title: Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis.
abstract: Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.
authors: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany.
authors: Marina
authors: M
authors: Berditsch
authors: Karlsruhe Institute of Technology, Institute of Biological Interfaces (IBG-2), Karlsruhe, Germany.
authors: Sergii
authors: S
authors: Afonin
authors: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany.
authors: Anna
authors: A
authors: Steineker
authors: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany.
authors: Nataliia
authors: N
authors: Orel
authors: Karlsruhe Institute of Technology, Institute of Biological Interfaces (IBG-2), Karlsruhe, Germany.
authors: Igor
authors: I
authors: Jakovkin
authors: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany.
authors: Christian
authors: C
authors: Weber
authors: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany Karlsruhe Institute of Technology, Institute of Biological Interfaces (IBG-2), Karlsruhe, Germany Anne.Ulrich@kit.edu.
authors: Anne S
authors: AS
authors: Ulrich
mesh_terms: D056449:Bacillales / Q000378:metabolism*; D002247:Carbon Isotopes / Q000378:metabolism*; D003362:Cost-Benefit Analysis; D003470:Culture Media / Q000737:chemistry; D005285:Fermentation; D006096:Gramicidin / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D003470:Culture Media; D009587:Nitrogen Isotopes; D006096:Gramicidin