Discovery, Biological Activity, and Biosynthesis of Pinocicolin A, an Antibiotic Isocyanide Metabolite Produced by <i>Penicillium pinophilum</i>

We discovered new natural products pinocicolins A (<b>1</b>) and B (<b>2</b>) from <i>Penicillium pinophilum</i> and determined the stereochemistry of these compounds by degradation and derivatization. Compound <b>1</b>, containing two isocyanide groups, exhibited bacteriostatic activity against Gram-positive bacteria depending upon its copper-chelating activity. The biosynthesis of compound <b>1</b> was reconstructed in <i>Aspergillus nidulans</i> by heterologous expression of multidomain isocyanide synthase-nonribosomal peptide synthetase <i>Pp</i>_<i>crmA</i> and efflux pump <i>Pp</i>_<i>crmE</i>. This is the first report of the heterologous production of a fungal diisocyanide natural product, highlighting the multistep catalysis of CrmA family enzymes.