Discovery and Biosynthesis of Rare Fungal Terpenoid‐Isonitrile Amino Acid Hybrid Natural Products

Abstract Terpenoid nonproteinogenic amino acid hybrid natural products usually have complex chemical structures and important biological functions. Herein, we discovered a group of conserved and widely distributed clusters of sesquiterpenoid‐isonitrile isoleucine hybrid compounds from Fusarium through a two‐step genome mining strategy. An investigation of the function of the isc cluster from Fusarium chlamydosporum revealed a variety of unusual enzymatic transformations, which importantly include 1) a single‐module nonribosomal peptide synthase (NRPS) IscF featuring an unusual C ‐terminal transferase domain that catalyses the esterification reaction on C2─OH of a highly oxidised guaiane‐type 5/7‐bicyclic sesquiterpene precursor 4 with T domain‐bound l ‐isonitrile isoleucine 11a to yield terpenoid‐isonitrile isoleucine hybrid natural product 9a (chlamonitrile A) and 2) cytochrome P450 (CYP450) IscD, which unexpectedly catalyses the Cα–Cβ dehydrogenation of the isonitrile isoleucine moiety of 9a , resulting in the final products 13a / 13b (chlamonitriles E/F). In addition, phytotoxic evaluation experiments revealed that 9a and its structural analogues are new phytotoxins of Fusarium . Our work provides the first example of the discovery of terpenoid‐isonitrile amino acid hybrid compounds from fungi, expands our knowledge of the new functions of NRPS domains and fungal CYP450s, and uncovers the possible biological functions of these compounds in Fusarium .