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Miyanaga A.,Tokyo Institute of Technology | Hayakawa Y.,Tokyo Institute of Technology | Numakura M.,Tokyo Institute of Technology | Hashimoto J.,Japan Biological Informatics Consortium | And 6 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2016

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for L-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2. © 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry.

Hashimoto T.,University of Tokyo | Hashimoto J.,Japan Biological Informatics Consortium | Teruya K.,Okinawa Institute of Advanced science | Hirano T.,Okinawa Institute of Advanced science | And 5 more authors.
Journal of the American Chemical Society | Year: 2015

Versipelostatin (VST) is an unusual 17-membered macrocyclic polyketide product that contains a spirotetronate skeleton. In this study, the entire VST biosynthetic gene cluster (vst) spanning 108 kb from Streptomyces versipellis 4083-SVS6 was identified by heterologous expression using a bacterial artificial chromosome vector. Here, we demonstrate that an enzyme, VstJ, catalyzes the stereoselective [4+2]-cycloaddition between the conjugated diene and the exocyclic olefin of a newly identified tetronate-containing intermediate to form the spirotetronate skeleton during VST biosynthesis. © 2014 American Chemical Society.

Yohda M.,Tokyo University of Agriculture and Technology | Yagi O.,Nihon University | Takechi A.,Tokyo University of Agriculture and Technology | Kitajima M.,Tokyo University of Agriculture and Technology | And 18 more authors.
Journal of Bioscience and Bioengineering | Year: 2015

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. © 2014 The Society for Biotechnology, Japan.

Murata R.,University of Ryukyus | Kobayashi Y.,University of Ryukyus | Kobayashi Y.,Okinawa Institute of Advanced science | Kobayashi Y.,Okayama University | And 4 more authors.
General and Comparative Endocrinology | Year: 2012

The aim of this study was to clarify the roles of 2 gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus. To do this, the mRNA expression patterns of GTH subunits (cga, fshb, and lhb) in the fish pituitary throughout gonadal sex differentiation were investigated. Real-time reverse transcriptase (RT)-PCR showed that cga and fshb were present in the undifferentiated and ovarian differentiation stages, and that the expression levels significantly increased after ovarian differentiation (AOD). However, lhb was not expressed before ovarian differentiation (BOD) and was first detected AOD. Next, to investigate the differentiation and distribution of Fshb and Lhb-producing cells in the pituitary of fish throughout gonadal sex differentiation, immunohistochemical analysis was used to detect teleost GTH subunits. Positive immunoreactivity against Fshb and Lhb was not detected in the pituitary BOD; Fshb and Lhb-positive cells first appeared in the pituitary AOD. It therefore seems unlikely that pituitary gonadotropins play a major role in the control of gonadal sex differentiation in the Malabar grouper. © 2012 Elsevier Inc..

Shrestha S.,Tribhuvan University | Tada T.,National Center for Global Health and Medicine | Miyoshi-Akiyama T.,National Health Research Institute | Ohara H.,National Center for Global Health and Medicine | And 10 more authors.
International Journal of Antimicrobial Agents | Year: 2015

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as blaNDM-1, blaOXA-23 or blaOXA-58. MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA). © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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