Molecular networking assisted discovery and combinatorial biosynthesis of new antimicrobial pleuromutilins.

Pleuromutilins, the unique fungal metabolites possessing 5/6/8 tricyclic skeleton, are potent antibacterial leading compounds for the development of new antibiotics. We applied the MS/MS molecular networking technique and the combinatorial biosynthesis approach to discover new pleuromutilin analogues. Ten pleuromutilin derivatives including seven new compounds (1-7) were obtained from the solid culture of Omphalina mutila. The gene cluster for the biosynthesis of pleuromutilins in the mushroom of O. mutila was identified and further expressed in yeast. Nine pleuromutilin-type diterpenes including three new "unnatural" pleuromutilins (16-18) were generated in a GGPP-engineered Saccharomyces cerevisiae. The antimicrobial bioassays indicated that compounds 3, 9, 10, 15, and 17 exhibited potent inhibition against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Several pleuromutilins were found to show immunomodulatory activities by promoting the cell viability, enhancing the ROS and NO production, or increasing the levels of proinflammatory cytokines IL-6 and TNF-α in the macrophage RAW 264.7. The structure-activity relationship for pleuromutilins was analyzed.