrial Application ◽

Strain Improvement ◽

Brewing Yeast ◽

Yeast Strains ◽

Worldwide Production ◽

Saccharomyces Pastorianus ◽

Saccharomyces Eubayanus ◽

Brewing Yeasts

ABSTRACT The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains.

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Impaired Uptake and/or Utilization of Leucine by Saccharomyces cerevisiae Is Suppressed by the SPT15-300 Allele of the TATA-Binding Protein Gene

Applied and Environmental Microbiology ◽

10.1128/aem.00989-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6055-6061 ◽

Cited By ~ 19

Author(s):

Richard J. S. Baerends ◽

Jin-Long Qiu ◽

Simon Rasmussen ◽

Henrik Bjørn Nielsen ◽

Anders Brandt

Keyword(s):

Saccharomyces Cerevisiae ◽

Mutant Allele ◽

Binding Protein ◽

Growth Conditions ◽

Tata Binding Protein ◽

Brewing Yeast ◽

Yeast Extract Peptone Dextrose ◽

Expression Of Genes ◽

Low Concentrations ◽

Saccharomyces Pastorianus

ABSTRACT Successful fermentations to produce ethanol require microbial strains that have a high tolerance to glucose and ethanol. Enhanced glucose/ethanol tolerance of the laboratory yeast Saccharomyces cerevisiae strain BY4741 under certain growth conditions as a consequence of the expression of a dominant mutant allele of the SPT15 gene (SPT1 5-3 00) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). The SPT15 gene codes for the TATA-binding protein. This finding prompted us to examine the effect of expression of the SPT1 5-3 00 allele in various yeast species of industrial importance. Expression of SPT1 5-3 00 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance to ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT1 5-3 00 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was indeed associated with enhanced uptake and/or utilization of leucine. Reexamination of the microarray data published by Alper and coworkers likewise suggested that expression of genes coding for the leucine permeases, Tat1p and Bap3p, were upregulated in the SPT1 5-3 00 mutant, as was expression of the genes ARO10, ADH3, ADH5, and SFA1, involved in leucine degradation.

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Optimization of the Fermentation Conditions for Brewing Yeast Biomass Production Using the Response Surface Methodology and Taguchi Technique

Agriculture ◽

10.3390/agriculture11121237 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1237

Author(s):

Iuliana Diana Bărbulescu ◽

Mihaela Violeta Ghica ◽

Mihaela Begea ◽

Mădălina Georgiana Albu Kaya ◽

Răzvan Ionuț Teodorescu ◽

...

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Performance Indicator ◽

Feed Additives ◽

Dry Matter Content ◽

Taguchi Technique ◽

Experimental Conditions ◽

Brewing Yeast ◽

Yeast Biomass ◽

Saccharomyces Pastorianus

Yeast (including brewing yeast) and yeast-based preparations derived from bioprocesses or agroindustrial byproducts represent valuable feed additives and ingredients for ruminants. The optimization of brewing yeast biotechnological processing through fermentation mediated by the brewing yeast strain Saccharomyces pastorianus ssp. carlsbergensis W34/70 was investigated. The cultivation conditions (temperature, pH, carbon source, and nitrogen source) were selected and designed according to a Taguchi fractional experimental plan, with four factors on three levels, and their influence on the evolution of the bioprocess of obtaining the brewing yeast biomass was evaluated. The dependent variables were the yeast biomass amount in wet form, yeast biomass amount in dried form after lyophilization, dried yeast biomass wettability assayed through the contact angle (CA), protein content (PC), and dry matter content (DS). The effects that the experimental conditions had on the system responses were visualized in tridimensional space using the response surface methodology, and the combination of biotechnological parameters that ensured process quality and robustness was then determined using the Taguchi technique through its performance indicator, i.e., the signal-to-noise ratio. By optimizing the biotechnological parameters, this study provides a valuable contribution in the area of brewing yeast biomass processing, with the aim of producing probiotic yeast for ruminant nutrition.

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Improving the Utilization of Isomaltose and Panose by Lager Yeast Saccharomyces pastorianus

Fermentation ◽

10.3390/fermentation7030107 ◽

2021 ◽

Vol 7 (3) ◽

pp. 107

Author(s):

Javier Porcayo Loza ◽

Anna Chailyan ◽

Jochen Forster ◽

Michael Katz ◽

Uffe Hasbro Mortensen ◽

...

Keyword(s):

Efficient Utilization ◽

Lager Yeast ◽

Brewing Yeast ◽

Stable Integration ◽

Yeast Strains ◽

Saccharomyces Pastorianus ◽

Organoleptic Traits

Approximately 25% of all carbohydrates in industrial worts are poorly, if at all, fermented by brewing yeast. This includes dextrins, β-glucans, arabinose, xylose, disaccharides such as isomaltose, nigerose, kojibiose, and trisaccharides such as panose and isopanose. As the efficient utilization of carbohydrates during the wort’s fermentation impacts the alcohol yield and the organoleptic traits of the product, developing brewing strains with enhanced abilities to ferment subsets of these sugars is highly desirable. In this study, we developed Saccharomyces pastorianus laboratory yeast strains with a superior capacity to grow on isomaltose and panose. First, we designed a plasmid toolbox for the stable integration of genes into lager strains. Next, we used the toolbox to elevate the levels of the α-glucoside transporter Agt1 and the major isomaltase Ima1. This was achieved by integrating synthetic AGT1 and IMA1 genes under the control of strong constitutive promoters into defined genomic sites. As a result, strains carrying both genes showed a superior capacity to grow on panose and isomaltose, indicating that Ima1 and Agt1 act in synergy to consume these sugars. Our study suggests that non-GMO strategies aiming to develop strains with improved isomaltose and panose utilization could include identifying strains that overexpress AGT1 and IMA1.

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Mitochondrial DNA and temperature tolerance in lager yeasts

10.1101/391946 ◽

2018 ◽

Cited By ~ 3

Author(s):

EmilyClare P. Baker ◽

David Peris ◽

Ryan V. Moriarty ◽

Xueying C. Li ◽

Justin C. Fay ◽

...

Keyword(s):

Mitochondrial Genome ◽

Temperature Tolerance ◽

Temperature Adaptation ◽

Relative Temperature ◽

Brewing Yeast ◽

Saccharomyces Pastorianus ◽

Fermented Beverage ◽

Temperature Preferences ◽

High Or Low Temperature ◽

Saccharomyces Eubayanus

AbstractA growing body of research suggests that the mitochondrial genome (mtDNA) is important for temperature adaptation. In the yeast genusSaccharomyces, species have diverged in temperature tolerance, driving their use in high or low temperature fermentations. Here we experimentally test the role of mtDNA in temperature tolerance in synthetic and industrial hybrids (Saccharomyces cerevisiaexSaccharomyces eubayanus, orSaccharomyces pastorianus), which cold-brew lager beer. We find that the relative temperature tolerances of hybrids correspond to the parent donating mtDNA, allowing us to modulate lager strain temperature preferences. The strong influence of mitotype on the temperature tolerance of otherwise identical hybrid strains provides support for the mitochondrial climactic adaptation hypothesis in yeasts and demonstrates how mitotype has influenced the world's most commonly fermented beverage.One Sentence SummaryMitochondrial genome origin affects the temperature tolerance of synthetic and industrial lager-brewing yeast hybrids.

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

10.1101/2020.07.17.209171 ◽

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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Novel Assessment Method of the Critical Parameters for Brewing Yeast Formulation as a Nutraceutical in Animal Diets

Revista de Chimie ◽

10.37358/rc.20.2.7911 ◽

2020 ◽

Vol 71 (2) ◽

pp. 167-174

Author(s):

Mihaela Begea ◽

Iuliana Diana Barbulescu ◽

Lacramioara Popa ◽

Carmen Otilia Rusanescu ◽

Madalina Georgiana Albu Kaya ◽

...

Keyword(s):

Yeast Strain ◽

Carbon Sources ◽

Assessment Method ◽

Critical Parameters ◽

Aggregative Stability ◽

Brewing Yeast ◽

Yeast Biomass ◽

Saccharomyces Pastorianus ◽

Ruminant Diets ◽

C 30

Brewing yeast biomasses may be converted into valuable products, including supplements for ruminant diets. This paper presents experimental data on a nutraceutical suspension formulation containing post-fermentation brewing yeasts and the brewing yeast strain, Saccharomyces pastorianus ssp. carlsbergensis W34/70, which was intended to be administered to ruminants. The brewing yeast biomass is a component of oral suspensions designed as dietary supplements that prevent digestive acidosis in ruminants. The brewing yeast strain W34/70 was cultivated using molasses (M) and glucose (G) as carbon sources and was lyophilized at different freezing temperatures (0�C, �10�C, �20�C, �30�C, and �40�C). The post-fermentation brewing yeast biomass was spray dried (AAY) and lyophilized (ALY). Both dried yeast biomasses were analyzed in terms of wettability using a contact angle (CA�) evaluation at the solid/liquid interface. Suspensions were subsequently formulated and tested. Their physical and aggregative stability was established in connection with controlled sedimentation and flocculation. The assays were performed following quality by design (QbD) principles. The critical process parameters (CPPs) corresponding to the technological process of yeast production, as well as the critical quality attributes (CQAs) for suspension formulations, were pointed out as preamble determinants when designing oral nutraceuticals destined to be included in ruminant diets.

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Isolation and Characterization of a Gene Specific to Lager Brewing Yeast That Encodes a Branched-Chain Amino Acid Permease

Applied and Environmental Microbiology ◽

10.1128/aem.67.8.3455-3462.2001 ◽

2001 ◽

Vol 67 (8) ◽

pp. 3455-3462 ◽

Cited By ~ 34

Author(s):

Yukiko Kodama ◽

Fumihiko Omura ◽

Toshihiko Ashikari

Keyword(s):

Amino Acid ◽

Northern Analysis ◽

Amino Acid Permease ◽

Brewing Yeast ◽

Weak Organic Acid ◽

Branched Chain Amino Acid ◽

Isolation And Characterization ◽

Saccharomyces Pastorianus ◽

Saccharomyces Bayanus ◽

Branched Chain

ABSTRACT We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that ofSaccharomyces bayanus (by-BAP2-1). The otherBAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.

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