Toshiro Omori

Genome sequence of the white Koji mold aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu

ABSTRACT The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications.

Enhancement of Glycerol Production by Brewing Yeast (Saccharomyces cerevisiae) with Heat Shock Treatment

Abstract The glycerol 3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) activity of cells subjected to heat shock treatment increased 1.15- to 1.25-fold over that of cells not heat shocked. The increased GPDH activity induced by heat shock treatment extended over several generations, but gradually decreased through subcultures. The effect of heat shock treatment during the pre culture stage on glycerol production in the main culture was investigated with the shochu yeast BAW-6 (Saccharomyces cerevisiae). Heat shock at 45 and 50°C was effective for glycerol production, but there was no influence at 35°C. The timing of the heat shock treatment did not influence glycerol production. Enhancement of glycerol production induced by heat shock was also observed in the sake yeast K-7, wine yeast W-3, beer yeast IFO1167, and whisky yeast IFO2363. Shochu was produced by using heat shocked BAW-6. The glycerol concentration in the mash was increased by heat shock treatment, thereby improving the aroma and taste of shochu produced from this mash. In addition to shochu, glycerol is a key factor influencing the taste of other brewed brewages such as sake or wine. The results suggest that the heat shock treatment during pre culture may be an effective and simple method for improving the quality of shochu, sake, and wine.

Putative Stress Sensors WscA and WscB Are Involved in Hypo-Osmotic and Acidic pH Stress Tolerance in Aspergillus nidulans

ABSTRACT Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in Aspergillus nidulans. Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be N- and O-glycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The wscA disruptant (ΔwscA) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the ΔwscA strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the ΔwscA strain. A similar inhibition was observed in the wscB disruptant strain, but to a lesser extent. In addition, a double wscA and wscB disruptant (ΔwscA ΔwscB) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of α-1,3-glucan synthase genes (agsA and agsB) was significantly altered in the wscA disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of wsc disruption. Moreover, the transient transcriptional upregulation of the agsB gene via MpkA signaling was observed in the ΔwscA ΔwscB strain to the same degree as in the wt strain. These results indicate that A. nidulans Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast Saccharomyces cerevisiae and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.

Breeding of high glycerol-producing shochu yeast (Saccharomyces cerevisiae) with acquired salt tolerance

Abstract We have obtained a high glycerol-producing strain of shochu yeast ( Saccharomyces cerevisiae ) which exhibited acquired salt tolerance, and have produced barley shochu using this strain. The shochu yeast BAW-6 was sensitive to sodium chloride, and did not grow in a medium which contained more than 12% sodium chloride. We have frequently found high glycerol-producing strains among salt tolerant mutants of BAW-6. The amount of glycerol produced by a mutant TK-2, which was tolerant to 18% sodium chloride, was approximately 130% that produced by the parent strain BAW-6. Although alcohol dehydrogenase activities of the two strains were the same, glycerophosphatase activity in TK-2 was higher than that in BAW-6. The amount of glycerol produced by TK-2 was approximately 114% that produced by BAW-6 during the fermentation of barley shochu . The difference in the amount of glycerol produced between TK-2 and BAW-6 was smaller in shochu mash containing citric acid than in the medium in the absence of citric acid. These results may be due to the presence of citric acid. We investigated the behavior of the flavor components during distillation under vacuum. The distillation ratios of isoamyl acetate and β-phenylethyl acetate of TK-2 were higher than those of BAW-6 although the alcohol distillation ratios were the same. It was considered that the glycerol level in both mashes influenced the distillation of esters.

High glycerol producing amino acid analogue-resistant Saccharomyces cerevisiae mutant

Abstract The alcohol dehydrogenase (ADH) activity of a yeast was lower while glycerol production of the same yeast was higher in media containing inorganic nitrogen than in media containing amino nitrogen. From this result, we developed the working hypothesis that activation of amino acid biosynthesis might enhance glycerol production. We isolated mutants which were resistant to 5,5,5-trifluoro-leucine (TFL), p -fluoro- dl -phenylalanine (FPA), canavanine (CAN), or cerulenin (CER) from shochu yeast BAW-6 ( Saccharomyces cerevisiae ). The amount of glycerol produced by 47 out of 50 TFL-mutant strains and 37 out of 50 FPA-mutant strains were at least 110% that produced by BAW-6. On the other hand, the amounts of glycerol produced by CAN and CER mutants were almost the same as that produced by BAW-6. TFL and FPA mutants produced larger amounts of glycerol and smaller amounts of ethanol than BAW-6 did. The ADH activities in TFL and FPA mutants were lower than that in BAW-6. The amounts of isoamyl alcohol produced by TFL-mutants and β-phenylethyl alcohol produced by FPA-mutants increased with increasing amount of glycerol produced by the mutants. We obtained a large number of glycerol-producing strains exhibiting leucine or phenylalanine analogue resistance at a high frequency. This is a new method for the isolation of high glycerol producing yeast.

Enhanced glycerol production in Shochu yeast by heat-shock treatment is due to prolonged transcription of GPD1.

Enhancement of glycerol production in Shochu yeast, which was induced by heat-shock treatment, was studied. Although heat-shock treatment (45 degrees C, 1 h) caused a transient delay in cell growth, the amount of glycerol produced by heat-shock-treated cells was 20% higher than that by control cells. During the glycerol-production phase, the NAD+-dependent glycerol-3-phosphate dehydrogenase (GPDH) activity of heat-shock-treated cells was much higher than that of control cells, suggesting that a higher GPDH activity enhances glycerol production. The level of NAD+-dependent glycerol dehydrogenase (GDH) activity was almost the same between heat-shock-treated cells and control cells. The results of Northern blot analysis of GPD genes (GPD1 and GPD2) encoding the GPDH enzyme showed that the transcription of GPD genes was not affected by heat-shock treatment but the period of intensive transcription of GPD1 was prolonged.

A novel method for screening high glycerol- and ester-producing brewing yeasts (Saccharomyces cerevisiae) by heat shock treatment

Abstract The screening of high glycerol-producing brewing yeasts ( Saccharomyces cerevisiae ) was attempted by heat shock treatment. The shochu yeast BAW-6 was grown in HS-medium, and the culture was treated by heat shock (45°C, 20 min). Surviving cells were isolated as heat shock-resistant (HSR) mutants. Glycerol production in the HSR mutants increased 1.8- to 2.0-fold over that in the parental strain. In some HSR mutants, the production of isoamyl acetate, ethyl caproate, ethyl caprylate, and ethyl capriate also increased significantly compared to their production in the parental strain. The glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) and alcohol acetyltransferase (EC 2.3.1.84) activities in selected HSR mutants were about 2-fold those in the parental strain. By contrast, NAD + -dependent glycerol dehydrogenase (EC 1.1.1.6) activity showed no difference. The ratio of oleic to stearic acid in the intracellular fatty acids was lower in the mutant strain HSR6-7 (6.52) than in the parental strain BAW-6 (8.88), and the incorporation of fatty acids from the medium was less in the HSR mutant than in the parental strain. High glycerol-producing sake and wine yeasts were also obtained from the sake yeast K-7 and wine yeast OC-2 by heat shock treatment. These results showed that heat shock treatment is an effective method for the improvement of brewing yeasts.

Utilization of Fermented Barley Extract Obtained from a By-product of Barley Shochu for Nisin Production

Fermented barley extract (FBE) obtained from a barley shochu by-product (shochu kasu) and its ethanol fractions were evaluated as a medium and supplement, respectively, for nisin A production by Lactococcus lactis subsp. lactis ATCC 11454. A Brix 2.5 FBE medium supplemented with glucose provided a high level of nisin A production with a nisin yield comparable to that from a nutritionally rich laboratory medium (basal medium). By adding the ethanol-insoluble (EI) fraction of FBE to the basal medium, nisin A production was enhanced concomitant with an increase in bacterial cell growth, while the ethanol-soluble (ES) fraction had a negative effect on nisin A production. These findings indicate that FBE obtained from shochu kasu can be utilized as a preferable medium for nisin A production and could be converted into a value-added food product having preservative functions. The procedure developed in this study would promote recycling of shochu kasu.

Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production

ABSTRACT The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.

Intracellular Fatty Acid Formation and Alcohol Acetyl Transferase Gene Expression in Brewing Yeast (Saccharomyces cerevisiae) Treated with Heat Shock

Abstract The proportion of palmitic acid in phosphatidyl-choline in brewing yeast cells pre-treated with heat shock was 5.2% higher, and that of palmitoleic acid was 5.7% lower than the proportions in untreated cells after 24-h incubation. Alcohol acetyl transferase (AATase) activity was 1.4-fold higher than that in untreated cells at 24-h. Heat-shocked cells were tested for use in shochu fermentation. On the 2nd day, the expression of the AATase gene (ATF1) was approximately three-fold higher than that in untreated cells. AATase activity in the cells and the isoamyl acetate concentration in the mash were higher in shochu fermentation using cells pre-treated with heat shock than when untreated cells were used.