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Living organisms are frequently exposed to multiple biotic and abiotic stress forms during their lifetime. Organisms cope with stress conditions by regulating their gene expression programs. In response to different environmental stress conditions, yeast cells activate different tolerance mechanisms, many of which share common signaling pathways. Flocculation is one of the key mechanisms underlying yeast survival under unfavorable environmental conditions, and the Tup1-Cyc8 corepressor complex is a major regulator of this process. Additionally, yeast cells can utilize different mitogen-activated protein kinase (MAPK) pathways to modulate gene expression during stress conditions. Here, we show that the high osmolarity glycerol (HOG) MAPK pathway is involved in the regulation of yeast flocculation. We observed that the HOG MAPK pathway was constitutively activated in flocculating cells, and found that the interaction between phosphorylated Hog1 and the FLO genes promoter region increased significantly upon sodium chloride exposure. We found that treatment of cells with cantharidin decreased Hog1 phosphorylation, causing a sharp reduction in the expression of FLO genes and the flocculation phenotype. Similarly, deletion of HOG1 in yeast cells reduced flocculation. Altogether, our results suggest a role for HOG MAPK signaling in the regulation of FLO genes and yeast flocculation.

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Malt produced from barley contaminated with Fusarium graminearum can cause premature yeast flocculation (PYF) in beer fermentation, which ultimately affects the production efficiency and flavor quality of beer. In this study, a strain of Bacillus velezensis B-3 that was isolated from soil displayed strong antifungal activity against F. graminearum. The antifungal substances were extracted and separated by ammonium sulfate fractional precipitation and semipreparative high-performance liquid chromatography to obtain fraction VI with the strongest antifungal activity. Ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry was used to identify that the fraction contained lipopeptides such as iturin A, surfactant B, and surfactant C. The minimum inhibitory concentration of the fraction was 0.2 mg/mL, and confocal laser scanning microscopy showed that the morphology of F. graminearum hyphae was obviously abnormal, with most of them were dead. Antifungal fraction VI could inhibit the growth of F. graminearum and avoid the production of PYF factor during malting but did not affect other qualities of malt. Therefore, the development of antimicrobial agents against Fusarium provides an effective potential strategy to control the production of PYF factors for malt-manufacturing enterprises. PRACTICAL APPLICATION: Antifungal fraction VI could inhibit the growth of F. graminearum and the formation of DON during malting but did not affect other qualities of malt. The levels of soluble nitrogen, free amino nitrogen, and color experienced a substantial reduction compared to the malt infected by F. graminearum. The PYF phenomenon was significantly improved, the number of suspended yeast cells and the degree of fermentation were increased, and the level of residual extract in the wort was low, close to that of control malt.

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Microbial activity is present at every step of the malting process. It is, therefore, critical to manage the grain-associated microbial communities for the production of high-quality malts. This study characterized barley and malt epiphytic microbiota by metabarcoding the internal transcribed spacer (ITS) 2 region and the 16S rRNA gene V1-V4 metabarcodes, respectively. We elucidated the changes in the diversity and the compositional and functional changes of the grain-associated microbiota and inferred the impact of such changes on malting efficiency and premature yeast flocculation (PYF) of the commercial malt end product. Through the malting process, the fungal diversity decreased while bacterial community diversity increased. Lactic acid bacteria (LAB) and some mycotoxin-producing fungi (e.g. Fusarium spp.) were found to be significantly enriched in malts. Most potential fungal pathogens, however, did not change in abundance through the malting process. Fungi (e.g. Aureobasidium, Candida) and bacteria (e.g. LAB, Arthrobacter, Brachybacterium) with the potential to generate organic acids or exhibit high hydrolytic enzymatic activity for degrading the endosperm cell walls and storage proteins were detected in greater abundance in kilned malt, suggesting their contribution to malting efficiency. Bacterial and fungal operational taxonomic units (OTUs) associated with PYF-positive malt were mainly identified as Aureobasidium, Candida, and Leuconostoc, while Pleosporaceae, Steptococcus, and Leucobacter were associated with PYF-negative malt. The ecological networks of the field and steeped barley samples were found to be larger and denser, while that of the malt microbiome was smaller and less connected. A decrease in the proportion of negative interactions through the malting process suggested that malting destabilized the microbial networks. In summary, this study profiled the microbiota of commercial malting barley and malt samples in western Canada; the findings expanded our knowledge in the microbiology of malting while providing potential insights regarding the management of microbial-associated problems, such as PYF, in commercial malting.

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Premature yeast flocculation (PYF) is one of the pivotal problems affecting beer flavor and production. PYF is induced by certain non-starch polysaccharides produced by the degradation of malted barley husks upon the growth of contaminated microorganisms, such as Fusarium graminearum. In this research, the formation mechanism of PYF was uncovered by investigating the secretome of F. graminearum MH1 inoculated to the barley husk. The polysaccharide extract of degraded husk was ultrafiltrated into four fractions and characterized by the minimum PYF concentration, molecular mass distribution, monosaccharide composition, and zeta potential. Among the four fractions, the high-molecular-weight polysaccharide fraction had the highest content of uronic acid and the most negative zeta potential, which contributed to the most severe PYF phenomenon. In addition, the PYF yeast showed a more negative zeta potential than the control yeast during the small-scale brewing process. This is aligned to the negatively charged polysaccharides potentially bonded to the surface of yeast cells through the calcium cation in the same fermentation system, which results in rapid flocculation and precipitation. Approximately 12% of the 214 proteins identified in the Fusarium graminearum MH1 secretome were hemicellulases, which substantially interpreted the mechanism of polysaccharides inducing PYF yeast during beer brewing.

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Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.

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Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

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