Summary of Study ST001994
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001267. The data can be accessed directly via it's Project DOI: 10.21228/M8XX3Q This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001994 |
| Study Title | Mutasynthetic production and antimicrobial characterisation of Darobactin darobactin analogs (NMR analysis) |
| Study Summary | There is great need for therapeutics against multi-drug resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer-membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding for the synthesis of darobactin A can also be found in other γ-proteobacterial families. Therein, the precursor peptides DarB-F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The generated analogs were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for co-crystallization with the target BamA, revealing an identical binding site to darobactin A. Besides its potency, darobactin B did not exhibit cytotoxicity and was slightly more active against Acinetobacter baumanii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotics class, which is likely to expand to several promising therapeutic candidates |
| Institute | Justus-Liebig-University Giessen |
| Laboratory | Schäberle Laboratory |
| Last Name | Mettal |
| First Name | Ute |
| Address | Ohlebergsweg 12, 35392 Giessen, Germany |
| Ute.Mettal@chemie.uni-giessen.de | |
| Phone | +49 641 97219 142 |
| Submit Date | 2021-11-04 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2022-11-21 |
| Release Version | 1 |
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Collection:
| Collection ID: | CO002068 |
| Collection Summary: | E. coli strains for cloning and expression were grown in LB broth or on agar medium supplemented with appropriate antibiotics or supplements at 37° C or 30° C using standard working concentrations. Plasmid DNA was isolated using the innuPREP plasmid mini kit 2.0 (AnalytikJena, Jena, Germany) according to the manufacturer’s protocol. Genomic DNA was extracted using the innuPREP bacteriaDNA kit (AnalytikJena, Jena, Germany). PCR amplification for cloning purposes was performed using Q5 DNA polymerase (NEB Biolabs, New Brunswick, USA) according to the given instruction. Restriction digestion was performed using standard techniques and employing NEB enzymes (NEB Biolabs, New Brunswick, USA). DNA fragments were analysed on and excised from 1% or 2% TAE-agarose with GeneRuler 1kb Plus (ThermoFisher, Waltham, USA) as marker. DNA for cloning purposes was purified using the Zymoclean large fragment DNA recovery kit according to manufacturer’s instruction. DNA concentrations were determined photometrically with an Eppendorf BioSpectrometer (Eppendorf AG, Hamburg, Germany) using a 1 mm light path UV cuvette. DNA fragments to be fused by isothermal assembly were gel purified and fused using self-made isothermal assembly master mix (Nat Methods 2009, 6, 343–345) using NEB enzymes (NEB Biolabs, New Brunswick, USA). Assembled plasmids were transferred to E. coli cells using standard electroporation protocols (Nature 2019, 576, 459-464, Metab Eng 2021, 66, 123-136). Construcion of pNBDaroMod for modification of the precursor peptide was performed by linearising pNB03 (Nature 2019, 576, 459-464) by PCR using 5’ TCCCTTAACGTGAGTTTTCG-3’/ 5’-TTTTATAACCTCCTTAGAGCTCGAA-3’, amplification of truncated (3’ minus 50 nt) darA using 5’ GCTCTAAGGAGGTTATAAAAATGCATAATACCTTAAATGAAACCGTTAAA-3’/ 5’-TAGGTTTATTGCTTAATTCGTTTAGTGCTT-3’, the lacZ spacer from pCRISPOMYCES-2 (5’ CGAATTAAGCAATAAACCTAAAGTCTTCTCAGCCGCTACA-3’/ 5’ ACCTGATGGGATAAGCTTTAATGTCTTCACCGGTGGAAAG-3’) and the rest of the P. khanii DSM3369 BGC using 5’-TAAAGCTTATCCCATCAGGTTATTT-3’/ 5’ CGAAAACTCACGTTAAGGGATTACGCCGCGATGGTTTGTTTTATT-3’ and subsequent isothermal assembly of the plasmid. After transformation and selection on LBKan/Apra/IPTG/X-gal, blue colonies were picked and the correct assembly of the plasmid was corroborated by test restriction. AA modifications were designed in silico and ordered as complementary oligonucleotides with 4 nt overlap to the pNBDaroMod backbone. Oligonucleotides were annealed and assembled into pNBDaroMod using the protocol described in ACS Synth Biol 2015, 4, 723-728 and the resulting plasmids were transferred to E. coli BW25113 and selected on LBKan/Apra/IPTG/X-gal. White colonies were picked and grown in LBKan/IPTG for three days at 220 rpm and 30° C. The correct assembly of the plasmid was corroborated by UHPLC-MS profiling, i.e. detection of the expected product ion. For increased production titter, the modified BGCs were recloned into pRSF-duett using the primers 5’-GTATAAGAAGGAGATATACAATGCATAATACCTTAAATGA-3’/ 5’ TGCTCAGCGGTGGCAGCAGCTTACGCCGCGATGGTTTGTT-3’ for all constructs to match the layout of pRSF-ADC5 and produced in E. coli Bap1 (Metab Eng 2021, 66, 123-136). |
| Collection Protocol Filename: | Collection_Protocol_Mutasynthetic_production_of_darobactin_analogs.docx |
| Sample Type: | Bacterial cells |
