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Investigation for sulfur metabolism in microorganisms and its application for microbial production of sulfur-containing amino acids.

Research Project

Project/Area Number 09660100
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionFukui Prefectural University

Principal Investigator

NAKAMORI Shigeru  Fukui Prefectural University, Bioscience, Professor, 生物資源学部, 教授 (00254243)

Co-Investigator(Kenkyū-buntansha) WADA Masaru  Fukui Prefectural University, Bioscience, Assistant, 生物資源学部, 助手 (00301416)
TAKAGI Hiroshi  Fukui Prefectural University, Bioscience, Assistant Professor, 生物資源学部, 助教授 (50275088)
Project Period (FY) 1997 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Keywordssulfur-containing amino acids / cysteine / cystine / methionine / Escherichia coli / Arabidopsis thaliana / serine acetyltransferase / fermentative production / トリプトファナーゼ / システインデスルフヒドリラーゼ / アンチセンスDNA / フィードバック阻害 / PCR / ランダム変異
Research Abstract

We have succeeded first in obtaining L-cysteine (plus L-cystine) producers from E.coli strains by the application of molecular breeding techniques, as follows :
1)E.coli serine acetyltransferase (SAT), which is sensitive to the feedback inhibition by L-cysteine, was converted to feedback inhibition-insensitive by the replacement of methionine residue at 256 with 19 amino acid residues. E.coli strains with low cysteine-degradative activity were transformed with plasmids containing genes coding for these altered SAT.Transformants thus obtained produced about 1.0 g/l of L-cysteine (plus L-cystine) by the cultivation in media containing 3% glucose and others.
2) Random mutations, which confered L-cysteine overproduction, were introduced in wild type SAT with error-prone PCR.These mutations were found in C-terminal region of SAT.Production of L-cysteine (plus L-cystine) by the best strain was 1.2 g/l.
3) Arabidopsis thaliana has three type of SAT, among which SAT-m and SAT-p are feedback inhibition-insensitive to L-cysteine. Genes coding for SAT-m and SAT-p were synthesized and expressed in E.coli. Transformants thus obtained produced about 2.0 g/l of L-cysteine (plus L-cystine).
4) Overproduction of L-methionine by a methionine analog-resistant mutant of E.coli was shown by the replacement of Ser with Asn in the 54 amino acid of MetJ protein.
5)Degradation of L-cysteine in E.coli was shown to mainly carry out by tryptophanase and cystationine-β-lyase. Disruption of genes coding these enzymes contributed to improved L-cysteine production.

Report

(5 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (17 results)

All Other

All Publications (17 results)

  • [Publications] S.Nakamori ほか: "Overproduction of L-cysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase."Appl.Environ.Microbiol.. 64. 1607-1611 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] H.Takagi ほか: "PCR random mutagenesis into Escherichia coli serine acetyltransferase : isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine*"FEBS Lett.. 452. 323-327 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Nakamori ほか: "Mechanism of L-methionine overproduction by Escherichia coli : the replacement of Ser54 by Asn in the MetJ protein causes the derepression of L-methionine**"Appl.Microbiol.Biotechnol.. 52. 179-185 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] H.Takagi ほか: "Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli."FEMS Microbiol.Lett.. 179. 453-459 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 中森茂,高木博史: "システイン/シスチン発酵の工業化は可能か"バイオサイエンスとインダストリー. 56. 29-30 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shigeru Nakamori, Sin-ichiro Kobayashi, Chitose Kobayashi, and Hiroshi Takagi: "Overproduction of L-cysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase."Appl.Environ.Microbiol.. 64. 1607-1611 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hiroshi Takagi, Chitose Kobayashi, Sin-ichiro Kobayashi, Shigeru Nakamori: "PCA random mutagenesis into Escherichia coli serine acetyltransferase : isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition."FEBS Letters. 452. 323-327 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Nakamori, S.Kobayashi, T.Nishimura, H.Takagi: "Mechanism of L-methionine overproduction by Escherichia coli the replacement of Ser-54 by Asn in the MetJ proteine causes the derepression of L-methionine biosynthetic enzymes."Appl.Microbiol.Biotechnol.. 52. 179-185 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hiroshi Takagi, Naoki Awano, Sin-ichiro Kobayashi, Masaaki Noji, Kazuki Saito, Shigeru Nakamori: "Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli."FEMS Microbiology Letters. 179. 453-459 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shigeru Nakamori and Hiroshi Takagi: "Expectation for industrialization of L-cysteine/cystine fermentation"Biosxience and Industry (in Japanese). 56. 29-30 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hiroshi Takagi ほか: "PCR random mutagenesis into Escherichia coli serine acetyltransferase"FEBS Letters. 452. 323-327 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Shigeru Nakamori ほか: "Mechanism of L-methionine overproduction by Escherichia coli :"Applid Microbiology and Biotechnology. 52. 179-185 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] SHIGERU NAKAMORIほか: "Overproduction of L-cysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase" Applied and Environmental Microbiology. 64(5). 1607-1611 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 小林千登勢ほか: "PCRランダム変異導入によるセリンアセチルトランスフェラーゼ遺伝子の改変と変異型遺伝子の導入によるシステイン生産" 日本農芸化学会関西支部第408回講演会 講演要旨集. 3 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 小林慎一郎ほか: "E.coliによるメチオニン生産のメカニズム" 平成10年度日本生物工学会大会講演要旨集. 255 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 小林慎一郎ほか2名: "E.coliのフィードバック阻害解除型Serine Acetyltransferase遺伝子の導入とシステイン生産" 日本農芸化学会誌(大会講演要旨集). 71・増刊. 224 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 小林慎一郎ほか3名: "システイン分解欠損株への変異型セリンアセチルトランスフェラーゼ遺伝子導入によるシステイン生産の向上と安定化" 日本生物工学会大会講演要旨集. 447 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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