Unusual and highly bioactive sesterterpenes synthesized by Pleurotus ostreatus during the co-culture with Trametes robiniophila Murr

Candida albicans and Cryptococcus neoformans , worldwide human pathogenic fungi, are receiving increasing attention due to the high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by co-culturing 16 wood-decaying basidiomycetes, among which the co-culture of Trametes robiniophila Murr and Pleurotus ostreatus was identified to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis discovered that 44 features were either newly synthesized or highly produced in this co-culture system, six of which belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with minimum inhibitory concentrations of 1 to 32 μg/mL against pathogenic fungi. Furthermore, dynamic 13 C-labeling analysis revealed an association between induced features and corresponding fungus. Unusual sesterterpenes were 13 C-labeled only in P. ostreatus in a time course after stimulation by the co-culture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr . Sesterterpenes 1 to 3 were renamed as postrediene A-C. qRT-PCR analysis revealed that transcriptional levels of three genes encoding for terpene synthase, farnesyl-diphosphate farnesyltransferase and oxidase were identified respectively to be 8.2, 88.7 and 21.6-fold higher in the co-culture than that in the mono-culture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A-C was then proposed based on structures of sesterterpenes and molecular network analysis. IMPORTANCE A number of gene clusters involved in secondary metabolites biosynthesis are presumably silent or low expressed under standard laboratory cultivation, resulting in a large gap between discovered metabolites and genome capability. This work mimicked naturally occurring competition by constructing an artificial co-culture of basidiomycete fungi for the identification of secondary metabolites with novel scaffolds and excellent bioactivity. Unusual linear sesterterpenes of postrediene A-C synthesized by P. ostreatus were not only promising lead drugs against human pathogenic fungi, but also highlighted a distinct pathway for sesterterpene biosynthesis in basidiomycetes. Current work provided an important basis for uncovering novel gene functions involved in sesterterpene synthesis and for gaining insights into the mechanism of silent gene activation in fungal defense.