Biosynthesis of Physcion and Identification of an O-Methyltransferase with C6–OH Selectivity in <i>Aspergillus chevalieri</i> 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 <i>Phy</i> biosynthetic gene cluster responsible for physcion biosynthesis was identified from the genome of high-yield physcion strain <i>Aspergillus chevalieri</i> BYST01. We reconstructed the biosynthesis of physcion via heterologous expression of <i>PhyFGL</i> in <i>Aspergillus oryzae</i> NSAR1. Of note, the PT domain of <i>PhyG</i> catalyzes the selective ring closure to form two distinct polyketide scaffolds (<b>1</b> and <b>7</b>) and for the first time to report the biosynthetic pathway of compound pannorin C (<b>1</b>). 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 <i>AO-PhyGF</i> and <i>AO-PhyGFL</i> revealed that the expression of the <i>PhyL</i> gene led to extensive alterations in the secondary metabolism of <i>A. oryzae</i> 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.