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.     

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.     

ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors

The ERG6 gene encodes an S‐adenosylmethionine dependent sterol C‐24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Lipid composition and cell surface hydrophobicity of Candida albicans influence the efficacy of fluconazole–gentamicin treatment

Adherence of the fungus, Candida albicans, to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans. The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain. Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole–gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment.     

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.     

Determinants of flocculence of brewer's yeast during fermentation in wort

Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.     

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.     

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.     

A new family of yeast genes implicated in ergosterol synthesis is related to the human oxysterol binding protein

We have identified three yeast genes, KES1, HES1 and OSH1, whose products show homology to the human oxysterol binding protein (OSBP). Mutations in these genes resulted in pleiotropic sterol-related phenotypes. These include tryptophan-transport defects and nystatin resistance, shown by double and triple mutants. In addition, mutant combinations showed small but apparently cumulative reductions in membrane ergosterol levels. The three yeast genes are also functionally related as overexpression of HES1 or KES1 alleviated the tryptophan-transport defect in kes1Δ or osh1Δ mutants, respectively. Our study implicates this new yeast gene family in ergosterol synthesis and provides comparative evidence of a role for human OSBP in cholesterol synthesis.     

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.     

II. Yeast sequencing reports. A putative P-type Cu2+-transporting ATPase gene on chromosome II of Saccharomyces cerevisiae

We have sequenced a gene on the right arm near the telomere of chromosome II of Saccharomyces cerevisiae which codes for a putative P-type cation-transporting ATPase (PCA1). The gene codes for a 1216 amino acids protein. The PCA1 gene expresses a 3·5 kb message in both haploid and diploid cells when grown in glucose-based rich medium YPD. The gene product is most similar at the C-terminal region to a human copper-transporting ATPase and Enterococcus hirae copper-transporting ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. Cells lacking PCA1 display no obvious phenotype when tested under standard conditions; whereas they cease growth much earlier than the isogenic wild-type cells in a minimal medium with high copper concentration. Overexpression of PCA1 under GAL1/10 promoter in yeast cells causes poor growth. We also show that yeast strains carrying PCA1 in multiple copies grow slower than isogenic wild-type strains in a minimal synthetic medium containing 0·3 mM-CuSO₄. The sequence has been deposited in the EMBL data library under Accession Number Z29332.     

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium–glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2⁺ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat-shocked cells (38°C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.     

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.     

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.     

XIII. Yeast sequencing reports. SED6 is identical to ERG6, and encodes a putative methyltransferase required for ergosterol synthesis

Luminal endoplasmic reticulum (ER) proteins carry a sorting signal that allows them to be retrieved from the Golgi apparatus by a specific receptor. In yeast, this receptor is encoded by the ERD2 gene. Although retrieval of ER proteins does not appear to be an essential process, cells lacking ERD2 do not grow. Several multicopy suppressors of this growth defect have been isolated. The sequence of one of these, SED6, is presented here. Its product contains motifs characteristic of methyltransferases, and it is identical to ERG6, the presumed structural gene for S-adenosylmethionine:Δ²⁴-sterol-C-methyltransferase. The gene is located adjacent to PDR4, near the centromere of chromosome XIII.     

Ergosterol content as a measure of biomass of potential biological control fungi in liquid cultures

Gliocladium roseum 784A and G. solani 810A exhibited the highest and lowest levels of ergosterol, respectively, under test conditions. Based upon relative amounts of ergosterol produced per mycelial dry weight, three groups namely high (G. roseum 784A), medium (Mariannaea elegans 386E, G. roseum 321M, G. viride 623E, G. virens 258C and G. virens 258D) and low (Trichoderma pseudokoningii 228B, G. solani 810A) were identified. Concentration of ergosterol do not always correlate with absolute fungal biomass and is influenced by both internal and external factors. Test growth conditions should therefore closely mimic that of anticipated environment. Concentrations of ergosterol of eight biological control fungi reported in this study fall within the range of other fungi reported in the literature. Under control conditions, ergosterol concentration of the eight potential biological control fungi may be used as a relative index of their biomass and their growth in liquid cultures.

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Gliocladium roseum 784A und G. solani 810A zeigten die h?chsten bzw. niedrigsten Gehalte unter den Testbedingungen. Das produzierte Ergosterol wurde auf die Trockenmasse des Mycels bezogen; daraus ergaben sich drei Pilzgruppen, und zwar mit hohem Gehalt (G. roseum 784A), mittlerem Gehalt (Mariannaea elegans 386E, G. roseum 321M, G. viride 623E, G. virens 258C und G. virens 258D) und niedrigem Gehalt (Trichoderma pseudokoningii 228B, G. solani 810A). Die Konzentration an Ergosterol korreliert nicht immer mit der tats?chlichen Biomasse der Pilze und wird durch interne und externe Faktoren beeinflu?t. Die Wachstumsbedingungen der Tests sollten daher die vermuteten Umgebungsbedingungen m?glichst genau nachahmen. Die hier ermittelten Ergosterolgehalte der acht Pilzst?mme liegen im Bereich anderer in der Literatur beschriebener Pilze. Unter Kontrollbedingungen kann der Ergosterolgehalt dieser Pilze als relativer Index der Biomasse und des Wachstums in flüssigen Kulturen benutzt werden.

     

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374–376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33·4%, 26·7%, 23·4% and 29·2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68·4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L37111.