Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria

The transmission of antibiotic-resistance genes, comprising mobilization and relocation events, orchestrates the dissemination of antimicrobial resistance. Inspired by this evolutionarily successful paradigm, we developed ACTIMOT, a CRISPR-Cas9–based approach to unlock the vast chemical diversity concealed within bacterial genomes. ACTIMOT enables the efficient mobilization and relocation of large DNA fragments from the chromosome to replicative plasmids within the same bacterial cell. ACTIMOT circumvents the limitations of traditional molecular cloning methods involving handling and replicating large pieces of genomic DNA. Using ACTIMOT, we mobilized and activated four cryptic biosynthetic gene clusters from Streptomyces, leading to the discovery of 39 compounds across four distinct classes. This work highlights the potential of ACTIMOT for accelerating the exploration of biosynthetic pathways and the discovery of natural products., , , # Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria

[https://doi.org/10.5061/dryad.2280gb60t](https://doi.org/10.5061/dryad.2280gb60t)

## Description of the data and file structure

We have submitted our liquid chromatography-mass spectrometry (LC-MS) data for creating figures in the main text (Figs._in_maintext.zip) and supplementary materials (figs._in_SM_part_1.zip and figs._in_SM_part_2.zip).

We have also submitted our nuclear magnetic resonance (NMR) data for structure elucidation of avidistatin (Avidistatin-NMR.7z), avidilipopeptins (Avidilipopeptins-NMR.7z), mobilipeptins (Mobilipeptins-NMR.7z), and actimotins (Actimotins-NMR.7z).

The data are structured in seven compressed archives. The files Figs._in_maintext.zip, figs._in_SM_part_1.zip and figs._in_SM_part_2.zip contains LC-MS data. The files Actimotins-NMR.7z, Avidistatin-NMR.7z, Mobilipeptins-NMR.7z, and Avidilipopeptins-NMR.7z contain NMR files. LC-MS data files are organiz...