English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene ( ato) gene cluster led to the characterization of two unprecedented bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly- in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.

Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach