Characterization of metabolites in intact Streptomyces citricolor culture supernatants using high-resolution nuclear magnetic resonance and directly coupled high-pressure liquid chromatography-nuclear magnetic resonance spectroscopy

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional H-1 NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures of Streptomyces citricolor. By applying H-1 NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC-NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the H-1 NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as H-1-H-1 correlation spectroscopy, H-1-C-13 heteronuclear single quantum correlation, and H-1-C-13 heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on H-1-H-1 coupling networks and C-13 chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment. (C) 1999 Academic Press.