Over the past decade, the One Strain Many Compounds (OSMAC) approach has been established for silent gene cluster activation and elicitation of secondary metabolite production, but so far the full secondary metabolome of a biosynthetically promising bacterium has not been elucidated yet. Here, we investigate the ability of seven categories of OSMAC conditions to enhance the diversity of new mass features from bacterial strains with little literature coverage but high biosynthetic potential. The strains Bacillus. amyloliquefaciens DSM7, Corallococcus. coralloides DSM2259, Pyxidicoccus. fallax HKI727, Rhodococcus. jostii DSM44719, and Streptomyces. griseochromogenes DSM40499 were selected after genome mining with antiSMASH. After cultivation under OSMAC conditions, the generated extracts were subjected to LC-MS and MZmine analysis to determine new mass features and evaluate the tested culture conditions. 4 predicted compounds, bacillibactin, desferrioxamine B, myxochelin A, and surfactin, were identified and up to 147 new mass features were detected in the generated extracts, which greatly surpasses the number of predicted gene clusters. Among the new mass features are bioactive compounds that were so far unreported for the strains such as cyclo-(Tyr-Pro) from DSM7 and nocardamin from DSM2259. Furthermore, the tested culture conditions were evaluated regarding their suitability for the generation of new mass features from the selected strains and promising new starting points for further screenings are postulated. Especially culture conditions with little prior literature coverage are responsible for the activation of predicted gene clusters

The scarcely investigated myxobacterium Corallococcus coralloides holds a large genome containing many uncharacterized biosynthetic gene clusters (BGCs) that potentially encode the synthesis of entirely new natural products. Despite its promising genomic potential, suitable cultivation conditions have not yet been found to activate the synthesis of new secondary metabolites (SMs). Finding the right cultivation conditions to activate BGCs in the genome remained a major bottleneck and its full biosynthetic potential was so far not retrieved. Here, we therefore applied a bivariate OSMAC approach, using a combination of two elicitor changes at once for activation of BGCs and concomitant SM production by C. coralloides. The bivariate OSMAC screening was carried out in 24-well System Duetz-plates, applying univariate and bivariate OSMAC conditions. We combined biotic additives and organic solvents with minimal media and complex growth medium. The success of the method was evaluated by the number of new mass features detected in the respective extracts. We found synergistic effects in bivariate OSMAC designs. The number of new mass features detected in bivariate OSMAC exceeded the sum of new mass features found in the respective univariate OSMAC with only one elicitor. Overall, the bivariate OSMAC screening led to 26 new mass features, which were not detected in the univariate OSMAC design. Hence, the presence of multiple elicitors in the bivariate OSMAC designs successfully activated the biosynthetic potential in C. coralloides. We propose the bivariate OSMAC designs with a complex combination of elicitors as a straightforward strategy to robustly expand the SM space of microorganisms with large genomes.