Search Results (12)

This study provides a comprehensive analysis of the microbial dynamics involved in the fermentation process of traditional Musalais wine, an intangible cultural heritage of Xinjiang. Utilizing metagenomic sequencing, we identified 2894 microbial species, of which 494 persisted throughout the fermentation process. Saccharomyces cerevisiae was the dominant species, with its prevalence increasing from 97.35% in the early phase to 99.38% in the mid phase, before slightly decreasing to 98.79% in the late phase. Additionally, 24 non-Saccharomyces yeast species, including Hanseniaspora uvarum, Lachancea thermotolerans, and Torulaspora delbrueckii, were detected. Common species associated with other fermented foods, including Wickerhamomyces anomalus, Kluyveromyces marxianus, Saccharomyces eubayanus, and Zygosaccharomyces parabailii, were also identified. Notably, species not previously used in food fermentation, such as Saccharomyces jurei, Sodiomyces alkalinus, Vanrija pseudolonga, and Moesziomyces antarcticus, were also identified in this study. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KO) and Gene Ontology (GO) revealed notable variations in metabolic pathways and enriched functional genes. In addition, a total of 82 volatile compounds were detected in the final product, with higher alcohols (60.12%), esters (37.80%), and organic acids (1.80%) being the most prevalent. These results offer important insights into microbial interactions and their influence on Musalais wine quality, laying the groundwork for optimizing the fermentation process. Full article

The aim of the present study was to evaluate the use of different chemical treatments of apple pomace in order to produce an economical culture medium for the propagation of two non-conventional yeast strains. An experimental design approach was used for the optimization of the hydrolysis conditions of apple pomace. Both acid and alkaline treatment conditions were tested. The optimal hydrolysis conditions to disrupt the pomace lignocellulosic structure were 1% w/v of H₃PO₄, 121 °C, and 40 min for acid treatment, while 6% w/v of NH₄OH, 20 °C, and 2 h were optimal for the alkaline condition. Saccharomyces uvarum NPCC 1420 and Saccharomyces eubayanus NPCC 1292 yeasts were able to grow in the liquid fraction obtained from both acid and alkaline treatments. However, the medium with the acid treatment was found to be more suitable for yeast growth, showing, for both strains, higher µmax and biomass production and lower td and λ than that observed for the medium with the alkaline treatment. According to the growth parameter analysis for both strains, the acid treatment was selected for further studies. By taking advantage of this agroindustrial by-product, a circular economy approach is promoted, reducing environmental impact and fostering sustainable development. Full article

Hybrid formation and introgressions had a profound impact on fermentative yeasts domesticated for beer, wine and cider fermentations. Here we provide a comparative genomic analysis of a British cider yeast isolate (E1) and characterize its fermentation properties. E1 has a Saccharomyces uvarum genome into which ~102 kb of S. eubayanus DNA were introgressed that replaced the endogenous homologous 55 genes of chromosome XIV between YNL182C and YNL239W. Sequence analyses indicated that the DNA donor was either a lager yeast or a yet unidentified S. eubayanus ancestor. Interestingly, a second introgression event added ~66 kb of DNA from Torulaspora microellipsoides to the left telomere of SuCHRX. This region bears high similarity with the previously described region C introgression in the wine yeast EC1118. Within this region FOT1 and FOT2 encode two oligopeptide transporters that promote improved nitrogen uptake from grape must in E1, as was reported for EC1118. Comparative laboratory scale grape must fermentations between the E1 and EC1118 indicated beneficial traits of faster consumption of total sugars and higher glycerol production but low acetic acid and reduced ethanol content. Importantly, the cider yeast strain produced high levels of fruity ester, including phenylethyl and isoamyl acetate. Full article

The multicomponent polymerase associated factor 1 (Paf1) complex (PAF1C) is an important transcription elongation factor that upregulates RNA polymerase II-mediated genome-wide transcription. PAF1C can regulate transcription through direct association with the polymerase or by impacting the chromatin structure epigenetically. In recent years, significant progress has been made in understanding the molecular mechanisms of PAF1C. However, high-resolution structures that can clarify the interaction details among the components of the complex are still needed. In this study, we evaluated the structural core of the yeast PAF1C containing the four components Ctr9, Paf1, Cdc73 and Rtf1 at high resolution. We observed the interaction details among these components. In particular, we identified a new binding surface of Rtf1 on PAF1C and found that the C-terminal sequence of Rtf1 dramatically changed during evolution, which may account for its different binding affinities to PAF1C among species. Our work presents a precise model of PAF1C, which will facilitate our understanding of the molecular mechanism and the in vivo function of the yeast PAF1C. Full article

Iron is an indispensable element that participates as an essential cofactor in multiple biological processes. However, when present in excess, iron can engage in redox reactions that generate reactive oxygen species that damage cells at multiple levels. In this report, we characterized the response of budding yeast species from the Saccharomyces genus to elevated environmental iron concentrations. We have observed that S. cerevisiae strains are more resistant to high-iron concentrations than Saccharomyces non-cerevisiae species. Liquid growth assays showed that species evolutionarily closer to S. cerevisiae, such as S. paradoxus, S. jurei, S. mikatae, and S. arboricola, were more resistant to high-iron levels than the more distant species S. eubayanus and S. uvarum. Remarkably, S. kudriavzevii strains were especially iron sensitive. Growth assays in solid media suggested that S. cerevisiae and S. paradoxus were more resistant to the oxidative stress caused by elevated iron concentrations. When comparing iron accumulation and sensitivity, different patterns were observed. As previously described for S. cerevisiae, S. uvarum and particular strains of S. kudriavzevii and S. paradoxus became more sensitive to iron while accumulating more intracellular iron levels. However, no remarkable changes in intracellular iron accumulation were observed for the remainder of species. These results indicate that different mechanisms of response to elevated iron concentrations exist in the different species of the genus Saccharomyces. Full article

Chiral dimethyl 2-methylsuccinate (1) is a very important building block for the manufacturing of many active pharmaceutical ingredients and fine chemicals. The asymmetric reduction of C=C double bond of dimethyl citraconate (2), dimethyl mesaconate (3) or dimethyl itaconate (4) by ene-reductases (ERs) represents an attractive straightforward approach, but lack of high-performance ERs, especially (S)-selective ones, has limited implementing this method to prepare the optically pure dimethyl 2-methylsuccinate. Herein, three ERs (Bac-OYE1 from Bacillus sp., SeER from Saccharomyces eubayanus and AfER from Aspergillus flavus) with high substrate tolerance and stereoselectivity towards 2, 3 and 4 have been identified. Up to 500 mM of 3 was converted to (S)-dimethyl 2-methylsuccinate ((S)-1) by SeER in high yields (80%) and enantioselectivity (98% ee), and 700 mM of 2 and 400 mM of 4 were converted to (R)-1 by Bac-OYE1 and AfER, respectively, in high yields (86% and 77%) with excellent enantioselectivity (99% ee). The reductions of diethyl citraconate (5), diethyl mesaconate (6) and diethyl itaconate (7) were also tested with the three ERs. Although up to 500 mM of 5 was completely converted to (R)-diethyl 2-methylsuccinate ((R)-8) by Bac-OYE1 with excellent enantioselectivity (99% ee), the alcohol moiety of the esters had a great effect on the activity and enantioselectivity of ERs. This work provides an efficient methodology for the enantiocomplementary production of optically pure dimethyl 2-methylsuccinate from dimethyl itaconate and its isomers at high titer. Full article

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. Full article

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. Full article

The recent isolation of the yeast Saccharomyces eubayanus has opened new avenues in the brewing industry. Recent studies characterized the production of volatile compounds in a handful set of isolates, utilizing a limited set of internal standards, representing insufficient evidence into the ability of the species to produce new and diverse aromas in beer. Using Headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME-GC-MS), we characterized for the first time the production of volatile compounds in 10 wild strains under fermentative brewing conditions and compared them to a commercial lager yeast. S. eubayanus produces a higher number of volatile compounds compared to lager yeast, including acetate and ethyl esters, together with higher alcohols and phenols. Many of the compounds identified in S. eubayanus are related to fruit and floral flavors, which were absent in the commercial lager yeast ferment. Interestingly, we found a significant strain × temperature interaction, in terms of the profiles of volatile compounds, where some strains produced significantly greater levels of esters and higher alcohols. In contrast, other isolates preferentially yielded phenols, depending on the fermentation temperature. This work demonstrates the profound fermentation product differences between different S. eubayanus strains, highlighting the enormous potential of this yeast to produce new styles of lager beers. Full article

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. Full article

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. Full article

The characteristic flavour and aroma of any beer is, in large part, determined by the yeast strain employed and the wort composition. In addition, properties such as flocculation, wort fermentation ability (including the uptake of wort sugars, amino acids, and peptides), ethanol and osmotic pressure tolerance together with oxygen requirements have a critical impact on fermentation performance. Yeast management between fermentations is also a critical brewing parameter. Brewer’s yeasts are mostly part of the genus Saccharomyces. Ale yeasts belong to the species Saccharomyces cerevisiae and lager yeasts to the species Saccharomyces pastorianus. The latter is an interspecies hybrid between S. cerevisiae and Saccharomyces eubayanus. Brewer’s yeast strains are facultative anaerobes—they are able to grow in the presence or absence of oxygen and this ability supports their property as an important industrial microorganism. This article covers important aspects of Saccharomyces molecular biology, physiology, and metabolism that is involved in wort fermentation and beer production. Full article