DOI: 10.5061/dryad.2280gb60t
title: Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria

description: 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.
description: # 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 organized into folders corresponding to each figure in the main text or SM, while NMR data files are grouped into folders based on each compound family characterized in the manuscript. LC-MS data (all archives starting named Figs*) The LC-MS files are organized into subfolders named according to the specific figure they correspond to. Each subfolder contains LC-MS files (.d) for related figures included in this manuscript submission. The name of each file indicates the figure number referenced in the main text and/or supplementary materials (SM). The .d folder is the standard format produced by Bruker Mass Spectrometers, and the files within the .d folder are accessory files necessary for opening the .d file using the DataAnalysis software (detailed in the Usage Notes). The users can treat the .d file as a single entity and open it directly with DataAnalysis. NMR data This dataset includes both raw and processed NMR data files for all NMR spectra presented in this manuscript. Raw data files are stored in subfolders named numerically, while processed NMR data files (.spectrus) are provided in the same folder as the corresponding compound name. The processed files (.spectrus) were generated using ACD/Labs software. All related files are named for the compound's name and the solvent used for the measurement. Each numerically named subfolder represents the data for a single spectrum and can be directly opened with the Bruker’s TopSpin software mentioned in the following “Usage Notes.” All these folders should contain all the required information to access the data and interactively view the LC-MS and NMR spectra. A more detailed list of the files is provided at the end of this README file. Data was derived from the following sources: * \[LC-MS source data](LC-MS source data was derived from experiments for compound identification and structure elucidation of avidistatins, avidilipopeptins, mobilipeptins, and actimotins.) * \[NMR source data](NMR source data was derived from experiments for compound structure elucidation of avidistatin, avidilipopeptins, mobilipeptins, and actimotins.) ## Usage notes For viewing NMR raw data, the Bruker’s TopSpin 4.4.1 release software package can be used. For viewing .spectrus files, the ACD/Labs software can be used. All LC-MS files (.d) can be viewed in the Compass DataAnalysis 5.3. To view in Compass DataAnalysis follow these steps: 1. Register for a Bruker users account. 2. Download the Compass DataAnalysis (without Compound Crawler) software. 3. Install the Software by opening the executable in the ISOOut folder. 4. Open the CD_Start executable. 5. Follow instructions for installation. 6. Open DataAnalysis and choose File - open to choose the desired .d folder for viewing.  ### Files and variables #### File: figs.\_in\_SM\_part\_2.zip **Description:** This compressed archive contains fourteen folders. Each fold contains the LC-MS data used to create the corresponding figures S17-S20, S25-S20, S28-S31, S33, S38, and S46-S48, in the supplementary materials. The file names within the folder adhere to the following naming convention: "AnalysisType_SampleName.d" or "SampleName_Media.d". AnalysisType indicates the type of analysis performed, such as Marfey. SampleName: Refers to the sample being analyzed, such as avidilipopeptinB2, L-Arg_D-FDLA or Del14_pCap-avl. Media indicates the culture media used, such as M1 or ISP4. Folder fig.S17: LC-MS data of Marfey's method analysis for avidilipopeptin B2 and corresponding standard amino acids (fig. S17). Folder fig.S18 and S19: LC-MS/MS data of avidistatins and avidilipopeptins (fig. S18). (MS/MS: Tandem mass spectrometry) Folder fig.S20: LC-MS data of crude extracts of S. albus Del14 harboring pCap-Sav11, pCap-avs, or pCap-avl (fig. S20). Folder fig.S25: LC-MS data of crude extracts of S. armeniacus/pCap-Sar13 and S. armeniacus WT (fig. S25). (WT: wild-type) Folder fig.S26: LC-MS data of mobilipeptins A, D, E, F (fig. S26). Folder fig.S28: LC-MS data of Marfey's method analysis for mobilipeptin A and corresponding standard amino acids (fig. S28). Folder fig.S29: LC-MS/MS data of mobilipeptins A, C, D (fig. S29). Folder fig.S30: LC-MS data of crude extracts of DSM43125/pCap-Sar13 (A) and S. albus Del14/pCap-Sar13 at different time points (fig. S30). Folder fig.S31: LC-MS data of mobilipeptins E and mobilipeptins E incubated with acetone (fig. S31). Folder fig.S33: LC-MS data of Marfey's method analysis for mobilipeptin D and corresponding standard amino acids (fig. S33). Folder fig.S38: LC-MS data of actimotins A-H, J (fig. S38). Folder fig.S46: LC-MS/MS data of actimotins D-E, H-L (fig. S46). Folder fig.S47: LC-MS data of crude extracts of S. albus Del14 harboring pCap-Sav17/ΔNRPS, S. albus Del14 harboring pCap-Sav17/Δamo, S. albus Del14 harboring pCap-Sav17, and S. albus Del14 (fig. S47). Folder fig.S48: LC-MS data of actimotins A when feeding with l-aspartic acid-13C4,15N or l-cysteine-13C3,15N (fig. S48). #### File: figs.\_in\_SM\_part\_1.zip **Description:** This compressed archive contains five folders. Each fold contains the LC-MS data used to create the corresponding figures S11, S13-S16 in the supplementary materials. The file names within the folder adhere to the following naming convention: "AnalysisType_SampleName.d". AnalysisType indicates the type of analysis performed, such as Marfey. SampleName: Refers to the sample being analyzed, such as avidistatinA1 or L-Arg_D-FDLA. Folder fig.S11: LC-MS data of avidistatin A1 (fig. S11). Folder fig.S13: LC-MS data of avidilipopeptins (fig. S13). Folder fig.S14: LC-MS data of Marfey's method analysis for avidistatin A1 and corresponding standard amino acids (fig. S14). Folder fig.S15: LC-MS data of Marfey's method analysis for avidilipopeptin A1 and corresponding standard amino acids (fig. S15). Folder fig.S16: LC-MS data of Marfey's method analysis for avidilipopeptin A5 and corresponding standard amino acids (fig. S16). #### File: Figs.\_in\_maintext.zip **Description:** This compressed archive contains three folders. Each fold contains the LC-MS data used to create the corresponding figures 3-5 in the main text. The file names within the folder adhere to the following naming convention: "SampleName_Media.d". SampleName: Refers to the sample being analyzed, such as Del14-control. Media indicates the culture media used, such as M1 or M3. Folder Fig.3: LC-MS data of crude extracts of S. albus Del14/pCap-Sav11 and S. albus Del14 (Fig. 3). Folder Fig.4: LC-MS data of crude extracts of S. armeniacus/pCap-Sar13, S. armeniacus, S. armeniacus/pCap-Sar13 and S. albus Del14/pCap-Sar13-int (Fig. 4). Folder Fig.5: LC-MS data of crude extracts of S. avidinii/pCap-Sav17, S. avidinii, S. albus Del14/pCap-Sav17, and S. albus Del14 (Fig. 5) #### File: Actimotins-NMR.7z **Description:** This compressed archive contains fourteen folders. Each fold contains the raw and ACD Labs software processed NMR data of actimotins A-H, J. The file names within the folder adhere to the following naming convention: "CompoundName_Solvent.d". CompoundName: Refers to the compound being analyzed, such as Actimotin-A. Solvent indicates the NMR solvent used, such as pyridine or DMSO. Folder Actimotin-A-pyridine: NMR data of actimotin A acquired using pyridine-*d*5 as solvent. Folder Actimotin-A-DMSO: NMR data of actimotin A acquired using DMSO-*d*6 as solvent. Folder Actimotin-B-pyridine: NMR data of actimotin B acquired using pyridine-*d*5 as solvent. Folder Actimotin-C-DMSO: NMR data of actimotin C acquired using DMSO-*d*6 as solvent. Folder Actimotin-C_R-MTPA-Cl-DMSO: NMR data of *R*-Mosher ester of actimotin C acquired using DMSO-*d*6 as solvent. Folder Actimotin-C_S-MTPA-Cl-DMSO: NMR data of *S*-Mosher ester of actimotin C acquired using DMSO-*d*6 as solvent. Folder Actimotin-D-DMSO: NMR data of actimotin D acquired using DMSO-*d*6 as solvent. Folder Actimotin-E-DMSO: NMR data of actimotin E acquired using DMSO-*d*6 as solvent. Folder Actimotin-F-DMSO: NMR data of actimotin F acquired using DMSO-*d*6 as solvent. Folder Actimotin-G-DMSO: NMR data of actimotin G acquired using DMSO-*d*6 as solvent. Folder Actimotin-G_R-MTPA-Cl-DMSO: NMR data of *R*-Mosher ester of actimotin G acquired using DMSO-*d*6 as solvent. Folder Actimotin-G_S-MTPA-Cl-DMSO: NMR data of *S*-Mosher ester of actimotin G acquired using DMSO-*d*6 as solvent. Folder Actimotin-H-DMSO: NMR data of actimotin H acquired using DMSO-*d*6 as solvent. Folder Actimotin-J-DMSO: NMR data of actimotin J acquired using DMSO-*d*6 as solvent. #### File: Avidistatin-NMR.7z **Description:** This compressed archive contains two folders. Each fold contains the raw and ACD Labs software processed NMR data of avidistatin A1. The file names within the folder adhere to the following naming convention: "CompoundName_Solvent.d". CompoundName: Refers to the compound being analyzed, such as Avidistatin-A1. Solvent indicates the NMR solvent used, such as pyridine or D2O. Folder Avidistatin-A1-D2O: NMR data of avidistatin A1 acquired using D2O as solvent (D2O: Deuterium oxide). Folder Avidistatin-A1-DMSO: NMR data of avidistatin A1 acquired using DMSO-*d*6 as solvent. (DMSO: Dimethylsulfoxid-*d*6). #### File: Mobilipeptins-NMR.7z **Description:** This compressed archive contains three folders. Each fold contains the raw and ACD Labs software processed NMR data of mobilipeptins A, B, F. The file names within the folder adhere to the following naming convention: "CompoundName_Solvent.d". CompoundName: Refers to the compound being analyzed, such as Mobilipeptin-A. Solvent indicates the NMR solvent used, such as DMSO. Folder Mobilipeptin-A-DMSO: NMR data of mobilipeptin A acquired using DMSO-*d*6 as solvent. Folder Mobilipeptin-B-DMSO: NMR data of mobilipeptin B acquired using DMSO-*d*6 as solvent. Folder Mobilipeptin-F-DMSO: NMR data of mobilipeptin F acquired using DMSO-*d*6 as solvent. #### File: Avidilipopeptins-NMR.7z **Description:** This compressed archive contains nine folders. Each fold contains the raw and ACD Labs software processed NMR data of avidilipopeptins A1-A5, B1-B4. The file names within the folder adhere to the following naming convention: "CompoundName_Solvent.d". CompoundName: Refers to the compound being analyzed, such as Avidilipopeptin-A1. Solvent indicates the NMR solvent used, such as MeOD. Folder Avidilipopeptin-A1-MeOD: NMR data of avidilipopeptin A1 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-A2-MeOD: NMR data of avidilipopeptin A2 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-A3-MeOD: NMR data of avidilipopeptin A3 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-A4-MeOD: NMR data of avidilipopeptin A4 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-A5-MeOD: NMR data of avidilipopeptin A5 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-B1-MeOD: NMR data of avidilipopeptin B1 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-B2-MeOD: NMR data of avidilipopeptin B2 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-B3-MeOD: NMR data of avidilipopeptin B3 acquired using methanol-*d*4 as solvent. Folder Avidilipopeptin-B4-MeOD: NMR data of avidilipopeptin B4 acquired using methanol-*d*4 as solvent.