Mass spectrometric pathway monitoring of secondary metabolites: systematic analysis of culture extracts of Streptomyces species

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A(1), and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects. The combination of full scan mass spectrometry (MS), parent ion scan MS, product ion scan MS, and in-source collision-induced fragmentation prior to product ion scans (pseudo-MS(3) scan), using characteristic fragmentation of the central aminocoumarin unit, was employed for the detection and structural interpretation of expected and new intermediates. We were able to show the applicability of this methodology to the three culture extracts, where the main intermediates could be found, and to demonstrate its use for interpretation of secondary metabolite biosynthesis. Some new compounds were discovered, including bis-carbamoylated novobiocin, hydroxylated clorobiocin, and several structurally and not yet fully elucidated coumermycin derivatives or precursors.