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.     

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.     

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.     

The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol Δ⁵ desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13·7 centimorgans from GAL2 toward SPT8. The essentially of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without ‘sparking’ levels of Δ⁵ sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.     

Simultaneous determination of free ergosterol and ergosteryl esters in Cordyceps sinensis by HPLC

The caterpillar-shaped Chinese medicinal mushroom Cordyceps sinensis consists of the fruiting body (CsA) and the host caterpillar (CsB). Ergosterol is a principal sterol in fungi and can indicate the level of mycelia of C. sinensis. Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, which have different physiological functions. The relative abundances of free to esterified ergosterol are different among the various species. In the present study, a gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of free and esterified ergosterol in CsA and CsB of C. sinensis. The results showed that CsA and CsB had similar ergosterol compositions, but the level of ergosteryl esters in CsB was much higher than that in CsA, indicating that CsA and CsB might be in different growth phase or have different physiological functions for the growth and multiplication of C. sinensis.     

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。     

Evaluation of a fluorodensitometric method for analysis of ergosterol as a fungal marker in compound feeds.

Ergosterol is the principal sterol of fungi and plays an essential role as a component of the cell membrane and other cell constituents. This molecule is considered a good marker of fungal contamination in foods and feeds. This paper reports a rapid and sensitive method to test ergosterol content in compound feeds based on fluorodensitometry after thin-layer chromatography (TLC) separation. This method involves a thermal treatment of TLC plates that leads to the formation of a highly fluorescent ergosterol derivative. Such a dosage allows ergosterol testing in any naturally contaminated samples (limit of detection: 1 ppm of ergosterol) and gives results in close agreement with high-pressure liquid chromatography determination. Moreover, values obtained on mixed feeds for animals at different steps of fungal contamination are linked to quantitative development of storage fungi, evaluated by mycological technique, reinforcing the interest of a rapid method for measuring this fungal marker.     

Determination of ergosterol as a measure of fungal growth using Si 60 HPLC

In order to determine to fungal growth of Fusarium graminearum 480, a method was developed for the extraction and estimation of ergosterol, a sterol specific for fungi. This method includes the direct saponification of bound ergosterol to fungal mycelia followed by n-hexane extraction and quantification using. High performance liquid chromatography (HPLC) with UV-detection. This procedure proved to be superior compared with other methods, since the yield of ergosterol yields was higher (up to 40%). n-Hexane extracts contained minor impurities which interfered with the UV-detection and the retention time of the compound was halved using Si 60 HPLC. The protein and ergosterol contents in F. graminearum cultures increased proportionally over a 3-week incubation period. The fungal formation of the mycotoxin zearalenone started at a level of 50 mg/kg ergosterol and increased rapidly in the stationary phase of growth, which was characterized by decreasing rates of ergosterol formation.     

Chemical constituents of some basidiomycetes

The sterol, fatty acid and free amino acid (FAA) contents of some Basidiomycetes were determined. The overall sterol distribution was similar to that of other mushrooms, with ergosterol as the predominant sterol accompanied by the other closely related sterols. The mushrooms examined also contained high levels of fatty acids of which linoleic is the most prominent. Glutamic acid, valine and proline were the dominant FAA.     

Correlation of total ergosterol levels in stored canola with fungal deterioration

Deterioration of canola caused by fungal growth during storage is a problem at moisture contents exceeding 8%. Secondary indicators of fungal growth, such as ergosterol, the main sterol found in fungal cell membranes, offer a way of relating fungal biomass to deterioration. The objective of this research was to determine ergosterol accumulation in stored canola and to correlate it with seed deterioration measured by germination, fungal infection, fat acidity values (FAV), and carbon dioxide production. Total ergosterol levels increased with storage time, temperature, and seed moisture content. Aggressive and destructive fungal species such as Aspergillus candidus and Penicillium spp. contributed more to ergosterol than Eurotium spp. Initial total ergosterol concentrations between 1.46 and 1.67 ppm may be taken as the background levels in sound canola, and levels greater than 2 ppm were related to significant levels of spoilage. Results showed that germination and FAV had a strong correlation with total ergosterol (Spearman rank order correlation coefficients of −0.826 and 0.800, respectively) but the relationship was weaker for CO₂ production (Spearman rank order correlation coefficient of 0.650).     

Determination of ergosterol in paddy rice using solid phase extraction

Ergosterol, the predominant sterol found in most fungi, is considered as an indicator of fungal invasion in grain. An extraction method based on solid phase extraction (SPE) has been developed for determination of ergosterol in unmilled rice, generally called paddy. The samples obtained by extraction through SPE have been analysed by HPLC. The method has been validated in experiments with paddy subjected to different drying treatments and recoveries above 85% have been obtained. SPE appeared to be simple, quick, reliable and consistent for ergosterol analysis. The ergosterol levels obtained in the paddy samples were found to be within the expected range and were comparable with those found using conventional methods. Copyright © 2004 Society of Chemical Industry     

Mutations sensitizing yeast cells to the start inhibitor nalidixic acid

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected. These aro7 alleles demonstrate ‘recruitment’, by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.     

Effects of genetic variability,parts and seasons on the sterol content and composition in bamboo shoots

Bamboo shoots are regarded as potential sources of sterols. The effects of genetic variability, parts and harvest seasons on the sterol content and composition in the bamboo shoots have been determined using a novel ultra-performance liquid chromatographic atmospheric pressure chemical ionisation mass spectrometer method. The results showed that the representative sterols in bamboo shoots were β-sitosterol, campesterol, stigmasterol, ergosterol, cholesterol and stigmastanol; exception stigmastanol, the significant differences were observed in the sterol content of different species (112.4–279.6 mg/100 g dry wt), different harvest seasons (195.3–279.6 mg/100 g dry wt) and different parts (253.6–321.8 mg/100 g dry wt); the sterol composition was similar in different species and different harvest seasons, however, it was significantly different between shoot bodies and shoot shell. The genetic variability, parts and harvest seasons could significantly affect the sterol composition in the bamboo shoots. The spring shoot shell of Phyllostachys pubescens contained the highest sterol content (321.8 mg/100 g dry wt).     

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.     

Development of a Novel Methodology for the Analysis of Ergosterol in Mushrooms

Sterols are important molecules in the unsaponifiable fraction of several matrices. Ergosterol, which is an important vitamin D₂ precursor, is clearly the main sterol in mushrooms. Herein, an analytical method for ergosterol determination in cultivated and wild mushrooms was developed using high-performance liquid chromatography coupled to ultraviolet detection. The chromatographic separation was achieved in an Inertsil 100A ODS-3 reversed-phase column using an isocratic elution with acetonitrile/methanol (70:30, v/v) at a flow rate of 1 mL/min. Different extraction methodologies were tested, using n-hexane, methanol/dichloromethane (75:25, v/v) or chloroform/methanol (20:10, v/v). The method was optimised using Fistulina hepatica and proved to be reproductive and accurate. Ergosterol was the most abundant sterol by a greater extent in all mushrooms. In general, the cultivated species showed higher contents, mainly Agaricus bisporus and Lentinus edodes. Among wild species, Boletus edulis was the mushroom with the highest content in ergosterol. Results were expressed in fat content, dry weight and fresh weight bases. The assessment of ergosterol amounts might be very useful due to the bioactive potential that has been attributed to this molecule and its derivatives.     

The wide-ranging phenotypes of ergosterol biosynthesis mutants,and implications for microbial cell factories

Yeast strains have been used extensively as robust microbial cell factories for the production of bulk and fine chemicals, including biofuels (bioethanol), complex pharmaceuticals (antimalarial drug artemisinin and opioid pain killers), flavours, and fragrances (vanillin, nootkatone, and resveratrol). In many cases, it is of benefit to suppress or modify ergosterol biosynthesis during strain engineering, for example, to increase thermotolerance or to increase metabolic flux through an alternate pathway. However, the impact of modifying ergosterol biosynthesis on engineered strains is discussed sparsely in literature, and little attention has been paid to the implications of these modifications on the general health and well-being of yeast. Importantly, yeast with modified sterol content exhibit a wide range of phenotypes, including altered organization and dynamics of plasma membrane, altered susceptibility to chemical treatment, increased tolerance to high temperatures, and reduced tolerance to other stresses such as high ethanol, salt, and solute concentrations. Here, we review the wide-ranging phenotypes of viable Saccharomyces cerevisiae strains with altered sterol content and discuss the implications of these for yeast as microbial cell factories.     

SOME ASPECTS OF THE STRUCTURE AND FUNCTION OF THE YEAST PLASMA MEMBRANE

The permeation of many compounds into the yeast cell is largely regulated by the plasma membrane according to their lipid solubility and degree of dissociation. Thus the permeation rate of the fatty acids and α-keto acids is proportional to their relative lipid solubility. The highly dissociated and poorly lipid-soluble di- and tri-carboxylic acids and halogenated acetic acids permeate only very slowly; the impermeable α-ketoglutaric acid becomes easily permeable when made lipid-soluble by esterification. The lipid composition of the plasma membrane can thus be of decisive importance in regulating the movement of different compounds into and out of the yeast cell. Lipid analyses revealed that anaerobiosis clearly affected the neutral lipid composition of the plasma membrane. The aerobic membrane contained more unsaturated fatty acids, mainly palmitoleic and oleic acids, more total sterol, much more ergosterol and much less squalene. The principal sterol in the aerobic membrane, ergosterol, was mainly in the free form, whereas zymosterol and other minor sterols were predominantly esterified. In contrast, the anaerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol, and was clearly richer in saturated fatty acids having a greater variation in chain length. Both plasma membranes contained a considerable amount of triacylglycerols.     

Chemical constituents of some mushrooms

The free amino acids, fatty acids and sterol contents of five Basidiomycetes were determined. The most abundant free amino acids were alanine, proline, serine and valine. The mushrooms examined also contained high levels of linoleic and palmitic acid. The most abundant sterol was ergosterol accompanied by its closely related sterols.