Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.     

Allelism of Saccharomyces cerevisiae genes PSO6, involved in survival after 3-CPs+UVA induced damage, and ERG3, encoding the enzyme sterol C-5 desaturase.

Sequencing of the yeast gene that complemented the sensitivity to the photoactivated monofunctional 3-carbethoxypsoralen of the pso6-1 mutant strain revealed that the ERG3 locus, encoding sterol C-5 desaturase involved in biosynthesis of ergosterol, is allelic to PSO6. Disruption of the ERG3 gene yielded an erg3Delta mutant viable in ergosterol-containing YEPD media with the same pleiotropic mutant phenotype known for pso6-1 and erg3 mutants, including sensitivity to hydrogen peroxide and paraquat. Thus, the erg3/pso6 yeast mutant seems to be more sensitive than the WT to 3-CPs+UVA because of the oxidative damage contributed by this treatment and not because of an impaired repair of the furocoumarin-thymine monoadducts formed in the DNA. We found a significant increase of petites amongst erg3Delta and pso6-1 yeast mutant strains grown in conditions where respiration was mandatory. Mutant pso6-1, with its lowered content of ergosterol, exhibited enhanced synthesis of chitin that was maldistributed and not confined to the bud scars. Chitin overproduction in pso6/erg3 mutants resulted in hypersensitivity to Calcofluor White.     

Engineering of Saccharomyces cerevisiae for the production of (+)-ambrein

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.     

Mutations in the nucleotide-binding domain of putative sterol importers Aus1 and Pdr11 selectively affect utilization of exogenous sterol species in yeast

Sterol uptake in the yeast Saccharomyces cerevisiae is mediated by two plasma membrane ATP-binding cassette transporters, Aus1 and Pdr11. Their expression is regulated by oxygen and is triggered by anaerobic growth conditions. Under these conditions, internal ergosterol synthesis is arrested and utilization of exogenous sterol is vital for yeast cells. Here, we demonstrate that Aus1 is the major importer of non–yeast sterols, mammalian cholesterol, and plant sterols under anaerobic conditions. In contrast, uptake of yeast native sterol, ergosterol, is relatively low. This uptake could not be enhanced by overexpression of either of the transporters. Interestingly, overexpression of the minor importer Pdr11 resulted in a substantial import of non–yeast sterols. We show that mutation of the conserved residue in one of the ABC characteristic motifs—the H-loop in Aus1 and Pdr11—lowered their ATPase activity. The residual activity was sufficient to import exogenous sterols and to preserve cell viability. Importantly, the reduction of sterol import was dramatic for mammalian cholesterol and plant sterols, whereas import of yeast ergosterol was decreased only slightly indicating substrate selectivity of the sterol utilization process.     

Direct Evidence That Maltose Transport Activity Is Affected by the Lipid Composition of Brewer's Yeast

A brewer's yeast strain was grown with maltose as sole carbon source under strictly anaerobic conditions with and without ergosterol and/or unsaturated fatty acid (Tween 80) supplements. Under all these conditions the MALx1 genes for maltose transporters were strongly expressed during growth. The fatty acid unsaturation indices of growing and stationary phase yeast were increased from about 20% to 56–69% by supplementation with Tween 80. Ergosterol contents were increased up to at least 4‐fold by supplementation with ergosterol and Tween 80. Maltose transport activity measured at 20°C was not increased by supplementation with Tween 80 alone, but was increased 2‐fold and 3‐fold, respectively, in growing and stationary phase yeast by supplementation with ergosterol together with Tween 80. The stimulation of maltose transport by ergosterol was greater when the transport was measured at temperatures (10°C and 0°C) lower than 20°C. The results show that proper function of maltose transporters requires adequate amounts of ergosterol in the yeast. This effect may partly explain the low maltose (and maltotriose) uptake rates both in the second half of brewery fermentations, when the sterol content of yeast has fallen, and when fresh wort is pitched with sterol‐deficient cropped yeast.     

产麦角固醇酵母的选育及其发酵条件的研究

从酵母中提取麦角固醇并经紫外线光化作用生产维生素D_2是当前获得维生素D_2的主要来源之一。本文介绍了高产麦角固醇酵母菌的选育。从数十株酿酒酵母、贝氏酵母、葡萄汁酵母和意大利酵母等菌株中筛选出优良的产麦角固醇的酵母,麦角固醇的产率最高可达4.0％以上。试验了发酵培养基中氮源,不同培养时间以及酵母自溶等条件对麦角固醇形成的影响。结果表明,当培养基中含氮量增加时,酵母细胞内竞角固醇的积累减少。当培养时间达16小时,酵母细胞内麦角固醇量的积累达高峰。鲜酵母经50～55℃在水溶液中自溶12小时,麦角固醇量可增加20％左右。     

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol-binding proteins in Saccharomyces cerevisiae

Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional—as yet unidentified—sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.     

麦角甾醇的研究进展

麦角甾醇主要存在于酵母菌、霉菌等真菌和某些植物中,是一种重要的植物甾醇,具有重要的生理作用,是食品、饲料及医药等工业中常用的一种原料。文章总结近十年来国内外对麦角甾醇的研究状况,分别对麦角甾醇的生物合成途径、高产麦角甾醇菌株的选育和麦角甾醇的萃取方法予以综述,为进一步研究麦角甾醇提供参考。     

Involvement of yeast YOL151W/GRE2 in ergosterol metabolism

The Saccharomyces cerevisiae gene YOL151W/GRE2 is widely used as a model gene in studies on yeast regulatory responses to osmotic and oxidative stress. Nevertheless, information concerning the physiological role of this enzyme, a distant homologue of mammalian 3-beta-hydroxysteroid dehydrogenases, is scarce. Combining quantitative phenotypic profiling and protein expression analysis studies, we here report the involvement of yeast Gre2p in ergosterol metabolism. Growth was significantly and exclusively reduced in gre2Delta strains subjected to environmental stress straining the cell membrane. Furthermore, whereas no compensatory mechanisms were activated due to loss of Gre2p during growth in favourable conditions (synthetic defined media, no stress), a striking and highly specific induction of the ergosterol biosynthesis pathway, represented by the enzymes Erg10p, Erg19p and Erg6p, was observed in gre2Delta during growth in a stress condition in which lack of Gre2p significantly affects growth. Involvement of Gre2p in ergosterol metabolism was confirmed by application of an array of selective inhibitors of lipid biosynthesis, as gre2Delta displayed vastly impaired tolerance exclusively to agents targeting the ergosterol biosynthesis. The approach outlined here, combining broad-spectrum phenotypic profiling, expression analysis during conditions reducing the growth of the mutant and functional confirmation by application of highly selective inhibitors, may prove a valuable tool in gene functional analysis.     

STEROL BIOSYNTHESIS BY STRAINS OF SACCHAROMYCES CEREVISIAE IN THE PRESENCE AND ABSENCE OF DISSOLVED OXYGEN

The content of sterols in Saccharomyces cerevisiae which has been harvested after anaerobic growth and then added to a complex nutrient medium, rises rapidly from ca. 1 mg/g dry yeast to ca. 10 mg in the presence of dissolved oxygen. A range of sterols, present principally as sterol esters, is formed during this period. The concentration of free sterols does not rise above 3 mg/g and esters are thought to form a reserve sterol pool. Cyclization of squalene to lanosterol in the presence of oxygen seems not to be markedly affected by oxygen concentration in contrast to demethylation and desaturation reactions on the pathway to ergosterol. When oxygen concentration falls to zero, further metabolism of preformed sterols continues, with the accumulation of episterol and ergosterol and reduction in the concentration of zymosterol and 24(28)-dehydroergosterol. During anaerobic growth a marked hydrolysis of sterol esters occurs and free sterols eventually predominate.     

EFFECT OF LIPIDS AND OXYGEN ON YEAST GROWTH AND THE BIOSYNTHESIS OF ACETOIN DURING FERMENTATION

Brewery yeast needs traces of oxygen for the biosynthesis of unsaturated fatty acids and ergosterol. Owing to the increase in cell mass during primary fermentation the concentrations of these essential lipids decrease and thereby affect the physiological condition of the yeast. When the sterol concentration of whole cells has decreased to 0.2 to 0.3 mg per 100 mg dry yeast, the yeast changes its metabolism. This metabolic change is revealed by a decrease in acetoin concentration. The absorption of wort nutrients and consequently the efficiency of growth is at this point also greatly reduced. The ratio between yeast growth and mole ethanol formed (i.e. the molar growth yield) decreases greatly during wort fermentation. A close correlation between molar growth yield and the change in acetoin metabolism can be observed. This metabolic change occurs when the ratio between yeast growth and ethanol formed is in the range of 8.3 to 9.1, averaging 8.7 g/mole.     

Quality control method based on quartz crystal microbalance and WGA for flour milled from germinated wheat

Plasma membrane is the initial sensor of different stress conditions and its composition is modified with response to environmental changes. In the present study, we have modified the lipid composition of the membrane by growing Saccharomyces cerevisiae in the presence of different fatty acids and ergosterol. All supplemented fatty acids were incorporated into the cell and this incorporation produced significant changes in the lipid composition. The incubation with ergosterol also modified the lipid composition of the cells; however, these cells presented a strong reduction in the content of this sterol. The different cellular lipid composition has been related to viability and fermentation performance at low temperature (13 °C). The cells incubated with palmitoleic acid (C16:1) showed higher viability and significant reduction in the fermentation length. These cells presented higher C16:1 and ergosterol content, shorter chain length of the fatty acids and higher ratio of sterols/phospholipids. Therefore redesigning the composition of cellular membranes during industrial yeast propagation seems to be a promising strategy for improving fermentation performance in the winery.     

FEN2: A gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast

We have isolated and characterized a pleiotropic recessive mutation, fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5·5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.     

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.     

浓香型大曲中功能性酵母菌的筛选、鉴定及发酵特性研究

以浓香型大曲为研究对象,采用稀释涂布平板法从中分离酵母菌,对其形态特征进行观察;采用2,3,5-氯化三苯基四氮唑（TTC）显色法初筛,通过发酵力、产酒能力、耐高温、耐酒精能力实验进行复筛,筛选一株优良的功能性酵母菌,利用Biolog微生物自动分析系统对其进行鉴定,并利用顶空固相微萃取气质联用（HS-SPME-GC-MS）法对其液态发酵产物进行分析。结果表明,从浓香型大曲中共分离出9株酵母菌,其中菌株NDY-06性能最突出,可耐受42 ℃的高温,16%vol的酒精度,且具有一定的发酵产酒能力;该菌株被鉴定为产香酵母Zygosaccharomyces cidri,从其液态发酵产物中共检测到35种挥发性风味物质,包括醇类物质7种,酯类物质13种,酸类物质7种,吡嗪类物质3种,对白酒香气成分有一定的贡献。     

五株酿酒醇母发酵特性比较

通过对安琪牌葡萄活性干酵母及菌株B、C、D的基本性能测试，得出安琪牌葡萄酒活性干酵母不仅应用方便，而且其耐低温、高酒精、高糖含量、高酸度的特性，使其应用更广泛。     

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.