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Development of novel expression vectors utilizing gene promoters involved in nitrile metabolism

Research Project

Project/Area Number 07556073
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section試験
Research Field Applied molecular and cellular biology
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

KOBAYASHI Michihiko  Kyoto Univ.Agr.Senior Lecturer, 農学部, 講師 (70221976)

Co-Investigator(Kenkyū-buntansha) KATAOKA Michihiko  Kyoto Univ.Assist., 農学部, 助手 (90252494)
SHIMIZU Sakayu  Kyoto Univ.Agr.Prof., 農学部, 教授 (70093250)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥4,300,000 (Direct Cost: ¥4,300,000)
KeywordsNitrile hydratase / promoter / Gene / Induction / Expression / Amide / イソバレロニトリル / ニトリルヒドラターゼ / Rhodococcus / 塩基配列
Research Abstract

High-Mr-(H-) and low-Mr-(L-) nitrile hydratases (NHases) in Rhodococcus rhodochrous J1 were analyzed at both protein and gene levels. The H-NHase gene (nhhAB) was expressed as an active form in R.rhodochrous ATCC12674 as a host using a Rhodococcus-Escherichia coli host-vector (pK4) system, whereas it was not in E.coli.SDS/PAGE of the R.rhodochrous transformant cell-extracts has shown that the amount of alpha-and beta-subunits of H-NHase is about 50% of the total soluble protein. The 5'-upstream region of nhhAB was found to be required for nhhAB expression with the host. In this region, there are four open reading frames (nhhC,nhhD,nhhE,nhhF). Both nhhC and nhhD play positive regulatory roles in nhhAB expression. On the other hand, the 5'-upstream region from the L-NHase gene (nhlAB) was required for the amide-dependent expression of nhlBA.There are two open reading frames (nhlD and nhlC) in this region. In the process of the L-NHase formation, nhlD and nhlC play negative and positive regulatory roles, respectively.
On the other hand, the 1.4 kb downstream from a nitrilase (nitA) of R.rhodochrous J1 was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis. Sequence analysis of this region revealed the existence of an open reading frame (nitR). The nitR codes for a transcriptional positive regulator in nitA expression. The transcriptional start site for nitA was mapped to a C residue located 26 bp upsteam of its translational start site.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] Komeda, H., Kobayashi, M. & Shimizu, S.: "A novel gene cluster including the Rhodococcus rhodochrous Jl nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product" J. Biol. Chem.271. 15796-15802 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda, H., Kobayashi, M. & Shimizu, S.: "Characterization of the gene cluster of a high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous Jl" Proc. Natl. Acad. Sci. USA. 93. 4267-4272 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda, H., Hori, Y., Kobayashi, M. & Shimizu, S.: "Transcriptional regulation of the Rhodococcus rhodochrous Jl nitA gene encoding a nitrilase" Proc. Natl. Acad. Sci. USA. 93. 10572-10577 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kobayashi, M., Fujita, T. & Shimizu, S.: "Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens" Appl. Microbiol. Biotechnol.45. 176-181 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Yamada, H. & Kobayashi, M.: "Nitrile hydratase and its application to industrial production of acrylamide" Biosci. Biotech. Biochem.60. 1391-1400 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kawashima, H., Kouzaki, N., Kobayashi, M. & Shimizu, S.: "Biosynthesis of trans fatty acids in a fungus, Cladosporium sphaerospermum, and some bacteria isolated from fish viscera" Biosci. Biotech. Biochem.60. 1888-1890 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kobayashi, M., Suzuki, T., Fujita, T., Masuda, M.& Shimizu, S: "Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium." J.Biol.Chem.271. 15796-15802 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda, H., Kobayashi, M.& Shimizu, S: "Characterization of the gene cluster of a high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1." Proc.Natl.Acad.Sci.USA. 93. 4267-4272 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda, H., Kobayashi, M.& Shimizu, S: "A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction Product." Proc.Natl.Acad.Sci.USA. 93. 10572-10577 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda, H., Hori, Y., Kobayashi, M.& Shimizu, S: "Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase." Appl.Microbiol.Biotechnol.45. 176-181 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Yamada H.& Kobayashi, M: "Nitrile hydratase and its application to industrial production of acrylamide." Biosci.Biotech.Biochem.60. 1391-1400 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kobayashi, M., Fujita, T.& Shimizu, S: "Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens." Biosci.Biotech.Biochem.60. 1888-1890 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kawashima, H., Kouzaki, N., Kobayashi, M.& Shimizu, S: "Biosynthesis of trans fatty acids in a fungus, Cladosporium sphaerospermum, and some bacteria isolated from fish visacera."

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Komeda,H.,Kobayashi,M.& Shimizu,S: "A novel gene cluster including the Rhodococus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product" J.Biol.Chem.271. 15796-15802 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Komeda,H.,Kobayashi,M.& Shimizu,S: "Characterization of the gene cluster of a high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1" Proc.Natl.Acad.Sci.USA. 93. 4267-4272 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Komeda,H.,Hori,Y.,Kobayashi,M.& Shimizu,S.: "Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase" Proc.Natl.Acad.Sci.USA. 93. 10572-10577 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Komeda,H.,Kobayashi,M.& Shimizu,S: "Hyperiinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens" Appl.Microbiol.Biotechnol.45. 176-181 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yamada,H.& Kobayashi,M.: "Nitrile hydratase and its application to indusuction Production of acrylamide" Biosci.Biotech.Biochem.60. 1391-1400 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kawashima,H.,Kozuki,N.,Kobayashi,M.& Shimizu,S.: "Biosynthesis of trans fatty acids in a fungus,Cladosproium sphaerospermum,and some bacteria isolated from fish viscera" Biosci.Biotech.Biochem.60. 1888-1890 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] M.Kobayashi, T.Suzuki, T.Fujita, M.Masuda & S.Shimizu: "Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium" Proc. Natl. Acad. Sci. USA. 92. 714-718 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 小林達彦、清水 昌: "ニトリル代謝関連酵素" 微生物機能の多様性(別府輝彦編;学会出版センター). 139-157 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 小林達彦、清水 昌: "植物ホルモン・インドール酢酸生合成研究の最前線:インドールアセトニトリル経路" 化学と生物. 33. 479-483 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 小林達彦、山田秀明: "ニトリルヒドラターゼと化学工業への応用" 現代化学増刊28 新しい酵素研究法(一島英治、小野寺一清編;東京化学同人). 152-165 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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