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Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster
Seong-Hwan Kim1, Wanli Lu1, Mahmoud Kamal Ahmadi1
1Laboratory of Genetically Encoded Small Molecules , The Rockefeller University , 1230 York Avenue , New York , New York 10065 , United States.
ACS Synthetic Biology
|December 22, 2018
Summary
Scientists developed a new method to activate silent gene clusters in bacteria. This technique enables the discovery of novel natural products, including cytotoxic compounds, from previously inaccessible bacterial genes.
Area of Science:
- Microbiology
- Synthetic Biology
- Natural Product Discovery
Background:
- Many bacterial biosynthetic gene clusters are silent under standard laboratory conditions, limiting natural product discovery.
- Activating these silent clusters is crucial for uncovering novel bioactive compounds.
Purpose of the Study:
- To develop a scalable and cost-effective method for activating silent biosynthetic gene clusters.
- To facilitate the discovery of new natural products from bacterial genomes and metagenomes.
Main Methods:
- A novel in vitro disassembly-TAR (transformation-assisted recombination) in yeast reassembly method was employed.
- CRISPR/Cas9 was used for in vitro gene cluster disassembly at interoperonic regions.
- Synthetic promoters were introduced during reassembly to create combinatorial libraries of refactored gene clusters.
Main Results:
- The method successfully activated silent gene clusters, enabling secondary metabolite production.
- Two new bacterial cyclic sesterterpenes, atolypene A and B, were characterized.
- These compounds exhibited moderate cytotoxicity against human cancer cell lines.
Conclusions:
- The streamlined in vitro disassembly-in vivo reassembly method simplifies silent gene cluster refactoring.
- This approach accelerates the discovery of natural products from both cultured bacteria and metagenomic sources.
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