2020 Fiscal Year Research-status Report
Characterization of novel biosynthetic gene clusters for combinatorial synthesis of thiopeptides
| Project/Area Number |
20K15407
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| Research Institution | The University of Tokyo |
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Principal Investigator |
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| Project Period (FY) |
2020-04-01 – 2022-03-31
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| Keywords | Biosynthesis / Biochemistry / Bioengineering / Natural products |
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| Outline of Annual Research Achievements |
In the first year, we successfully performed bioinformatic analysis and identified 4 biosynthetic gene clusters of interest. Next, we focused on heterologous overexpression and characterization of the biosynthetic enzymes. Work so far has primarily focused on the study of glutamylation domain enzymes. Four enzymes were cloned and heterologously expressed in E. Coli. Three enzymes were found active, one of which displayed a unique substrate scope as compared to LazB (the parent lactazole enzyme) and other homologs. The ongoing work in this direction involves further biochemical characterization of the enzymes. In parallel, cloning and expression of YcaO heterocyclaze enzymes from the same biosynthetic gene clusters is also ongoing.
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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
For the most part, the work is progressing according to the expectations. Several challenges, which hindered the progress, were encountered during heterologous enzyme expression (low enzyme yields, low solubility and/or no enzymatic activity), but these were overcome by optimizing expression conditions.
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| Strategy for Future Research Activity |
The plans for ongoing research adhere to the original research proposal. This year, we will finish heterologous expression of other biosynthetic enzymes from the identified biosynthetic gene clusters, and will move on to establishing a combinatorial system for the production of novel lactazole-like thiopeptides.
If the work can progress beyond this stage, overexpression and characterization of the underlying natural products, as well as work toward de novo discovery of bioactive thiopeptides using the system established above will be also pursued.
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