Compartmentalized biosynthesis of mycophenolic acid

Abstract

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. 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 the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC' and O-methyltransferase MpaG', the Golgi apparatus-associated prenyltransferase MpaA', the endoplasmic reticulum-bound oxygenase MpaB' and P450-hydrolase fusion enzyme MpaDE', and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH'. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.

Keywords: biosynthesis; compartmentalization; fungal natural product; mycophenolic acid; peroxisomal β-oxidation.

Fig. 1.

MPA biosynthetic pathway. Bold solid, solid, and dashed arrows indicate the major, minor, and shunt pathways, respectively. The newly installed functional groups are colored red.

Fig. 2.

HPLC analysis (254 nm) of Ao_M-2-3 precursor feeding experiments and Pb₈₆₄ knockout mutants. (A) i, standards; ii, the extracellular extract of Ao_M-2-3-mpaDE′/2; iii, the extracellular extract of Ao_M-2-3-pTAex3/2 as the control of ii; iv, the intracellular extract of Ao_M-2-3-mpaA′/4; v, the intracellular extract of Ao_M-2-3-pTAex3/4 as the control of iv; vi, the extracellular extract of Ao_M-2-3-mpaA′/4; vii, the extracellular extract of Ao_M-2-3-pTAex3/4 as the control of vi. (B) i, standards; ii, the extracellular extract of Pb₈₆₄; iii, the intracellular extract of PB₈₆₄; iv, the extracellular extract of Pb₈₆₄-ΔmpaB′; v, the intracellular extract of Pb₈₆₄-ΔmpaB′; vi, the extracellular extract of Pb₈₆₄-ΔmpaH′; vii, the intracellular extract of Pb₈₆₄-ΔmpaH′. (C) i, standards; ii, the extracellular extract of Ao_M-2-3-mpaA′-mpaB′/4; iii, the extracellular extract of Ao_M-2-3-mpaA′-mpaB′-mpaH′/4; iv, the extracellular extract of Ao_M-2-3-mpaA′-mpaB′-mpaH′^△GKL/4. (D) Quantitative analysis of the production of 6 and the derivatives of 5. i, Ao_M-2-3-mpaA′; ii, Ao_M-2-3-mpaA′-mpaB′; iii, Ao_M-2-3-mpaA′-mpaB′-mpaH′; iv, Ao_M-2-3-mpaA′-mpaB′-mpaH′^△GKL. The amount of each compound was calculated by plotting its integrated peak area to the corresponding standard curve, and chloramphenicol was used as an internal standard.

Fig. 3.

High-resolution confocal images for subcellular localization of MpaH′, MpaB′, MpaDE′, and MpaA′ in Ao_M-2-3. (A) GFP-MpaH′ localization. (B) Peroxisomal localization of RFP^SKL. (C) Merged images of A and B in bright field. (D) GFP-MpaH′^△GKL localization. (E) MpaB′-GFP localization. (F) Localization of ER by ER-Tracker Red. (G) Localization of multiple nuclei by DAPI. (H) Merged images of E–G in bright field. (I) MpaDE′-GFP localization. (J) Localization of ER by ER-Tracker Red. (K) Localization of multiple nuclei by DAPI. (L) Merged images of I–K in bright field. (M) GFP-MpaA′ localization. (N) Localization of Golgi complex with CellLight Golgi-RFP. (O) Merged images of M and N in bright field. (Magnification: 20 μm.)

Fig. 4.

Schematic compartmentalized MPA biosynthesis (solid arrows indicate the major pathway, and dashed arrows indicate the shunt pathways), which is sequentially mediated by the cytosolic polyketide synthase MpaC′, the ER-bound P450-hydrolase fusion enzyme MpaDE′, the Golgi apparatus-associated prenyltransferase MpaA′, the ER-bound oxygenase MpaB′, the cytosolic O-methyltransferase MpaG′, and the long-chain fatty acid acyl-CoA ligase PbACL891, the β-oxidation machinery, and the acyl-CoA hydrolase MpaH′ in peroxisomes.