Goro Taguchi

Shinshu University, Shonai, Nagano, Japan

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Publications (37)91.36 Total impact

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    ABSTRACT: C-glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C-glycosylflavonoids; however, the genes encoding for enzymes that catalyze C-glycosylation of flavonoids have been identified only from rice and maize, and have not been identified from dicot plants. In this study, we identified C-glucosyltransferase gene from a dicot plant, buckwheat (Fagopyrum esculentum M.). We purified two isozymes from buckwheat seedlings that catalyze C-glucosylation of 2-hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C-glucosylation activity toward 2-hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones, and other related compounds with chemical structures similar to 2',4',6'-trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C-glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases. This article is protected by copyright. All rights reserved.
    The Plant Journal 08/2014; · 6.58 Impact Factor
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    ABSTRACT: A full-length cDNA coding for a putative adenosine deaminase (Fv-ada) was isolated from the basidiomycete Flammulina velutipes. Fv-ada encodes a polypeptide consisting of 537 amino acid residues, which has a consensus sequence conserved among adenosine deaminase-related growth factors (ADGF) found in several metazoa, including chordates and insects. Fv-ada transcript was detected at all stages of growth in dikaryotic F. velutipes cells, with a peak at the primordial stage. Heterologous expression of Fv-ada in the yeast Pichia pastoris produced recombinant Fv-ADA that catalyzed the conversion of adenosine to inosine. Dikaryotic mycelia from F. velutipes were transformed with the binary plasmid pFungiway-Fv-ada, which was designed to suppress the expression of Fv-ada through RNA interference. The growth rates of the resulting transformants were retarded in response to the degree of suppression, indicating that Fv-ada plays an important role in the mycelial growth of F. velutipes. These results suggested that ADGF could function as growth factors in fungi, as is seen in other eukaryotes.
    Journal of Bioscience and Bioengineering 11/2012; · 1.74 Impact Factor
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    ABSTRACT: Plant HXXXD acyltransferase-catalyzed malonylation is an important modification reaction in elaborating the structural diversity of flavonoids and anthocyanins, and a universal adaptive mechanism to detoxify xenobiotics. Nicotiana tabacum malonyltransferase 1 (NtMaT1) is a member of anthocyanin acyltransferase subfamily that uses malonyl-CoA (MLC) as donor catalyzing transacylation in a range of flavonoid and naphthol glucosides. To gain insights into the molecular basis underlying its catalytic mechanism and versatile substrate specificity, we resolved the X-ray crystal structure of NtMaT1 to 3.1 Å resolution. The structure comprises two α/β mixed subdomains, as typically found in the HXXXD acyltransferases. The partial electron density map of malonyl-CoA allowed us to reliably dock the entire molecule into the solvent channel and subsequently define the binding sites for both donor and acceptor substrates. MLC bound to the NtMaT1 occupies one end of the long solvent channel between two subdomains. On superimposing and comparing the structure of NtMaT1 with that of an enzyme from anthocyanin acyltransferase subfamily from red chrysanthemum (Dm3Mat3) revealed large architectural variation in the binding sites, both for the acyl donor and for the acceptor, although their overall protein folds are structurally conserved. Consequently, the shape and the interactions of malonyl-CoA with the binding sites' amino acid residues differ substantially. These major local architectural disparities point to the independent, divergent evolution of plant HXXXD acyltransferases in different species. The structural flexibility of the enzyme and the amendable binding pattern of the substrates provide a basis for the evolution of the distinct, versatile substrate specificity of plant HXXXD acyltransferases.
    Planta 05/2012; 236(3):781-93. · 3.38 Impact Factor
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    ABSTRACT: Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.
    Bioscience Biotechnology and Biochemistry 01/2012; 76(3):517-22. · 1.27 Impact Factor
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    ABSTRACT: A gene coding for a major extracellular chitosanase was isolated from Aspergillus oryzae IAM2660. It had a multi-domain structure composed of a signal peptide, a catalytic domain, Thr- and Pro-rich linkers, and repeated peptides (the R3 domain) from the N-terminus. The R3 domain bound to insoluble powder chitosan, but it did not promote the hydrolysis rate of the chitosanase to any extent.
    Bioscience Biotechnology and Biochemistry 01/2012; 76(1):193-5. · 1.27 Impact Factor
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    ABSTRACT: Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.
    Journal of Bioscience and Bioengineering 12/2011; 113(3):293-9. · 1.74 Impact Factor
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    ABSTRACT: Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.
    Bioscience Biotechnology and Biochemistry 11/2010; 74(11):2327-9. · 1.27 Impact Factor
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    ABSTRACT: Tobacco cells (Nicotiana tabacum L.) accumulate harmful naphthols in the form of malonylated glucosides (Taguchi et al., 2005). Here, we showed that the malonylation of glucosides is a system to metabolize xenobiotics and is common to higher plants. Moreover, some plantlets including Arabidopsis thaliana excreted some of the incorporated naphthols into the culture media as their glucosides. In order to analyze the function of malonylation in the metabolism of these xenobiotics, we identified a malonyltransferase gene (At5g39050) responsible for the malonylation of these compounds in A. thaliana. The recombinant enzyme had malonyltransferase activity toward several phenolic glucosides including naphthol glucosides. A knockout mutant of At5g39050 (pmat1) exposed to naphthols accumulated only a few malonylglucosides in the cell, and released larger amounts of simple glucosides into the culture medium. In contrast, forced expression of At5g39050 in the pmat1 mutant resulted in increased malonylglucoside accumulation and decreased glucoside excretion to the media. The results provided clear evidence of whether the release of glucosides or the storage of malonylglucosides was determined by the At5g39050 expression level. A similar event in naphthol metabolism was observed in the tobacco mutant with a suppressed malonyltransferase gene (NtMaT1). These results suggested that malonylation could be a key reaction to separate the way of xenobiotics disposition, that is, release from cell surface or storage in vacuoles.
    The Plant Journal 09/2010; 63(6):1031-41. · 6.58 Impact Factor
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    ABSTRACT: The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments.
    Journal of Bioscience and Bioengineering 05/2010; 109(5):472-8. · 1.74 Impact Factor
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    ABSTRACT: A stable bacterial community expressing strong chitinolytic activity was constructed by mixing and cultivating chitinolytic bacteria collected from different natural sources. The DNA fragment pattern, after PCR-denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes using total DNA prepared from whole cells, indicated that the community was composed of four dominant bacterial species. All four species were isolated on agar medium, and one strain, SAY3, was deduced to be a novel species belonging to a new genus based on the 16S rRNA nucleotide sequence. The other strains showed high similarity in their 16S rRNA sequences to those of previously identified bacteria (Acinetobacter and Microbacterium). Strain SAY3 degraded and utilized larger particles of chitin faster than the community, indicating that it plays an important role in the chitin degradation conferred by the community.
    Bioscience Biotechnology and Biochemistry 03/2010; 74(3):636-40. · 1.27 Impact Factor
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    ABSTRACT: The filamentous fungus Coleophoma empetri F-11899 produces an echinocandin-like compound FR901379, the original source for micafungin which is prescribed to treat deep-seated mycoses. Despite its industrial importance, no genetic information on C. empetri F-11899 is currently available. To characterize FR901379 biosynthetic genes by insertional mutagenesis and to improve the compound production genetically, Agrobacterium tumefaciens-mediated transformation (ATMT) was attempted to make genetic manipulation possible in this strain. The optimum conditions for ATMT of C. empetri were determined for the cell density of bacteria, time period of co-cultivation and types of filters in co-cultivation. Using the established ATMT method, the hygromycin B resistant gene was successfully transferred into the genome of C. empetri F-11899 and stably maintained even after a serial passage. Some of these results will be applicable for ATMT of various filamentous fungi.
    Current Genetics 10/2009; 55(6):623-30. · 2.41 Impact Factor
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    ABSTRACT: A bacterial strain capable of degrading chitin, strain SAY3T, was isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. The strain was gram-negative, curved rod-shaped, facultatively anaerobic, and motile with a single polar flagellum. It grew well with chitin as a sole carbon source. The cellular fatty acids profiles showed the presence of C16:1 omega7c and C16:0 as the major components. The G+C content of DNA was 67.6 mol% and Q-8 was the major respiratory quinone. A 16S rRNA gene sequence-based phylogenetic analysis showed the strain belonged to the family Neisseriaceae but was distantly related (94% identity) to any previously known species. Since the strain was clearly distinct from closely related genera in phenotypic and chemotaxonomic characteristics, it should be classified under a new genus and a new species. We propose the name Chitiniphilus shinanonensis gen. nov., sp. nov. The type strain is SAY3T (=NBRC 104970T=NICMB 14509T).
    The Journal of General and Applied Microbiology 05/2009; 55(2):147-53. · 0.74 Impact Factor
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    ABSTRACT: Fv-pda, a gene coding for chitin deacetylase (CDA), was isolated from the basidiomycete Flammulina velutipes by differential display targeted for genes specifically expressed during fruiting body development. The fv-pda ORF comprises 250 amino acid residues and is interrupted by 10 introns. The fv-pda cDNA was expressed in the yeast Pichia pastoris, and the resulting recombinant FV-PDA was used for enzymatic characterization. The recombinant FV-PDA catalyses deacetylation of N-acetyl-chitooligomers, from dimer to pentamer, glycol chitin and colloidal chitin. The fv-pda was specifically expressed through the entire stage of fruiting body development, and the transcript was abundant in stipes of mature fruiting bodies. These results suggest that CDA plays an important role in the process of fruiting of F. velutipes.
    FEMS Microbiology Letters 01/2009; 289(2):130-7. · 2.05 Impact Factor
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    ABSTRACT: A gene (cob) conferring red fluorescence on Escherichia coli cells was isolated from an environmental DNA library constructed from soil bacteria. The nucleotide sequence showed a single open reading frame (ORF) encoding a polypeptide of 257 amino acid residues responsible for the red fluorescence. The deduced amino acid sequence of the ORF showed significant similarity (less than 75% identity) to uroporphyrinogen III methyltransferases from various bacteria. Cell extracts from the E. coli transformant had a spectrum pattern of fluorescence corresponding to trimethylpyrrocorphin or sirohydrochlorin that was absent in the control cells harboring the vector alone. Expression of the cob gene in the fungus Fusarium oxysporum conferred red fluorescence on the host cells, indicating that it is a promising transcriptional reporter in fungi as well as bacteria.
    Bioscience Biotechnology and Biochemistry 08/2008; 72(7):1908-14. · 1.27 Impact Factor
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    ABSTRACT: A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines--T136-8, K10, and C9--which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.
    Theoretical and Applied Genetics 08/2008; 117(5):759-67. · 3.66 Impact Factor
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    ABSTRACT: A restriction fragment length polymorphism (RFLP) marker, HC352b, has identified as closely linked to the CRa locus responsible for clubroot resistance (CR) in a study dissecting CR loci in Chinese cabbage (Brassica rapa L. ssp pekinensis). Unfortunately, the RFLP pattern, including HC352b detected by the cDNA clone HC352, was complicated and confused in its interpretation because it represented multiple-copy loci. To provide a practical HC352b marker for CR breeding programs, a sequence characterized amplified region (SCAR) marker was constructed along the analysis of HC352 genes, followed by its evaluation for CR selection. Schematic characterization of signals was achieved by identification of HC352 homologous genes with their restriction sites, employing CRa-positive and -negative doubled haploid (DH) lines. Genomic sequence information from a set of HC352 homologous genes was analyzed to identify the CRa linked paralog HC352b, followed by the successful designation of a SCAR marker. This correctly predicted the CR phenotypes of all tested individuals of F2 and back-crossed progenies.
    01/2008;
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    ABSTRACT: Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.
    The Plant Journal 05/2007; 50(4):678-95. · 6.58 Impact Factor
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    ABSTRACT: Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.
    FEMS Microbiology Letters 02/2006; 254(1):165-72. · 2.05 Impact Factor
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    ABSTRACT: Tobacco cells (Nicotiana tabacum L. Bright Yellow T-13) exposed to harmful naphthols accumulate them as glucosylated and further modified compounds [Taguchi et al. (2003a) Plant Sci. 164, 231-240]. In this study, we identified the accumulated compounds to be 6'-O-malonylated glucosides of naphthols. Cells treated with various phenolic compounds accumulated the flavonoids mainly as malonylglucosides. To clarify the function of this malonylation in tobacco, we isolated the cDNA encoding a malonyltransferase (NtMaT1) from a cDNA library derived from tobacco cells. The heterologous expression of the gene in Escherichia coli revealed that the recombinant enzyme had malonyltransferase activity against several phenolic glucosides such as flavonoid 7-O-glucosides, flavonoid 3-O-glucosides and naphthol glucosides. The substrate preference of the enzyme was similar to that of the tobacco cell extract. Malonylation activity in the transgenic cells markedly decreased with the suppression of the expression of NtMaT1 mRNA in tobacco BY-2 cells by RNA interference. The compounds administered to the transgenic cells were accumulated in the cells as glucosides or other modified compounds in place of malonylglucosides. These results show that NtMaT1 is the main catalyst of malonylation on glucosides of xenobiotic flavonoids and naphthols in tobacco plants.
    The Plant Journal 06/2005; 42(4):481-91. · 6.58 Impact Factor
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    ABSTRACT: We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that the pcb2 mutant accumulated divinyl chlorophylls, and the relative amount of divinyl chlorophyll b was remarkably less than that of divinyl chlorophyll a. The responsible gene was mapped in an area of 190 kb length at the upper arm of the 5th chromosome, and comparison of DNA sequences revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis confirmed that the wild-type of this gene suppressed the phenotypes of the mutation. Antisense transformants of the gene also accumulated divinyl chlorophylls. The genes homologous to At5g18660 are conserved in a broad range of species in the plant kingdom, and have similarity to reductases. Our results suggest that the PCB2 product is divinyl protochlorophyllide 8-vinyl reductase.
    Plant and Cell Physiology 04/2005; 46(3):467-73. · 4.98 Impact Factor