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.     

Molecular and functional analysis of a MIG1 homologue from the yeast Schwanniomyces occidentalis

A putative glucose repressor MIG1-homologue (SoMIG1) was isolated from the amylolytic yeast Schwanniomyces occidentalis. Degenerate primers were designed from the conserved zinc finger regions of Mig1 and CreA proteins from different organisms. PCR using these primers and S. occidentalis genomic DNA as template yielded a single 128 bp product. This fragment was used as a DNA probe to screen a S. occidentalis genomic library. Analysis of the positive clones led to the isolation by PCR of a DNA fragment, which contained an open reading frame (ORF) that would encode a 458 amino acid polypeptide. The DNA binding and effector domains of this putative protein showed an identity of 71% and 15%, respectively, to those of the Mig1 protein from Saccharomyces cerevisiae. The SoMIG1 gene complemented a mig1 mutant of this yeast, which suggests that in S. occidentalis SoMIG1 is a glucose repressor. The Accession No. is AJ417892.     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR⁺] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR⁺] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR⁺] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR⁺] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR⁺] cells in the industrial sake strains but not in the kimoto strains.     

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.     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.     

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.     

The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain and effects on nitrogen catabolite repression in Chinese rice wine production

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small‐scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non‐preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT‐PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling     

超高浓酿造下抗葡萄糖阻遏啤酒酵母的筛选

根据高浓发酵下(16°P)发酵度的高低,挑选下面啤酒酵母C12作为出发菌株。经过2-去氧-D-葡萄糖的定向驯养、抗性平板分离初筛以及复筛验证等步骤,筛选出一株抗葡萄糖阻遏效应的菌株CM23。将该菌株在18°P麦汁15℃条件下进行3L的EBC小型啤酒发酵实验并测定发酵指标。结果表明:与出发菌株相比,CM23的降糖速度提高了37%,达到1.8°P/d,真正发酵度达到66%,且双乙酰还原能力以及啤酒中主要风味物质含量基本不变。CM23是一株具有工业应用前景的啤酒超高浓酿造酵母菌株。     

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis

An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast.     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

Chromatin structure of the yeast FBP1 gene: Transcription-dependent changes in the regulatory and coding regions

We have studied the chromatin structure of the Saccharomyces cerevisiae FBP1 gene, which codes for fructose-1,6-bisphosphatase. A strong, constitutive, DNase I, micrococcal nuclease and S1 nuclease hypersensitive site is present close to the 3′ end of the coding region. In the repressed state, positioned nucleosomes exist around this site, and subtle changes occur in this nucleosomal organization upon derepression. A DNase I hypersensitive region is located within the promoter between positions ?540 and ?400 and it extends towards the gene in the derepressed state, leading to an alteration of nucleosomal positioning. Psoralen crosslinking of chromatin, which is used for the first time to study the mobility of restriction fragments from an RNA polymerase II gene, revealed that part of the promoter is nucleosome-free, in accordance with the results of DNase I digestion. A model is presented that, based on the chromatin structure, puts forward the hypothesis that the promoter UAS is located between ? 540 and ? 340. Finally, psoralen crosslinking, as well as digestions with micrococcal nuclease or restriction endonucleases suggests that most if not all of the copies of the active FBP1 gene are covered by nucleosomes.