2021 Fiscal Year Annual Research 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 / mRNA display / Enzymes |
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| Outline of Annual Research Achievements |
The work focused around two major directions. 1. Development of an mRNA-display based platform for de novo discovery of pseudo-natural lactazole-like thiopeptides with novel bioactivities.
We have successfully established the proposed platform and showed its utility by discovering dozens of pseudo-natural products with designed biological activities. The development entailed reengineering of in vitro lactazole biosynthesis, development of library construction protocols, establishment of a general synthetic strategy for rapid and modular access to the discovered structures, and finally, thorough biochemical characterization of the synthesized selection hits. Using the established platform, we performed an in vitro selection of a thiopeptide library against TNIK, a kinase involved in several forms of cancer, and identified numerous potent and selective kinase inhibitors (the best compound had Kd of 1 nM).
2. In vitro reconstitution of lactazole-like biosynthetic enzymes. So far, we have successfully produced 12 enzymes from 4 biosynthetic gene clusters (BGCs) homologous to the prototypical laz BGC. To do this, we established an E. coli expression system that greatly facilitated heterologous protein expression from these BGCs. All expressed enzymes were active, and catalyzed reactions analogous to Laz enzymes, which simplified the analysis, but crucially, had a divergent substrate specificity profiles. The ongoing work is directed toward establishing hybrid biosynthetic pathways by combining the most active enzymes from their respective BGCs with Laz enzymes.
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| Remarks |
One study pertaining to this project has been submitted to a peer reviewed journal, but not yet published. A pre-print of the manuscript is available on bioRxiv as listed above.
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