Enhancing the <i>β</i> ‐Oxidation‐Like Pathway for the Optimal Production of the Immunosuppressant Mycophenolic Acid

The recruitment of catabolic β-oxidation enzyme cascades and their reaction logic for natural product biosynthesis remains underexplored, representing a significant opportunity for synthetic biology to engineer novel pathways for structurally unique metabolites. In this study, the first functional reconstitution of the fungal β-oxidative cascade responsible for assembling the immunosuppressant mycophenolic acid (MPA) is reported. Through in vitro enzyme assays, five peroxisomal enzymes are identified that cooperatively mediate two iterative rounds of side-chain cleavage of the biosynthetic precursor MFDHMP-3C and revealed a key oxidative strategy for pharmacophore formation of MPA. These enzymes catalyzed sequential oxidation, dehydrogenation, hydration, reduction, isomerization, and reverse Claisen condensation reactions, mirroring canonical β-oxidation while adapting it for biosynthetic purposes. Furthermore, integrated overexpression of the rate-limiting peroxisomal acyl-CoA oxidase PbACOX323, peroxisomal biogenesis factor PbPex337, and endoplasmic reticulum (ER)-localized oxygenase MpaB' in Penicillium brevicompactum NRRL864 increased MPA production by 50% (from 0.77 to 1.15 g L^-1), demonstrating the biotechnological efficacy of pathway optimization. This work establishes the first example of a full β-oxidation-like enzyme cascade in fungal natural product biosynthesis, providing a paradigm for the evolutionary repurposing of catabolic modules to drive synthetic innovation.