Spirolactone, an unprecedented antifungal β-lactone spiroketal macrolide from Streptomyces iranensis

Fungal infections pose a great threat to public health. There are only four classes of antifungals that have limitations due to high toxicity, drug-drug interactions, and emerging drug-resistance. Streptomyces spp. represent an important source of antimicrobial substances, notably including the antifungal agent amphotericin B. The rapamycin-producer Streptomyces iranensis displayed strong antifungal activities against Aspergillus. Revisiting its genome revealed several intriguing biosynthetic gene clusters, including one unparalleled Type I polyketide synthase, which codes for uncharacterized metabolites. The identification of a novel macrolide spirolactone (1) and its biosynthetic gene cluster was facilitated through CRISPR-based gene editing, HR-ESI-MS analysis, followed by fermentation and purification processes. Their structures and absolute configurations were confirmed by NMR, MS and X-ray crystallography. Spirolactone harbors an undescribed carbon skeleton with 13 chiral centers, featuring a rare β-lactone moiety, a [6,6]-spiroketal ring, and an unprecedented 7-oxo-octylmalonyl-CoA extender unit incorporated by a potential novel Crotonyl-CoA carboxylase/reductase. Spirolactone displayed profound antifungal effects against numerous fungal pathogens, e.g. the genus Talaromyces and several sections of Aspergillus including clinically relevant species such as Aspergillus niger and A. tubingensis (section Nigri), A. terreus (section Terrei) and the azol-resistant A. calidoustus (section Usti). Proteomics analysis revealed spirolactone potentially disrupted the integrity of fungal cell walls and induced the expression of stress-response proteins in A. niger. Spirolactone represents a new class of potential drug candidate to combat fungal infections. ### Competing Interest Statement Two patent applications were filed with the numbers of PCT/EP2023/067953 and EP24150176.6.