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ABSTRACT: The basidiomycetous yeast Pseudozyma antarctica T-34 is an excellent producer of mannosylerythritol lipids (MELs), members of the multifunctional extracellular glycolipids, from various feedstocks. Here, the genome sequence of P. antarctica T-34 was determined and annotated. Analysis of the sequence might provide insights into the properties of this yeast that make it superior for use in the production of functional glycolipids, leading to the further development of P. antarctica for industrial applications.
Genome announcements. 01/2013; 1(2):e0006413.
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ABSTRACT: 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.
Applied Microbiology and Biotechnology 12/2012; · 3.42 Impact Factor
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Myco Umemura,
Hideaki Koike,
Noriko Yamane,
Yoshinori Koyama,
Yuki Satou,
Ikuya Kikuzato,
Morimi Teruya,
Masatoshi Tsukahara,
Yumi Imada,
Youji Wachi,
Yukino Miwa,
Shuichi Yano,
Koichi Tamano,
Yutaka Kawarabayasi,
Kazuhiro E Fujimori, Masayuki Machida,
Takashi Hirano
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ABSTRACT: Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.
DNA Research 08/2012; 19(5):375-82. · 5.16 Impact Factor
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ABSTRACT: Microbial production of fats and oils is being developed as a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillus oryzae. Examination of the A. oryzae genome demonstrates that it contains two fatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhanced the expression of fatty acid synthesis-related genes by replacing their promoters with the promoter from the constitutively highly expressed gene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthase genes we successfully increased the production of fatty acids and triglycerides by more than two-fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesterase increased productivity to a lesser extent. Increasing expression of acetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored using quantitative real-time reverse transcription polymerase chain reaction. Our data demonstrate that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.
Applied Microbiology and Biotechnology 06/2012; · 3.42 Impact Factor
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ABSTRACT: Aspergillus oryzae is considered to be an attractive host for heterologous protein production because of its safety and ability to secrete large
amounts of proteins. In order to obtain high productivity, thus far promoters of amylases have been most widely used inA. oryzae. Recent progress in cloning and expression analysis, including EST sequencing, revealed that glycolytic genes represent some
of those most strongly expressed inA. oryzae. Therefore, promoters of glycolytic genes could be important alternatives to promoters of amylases because lower amounts
of proteases are produced in the presence of glucose. SeveralA. oryzae transcription factors responsible for the induction and/or maximum expression of many industrially important genes encoding
amylases and proteases have been cloned and characterized. In addition to the transcriptional regulatory factors, the gene
encoding the largest subunit of RNA polymerase II, constituting the basic transcription machinery, has also been cloned fromA. orzae. This recently acquired understanding of the details of transcriptional regulatory mechanisms and factors will facilitate
engineering flexible controls for the expression of proteins important for the fermentation industries.
Biotechnology and Bioprocess Engineering 04/2012; 5(4):253-262. · 1.28 Impact Factor
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ABSTRACT: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method "cDNA display". In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.
Biochemical and Biophysical Research Communications 04/2012; 421(1):129-33. · 2.48 Impact Factor
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Junichiro Marui,
Noriko Yamane,
Sumiko Ohashi-Kunihiro,
Tomohiro Ando,
Yasunobu Terabayashi,
Motoaki Sano,
Shinichi Ohashi,
Eiji Ohshima,
Kuniharu Tachibana,
Yoshitaka Higa,
Marie Nishimura,
Hideaki Koike, Masayuki Machida
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ABSTRACT: A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA.
Journal of Bioscience and Bioengineering 04/2011; 112(1):40-3. · 1.79 Impact Factor
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ABSTRACT: Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution.
We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function.
This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.
Molecular Brain 01/2011; 4:2.
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Yasunobu Terabayashi,
Motoaki Sano,
Noriko Yamane,
Junichiro Marui,
Koichi Tamano,
Junichi Sagara,
Mitsuko Dohmoto,
Ken Oda,
Eiji Ohshima,
Kuniharu Tachibana,
Yoshitaka Higa,
Shinichi Ohashi,
Hideaki Koike, Masayuki Machida
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ABSTRACT: Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism.
Fungal Genetics and Biology 12/2010; 47(12):953-61. · 3.74 Impact Factor
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ABSTRACT: Aspergillus oryzae penicillin biosynthetic genes were clustered. The penicillin production was positively regulated by VeA, a global gene regulator required for transcriptional expression of the penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production.
Journal of Bioscience and Bioengineering 07/2010; 110(1):8-11. · 1.79 Impact Factor
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Junichiro Marui,
Akira Yoshimi,
Daisuke Hagiwara,
Yoshimi Fujii-Watanabe,
Ken Oda,
Hideaki Koike,
Koichi Tamano,
Tomoko Ishii,
Motoaki Sano, Masayuki Machida,
Keietsu Abe
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ABSTRACT: Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.
Applied Microbiology and Biotechnology 05/2010; 87(5):1829-40. · 3.42 Impact Factor
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ABSTRACT: We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.
Applied and environmental microbiology 08/2009; 75(18):5943-51. · 3.69 Impact Factor
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ABSTRACT: We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.
Nucleic Acids Research 07/2009; 37(16):e108. · 8.03 Impact Factor
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Kiyoshi Migita,
Kazumi Sawakami-Kobayashi,
Yumi Maeda,
Kazuhiko Nakao,
Susumu Kondoh,
Mika Sugiura,
Ryoko Kawasumi,
Osamu Segawa,
Hideji Tajima, Masayuki Machida,
Minoru Nakamura,
Koji Yano,
Seigo Abiru,
Eiji Kawasaki,
Hiroshi Yatsuhashi,
Katsumi Eguchi,
Hiromi Ishibashi
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ABSTRACT: In this study, we aimed to explore whether interleukin-18 (IL-18) gene-promoter polymorphisms are associated with the outcome of hepatitis B virus (HBV) infection. In all, 204 chronically HBV-infected patients were recruited in this study. Of the 204 HBV-infected patients, 43 were considered to be inactive HBV carriers based on the sustained normalization of serum alanine aminotransferase (ALT) together with seropositivity for the antibody to hepatitis B e-antigen (anti-HBe). A total of 161 patients were found to have chronic progressive liver disease, which included cirrhosis. In these HBV-infected patients, the frequencies of AA genotype of IL-18 gene-promoter polymorphisms at position -607 and C allele at position -137 were significantly higher in inactive HBV carriers compared with those in patients with chronic progressive liver disease. These polymorphisms of the IL-18 promoter regions (-607 and -137) could be associated with different outcomes of HBV infection.
Translational Research 03/2009; 153(2):91-6. · 2.99 Impact Factor
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ABSTRACT: We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70(+) strain due to the low frequency of homologous recombination were efficiently obtained from Delta ku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.
Applied and environmental microbiology 11/2008; 74(24):7684-93. · 3.69 Impact Factor
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ABSTRACT: Aspergillus flavus is a weak pathogen that infects plants, animals and humans. When it infects agricultural crops, however, it produces one of the most potent carcinogens known (aflatoxins). To devise strategies to control aflatoxin contamination of pre-harvest agricultural crops and post-harvest grains during storage, we launched the A. flavus genomics program. The major objective of this program is the identification of genes involved in aflatoxin biosynthesis and regulation, as well as in pathogenicity, to gain a better understanding of the mechanism of aflatoxin formation. The sequencing of A. flavus whole genome has been completed. Initial annotation of the sequence revealed that there are about 13,071 genes in the A. flavus genome. Genes which potentially encode for enzymes involved in secondary metabolite production in the A. flavus genome have been identified. Preliminary comparative genome analysis of A. flavus with A. oryzae is summarized here.
Food Additives & Contaminants: Part A 10/2008; 25(9):1152-7. · 1.76 Impact Factor
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ABSTRACT: Aspergillus oryzae has been used in Japanese fermentation industries for more than a thousand years. The species produces large amounts of various hydrolytic enzymes and has been successfully applied to modern biotechnology. The size of the A. oryzae genome (37.5 Mb) is very close to that of A. flavus and A. niger, and 20-30% larger than that of either A. nidulans or A. fumigatus. A. oryzae and A. flavus have exactly the same number of aspartic proteinase genes, of which each orthologous pair shares highly conserved amino acid sequences. Synteny analysis with A. fumigatus and A. nidulans showed that the A. oryzae genome has a mosaic structure consisting of syntenic and non-syntenic blocks. In the microorganisms to be compared, the density of the genes having homologs was obviously higher on the syntenic than on the non-syntenic blocks. Expression analysis by the DNA microarray supported the significantly lower expression of genes on the non-syntenic than on the syntenic blocks.
Food Additives & Contaminants: Part A 10/2008; 25(9):1147-51. · 1.76 Impact Factor
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ABSTRACT: At a time when the notion of microorganisms did not exist, our ancestors empirically established methods for the production of various fermentation foods: miso (bean curd seasoning) and shoyu (soy sauce), both of which have been widely used and are essential for Japanese cooking, and sake, a magical alcoholic drink consumed at a variety of ritual occasions, are typical examples. A filamentous fungus, Aspergillus oryzae, is the key organism in the production of all these traditional foods, and its solid-state cultivation (SSC) has been confirmed to be the secret for the high productivity of secretory hydrolases vital for the fermentation process. Indeed, our genome comparison and transcriptome analysis uncovered mechanisms for effective degradation of raw materials in SSC: the extracellular hydrolase genes that have been found only in the A. oryzae genome but not in A. fumigatus are highly induced during SSC but not in liquid cultivation. Also, the temperature reduction process empirically adopted in the traditional soy-sauce fermentation processes has been found to be important to keep strong expression of the A. oryzae-specific extracellular hydrolases. One of the prominent potentials of A. oryzae is that it has been successfully applied to effective degradation of biodegradable plastic. Both cutinase, responsible for the degradation of plastic, and hydrophobin, which recruits cutinase on the hydrophobic surface to enhance degradation, have been discovered in A. oryzae. Genomic analysis in concert with traditional knowledge and technology will continue to be powerful tools in the future exploration of A. oryzae.
DNA Research 09/2008; 15(4):173-83. · 5.16 Impact Factor
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Koichi Tamano,
Motoaki Sano,
Noriko Yamane,
Yasunobu Terabayashi,
Tomomi Toda,
Misao Sunagawa,
Hideaki Koike,
Osamu Hatamoto,
Genryou Umitsuki,
Tadashi Takahashi,
Yasuji Koyama,
Ryoichi Asai,
Keietsu Abe, Masayuki Machida
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ABSTRACT: Transcriptome analysis revealed close relationship between solid-state cultivation and the transcriptional regulation of the genes on the non-syntenic blocks (NSBs), which were characterized by the comparison of Aspergillus oryzae genome with those of Aspergillus fumigatus and Aspergillus nidulans. Average expression ratio of the genes on NSBs in solid-state cultivation was significantly higher than that on the syntenic blocks (SBs). Of the induced genes, the genes relating to metabolism, which are highly enriched on NSBs, most contributed to the NSB-specific induction. The analysis using the SB- and NSB-genes that had sequence similarity between the two blocks significantly decreased the difference of average expression ratios between the two blocks. In spite of remarkably high averaged expression ratio of the NSB genes encoding extracellular enzymes, no induction of PKS and NRPS genes on NSBs were observed in solid-state cultivations. These results strongly suggest that the genes on NSBs play an important role on solid-state fermentation.
Fungal Genetics and Biology 03/2008; 45(2):139-51. · 3.74 Impact Factor
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ABSTRACT: The catalase-encoding gene (catB) is expressed strongly in Aspergillus oryzae. To identify the transcription regulatory elements involved in strong expression, we did promoter deletion analysis using beta-glucuronidase (GUS) as a reporter and an electrophoretic gel mobility shift assay (EMSA) systematically. The deletion 200-bp sequence from -1,000 to -800 in the 1,400-bp catB promoter caused a drastic decrease in GUS activity. In addition, EMSA implicated a 45-bp element from -1,000 to -956 containing cis-elements. According to detailed promoter deletion analysis, a region from -1,000 to -975, which contains putative heat shock element (HSE) and the CCAAT-box, was involved in strong expression.
Bioscience Biotechnology and Biochemistry 02/2008; 72(1):48-53. · 1.28 Impact Factor
