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Development of Rhodococcus promoter for utilization in actinomycetes

Research Project

Project/Area Number 13556011
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 応用微生物学・応用生物化学
Research InstitutionUniversity of Tsukuba

Principal Investigator

KOBAYASHI Michihiko  University of Tsukuba, Institute of Applied Biochemistry, Professor, 応用生物化学系, 教授 (70221976)

Co-Investigator(Kenkyū-buntansha) GODA Masahiko  JSPS, Research Fellow, 応用生物化学系, 特別研究員(PD)
HIGASHIBATA Hiroki  University of Tsukuba, Institute of Applied Biochemistry, Research associate, 応用生物化学系, 助手 (20344864)
HASHIMOTO Yoshiteru  University of Tsukuba, Institute of Applied Biochemistry, Research associate, 応用生物化学系, 助手 (00323254)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2003: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2001: ¥6,200,000 (Direct Cost: ¥6,200,000)
KeywordsPromoter / Gene / Expression / Rhodococcus
Research Abstract

The microbial degradation of nitrites proceeds through two enzymatic pathways : nitrite hydratase (NHase} catalyzes hydration of nitrite to amide, whereas nitrilase catalyzes hydrolysis of nitrite to acid and ammonium. Nitrilase is strongly induced by the addition of ε-caprolactam or isovaleronitrile to the culture medium ; the formed nitrilase corresponds to 35ーIo of aft soluble protein in Rhodococcus rhodochrous J1. On the other hand, NHase is strongly induced by the addition of urea to the culture medium ; the formed NHase corresponds to 50% of all soluble protein in this strain. At first, we trimmed the DNA region containing the nitrilase gene cluster, and constructed several plasmids where the upstream or downstream region of the gene cluster was deleted. We transformed each plasmid into Escherichia coli, and examined the nitrilase formation in the resultant transformants. SDS-PAGE and enzyme assay by HPLC revealed that the Rhodococcus nitrilase expression system was not able to function in E. coli. Next, we tried to investigate whether the nitrilase regulatory system is active or not in other Rhodococcus actinomycetes, and found that the nitrilase expression system functioned, indicating the development of a hyper-expression vector. We also obtained similar results for the NHase expression system. Furtheremore, we tried to construct a Rhodococcus host-vector system, considering the useful characteristics of this genus. We bought more than 100 Rhodococcus strains from microorganism stock centers, and searched for Rhodococcus strains carrying cryptic plasmids. We found three strains. with a cryptic plasmid, and succeeded in the construction of a Rhodococcus-E.coli shuttle-vector using one of their plasmids

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report

Research Products

(30 results)

All Other

All Publications

  • [Publications] Yamada, H. et al.: "Hydratases involved in nitrile conversion : Screening, characterization and application"Chemical Records. 1. 152-161 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Goda, M. et al.: "Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage"J.Biol.Chem.. 276. 23480-23485 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hashimoto, Y. et al.: "Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase"J.Inorg.Biochem. 91. 70-77 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Goda, M. et al.: "Isonitrile Hydratase from Pseudomonas putida N19-2 : Cloning, sequencing, gene expression, and identification of its active amino acid residu"J.Biol.Chem. 277. 45860-45865 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Xu, J.et al.: "Novel mutations in the ribosomal L11 protein gene (rplK=relC) of Streptomyces coelicolor A3(2)"Actinomycetol. 16. 32-36 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Oinuma, K-I: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification, and characterization"J.Biol.Chem. 278. 29600-29608 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kojima, T. et al.: "A novel lipase from Pseudomonas fluorescens HU380 : Gene cloning, overproduction, renaturation-activation, two-step purification, and characterization"J.Biosci.Bioeng. 96. 242-249 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Omura, H. et al.: "Purification, characterization and gene cloning of thermostable O-acetyl-L-homoserine sulfhydrylase forming γ-cyano-α-aminobutyric acid"J.Biosci.Bioeng. 96. 53-58 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Omura, H. et al.: "Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine"J.Biosci.Bioeng. 96. 470-475 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yamada, H., Shimizu, S., Kobayashi, M.: "Hydratases involved in nitrile conversion : Screening, characterization and application"Chemical Records. 1. 152-161 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Goda, M., Hastiimoto, Y., Shimizu, S., Kobbyashi, M.: "Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage"J.Biol.Chem.. 276. 23480-23485 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Goda, M., Hasliimoto, Y., Takase M., Herai, S., Iwahara, Y., Higashibata, H., Kobayashi, M.: "Isonitrile hydratase from Pseudomonas putida N19-2 : Cloning, sequencing, gene expression, and identificatian of its active amino acid residue"J.Biol.Chem.. 277. 45860-45865 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hashimuto, Y., Sasaki, S., Herai.S., Oinuma, K., Shimizu, S., Kubayastii, M.: "Site-directed mutagenesis for cysteine residues, of cobalt-containing nitrile hydratase"J.Inorg.Biochem.. 91. 70-77 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] XU, J., Saito, N., Kurosawa, K., Okamoto, S., Takaya, N., Kobayashi, M., Ochi K.: "Novel mutations in the ribosomal L11 protein gene (rplK relC) of Streptomyces coelicolor A3(2)"Actinomycetol. 16. 32-36 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification and characterization"J.Biol.Chem.. 278. 29600-29608 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kojima, Y., Kobayashi, M., Shimizu, S.: "A navel lipase from Pseudomonas fluorescens HU380 : Gene cloning, overproduction, renaturation-activation, two-step purification and characterization"J.Biosci.Bioeng.. 96. 242-249 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Omura, H., Ikemoto, M., Kobayashi, M., Shimizu, S., Yoshida, T., Nabasawa, T.: "Purification, characterization And gene cloning of thermostable U-acetyl-L-homoserine sulfhydrylase forming γ-cyano-α-aminobutyric acid"J.Biosci.Bioeng.. 96. 53-58 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Omura, H., Kuroda, M., Kobayashi, M., Shimizu, S., Yoshida, T., Nabasawa, T.: "Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanin"J.Biosci.Bioeng.. 96. 470-475 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Oinuma, K-I. et al.: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of pseudomonas, chlororaphis B23 : Sequencing, gene expression purification and characterization"J.Biol.Chem.. 278. 29600-29608 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kojima, Y. et al.: "A novel lipase from pseudomonas fluorescens HU380 : Gene cloning, overproduction, renaturation-activation, two-step purification and characterization"J.Biosci.Bioeng.. 96. 242-249 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Shiraki, K. et al.: "Genetic, enzymatic and structural analyses of phenylalanyl-tRNA Synthetase from Thermococcus Kodakaraensis KOD1"J.Biochem.. 134. 567-574 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Higashibata, H. et al.: "Helicase and maclease activities of hyperthermophile pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments of 5'-end"J.Biol.Chem.. 278. 15983-15990 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Higashibata, H. et al.: "Surface histidine residue of archaeal histone affects DNA compaction and thermostability"FEMS.Microbiol.Lett.. 224. 17-22 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Goda, M.et al.: "Isonitrile hydratase from Pseudomonas putida N19-2"J.Biol.Chem.. 277. 45860-45865 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hashimoto, Y.et al.: "Site-divected mutageuesis for cysteine residues of cobalt-containing nitnile hydratase"J.Inorg.Biochem.. 91. 70-77 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Higashibata, H.et al.: "Helicase and endonuclease activities of hyperthermophile Pyrococcus horikoshii Dua2 inhibited by substrates with RNA sequence at 5'end"J.Biol.Chem.. (印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Yamada, H. et al.: "Hydratases involved in nitrile conversion: Screening, Characterization and application"Chemical Records. 1. 152-161 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Goda, M. et al.: "Discovery of a novel enzyme, isonitrile hydratase, invdved in nitrogen-carbon triple bond cleavage"J. Biol. Chem.. 276. 23480-23485 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hashimoto, Y. et al.: "Extracellular synthesis, specific recognition and intracellular degradation of cyclomaltodextrins by hyperthermophilic archaeon, Thermococcus sp.Blool"J. Bacteriol.. 183. 5050-5057 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hashimoto, T. et al.: "Site-directed mutaseresis for cystein residnes of cobalt-containing nitrile hydratase"J. lnorg. Biochem.. (印刷中). (2002)

    • Related Report
      2001 Annual Research Report

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Published: 2001-03-31   Modified: 2016-04-21  

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