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Mizutani O.,Japanese National Research Institute of Brewing | Masaki K.,Japanese National Research Institute of Brewing | Gomi K.,Tohoku University | Iefuji H.,Japanese National Research Institute of Brewing
Applied and Environmental Microbiology | Year: 2012

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme. © 2012, American Society for Microbiology.


Koyama K.,Japanese National Research Institute of Brewing | Koyama K.,Hiroshima University | Sadamatsu K.,Hiroshima University | Goto-Yamamoto N.,Japanese National Research Institute of Brewing | Goto-Yamamoto N.,Hiroshima University
Functional and Integrative Genomics | Year: 2010

We investigated the effect of exogenous abscisic acid (ABA) application on the transcriptome as well as the phenolic profiles in the skins of Vitis vinifera cv. Cabernet Sauvignon grape berries grown on the vine and cultured in vitro. ABA application rapidly induced the accumulation of anthocyanin and flavonol. Correlatively, the structural genes in the phenylpropanoid and flavonoid pathways, their transcriptional regulators, as well as genes considered to be involved in the acylation and transport of anthocyanin into the vacuole, were upregulated by ABA treatment. The Genechip analysis showed that the ABA treatment significantly up- or downregulated a total of 345 and 1,482 transcripts in the skins of berries grown on the vine and cultured in vitro, respectively. Exogenous ABA modulated the transcripts associated with osmotic responses, stress responses, cell wall modification, auxin and ethylene metabolism and responses, in addition to the induction of anthocyanin biosynthetic genes, and reduced those associated with photosynthesis; approximately half of these transcripts were identical to the previously reported ripening-specific genes. © 2009 Springer-Verlag.


Kume K.,Hiroshima University | Koyano T.,Hiroshima University | Kanai M.,Japanese National Research Institute of Brewing | Toda T.,Cancer Research UK Research Institute | Hirata D.,Hiroshima University
Nature Cell Biology | Year: 2011

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis. © 2011 Macmillan Publishers Limited. All rights reserved.


Thongekkaew J.,Ubon Ratchathani University | Ikeda H.,Japanese National Research Institute of Brewing | Iefuji H.,Japanese National Research Institute of Brewing
Biochemical and Biophysical Research Communications | Year: 2012

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27. kDa, respectively. The fusion enzyme was stable at 80°C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization. © 2012 Elsevier Inc.


Koyama K.,Japanese National Research Institute of Brewing | Ikeda H.,Japanese National Research Institute of Brewing | Poudel P.R.,Japanese National Research Institute of Brewing | Goto-Yamamoto N.,Japanese National Research Institute of Brewing
Phytochemistry | Year: 2012

Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49 days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related gene more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed. © 2012 Elsevier Ltd. All rights reserved.


Hong S.-B.,National Academy of Agricultural Science | Yamada O.,Japanese National Research Institute of Brewing | Samson R.A.,Fungal Biodiversity Center
Applied Microbiology and Biotechnology | Year: 2014

Black koji molds including its albino mutant, the white koji mold, have been widely used for making the distilled spirit shochu in Northeast Asia because they produce citric acid which prevents undesirable contamination from bacteria. Since Inui reported Aspergillus luchuensis from black koji in Okinawa in 1901, many fungal names associated with black koji molds were reported. However, some species are similar and differentiation between species is difficult. Fungal taxonomists tried to arrange a taxonomic system for black koji molds, but the results were not clear. Recently, multi-locus sequence typing has been successfully used to taxonomy of black Aspergillus. According to β-tubulin and calmodulin gene sequences, black koji molds can be subdivided in three species, A. luchuensis, Aspergillus niger, and Aspergillus tubingensis. Aspergillus awamori, Aspergillus kawachii, Aspergillus inuii, Aspergillus nakazawai, and Aspergillus coreanus are synonyms of A. luchuensis, Aspergillus batatae, Aspergillus aureus (or Aspergillus foetidus), Aspergillus miyakoensis, and Aspergillus usamii (including A. usamii mut. shirousamii) are synonyms of A. niger and Aspergillus saitoi and A. saitoi var. kagoshimaensis are synonyms of A. tubingensis. A. luchuensis mut. kawachii was suggested particular names for A. kawachii because of their industrial importance. The history and modern taxonomy of black koji molds is further discussed. © 2013 Springer-Verlag Berlin Heidelberg.


Takahashi K.,Japanese National Research Institute of Brewing | Goto-Yamamoto N.,Japanese National Research Institute of Brewing
Journal of Chromatography A | Year: 2011

Free medium-chain fatty acids (MCFAs) can negatively influence the fermentation process and taste quality in alcoholic beverages. Ethyl hexanoate is important in providing a fruit-like flavour to drinks, particularly in Japanese sake. In this study, we developed a direct injection method for a gas chromatography-flame ionization detector following the semi-purification of chemical components, such as esters, alcohols and MCFAs in alcoholic beverages. Evaluation of MCFAs by this method gave a limit of detection on the order of sub-ppm and relative standard deviations less than 10% in standard solution. Good repeatability and recovery rates against MCFAs and ethyl hexanoate were also obtained in non-distilled real alcoholic beverages. Because this method enabled us to simultaneously quantify the concentrations of MCFAs and ethyl hexanoate, the proportion of ester against MCFAs was proposed as a quality control index. This method could be suitable for routine analysis in the alcohol beverage industry. © 2011 Elsevier B.V.


Thongekkaew J.,Ubon Ratchathani University | Ikeda H.,Japanese National Research Institute of Brewing | Masaki K.,Japanese National Research Institute of Brewing | Iefuji H.,Ehime University
Enzyme and Microbial Technology | Year: 2013

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application. © 2013 Elsevier Inc.


Horii S.,Japanese National Research Institute of Brewing | Goto K.,Japanese National Research Institute of Brewing
Journal of the Institute of Brewing | Year: 2010

An analytical method for the determination of ethyl carbamate in alcoholic beverages has been developed and optimized. A combination of headspace solid phase microextraction (HS-SPME), as the extraction technique, and GC/MS, as the determination technique, was utilized. Analytical grade ethyl carbamate dissolved in ethanol solution was analyzed to determine the optimum analytical conditions. Ethyl carbamate-d5 was added as an internal standard. The following HS-SPME conditions were investigated: type of stationary phase of the fibre, ethanol content, sample volume and extraction time. The optimized procedure showed that the detection limit, relative standard deviation, and recovery were 6.7 μg/L, 0.4-2.0%, and 103.6-107.6%, respectively. The precision of this new method was equivalent to previous analyses. Finally, the developed method was applied to the analysis of ethyl carbamate in sake. © 2010 The Institute of Brewing & Distilling.


Tsuji H.,Japanese National Research Institute of Brewing | Mizuno A.,Japanese National Research Institute of Brewing
Journal of Food Science | Year: 2010

Volatile compounds in beers brewed with different amounts of malt were analyzed by using the stir bar sorptive extraction-gas chromatography-mass spectrometry method. We identified 90 compounds - 25 esters, 17 terpenes, 14 alcohols, 11 acids, 6 furans, 6 aroma compounds, 5 carbonyls, and other compounds. An analysis of aged beer suggested that the concentration levels of stale flavor compounds - β-damascenone, γ-nonalactone, ethyl cinnamate, and 2-methoxy-4-vinylphenol - in nonmalt beer were different from those in all-malt and standard beer. Additionally, concentrations of these compounds did not increase during storage in most nonmalt beer analyzed in this study. Nerolidol may be a good marker candidate regardless of the malt content. © 2009 Institute of Food Technologists®.

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