MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses.

We have isolated a new Saccharomyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the MOL1 gene (for MOLasses-inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M(r) 35 kDa that is closely related to stress-inducible proteins of similar size from two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 disruption strain showed no apparent phenotype under a variety of growth conditions. The 5' region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA-gly (UCC) gene, which has been mapped to chromosome VII.     

GSG1, a yeast gene required for sporulation

We have identified a gene, GSG1 (g eneral s porulation g ene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and α haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4–6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication. The sequence reported here has the GenBank Accession Number U26674.     

The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1·2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25 286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H⁺-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM -CaCl₂ or 4 mM -ZnCl₂ or buffered to pH 7·5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H⁺-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

Isolation and characterization of sake yeast mutants deficient in gamma-aminobutyric acid utilization in sake brewing

Sake yeasts take up gamma-aminobutyric acid (GABA) derived from rice-koji in the primary stage of sake brewing. The GABA content in sake brewed with the UGA1 disruptant, which lacked GABA transaminase, was higher than that brewed with the wild-type strain K701. The UGA1 disruptant derived from sake yeast could not grow on a medium with GABA as the sole nitrogen source. We have isolated the sake yeast mutants of K701 that were unable to grow on a medium containing GABA as the sole nitrogen source. The growth defect of GAB7-1 and GAB7-2 mutants on GABA plates was complemented by UGA1, which encodes GABA transaminase, and UGA2, which encodes succinic semialdehyde dehydrogenase (SSADH), respectively. DNA sequence analysis revealed that GAB7-1 had a homozygous nonsense mutation in UGA1 and GAB7-2 had a heterozygous mutation (G247D) in UGA2. The GABA transaminase activity of GAB7-1 and the SSADH activity of GAB7-2 were markedly lower than those of K701. These GAB mutants displayed a higher intracellular GABA content. The GABA contents in sake brewed with the mutants GAB7-1 and GAB7-2 were 2.0 and 2.1 times higher, respectively, than that brewed with the wild-type strain K701. These results suggest that the reduced function of the GABA utilization pathway increases the GABA content in sake.     

Isolation and quantitation of M-factor,a diffusible mating factor from the fission yeast Schizosaccharomyces pombe

Although there have been several reports demonstrating the existence of mating factors in the fission yeast Schizosaccharomyces pombe it has not been possible to isolate these factors as cell-free preparations. Such an ability is the first requirement towards a molecular characterization of these factors and here I report the successful isolation of a mating factor from S. pombe. This factor, termed M-factor, is released by cells of the cellular mating type M (Minus) and induces mating-specific changes in P-type cells. A reliable and accurate assay for the quantitation of the M-factor, based upon changes in cell volume following exposure to the factor, is also described.     

米酒生香酵母的分离筛选鉴定及其性能研究

为获得米酒酿造中优良的生香酵母,通过嗅闻法初筛得到6株香气突出的菌株,通过测定菌株发酵米酒过程中乙酸乙酯、β-苯乙醇、乳酸乙酯和乙醇的积累量,复筛获得菌株11Z1,其积累量分别为(2.883±0.260)、(0.301±0.028)、(0.080±0.008)和(37.296±1.036)g/L.经形态学及分子生物学方...     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: Subcellular distribution of a protein required for yeast protein secretion

Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37°C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic tests confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.     

Isolation and characterization of a Δ-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the Δ-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned Δ-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the Δ-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.     

Effects of temperature, ammonium and glucose concentrations on yeast growth in a model wine system

In enology, alcoholic fermentation is a complex process involving several mechanisms. Slow and incomplete alcoholic fermentation is a chronic problem for the wine industry and factors leading to sluggish and stuck fermentations have been extensively studied and reviewed. The most studied cause of sluggish and stuck fermentation is the nitrogen content limitation. Nevertheless, other factors, such as temperature of fermentation and sugar concentration can affect the growth of yeasts. In this study we modelled the yeast growth‐cycle in wine model system as a function of temperature, sugar and ammonium concentrations; the individual effects and the interaction of these factors were analysed by means of a quadratic response surface methodology. Cell concentrations and weight loss were monitored in the whole wine fermentation process. The results of central composite design show that lower is the availability of nitrogen, higher is the cell growth rate; moreover, initial nitrogen concentration also influences survival time of Saccharomyces cerevisiae.     

An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme

The lipase-encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N-terminal 28 prepro-secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34-38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 degrees C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle-sized chain fatty acids are more efficiently hydrolysed than short- and long-chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP-caprylate, pNP-caprate and tricaprylin.     

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).     

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase‐encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587‐amino acid protein, preceded by an N‐terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild‐type and recombinant strains were essentially indistinguishable. A molecular mass of ～320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35–40 °C and a pH optimum at ～6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild‐type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single‐copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO₃. We quantitatively analysed the promoter activities by qRT–PCR and after fusing individual promoter fragments to the phytase (phyK) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO₃ are compatible with these results. Both enzymes show optimal activity at around 42°C and near‐neutral pH, and require FAD as a co‐factor and NADPH as electron donor. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and characterization of a novel fermented substance produced by edible Aspergillus oryzae AO-1 that inhibits DPP-IV activity

We identified a novel fermented substance (FKA) produced by the edible Aspergillus oryzae strain AO-1 that inhibited dipeptidyl peptidase IV (DPP-IV) (IC₅₀ = 3.41 mg·mL^− 1). HPLC analysis of FKA showed specific one metabolite (WYK-1), which inhibited DPP-IV (IC₅₀ = 6.98 μM). WYK-1 was identified as a tetrahydroxyisoquinoline derivative. Interestingly, we examined 60 strains of A. oryzae, AO-1 was the only A. oryzae strain that produces WYK-1. This is the first report that an edible A. oryzae strain produces a DPP-IV inhibitor such as WYK-1. This study also suggests that FKA has applications in the development of novel antihyperglycemic therapeutics or functional foods.     

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames,including a homologue of the KIN1/KIN2 and SNF1 protein kinases

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5′ part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.