Hi-C metagenomics facilitate comparative genome analysis of bacteria and yeast from spontaneous beer and cider.

Sequence-based analysis of fermented foods and beverages' microbiomes offers insights into their impact on taste and consumer health. High-throughput metagenomics provide detailed taxonomic and functional community profiling, but bacterial and yeast genome reconstruction and mobile genetic elements tracking are to be improved. We established a pipeline for exploring fermented foods microbiomes using metagenomics coupled with chromosome conformation capture (Hi-C metagenomics). The approach was applied to analyze a collection of spontaneously fermented beers and ciders (n = 12). The Hi-C reads were used to reconstruct the metagenome-assembled genomes (MAGs) of bacteria and yeasts facilitating subsequent comparative genomic analysis, assembly scaffolding and exploration of "plasmid-bacteria" links. For a subset of beverages, yeasts were isolated and characterized phenotypically. The reconstructed Hi-C MAGs primarily belonged to the Lactobacillaceae family in beers, along with Acetobacteraceae and Enterobacteriaceae in ciders, exhibiting improved quality compared to conventional metagenomic MAGs. Comparative genomic analysis of Lactobacillaceae Hi-C MAGs revealed clustering by niche and suggested genetic determinants of survival and probiotic potential. For Pediococcus damnosus, Hi-C-based networks of contigs enabled linking bacteria with plasmids. Analyzing phylogeny and accessory genes in the context of known reference genomes offered insights into the niche specialization of beer lactobacilli. The subspecies-level diversity of cider Tatumella spp. was disentangled using a Hi-C-based graph. We obtained highly complete yeast Hi-C MAGs primarily represented by Brettanomyces and Saccharomyces, with Hi-C-facilitated chromosome-level genome assembly for the former. Utilizing Hi-C metagenomics to unravel the genomic content of individual species can provide a deeper understanding of the ecological interactions within the food microbiome, aid in bioprospecting beneficial microorganisms, improving quality control and improving innovative fermented products.

Assess different fermentation characteristics of 54 lager yeasts based on group classification.

Saccharomyces pastorianus, hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus, were generally regarded as authentic lager beer yeasts. In recent years, with more new findings of other Saccharomyces genus hybrids, yeasts used in lager beer brewing have been proved much more complicated than previous cognition. In this study, we analyzed the different fermentation characteristics of 54 yeast strains used for lager brewing in normal and very high gravity brewing based on group classification. The difference between Group Ⅰ and Group Ⅱ lager yeasts were more striking in very high gravity brewing. However, during our research progress, we realized that some yeasts used in this study were actually hybrids of S. cerevisiae and Saccharomyces kudriavzevii. Features of these hybrids could be beneficial to very high gravity brewing. We further discussed about the mechanism behind their outstanding characteristics and the reason why group classification methods of lager beer yeasts had limitations. Hybridization in yeasts is constantly getting richer. Lager yeasts could have more possibilities based on better understandings of their genetic background and roles of other Saccharomyces genus hybrids. Their heterosis shed light on innovation in brewing and other diverse fermentation industries.

Targeted 1-H-NMR wine analyses revealed specific metabolomic signatures of yeast populations belonging to the Saccharomyces genus.

This study aimed to explore the non-volatile metabolomic variability of a large panel of strains (44) belonging to the Saccharomyces cerevisiae and Saccharomyces uvarum species in the context of the wine alcoholic fermentation. For the S. cerevisiae strains flor, fruit and wine strains isolated from different anthropic niches were compared. This phenotypic survey was achieved with a special focus on acidity management by using natural grape juices showing opposite level of acidity. A 1H NMR based metabolomics approach was developed for quantifying fifteen wine metabolites that showed important quantitative variability within the strains. Thanks to the robustness of the assay and the low amount of sample required, this tool is relevant for the analysis of the metabolomic profile of numerous wines. The S. cerevisiae and S. uvarum species displayed significant differences for malic, succinic, and pyruvic acids, as well as for glycerol and 2,3-butanediol production. As expected, S. uvarum showed weaker fermentation fitness but interesting acidifying properties. The three groups of S. cerevisiae strains showed different metabolic profiles mostly related to their production and consumption of organic acids. More specifically, flor yeast consumed more malic acid and produced more acetic acid than the other S. cerevisiae strains which was never reported before. These features might be linked to the ability of flor yeasts to shift their metabolism during wine oxidation.

Divergence in the Saccharomyces Species' Heat Shock Response Is Indicative of Their Thermal Tolerance.

The Saccharomyces species have diverged in their thermal growth profile. Both Saccharomyces cerevisiae and Saccharomyces paradoxus grow at temperatures well above the maximum growth temperature of Saccharomyces kudriavzevii and Saccharomyces uvarum but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature, we measured changes in gene expression in response to a 12 °C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families, we sequenced, assembled, and annotated a complete S. uvarum genome. In response to heat, the cryophilic species showed a stronger stress response than the thermophilic species, and the hybrids showed a mixture of parental responses that depended on the time point. After an initial strong response indicative of high thermal stress, hybrids with a thermophilic parent resolved their heat shock response to become similar to their thermophilic parent. Within the hybrids, only a small number of temperature-responsive genes showed consistent differences between alleles from the thermophilic and cryophilic species. Our results show that divergence in the heat shock response is mainly a consequence of a strain's thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat-induced changes are relevant to thermal divergence in the Saccharomyces species.

Revealing Potential Genes Affecting Flocculation and/or Viability of Saccharomyces pastorianus by Comparative Genomic Analysis.

Yeast flocculation and viability are critical factors in beer production. Adequate flocculation of yeast at the end of fermentation helps to reduce off-flavors and cell separation, while high viability is beneficial for yeast reuse. In this study, we used comparative genomics to analyze the genome information on Saccharomyces pastorianus W01, and its spontaneous mutant W02 with appropriate weakened flocculation ability (better off-flavor reduction performance) and unwanted decreased viability, to investigate the effect of different gene expressions on yeast flocculation or/and viability. Our results indicate that knockout of CNE1, CIN5, SIN3, HP-3, YPR170W-B, and SCEPF1_0274000100 and overexpression of CNE1 and ALD2 significantly decreased the flocculation ability of W01, while knockout of EPL1 increased the flocculation ability of W01. Meanwhile, knockout of CIN5, YPR170W-B, OST5, SFT1, SCEPF1_0274000100, and EPL1 and overexpression of SWC3, ALD2, and HP-2 decreased the viability of W01. CIN5, EPL1, SCEPF1_0274000100, ALD2, and YPR170W-B have all been shown to affect yeast flocculation ability and viability.

Addition of Saccharomyces eubayanus to SCOBY fermentations modulates the chemical and volatile compound profiles in kombucha.

Kombucha is a fermented beverage derived from a sweetened tea fermentation inoculated with a bacteria-yeast consortium referred to as Symbiotic Culture of Bacteria and Yeast (SCOBY). Different SCOBY cultures can impact the beverage's quality and make the whole process highly variable. Adding Saccharomyces yeast cultures to the fermentation process can avoid stalled fermentations, providing a reproducible beverage. Here, we explored using different Saccharomyces eubayanus strains together with SCOBY in the context of kombucha fermentation. Our results show that yeast x SCOBY co-cultures exhibited a robust fermentation profile, providing ethanol and acetic acid levels ranging from 0,18-1,81 %v/v and 0,35-1,15 g/L, respectively. The kombucha volatile compound profile of co-cultures was unique, where compounds such as Isopentyl acetate where only found in yeast x SCOBY fermentations. Metabarcoding revealed that the SCOBY composition was also dependent on the S. eubayanus genotype, where besides Saccharomyces, amplicon sequence variants belonging to Brettanomyces and Starmerella were detected. These differences concomitated global changes in transcript levels in S. eubayanus related to the metabolism of organic molecules used in kombucha fermentation. This study highlights the potential for exploring different S. eubayanus strains for kombucha fermentation, and the significant yeast genotype effect in the profile differentiation in this process.

Understanding the role of GRE3 in the erythritol biosynthesis pathway in Saccharomyces uvarum and its implication in osmoregulation and redox homeostasis.

Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.

Improving ribonucleic acid production in Saccharomyces pastorianus via in silico genome-scale metabolic network model.

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.

Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach.

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

Genetic basis for probiotic yeast phenotypes revealed by nanopore sequencing.

Probiotic yeasts are emerging as preventative and therapeutic solutions for disease. Often ingested via cultured foods and beverages, they can survive the harsh conditions of the gastrointestinal tract and adhere to it, where they provide nutrients and inhibit pathogens like Candida albicans. Yet, little is known of the genomic determinants of these beneficial traits. To this end, we have sequenced 2 food-derived probiotic yeast isolates that mitigate fungal infections. We find that the first strain, KTP, is a strain of Saccharomyces cerevisiae within a small clade that lacks any apparent ancestry from common European/wine S. cerevisiae strains. Significantly, we show that S. cerevisiae KTP genes involved in general stress, pH tolerance, and adherence are markedly different from S. cerevisiae S288C but are similar to the commercial probiotic yeast species S. boulardii. This suggests that even though S. cerevisiae KTP and S. boulardii are from different clades, they may achieve probiotic effect through similar genetic mechanisms. We find that the second strain, ApC, is a strain of Issatchenkia occidentalis, one of the few of this family of yeasts to be sequenced. Because of the dissimilarity of its genome structure and gene organization, we infer that I. occidentalis ApC likely achieves a probiotic effect through a different mechanism than the Saccharomyces strains. Therefore, this work establishes a strong genetic link among probiotic Saccharomycetes, advances the genomics of Issatchenkia yeasts, and indicates that probiotic activity is not monophyletic and complimentary mixtures of probiotics could enhance health benefits beyond a single species.

Beyond S. cerevisiae for winemaking: Fermentation-related trait diversity in the genus Saccharomyces.

Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces.

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

Specific targeting of inflammatory osteoclastogenesis by the probiotic yeast S. boulardii CNCM I-745 reduces bone loss in osteoporosis.

Bone destruction is a hallmark of chronic inflammation, and bone-resorbing osteoclasts arising under such a condition differ from steady-state ones. However, osteoclast diversity remains poorly explored. Here, we combined transcriptomic profiling, differentiation assays and in vivo analysis in mouse to decipher specific traits for inflammatory and steady-state osteoclasts. We identified and validated the pattern-recognition receptors (PRR) Tlr2, Dectin-1, and Mincle, all involved in yeast recognition as major regulators of inflammatory osteoclasts. We showed that administration of the yeast probiotic Saccharomyces boulardii CNCM I-745 (Sb) in vivo reduced bone loss in ovariectomized but not sham mice by reducing inflammatory osteoclastogenesis. This beneficial impact of Sb is mediated by the regulation of the inflammatory environment required for the generation of inflammatory osteoclasts. We also showed that Sb derivatives as well as agonists of Tlr2, Dectin-1, and Mincle specifically inhibited directly the differentiation of inflammatory but not steady-state osteoclasts in vitro. These findings demonstrate a preferential use of the PRR-associated costimulatory differentiation pathway by inflammatory osteoclasts, thus enabling their specific inhibition, which opens new therapeutic perspectives for inflammatory bone loss.

Synthetic two-species allodiploid and three-species allotetraploid Saccharomyces hybrids with euploid (complete) parental subgenomes.

Combination of the genomes of Saccharomyces species has great potential for the construction of new industrial strains as well as for the study of the process of speciation. However, these species are reproductively isolated by a double sterility barrier. The first barrier is mainly due to the failure of the chromosomes to pair in allodiploid meiosis. The second barrier ensures that the hybrid remains sterile even after genome duplication, an event that can restore fertility in plant interspecies hybrids. The latter is attributable to the autodiploidisation of the allotetraploid meiosis that results in sterile allodiploid spores (return to the first barrier). Occasionally, mating-competent alloaneuploid spores arise by malsegregation of MAT-carrying chromosomes. These can mate with cells of a third species resulting in aneuploid zygotes having at least one incomplete subgenome. Here we report on the construction of euploid three-species hybrids by making use of "rare mating" between a sterile S. kudriavzevii x S. uvarum allodiploid hybrid and a diploid S. cerevisiae strain. The hybrids have allotetraploid 2nScnSk nSu genomes consisting of complete sets of parental chromosomes. This is the first report on the production of euploid three-species Saccharomyces hybrids by natural mating, without genetic manipulation. The hybrids provide possibilities for studying the interactions of three allospecific genomes and their orthologous genes present in the same cell.

Saccharomyces genome database update: server architecture, pan-genome nomenclature, and external resources.

As one of the first model organism knowledgebases, Saccharomyces Genome Database (SGD) has been supporting the scientific research community since 1993. As technologies and research evolve, so does SGD: from updates in software architecture, to curation of novel data types, to incorporation of data from, and collaboration with, other knowledgebases. We are continuing to make steps toward providing the community with an S. cerevisiae pan-genome. Here, we describe software upgrades, a new nomenclature system for genes not found in the reference strain, and additions to gene pages. With these improvements, we aim to remain a leading resource for students, researchers, and the broader scientific community.

Functional diversity and plasticity in the sugar preferences of Saccharomyces MALT transporters in domesticated yeasts.

Maltose and maltotriose, together with glucose, are the major carbohydrates found in malts. Thus, brewing yeasts grown in malt-based brewing processes with serial re-pitching have likely increased their ability to uptake these sugars during domestication by modulating the expression and copy number of maltose transporter genes (MALT, also known as Malx1). However, the molecular basis for and structural insights into the sugar preferences of MALT proteins remain to be elucidated. Here we report the functional evaluation of two novel Saccharomyces cerevisiae MALT proteins, ScMalt#2p and ScMalt#5p, from industrial brewing yeasts, focusing on their maltose and maltotriose preferences. Structural models of the MALT proteins generated by AlphaFold2 and functional analyses of substitution mutants revealed that a very small number of amino acid residues in two spatially adjacent transmembrane helixes, TMH7 and TMH11, appear to be crucial for sugar preference. Thus, subtle conformational alterations conferred by a small number of amino acid polymorphisms within MALTs would contribute to the adaptation of domesticated brewing yeasts to the constrained carbohydrate environment of industrial wort during beer brewing.

A widespread inversion polymorphism conserved among Saccharomyces species is caused by recurrent homogenization of a sporulation gene family.

Saccharomyces genomes are highly collinear and show relatively little structural variation, both within and between species of this yeast genus. We investigated the only common inversion polymorphism known in S. cerevisiae, which affects a 24-kb 'flip/flop' region containing 15 genes near the centromere of chromosome XIV. The region exists in two orientations, called reference (REF) and inverted (INV). Meiotic recombination in this region is suppressed in crosses between REF and INV orientation strains such as the BY x RM cross. We find that the inversion polymorphism is at least 17 million years old because it is conserved across the genus Saccharomyces. However, the REF and INV isomers are not ancient alleles but are continually being re-created by re-inversion of the region within each species. Inversion occurs due to continual homogenization of two almost identical 4-kb sequences that form an inverted repeat (IR) at the ends of the flip/flop region. The IR consists of two pairs of genes that are specifically and strongly expressed during the late stages of sporulation. We show that one of these gene pairs, YNL018C/YNL034W, codes for a protein that is essential for spore formation. YNL018C and YNL034W are the founder members of a gene family, Centroid, whose members in other Saccharomycetaceae species evolve fast, duplicate frequently, and are preferentially located close to centromeres. We tested the hypothesis that Centroid genes are a meiotic drive system, but found no support for this idea.

Adaptive evolution in the Saccharomyces kudriavzevii Aro4p promoted a reduced production of higher alcohols.

The use of unconventional yeast species in human-driven fermentations has attracted a lot of attention in the last few years. This tool allows the alcoholic beverage industries to solve problems related to climate change or the consumer demand for newer high-quality products. In this sense, one of the most attractive species is Saccharomyces kudriavzevii, which shows interesting fermentative traits such as the increased and diverse aroma compound production in wines. Specifically, it has been observed that different isolates of this species can produce higher amounts of higher alcohols such as phenylethanol compared with Saccharomyces cerevisiae. In this work, we have shed light on this feature relating it to the S. kudriavzevii aromatic amino acid anabolic pathway in which the enzyme Aro4p plays an essential role. Unexpectedly, we observed that the presence of the S. kudriavzevii ARO4 variant reduces phenylethanol production compared with the S. cerevisiae ARO4 allele. Our experiments suggest that this can be explained by increased feedback inhibition, which might be a consequence of the changes detected in the Aro4p amino end such as L26 Q24 that have been under positive selection in the S. kudriavzevii specie.