Microtiter Plate Cultivation Systems Enable Chemically Diverse Metabolic Footprints During Bacterial Natural Product Discovery

Rediscovery of known structures is a frequent problem in screening for bioactive bacterial natural products (NPs). Highly parallelized microtiter plate cultivation systems (MPCS) can improve the chance to discover novel NPs by testing a multitude of cultivation conditions simultaneously. An in‐depth analysis and comparison of cultivation systems for NP discovery, however, has not been carried out so far. We compared the growth and metabolic footprint of four distinct bacterial species in three MPCS, shake flasks, and stirred tank bioreactors (STR). While the big majority of the cultivation systems provided good growth, we found a considerable divergence in secondary metabolite (SM) formation. The SM space was approximated by the appearance of unique mass features (MFs) in the supernatant extracts throughout the cultivation period. Molecular network analysis was applied to visualize the changes from detected MFs at the molecular level. The cultivation systems had a minor impact on the unicellular growing Bacillus amyloliquefaciens. This impact was more pronounced for the tested filamentous bacteria, resulting in a diversified metabolic footprint. The maximal overlap of 31% of produced MFs indicates a lack of comparability between the cultivation systems, resulting in different entries of growth phases and the formation of associated SMs. The detected SMs and its derivatives exhibited structural modification depending on the cultivation system. A comparison of Streptomyces griseochromogenes NP profile revealed that MPCS yielded less divergent SM formation than shake flasks. Our comprehensive assessment is the first to demonstrate the impact of cultivation systems on the bacterial metabolic footprint, confirming that MPCS provide a robust platform for the parallelization of bacterial cultivations for the discovery of bacterial NPs and accessing the chemical NP space more broadly.