Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Abstract

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.