# Enhancing the β‐Oxidation‐Like Pathway for the Optimal Production of the Immunosuppressant Mycophenolic Acid

## TL;DR

This study explores how fungi use a modified β-oxidation pathway to produce the immunosuppressant mycophenolic acid, offering new strategies for synthetic biology.

## Contribution

The first functional reconstitution of a fungal β-oxidation-like pathway for mycophenolic acid biosynthesis is demonstrated.

## Key findings

- Five peroxisomal enzymes mediate two rounds of side-chain cleavage to form mycophenolic acid.
- Overexpression of key enzymes increased MPA production by 50% in Penicillium brevicompactum.
- The pathway mirrors β-oxidation but is adapted for biosynthesis, offering a new model for metabolic engineering.

## Abstract

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.

The most elaborate known example of β‐oxidation‐like pathway recruitment in fungi is reported, involving a 10‐step oxidative chain‐shortening cascade catalyzed by five dedicated enzymes to generate the pharmacophore of mycophenolic acid (MPA)—a novel clinical immunosuppressant. Our findings illustrate how nature repurposes catabolic enzymes for complex biosynthesis, offering fundamental insights into biocatalysis and metabolic engineering for unnatural pathway design.