The genetic diversity of bottom-fermenting yeast classified as Saccharomyces pastorianus is poor because strains are restricted to a few genetically distinct groups. Crossbreeding is an effective ...approach to construct novel yeast strains, but it is difficult because of inefficiency to obtain mating-competent cells (MCCs) of bottom-fermenting yeast. By using mating pheromone-supersensitive mutants, we previously isolated several mating-competent meiotic segregants from two bottom-fermenting yeast strains: high isoamyl acetate-producing KY1247, and low diacetyl-producing KY2645. Here, we constructed novel non-GM hybrids carrying preferable characteristics from both parents by crossbreeding these bottom-fermenting strains for the first time. Sixteen a/a-type meiotic segregants from KY2645 and 12 α/α-type meiotic segregants from KY1247 were mixed, and cells resembling zygotes were isolated via micromanipulation. In total, 149 hybrids were obtained and verified by examining known single-nucleotide polymorphisms (SNPs) between the parental strains. A sporulation test showed that some of the hybrids were able to sporulate. Moreover, fermentation tests on a test-tube and pilot-plant scale identified two hybrids with production levels of isoamyl acetate and diacetyl that were almost the same as KY1247 and KY2645, respectively. Both of these hybrids produced satisfactory beer in terms of taste, flavor, and overall quality, comparable to that produced by the parental strains. Collectively, our results suggest that crossbreeding between bottom-fermenting yeast strains has the potential to increase the diversity of yeast strains available for brewing, and our method of isolating MCCs provides a huge advance for crossbreeding of bottom-fermenting yeast without using DNA recombination techniques.
Brewing practice uses the same yeast to inoculate the following fermentation (repitching). Saccharomyces pastorianus, used to produce Lager beer, is widely reused, not changing its fermentation ...performance. However, S. cerevisiae, used to produce Ale beer, is partial or not even reused, due to its poor performance. It is hypothesized that cells modulate their wall polysaccharides to increase the cell-wall strength. In this work industrial S. cerevisiae and S. pastorianus brewer's spent yeasts with different repitching numbers were studied. Glucans were the main component of S. cerevisiae whereas mannoproteins were abundant in S. pastorianus. The major changes were noticed on glucans of both species, β1,3-glucans decrease more pronounced in S. cerevisiae. The increase of α1,4-Glc, related with osmotolerance, was higher in S. cerevisiae while β1,4-Glc, related with cell-wall strength, had a small increase. In addition, these structural details showed different binding profiles to immune receptors, important to develop tailored bioactive applications.
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•Brewer’s spent yeast extract a new ingredient for food and nutraceutical industries.•High nutritional composition and antioxidant activity of spent yeast extracts.•Brewer’s spent yeast extracts ...contain free and both phenolic compounds.
Spent yeast is the second major by-product from brewing. Mechanical disruption of yeast cell wall can be used to obtain β-glucan rich ingredients and separate inner yeast content, both with potential applications as food and nutraceutical ingredients. In this work, the nutritional composition, including minerals and B-complex vitamins, together with the antioxidant activity and phenolic compounds profile of yeast extract, prepared by mechanic disruption of brewer’s spent yeast and removal of yeast cell wall, was investigated. The lyophilised yeast extract presented 64% of proteins with high proportion of essential amino acids and 4% of RNA; macrominerals (Na, K, Ca, Mg), trace elements such as Zn (11.9mg/100g dw), Fe (1.76mg/100g dw), and Mn (0.564mg/100g dw), and vitamins B3 (77.2mg/100mg dw), B6 (55.1mg/100g dw) and B9 (3.01mg/100g dw). Two phenolic compounds were quantified as free forms, gallic acid and (±) catechin, whereas other bounded phenolic compounds were also quantified. The nutrients content, antioxidant properties and phenolic composition of the lyophilised brewer’s spent yeast extract indicates that it can be an interesting food or nutraceutical ingredient. Thus, its recovery will be beneficial in terms of sustainability and environmental impact.
lager-brewing yeasts are domesticated hybrids of
x
that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome ...rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of
and
was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid
brewing strains. In contrast to the extensive aneuploidy of domesticated
strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated
strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of
×
hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.
Domestication of plants and animals promoted humanity's transition from nomadic to sedentary lifestyles, demographic expansion, and the emergence of civilizations. In contrast to the well-documented ...successes of crop and livestock breeding, processes of microbe domestication remain obscure, despite the importance of microbes to the production of food, beverages, and biofuels. Lager-beer, first brewed in the 15th century, employs an allotetraploid hybrid yeast, Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis), a domesticated species created by the fusion of a Saccharomyces cerevisiae ale-yeast with an unknown cryotolerant Saccharomyces species. We report the isolation of that species and designate it Saccharomyces eubayanus sp. nov. because of its resemblance to Saccharomyces bayanus (a complex hybrid of S. eubayanus, Saccharomyces uvarum, and S. cerevisiae found only in the brewing environment). Individuals from populations of S. eubayanus and its sister species, S. uvarum, exist in apparent sympatry in Nothofagus (Southern beech) forests in Patagonia, but are isolated genetically through intrinsic postzygotic barriers, and ecologically through host-preference. The draft genome sequence of S. eubayanus is 99.5% identical to the non-S. cerevisiae portion of the S. pastorianus genome sequence and suggests specific changes in sugar and sulfite metabolism that were crucial for domestication in the lager-brewing environment. This study shows that combining microbial ecology with comparative genomics facilitates the discovery and preservation of wild genetic stocks of domesticated microbes to trace their history, identify genetic changes, and suggest paths to further industrial improvement.
The contribution of polyfunctional thiols (PFTs) to the overall flavor of a lager beer has been well documented, but their origin remains unclear. In comparison with Saccharomyces cerevisiae strains ...previously investigated (same conditions: 7 days at 24 °C, 3 days at 4 °C), we examined how Saccharomyces pastorianus yeasts are able to produce these PFTs from cysteinylated (Cys-) and glutathionylated (G-) conjugates. Up to 0.35% release was observed from G-conjugates against less than 0.08% for all yeasts studied so far. Lowering the wort nitrogen level and the fermentation temperature (12 °C) strongly increased the release efficiency and the ester/alcohol ratio from Cys-conjugates. However, it did not improve the release from G-conjugates and even prevented the yeast from producing any acetate. Although poor in free PFTs and their Cys-precursors, both Saaz hop and pale malt (classical ingredients for premium lager beers) confirmed to be significant sources of G-conjugates (especially those of 3-sulfanylhexanol and 3-sulfanyl-4-methylpentanol).
Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we ...used a genome‐scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03). Yeast 8.5.0 was used as the base model, which accurately predicted G03's growth. Using OptForce, we found that overexpression of ARO8 and ATP1 among six different strategies increased the RNA content of G03 by 58.0% and 74.8%, respectively. We also identified new metabolic targets for improved RNA production using a modified GSMM called TissueModel, constructed using the GIMME transcriptome constraint tool to remove low‐expressed reactions in the model. After running OptKnock, the RNA content of G03‐△BNA1 and G03‐△PMA1 increased by 44.6% and 39.8%, respectively, compared to G03. We suggest that ATP1, ARO8, BNA1, and PMA1 regulate cell fitness, which affects RNA content. This study is the first to identify strategies for RNA overproduction using GSMM and to report that regulation of ATP1, ARO8, BNA1, and PMA1 can increase RNA content in S. pastorianus. These findings also provide valuable knowledge on model reconstruction for S. pastorianus.
Graphical and Lay Summary
Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we used a genome‐scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03) which also provide valuable knowledge on model reconstruction for S. pastorianus.
Lager 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 of Saccharomyces ...pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces 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 of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. 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.
The concentrations and relative ratios of various aroma compounds produced by fermenting yeast cells are essential for the sensory quality of many fermented foods, including beer, bread, wine, and ...sake. Since the production of these aroma-active compounds varies highly among different yeast strains, careful selection of variants with optimal aromatic profiles is of crucial importance for a high-quality end product. This study evaluates the production of different aroma-active compounds in 301 different Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus yeast strains. Our results show that the production of key aroma compounds like isoamyl acetate and ethyl acetate varies by an order of magnitude between natural yeasts, with the concentrations of some compounds showing significant positive correlation, whereas others vary independently. Targeted hybridization of some of the best aroma-producing strains yielded 46 intraspecific hybrids, of which some show a distinct heterosis (hybrid vigor) effect and produce up to 45% more isoamyl acetate than the best parental strains while retaining their overall fermentation performance. Together, our results demonstrate the potential of large-scale outbreeding to obtain superior industrial yeasts that are directly applicable for commercial use.
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