Shodo Hara

Specific Expression and Temperature-Dependent Expression of the Acid Protease-Encoding Gene (pepA) in Aspergillus oryzae in Solid-State Culture (Rice-Koji).

The synthesis of acid protease in rice-koji is important for sake brewing. Northern blot analysis was carried out to study the transcriptional regulation of acid protease-encoding gene (pepA in Aspergillus orytae. The pepA gene was not expressed in submerged culture, while it was expressed when cultured on steamed rice. Additionally, the culture at high temperature (>38 degrees C) caused a marked decrease in transcription level of pepA, although the alpha-amylase (amyB) and actin genes were expressed regardless of the temperature. To examine whether the pepA promoter controlled the temperature-dependent expression, the promoter regions of pepA and amyB were introduced into a vector containing the GUS reporter gene (uidA gene). Northern blot analysis showed that the elevation of culture temperature caused the loss of uidA expression in the pepA promoter-uidA transformant but not in the amyB promoter-uidA transformant. These results suggest that its promoter controlled the temperature-dependent expression of pepA.

Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.

SSU1-R, a sulfite resistance gene of wine yeast, is an allele of SSU1 with a different upstream sequence

Abstract A sulfite resistance gene, SSU1-R, was cloned from a highly sulfite-resistant strain of Saccharomyces cerevisiae, Y-9, which was isolated as a contaminant from a wine must. The coding region sequence of SSU1-R was almost identical to that of SSU1, the gene that has been shown to be responsible for sulfite resistance. Although SSU1 is located on chromosome XVI of S. cerevisiae, the upstream region of SSU1-R was homologous with a sequence of chromosome VIII. In our Northern analysis, an intense band was detected in Y-9 and K1-V1116 strains, both of which exhibit strong sulfite resistance, with and without sulfite in the medium. On the other hand, under the same experimental conditions, no band was detected in OC-2, a strain that exhibits weak sulfite resistance. Physical mapping of SSU1/SSU1-R showed that this sequence was on chromosome VIII of Y-9 and the two wine strains, K1-V1116 and WE14. OC-2 and twenty-three other wine, sake, beer, shochu (Japanese distilled liquor), alcohol, bakery, and laboratory strains, had this sequence on chromosome XVI, and four other wine strains had it on both chromosomes. Thus, the difference in the upstream sequence of SSU1/SSU1-R seems to cause differences in the transcription rates and degree of sulfite resistance.

Increased alcohol acetyltransferase activity by Inositol limitation in Saccharomyces cerevisiae in sake mash

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of the OPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino-) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26-55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.

Effect of fatty acid saturation in membrane lipid bilayers on simple diffusion in the presence of ethanol at high concentrations

When multilamellar liposome trapping calcein, the fluorescent dye, was suspended in buffered ethanol containing the quencher, cobalt (II) ions, the increase in ethanol concentration in the range of 0–17.5% was accompanied with a striking decrease in fluorescence intensity, suggesting that permeability to the liposome membrane depended upon the ethanol concentration. In buffer containing 17.5% ethanol, as the percentage of phospholipid in which the 1- and 2-positions were occupied by saturated fatty acids increased in PC-PE liposome, there was a progressive decrease in velocity coefficient in quenching fluorescence, and the ratio of PCPE appeared to have little effect on velocity coefficient. Intracellular nucleotide in cell suspension in 20% ethanol at 15°C appeared to diffuse to the liquid phase by simple diffusion, and palmitic acid-enriched cells showed a lower permeability coefficient P′, compared with linoleic acid-enriched cells, regardless of intracellular ergosterol content. The survival in linoleic acid-enriched cell was less than that of palmitic acid-enriched cells, suggesting that cell viability corresponded with P′.

Ethanol-Induced Alterations in Lipid Composition of Saccharomyces cerevisiae in the Presence of Exogenous Fatty Acid

Abstract Ethanol-induced alterations in the lipid composition of Saccharomyces cerevisiae grown in the presence of exogenous fatty acids were studied. The addition of both palmitic acid and ethanol (4–8%) to the basal medium resulted in a striking increase in the palmitic acid content and decreases in the content of myristoleic, palmitoleic, and oleic acids in the phospholipid fatty acid composition, compared with their contents in the absence of ethanol. On the other hand, the addition of linoleic acid to the medium containing ethanol (4–8 %) resulted in an increase in oleic acid and corresponding decreases in myristoleic, palmitoleic acid, and palmitic acid, while the linoleic acid levels were maintained in excess of 14%. Generally, growth in the presence of ethanol resulted in a reduction in the amount of phosphatidylcholine (PC) and more obviously, in the amount of phosphatidylethanolamine (PE), leading to an increase in the PC PE ratio. A good cell survival rate in replacement cultures containing 18% ethanol was attained when cells were preincubated in the presence of ethanol (4–8%). Under the same conditions, palmitic acid-enriched cells had additional ethanol-endurability and a higher cell-membrane integrity than linoleic acid-enriched cells.

Cloning and Nucleotide Sequence of the Glutamate Decarboxylase-encoding Gene gadA from Aspergillus oryzae

We cloned a genomic DNA encoding the glutamate decarboxylase (GAD) from Aspergillus oryzae using a 200-bp DNA fragment as the probe. This DNA fragment was amplified by the reverse transcription polymerase chain reaction with mRNA of A. oryzae as the template and degenerate primers designed from the conserved amino acid sequence of Escherichia coli GAD and Arabidopsis thaliana GAD. Nucleotide sequencing analysis showed that the cloned gene (designated gadA) encoded 514 amino acid residues and contained three introns. Southern hybridization showed that the gadA gene was on a 6.0-kb SacI fragment and that there was a single copy in the A. oryzae chromosome. The cloned gene was functional, because one transformant of A. oryzae containing multiple copies of the gadA gene had 10-fold the GAD activity and a 12-fold increase in gamma-aminobutyric acid production compared with the control strain.

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Permeability barrier of the yeast plasma membrane induced by ethanol

Abstract An increase in extracellular nucleotides appeared to conform to simple diffusion, when Saccharomyces cerevisiae cells were suspended in 20% ethanol at 15°C. However, in the case of cells grown in the presence of 8% ethanol, the leakage of nucleotide appeared to be repressed significantly in the initial phase. The addition of glucose led to a continuation of the repression of the leakage. Under this condition, the addition of iodoacetamide as an inhibitor in glycolytic pathway, or stilbestrol as an inhibitor of plasma membrane ATPase, resulted in a rapid increase in the leakage of nucleotides, indicating that the membrane permeability barrier is dependent on membrane ATPase. Ouabain, an inhibitor of the Na + K + -ATPase , had no effect. The addition of 10 mM CaCl2 for inducing lateral phase separation in the inner membrane, which caused a faster release of nucleotides in general, had a little effect on nucleotide leakage, when cells grown in the presence of 8% ethanol were suspended in 20% ethanol containing glucose. Moreover, the addition of 10 mM KCl, together with CaCl2, had almost no effect on leakage. Ca2+ influx was found to be smaller in cells grown in the presence of ethanol when compared to cells grown in the absence of ethanol, when fura-2 loaded cells were suspended in K-MOPS buffer containing 50 mM CaCl2. Divalent cations such as Ba2+, Mg2+ and Mn2+ had effects similar to Ca2+ on membrane permeability. The decrease in cell viability corresponded to the amount of leakage of nucleotides over the experimental time course. These results suggest that the high activity of membrane ATPase which is induced by growth in the presence of 8% ethanol ensures homeostasis of ions in the cytoplasm at high concentrations of ethanol, and reduces the effects of membrane surface-acting substances such as divalent cations on the membrane integrity. Thus the maintenance of the cell membrane as a permeability barrier appears to lead to a high ethanol-endurability.

Isolation and properties of a chromosome-dependent KHR killer toxin in Saccharomyces cerevisiae.

A strain of the yeast Saccharomyces cerevisiae coding for KHR on the chromosome secreted a toxin that kills sensitive yeasts. The transformants of multicopy vectors carrying the KHR gene could secrete 3-4-fold the killer toxin of the donor strain. This toxic substance was purified 80-fold in specific activity from the culture filtrate by gel filtration and hydrophobic column chromatography. The purified toxin gave a single protein band with molecular mass of 20 kDa on SDS-PAGE and had an isoelectric point of pH 5.3. The toxin had novel killer activity against Candida glabrata and S. cerevisiae, but did not affect bacteria, fungi, or other yeasts.