Publications by authors named "Hiroki Ishida"

39 Publications

Mutation in gene coding for glucose-induced degradation-deficient protein contributes to high malate production in yeast strain No. 28 and No. 77 used for industrial brewing of sake.

Biosci Biotechnol Biochem 2021 Apr;85(5):1283-1289

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, Japan.

Saccharomyces cerevisiae produces organic acids including malate during alcohol fermentation. Since malate contributes to the pleasant flavor of sake, high-malate-producing yeast strain No. 28 and No. 77 have been developed by the Brewing Society of Japan. In this study, the genes responsible for the high malate phenotype in these strains were investigated. We had previously found that the deletion of components of the glucose-induced degradation-deficient (GID) complex led to high malate production in yeast. Upon examining GID protein-coding genes in yeast strain No. 28 and No. 77, a nonsense homozygous mutation of GID4 in strain No. 28 and of GID2 in strain No. 77 were identified as the cause of high malate production. Furthermore, complementary tests of these mutations indicated that the heterozygous nonsense mutation in GID2 was recessive. In contrast, the heterozygous nonsense mutation in GID4 was considered semidominant.
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http://dx.doi.org/10.1093/bbb/zbab031DOI Listing
April 2021

Polyoxometalates in Imidazolim-based Ionic Liquids: Acceptor Number and Polarity estimated from their Voltammetric Behaviour.

Anal Sci 2021 Jan 22. Epub 2021 Jan 22.

Department of Marine Resource Science, Faculty of Agriculture and Marine Sciences, Kochi University.

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http://dx.doi.org/10.2116/analsci.20P412DOI Listing
January 2021

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Cell 2020 11 6;183(5):1420-1435.e21. Epub 2020 Nov 6.

Department of Pulmonary Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.
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http://dx.doi.org/10.1016/j.cell.2020.10.023DOI Listing
November 2020

A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

Commun Biol 2020 10 20;3(1):592. Epub 2020 Oct 20.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-Ku, Sapporo, Hokkaido, 060-8638, Japan.

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.
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http://dx.doi.org/10.1038/s42003-020-01328-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576197PMC
October 2020

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing.

J Ind Microbiol Biotechnol 2020 Oct 3;47(9-10):715-723. Epub 2020 Aug 3.

Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.
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http://dx.doi.org/10.1007/s10295-020-02297-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658068PMC
October 2020

Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.

J Biosci Bioeng 2020 Sep 31;130(3):227-232. Epub 2020 May 31.

Research Institute, Gekkeikan Sake Co., Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto 612-8385, Japan.

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.
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http://dx.doi.org/10.1016/j.jbiosc.2020.04.013DOI Listing
September 2020

Effects of mutations of GID protein-coding genes on malate production and enzyme expression profiles in Saccharomyces cerevisiae.

Appl Microbiol Biotechnol 2020 Jun 4;104(11):4971-4983. Epub 2020 Apr 4.

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.

During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex-mediated degradation. Next, we investigated the effects of GID protein-coding genes (GID1-GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle-related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation. KEY POINTS: An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production. The protein expression profiles in the GID disruptants differed from one another.
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http://dx.doi.org/10.1007/s00253-020-10573-4DOI Listing
June 2020

Metabolism of Natural Highly Unsaturated Fatty Acid, Tetracosahexaenoic Acid (24:6n-3), in C57BL/KsJ-db/db Mice.

J Oleo Sci 2018 Dec 15;67(12):1597-1607. Epub 2018 Nov 15.

Department of Health and Nutrition Science, Nishikyushu University.

Tetracosahexaenoic acid (THA; 24:6n-3) is a natural, n-3 highly unsaturated fatty acid (n-3HUFA) that exists in fish, including Baltic herring (Clupea harengus) and the flathead flounder (Hippoglossoides dubius). In this study, natural n-3HUFAs, i.d. eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and THA were administrated to C57BL/KsJ-db/db mice for 4 weeks and the liver and serum lipid profiles, hepatic enzyme activity, expression of mRNA related to lipid metabolism, and adiponectin serum levels were then analyzed. The results showed that THA had the highest activity in suppressing hepatic triglyceride (TG) accumulation and increase in liver weight among the test groups. Furthermore, THA increased adiponectin levels in serum. These results indicate that THA is an excellent natural n-3HUFA that can suppress the development of metabolic syndromes and circulatory system diseases. The order of the n-3HUFA activity was THA > DHA > EPA in almost all the factors examined here. In a previous study of ours, the order was DHA > DPA > EPA, so the final order was summarized as THA > DHA > DPA > EPA. This order clearly translates to the rule that "the number of double bonds and carbon atoms in the n-3HUFA structure relates to their clinical functions".
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http://dx.doi.org/10.5650/jos.ess18167DOI Listing
December 2018

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Cell 2018 08;174(4):856-869.e17

Department of Gastroenterology, Keio University School of Medicine, Tokyo, 160-8582, Japan. Electronic address:

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.
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http://dx.doi.org/10.1016/j.cell.2018.07.027DOI Listing
August 2018

Super-low-frequency wireless power transfer with lightweight coils for passing through a stainless steel plate.

Rev Sci Instrum 2018 Mar;89(3):034706

Department of Electrical and Control Systems Engineering, National Institute of Technology, Toyama College, 13 Hongo, Toyama 939-8630, Japan.

To achieve wireless power transfer (WPT) through a stainless-steel plate, a super-low frequency (SLF) was used as a resonance frequency. In our previous study of SLF-WPT, heavy coils were prepared. In this study, we designed lightweight coils using a WPT simulator that we developed previously. As a result, the weight was reduced to 1.69 kg from 11.9 kg, the previous coil weight. At a resonance frequency of 400 Hz, the transmission efficiency and output power of advanced SLF-WPT reached 91% and 426 W, respectively, over a transmission distance of 30 mm. Furthermore, 80% efficiency and 317 W output were achieved when transmitting power through a 1 mm-thick stainless-steel plate. This performance is much better than that in previous reports. We show using both calculations and experimental results that a power-to-weight ratio of 252 W/kg is possible even when using a 400 Hz power supply frequency.
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http://dx.doi.org/10.1063/1.5010855DOI Listing
March 2018

A novel non-thermostable deuterolysin from Aspergillus oryzae.

Biosci Biotechnol Biochem 2016 Sep 6;80(9):1813-9. Epub 2016 Apr 6.

a Department of Applied Biological Chemistry, The Graduate School of Agriculture , Tokyo University of Agriculture and Technology , Fuchu , Tokyo , Japan.

Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.
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http://dx.doi.org/10.1080/09168451.2016.1166933DOI Listing
September 2016

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

Appl Microbiol Biotechnol 2016 Jun 5;100(11):4947-58. Epub 2016 Feb 5.

Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 1838509, Japan.

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.
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http://dx.doi.org/10.1007/s00253-016-7339-5DOI Listing
June 2016

Photochromic Properties of Tungsten Oxide/Methylcellulose Composite Film Containing Dispersing Agents.

ACS Appl Mater Interfaces 2015 Dec 17;7(47):26326-32. Epub 2015 Nov 17.

Division of Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University , Yamaguchi 753-8512, Japan.

Tungsten oxide-based photochromic films which changed reversibly in air between colorless- transparent in the dark and dark blue under UV irradiation were prepared by using methylcellulose as a film matrix and polyols such as ethylene glycol (EG), propylene glycol (PG), and glycerin (Gly) as dispersing agents. Influence of the dispersing agents and water in the films on the photochromic behavior was systematically studied. Under UV irradiation, absorption bands around 640 and 980 nm increased and the coloring rate was the following order: Gly > EG > PG. An increase in the amounts of dispersing agents or water accelerated the coloring rate. By increasing the water content of the film, a new absorption peak appeared at ca. 775 nm and the Raman spectra indicated a shift of W-O-W stretching vibration to lower wavenumber which was due to the formation of hydrogen bonding. All absorption spectra were fit by three Lorentz functions, whose bands were ascribed to various packing of WO6 octahedra. After the light was turned off, the formation of W(5+) was stopped and bleaching occurred by the reaction with O2 in air to recover its original transparent state. We anticipate that the biodegradable photochromic films developed in this study can be applied in recyclable display medium and especially in detachable films for glass windows whose light transmission properties are changed by sunlight, i.e., for usage as an alternative of smart windows without applying voltage.
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http://dx.doi.org/10.1021/acsami.5b09310DOI Listing
December 2015

Comparison of the lipid-lowering effects of four different n-3 highly unsaturated fatty acids in HepG2 cells.

J Oleo Sci 2014 10;63(10):979-85. Epub 2014 Sep 10.

Department of Applied Biochemistry and Food Science, Saga University.

The effects on lipid metabolism of four different n-3 highly unsaturated fatty acids (n-3HUFA) including eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and tetracosahexaenoic acid (THA, 24:6n-3) were compared in the HepG2 cell model. None of the n-3HUFAs affected the viability of the cells. THA exerted the strongest suppression on the synthesis of triacylglycerol and cholesteryl ester (ChE), and the order of the strength of suppression was found to be THA > DHA > DPA > EPA. The mRNA level of fatty acid synthase was suppressed by the n-3HUFAs and the order of the strength of suppression by n-3HUFAs was the same in both triacylglycerol and ChE synthesis. These findings support previous animal test results using EPA, DPA, and DHA. In conclusion, both the number of carbon atoms and double bonds in an n-3HUFA structure has an effect on lipid metabolism in HepG2 cells.
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http://dx.doi.org/10.5650/jos.ess14118DOI Listing
April 2015

Characterization of a (D)-stereoselective aminopeptidase (DamA) exhibiting aminolytic activity and halophilicity from Aspergillus oryzae.

Appl Biochem Biotechnol 2013 Sep 3;171(1):145-64. Epub 2013 Jul 3.

National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.
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http://dx.doi.org/10.1007/s12010-013-0330-zDOI Listing
September 2013

Identification of regulatory elements in the glucoamylase-encoding gene (glaB) promoter from Aspergillus oryzae.

Appl Microbiol Biotechnol 2013 Jun 9;97(11):4951-6. Epub 2012 Dec 9.

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, Kyoto 612-8385, Japan.

The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from -382 to -353 and from -332 to -313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using β-glucuronidase as a reporter. The results of the deletion analysis were consistent with the EMSA results. The promoter missing the -332 to -313 element was not induced by low water activity stress during SSC.
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http://dx.doi.org/10.1007/s00253-012-4622-yDOI Listing
June 2013

Serine-type carboxypeptidase KexA of Aspergillus oryzae has broader substrate specificity than Saccharomyces cerevisiae Kex1 and is required for normal hyphal growth and conidiation.

Appl Environ Microbiol 2012 Nov 7;78(22):8154-7. Epub 2012 Sep 7.

Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.

Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1, termed kexA. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.
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http://dx.doi.org/10.1128/AEM.01601-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485961PMC
November 2012

Comparison of expression and enzymatic properties of Aspergillus oryzae lysine aminopeptidases ApsA and ApsB.

World J Microbiol Biotechnol 2012 Aug 11;28(8):2643-50. Epub 2012 May 11.

Applied Microbiology Division, NARO, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.

The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.
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http://dx.doi.org/10.1007/s11274-012-1074-6DOI Listing
August 2012

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Appl Microbiol Biotechnol 2013 Jan 3;97(2):761-6. Epub 2012 Jul 3.

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, Kyoto 612-8385, Japan.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.
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http://dx.doi.org/10.1007/s00253-012-4211-0DOI Listing
January 2013

Actual ratio of triacylglycerol positional isomers in milk and cheese.

J Oleo Sci 2012 ;61(4):173-80

Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan.

Actual ratios of triacylglycerol (TAG) positional isomers in human, rat, and cow milk fat and cow, buffalo, goat, and sheep cheese fat were analyzed using HPLC-UV-atmospheric pressure chemical ionization-MS/MS system equipped with an octacosyl silylation column or polymeric ODS column. We substituted cheese fats for milk fats in parts of our study because milks from ruminants, with the exception of cows, are difficult to get in Japan. The actual ratio of β-PPC (the TAG consisting of two palmitic acids (P) and one capric acid (C), with the palmitic acid located at the β position) and β-PCP in human milk was different from those in ruminants, with more than half of the medium-chain fatty acids located at the β position even though other fats possessed it mainly at the α position. Palmitic acid was mainly located at the β position for human milk and rat milk; however, the location in ruminant cheese fat was mainly at the α position. The location of fatty acids is thought to be very important for infant nutrition. Particularly, the location of palmitic acid in case of human milk and of medium-chain fatty acids in case of ruminant milk was very characteristic and is considered to be very important to the fatty acids in milk fat.
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http://dx.doi.org/10.5650/jos.61.173DOI Listing
July 2012

Enzymatic properties of the glycine D-alanine [corrected] aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji).

Appl Microbiol Biotechnol 2012 Jan 18;93(2):655-69. Epub 2011 Oct 18.

Applied Microbiology Division, National Food Research Institute, 2-1-12 Kan-nondai, Tsukuba, Ibaraki, 305-8642, Japan.

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.
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http://dx.doi.org/10.1007/s00253-011-3610-yDOI Listing
January 2012

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination.

Plant Cell 2011 Sep 6;23(9):3215-29. Epub 2011 Sep 6.

National Institute of Crop Science, Tsukuba 305-8518, Japan.

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.
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http://dx.doi.org/10.1105/tpc.111.088492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203438PMC
September 2011

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

Appl Microbiol Biotechnol 2011 Nov 6;92(3):561-9. Epub 2011 Jul 6.

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.
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http://dx.doi.org/10.1007/s00253-011-3446-5DOI Listing
November 2011

Enzymatic properties of the recombinant serine-type carboxypeptidase OcpC, which is unique to Aspergillus oryzae.

Biosci Biotechnol Biochem 2011 22;75(4):662-8. Epub 2011 Apr 22.

Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Japan.

Gene AO090103000153 is unique to Aspergillus oryzae RIB40 and A. flavus NRRL3357, and is speculated to encode a serine-type carboxypeptidase. In this study, we purified and characterized a heterologously expressed gene product of AO090103000153. 5'-Rapid amplification of cDNA ends indicated that the translation start site of the gene is located 1,586 bp downstream of the translation start site predicted by the genome sequencing project. The gene, starting from the revised translation start codon, termed ocpC, was transcribed constantly in A. oryzae RIB40. Purified recombinant OcpC exhibited the enzymatic properties of a serine-type carboxypeptidase. This protease was stable at temperatures below 45°C and a low pH, and had broad substrate specificity for N-acylpeptides, but it exhibited significantly lower specific activity and a lower k(cat) value for substrates than previously reported serine-type carboxypeptidases from A. oryzae.
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http://dx.doi.org/10.1271/bbb.100749DOI Listing
September 2011

Characterization of an Aspergillus oryzae cysteinyl dipeptidase expressed in Escherichia coli.

Biosci Biotechnol Biochem 2011 7;75(1):159-61. Epub 2011 Jan 7.

National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.

Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.
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http://dx.doi.org/10.1271/bbb.100604DOI Listing
April 2011

Overexpression and characterization of an extracellular leucine aminopeptidase from Aspergillus oryzae.

Curr Microbiol 2011 Feb 28;62(2):557-64. Epub 2010 Aug 28.

National Food Research Institute, Tsukuba, Ibaraki, 305-8642, Japan.

Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.
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http://dx.doi.org/10.1007/s00284-010-9744-9DOI Listing
February 2011

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module.

Appl Microbiol Biotechnol 2010 Aug 25;87(5):1783-9. Epub 2010 May 25.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.
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http://dx.doi.org/10.1007/s00253-010-2664-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903697PMC
August 2010

Molecular cloning of ocpO encoding carboxypeptidase O of Aspergillus oryzae IAM2640.

Biosci Biotechnol Biochem 2010 7;74(5):1000-6. Epub 2010 May 7.

Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.

Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.
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http://dx.doi.org/10.1271/bbb.90863DOI Listing
September 2010

Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae.

Appl Microbiol Biotechnol 2009 Nov 26;85(2):335-46. Epub 2009 Jun 26.

Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45 degrees C, 55 degrees C, and 55 degrees C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.
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http://dx.doi.org/10.1007/s00253-009-2087-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773364PMC
November 2009

Highly sensitive and accurate profiling of carotenoids by supercritical fluid chromatography coupled with mass spectrometry.

J Sep Sci 2009 May;32(9):1459-64

Department of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences Osaka University, Suita, Osaka, Japan.

We attempted to establish a high-speed and high-resolution profiling method for a carotenoid mixture as a highly selective and highly sensitive detection method; the analysis was carried out by supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS). When an octadecyl-bonded silica (ODS) particle-packed column was used for separation, seven carotenoids including structural isomers were successfully separated within 15 min. This result indicated not only improved separation but also improved throughput compared to the separation and throughput in RP-HPLC. The use of a monolithic ODS column resulted in additional improvement in both the resolution and the throughput; the analysis time was reduced to 4 min by increasing the flow rate. Furthermore, carotenoids in biological samples containing the complex matrices were separated effectively by using several monolithic columns whose back pressure was very low. The mass spectrometer allowed us to perform a more sensitive analysis than UV detection; the detection limit of each carotenoid was 50 pg or below. This is the first report of carotenoid analysis carried out by SFC-MS. The profiling method developed in this study will be a powerful tool for carrying out accurate profiling of biological samples.
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http://dx.doi.org/10.1002/jssc.200800699DOI Listing
May 2009