Yasuhiro Kajiwara

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.

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.

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.

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.

The putative stress sensor protein MtlA is required for conidia formation, cell wall stress tolerance, and cell wall integrity in Aspergillus nidulans

The Mid2-like protein MtlA is a putative sensor of the cell wall integrity (CWI) signaling pathway in Aspergillus nidulans. An MtlA-EGFP fusion protein was localized at the cell surface and septa. The mtlA disruptant (∆mtlA) showed radial colony growth similar to the wild-type (wt) strain, but showed reduced conidia formation. The ∆mtlA mutant showed growth deficiency in the presence of inhibitors of cell wall synthesis. Moreover, mtlA disruption resulted in a reduction in the glucan and chitin content in the cell wall. These results suggest that MtlA plays a significant role in asexual sporulation, cell wall stress tolerance, and the maintenance of CWI in A. nidulans, but transcriptional upregulation of α-1,3-glucan synthase gene agsB induced by micafungin was observed in the ∆mtlA strain as well as the wt strain. Thus, MtlA is not essential for activation of the downstream CWI signaling pathway components identified in previous studies of Saccharomyces cerevisiae. Graphical Abstract The MtlA is a putative sensor protein of the cell wall integrity signaling pathway and required for conidia formation and cell wall stress tolerance in Aspergillus nidulan

Production of acid-stable α-amylase by Aspergillus kawachii during barley Shochu-Koji production

Abstract Acid-stable α-amylase (asAA) produced by Aspergillus kawachii (IFO4308) during shochu-koji production was investigated. Two types of asAA (asA-1 and asA-2) were purified from barley shochu-koji and their molecular weights were estimated to be 108,000 and 88,000, respectively. The characteristics of the adsorption of asA-1 and asA-2 onto raw starch were different. In barley shochu-koji production, the asA-1 activity increased gradually, but decreased after 24 h incubation. In contrast, the asA-2 activity increased with incubation time. The ratio of asA-1 to total asAA activity ( A1 AA ratio) was influenced by temperature. asA-1 was incubated with the acid protease of A. kawachii, and the mixture was subjected to SDS-PAGE analysis. The band corresponding to asA-1 disappeared at 12 h into the incubation time, and an unknown band appeared at this time. The unknown protein possessed almost the same characteristics with respect to α-amylase activity and molecular weight as did asA-2. From these results, acid-stable α-amylase is concluded to have many forms similar to the glucoamylases of Aspergillus awamori var. kawachi and it is thought that it is formed during shuchu-koji production.

Genetic instability of constitutive acid phosphatase in shochu and sake yeast

Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewers yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

Competitive advantage and tolerance of selected shochu yeast in barley shochu mash

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the fermentation superiority of BAW-6. Here, we demonstrate the growth advantage of the BAW-6 strain over the Ko strain by competitive cultivation in barley shochu preparation, where alcohol yield and nihonshudo of the former strain were higher than those of the latter strain. In addition, the maximum growth rate of BAW-6 was less affected than that of Ko by high Brix values of barley koji medium, suggesting that BAW-6 is less sensitive to growth inhibitory compounds derived from barley or barley koji. The tolerance of BAW-6 to growth inhibitory compounds, cerulenin and diethylstilbestrol (an H⁺-ATPase inhibitor), was also higher than that of other yeast strains. Consistent with BAW-6s tolerance to diethylstilbestrol in the presence of 8% ethanol (pH 4.5), H⁺-ATPase activity, but not transcription of its gene, was higher in BAW-6 than in Ko. We conclude that the BAW-6 strain is associated with certain gene alterations other than PHO3, such that it can maintain cellular ion homeostasis under conditions of ethanol stress during the latter phase of fermentation.