| Project/Area Number |
11556015
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SAKAI Yashuyoshi Graduate School of Agriculture, Kyoto University, Associate Professor, 農学研究科, 助教授 (60202082)
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| Co-Investigator(Kenkyū-buntansha) |
KONDO Keiji Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., Senior Research Scientist, 基盤技術研究所, 主任研究員
YURIMOTO Hiroya Graduate School of Agriculture, Kyoto University, Instructor, 農学研究科, 助手 (00283648)
KATO Nobuo Graduate School of Agriculture, Kyoto University, Professor, 農学研究科, 教授 (50026556)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥7,500,000 (Direct Cost: ¥7,500,000)
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| Keywords | methylotrophic yeast / Candida boidinii / alcohol oxidase / dihydroxyacetone synthase / formate dehydrogenase / methanol-indiicible promoter / peroxisome / Pichia methanolica / Pichia methanolica / ペルオキシーム |
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| Research Abstract |
In this resarch project, we studied on the molecular mechanism of methanol-inducible gene expression and the development of high-level gene expression system in the methylotrophic yeast.
1. Evaluation of methanol-induicible promoters Among the five methanol-inducible promoters in Candida boidinii, dihydroxyacetone synthase gene promoter (PDAS1) was the strongest promoter whose strength was 1.5 times higher than that of commonly used alcohol oxidase gene promoter (PAOD1). We also found that PDAS1 was activated earlier than "PAOD1 at the initial stage of methanol induction, which minimizes the accumulation of formaldehyde generated by alcohol oxidase.
2. Identification of di-element in PAOD1 and PDAS1 Through the experiments using the aod Δdas1Δ strain, we clarified that PAOD1 and PDAS1 Were activated by methanol and formaldehyde. In contrast, while PAOD1 was activated by methanol and formaldenhyde. In contrest,while PDOD1 was activeted by their anabolites, e.g.,dihydroxyacetone and glyceraldefeyde, PDAS1 was not. We identified the methanol response elements within PDAS1, and confirmed the existence of the protein which specifically bound to these elements. Within PAOD1, we found the cis-elements responding specifically to either methanol or glyceraldehyde.
3. Developmeat of High-level heterologous protein production System Using PAOD1 and formate dehydrogenase gene promother and optimizing host strains, cultivation conditions, localization of produced proteins, and so on, we established a high-level production sys-tem for useful proteins.
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