Targeted Discovery and Characterization of Type‐II PKS‐NRPS Hybrid DNA‐Alkylating Antibiotics

DNA alkylating natural products usually exhibit diverse bioactivity and serve as a crucial source of drug leads. Here, we employed genome mining guided by HTH-42 superfamily resistance gene to precisely discover a new class of DNA-alkylating antibiotics, xinghaicarcins, from Streptomyces xinghaiensis. They possess an intricate spiro-epoxide-bearing spiroketal heptacyclic scaffold fused with a pipecolic acid, assembled by a type II polyketide synthase-nonribosomal peptide synthetase hybrid system. An aminotransferase XhnB1 and a methyltransferase XhnM are identified to catalyze the formation of N-methylated pipecolic acid building block, leading to the completion of the polyketide-peptide backbone. The identification of XhnM facilitated stereochemical determination of six chiral centers in xinghaicarcins by co-crystallization. Notably, xinghaicarcins exhibit potent antibacterial activity against drug-resistant pathogens and cytotoxicity against multiple cancer cell lines. Additionally, the HTH-42 superfamily resistant protein, XhnU2, was characterized to mitigate xinghaicarcin-induced genotoxicity. This work provides comprehensive insights into structure, biosynthesis, bioactivity, and self-resistance mechanisms of xinghaicarcins, expanding diversity of DNA alkylating natural products.