Deciphering the Regioselective Acylation Modification in Bipentaromycin

Comprehensive Summary Aromatic polyketides (APKs) are renowned for their structural diversity and potent biological activities, making them valuable resources for drug discovery in antibiotics, anticancer agents, and antiparasitic therapies. Bipentaromycins, a unique class of dimeric APKs with a cyclic head‐to‐tail linkage, exhibit broad‐spectrum antibacterial activities. Acylation modifications in bipentaromycins C–F enhance their pharmacological properties, yet the responsible acyltransferase remains enigmatic. Herein, we present REGAIN, an innovative strategy combining r estriction e nzyme digestion, G ibson a ssembly, and i n vivo Cre‐ lox recombinatio n , enabling rapid and efficient cloning of biosynthetic gene clusters (BGCs). Using REGAIN, we successfully cloned a 40 kb bipentaromycin BGC ( bpm ). By integrating genetic experiments and computational modeling, we speculated BpmP as the acyltransferase responsible for regioselective acylation. This study establishes a robust platform for exploring novel bioactive molecules and advances the understanding of bipentaromycin biosynthesis.