Discovery of a Unique NRPS Gene Cluster San for the Biosynthesis of Anti-Phytopathogenic and Cytotoxic Sansalvamides in the Endophytic Fusarium sp. R1

Sansalvamides are a class of bioactive cyclodepsipeptides recognized for their potent antitumor properties. In this study, genome mining of an endophytic fungus Fusarium sp. R1 led to the identification of a unique nonribosomal peptide synthetase (NRPS) gene cluster ( san ), which consists of 5 core genes and 14 auxiliary tailoring genes. Chemical investigation of strain R1 resulted in discovery of one new noncyclic sansalvamide derivative 1 together with three analogues ( 2 – 4 ). Using CRISPR-Cas9-mediated gene knockout, we disrupted the san cluster for the first time, abolishing production of compounds 1 – 4 and confirming its functional indispensability. Transcriptomic analysis unveiled a GDSL-like lipase/acylhydrolase ( sanP ) responsible for catalysis of a key ring-opening step followed by the production of 1 and 2 . Antifungal assays revealed that compounds 3 and 4 possessed pronounced activity against the phytopathogenic fungus Alternaria solani with inhibition rates of 66.7 and 77.0%, respectively. In addition, compound 4 exhibits excellent inhibitory activity against tumor cell lines HCT-116 and MCF-7/ADR with IC 50 values of 0.71 and 12.50 μM, respectively, indicating its potential application in agrochemical and pharmaceutical industry. Simultaneously, these results showed that the role of the macrocyclic scaffold of sansalvamides is essential for antifungal effect and the N -methylation of these chemicals could improve efficiency.