Unravelling the Biosynthetic Flexibility of UK-2A Enables Enzymatic Synthesis of Its Structural Variants

Emerging antimicrobial resistant fungal pathogens are a growing threat, and fungicides with novel modes of action are urgently needed to prevent critical failures in global food security. Fenpicoxamid, the prodrug of UK-2A, is a member of a new class of antifungal agents that displays no cross-resistance to other fungicides. Rational engineering of its structure using a biosynthetic approach is a promising avenue for developing more potent fungicides. Herein, through in vitro enzymatic reconstitution, we elucidate the biosynthetic pathway of UK-2A. Its biosynthesis involves a flexible AMP-binding protein and dilactone formation assembly enzymes that are able to select and incorporate highly diverse substituted salicylic acids into the dilactone scaffold. By introducing diverse salicylic acids into the in vitro biosynthetic pathway, we successfully generate 14 novel deacyl UK-2A analogues. This study reveals the flexibility of the biosynthetic pathway of UK-2A and provides an effective solution to rationally engineer its crucial C3 moiety.