Expanding the Scope of Detectable Microbial Natural Products by Complementary Analytical Methods and Cultivation Systems

Recent advances in genome sequencing unveiled a large discrepancy between the genome-encoded capacity of micro-organisms to produce secondary metabolites and the number of natural products (NP) actually detected in their extracts. In order connect biosynthetic gene cluster diversity to NPs, common approaches include genetic manipulation of producers and diversifying the cultivation conditions. However, the range of detectable metabolites is fundamentally limited by the applied analytical method. In this work, we present a two-platform mass spectrometry analysis for the comprehensive secondary metabolomics characterization of nine myxobacterial strains. We evaluated direct infusion (DI) measurements of crude extracts on a Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer and compared them to measurements conducted on a Time-Of-Flight (TOF) device coupled to liquid chromatography (LC), a setup widely used in NP laboratories. We demonstrate that both methods are likewise successful to detect known metabolites, whereas statistical analysis of unknowns highlights their complementarity: strikingly, 82-99% molecular features were only found with one of the analytical setups. In addition, we evaluated NP profile differences seen from our set of strains grown in liquid culture versus their swarming colonies on agar plates. The detection of 21-96% more molecular features when both liquid and plate cultures were ana-lyzed (compared to only one of them) translates into increased chances to identify new NPs. Determination of strain-specific compounds in combination with GNPS molecular networking revealed strain Mx3 as particularly promising in terms of isolation and structure elucidation of novel secondary metabolites.