SciCombinator

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Concept: Saccharomycetes

28

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions.

Concepts: Yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Yeasts, Saccharomycetes, Saccharomycetaceae, Sauvignon blanc

28

In order to better understand the differences in xylose metabolism between natural xylose-utilizing Pichia stipitis and metabolically engineered Saccharomyces cerevisiae, we constructed a series of recombinant S. cerevisiae strains with different xylose reductase/xylitol dehydrogenase/xylulokinase activity ratios by integrating xylitol dehydrogenase gene (XYL2) into the chromosome with variable copies and heterogeneously expressing xylose reductase gene (XYL1) and endogenous xylulokinase gene (XKS1). The strain with the highest specific xylose uptake rate and ethanol productivity on pure xylose fermentation was selected to compare to P. stipitis under oxygen-limited condition. Physiological and enzymatic comparison showed that they have different patterns of xylose metabolism and NADPH generation.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Brewing, Beer, Yeasts, Saccharomycetes, Saccharomycetaceae

27

Atg17, in complex with Atg29 and Atg31, constitutes a key module of the Atg1 kinase signaling complex and functions as an important organizer of the phagophore assembly site in the yeast Saccharomyces cerevisiae. We have determined the three-dimensional reconstruction of the full S. cerevisiae Atg17-Atg31-Atg29 complex by single-particle electron microscopy. Our structure shows that Atg17-Atg31-Atg29 is dimeric and adopts a relatively rigid and extended “S-shape” architecture with an end-to-end distance of approximately 345 Å. Subunit mapping analysis indicated that Atg17 mediates dimerization and generates a central rod-like scaffold, while Atg31 and Atg29 form two globular domains that are tethered to the concave sides of the scaffold at the terminal regions. Finally, our observation that Atg17 adopts multiple conformations in the absence of Atg31 and Atg29 suggests that the two smaller components play key roles in defining and maintaining the distinct curvature of the ternary complex.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Saccharomyces, Saccharomycetes, Saccharomycetaceae

27

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Ascomycetes in either the Saccharomycotina or the Taphrinomycotina have one to three GGTs, whereas members of the Pezizomycotina have two to four GGTs. A Bayesian analysis indicates there are three well-supported main clades of GGTs in the Ascomycota. 1) A Saccharomycotina and a Taphrinomycotina-specific GGT sub-clade form a yeast main clade. This clade has the three relatively well-characterized fungal GGTs: (Saccharomyces cerevisiae CIS2 and Schizosaccharomyces pombe Ggt1 and Ggt2) and most of its members have all 14 of the highly conserved and critical amino acids that are found in GGTs in the other kingdoms. 2) In contrast, a main clade (GGT3) differs in 11 of the 14 highly conserved amino acids that are found in GGTs in the other kingdoms. All of the 44 Pezizomycotina analyzed have either one or two GGT3s. 3) There is a Pezizomycotina-only GGT clade that has two well-supported sub-clades (GGT1 and GGT2); this clade differs in only two of the 14 highly conserved amino acids found in GGTs in the other kingdoms. Because the Pezizomycotina GGTs differ in apparently critical amino acids from the cross-kingdom consensus, a putative GGT from Colletotrichum graminicola, a member of the Pezizomycotina, was cloned and the protein product was expressed as a secreted protein in Pichia pastoris. A GGT enzyme assay of the P. pastoris supernatant showed that the recombinant protein was active, thereby demonstrating that CgGGT1 is a bona fide GGT.

Concepts: Amino acid, Fungus, Yeast, Saccharomyces cerevisiae, Ascomycota, Yeasts, Saccharomycetes, Saccharomycotina

27

The ability of Candida shehatae, Saccharomyces cerevisiae, or the combination of these two yeasts in converting the mixed sugar composition of rice hull hydrolysate (RHH) as substrate for ethanol production is presented. In shake flask experiments, co-cultures showed ethanol yields (Y) of 0.42 and 0.51 in synthetic medium simulating the sugar composition of RHH and in RHH, respectively, with both glucose and xylose being completely depleted, while pure cultures of C. shehatae produced slightly lower ethanol yields (0.40). Experiments were scaled-up to bioreactors, in which anaerobiosis and oxygen limitation conditions were tested. Bioreactor co-cultures produced similar ethanol yields in both conditions (0.50-0.51) in synthetic medium, while in RHH, yields of 0.48 and 0.44 were obtained, respectively. The results showed near-theoretical yields of ethanol. Results suggest the feasibility of co-cultures of C. shehatae, a newly isolated strain, and S. cerevisiae in RHH as substrate for second-generation ethanol production.

Concepts: Fungus, Yeast, Model organism, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Yeasts, Saccharomycetes

27

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.

Concepts: Enzyme, Fungus, Yeast, Saccharomyces cerevisiae, Brewing, Beer, Saccharomycetes, Saccharomycetaceae

25

Due to increased occurrence of infections and food spoilage caused by yeast, there is an unmet need for new antifungal agents. The arginine-β-(2,5,7-tri-tert-butylindol-3-yl)alanine-arginine (R-Tbt-R) motif was previously proved useful in the design of an antifungal tripeptide. Here, an array of peptidomimetics based on this motif was investigated for antifungal and hemolytic activity. The five most promising modified tetrapeptide analogues ( 6: and 9-12: contain an additional C-terminal hydrophobic residue, and these were found to exhibit antifungal activity against Saccharomyces cerevisiae (MIC 6 and 12 μg mL(-1)) and Zygosaccharomyces bailii (MIC 6-25 μg mL(-1)). Four compounds ( 6: and 9-11: , had limited hemolytic activity (<10% hemolysis at 8 × MIC). Determination of their killing kinetics revealed that compound 9: displayed fungicidal effect. Testing against cells from an S. cerevisiae deletion mutant library indicated that interaction with yeast-specific fungal sphingolipids, most likely constitutes a crucial step in the mode of action of these. Interestingly, a lack of activity of peptidomimetics 6: and 9-11: towards Candida spp. was shown to be due to degradation or sequestering by the yeast. Due to their ultrashort nature, antifungal activity and low toxicity the four compounds may have potential as leads for novel preservatives.

Concepts: Fungus, Yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Brewing, Yeasts, Saccharomycetes, Saccharomycetaceae

8

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species, Yarrowia histolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisae. The subunit compositions of the purified enzyme complexes depended upon the detergent used to solubilise and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

Concepts: Adenosine triphosphate, Enzyme, Fungus, Yeast, Saccharomyces cerevisiae, Saccharomyces, ATP synthase, Saccharomycetes

2

G protein-coupled receptors (GPCRs) must discriminate between hundreds of related signal molecules. In order to better understand how GPCR specificity can arise from a common promiscuous ancestor, we used laboratory evolution to invert the specificity of the Saccharomyces cerevisiae mating receptor Ste2. This GPCR normally responds weakly to the pheromone of the related species Kluyveromyces lactis, though we previously showed that mutation N216S is sufficient to make this receptor promiscuous. Here, we found that three additional substitutions, A265T, Y266F and P290Q, can act together to confer a novel specificity for K. lactis pheromone. Unlike wild-type Ste2, this new variant does not rely on differences in binding affinity to discriminate against its non-preferred ligand. Instead, the mutation P290Q is critical for suppressing the efficacy of the native pheromone. These two alternative methods of ligand discrimination were mapped to specific amino acid positions on the peptide pheromones. Our work demonstrates that changes in ligand efficacy can drive changes in GPCR specificity, thus obviating the need for extensive binding pocket re-modeling.

Concepts: Protein, Amine, Signal transduction, Fungus, G protein-coupled receptor, Metabotropic glutamate receptor, Saccharomycetes, Saccharomycetaceae

2

Many species of fungi have been detected in the healthy human gut; however, nearly half of all taxa reported have only been found in one sample or one study. Fungi capable of growing in and colonizing the gut are limited to a small number of species, mostly Candida yeasts and yeasts in the family Dipodascaceae (Galactomyces, Geotrichum, Saprochaete). Malassezia and the filamentous fungus Cladosporium are potential colonizers; more work is needed to clarify their role. Other commonly-detected fungi come from the diet or environment but either cannot or do not colonize (Penicillium and Debaryomyces species, which are common on fermented foods but cannot grow at human body temperature), while still others have dietary or environmental sources (Saccharomyces cerevisiae, a fermentation agent and sometime probiotic; Aspergillus species, ubiquitous molds) yet are likely to impact gut ecology. The gut mycobiome appears less stable than the bacterial microbiome, and is likely subject to environmental factors.

Concepts: Gut flora, Fungus, Yeast, Saccharomyces cerevisiae, Brewing, Ascomycota, Yeasts, Saccharomycetes