Effect of low temperature upon vitality of Saccharomyces cerevisiae phospholipid mutants

The phospholipid metabolism of Saccharomyces cerevisiae plays a central role in its adaptation to low temperatures. In order to detect the key genes in this adaptation, various phospholipid mutants from the EUROSCARF collection of Saccharomyces cerevisiae BY4742 were tested to ascertain whether the suppression of some genes could improve the fermentation vitality of the cells at low temperature. The cell vitality and phospholipid composition of these mutants were analysed. Some knockouts improved (hmn1Δ) or impaired (cho2Δ and psd1Δ) their vitality at low temperature (13 °C) but were not affected at optimum temperature (25 °C). A common trait of the mutants that had some defect in vitality was a lower concentration of phosphatidylcholine and/or phosphatidylethanolamine. The supplementation with choline allowed them to recover viability, probably by synthesis through the Kennedy pathway. Hmn1Δ showed a lower concentration of phosphatidylcholine, which explains the dominant role of the de novo pathway in cellular phosphatidylethanolamine and phosphatidylcholine vs the Kennedy pathway. The absence of such genes as CRD1 or OPI3 produced important changes in phospholipid composition. Cardiolipin was not detected in crd1Δ but phosphatidylglycerol circumvents most of the functions assigned to CL. The considerable reduction in PC diminished the cell vitality of opi3Δ at both temperatures, although the decrease at 13 °C was more marked. Copyright © 2012 John Wiley & Sons, Ltd.     

Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.     

Yeast mutants affected in viability upon starvation have a modified phospholipid composition

Selection of mutations, based on the suppression of rvs161Δ defects, was performed. Ten mutants were obtained, ranged amongst four complementation groups, named SUR1, SUR2, SUR3 and SUR4. All sur mutations also suppress a mutation in another gene, RVS167, indicating that all six genes are involved in the same biological pathway. The sur mutant cells have abnormal morphologies in stationary phase, i.e. dumbbell-like in sur1, sur2 or sur3 strains and multibudded in sur4 strains. Several phenotypic characteristics of the physiological suppressors as well as the rvs161Δ strain itself led us to analyse the phospholipid composition of the mutants. The assays show an overall decrease of the phospholipid amounts and modifications in the relative contents of some phospholipid classes in sur mutant cells.     

Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis

The inositol/choline responsive element (ICRE) functions as a UAS element mediating coordinate expression of structural genes required for yeast phospholipid biosynthesis. However, ICRE motifs could be detected upstream of various genes apparently not involved in lipid metabolism. In this work we investigated the expression pattern of selected genes containing ICRE promoter motifs, as identified by in silico analysis (ARG4, ERG20, FAR8, GPD2, RSF1, URA8, VHT1 and YEL073C). It turned out that the presence of an ICRE upstream of a gene of unknown function indeed allows to conclude for regulation by phospholipid precursors, which is mediated by activators Ino2/Ino4 and the repressor Opi1. We also demonstrated in vitro binding of Ino2/Ino4 heterodimers to promoter regions. Thus, our analysis supports the view that identification of regulatory elements by a database search provides evidence for a specific pattern of gene expression. Activation by pathway-specific regulators may suggest a physiological function for as yet uncharacterized genes.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae

Three regulatory genes, known to be required for glucose repression/derepression of some genes in Saccharomyces cerevisiae, were disrupted to study their effects on the carbon-source regulation of the STA2 glucoamylase gene expression. Using a STA2-lacZ fusion it was found that: (1) the MIG1 gene is dispensable for the repression of the STA2 gene; (2) there are two components in the carbon-source repression of STA2: HXK2-dependent and HXK2-independent; and (3) the HAP2 gene seems to be involved in repression rather than activation of the STA2 expression.     

Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions

Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [¹⁴C]palmitic acid (90%) and [³H]oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A₂-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.     

Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline‐responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE‐dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10‐fold. Repression of ICRE‐dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE‐dependent gene expression by phosphorylation‐dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation

The biochemical formation of yeast-derived sensory-active metabolites like higher alcohols and esters determines the different characteristics of aroma and taste in fermented beverages. In yeast fermentation process, a large number of environmental factors affecting the production of volatile aroma compounds are abundant. Factors like substrate composition in fermentation media as well as process parameters influencing these flavor-active metabolites have already been described. These factors can act on the expression of yeast genes involved in aroma metabolism resulting in concentration differences in esters and higher alcohols important for flavor and taste. The understanding of the function of genes involved in biosynthetic pathways of aroma-active substances as well as their regulatory mechanisms is needed to control the production of ester and higher alcohol synthesis to create specific aroma profiles in fermented beverages. This review discusses the known regulation and function of several individual genes (ATF1, ATF2, EEB1, EHT1, BAT1, BAT2 and BAP2) described in fusel alcohol and ester synthesis mainly in S. cerevisiae and S. pastorianus var. carlsbergensis. Also, different factors like oxygen and temperature that allow ester and higher alcohol synthesis to be controlled during yeast fermentation are described.     

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10^?5 M ) or spermine (10^?5 M ) compared to a control culture containing 10^?8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.     

Functional analysis of YCL09C: Evidence for a role as the regulatory subunit of acetolactate synthase

We have analysed the function of the open reading frame (ORF) YCL09C. The deletion of this ORF from chromosome III does not affect the physiology of the corresponding yeast strain enough to give a distinct phenotype. Nevertheless a computational analysis reveals high homology between this ORF and the enterobacterial genes encoding the regulatory subunit of acetolactate synthase. We have therefore tested the possibility that ycl09cp is the regulatory subunit of yeast acetolactate synthase by in vitro enzymatic analysis. The acetolactate synthase was previously shown to be retroinhibited by its final product valine. In Escherichia coli this retro-control is assured by the regulatory subunit. Using a yeast strain carrying a complete deletion of YCL09C, we have observed the loss of such retro-inhibition. These results together with the computational predictions show that YCL09C encodes the regulatory subunit of yeast acetolactate synthase.     

Candida famata (Candida flareri)

Candida famata (Candida flareri) belongs to the group of so‐called ‘flavinogenic yeasts’, capable of riboflavin oversynthesis under condition of iron starvation. Some strains of C. famata belong to the most flavinogenic organisms known and were used for industrial production of riboflavin for a long time in the USA. C. famata is characterized by high salt tolerance, growing at NaCl concentrations of up to 2.5 m . Development of basic tools for the metabolic engineering of C. famata, such as a transformation system, selective markers, insertional mutagenesis, a reporter system and others, are described. The developed tools were used for cloning and identification of structural and regulatory genes of riboflavin synthesis. The construction of improved yeast strains producing riboflavin, FMN and FAD, based on the industrial riboflavin‐producing strain dep8 and its non‐reverting analogue AF4, is also described. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26S protease complex

There is accumulating evidence for a large, highly conserved gene family of putative ATPases. We have identified 12 different members of this novel gene family (the YTA family) in yeast and determined the nucleotide sequences of nine of these genes. All of the putative gene products are characterized by the presence of a highly conserved domain of 300 amino acids containing specialized forms of the A and B boxes of ATPases. YTA1, YTA2, YTA3 and YTA5 exhibit significant similarity to proteins involved in human immunodeficiency virus Tat-mediated gene expression but more significantly to subunits of the human 26S proteasome. YTA1 and YTA2 are essential genes in yeast. Remarkably, the cDNA of human TBP-1 can compensate for the loss of YTA1. Preliminary experiments indicate that YTA1 is a component of the 26S protease complex from yeast. Our findings lead us to propose that YTA1, YTA2, YTA3 and YTA5 function as regulatory subunits of the yeast 26S proteasome. YTA10, YTA11 and YTA12 share significant homology with the Escherichia coli FtsH protein, and together with YTA4 and YTA6 may constitute a separate subclass within this family of putative ATPases.     

Alcohol oxidase hybrid oligomers formed in vivo and in vitro

Cell‐free extract prepared from methanol‐grown cells of the methylotrophic yeast Pichia methanolica showed nine multiple alcohol oxidase (AOD) bands on active staining in native polyacrylamide gel electrophoresis. Their molecular basis was investigated and two AOD‐encoding genes, MOD1 and MOD2, were cloned from P. methanolica genome. When the two genes were expressed in a heterologous host, an alcohol oxidase‐depleted strain of Candida boidinii(aod1Δ strain), both MOD1 and MOD2 partially complemented growth defect of the host strain on methanol. While expression of either MOD1 or MOD2 in C. boidinii aod1Δ strain gave a single AOD band corresponding to the band with the largest and smallest mobility among the nine AOD bands, respectively, co‐expression of MOD1 and MOD2 resulted in multiple band formation. Mixed oligomerization of Mod1p and Mod2p in vitro also gave nine multiple bands. From these results, we concluded that the nine multiple forms of AOD observed on native–PAGE represent two homo‐octamers and seven hetero‐octamers of Mod1p and Mod2p. Using this zymogram analysis, we also found that Mod1p was preferably produced at low methanol concentrations in the media, while Mod2p was produced at higher methanol concentrations. This shows distinct regulatory features of the two AOD‐encoding genes in this methylotrophic yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

Genome‐wide expression profiling of the osmoadaptation response of Debaryomyces hansenii

The euryhaline marine yeast Debaromyces hansenii is a model system for the study of processes related to osmoadaptation. In this study, microarray‐based gene expression analyses of the entire genome of D. hansenii was used to study its response to osmotic stress. Differential gene expression, compared to control, was examined at three time points (0.5, 3 and 6 h) after exposure of D. hansenii cultures to high salt concentration. Among the 1.72% of genes showing statistically significant differences in expression, only 65 genes displayed at least three‐fold increases in mRNA levels after treatment with 2 M NaCl. On the other hand, 44 genes showed three‐fold repression. Upregulated as well as the downregulated genes were grouped into functional categories to identify biochemical processes possibly affected by osmotic stress and involved in osmoadaptation. The observation that only a limited number of genes are upregulated in D. hansenii in response to osmotic stress supports the notion that D. hansenii is pre‐adapted to survive in extreme saline environments. In addition, since more than one‐half of the upregulated genes encode for ribosomal proteins, it is possible that a translational gene regulatory mechanism plays a key role in D. hansenii's osmoregulatory response. Validation studies for ENA1 and for hyphal wall/cell elongation protein genes, using real‐time PCR, confirmed patterns of gene expression observed in our microarray experiments. To our knowledge, this study is the first of its kind in this organism and provides the foundation for future molecular studies assessing the significance of the genes identified here in D. hansenii's osmoadaptation. Copyright © 2009 John Wiley & Sons, Ltd.