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.

Show all samples  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST001994
Study TitleMutasynthetic production and antimicrobial characterisation of Darobactin darobactin analogs (NMR analysis)
Study SummaryThere 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
LaboratorySchäberle Laboratory
Last NameMettal
First NameUte
AddressOhlebergsweg 12, 35392 Giessen, Germany
EmailUte.Mettal@chemie.uni-giessen.de
Phone+49 641 97219 142
Submit Date2021-11-04
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2022-11-21
Release Version1
Ute Mettal Ute Mettal
https://dx.doi.org/10.21228/M8XX3Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Treatment:

Treatment ID:TR002087
Treatment Summary:Purification of DaroB from the producer strain was achieved with a modified purification strategy from DaroA. Briefly, E. coli production strains were incubated for 5 days in a 2 L Erlenmeyer flask with 1 L LB medium supplemented with 50 μg/mL kanamycin at 30 °C. Cells were removed via centrifugation and the culture supernatant was mixed with XAD16N resin (Sigma-Aldrich) overnight under agitation. DaroB was subsequently eluted from the resin with a 50/50 solution of methanol and water, containing 0.1% formic acid. The eluate was then concentrated via rotary evaporator and loaded onto a cation-exchange column (SP Sepharose XL). DaroB was eluted by step gradients of 50 mM ammonium acetate pH 7, pH 8, and pH 10. Eluates were then concentrated by freeze drying, resuspended in Milli-Q water 0.1% (v/v) formic acid, and loaded onto a C18 reversed-phase high-performance liquid chromatography (RP-HPLC) column (Agilent, C18 5 µm: 250 x10mm, Restek). HPLC conditions for purification of DaroB are: solvent A, Milli-Q water and 0.1% (v/v) formic acid; solvent B, acetonitrile and 0.1% (v/v) formic acid. The initial concentration of 2% solvent B is maintained for 2 min, followed by a linear gradient to 26% B over 12 min with a flow rate of 5 mL min−1; UV detection by diode-array detector from 210 to 400 nm. Pure DaroB was then collected at 11.5 min. For purification of DaroE, fermentation broth was pelleted by centrifugation. The cell pellet was extracted using 80% acetonitrile and water by sonification. The resulting crude extract was fractionated by flash chromatography using a C18 F0120 column with the following gradient: 1) 0-28 min 5% ACN, 2) 28-37 min increased to 15% ACN, 3) 37-50 min, keeping 15% ACN, 4) 50-60 min, increased to 30% ACN, 5) 60-80 min, increased to 100% ACN and keeping 100% ACN for 15 min. By LCMS guided isolation, the DaroE-containing fraction was identified and further separated by HPLC using the following gradient: 1) 0-10 min 23% MeOH, 2)10-20 min increased to 50% MeOH, 3) 20-30 min increased to 100% MeOH, 4) 30-37 min 100% MeOH. Afterwards, the DaroE fraction was further purified by HPLC (gradient: 1) 0-5 min 25 %MeOH, 2) 5-45 min increased to 42.5% MeOH, 3) 45-52 min keeping 100% MeOH to obtain pure compound. For DaroD the same procedure via flash chromatography was followed. Then, the following HPLC gradient was applied: 1) 0-5 min 15% ACN, 2) 5-25 min increased to 25% ACN, 3) 25-30 min increased to 60% ACN, 4) 30-39 min 100% ACN. As before, a further HPLC separation followed to obtain DaroD as pure compound.
Treatment Protocol Filename:Treatment_Protocol_Isolation_of_compounds.docx
  logo