Elucidation of the Verrucofortine Biosynthetic Pathway Enables Identification of a Cyclodipeptide Prenyltransferase with High Catalytic Efficiency

<i>Penicillium polonicum</i>, a common spoilage fungus affecting stored grains, nuts, and dried fruits, produces diverse secondary metabolites, including the indole-diketopiperazine derivative verrucofortine. Here, we elucidate its biosynthetic pathway through targeted gene deletion in a constructed low-metabolite-background strain and biochemical characterization with recombinant proteins. A nonribosomal peptide synthetase (VftPS) is responsible for assembling the cyclodipeptide scaffold, which is then modified by the prenyltransferase VftPT and the acetyltransferase VftAT. Notably, VftPT shows higher catalytic efficiency toward all the tested tryptophan-containing cyclodipeptides for <i>C3ß</i>-prenylation at the indole ring than its known homologues, highlighting its potential for biocatalysis and small molecule modification.