| Project/Area Number |
22K14828
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 38030:Applied biochemistry-related
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| Research Institution | Nagoya University |
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Principal Investigator |
ダムナニョヴィッチ ヤスミナ 名古屋大学, 生命農学研究科, 講師 (00754673)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2024: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2023: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2022: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | protein engineering / enzyme engineering / single-molecule display / in vitro translation / activity-based selection / NGS / bioinformatics / in vitro display / cDNA display |
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| Outline of Research at the Start |
This research aims to develop a system for the rapid development of enzymes, as environmentally friendly, safe, and inexpensive bio-tools used to drive the conversions of various compounds into useful products. Enzymes with improved properties have a wide range of biotechnological applications (industry, pharma, diagnostics).
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| Outline of Annual Research Achievements |
DAAO engineering for specific detection of D-Ala: We mapped the sites of interest for mutagenesis and conducted DAAO random library screening, as well as established a reliable assay to monitor the selection progress across multiple selection rounds. To increase the likelihood of selecting improved variants, we introduced negative selection steps aimed at eliminating non-specific DAAO variants.
MTG engineering for site-specific antibody-drug conjugation: The selection system for sequence-specific MTG has been established, and its first evaluation completed.
PLD engineering for improved synthesis of designer phospholipids: We discovered ways to improve the efficiency of enzyme display formation, and are currently evaluating the PLD selection system.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In the AY2023, the project continued for all three set themes. Engineering of DAAO is proceeding as planned. We created several mutant libraries to probe different parts of the enzyme structure for substrate specificity change. We have also introduced a control library where we mutated one residue of the active site and performed selection to verify the enrichment of the active DAAO by NGS.
Engineering of TG continues with selection evaluation using model binary libraries. Engineering of PLD proceeds at the slowest pace, since we encountered problems with PLD display formation efficiency, which we are addressing by changing its sequence, and since recently, by mutagenesis using structural bioinformatics.
The progress of all three themes has been reported at local and international meetings.
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| Strategy for Future Research Activity |
In AY2024, we plan to continue according to the experimental plan. After completing the DAAO selection, we will analyze the genes of selected DAAO variants and design second-generation libraries to fine-tune the specificity of DAAO for D-Ala.
MTG engineering will continue with the preparation and selection of mutant MTG libraries, followed by NGS analysis.
We will use structural bioinformatics to predict mutation sites in PLD, and proceed to evaluate the PLD selection system.
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