Isolation of copper-tolerant mutants of sake yeast with defective peptide uptake.

Mutants of Saccharomyces cerevisiae that have a decreased peptide uptake ability were isolated from sake yeast. A copper medium containing copper sulfate, histidylleucine and sodium lactate as a carbon source was used for the isolation. One mutant showed a decreased peptide uptake ability due to PTR2 transcriptional repression, but for other mutants, the decrease was not due to the transcriptional repression. The peptide concentration in sake brewed with these mutants increased to about 1.5-fold that of sake brewed with the parental strain. The angiotensin I-converting enzyme (ACE) inhibitory activity of sake brewed with the mutant was about 3.6-fold that of sake brewed with the parental strain.

Effect of amino acids on peptide transport in sake yeast.

The PTR 2 gene of Saccharomyces cerevisiae encodes a major peptide permease responsible for the uptake of low-molecular-weight peptides consisting of two or three amino acids. We show that the PTR 2 gene of sake yeast encodes a major peptide permease in the main mash of sake brewing. The peptide uptake activity in sake yeast is decreased by the addition of certain types of amino acids, particularly asparagine, serine and lysine. Northern blot analysis suggested that asparagine and serine repress the expression of the PTR 2 gene, but lysine decreases the peptide transport activity without repressing PTR 2 gene transcription. The deletion analysis of the PTR 2 promoter region confirmed these suggestions and revealed that the cis-element involved in the regulation of the PTR 2 gene by amino acids is located in the region from residue --400 to the start codon.

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.

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.

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.

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.

High fidelity segregation of a YEp vector in [cir0] strains of the yeast Saccharomyces cerevisiae.

YEp vectors carrying the GPD promoter and PGK terminator for constitutive expression showed high partition efficiencies specifically in [cir(0)] strains, when DNA fragments were inserted into the cloning site. These plasmids appeared to be more stable than yeast centromeric plasmids, being lost in less than 10(-2) cells after each generation, and more than 90% of the cells carried the plasmids after 20 generations of growth in the absence of selective pressure. The REP3 region was essential together with the GPD promoter and PGK terminator for plasmid equipartitioning in [cir0] strains. The present results suggest that this host-vector system would be useful for astute observation of the phenotype caused by gene overexpression, and for heterogeneous protein production using natural medium, because of efficient partitioning of the plasmid without selective pressure.

Influence of Ras function on ethanol stress response of sake yeast.

Reporter assay and Northern hybridization analysis revealed that the deletion of the RAS2 gene induced the expression of stress-responsive genes such as YAK1, CTT1, HSP12, and TSA2 in the laboratory yeast YNN27, but not in the sake yeast UT-1, suggesting that the Ras-cAMP-PKA signaling pathway does not play a very important part in the regulation of transcription of general stress-responsive genes in sake yeasts. However, these analyses showed that ethanol induces other stress-response element (STRE)-driven genes in the strain UT-1, with the exception of YAK1 which encodes a growth inhibitory protein, implying an ethanol-specific response. The good growth of the sake yeast in the presence of ethanol could be partially explained by YAK1 mRNA levels being unaffected by ethanol. Although the ras2 disruption of strain UT-1 did not potentiate ethanol tolerance, the disruptant could grow well in the presence of ethanol, and acquired ethanol tolerance, as is the case with the wild-type strain. These results suggest that specific stress responses of the sake yeast, which are different from those of the laboratory yeast, result in high ethanol tolerance and hence good growth in the presence of ethanol.

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.

A role of Saccharomyces cerevisiae fatty acid activation protein 4 in palmitoyl-CoA pool for growth in the presence of ethanol.

It is well known that sake yeast has a high tolerance for ethanol, as compared to baker's yeast. To investigate the relationship between the ethanol tolerance of sake yeast and the palmitoyl-CoA pool for protein modification, the growth of yeast cells with depletion of the palmitoyl-CoA pool was monitored in the presence of ethanol. The overexpression of SNC1 was used to achieve the depletion of the palmitoyl-CoA pool, because the loss of Snc palmitoylation does not affect the general growth characteristics of yeast and does not interfere with the secretory processes (Couve, A. et al., Proc. Natl. Acad. Sci. USA, 92, 5987-5991 (1995)). Although the sake yeast UT-1 exhibited much better growth in the presence of ethanol than laboratory strains, the overexpression of Snc1 was accompanied by sparse growth with increasing ethanol concentration. Exogenous palmitic acid rescued the poor growth caused by Snc1 overexpression, and the overexpression of Snc1(ser95) (which could not palmitoylated) had no effect on the growth characteristics of strain UT-1, suggesting that the poor growth with Snc1 overexpression was due to an overall increase in proteins in the unpalmitoylated form. To ascertain that fatty acid activation has a distinct role in the growth of yeast in the presence of ethanol, FAA genes encoding long chain acyl-CoA synthetases were overexpressed in combination with snc1 overexpression. Interestingly, the growth defect caused by snc1 overexpression was rescued by the overexpression of FAA4, but not of FAA1, which plays a predominant role in laboratory strains. On the contrary, disruption of faa1 led to faster growth in the presence of ethanol. These results suggest that Faa1p and Faa4p play reciprocal roles in regulating protein modification during growth in the presence of ethanol, since Faa1p and Faa4p both function to incorporate palmitic acid into phospholipids and neutral lipids. Moreover, Northern hybridization analysis revealed that faa1 mRNA was expressed strongly in a laboratory strain, and weakly in the sake yeast strain K-7 which exhibited good growth in the presence of ethanol. The combination of the disruption of faa1 and exogenously supplied palmitic acid was highly effective for growth in the presence of ethanol even under the normal snc1 expression level, implying that activation of exogenous palmitic acid by Faa4p is of particular importance in growth in ethanol.

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.