Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains as well as the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents like MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622 which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as part of future microbial natural products screening workflows. File list (7) download file view on ChemRxiv SFE_Preprint_2020-07-10.pdf (1.28 MiB) download file view on ChemRxiv SFE_SI.pdf (2.23 MiB) download file view on ChemRxiv MSr11367.zip (2.43 GiB) download file view on ChemRxiv MSr10575.zip (2.67 GiB) download file view on ChemRxiv DK1622.zip (2.91 GiB) download file view on ChemRxiv CBD_DK1622_Cl_295.zip (68.60 MiB) download file view on ChemRxiv Blanks.zip (2.43 GiB)

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2S,4R)-4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from the 4-methylproline pathway. We isolate and structurally elucidate four mycoplanecins comprising scarce homo-amino acids and 4-alkylprolines. Evaluating mycoplanecin E against Mycobacterium tuberculosis surprisingly reveals an excitingly low minimum inhibition concentration at 83 ng/mL, thus outcompeting griselimycin by approximately 24-fold. We show that mycoplanecins bind DnaN with nanomolar affinity and provide a co-crystal structure of mycoplanecin A-bound DnaN. Additionally, we reconstitute the biosyntheses of the unusual L-homoleucine, L-homonorleucine, and (2S,4R)-4-ethylproline building blocks by characterizing in vitro the full set of eight enzymes involved. The biosynthetic study, bioactivity evaluation, and drug target validation of mycoplanecins pave the way for their further development to tackle multidrug-resistant mycobacterial infections.