Insights into the mechanisms driving microbial community succession during pepper fermentation: Roles of microbial interactions and endogenous environmental changes.

Elucidating the driving mechanism of microbial community succession during pepper fermentation contributes to establishing efficient fermentation regulation strategies. This study utilized three-generation high-throughput sequencing technology, microbial co-occurrence network analysis, and random forest analysis to reveal microbial community succession processes and driving mechanisms during pepper fermentation. The results showed that more positive correlations than negative correlations were observed among microorganisms, with positive correlation proportions of 60 %, 51.03 %, and 71.43 % between bacteria and bacteria, fungi and fungi, and bacteria and fungi in sipingtou peppers, and 69.23 %, 54.93 %, and 79.44 % in zhudachang peppers, respectively. Microbial interactions, mainly among Weissella hellenica, Lactobacillus plantarum, Hanseniaspora opuntiae, and Kazachstania humillis, could drive bacterial and fungal community succession. Notably, the bacterial community successions during the fermentation of two peppers were similar, showing the transition from Leuconostoc pseudomesenteroides, Lactococcus lactis, Weissella ghanensis to Weissella hellenica and Lactobacillus plantarum. However, the fungal community successions in the two fermented peppers differed significantly, and the differential biomarkers were Dipodascus geotrichum and Kazachstania humillis. Differences in autochthonous microbial composition and inherent constituents brought by pepper varieties resulted in different endogenous environmental changes, mainly in fructose, malic acid, and citric acid. Furthermore, endogenous environmental factors could also drive microbial community succession, with succinic acid, lactic acid, and malic acid being the main potential drivers of bacterial community succession, whereas fructose, glucose, and succinic acid were the main drivers of fungal community succession. These results will provide insights into controlling fermentation processes by raw material combinations, optimization of environmental parameters, and microbial interactions.

Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum.

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage.

Open (non-sterile) cultivations of Debaryomyces hansenii for recombinant protein production combining industrial side-streams with high salt content.

The halotolerant non-conventional yeast Debaryomyces hansenii can grow in media containing high concentrations of salt (up to 4 M), metabolize alternative carbon sources than glucose, such as lactose or glycerol, and withstand a wide range of temperatures and pH. These inherent capabilities allow this yeast to grow in harsh environments and use alternative feedstock than traditional commercial media. For example, D. hansenii could be a potential cell factory for revalorizing industrial salty by-products, using them as a substrate for producing new valuable bioproducts, boosting a circular economy. In this work, three different salty by-products derived from the dairy and biopharmaceutical industry have been tested as a possible feedstock for D. hansenii's growth. The yeast was not only able to grow efficiently in all of them but also to produce a recombinant protein (Yellow Fluorescent Protein, used as a model) without altering its performance. Moreover, open cultivations at different laboratory scales (1.5 mL and 1 L) were performed under non-sterile conditions and without adding fresh water or any nutritional supplement to the cultivation, making the process cheaper and more sustainable.

Insights into the identification and evolutionary conservation of key genes in the transcriptional circuits of meiosis initiation and commitment in budding yeast.

Initiation of meiosis in budding yeast does not commit the cells for meiosis. Thus, two distinct signaling cascades may differentially regulate meiosis initiation and commitment in budding yeast. To distinguish between the role of these signaling cascades, we reconstructed protein-protein interaction networks and gene regulatory networks with upregulated genes in meiosis initiation and commitment. Analyzing the integrated networks, we identified four master regulators (MRs) [Ume6p, Msn2p, Met31p, Ino2p], three transcription factors (TFs), and 279 target genes (TGs) unique for meiosis initiation, and three MRs [Ndt80p, Aro80p, Rds2p], 11 TFs, and 948 TGs unique for meiosis commitment. Functional enrichment analysis of these distinct members from the transcriptional cascades for meiosis initiation and commitment revealed that nutritional cues rewire gene expression for initiating meiosis and chromosomal recombination commits cells to meiosis. As meiotic chromosomal recombination is highly conserved in eukaryotes, we compared the evolutionary rate of unique members in the transcriptional cascade of two meiotic phases of Saccharomyces cerevisiae with members of the phylum Ascomycota, revealing that the transcriptional cascade governing chromosomal recombination during meiosis commitment has experienced greater purifying selection pressure (P value = 0.0013, 0.0382, 0.0448, 0.0369, 0.02967, 0.04937, 0.03046, 0.03357 and < 0.00001 for Ashbya gossypii, Yarrowia lipolytica, Debaryomyces hansenii, Aspergillus fumigatus, Neurospora crassa, Kluyveromyces lactis, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, and Schizosaccharomyces octosporus, respectively). This study demarcates crucial players driving meiosis initiation and commitment and demonstrates their differential rate of evolution in budding yeast.

Interaction and Metabolic Function of Microbiota during the Washed Processing of Coffea arabica.

Coffee fermentation is crucial for flavor and aroma, as microorganisms degrade mucilage and produce metabolites. This study aimed to provide a basis for understanding the impact of microorganisms on Coffea arabica from Yunnan, China, during washed processing. The microbial community structure and differentially changed metabolites (DCMs) of C. arabica beans during washed processing were analyzed. The results indicated that the top five predominant microorganisms at the genera level were Achromobacter, Tatumella, Weissella, Streptococcus, and Trichocoleus for bacteria and Cystofilobasidium, Hanseniaspora, Lachancea, Wickerhamomyces, and Aspergillus for fungi. Meanwhile, the relative content of 115 DCMs in 36 h samples decreased significantly, compared to non-fermentation coffee samples (VIP > 1, p < 0.05, FC < 0.65), and the relative content of 28 DCMs increased significantly (VIP > 1, p < 0.05, FC > 1.5). Furthermore, 17 DCMs showed a strong positive correlation with microorganisms, and 5 DCMs had a strong negative correlation (p < 0.05, |r| > 0.6). Therefore, the interaction and metabolic function of microbiota play a key role in the formation of coffee flavor, and these results help in clarifying the fermentation mechanisms of C. arabica and in controlling and improving the quality of coffee flavor.

Hanseniaspora menglaensis f.a., sp. nov., a novel apiculate yeast species isolated from rotting wood.

Two apiculate strains (NYNU 181072 and NYNU 181083) of a bipolar budding yeast species were isolated from rotting wood samples collected in Xishuangbanna Tropical Rainforest in Yunnan Province, southwest PR China. On the basis of phenotypic characteristics and the results of phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA, internal transcribed spacer (ITS) region and the actin (ACT1) gene, the two strains were found to represent a single novel species of the genus Hanseniaspora, for which the name Hanseniaspora menglaensis f.a., sp. nov. (holotype CICC 33364T; MycoBank MB 847437) is proposed. In the phylogenetic tree, H. menglaensis sp. nov. showed a close relationship with Hanseniaspora lindneri, Hanseniaspora mollemarum, Hanseniaspora smithiae and Hanseniaspora valbyensis. H. menglaensis sp. nov. differed from H. lindneri, the most closely related known species, by 1.2 % substitutions in the D1/D2 domain, 2.5 % substitutions in the ITS region and 5.4 % substitutions in the ACT1 gene, respectively. Physiologically, H. menglaensis sp. nov. can also be distinguished from H. lindneri by its ability to assimilate d-gluconate.

Co-inoculations of Lachancea thermotolerans with different Hanseniaspora spp.: Acidification, aroma, biocompatibility, and effects of nutrients in wine.

The use of non-Saccharomyces yeast in the winemaking industry and even more their co-inoculations to maximize their growth and to express phenotypic characteristic is gaining more and more relevance. This study aimed to shed light on the biocompatibilities between Lachancea thermotolerans and Hanseniaspora spp., using different types of nutrients and considering the effect on Yeast Assimilable Nitrogen (YAN), at low temperature (16 °C) and medium SO2 (50 mg/L), in white must. L. thermotolerans has been used for its positive effect on pH reduction and Hanseniaspora spp. for improving the sensory profile. The behaviour of these yeasts was evaluated in co-inoculation, always finishing the fermentation with the sequential inoculation of S. cerevisiae. Significant results were obtained on the population count (CFU/mL) in CHROMagar™, with higher populations of Hanseniaspora spp. with respect to L. thermotolerans. Fermentations with L. thermotolerans/H. vineae, showed inhibition of acidification, generating up to 0.41 g/L of lactic acid. On the contrary, a synergistic effect when L. thermotolerans/H. opuntiae was used, achieved 2.44 g/L of lactic acid and a pH reduction of up to 0.16 and always more significant with Nutrient Vit BlancTM. At the same time ethanol concentration decreased by 3.4 % and volatile acidity never exceeded 0.5 g/L. Aromatic composition was analysed and it was found that all fermentations retained more aromatic esters and that on day 7 the amount of 2-phenylethyl acetate was at least 3 times higher in all fermentations compared to the control (Sc + Nutrient Vit BlancTM) which had 5.96 mg/L. Less yellow intensity (-17.3 %) typical of oxidation were observed in all fermentations in which Nutrient Vit BlancTM had been used and in the sensory analysis the co-inoculations with H. vineae generated better scores.

Xylose and yeasts: A story beyond xylitol production.

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Evaluation of the Potential Probiotic Yeast Characteristics with Anti-MRSA Abilities.

Methicillin-resistant Staphylococcus aureus (MRSA) is a disreputable pathogenic bacterium that has been proven to colonize the intestinal tract. The goal of this study is to find anti-MRSA probiotic yeast from food and evaluate its probiotic characteristics and safety. Finally, 15 strains were isolated from fruit peel with anti-MRSA ability. Using DNA sequence analysis, they were identified as the genus Hanseniaspora (7 strains) and Starmerella (8 strains). Starmerella bacillaris CC-PT4 (CGMCC No. 23573) that was isolated from the grape peel has good auto-aggregation ability and hydrophobicity, and can tolerate 0.3% bile, pH 2, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Strikingly, Starmerella bacillaris CC-PT4, like commercial probiotic Saccharomyces boulardii CNCM I-745 (Florastor ®), can adapt to the temperature of the human body (37 ℃). After safety assessment, this strain is sensitive to amphotericin B and cannot produced ß-hemolytic activities. Overall, this study provides a new candidate for probiotic yeast with anti-MRSA ability.

Debaryomyces hansenii: an old acquaintance for a fresh start in the era of the green biotechnology.

The halophilic yeast Debaryomyces hansenii has been studied for several decades, serving as eukaryotic model for understanding salt and osmotic tolerance. Nevertheless, lack of consensus among different studies is found and, sometimes, contradictory information derived from studies performed in very diverse conditions. These two factors hampered its establishment as the key biotechnological player that was called to be in the past decade. On top of that, very limited (often deficient) engineering tools are available for this yeast. Fortunately Debaryomyces is again gaining momentum and recent advances using highly instrumented lab scale bioreactors, together with advanced -omics and HT-robotics, have revealed a new set of interesting results. Those forecast a very promising future for D. hansenii in the era of the so-called green biotechnology. Moreover, novel genetic tools enabling precise gene editing on this yeast are now available. In this review, we highlight the most recent developments, which include the identification of a novel gene implicated in salt tolerance, a newly proposed survival mechanism for D. hansenii at very high salt and limiting nutrient concentrations, and its utilization as production host in biotechnological processes.

The Role of Plasma Membrane Pleiotropic Drug Resistance Transporters in the Killer Activity of Debaryomyces hansenii and Wickerhamomyces anomalus Toxins.

The killer strains of Debaryomyces hansenii and Wickerhamomyces anomalus species secrete antimicrobial proteins called killer toxins which are active against selected fungal phytopathogens. In our research, we attempted to investigate the role of plasma membrane pleiotropic drug resistance (PDR) transporters (Pdr5p and Snq2p) in the mechanism of defense against killer toxins. Saccharomyces cerevisiae mutant strains with strengthened or weakened pleiotropic drug resistance due to increased or reduced number of mentioned PDR efflux pumps were tested for killer toxin susceptibility. The present study demonstrates the influence of the Snq2p efflux pump in immunity to W.anomalus BS91 killer toxin. It was also shown that the activity of killer toxins of D. hansenii AII4b, KI2a, MI1a and CBS767 strains is regulated by other transporters than those influencing W. anomalus killer toxin activity. In turn, this might be related to the functioning of the Pdr5p transporter and a complex cross-talk between several regulatory multidrug resistance networks. To the best of our knowledge, this is the first study that reports the involvement of PDR transporters in the cell membrane of susceptible microorganisms in resistance to killer yeasts' toxins.

Recent developments in the biology and biotechnological applications of halotolerant yeasts.

Natural hypersaline environments are inhabited by an abundance of prokaryotic and eukaryotic microorganisms capable of thriving under extreme saline conditions. Yeasts represent a substantial fraction of halotolerant eukaryotic microbiomes and are frequently isolated as food contaminants and from solar salterns. During the last years, a handful of new species has been discovered in moderate saline environments, including estuarine and deep-sea waters. Although Saccharomyces cerevisiae is considered the primary osmoadaptation model system for studies of hyperosmotic stress conditions, our increasing understanding of the physiology and molecular biology of halotolerant yeasts provides new insights into their distinct metabolic traits and provides novel and innovative opportunities for genome mining of biotechnologically relevant genes. Yeast species such as Debaryomyces hansenii, Zygosaccharomyces rouxii, Hortaea werneckii and Wallemia ichthyophaga show unique properties, which make them attractive for biotechnological applications. Select halotolerant yeasts are used in food processing and contribute to aromas and taste, while certain gene clusters are used in second generation biofuel production. Finally, both pharmaceutical and chemical industries benefit from applications of halotolerant yeasts as biocatalysts. This comprehensive review summarizes the most recent findings related to the biology of industrially-important halotolerant yeasts and provides a detailed and up-to-date description of modern halotolerant yeast-based biotechnological applications.

Effects of three indigenous non-Saccharomyces yeasts and their pairwise combinations in co-fermentation with Saccharomyces cerevisiae on volatile compounds of Petit Manseng wines.

The combined use of selected Saccharomyces cerevisiae and non-Saccharomyces strains is becoming an effective way to achieve wine products with distinctive aromas. The purpose of this study was to further improve the wine aroma complexity through optimizing inoculation protocols of multi-starters. The three indigenous non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora vineae, and Lachancea thermotolerans) and their pairwise combinations (co-inoculation) were sequentially inoculated with S. cerevisiae in Petit Manseng grape must, respectively. Results evidenced a higher divergence in aroma compounds produced by two different non-Saccharomyces species compared to single species. Especially for the combination of T. delbrueckii and L. thermotolerans, the concentrations of most ethyl esters were further increased, contributing to a higher score of 'pineapple' note in agreement with sensory analysis. Our results highlighted that the inoculation of more than one non-Saccharomyces species is a potential strategy to improve the aroma diversity and quality of industrial wines.

Microbiota dynamics and volatile compounds in lupine based Moromi fermented at different salt concentrations.

Fermented soy sauces are used as food seasonings in Eastern countries and all over the world. Depending on their cultural origins, their production differs in parameters such as wheat addition, temperature, and salt concentration. The fermentation of lupine seeds presents an alternative to the use of soybeans; however, the microbiota and influencing factors are currently unknown. In this study, we analyse the microbiota of lupine Moromi (mash) fermentations for a period of six months and determine the influence of different salt concentrations on the microbiota dynamics and the volatile compound composition. Cultured microorganisms were identified by protein profiling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene amplicon sequencing provided an overview of the microbiota including non-cultured bacteria. The volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS). At all salt concentrations, we found that Tetragenococcus halophilus (up to 1.4 × 109 colony forming units (CFU)/mL on day 21) and Chromohalobacter japonicus (1.9 × 109 CFU/mL, day 28) were the dominating bacteria during Moromi fermentation. Debaryomyces hansenii (3.6 × 108 CFU/mL, day 42) and Candida guilliermondii (2.2 × 108 CFU/mL, day 2) were found to be the most prevalent yeast species. Interestingly, Zygosaccharomyces rouxii and other yeasts described as typical for soy Moromi were not found. With increasing salinity, we found lower diversity in the microbiota, the prevalence-gain of typical species was delayed, and ratios differed depending on their halo- or acid tolerance. GC-MS analysis revealed aroma-active compounds, such as pyrazines, acids, and some furanones, which were mostly different from the aroma compounds found in soy sauce. The absence of wheat may have caused a change in yeast microbiota, and the use of lupine seeds may have led to the differing aromatic composition. Salt reduction resulted in a more complex microbiome, higher cell counts, and did not show any spoiling organisms. With these findings, we show that seasoning sauce that uses lupine seeds as the sole substrate is a suitable gluten-free, soy-free and salt reduced alternative to common soy sauces with a unique flavour.

Fungus fuels mucosal wounds in Crohn's disease.

Intestinal microbiome perturbation characterizes Crohn's disease (CD), though specific contributors to pathophysiology remain elusive. In a recent issue of Science, Jain et al. show that Debaryomyces hansenii impairs intestinal healing in mice via effects on type I interferon signaling and chemokine CCL5 expression in macrophages and that it is also prevalent in the inflamed mucosa of CD patients.

Cultivable microbial ecology and aromatic profile of "mothers" for Vino cotto wine production.

The aim of the present study was to assess the cultivable microbiota of "mothers" of Vino cotto collected from production of different years 1890, 1895, 1920, 1975, 2008. A total of 73 yeasts and 81 bacteria were isolated. Starmerella lactis-condensi, Starmerella bacillaris, Hanseniaspora uvarum, Saccharomyces cerevisiae, Hanseniaspora guillermondi and Metschnikowia pulcherrima were identified. Bacteria isolates belonged to lactic acid bacteria (Lactiplantibacillus plantarum and Pediococcus pentosaceus) and acetic acid bacteria (Gluconobacter oxydans). Remarkable biodiversity was observed for Starm. bacillaris, as well as L. plantarum and G. oxydans. Organic acids and volatile compounds were also determined. Malic and succinic acids were the main ones with values ranging from 8.49 g/L to 11.76 g/L and from 4.15 g/L to 7.73 g/L respectively, while citric acid was present at low concentrations (<0.2 g/L) in all samples. Esters and higher alcohols were the main volatile compounds detected followed by alkanes. This study permits to better understand the microbial communities associated to this product and could be considered a starting point for the definition of tailored starter cultures to improve the quality of Vino cotto preserving its typical traits.

Impact of Background Fruit Odors on Attraction of Drosophila suzukii (Diptera: Drosophilidae) to Its Symbiotic Yeast.

Background odors produced by plants in the environment can interfere with the response of insects to a point-releasing attractant, especially when their compositions overlap. In this study, a series of binary choice tests was conducted in a wind tunnel to investigate whether background odors emitted from cherry, blueberry, blackberry, or raspberry fruits would affect the level of Drosophila suzukii (Matsumura) attraction to its symbiotic yeast, Hanseniaspora uvarum (Niehaus) (Saccharomycetales: Saccharomycetaceae). Whether an increase in the intensity of background odors would affect the attractiveness of H. uvarum to D. suzukii was also investigated, either by increasing the number of cherry or raspberry fruit per cup or by increasing the number of fruit cups surrounding the cup baited with the yeast. In wind tunnel assays, background fruit odors interfering with D. suzukii attraction to the yeast varied among fruit types. Raspberry odor inhibited the attractiveness of H. uvarum to the fly the most, followed by blackberry odor, whereas cherry and blueberry odors had no significant impact on the attraction. An increase in the intensity of odors by adding more cherry or raspberry fruit per cup did not increase the impact of fruit odor on the attraction; however, adding more raspberry cups around H. uvarum linearly decreased its attractiveness, suggesting that background host fruit abundance and likely increase in host odor may influence D. suzukii attraction to yeast odor depending on host species.

Genome sequencing, annotation and exploration of the SO2-tolerant non-conventional yeast Saccharomycodes ludwigii.

BACKGROUND: Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome. RESULTS: Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall ß-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome. CONCLUSIONS: This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.

[Molecular Polymorphism of ß-Galactosidase LAC4 Genes in Dairy and Natural Strains of Kluyveromyces Yeasts].

The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this disaccharide. Molecular polymorphism of LAC4 genes encoding ß-galactosidase controlling lactose fermentation is not well studied, and the published data concern only a single strain (K. lactis var. lactis NRRL Y-1140) isolated from cream in the United States. We studied ß-galactosidase genes in lactose-fermenting К lactis strains isolated from dairy products and natural sources in different regions of the world using molecular karyotyping, Southern hybridization, and sequencing. It was established that the ability to ferment lactose in К. lactis var. lactis dairy yeasts is controlled by at least three polymeric LAC loci with different chromosomal localization: LAC1 (chromosome III), LAC2 (II), and LAC3 (IV). Most of the strains we studied had the LAC2 locus. A comparative analysis of ß-galactosidases of the Kluyveromyces genus yeasts and these enzymes from other yeasts was conducted for the first time. Phylogenetic analysis detected significant differences between the LAC4 proteins of yeasts of the Kluyveromyces genus (K. lactis, К. marxianus, К. aestuarii, К. nonfermentans, К. wickerhamii), Scheffersomyces stipitis, Sugiyamaella lignohabitans, and Debaryomyces hansenii. A correlation between ß-galactosidase sequences and ecological origin (dairy products and natural sources) of Kluyveromyces strains was found. The group of dairy strains is heterogeneous and includes К. lactis var. lactis and К. marxianus yeasts (99.80-100% similarity), which indicates a common origin of their LAC4 genes. Phylogenetic analysis of ß-galactosidases indicates a close genetic relationship of dairy and hospital strains of К. lactis var. lactis and К. marxianus. Clinical isolates are able to ferment lactose and appear to originate from the dairy yeasts.