Transcriptomic analysis of temperature responses of Aspergillus kawachii during barley koji production.

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.

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.

Crystal structure of the rice branching enzyme I (BEI) in complex with maltopentaose.

Starch branching enzyme (SBE) catalyzes the cleavage of α-1,4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. We determined the crystal structure of the rice branching enzyme I (BEI) in complex with maltopentaose at a resolution of 2.2Å. Maltopentaose bound to a hydrophobic pocket formed by the N-terminal helix, carbohydrate-binding module 48 (CBM48), and α-amylase domain. In addition, glucose moieties could be observed at molecular surfaces on the N-terminal helix (α2) and CBM48. Amino acid residues involved in the carbohydrate bindings are highly conserved in other SBEs, suggesting their generally conserved role in substrate binding for SBEs.

Genome sequence of the white koji mold Aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu.

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.

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.

Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding.

Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 Å by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.

Effects of a fermented barley extract on subjects with slightly high serum uric acid or mild hyperuricemia.

The uric acid-lowering effect and safety of a fermented barley extract P (FBEP) prepared from barley-shochu distillery by-products were investigated in a randomized, placebo-controlled, parallel-group, double-blinded study. A total of 111 subjects with serum uric acid levels of 6.0-7.9 mg/dl were provided with either a drink containing 2 g/d of FBEP (test group) or a placebo drink. After 12 weeks, the serum uric acid levels changed by -0.21+/-0.56 mg/dl in the test group, showing a significant decrease in comparison to those of the placebo group (+0.02+/-0.54 mg/dl). Additionally, the uric acid clearance in the test group showed a tendency to increase after 12 weeks more than in the placebo group (p=0.054). No abnormalities in the physical and clinical tests were observed, and no adverse diagnostic findings were attributed to the intake of the test meal. These results demonstrated the benefits and safety of the FBEP treatment to subjects with slightly high serum uric acid or mild hyperuricemia.

Effects of single and combined administration of fermented barley extract and gamma-aminobutyric acid on the development of atopic dermatitis in NC/Nga mice.

We examined the effects single and combined administration of fermented barley extract P (FBEP), prepared from barley-shochu distillery by-products, and gamma-aminobutyric acid (GABA) on the development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice. Single administration of FBEP and GABA dose-dependently reduced the development of AD-like skin lesions in mice. GABA reduced the development of AD-like skin lesions by suppressing serum immunoglobulin E (IgE) and splenocyte interleukin (IL)-4 production, while FBEP reduced skin lesions without affecting the IgE or cytokine production. However, in mice with induced AD-like skin lesions, combined administration of FBEP and GABA decreased serum IgE levels and splenic cell IL-4 production, and increased splenic cell interferon-gamma production. These results suggest that combined administration of FBEP and GABA alleviated AD-like skin lesions in the NC/Nga mice by adjusting the Th1/Th2 balance to a Th1-predominant immune response.

The action of rice branching enzyme I (BEI) on starches.

The rice branching enzyme I (BEI) overproduced in Escherichia coli cells was investigated with respect to action on starches. BEI treatment decreased the turbidity of starch suspensions with distinct pasting behaviors from a native starch. This result suggests the great potential of BEI as a molecular tool for the production of a novel glucan polymer.

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.

Biochemical and crystallographic characterization of the starch branching enzyme I (BEI) from Oryza sativa L.

Starch branching enzyme (SBE) catalyzes the cleavage of alpha-1.4-linkages and the subsequent transfer of alpha-1.4 glucan to form an alpha-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the alpha-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other alpha-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 A on a synchrotron X-ray source.

Inhibition of bcr-abl and/or c-abl gene expression by small interfering, double-stranded RNAs: cross-talk with cell proliferation factors and other oncogenes.

BACKGROUND: Short, 21-mer, double-stranded/small interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia (CML) with a potential also to target c-abl mRNA. METHODS: ds/siRNAs were transfected into bcr-abl-positive K-562 cells (derived from blast-crisis) or bcr-abl-negative/c-abl-positive Jurkat cells (derived from acute lymphoblastic leukemia) using lipofectamine. ds/siRNAs intracellular uptake was detected by fluorescent confocal microscopy using fluorescein-labeled ds/siRNAs. The treatment was performed over 6 days with repetitive siRNA transfections. Efficiency of the siRNAs was determined 24 hours after single siRNA transfection and 6 days after repetitive siRNA transfections. RESULTS: Two of the designed ds/siRNAs decreased the target mRNA levels markedly (determined by reverse transcriptase-polymerase chain reaction analysis) and bcr-abl/c-abl oncoproteins (determined by flow cytometry using Fluor-488-labeled, anti-c-abl antibody as well as by Western blot analysis). These sequences also inhibited protein tyrosine kinase activity significantly and suppressed cell proliferation. One of the three selected ds/siRNAs expressed only slight effects on the bcr-abl/c-abl mRNA in K-562 cells (but not on the oncoprotein level), on protein tyrosine kinase activity, and on cell proliferation. The combination of the three ds/siRNA constructs provoked stronger decreases in bcr-abl/c-abl mRNAs and their respective oncoproteins and produced the strongest suppression of cell proliferation. CONCLUSIONS: The cross-talk between siRNA interference of bcr-abl oncogene and the expression of several apoptotic/antiapoptotic factors, cell proliferation factors, and other oncogenes exists and it was determined by microarray analysis in K-562 cells that were treated over 6 days.