| Project/Area Number |
17560688
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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 |
Biofunction/Bioprocess
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| Research Institution | Nagoya University |
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Principal Investigator |
OKOCHI Mina Nagoya University, Graduate School of Engineering, Associate Professor, 大学院工学研究科, 講師 (70313301)
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| Co-Investigator(Kenkyū-buntansha) |
HONDA Hiroyuki Nagoya University, Graduate School of Engineering, Professor, 大学院工学研究科, 教授 (70209328)
井藤 彰 名古屋大学, 大学院工学研究科, 助手 (60345915)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | organic solvent tolerance / microorganisms / prefoldin / molecular chaperones |
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| Research Abstract |
The molecular mechanisms in organic solvent tolerance of microorganisms have become increasingly important in whole-cell bioprocesses involving an organic solvent-aqueous phase for the bioconversion of substrates that have relatively low solubility in water. In the present study, organic solvent tolerant mutants were constructed by introducing molecular chaperones.
(1) Prefoldin from the hyperthermophilic archaea increased organic solvent tolerance.
Molecular stability of extremophile enzymes generally reflects the growth temperatire of parent microorganisms and positively correlated with the stability in the presence of denaturing enzymes including organic solvents. Expression of prefoldin from hyperthermophilic archaeum, Pyrococcus horikoshii OT3 (PhPfd) resulted in increased organic solvent tolerance in Escherichia coli cells. The cells overexpressing PhPfd could grow continuously after addition of organic solvent mixture of octane and hexane at 10% volume. The intracellular level of
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cells overexpressing PhPfd was kept lower than the control cells. Archaeal prefoldin consists of two species of subunits and exists as a hexamer of α2β4 in jellyfish like structure. Overexpression of PhPFD truncation mutants that have low chaperone activities resulted in decreased organic solvent tolerance. Therefore, it was shown that molecular chaperone activity of prefoldin resulted in increased organic solvent tolerance in E.coli.
(2) Construction of organic solvent tolerant mutants by overexpression of upregulated genes in transcriptional analysis when addition of organic solvents.
Overexpression of manXYZ that was selected from gene expression profile was shown to increase organic solvent tolerance level of E.coli JA300 strain. The cells could grow in an organic solvent mixture of hexane and cyclohexane at 10% volume. Since overexpression of ManXYZ in cell membrane changed cell surface property to obtain higher affinity to hydrocarbons, this strain would be useful in whole-cell bioprocesses in biphasic systems. In the analysis of global regulatory genes related to glucose catabolism, knockout mutants of cyclic AMP receptor protein (crp) and adenylate cyclase (cyaA) was found to increase their tolerance to organic solvents. In the transcriptional analysis of a crp knockout mutant, genes related to molecular chaperones were upregulated. Less
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