Overexpression of Geranylgeranyl Diphosphate Synthase and Cyclase Enhances Pleuromutilin Production in <i>Clitopilus Passeckerianus</i> T6

Pleuromutilin, a tricyclic diterpene compound with significant inhibitory activity against gram-positive bacteria and mycoplasmas, serves as a precursor for various veterinary and human medicines. Previous efforts have primarily focused on strain screening and fermentation process optimization to enhance pleuromutilin production in native pleuromutilin-producing strains, with the absence of genetic engineering strategies. In this study, we performed whole-genome sequencing of the pleuromutilin-producing strain Clitopilus passeckerianus T6 to identify the biosynthetic genes related to pleuromutilin production. Transcriptomic and metabolomic data were collected during the fermentation of C. passeckerianus T6, and gene transcription and metabolite accumulation in the pleuromutilin biosynthetic pathway were analyzed to identify the rate-limiting steps in pleuromutilin biosynthesis. Overexpression of the key genes ple-ggpps and ple-cyc increased pleuromutilin production by 50%, achieving a titer of 6.9 g/L. This study is the first to employ metabolic engineering to enhance pleuromutilin production in a native strain, providing a strategy for efficient pleuromutilin production.