| Project/Area Number |
09660100
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Fukui Prefectural University |
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Principal Investigator |
NAKAMORI Shigeru Fukui Prefectural University, Bioscience, Professor, 生物資源学部, 教授 (00254243)
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| 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)
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| Project Period (FY) |
1997 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| 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)
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| Keywords | sulfur-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 |
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| 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.
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