Biosynthesis of Physcion and Identification of an O‑Methyltransferase with C6–OH Selectivity in Aspergillus chevalieri BYST01

Physcion, a polyketide natural product derived from plants
and
microorganisms, has been commercially approved as an agricultural
fungicide for the prevention and treatment of powdery mildew. However,
the long planting period and complex extraction process from plants
limit the yield of physcion. Here, the Phy biosynthetic
gene cluster responsible for physcion biosynthesis was identified
from the genome of high-yield physcion strain Aspergillus
chevalieri BYST01. We reconstructed the biosynthesis
of physcion via heterologous expression of PhyFGL in Aspergillus oryzae NSAR1. Of note,
the PT domain of PhyG catalyzes the selective ring
closure to form two distinct polyketide scaffolds (1 and 7) and for the first time to report the biosynthetic pathway
of compound pannorin C (1). In addition, in vitro and
in vivo enzymatic assays demonstrated that PhyL had the capability
to catalyze the stereoselective methylation of C6–OH. The physiological
biosynthetic pathway was further rationally engineered by improving
the catalytic efficiency of O-methyltransferase (OMT)-PhyL by 2.64-fold
through site-directed mutagenesis. Subsequently, the titer of physcion
reached 152.81 mg/L in shake-flask fermentation through optimizing
the cultivation conditions and alkaline treatment of the fermentation
broth. Furthermore, the novel CYP-PhyE could with regioselectivity
catalyze symmetrically oxidative phenol coupling (OPC) of monomeric
polyketone to form 10,10′-dimers. Finally, differential expression
analysis of transcriptome between AO-PhyGF and AO-PhyGFL revealed that the expression of the PhyL gene led to extensive alterations in the secondary metabolism of A. oryzae NSAR1 and upregulating the expression level
of ABC transporters, promoting the translocation of host metabolites.
Thus, our study provides a foundation for further improving the production
of physcion via a highly efficient route.