Compartmentalized Biosynthesis of Mycophenolic Acid

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic in human history and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, its biosynthetic mechanisms have remained a long-standing mystery. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB′ as the long-sought key enzyme responsible for the oxidative cleavage of sesquiterpene side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC9 and O-methyltransferase MpaG9, the Golgi apparatus-associated prenyltransferase MpaA9, the endoplasmic reticulum-bound oxygenase MpaB9 and P450-hydrolase fusion enzyme MpaDE9, and the peroxisomal acyl-CoA hydrolase MpaH9. The whole pathway is elegantly co-mediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic pathway, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.