Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

Abstract

Cyanogramide ( 1 ) from marine‐derived Actinoalloteichus cyanogriseus WH1‐2216‐6 features a unique spirooxindole skeleton and exhibits significant bioactivity to efficiently reverse the multiple drug resistance of tumor cells. The biosynthetic gene cluster of 1 was identified and refactored by promoter engineering for heterologous expression in Streptomyces coelicolor YF11, enabling the production of 1 and five new derivatives. Interesting, four of them, including 1 , are identified as enantiomeric mixtures in different ratios. Functions of tailoring enzymes, including two methyltransferases CyaEF, and three cytochrome P450 monooxygenases CyaGHI, were confirmed by gene inactivation and feeding experiments, leading to the elucidation of a concise biosynthetic pathway for 1 . Notably, CyaH was biochemically verified to catalyze formation of the spirooxindole skeleton in 1 through an unusual carbocation‐mediated semipinacol‐type rearrangement reaction .