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Genetic engineering for Cholesterol Transformation System in Streptomyces

Research Project

Project/Area Number 01480036
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 発酵工学
Research InstitutionHiroshima University

Principal Investigator

MUROOKA Yosikatu  Hiroshima Univ. Faculty of Engineering, Prof., 工学部, 教授 (60029882)

Co-Investigator(Kenkyū-buntansha) YAMASHITA Mituo  Hiroshima Univ., Facul. of Engineering, Resh. Assoc., 工学部, 助手 (40220347)
ONO Kazuhisa  Hiroshima Univ., Facul. of Engineering, Assoc. Prof., 工学部, 助教授 (10144883)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥4,300,000 (Direct Cost: ¥4,300,000)
KeywordsStreptomyces / Cholesterol oxidase / Transformation of cholesterol / Genetic engineering / Cytochrome P-450 / コレステロ-ル酸化酵素 / コレステロ-ル転換系 / Pー450
Research Abstract

Streptomyces species produce a large variety of medically important metabolites and extracellular enzymes, in particular cholesterol oxidase. Cholesterol oxidase catalyzes the oxidation of cholesterol to 4-cholesten-3-one, with the reduction of oxygen to hydrogen peroxide. The enzyme is commonly used for the enzymatic transformation of cholesterol and is alo used for quantitation of levels of cholesterol in clinical specimens.
Recently, we cloned the gene involved in the production of cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO. Cells of Streptomyces lividans carrying a plasmid that includes the choA gene overproduce extracellular Cholesterol oxidase when cultured in appropriate medium.
We sequenced the complate cholesterol oxidase gene. The sequence of the choA gene revealed that the gene of cholesterol oxidase starts from a GTG initiation codon and terminates at a TAA termination codon. In the 3'-flanking region of choA, a long palin dromic sequence, which may act as … More a transcription terminator, was found. The gene encodes a mature cholesterol oxidase of 504 amino acids with a caluculated Mr of 54,913.
The nucleotide sequence of the promoter region of the gene for choA was determined. We found an open reading frame (choP) that has a high degree of similarity to the sequence of the gene for cytochrome P-450s from heman and Pseudomonas species. We determined the product of the choP gene was cytochrome P-450-like protein.
We have cloned the chromosomal DNA, which is adjoined upstream and down stream of the choP-choA operon, by guided genomic walking method developed by us. These clones may be involved in cholesterol metabolism.
During the study on the decomposition of cholesterol and secretion of heterologous gene products in Streptomyces, we found secretory overproduction of cholesterol oxidase in Streptomyces lividans by a recombinant multi-copy shuttle vector, the results of which are potential useful for production of heterologous gene product extracellularly bu using Streptomyces host-vector system. Less

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] Tomoyuki Ishizaki: "Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp." Jouranl of Bacteriology. 171. 596-601 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu Murooka: "A new operon involved in a sterol metabolism in Streptomyces sp." Actinomycetology. 3. 89-94 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Masami Horii: "An operon containing the gene for cholesterol oxidase and a cytochrome Pー450ーlike protein from a Streptomyces sp." Jouranal of Bacteriology. 172. 3644-3653 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Istvan Molnar: "“Guided genomic walking",a systematic approach to cloning intermediateーsized restriction fragments extending outside the boundaries of known sequences"

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Istvan Molanr: "Secretory overproduction of the Streptomyces cholesterol oxidase by Streptomyces lividans with a recombinant multiーcopy vector"

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu Murooka: "Biotechnology Advances (分担執筆)" Pergamon Press Plc.,Oxford,UK, 29 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu Murooka: "Fermentation Technologies: Industrial Applications (分担執筆)" Elsvier Applied Sciences,London,UK, 6 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tomoyuki, Ishizaki et al.: "Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp." J. Bacteriol.171. 596-601 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu, Murooka et al.: "A new operon in a sterol metabolism in Streptomyces sp." Actinomycetology. 3. 89-94 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Masami, Horii et al.: "An operon containing the gene for cholesterol oxidase and a cytochorome P-45-like protein from a Streptomyces sp." J. Bacteriol.172. 3644-3653 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Istvan Molnar et al.: ""Guided genomic walking", a systematic approach to cloning intermediate-sized restriction fragments extending outside the boundaryies of known sequences"

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Istvan, Molnar et al.: "Secretory overproduction of the Streptomyces cholesterol oxidase by Streptomyces lividans with a recombinant multi-copy vector"

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu, Murooka: "Biotechnology Advances" Pergamon Press Plc., Oxford, UK. Vol8. 29-57 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yoshikatsu, Murooka: "Fermentation Technologies : Industrial Applications" Elsvier Applied Science, London, Uk.40-45 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tomoyuki Ishizaki: "Nucleotide sequence of the gene for cholesterol oxidase from a streptomyces sp." Jouranal of Bacteriology. 171. 596-601 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yoshikatsu Murooka: "A new operon involved in a sterol metabolism in Streptomyces sp." Actinomycetology. 3. 89-94 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] Masami Horii: "An operon containing the gene for cholesterol oxidase and a cytochrome Pー450ーlike protein from a Streptomyces sp." Jouranal of Bacteriology. 172. 3644-3653 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Istv^^'an Moln^^'ar: "“Guided genomic walking",a systematic approach to cloning intermediateーsized restriction fragments extending outside the boundaries of known sequences"

    • Related Report
      1990 Annual Research Report
  • [Publications] Istv^^'an Molna^^'ar: "Secretory overproduction of the Streptomyces cholesterol oxidase by Streptomyces lividans with a recombinant multiーcopy vector"

    • Related Report
      1990 Annual Research Report
  • [Publications] Yoshikatsu Murooka: "Biotechnology Advances 分担執筆" Pergamon Press Plc.,Oxford,UK, 29 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yoshikatsu Murooka: "Fermentation Technologies:Industrial Applications 分担執筆" Elsvier Applied Sciences,London,UK, 6 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Ishizaki,T.,N.Hirayama,H.Shinkawa,O.Nimi and Y.Murooka: "Nucleotide sequence of the gene for cholesterol oxidase from Streptomyces sp." Journal of Bacteriology. 171. 596-601 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Horii,M.,T.Ishizaki S-Y.Paik,T.Manome and Y.Murooka: "An Operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from Streptomyces sp." Journal of Bacteriology. submitted.

    • Related Report
      1989 Annual Research Report
  • [Publications] Murooka,Y.,T.Ishizaki,M.Horii and S-Y.Paik: "A new operon involved in a sterol metabolism in Streptomyces sp." Actinomycetologia. 3. 89-94 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Murooka,Y.(ed.Spiechck): "Genetic desighns for product formation in recombinant microbes/in Biotechnology Advances" Pergamon Press plc,Oxford,England, 30 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Murooka,Y.(ed.P-L.Yu): "Secretory over-production of microbial enzymes using new host-vector systems/in Fermentation Technology;Industrial Applications" Elsevier Scientific Publishers Ltd, 6 (1990)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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