Fatty acid-containing lipids of the yeast Saccharomyces cerevisiae during post-fermentation decline in viability

On exhaustion of the sugar supply in the medium, cells of the yeast Saccharomyces cerevisiae maintained at 30°C lost most of their phospholipid content and their viability, as assessed by methylene blue staining, within a few days. Both processes occurred more rapidly in cells which had fermented glucose rather than maltose. During this period of decline the cell content of other fatty acid esters, mainly t riacylglycerols, increased. This seemed greater in cells grown with maltose than with glucose but the latter cells also synthesised distinct amounts of waxes. The total quantity of lipid per cell did not change significantly during loss of viability although the composition of the lipid fraction did.     

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright © 1999 John Wiley & Sons, Ltd.     

Incorporation of unsaturated fatty acids by Saccharomyces cerevisiae: conservation of fatty-acyl saturation in phosphatidylinositol

Saccharomyces cerevisiae was grown anaerobically in media supplemented with myristoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), gamma-linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid. Cells from exponential-phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids. The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid. Analysis of the fatty-acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation. The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is discussed.     

A family of laboratory strains of Saccharomyces cerevisiae carry rearrangements involving chromosomes I and III

In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.     

Acid Washing and Serial Repitching a Brewing Ale Strain of Saccharomyces cerevisiae in High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.     

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale‐down cultivations

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large‐scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production‐scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two‐ and three‐compartment scale‐down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab‐scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale‐down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three‐compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single‐cell size distribution was observed under oscillatory growth conditions with three‐dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single‐cell‐based analysis provides useful information about the homogeneity of responses among the population.     

Effect of sterol alterations on conjugation in Saccharomyces cerevisiae.

Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols. The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols. After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.     

烟台贵人香葡萄自然发酵醪中本土酿酒酵母的选育及酿造特性研究

自葡萄原料生产地选育优良酿造特性的本土酵母是开发地方特色葡萄酒品种的重要措施。本研究以烟台地区贵人香葡萄自然发酵醪为材料,筛选酿酒酵母并对其发酵特点和耐受能力进行测定,进而选育本土酿酒酵母用于霞多丽干白和赤霞珠干红葡萄酒的酿造试验。采用孟加拉红选择性培养基筛选本土酵母菌,并经WL鉴定培养基和5.8S ITS序列鉴定获得12株酿酒酵母。通过测定发酵后残糖量和酒精度及耐受能力(酒精度、SO2、酸、高糖),选育4株酵母用于酿造霞多丽干白葡萄酒,对酒样指标、主要挥发物和香气组分分析,显示菌株YGF2、YGF5和YGF10的酿造特性和发酵指标较好,并用于酿造赤霞珠干红葡萄酒。结果表明菌株YGF2酿造的葡萄酒质量较好,香气浓郁,部分指标优于商品酿酒酵母,具备生产区域特色葡萄酒的潜力。     

脂肪酸对酿酒酵母酒精耐性的影响

研究几种不同脂肪酸对酿酒酵母在存活率、生长速率和发酵方面的酒精耐性的影响。结果表明：培养基中添加两种不饱和脂肪酸（棕榈油酸和油酸）能显著增加酵母菌在酒精冲击下的存活率,其中棕榈油酸的效应更强。同时这些存活的酵母菌在含酒精培养基上的生长速率也比普通酵母菌更快,而两种饱和脂肪酸（棕榈酸和硬脂酸）在提高酵母菌存活率和生长速率方面几乎无贡献。同时在添加相同浓度不饱和脂肪酸的条件下,培养至稳定期的酵母菌比对数期酵母菌具有更高的存活率和更好的生长速率。但在发酵方面,添加短链脂肪酸（棕榈油酸和棕榈酸）能够使酵母菌发酵达到较高的酒精体积分数,这个结果与酵母菌生长耐酒精性的结果不一致,表明酵母菌生长和发酵的酒精耐性机制是不同的。     

Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae

The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences: Y', pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum.     

Induction of a heat-shock-type response in Saccharomyces cerevisiae following glucose limitation

The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat-shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells. The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at various stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.     

Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells,but not the activity of glucose transport

Incubation of starved galactose-grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period. The V_max for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s. After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.     

转录组测序分析3 种不同链长脂肪酸对酿酒酵母基因转录水平的影响

将不同链长的脂肪酸己酸（C6）、十二烷酸（C12）及十六烷酸（C16）添加到对数期酿酒酵母中,通过转录组测序分析其对酿酒酵母基因转录水平的影响。结果表明,在不同链长脂肪酸存在条件下,酿酒酵母基因的转录数量、基因转录水平均有所差异,其中短链脂肪酸C6对酵母基因转录水平的影响最为显著,C16对酿酒酵母的影响最小。此外,通过转录组测序数据分析对不同脂肪酸存在时的转录因子及转运蛋白进行预测,其中包括17 条转录因子及40 条转运蛋白基因。最后分析不同链长脂肪酸存在对脂肪酸合成途径的关键酶基因转录水平的影响,为进一步分析酿酒酵母中脂肪酸生物合成过程中的基因表达及调控机制提供支持。     

Cloning and sequencing of the Saccharomyces cerevisiae gene LYP1 coding for a lysine-specific permease

The LYP1 gene of Saccharomyces cerevisiae was cloned by complementation in lysine-permease-deficint recipient yeast cells, and its nucleotide sequence was determined. An open reading frame of 1833 nucleotides was found encoding a polypeptide of 611 amino acids, with a calculated molecular weight of 68 118. Analysis of the deduced primary structure of the protein revealed ten membrane-spanning regions and three potential N-glycosylation sites. Analysis of the deduced sequence of protein LYP1 indicates homology with other yeast amino-acid permeases, in particular with CAN1, and also the lysine-specific permease of Escherichia coli. The strain transformed by a multi-copy plasmid harbouring the LYP1 gene, showed a 20-fold increase in the maximum velocity of lysine uptake over that in the wild type, with no changes in the affinity of the permease for its substrate.     

Effect of Intramuscular Fat,Breed, and Age at Slaughter on Fatty Acid Composition in Green Hams

The evolution of fatty acid classes (ΣSFA, ΣMUFA, and ΣPUFA) in neutral lipids (NL) and phospholipids (PL) in pigs were studied. The M. biceps femoris in green hams from Norwegian breeds Landrace (LR), Duroc (DU), and Hampshire (HS) slaughtered at 6, 7.5, and 9 mo of age were examined. The parameters were analyzed for age and breed effects with and without adjustment for NL or PL content. The fatty acid class contents of NL were not significantly different between the age groups. However, when NL content was adjusted, deposition of ΣSFA and ΣMUFA in NL significantly increased and deposition of ΣPUFA in NL significantly decreased with age. Duroc had consistently higher fatty acid class contents, but after adjusting for NL content, only the deposition of ΣSFA proved to be genetically different. The nonlinear relationship between NL content and ΣMUFA and ΣPUFA, together with the genetic potential of HS to deposit less ΣSFA than LR, gave significantly different fatty acid class compositions between the breeds. The content and proportion of ΣMUFA in PL increased with age. Specific ratios were also determined. The fat firmness index, C18:0/C18:2n ? 6 in NL, increased with age and was lowest in HS due to genetics and highest in DU due to its high NL content. The sensory rancidity index C18:1n ? 9/Σn ? 6 increased in NL and decreased in PL between 7.5 and 9 mo. The C18:1n ? 9/Σn ? 6 in NL was highest in DU due to higher NL content. The Σn ? 6/Σn ? 3 in NL and PL increased between 7.5 and 9 mo.     

A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF.

In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S. servazzii. We find that the order of four genes surrounding the RE in S. cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii. However, there is an additional ORF in S. servazzii between PRD1 and KAR4 that is not homologous to any gene in S. cerevisiae or to genes in other organisms. Despite a 75-79% amino acid identity for KAR4 and SPB1, respectively, the S. servazzii sequence did not carry a well-conserved RE sequence and these sequences lacked RE function when introduced into S. cerevisiae. The S. servazzii region contains a sequence that supports autonomous DNA replication in S. cerevisiae and may represent a homologue of ARS304. The S. servazziii sequence has Genbank Accession No. BankIt359091 AF307954.     

The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity

New double‐ and triple‐hybrid Saccharomyces yeasts were characterized using PCR‐restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double‐hybrid S. cerevisiae × S. kudriavzevii in non‐fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in these hybrids revealed a high diversity of genome types. From these molecular characterizations, a reduction of the S. kudriavzevii fraction of the hybrid genomes is observed in most hybrids. Mitochondrial inheritance in hybrids was deduced from the analysis of mitochondrial COX2 gene sequences, which showed that most hybrids received the mitochondrial genome from the S. kudriavzevii parent. However, two strains inherited a S. cerevisiae COX2, being the first report of S. cerevisiae × S. kudriavzevii hybrids with S. cerevisiae mitochondrial genomes. These two strains are those showing a higher S. kudriavzevii nuclear genome reduction, especially in the wine hybrid AMH. This may be due to the release of selective pressures acting on the other hybrids to maintain kudriavzevii mitochondria‐interacting genes. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro characterization of the Mig1 repressor from Saccharomyces cerevisiae reveals evidence for monomeric and higher molecular weight forms

The Mig1 DNA-binding protein of Saccharomyces cerevisiae was expressed and purified from yeast and the physical properties were characterized by several methods, including gel filtration, sucrose gradient sedimentation and native gel electrophoresis. Purified Mig1 exists as a monomer with a Stokes' radius of 48 A and a sedimentation coefficient of 3.55 S. Mig1 has an elongated shape with a frictional coefficient of 1.83. The K(d) of purified Mig1 for the SUC2 A site is 2.8 nM and for SUC2 B site 25.8 nM; these values were similar for Mig1 purified from repressed and derepressed cells. Full-length Mig1 expressed in yeast binds more tightly to SUC2 B than bacterially expressed GST-Mig1. Sucrose gradient sedimentation resolved a larger molecular weight form of Mig1 in whole-cell extracts that was not seen in purified samples and may represent a complex with another protein. This complex is found within the nucleus and is seen only in repressed cells. Mig1 exists in multiple phosphorylation states and only less phosphorylated forms of Mig1 are associated with this complex.